Bone and mineral metabolism


Bone and mineral metabolism


Bone and mineral metabolism


Bone and mineral metabolism

6.1 Bone metabolism

Lothar Thomas

6.1.1 Bone structure

Bone is a special connective tissue hardened by mineralization with calcium phosphate in the form of hydroxyapatite [Ca5(PO4)3(OH)2]. Bone is composed of approximately 70% mineral and 30% organic matter (cells, collagen and non-collagenous proteins).

Bone has important functions, because it provides rigidity and shape, protection and support for body structures, and aids locomotion. Bone tissue is a dynamic structure undergoing constant remodeling. The bone turnover allows the bone tissue to repair itself and to adapt to forces placed on it. During childhood, bone turnover is very high and formation outweighs resorption. In young adulthood, formation and resorption are in equilibrium, but after age 35, there is a net loss. The bone’s overall mechanical properties are a combination of the rate of bone turnover, collagen matrix, structure, size, geometry, and the bone density. In order for the strength of the bone to be maintained, the bone turnover must be carefully regulated /1/.

The human skeleton is made up of long bones, such as humerus, tibia and femur, and flat bones, such as skull, ileum and scapula. There are two main histological types of mature bone /1/:

  • The cortical or compact bone, which comprises up to 85% of the bone mass has a dense ordered structure. Cortical bone is found mainly in the shaft of long bones and the surfaces of flat bones. It is composed of bone laid down concentrically around central canals (Haversian systems). The canals contain blood vessels, lymph vessels, nerves and connective tissue. A concentric layer of rings or lamellae of bone matrix surround each Haversian canal. Tiny spaces (lacunae) containing osteocytes are within the lamellae.
  • The cancellous or trabecular bone is lighter than the cortical bone and has an irregular structure. Trabecular bone forms the ends of long bones and the inner parts of flat bones. It contains interconnecting plates and bars called trabeculae, with intervening marrow lending it a honeycomb appearance. The trabeculae are aligned along lines of stress; this connectivity adds considerably to its strength. The collagen fibres are parallel to one another arranged.

In general, each bone has an outer layer of cortical bone overlying trabecular bone and the medullary cavity /1/. The cortical bone has an outer membrane called the periosteum. The periosteum has two layers:

  • The outer fibrous layer
  • The inner cell layer with osteogenic potential which lays down new bone allowing the bone to enlarge, a process known as periosteal apposition.

The inner surface of the cortex has another lining called the endosteum. Bone tends to undergo resorption from the endosteal surface.

Both the periosteum and the endosteum contain the multicellular unit of bone which comprises osteocytes, osteoblasts and osteoclasts and their precursors. Osteoblasts and osteoclasts function in a coordinated manner, by their respective bone forming and resorbing activities, to carryout remodeling, growth and repair /1/.

6.1.2 Bone turnover (remodeling)

Bone turnover is a dynamic process, which constantly renews the bone and maintains its mechanical competence. Each year, 5–10 % of the bone of the skeletal system is renewed in cycles. The term modeling is restricted to the period of skeletal growth, when the size and shape of the bone is determined. The process of bone turnover in adults is known as remodeling. Bone remodeling is part of the body’s way to manage mineral (i.e., calcium and phosphate) homeostasis, and a mechanism for repair and adaption /2/. The basic multi-cellular unit (Bone Remodeling Unit, or BRU) of remodeling, comprises the osteocytes, osteoblasts, and osteoclasts. The activity of the BRUs is regulated by mechanical forces, bone cell turnover, hormones (e.g., parathyroid hormone, growth hormone, calcitonin) and local factors /2/. The remodeling is partially initiated by the osteocytes (Fig. 6.1-1 – Sequence of bone remodeling), which detect mechanical stress and respond to biochemical stimuli. Activation results in the lining cells of the endosteal surface being retracted, and digestion by matrix metalloproteinases of the endosteal membrane /1/. Osteoclasts are then recruited, followed by fusion of activated osteoclasts to become multi-nucleated osteoclasts /1/. The activated osteoclasts cause resorption of the underlying bone forming a resorption cavity. The activation and resorption process takes 10–15 days. In the next step osteoblasts are recruited to the cavity, lay down new osteoid which becomes calcified. The entire process is completed after 3–6 months. The rate of bone turnover is dependent on the type of bone, being highest in sites where trabecular bone predominates (vertebrae) and lowest in cortical bone (hip) /1/.

6.1.3 Regulation of bone metabolism

Bone metabolism is regulated via central mechanisms and the local control of the osteoblasts, osteocytes and osteoclasts /12/. Osteoblast

During skeletal development osteoblast differentiation and bone matrix deposition occurs. The spatial and chronological coordination for this involves the interaction of endocrine, paracrine and autocrine factors. Included are the epidermal growth factor (EGF), the osteoblast-stimulating factor-1 (OSF-1), parathyroid hormone, growth hormone, prostaglandin and insulin-like growth factors (Fig. 6.1-2 – Osteoblast differentiation starting from the stem cell). The development of the skeleton requires the chronologically and spatially controlled molecular interaction between Wnt/catenin and transforming growth factor-β/BMP bone morphogenetic protein mediated signaling pathways /1/. The expression of Runx2 is required for multi potent stem cells to differentiate into osteoblastic lineage /1/. Runx 2 is a key osteoblast differentiation transcription factor. A suppressing effect on the Wnt/β-catenin signal pathway has the Dickkopf-1 protein. Other details are listed in Tab. 6.1-1 – Significant factors in the regulation of osteoblasts. Osteocyte

Osteoblasts, encased by the matrix that they themselves synthesize become osteocytes, terminally differentiated cells. Osteocytes have less matrix forming activity, and the alkaline phosphatase activity and basophilic nature of its cytoplasm is reduced. The cells occupy small lacunae within the bone matrix and constitute more than 90% of the bone cells. Osteocytes, isolated by bone matrix from each other communicate through protoplasmic extensions via lacuno-canaliculi with neighboring cells. These processes may have the potential to stimulate bone resorption. Osteocytes act as mechanosensory cells and are responsible for the maintenance of bone structure and mass. By far the most abundant matrix protein in the osteocyte environment is type-1 collagen. The phenotype of the osteocyte and the formation of osteocyte processes depend on cleavage of type-1 collagen /3/. Osteoclast

Osteoclasts are large end-differentiated multi nucleated cells derived from bone marrow macrophages of the hematopoietic lineage /12/. The unique function is resorbing bone matrix. Osteoclastogenesis is dependent on cytokines generated in the bone micro environment. Osteoblasts control osteoclast formation through the expression of cytokines like RANKL (receptor activator of nuclear factor-kB ligand) and M-CSF (monocyte-colony stimulation factor). M-CSF is important for the proliferation of osteoclast progenitors. RANKL controls the differentiation process by activating the RANK receptor, the osteoclast associated receptor (OSCAR) and the triggering receptor expressed on myeloid cells-2 (TREM-2). In contrast to this, osteoprotegerin, a receptor of the tumor necrosis factor receptor superfamily, suppresses the formation of osteoclasts (Fig. 6.1-3 – Mechanisms of osteoclast activation). Additional details on this are listed in Tab. 6.1-2 – Significant factors in the regulation of osteoclasts.

Osteoclast formation is also controlled by circulating hormones like parathyroid hormone, calcitriol, trans growth factor-β and estrogens.

The osteoclastic bone resorption is important for modeling and remodeling the bone and for the calcium homeostasis.

Normal bone physiology depends on the interaction of osteoblasts and osteoclasts. There are, however, diseases in which this interaction is disrupted, such as osteoporosis, Paget’s disease, inflammatory arthritis, as well as bone tumors and bone metastases. Bone matrix

The organic bone matrix produced by the osteoblastic cells contains (Fig. 6.1-4 – Collagen synthesis):

  • 90% collagen type 1
  • 5% proteoglycans (chondroitin sulfate, glucuronic acid, heparan sulfate, decorin, biglycan, versican), cell adhesion molecules (fibronectin, thrombospondin, vitronectin, osteopontin, bone sialoprotein), γ-carboxylated proteins (1% osteocalcin, matrix Gla protein, protein S)
  • 3% growth factors and 2% osteonectin.

Osteopontin (OPN)

OPN, also known as secreted phosphoprotein-1, is a large phosphoglycoprotein profibrotic adhesion molecule with several domains, one of which has binding affinities for integrins (e.g., the vitronectin receptor) /4/. The molecule is attacked at several points by thrombin and matrix metalloproteases, resulting in fragments which either promote or reduce cellular adhesion. OPN binds CD44, which interacts with the ERM (ezrin, radixin, moesin) family of adapter proteins that link to actin in the cytoskeleton.

Bone sialoprotein (BSP)

BSP is a cell adhesion molecule produced by osteoblasts and is stored in the organic bone matrix. BSP is part of the non-collagen connective tissue matrix of bones, dentin and calcifying cartilage. If BSP is increased in serum, this can best be correlated with bone-resorptive processes /5/. Since thrombocytes also contain BSP, changes in the thrombocyte count also affect the BSP concentration in serum. Mineralization of the organic bone matrix

Active osteoblasts synthesize the bone matrix which is composed of organic (osteoid) and inorganic (mineral) components /3/. The osteoid consists predominantly of type-1 collagen, with small amounts of proteoglycan, lipids and several non collagenous proteins (osteocalcin, fibronectin, osteonectin and beta-carboxyglutamic acid containing protein). The osteoid comprises about one third of the total skeleton weight.

The inorganic mineral phase consists of crystals of hydroxyl apatite. The crystal is small and in the order of 200 by 30 to 70 Angstroem. The mineralization of the osteoid takes place via the osteoblastic exocytotic release of vesicles, in which the hydroxyapatite nucleation takes place in the matrix. Together with the matrix components of the osteoid, crystallized matrix vesicles form the bone matrix outside the osteoblasts /6/. The speed of the of the hydroxyapatite crystallization from amorphous calcium phosphate is controlled by matrix components. The resorptive cavity created by the osteoclast is often the site of subsequent osteoblastic activity which fills the cavity with new bone.

In lamellar bone, the mineralization appears to occur directly mediated by the lining osteoblasts without the exocytotic release of vesicles.

6.1.4 Hormonal modulation of bone metabolism

During adulthood, bone is remodeled in order to adapt acute changes in calcium homeostasis, bio mechanical demands and to renew and repair old bone substance and osseous micro fractures. Hormones that systemically affect the bone metabolism are growth hormone/IGF-1, estrogens and testosterone (Tab. 6.1-3 – Effect of estrogens and androgens on bone metabolism), glucocorticoids, thyroid hormone, parathyroid hormone and 1,25(OH)2D. See Section 6.6.5 – Clinical significance). Growth hormone (GH)

In childhood, GH promotes skeletal growth via a direct effect on the mature chondrocytes of the epiphyseal plate (i.e., secretion of insulin-like growth factor 1 (IGF-1)) by these cells is increased. IGF-1 together with transforming growth factor-α (TGF-α), which is also produced by chondrocytes, exerts a mitogenic effect (in a paracrine manner) on the chondroblasts and promotes their differentiation with a resultant expansion of the cartilage layer in the epiphyseal growth plate. Preosteoblasts and preosteoclasts also have GH receptors. The cell proliferation and differentiated cell functions of osteoblasts (e.g., the synthesis of collagen type 1 and the excretion of IGF-1) are positively influenced by GH. In the adult skeleton, GH/IGF-1 thus maintains both the processes that are required for the continuous remodeling of bone tissue (i.e., both formation as well as the resorption of bone) /8/. Estrogens

Estrogens influence osteoblasts and osteoclasts via two estrogen receptors (ER). The ERα is an activator and the ERβ is an inhibitor of the estrogen effect, because ERβ uses the ERα to form a heterodimer and thus deactivates it. The relative expression of both ERs on the bone cell decides its estrogen response. The density of ERα is greater in the developing compact bone type and of ERβ in the cancellous (trabecular) type. In estradiol deficiency, the activity of the osteoclasts is higher than of the osteoblasts with the net effect of osteopenia /9/.

Estrogen deficiency is the most important factor in the pathogenesis of the post menopausal bone loss. Post menopausal women with estradiol values of > 10 ng/L have a normal bone fracture rate, with values of ≤ 10 ng/L a moderate increased rate and with < 5 ng/L a strong increased rate, especially in the spinal column. If, several years after menopause, a low dosage of transdermal estrogen is supplied (tape with 25 μg estradiol release per day), the serum value of estradiol maintains at about 20 ng/L. The benefit for the patient is an increase of bone mineral density (8% in 3 years), especially in the spine, which can scarcely be improved upon even by increasing the estradiol dosage (9% in 3 years with 50 μg estradiol release per day) /10/.

Hormonal contraceptives do not cause an increased risk of bone fracture, but they can disturb the development of peak bone mass if they are taken within the first 3 years after menarche /11/.

The supply of estrogen is also important for men in suspicion of developing osteoporosis, because they also have osteoblast and osteoclast estrogen receptors. Osteoporosis is cumulatively diagnosed for an estradiol concentration below 13 ng/L /11/. Androgens

Androgens stimulate the proliferation and differentiation of osteoblasts both directly and via the GH/IGF-1 axis. The signal is transmitted via androgen receptors, which are present in low densities on the osteoblasts. The effects of androgen are especially evident from puberty on when achieving the maximum bone mass of the skeleton, which depends on timely excretion of androgens. Thus, delayed puberty in men is associated with reduced bone mass /12/. Androgens decrease bone resorption of osteoclasts indirectly by inhibition the recruiting of osteoclast precursor cells. This happens due to reduced secretion of IL-6 and prostaglandin E2 by osteoblasts and stromal cells.

In addition to the androgen receptor, bone cells also have 5α-reductase activity, which is responsible for the conversion of testosterone to dihydrotestosterone (DHT). DHT is the most important biologically active metabolite of testosterone in the tissues. Testosterone can also be aromatized to estradiol catalyzed by aromatase; bone cells have aromatase activity.

Similar to ovarectomy, orchi ectomy will lead to increased bone resorption and rapid bone loss. Glucocorticoids

Physiological plasma glucocorticoid concentrations promote important osteoblastic functions (type I collagen synthesis, ALP synthesis) and simultaneously suppress the differentiation of osteoclast precursor cells into active osteoclasts. Therefore, physiological glucocorticoid concentrations exert a stabilizing effect one bone metabolism without any negative effects on calcium homeostasis /13/.

High glucocorticoid concentrations over a prolonged period of time have an unfavorable effect on calcium homeostasis. The cancellous bones are especially affected. After glucocorticoid treatment is started, there is a rapid phase of bone loss, followed by a slower phase of demineralization. The extent depends on the dosage and the duration of the treatment. However, there are also patients with normal bone density who undergo glucocorticoid treatment. In patients with Cushing’s syndrome, fractures are more frequent the longer the disease persists. Thyroid hormones

Physiological thyroid hormone concentrations promote bone turnover. This is caused by stimulation of osteogenic cells as mediated by the osteoblast triiodothyronine (T3) receptor. As a result of stimulation, these cells produce signal peptides which exert a positive effect on osteoclast differentiation /14/. Hence, bone resorption increases although no net loss in bone mass occurs because of the coupled new bone formation and the T3 induced stimulation of specialized osteoblast functions.

In the case of permanently increased thyroid hormone concentrations, the activation of osteoclasts may exceed the bone-producing capacity of the coupled bone formation since T3 also exerts an inhibitory effect on osteoblast proliferation; consequently, a loss in bone mass can occur. Juvenile hypothyroidism causes stunted growth. Parathyroid hormone, calcitriol, calcitonin

Bone is subject to constant remodeling in adulthood:

  • To adapt to acute changes in calcium homeostasis
  • To adapt to bio mechanical stress
  • To renew and repair old bone substance and bones with micro fractures.

The bone metabolism needs sufficient calcium supply from the diet and fluid to maintain these functions. The absorption of calcium can fluctuate between 25% and 70% of calcium in the diet, depending on the calcium content and components (complexing agent, pH) of the food and the presence of gastrointestinal disorders. Small changes in the plasma calcium concentration due to calcium malnourishment, vomiting, diarrhea, and malabsorption are registered by the parathyroid cells and are responded with an increased secretion parathyroid hormone (PTH) and of 1,25 (OH)2D (calcitriol). Calcium in bone is mobilized with a consecutive increase in the serum calcium level.

The mechanisms are /15/:

  • PTH increases the number and the resorptive capacity of the osteoclasts. PTH also suppresses the activity of the osteoblasts and thus prevents storage of calcium in the bones. The increase in osteoclast activity occurs indirectly via the PTH stimulation and directly due to the activation of the osteoclasts. The latter have PTH receptors like the osteoblasts.
  • Mediated by calcitonin, because this hormone constitutes to the stabilization of the calcium concentration in plasma (see Section 28.9 – Calcitonin (CT)). Its primary effect is the suppression of the osteoclast-conveyed bone resorption as a response to a systemic increase of the calcium level. Calcitonin mediates its effects via the adenylate cyclase pathway. In the small intestine, calcitonin promotes the transcellular absorption of calcium by increasing the calbindin expression of the enterocytes. In the kidneys calcitonin enhances the re absorptive capacity for calcium in the thick, ascending part of the loop of Henle.

6.1.5 Markers of bone metabolism

In metabolic bone disease (Paget’s disease, rickets and osteomalacia, primary and secondary hyperparathyroidism) and osteoporosis biomarkers of bone metabolism and bone turnover are important clinical tools in patient management. The biomarkers are supplementary to imaging procedures, bone mineral density measurements, and possible histomorphological examinations of bone aspirates. Routine investigations and bone markers for the diagnosis and monitoring of bone disorders are shown in Tab. 6.1-4 – Biomarkers for the diagnosis and monitoring of metabolic bone disease and osteoporosis.

Bone turnover markers reflect whole body rates of bone resorption and bone formation. The markers provide a dynamic assessment of the skeleton and can provide real-time assessment of bone remodeling. Bone turnover markers are differentiated in bone resorption and bone formation markers /16/.

Bone resorption markers (refer to Section 6.7 – Biochemical bone markers)

The markers that are preferably measured are those which show greater activation of the osteoclasts than of the osteoblasts (Fig. 6.1-5 – Bone formation and bone resorption markers):

The most important indication for bone resorption markers such as CTX, PYD and DPD is monitoring the course and assessment of the progress in treatment of osteoporosis. The markers give information:

  • Whether a high turnover bone metabolism exists
  • How high the extent of the bone resorption is
  • Whether there is a response to treatment.

Within the scope of monitoring osteoporosis, the optimal points in time of the response to treatment with bisphosphonates is 1 month after the start of the treatment and 6 months for estradiol substitution.

Bone resorption markers are also indicated:

  • For osteomalacia/rickets
  • For monitoring of Paget’s disease
  • For estimating the bone participation in hyperparathyroidism.

For chronic kidney disease (CKD) in stage 3–5, bone resorption markers are not recommended for diagnosing a CKD mineral bone disease (CKD-MBD) /17/.

Bone formation markers (see Section 6.7 – Biochemical bone markers)

In the serum, the markers that are preferably measured are those which show greater activation of the osteoblasts than the osteoclasts:

  • Alkaline phosphatase, especially the bone isoform (bone ALP). Thus, according to the KDIGO, the assay of the ALP or bone ALP in CKD patients in stages 3 to 5 is recommended annually for diagnosing CKD-MBD.
  • Osteocalcin (see Section 6.9 – Osteocalcin (OC)).
  • N-terminal pro peptide of type 1 collagen (P1NP).

These markers are important, especially the bone ALP in combination with the PTH assay, if adynamic bone disease is suspected in dialysis patients.

6.1.6 Clinical tabular part

Tables 6.1-5 to 6.1-14 list laboratory findings in physiological conditions and for diagnosing, differentiating and monitoring bone diseases:


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6.2 Calcium

Lothar Thomas

The hormonal regulation of calcium metabolism as well as the interrelated phosphate metabolism is complex. Reciprocal relations between the small intestine, skeleton, kidneys and the endocrine system, in particular the parathyroids, maintain calcium and phosphate homeostasis.

Without the exception of tetany, clinical symptoms caused by calcium status disorders are often initially misinterpreted. Therefore, the diagnosis is often established incidentally during the routine determination of serum calcium and phosphate or if secondary changes, especially of the kidney and bone are present.

The determination of serum calcium and phosphate is recommended for early detection and differentiation of bone disorders and in chronic renal disease. The determination of parathyroid hormone is an important follow-up investigation of hyper- and hypocalcemia.

6.2.1 Calcium and ionized calcium in blood

The calcium consists of three fractions:

  • Free or ionized calcium (iCa), it accounts for 50% of calcium
  • Protein-bound calcium, most of which is bound to albumin with only a small portion bound to globulins; it accounts for 45% of calcium
  • Complex bound calcium, the portion bound to anions, especially phosphate, citrate and bicarbonate accounts for 5% of calcium.

In routine diagnostics calcium is primarily determined in serum or plasma. The assay of iCa in anticoagulated whole blood or plasma is mostly requested secondarily to clarify hypocalcemia or, more rarely, hypercalcemia.

Total Calcium

Under routine diagnostic conditions the measurement of calcium is easier than that of iCa, but has the disadvantage that serum calcium is greatly influenced by the concentration of protein, especially albumin. Because albumin is responsible for 80–90% of the protein-bound calcium fraction. Accordingly, a decrease in albumin by 10 g/L results in a decline of serum calcium by 1 mg/dL (0.25 mmol/L).

Ionized Ca (iCa)

The iCa is an indicator of the biologically active calcium, because its plasma concentration is directly regulated by PTH and 1,25(OH)2D. Therefore, iCa responds sensitively to disturbances in calcium homeostasis, but it cannot be determined in every laboratory. The iCa is influenced by pH changes. A shift of the pH of 0.1 in either direction causes an inverse iCa change of 0.05 mmol/L. Pre-analytical sources of errors, which cause a pH shift in vitro, must therefore be taken into consideration. The most important biological causes which cause a change in pH in vitro are:

  • The loss of CO2 from the specimen into the surrounding air. Due to solution of CO2 in blood there is a rise in pH with a consecutive decrease of iCa. The sample must therefore collected under anaerobic conditions. The sample tubes must be completely filled and the sample must be hermetically sealed.
  • Formation of lactate in the specimen due to glycolysis. As a result, the pH decreases and the concentration of iCa increases. This can be prevented by the rapid separation of the blood cells from the plasma or by the addition of glycolysis inhibitors such as sodium fluoride. Indication

Screening: every 2 years after age 50, including recording height and weight (concern: osteoporosis; a decline in height by more than 1 cm every 2 years necessitates further examinations).

Critically ill patients: intensive-care patients, surgical patients during and after major operations.

Newborns: in the event of premature delivery, maternal diabetes, asphyxia, hypo tension, cramps, hypoglycemia, sepsis.

Tetanic syndrome: investigation of the hypocalcemic form.

Bone: spontaneous fractures, osteoporotic fractures (femoral neck, spine, radius), bone pain, radiographically detected bone changes, dental changes, growth disorders.

Kidney: nephro- or urolithiasis, nephro calcinosis, polydipsia, polyuria, chronic kidney disease, dialysis patients.

Neuromuscular: tetany, seizures, suspicion of hypoparathyroidism after thyroid surgery, headache, muscular weakness.

Parathyroids: suspicion of hyperparathyroidism, hypoparathyroidism and pseudohypoparathyroidism.

Genetic disorder: suspicion of autosomally dominant hypocalcemia.

Psyche: fatigue, loss of drive, lethargy, depression, anorexia.

Stomach and intestines: peptic ulcer disease, pancreatitis, gall stones, recurrent diarrhea, malabsorption, obstipation.

Skin and skin appendages: changes involving the skin, nails, hair, hyperpigmentation of skin.

Lung: sarcoidosis, tuberculosis, other granulomatous diseases.

Tumor: weight loss, malignoma, lymphoma, treatment with cytostatics, radiotherapy.

Endocrine: thyroid, testicular, ovarian, adrenocortical diseases.

Medications: intake of vitamin D and its metabolites or analogues, vitamin A, anti-epileptic drugs, corticosteroids, thiazides, digitalis. Method of determination

Total calcium is easier to determine than ionized calcium in clinical routines. Total calcium (Ca)

Atomic absorption spectroscopy (AAS)

AAS is recommended as the reference method for the measurement of calcium /1/.

Principle: in AAS calcium is not excited in the flame but dissociated from its chemical bonds and placed in an unexcited ground state as neutral atom. The neutral atom has low energy level and is capable of absorbing radiation corresponding to its own line spectrum. Energy of a line specific spectrum is absorbed when atoms with the same line-specific spectrum absorb radiation. In this way, AAS is the converse of the emission procedure for determining metals e.g., flame spectrophotometry. The components of the AAS are:

  • Hollow cathode lamp; this generates the element-specific linear spectrum and is different for the individual element
  • A gas flame or electrically heated graphite furnace cuvette for specimen atomization at high temperature
  • Photomultiplier that transforms the decrease of the light from the hollow cathode lamp, when a proportion is absorbed from the ground-state atoms in the flame, into an electrical signal.

In AAS, the absorption change measured at the signal display unit is proportional to the concentration of the element in the specimen.

Flame photometry

Principle: in flame emission photometry the specimen to be measured is diluted with A. bidest and exited by an acetylene gas flame. Light is emitted at a specific wavelength as calcium returns to its lower energy level. Due to a partial overlap of the emission bands of sodium and calcium at 622 nm, a compensation solution which contains a sodium concentration that is identical to that of normal serum is used to compensate the emission of sodium.


Principle: calcium forms chromophores with the metal complexing dyes o-cresolphthalein complexone /2/ and arsenazo III /3/. At pH 10–12, the reaction of o-cresolphthalein complexone with calcium forms a red complex, which is measured at 570–575 nm. The complex is stabilized by adding KCN; this also eliminates interferences caused by heavy metals. Interference due to magnesium ions is prevented by the presence of 8-hydroxyquinoline. Ionized calcium (iCa)

Calcium-selective electrode /4/

Principle: the concentration of iCa is measured using an electrode arrangement, which consists of a flow-through calcium-selective electrode and an Ag/AgCl reference electrode. An ion-exchanger as the active phase of the measurement electrode is separated from the specimen to be analyzed by a porous membrane and within the electrode it is in contact with a millimolar CaCl2solution; an electrode immersed in this solution leads the electrical potential to a measuring device. When the specimen flows through the calcium-selective electrode, the iCa reacts with the ion-exchanger.

By moving charged particles over the porous membrane, the active electrode phase becomes increasingly charged compared to the serum. A difference in potential forms, which is measured against the constant potential of the reference electrode. Between the potential recorded at the measuring device and the logarithm of iCa activity, there is a linear relation.

The measurement of iCa is influenced by the pH value of the blood, because iCa and H+ ions compete for protein binding sites. When the pH increases, the concentration of iCa decreases. Some analyzers simultaneously measure iCa and pH and correct the iCa to pH 7.4 for samples with a changed pH value. This is not recommended because 70% of the patients with low iCa are not detected /5/. Specimen

Total calcium

Serum, plasma (ammonium heparinate): 1 mL

Ionized Ca

  • Whole blood (calcium heparinate), the measurement should be carried out within 40 min. after sampling /6/
  • Serum or plasma (Ca-saturated heparin) if centrifugation and measurement are done immediately after sampling
  • Serum or plasma (Ca-saturated heparin). Sampling in vials with separator gel and then centrifugation if measurement is not earlier possible as 24–72 hours after sampling. Reference interval

Refer to Tab. 6.2-1 – Reference intervals for calcium in serum and plasma. Clinical significance

The suggested calcium intake for people without kidney disease is 1,000–1,300 mg per day (25 to 32 mmol).

The calcium metabolism can be assessed based on total calcium or iCa. Approximately 40% of total calcium is bound to albumin, but only the iCa is biologically active in the extracellular fluids. The total calcium level is clinically equivalent to the iCa if a change in protein concentration and dysproteinemia are ruled out.

The determination of iCa is always preferable in the following diseases and conditions /4/:

  • For critically ill patients and if an intravenous calcium substitution is considered
  • If hypoproteinemia or dysproteinemia is present (e.g., in pregnant women, newborns, renal or intestinal protein loss, malabsorption syndrome, multiple myeloma, chronic inflammation)
  • In the final stage of chronic kidney disease
  • In cases of hypercalcemia, especially if PTH is normal and there are no signs of tumor
  • To verify hypocalcemia postoperatively or intraoperatively (thyroid and parathyroid operation, open heart surgery, liver transplantation)
  • If there is a massive transfusion of citrated blood or of fresh frozen plasma
  • In cases of increased PTH and normal calcium as a result of a vitamin D deficiency, insufficient enteral calcium absorption or hypercalciuria.

According to a study /12/ approximately 72–76% of the iCa measurements can be spared if they are only performed in cases with total calcium levels below 8 mg/dL (2.0 mmol/L), because patients with clinically relevant iCa levels below 4 mg/dL (1.0 mmol/L) will still be detected.

If it is not possible to measure iCa, the calcium can be corrected to an albumin value of 40 g/L (recommended for dialysis patients) or a protein value of 77.6 g/L for a better assessment of hypocalcemia when there is a decreased concentration of albumin or total protein (Tab. 6.2-2 – Protein correction of serum calcium). This applies to children older than 1 year and to adults.However, Ca correction for hypoalbuminemia is not an accurate concept. Studies have shown that in patients with endocrine disorders or acid-base disturbances, patients who are critically ill, and patients undergoing hemodialysis the correction formula is not an acceptable substitute for iCa /13/.

The reference intervals for total calcium vary in literature by up to 1.6 mg/dL (0.40 mmol/L). The following is generally accepted for adults: a mean value of 9.4–9.5 mg/dL (2.35–2.38 mmol/L), an upper reference interval value of 10.5 mg/dL (2.62 mmol/L), and a lower reference interval value of 8.6–8.8 mg/dL (2.15–2.20 mmol/L).

The calcium concentration in serum is kept constant in a narrow range under the control of PTH. The relationship between iCa and PTH is transmitted via the calcium-sensitive receptor. This is expressed on the cell membrane of parathyroid cells and renal tubular cells, where it regulates the calcium reabsorption. Individual activating or deactivating mutations of the calcium-sensitive receptor genes can cause hypercalcemic or hypocalcemic disorders such as the familial hypocalciuric hypercalcemia (FHH), the neonatal severe primary hyperparathyroidism (NSHPT), or the autosomally dominant hypocalcemia with hypercalciuria (ADHH) /15/. Hypercalcemia

The prevalence of hypercalcemia in hospital patients is 0.6–1% /16/. Mild hypercalcemias up to 11.2 mg/dL (2.8 mmol/L) are mostly diagnosed incidentally and are asymptomatic. Severe hypercalcemia

If there is an increase in total calcium above 11.2 mg/dL (2.8 mmol/L) and rising values, organ systems can be negatively impacted. Since the effect of the antidiuretic hormone and the renal sodium transport are suppressed, the renal concentrating capacity is reduced. The result is sodium excretion with polyuria, exsiccation and polydipsia. The symptoms of chronic hypercalcemia are fatigue, depression, myasthenia, abdominal pain and bone pain. Hypercalcemia syndrome

In hypercalcemic syndrome a life-threatening crisis with values of more than 14 mg/dL (3.5 mmol/L) has developed due to dehydration. The consequences are limited consciousness up to a coma, exsiccosis, hyperpyrexia, oliguria preceded by polyuria, arrhythmia, encephalopathic symptoms, and bradyarrhythmias. Diseases associated with hypercalcemia

In more than 90% of cases with hypercalcemia, there is a clinically manifest disease or the hypercalcemia can be further differentiated by biochemical investigations. A small proportion of the remaining causes are related to medications. Thus, in one study /16/, in which two serum calcium values > 10.6 mg/dL (2.64 mmol/L) or at least one value ≥ 10.8 mg/dL (2,70 mmol/L) was the criterion, the underlying cause of hypercalcemia was in 46% of the cases a malignoma and in 35% the primary hyperparathyroidism. In the remaining 19% of the cases, the hypercalcemia was connected with the medication of thiazides, an increased 1,25 (OH)2D concentration or immobilization /17/.

Hypercalcemias result from the interaction of several mechanisms, such as increased intestinal absorption, increased bone resorption, exsiccosis, and reduced renal calcium excretion.

If the hypercalcemia is only temporary, the examinations must be repeated. In that case, preferably iCa should be determined in addition. Pseudo-hypercalcemia due to a hemoconcentration, often caused by a long period of venous occlusion when drawing blood, must be taken into consideration. Differentiation of hypercalcemia 

The differentiation of an unclear hypercalcemia can be possible by determining parathyroid hormone (PTH) level /18/. With few exceptions, the concentration of PTH allows the differentiation of the two most common causes of hypercalcemia (e.g., primary hyperparathyroidism (pHPT) and tumor hypercalcemia).

Increased PTH: if PTH has been increased to more than 30% of the upper reference interval value, this most likely indicates pHPT. Some rare causes are lithium-induced hypercalcemia, tertiary hyperparathyroidism, and ectopic PTH formation.

PTH slightly increased or near the upper reference interval value: in such cases the calcium excretion in 24-hour urine should be determined. If the excretion is low, familial hypocalciuric hypercalcemia must be taken into consideration.

PTH normal or suppressed: the search for a malignant tumor should be the primary aim:

  • If a tumor is diagnosed, the PTH-related peptide (PTHrP) should be determined to clarify the cause of the hypercalcemia. Increases indicate a paraneoplastic PTHrP formation which is often associated with adenocarcinoma of the lung, carcinoma in the head and neck area, of the pancreas, ovaries and the urogenital tract.
  • If a granulomatous disease is present (sarcoidosis, tuberculosis, lymphoma), the 1,25(OH)2D formation in the granulomas is often the cause of the hypercalcemia. If the serum concentration of 1,25(OH)2D is normal multiple myeloma, bone metastases, immobilization, hyperthyroidism or medication may be the cause. Biochemical findings for diseases with hypercalcemia are shown in Tab. 6.2-3 – Diseases that can cause hypercalcemia. Hypocalcemia

Hypocalcemia can be devided into causes associated with /11/:

  • Low parathyroid (PTH) levels e.g., genetic disorders that result in abnormal PTH concentrations and acquired causes that include infection, autoimmune disorders (autoimmune polyglandular syndrome type 1), destruction of the parathroid gland (after surgery or radiation therapy)
  • High PTH level e.g., vitamin D deficiency, PTH resistance, poor calcium intake or absorption, calcium loss.

Hypocalcemia is a status of serum calcium below 8.8 mg/dL (2.2 mmol/L) and of iCa below 4.0 mg/dL (1.0 mmol/L).

In cases of mild hypocalcemia, the calcium level is 8.0–8.7 mg/dL and iCa is 1.00–1.12 mmol/L.

In severe hypocalcemia, the calcium level is below 6.4 mg/dL and iCa is below 0.80 mmol/L. The calcium data applies in cases of normo proteinemia.

Hypocalcemia occurs physiologically in newborns in the first 3 days of life and is regarded as a natural stimulus for PTH secretion. The most frequent cause of a decreased calcium is hypoalbuminemia. Although in some of the cases iCa concentration is normal, hypoalbuminemia is often a symptom of diseases that are associated with a decrease in iCa. Examples for this are:

  • Proteinurias with a loss of calcium-binding protein, that result in a deficiency of 25(OH)D
  • Malabsorption syndrome, which also leads to a 25(OH)D deficiency.

In the presence of unclear hypocalcemia or hypoalbuminemia, preferably iCa can be determined separately. The measurement of calcium excretion in the 24-hour urine and of serum PTH is also important for differential diagnostics. Diseases associated with hypocalcemia

If the albumin concentration is in the reference interval, then hypocalcemia includes (Tab. 6.2-4 – Diseases that may cause hypocalcemia/11/:

  • PTH decreased; hypocalcemia is caused by a reduction of PTH-stimulated functions (i.e., the intestinal absorption of calcium, the mobilization of calcium from the bone, and the renal tubular reabsorption of calcium)
  • Pseudohypoparathyroidism (PTH elevated) which is characterized by end-organ resistance to PTH; its manifestations are comparable to those seen in hypoparathyroidism. To distinguish pseudohypoparathyroidism from secondary hyperparathyroidism, it may be necessary to perform the Ellsworth-Howard test.
  • Renal failure (PTH elevated); the following mechanisms are responsible for hypocalcemia:

a) renal phosphate retention if the glomerular filtration rate is less than 25 [ml × min–1 × (1.73 m2)–1] (PTH elevated)

b) decreased intestinal calcium absorption, caused by 1,25(OH)2D deficiency that in turn is a result of reduced 25-OHD-1α-hydroxylase activity which occurs as the mass of kidney tissue is reduced (PTH elevated)

c) resistance of the bone to PTH with reduced calcium release by osteoclasts (PTH elevated)

d) defects in the metabolism of vitamin D, and vitamin D deficiency (PTH elevated)

e) hypomagnesemia that results in decreased PTH secretion and end-organ resistance to PTH

f) hyperphosphatemia which causes 1,25(OH)2D deficiency because of the inhibition of 25-OHD-1α-hydroxylase (PTH elevated).

Hypocalcemias occur frequently in critically ill patients, especially in cases with sepsis, cardiac, renal or pulmonary failure, and after major surgery or serious burns. It may result from a shift of Ca to the intracellular compartment.

The determination of iCa is required /12/ e.g.,

  • If, in hypocalcemia, the cardiac function is to be improved by calcium infusions
  • After open heart surgery, if the high cardioplegic calcium concentration has to be normalized
  • During liver transplantation. Often many banked blood units containing citrate are administered and the serum calcium level can decrease below 2.0 mg/dL (0.5 mmol/L), because liver function can not metabolize the citrate which binds serum calcium.
  • Hyperventilation which causes metabolic alkalosis and the clinical symptom of obstetrician’s hand
  • In chronic kidney disease the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI) clinical practice guidelines advise maintaining serum levels of calcium, corrected for albumin level, within the normal range of the laboratory. Since albumin measurement is not standardized the use of albumin correction for calcium is likely to induce additional errors in bone mineral interpretation. Thus, in one study /19/ 32.6% errors of interpretation were registered. Clinical symptoms of hypocalcemia

The clinical symptoms of hypocalcemia depend on the speed of its appearance, the extent of decline, and its duration.

Symptoms depending on the calcium concentration:

  • If there is a decline of iCa below 1.0 mmol/L, slight neurological symptoms such as circumoral and acral (fingers, tongue) paresthesias will occur
  • If the concentration is below 0.60 mmol/L, cardiac arrest is likely
  • For patients with low blood pressure or low cardiac output and iCa levels of 0.8–0.9 mmol/L a calcium treatment is required /20/. Comments and problems

Sample collection

Especially if iCa is to be determined, the patient needs to be relaxed for at least 10 min. and should also be in a supine or sitting position for at least 5 min. prior to blood collection. The interval since the last food intake should be 4 hours. To determine iCa the blood must be collected anaerobically and tube must be completely filled. When the patient transitions from sitting to supine position, the serum total calcium after half an hour is 4.7% lower than in the sitting position /14/. The iCa shows no change due to the change in position.

The veins should be compressed for a short period of time only with little pressure since compression for several minutes may cause a rise of total calcium by up to 10%, especially if combined with opening and closing the fist /34/. As a result of this kind of action, there is also an increase in iCa due to acidosis.


Total calcium: serum or heparin plasma, no EDTA or citrate plasma.

Ionized calcium: lithium-heparinized whole blood, drawn in syringes or evacuated sample tubes with separator gel, are the best sample materials, if calcium-titrated heparin (40 IU heparin as the end concentration/mL of blood) is used /635/. Titration is needed because heparin binds iCa in proportion to its concentration, possibly unacceptably reducing measured concentration of iCa. One disadvantage of whole blood is that hemolysis or micro thrombi are not detected. Hemolysis above 3 g Hb/L causes a clear change in measured iCa /36/.

Serum, anaerobically collected in evacuated tubes, is the most stable specimen for iCa. However, a partly filled evacuated tube will cause pH and subsequent iCa changes due to the loss of CO2 from the specimen /36/.

Plasma has no analytical advantages over serum or whole blood /36/.

The iCa correction, which is done automatically by some analyzers to compensate for pH changes which are not the result of non-anaerobic sampling, is discussed controversial /6/.

Influence factors

Physical activity: the average rise of iCa is 0.45 mg/dL (0.11 mmol/L) after 10 min. of bicycling and 0.1 mg/dL (0.02 mmol/L) after 10 min. of stair walking /36/.

The following increases should be anticipated in the case of a change from a supine to a standing position: calcium 4.6%, iCa 1.7%, albumin and total protein 12% /13/.

Bed rest: Approximately 12 or more days of bed rest lead to an increase in iCa of 8%, but not of calcium /36/.

Intake of nourishment: after a diet, calcium falls temporarily by 5.4% due to an increase in the pH, an increase in the concentrations of protein, phosphorus and bicarbonate /36/.

Respiratory alkalosis: in the case of hyperventilation, for rise in pH by 0.1 unit, the iCa declines by 0.2 mg/dL (0.05 mmol/L).

Exsiccosis: for each increase in albumin of 1 g/dL above the upper reference interval value, 0.8 mg/dL (0.2 mmol/L) can be subtracted from the calcium level.

Circadian variation: the intraindividual changes of iCa are 4–10%. The changes are due to the effect of meals, daily variation of acid-base balance, and sleep /36/.


Calcium: storage at 9 °C for up to 1 week.

Ionized calcium: in whole blood at 4 °C for up to 4 hours, in plasma in a sealed sampling tube /5/:

  • Centrifuged but not separated from the erythrocytes up to 24 hours at room temperature
  • Separated from the erythrocytes with the use of a separator gel up to 48 hours in an anaerobic state.

6.2.2 Urinary calcium excretion Indication

Assessment of the calcium status, if serum calcium is:

  • Increased or decreased
  • Normal, but clinical symptoms are present, such as bone pain, urolithiasis, renal failure, chronic diarrhea and steatorrhea or prolonged cortisone therapy.
  • Differentiation between familial hypocalciuric hypercalcemia and primary hyperparathyroidism. Method of determination

Atomic absorption spectroscopy (AAS)

Principle, see Section – Method of determination.

Flame photometry

Initially sodium is determined in the urine specimen. The calcium is then measured using a compensation solution that contains the same sodium concentration as the urine. Specimen

Urine collection over a 24-hour period (acidification in the laboratory to pH < 6.5):

  • By adding 10 mL of concentrated HCl or
  • By adding 1 volume of 5% glacial acetic acid to 9 volumes of urine. More recent examinations only recommend neutral collection. During the collection, the urine should be stored at temperatures of approximately 20 °C.
  • 2-hour urine collection without additives collected by the patient in a fasting state between 8–10 a.m. or first morning urine for determining the calcium/creatinine ratio. Reference interval

Refer to: Clinical significance

In healthy people with a normal calcium balance, the kidneys excrete the intestinally absorbed calcium. This is 6–7% of the daily dietary calcium load. If the calcium load of the diet is more than 1.5 g/day, however, hypercalciuria may occur. Hypercalciuria

Hypercalciuria can be of primary or secondary origin. Based on their pathophysiology, primary hypercalciuria is subdivided into the following types /40/:

  • Increased intestinal calcium absorption. In this type of hypercalciuria, dietary restrictions of the calcium intake do not result in the normalization of urinary calcium excretion.
  • Increased resorption of calcium from the skeleton, even on a low-calcium diet (resorptive hypercalciuria). This type of hypercalciuria is not associated with a secondary hyperparathyroidism. The patients on a low-calcium diet have a negative calcium balance resulting in significant bone resorption and an increased risk of fracture.
  • Reduced renal-tubular calcium reabsorption. This type should be authoritative for a maximum of 5% of the hypercalciurias.

Diseases with hypercalciuria are listed in Tab. 6.2-7 – Diseases associated with increased urinary calcium excretion. Primary hypercalciuria (PH)

PH or idiopathic hypercalciuria is a form of Ca excretion, with which on a diet of 1.0 to 1.2 g of calcium and 1.0 to 1.5 g protein/kg of body weight per day there is increased Ca excretion. Most of these patients have absorptive hypercalciuria due to increased enteral calcium absorption, with resorptive hypercalcemia being the second most common cause. The latter is connected to a certain extent with demineralization of the skeleton, but the most frequent association is with calcium nephro lithiasis, thus, the incidence of hypercalciuria for kidney stone patients is 50–60%. Secondary hypercalciuria

In addition to racial, geographic and seasonal fluctuations, gender and weight, nutrition also influences calcium excretion. Important influence factors are the contents of sodium, potassium, phosphorus, carbohydrates and protein in food, and alcohol consumption. When the intake of sodium is increased, the calcium excretion in the urine increases by 20–40 mg/day for each 2.3 g of sodium intake (corresponding to 5.8 g of table salt) /40/.

Due to increased renal synthesis of 1,25 (OH)2D hypophosphatemia also leads to increased intestinal calcium absorption. An increased supply of protein also increases calcium excretion. A moderate supply of protein (1–1.5 g/kg of body weight per day) does not change the calcium metabolism. The causes of secondary hypercalciurias are listed in Tab. 6.2-8 – Causes of secondary hypercalciuria. Diagnosis of hypercalciuria

The following procedures are carried out to confirm an increased calcium excretion:

  • Assay of the calcium/creatinine ratio in the morning urine. A ratio ≥ 0.57 is indicative of hypercalciuria in adults
  • Investigation of one to two 24-hour collected urine samples. An excretion of ≥ 300 mg in men and ≥ 250 mg in women indicates hypercalciuria.

The patient should not alter his dietary habits 24 hours before and during the collection of urine samples.

A calcium excretion in the reference interval of the 24-hour collected urine does not rule out relative hypercalciuria because the calcium excretion can be too high relative to the calcium intake with the diet (Fig. 6.2-1 – Relationship between Ca excretion in the 24-hour urine and dietary intake of Ca).

When clarifying hypercalciuria, a differentiation must be made between primary and secondary causes, with the latter including primary hyperparathyroidism. Differentiation of primary hypercalciuria

If hypercalciuria has been diagnosed and secondary causes of the increased calcium excretion have been ruled out, it is necessary to differentiate metabolic causes from primary hypercalciuria /39/. To do this, the calcium/creatinine ratio is determined with a restricted diet of calcium, protein and sodium and evaluated as shown in Fig. 6.2-2 – Differentiation of hypercalciuria using the calcium/creatinine ratio:

Clinical assessment:

  • After 1 week on the diet (1.0 to 1.2 g of protein per kg of body weight, calcium ≤ 400 mg/day, sodium 100 to 150 mmol/day, corresponding to 5.8– 8.7 g table salt/day) and fasting overnight, the calcium/creatinine ratio is determined in the spontaneous morning urine.
  • If the calcium/creatinine ratio falls below 0.57 too much calcium is enterally absorbed with a regular diet. Absorptive hypercalciuria is the result.
  • If the calcium/creatinine ratio does not normalize (ratio ≥ 0.57) despite calcium restriction, the result is fasting hypercalciuria. There is increased calcium resorption from the bone (resorptive hypercalciuria). If PTH is also determined, its increase indicates renal hypercalciuria. Hypocalciuria

The calcium excretion must be evaluated relative to the amount of dietary calcium. For example, if the calcium excretion for a daily intake of 800 mg (20 mmol) of calcium is less than 50 mg (1.3 mmol) in 24 hours, the result is hypocalciuria. Such a finding is typical for osteomalacia related to a vitamin D deficiency.

In cases of hypercalcemia or calcium levels near the upper reference interval value and increased PTH concentration, the calcium excretion in the 24-hour urine must be determined to differentiate the familial hypocalciuric hypercalcemia (FHH) from primary hyperparathyroidism. Calcium excretion below 100 mg (2.5 mmol/L)/24 hours or a urine calcium/creatinine ratio (mmol/mmol) below 0.01 are indicative of FHH. Comments and problems

Method of determination

Accurate results are obtained by means of atomic absorption spectrometry. Results determined by flame photometry are only accurate if a compensation solution is used with a sodium concentration identical to that of the urine to be examined.

Urinary calcium excretion

The excretion has a minimum between 9 p.m. and 6 a.m. and a maximum before noon /43/.


Under alkaline conditions, the divalent cations calcium and magnesium have the tendency to precipitate in the urine, primarily as phosphates such as brushite (CaHPO4 × 2 H2O) and struvite (MgNH4PO4 × 6 H2O). As a result, magnesium, calcium and phosphate in the urine are determined to be too low. Therefore, the urine should be acidified (pH under 6.5) in the laboratory before the measurement. One recent examinations /44/ shows no difference between acidified and neutral urine.

6.2.3 Biochemistry and physiology

Approx. 1% of total body calcium is renewed daily. On a systemic level, a complex interaction between PTH, 1,25(OH)2D and calcium allows the organism to maintain the calcium homeostasis, despite variations in the daily intake.

PTH has a regulating effect as follows:

  • Stimulation of the kidneys for the reabsorption of calcium
  • Release of calcium from the skeletal system
  • Enteral absorption of calcium.

By activating the enzyme 25-OHD-1α-hydroxylase in the kidneys, PTH stimulates the synthesis of 1,25(OH)2D (calcitriol) from 25(OH)D.

Calcitriol has the following effects:

  • Increase of the Ca absorption in the duodenum and upper jejunum via calcium-binding proteins. The increase in the Ca concentration in the blood, on the other hand, suppresses PTH production.
  • Control of the PTH concentration by suppressing the genetic transcription of pre-pro-PTH.

Ca is excreted via the kidneys and the intestines. Glomerularly filtrated Ca is reabsorbed up to 94–96% (tubular). The Ca amount in urine is up to 300 mg (7.5 mmol)/24 hrs. Ca excreted together with the digestive juices in the intestines is reabsorbed up to 90% (Fig. 6.2-3 – The maintenance of calcium homeostasis).

The G protein-linked calcium-sensing receptor (CaSR), a guanine nucleotide-binding protein (G protein), is the main mediator on the cellular level of the calcium homeostasis. It is expressed on the cell membrane of the parathyroids and the renal tubular cells. Ca directly activates the CaSR, suppresses the PTH excretion, and reduces the renal-tubular reabsorption of Ca. The activation of the CaSR in hypercalcemia causes the G protein-mediated stimulation of the phospholipase C by the proteins Gq and G11. The result is an increase of inositol 1,4,5-triphosphate and an accumulation of intracellular calcium. These changes lead to a decrease in the PTH level and an increase in the renal excretion of calcium.. Thus, a high concentration of calcium suppresses the vasopressin-induced water reabsorption in the collecting ducts via the CaSR and thus declines the capacity of urine concentration. This is the case, for example, with hypercalcemias over 12 mg/dL (3.0 mmol/L), which cause hypercalcemic nephrogenic diabetes.

The G protein-mediated signal transmission of the PTH-sensitive receptor and of the CaSR is illustrated in the example in Fig. 6.2-4 – Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor (PTHSR) or of the calcium-sensitive receptor (CaSR).

Parathyroids of patients with terminal kidney failure have a reduced number of CaSR. Therefore, extracellular Ca is only insufficiently perceived, with the result of secondary hyperparathyroidism and its systemic effects. It is treated with the allosteric modifier of the CaSR, the cinacalcet, which causes activation of the CaSR. Its use leads to a decrease in the elevated PTH concentration and in the Ca × P product in dialysis patients.

Mutations in the gene that encodes the CaSR lead to a suppressing or activating effect with hypercalcemic or hypocalcemic disorders.

The following pathophysiology exists for the familial hypocalciuric hypercalcemia /31/:

  • The parathyroids are resistant to calcium and only respond to an increased calcium concentration with suppression of the PTH excretion. The cause is that the extracellular domain of CaSR is changed by mis- sense mutations in such a way that a reduced number of functional receptors is expressed on the cell membrane.
  • There is an increased reabsorption of calcium in the thick ascending limb of the nephron.

Hypocalcemias are caused by absent or hypofunctioning parathyroids, vitamin D deficiency due to reduced enteral absorption, UV light deficiency, or by hepatic or renal defects in the metabolism of vitamin D.

In chronic renal insufficiency with decreased phosphate clearance, the diminished synthesis of 1,25(OH)2D and the existing hyperphosphatemia with subsequent hypocalcemia are the causes for the development of secondary hyperpathyroidism (sHPT) and of hyperactive bone disease or so-called renal osteopathy (osteomalacia, osteodystrophia fibrosa) /43/. The renal insufficiency associated decline in 1,25(OH)2D diminishes gastrointestinal absorption of Ca; however, passive diffusion of Ca2+ continues and may lead to a positive Ca balance, aggravated by diminished urinary Ca excretion due to sHPT. Increased Ca release from bone in hyperactive renal bone disease enhances the positive Ca balance and may worsen vascular calcification /45/. Hyperactive bone disease leads to an increased Ca resorption from the bone because of increased secretion of PTH (secondary hyperparathyroidism).

Hypercalcemia is based on increased mobilization of calcium from the bone, increased intestinal absorption and/or increased renal tubular reabsorption.

In the case of pHPT, a singular adenoma or hyperplasia of all parathyroids is the cause of autonomous PTH excretion and of hypercalcemia. The result can be the formation of kidney stones, renal calcification, osteodystrophia fibrosa, pancreatitis, and duodenal ulcers. Metastatic calcifications are mainly observed with an increase of the calcium phosphate product to values above 60–75 (calculation based on values in mg/dL) /46/.

Hypercalcemia is a frequent complication of malignant tumors in the advanced stage. Two mechanisms are essentially responsible for tumor-associated hypercalcemia:

  • Metastatic tumor cells cause osteolysis by direct contact with the bone. Factors with a local effect such as prostaglandin E2, interleukin-1 as well as tumor necrosis factors are said to be released. This mechanism is common in cases of multiple myeloma and breast carcinoma.
  • Humoral factors, which lead to an effect like PTH (i.e., increased Ca mobilization from the bone, increase of the cyclic AMP and phosphaturia). However, unlike pHPT, there is no increase in 1,25(OH)2D, instead, it is rather decreased. One such humoral factor which is formed by the tumor and bound to the PTH receptors is the parathormone-related peptide (PTHrP). In cases of PTHrP forming metastases, circulus vitiosus develops. During this, PTHrP releases growth factors such as TGF-β from the bone matrix, which, in turn, increase the PTHrP excretion of the tumor tissue /47/.


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6.3 Phosphorus

Lothar Thomas

The terms phosphate and phosphorus are used interchangeably in laboratory medicine. This is meaningless for clinical purposes, because the phosphate content of a sample is specified as elementary, inorganic phosphorus (Pi). The Pi is present in serum in the form of orthophosphate which is derived from the sequential ionization of phosphoric acid (Fig. 6.3-1 – Sequential ionization of phosphoric acid). Pi occurs only in the organism as H2PO4 and HPO42– , because neither H3PO4 as a strong acid nor PO43– as a strong base is compatible with a physiologic pH. At a pH of 7.4, the molar ratio of HPO42–/H2PO4 is 4 : 1. It decreases in acidosis and increases in alkalosis /1/. It is customary for clinical laboratories to report phosphate concentration in terms of Pi. Thus a 1 mmol/L solution of phosphorus (atomic weight 31) is equivalent to a serum level of 3.1 mg/dL.

At neutral pH, the 4 : 1 molar ratio carries a total of nine negative charges, making the every charge per mmol –9/5 or –1.8. Thus, a 1 mmol/L solution of orthophosphate at pH 7.4 is equivalent to a 1.8 mEq/L solution /1/. Consequently, in serum, the following measurement units for Pi are equal to each other: 1 mmol/L = 3.1 mg/dL = 1 mEq/L /1/.

The determination of Pi in serum is often not sufficient for the assessment of the phosphate status, thus necessitating the determination of urinary phosphate.

6.3.1 Phosphorus (Pi) in serum Indication

  • Bone disease
  • Chronic kidney disease, dialysis patient
  • Status post thyroid operation
  • Parathyroid disease
  • Kidney stone patient
  • Chronic alcoholism
  • Intensive care medicine (parenteral nutrition, mechanical ventilation)
  • Cardiovascular disease
  • Suspicion of vitamin D deficiency (malabsorption)
  • Muscle weakness, bone pain. Method of determination

Phosphorus molybdate method /2/

Principle: reaction of Pi with ammonium molybdate to form a phospho molybdate complex. The colorless complex is measured after reduction by reducing reagents to produce molybdenum blue which is measured spectrophotometrically at 580 nm.

Enzymatic methods

Various methods have been described such as including the use of purine-nucleoside phosphorylase and xanthine oxidase /3/ or sucrose phosphorylase and phosphoglucomutase /4/. Specimen

Serum, heparin plasma (sample taken in fasting state in the morning before 10 a.m. based on circadian rhythms, see Comments and problems): 1 mL Reference interval

Refer to Tab. 6.3-1 – Reference intervals for phosphate. Clinical assessment

Approximately 85% of phosphorus is localized in the bones in connection with calcium and 14% is intracellular. Most intracellular phosphorus is organically bound to carbohydrate intermediates, lipids, and proteins, and a smaller fraction is inorganic /1/. Phosphate is the main intracellular anion and its metabolism is closely linked with that of calcium /5/. The daily phosphorus intake in diet is approximately 1 g (32 mmol). A low protein diet decreases phosphorus intake. However the quantity and bio availability of phosphorus differ according to the type of protein. The gastrointestinal absorption of phosphorus, mostly in the form of phytate, is lower from plants (along with fibers) than from meat (30 to 50% vs. 50 to 70%). Restricting dietary phosphorus intake to less than 800 mg (26 mmol) per day is recommended for moderate-to-advanced kidney disease /6/.

Although only 1% of the phosphorus content of the organism is present in plasma and other bodily fluids, the serum concentration correlates to the body content in most cases. Renal reabsorption of phosphate is the most important factor of the phosphorus level in serum. If the phosphorus reabsorption increases due to an elevated intake or a decrease in glomerular filtration rate, the renal reabsorption of phosphate is reduced. The renal reabsorption of phosphorus is regulated by the fibroblast growth factor 23 (FGF-23) and the parathyroid hormone. The concentration of both hormones increases with a rise in the serum phosphorus concentration. The renal reabsorption of phosphorus can be determined by measuring the TmP/GFR (see Section Hypophosphatemia

Hypophosphatemia mainly occurs due to /1/:

  • Redistribution disturbance between the intracellular and extracellular compartment
  • Decreased supply or reduced intestinal absorption of phosphorus
  • Renal loss of phosphorus because of insufficient vitamin D effect. Changes in the homeostasis of phosphorus

In the intracellular space, phosphorus is primarily involved in the carbohydrate and lipid metabolism or is bound to proteins. Only a small proportion is inorganic. Conditions that favor organic cellular phosphorus binding result in a shift of phosphorus from the extracellular to the intracellular space, thus lowering serum phosphorus concentration.

Such conditions are /1/:

  • Glucose-induced insulin release that promotes the transport of glucose and phosphate ions into liver and muscle cells /7/
  • Administration of fructose stimulates phosphorylation phosphate ions are metabolized in the intracellular compartment and replaced from outside the cell
  • Metabolic acidosis, respiratory alkalosis, recovery from hyperparathyroidism, and physiologic concentrations of catecholamines may cause mild and transient phosphorus shift to the intracellular compartment. The maldistribution may become severe if phosphate stores are depleted because of disease or malnutrition /1/.
  • Extended fasting
  • Fluid losses owing to surgical trauma
  • Rapidly proliferating tumors, which metabolize a lot of phosphate
  • Acute-phase reaction (e.g., sepsis, postoperative state)
  • Nonabsorbable antacids (e.g., aluminium hydroxide) which bind intestinal phosphate and limit the gastrointestinal absorption. Decreased supply or reduced intestinal absorption of phosphorus

The external supply of phosphorus is reflected by the urinary phosphate ion (P) excretion (UPV). The UPV is the product of the urinary P concentration (UP) in mg/dL and the urine volume (V). The UPV corresponds to the enteral absorption of phosphorus. In cases of malnutrition (alcoholism), malabsorption, in intensive care patients and in treatment with phosphate binders, enteral phosphorus uptake and UPV are reduced. The UPV is then below 600 mg (20 mmol)/24 h.

With normal glomerular filtration rate (GFR), the renal threshold of phosphate (i.e., UpV/GFR) is the dominating factor for the regulation of the phosphate status. With a decrease of GFR, the UpV increasingly gains importance in its influence on the serum concentration of phosphorus. Tubular phosphate reabsorption usually ranges from 80–90% and is essentially influenced by PTH, vitamin D, calcitonin, antidiuretic hormone, growth hormone, thyroid hormones, estrogens and glucocorticoids. Renal phosphate leak

The renal excretion of phosphate is dependent on the (TmP/GFR) which is dependent on the renal transport maximum of phosphate (TmP) and the glomerular filtration rate (GFR) /18/. TmP, also known as phosphate threshold is the proportion (%) of phosphate in primary urine which is reabsorbed in the proximal renal tubules (more than 80% in healthy individual). An increased TmP/GFR (mmol/L) can be found in acquired and hereditary bone mineralization disorders.

These metabolic disorders can exhibit similar characteristics /9/:

  • Hypophosphatemia due to increased renal phosphate clearance
  • An inadequate low value of 1,25(OH)2D
  • A variable bone disease (osteomalacia, rickets, renal bone mineralization disease). Clinical symptoms of hypophosphatemia

Mild hypophosphatemia is relatively common in hospitalized patients and is reported in up to 30% of the surgical cases /1/. Clinically relevant hypophosphatemia below 1.5 mg/dL (0.48 mmol/L) and serious forms with a level below 1 mg/dL (0.32 mmol/L) are rare. The prevalence of the serious form in hospitalized patients is 1–2 per thousand /10/. The symptoms of serious hypophosphatemia are muscular weakness, muscle pain, central nervous system symptoms such as confusion, convulsions, and coma. Muscular weakness can lead to respiratory failure. Hematological disorders such as hemolytic anemia and a dysfunction of the neutrophil granulocyte can also occur.

For causes of hypophosphatemia refer to: Hyperphosphatemia

Hyperphosphatemia reduces the concentration of 1,25 (OH)2D and increases the levels of PTH and fibroblast growth factor 23 (FGF23). Both hormones promote urinary phosphate excretion.

Elevated levels of PTH and FGF23 can cause renal bone disease, left ventricular hypertrophy, vascular calcification, and accelerated progression of kidney disease from vascular and tubulointerstitial injury /8/.

The effect of serum phosphorus on kidney disease itself remains uncertain. In non dialysis chronic kidney disease and non chronic kidney disease populations, small increases in phosphorus levels, even within normal ranges, have been shown to be associated with greater mortality and cardiovascular outcomes. In subjects with advanced chronic kidney disease, higher serum phosphorus levels have been shown to predict increased incidence of end-stage renal disease, suggesting that phosphorus elevations may adversely affect kidney function /11/.

Hyperphosphatemia can be related to /612/:

  • Disturbance of phosphate homeostasis between the intracellular and extracellular compartment
  • Acute and chronic renal failure
  • Increased renal phosphate reabsorption (hypoparathyroidism)
  • Increased dietary intake of phosphates, increased intestinal phosphate absorption or intravenous phosphate supply (e.g., during treatment with the antibiotic fosfomycin).

Disturbance of phosphate homeostasis between the compartments

An increased phosphate shift from the intracellular to the extracellular compartment occurs in cases of:

  • Acidosis such as respiratory acidosis, tissue ischemia, lactic acidosis, and diabetic ketoacidosis
  • Tissue trauma (rhabdomyolysis, hemolysis, cytostatic treatment, malignant pyrexia). Chronic kidney disease and hyperphosphatemia

The renal insufficiency associated decline in 1,25 (OH)2 and reduction of GFR in chronic kidney disease diminishes urinary phosphate excretion resulting in hyperphosphatemia. This causes hyperphosphatemia, a condition that results in the development of secondary hyperparathyroidism and mineral renal bone disease /12/.

The capacity of the kidneys for excreting phosphate is high with normal GFR. Overt hyperphosphatemia is infrequent in stages 1, 2, and 3 of chronic kidney disease (CKD), however in stages 4 and 5 the GFR is below 30 [mL × min–1 × (1.73 m2)–1] and overt hyperphosphatemia is frequent. Secondary hyperparathyroidism develops, caused by phosphate retention and reduced renal 1,25(OH)2D (calcitriol) synthesis. Calcitriol stimulates the intestinal calcium absorption and suppresses the secretion of PTH. Impaired renal production of calcitriol, the active form of vitamin D, contributes to the generation and maintenance of secondary hyperparathyroidism. Calcitrol represses parathyroid cell proliferation and PTH synthesis. Calcitriol deficiency results in direct secondary hyperparathyroidism due to decrease in intestinal absorption of calcium.

The Kidney Disease Outcomes Quality Initiative (NKF/KDOQ1) Clinical Practice Guidelines for Bone Metabolism and Disease recommend that the phosphate levels in the serum should be kept constant:

  • In stage 3–4 of CKD, in the range of 2.7–4.6 mg/dL (0.87–1.49 mmol/L)
  • In the range of 3.5–5.5 mg/dL (1.13–1.78 mmol/L) for dialysis patients.

High levels of PTH and phosphate result in systemic disorders such as cardiovascular disease (CVD) and increased mortality in patients with CVD. In chronic dialysis patients with phosphate levels in serum above 6.5 mg/dL (2.1 mmol/L) and a product Ca × phosphate above 72 mg2/dL2 the prevalence of CVD is 40% and of left ventricular hypertrophy 70%. Cardiovascular mortality is 10–20 times higher than in the general population of the same age.

One important regulator of the phosphate content and cause of hyper phosphate-related systemic defects is FGF23 (see Section 6.3.4 – Biochemistry and physiology). Clinical symptoms of hyperphosphatemia

The most common cause of hyperphosphatemia is CKD. If the GFR is above 30 [ml × min–1 × (1.73 m2)–1], other causes must also be considered. The consequences of hyperphosphatemia are /13/:

  • A decrease in calcium and 1,25(OH)2D concentration and a reduction of intestinal calcium absorption. For tumor calcinosis, pseudoxanthoma elasticum, cortical hyperostosis and thyrotoxicosis, there is no decrease of calcium.
  • Ectopic calcifications in various organs and vessels, particularly in end stage renal failure. The risk of calcifications begins with a Pi serum level above 5.5 mg/dL (1.8 mmol/L) and a product of Ca (mg/dL) × phosphate (mg/dL) above 60–75 with the presence of an alkaline pH. Calcium phosphates precipitate in the form of hydroxyl apatite crystals in the tissues.

According to clinical aspects, hyperphosphatemia is usefully subdivided according to /14/:

  • Whether phosphate is added to the extracellular compartment from a variety of exogenous or endogenous sources or
  • Whether the urinary excretion of phosphate is reduced from either decreased GFR or increased tubular reabsorption.

Severe hyperphosphatemia with a concentration ≥ 14 mg/dL (4.5 mmol/L) is always invariably multi factorial, usually resulting from addition of phosphate to the extracellular compartment together with decreased phosphate excretion /14/.

The behavior of phosphate in diseases and conditions with hyperphosphatemia is shown in Tab. 6.3-4 – Diseases and conditions associated with hyperphosphatemia.

6.3.2 Phosphorus excretion

The determination of urinary phosphate excretion by itself is often insufficient for assessing the phosphate status, because the excretion is dependent on dietary intake, bone metabolism, GFR, and renal phosphate reabsorption. Therefore, clearance methods are used for assessing the phosphate excretion. The following examinations are conducted depending on the clinical concerns:

  • Phosphate clearance
  • Percentage of tubular phosphate reabsorption
  • Tubular maximum for phosphate reabsorption. Phosphate clearance (Cp) Indication

Suspected tubular syndromes associated with phosphate losses.

Primary and secondary parathyroid dysfunctions. Test protocol

Principle: the object is to determine the plasma volume that is cleared of phosphate per minute. The phosphate clearance test is carried out in two 1-hour collection periods according to the following test protocol:

  • 7.00 a.m., the fasting patient drinks 500 ml of tea
  • 8.00 a.m., the patient empties the bladder in the toilette bowl and then drinks another 250 mL of tea
  • 9.00 a.m., the patient empties the bladder in the first container, blood is collected for the determination of phosphate.
  • 10.00 a.m., the patient empties the bladder in the second container.

Determination of phosphate (P) in serum and in both urine collection specimens, measurement of the urinary excretion over the course of both collection periods.

Calculation of phosphate clearance (Cp)

C p = (mL/min) = Urine P (mg/dL) × urine volume (mL) Serum P (mg/dL) × collection time (min)

The Cp of both collection periods is determined. Reference interval

Cp = 5.4–16.2 mL/min. /15/ Clinical assessment

The excretion of phosphate in the 24-hour urine in individuals with a normal phosphate supply is 0.6–1.55 g (20–50 mmol).

The Cp has an advantage over the determination of the phosphate excretion that the phosphate excretion is assessed relative to the serum phosphate level. Physiological increases of Cp are measured in increased alimentary supply of phosphate and NaCl and physiological decreases in growth spurt, gravidity and the lactation period. Diseases with increase in Cp are shown in Tab. 6.3-4 – Diseases and conditions associated with hyperphosphatemia.

The Cp does not take renal function into consideration. Thus, for primary hyperparathyroidism with limited renal function, the Cp is often normal.

For the differential diagnostic assessment of pathological Cp values the following further examinations are important:

  • Calcium, phosphate, serum protein, chloride, creatinine, and alkaline phosphatase in serum.
  • Estimated glomerular filtration rate and calcium excretion in urine.

Diseases with increased Cp: see Tab. 6.3-5 – Diseases with Cp-increase. Fractional tubular phosphate reabsorption, TRP (%) Indication

Detection of renal tubular phosphate reabsorption defect:

  • Primary and secondary disorders of the parathyroid function
  • Tubular syndromes with phosphate leak. Test protocol

Principle: the percentage of renal tubular phosphate reabsorption (TRP%) is determined as follows:

  • 2-hour urine collection in the morning after the bladder has been emptied. The patient should be in a fasting state. Measurement of urine volume, creatinine and phosphate.
  • Blood collection at the midpoint of the collection period for determination of creatinine and phosphate
  • Calculation of the tubular reabsorption of phosphate according to the following formula:
TRP (%) = 1 – C p × 100 C cr

Cp = Phosphate clearance, Ccr = Creatinine clearance Reference interval

TRP (%) = 82–90 /16/ Clinical assessment

The TRP (%) considers, in contrast to the Cp the renal function. It is used for differentiating hypophosphatemia. One disadvantage is the dependency on the supply of phosphate. Thus, if phosphate is withdrawn, a healthy individual can have a TRP (%) above 90%, compared to below 82% with a high supply of phosphate. The evaluation of the (TRP%) for diseases is shown in Tab. 6.3-6 – TRP(%) with diseases. Tubular maximum for phosphate reabsorption (TmP/GFR)

The TmP/GFR, also called renal phosphate threshold, describes the maximum phosphate concentration (TmP) in the glomerular filtrate, below which all of the filtered phosphate is tubularly reabsorbed. It is possible to determine the TmP/GFR directly, but is technically very demanding. Therefore, the renal phosphate threshold is generally determined according to the nomogram of Walton and Bijvoet /17/. Refer to Fig. 6.3-2 – Nomogram for the determination of the renal phosphate threshold. Indication

Detection of a renal tubular phosphate reabsorption defect. Test protocol

Principle: the percentage of renal phosphate reabsorption is determined.

The evaluation of TRP using the nomogram (Fig. 6.3-2) necessitates the following steps:

  • 2-hour urine collection in the morning after the bladder has been emptied. The patient should be in a fasting state. Measurement of urine volume, creatinine and phosphate.
  • Blood collection at the midpoint of the collection period for determination of creatinine and phosphate
  • Calculation of the tubular reabsorption of phosphate according to the following formula:
TRP = 1 – C p C cr

Cp = Phosphate clearance, Ccr = Creatinine clearance

  • The phosphate threshold (TmP/GFR) is read off the nomogram (Fig. 6.3-2), based on the serum phosphate concentration and the TRP value. The TmP/GFR is determined from the nomogram as follows: phosphate concentration of the serum and TRP or Cp/Ccr value are connected by using a ruler in such a manner that the right side intersects the TmP/GFR axis. This point of intersection corresponds to the phosphate threshold of the patient in mg/dL or mmol/L in the glomerular filtrate. Reference interval of the TmP/GFR

Refer to Tab. 6.3-7 – Reference intervals of the tubular maximum for phosphate reabsorption (TmP/GFR). Clinical significance

The TmP/GFR behaves in parallel with the phosphate concentration in serum, which means that it depends on the phosphate threshold as it changes throughout the day. Between 11 a.m. and 3 p.m., there is a simultaneous increase of phosphate in serum with the phosphate excretion in urine and the TmP/GFR /18/.

The TmP/GFR is decreased in cases of tubular syndromes with a loss of phosphate such as phosphate diabetes and the hyperparathyroidism /19/. A tubular phosphate threshold below 2.2 mg/dL (0.70 mmol/L) is seen in 20% of the patients who are developing kidney stones, but in only 5% of the control patients /20/.

6.3.3 Comments and problems

Sample collection

The phosphate content of food influences the serum phosphate concentration. To minimize this influence, the blood sample should be collected in the morning after fasting overnight. Serum phosphate shows a circadian rhythm with a nadir early in the morning, a plateau late in the afternoon, a slight decline late in the evening, and a plateau at night. The difference between the highest and lowest value is 30% and the absolute value is around 1.2 mg/dL (0.39 mmol/L) /1821/.


Serum is preferred over plasma; the serum level is 0.2–0.3 mg/dL (0.06–0.10 mmol/L) higher than in plasma.

The serum must be separated from the erythrocytes within 2 hours, because phosphate escapes from the blood cells depending on the temperature, causing falsely elevated values.

Hemolytic serum is not acceptable, because phosphate is cleaved from organic phosphate esters that are released in the serum. Theoretically, the measured phosphate can be increased by up to 20%, something which is not seen in practice, however.

Thrombocytosis is also associated with increased phosphate values.

Method of determination

In addition to hemolysis, hyperlipidemia and hyperbilirubinemia interfere with the phosphate assays. Obfuscations interfere due to spectral interference, because the phospho molybdate method used in most analysis systems measures at 340 nm /22/.

Interference factors

The unmodified acid ammonium molybdate method produces 19% spuriously high results. The false increase of Pi concentration is attributable to formation of precipitate in the reaction mixture. The precipitate is formed by interaction between immunoglobulins and the unmodified acid molybdate reagent. Diluting of the sample to about 40 g/L total protein reduces, but does not always eliminate the interference /23/.


Whole blood at room temperature for a maximum of 1 day, then an increase, at 9 °C increase after 4 days. Serum at room temperature for 2 days, at 9 °C for 7 days /24/.

6.3.4 Biochemistry and physiology

Phosphorus is one of the essential structural components of cells and organelles and it plays an active part in the generation, storage and release of metabolic energy. In particular, phosphate is involved:

  • As a part of nucleoproteins, nucleic acids and in the form of phospholipids in membranes
  • In the production of ATP during the oxidative phosphorylation in the mitochondria
  • In the glycogenolysis and glycolysis
  • In the form of 2.3 diphosphoglycerate in regulating the dissociation of oxygen from oxyhemoglobin
  • In enzymatic processes the phosphorylation of enzymes is an important control mechanism of enzymatic functions. In addition, phosphate is involved in many enzymatic reactions in the form of cyclic adenosine and guanine nucleotides as well as NADP.
  • In many functions the organism needs ATP such as muscle contraction, central nervous system processes, and the transport of electrolytes
  • AS an essential component of bones.

All of the above-mentioned functions are impacted by a phosphate deficiency. Intracellular phosphorus

The concentration of intracellular phosphorus is approximately 3.1 mg/dL (1 mmol/L). Most of the intracellular phosphorus exists in the form of organic compounds, (lipids, carbohydrate intermediary products) and plays a role in transport processes, and cell growth. Conditions that promote the binding of organic phosphorus, such as the administration of insulin for hyperglycemia, lead to a shift of phosphate from the extracellular to the intracellular compartment.

Metabolic disorders that involve acidosis lead to a decrease in the oxidative phosphorylation, and the glycolysis, and hydrolysis of intracellular phosphate esters. The released phosphate is shifted to extracellular and leads to an increase in phosphate. In cases of deficient phosphate supply or catabolic metabolism, the concentration of plasma phosphate remains constant for a long time despite the renal excretion of phosphate because intracellular phosphate is continuously resupplied. Hypophosphatemia only occurs if an anabolic metabolism is restored and phosphate is shifted from the extracellular to the intracellular compartment. In cases of severe malnutrition, high amounts of infused glucose can be fatal due to severe hypophosphatemia that can develop. Phosphate homeostasis

Phosphate is present in many foodstuffs such as meat and vegetables. The mean daily intake of dietary phosphate is 1,000 mg of which approximately 70% is absorbed in the small intestine. Approximately 200 mg of phosphate derived from endogenous sources is secreted into the intestines daily and are reabsorbed.

The tissue of a male person weighing 70 kg contains approximately 0.7 kg of phosphate, of which 85% is located in the skeleton, approximately 15% is contained in the soft tissues, and 0.3% is in the extracellular compartment. The bone is the body’s store for phosphate and the kidneys are the regulator of the phosphate homeostasis. With a phosphate plasma level of 3.4 mg/dL (1.1 mmol/L) and a GFR of 180 liters/24 hours, 6.3 kg of phosphate is filtered. Approximately 80% of the filtered load is reabsorbed in the proximal and 10% in the distal tubules. The capacity of the kidneys to reabsorb phosphate is determined by the tubular maximum (Tm) per mL of GFR (Tm/GFR). The kidneys regulate the excretion of phosphate depending on the intake. Reduced intestinal phosphate intake is responded to by an elevation of the TmP/GFR, increased intestinal phosphate intake by a decrease.

With chronic renal failure, hemodynamic and morphological changes occur in order to maintain the phosphate homeostasis /25/. These include hypertrophy of nephron segments and the tubular phosphate handling. The TmP/GFR of the remaining nephrons is increased, which allows the phosphate plasma concentration to be kept normal for a long time. Renal and intestinal handling of phosphate

The homeostasis of phosphate and thus the phosphate serum concentration is maintained by mechanisms which regulate the intestinal absorption of phosphate. If the concentration of plasma phosphate is increased due to increased intake with the diet or due to the decrease in GFR, the enteral phosphate resorption decreases. In this situation, the regulation is done via the increase of FGF23, which reduces the reabsorption of phosphate in the kidneys and causes phosphaturia. PTH has the same effect.

Between 60–80% of the phosphate is resorbed in the small intestine and the net excretion takes place via glomerular filtration minus the tubular reabsorption. Intestinal absorption and renal reabsorption occur via sodium-dependent co transporters (Fig. 6.3-3 – Renal reabsorption of phosphate).

The kidney tubular brush border membrane contains the co transporters NPT2a and NPT2c. The small intestine primarily contains NPT2b. NTP2a transports 3 sodium ions with 1 phosphate anion and NTP2c only 2 sodium ions with 1 phosphate ion. The expression of NPT2a and NPT2c in the brush border membrane of the kidneys is rapidly down regulated by an increase of phosphate, FGF-23 and PTH, but NPT2c of the small intestine only does this after several days /7/. Inactivity of the genes of the renal co transporters induces hypophosphatemia through increased excretion of phosphate. The consequences are demineralization of the bone and the formation of kidney stones. Serum phosphate

The phosphate level in serum is approximately 4 mmol/L, of which 70% is present in organic form, primarily in the form of phospholipids. The remainder is inorganic phosphate, of which 70% is in free form, 15% in protein-bound form, and 5% is present bound to magnesium or calcium.

Serum phosphate level is regulated by intestinal phosphate absorption, renal phosphate handling and equilibrium of extracellular phosphate with that in bone or intracellular fluid. PTH, 1,25(OH)2D and FGF 23 regulate serum phosphate by modulating intestinal phosphate absorption, renal phosphate reabsorption and/or bone metabolism. PTH

The effects of PTH on the kidneys are as follows: PTH binds to the PTH receptor 1 of the proximal tubular cells, stimulates the synthesis of cyclic AMP and phospholipase C, and reduces the renal phosphate reabsorption by preventing the expression of NPT2a (Fig. 6.3-3 – Renal reabsorption of phosphate). PTH in addition increases the renal phosphate excretion, but it is unknown whether PTH has a direct effect, because changes of the phosphate concentration always involve changes of calcium. It is assumed that PTH has no direct phosphaturic effect /7/. Fibroblast growth factor 23 (FGF23)

The FGF-23 protein consists of 251 amino acids, and is formed in osteocytes and also in other tissues during the developmental phase of the organism. It exerts its effect via FGF receptors (FGFRs), which are bound to the transmembrane protein klotho (Fig. 6.3-3). Klotho is a co receptor, which increases the sensitivity of the FGFRs for FGF23. FGF23 acts directly on the parathyroid to decrease serum PTH. In absence of functioning klotho, plasma concentration of FGF23 is high, but ineffective in decreasing the PTH level.

FGF23 induces phosphaturia by decreasing phosphate reabsorption in the proximal tubule and inhibits renal 25-OHD-1α-hydroxylase leading to decreased conversion of 25(OH)D to 1,25 (OH)2D. Excess of FGF 23 causes marked hypophosphatemia, renal phosphate wasting, and an inappropriate low 1,25 (OH)2D level for the degree of hypophosphatemia /26/. The plasma FGF23 concentration increases with a decline in the GFR and correlates to the phosphate concentration in serum and the fractional secretion of phosphate serves to differentiate patients with volume depletion who respond to saline intake with correction of the alkalosis from those who do not respond. The early increase of FGF23 in CKD prevents hyperphosphatemia through increased renal phosphate excretion and decreased intestinal absorption of phosphate. By suppressing the 25-OHD-1α-hydroxylase and its associated decrease of 1,25 (OH)2D increased levels of FGF23 can cause secondary hyperparathyroidism (sHPT). The relationships between calcium, phosphate, PTH, FGF23 and 1,25(OH)2D are shown in Fig. 6.3-4 – Inter-relationship between calcium, phosphorus, parathyroid hormone, fibroblast growth factor 23 and 1,25(OH)2D.

Dialysis patients have significantly increased FGF23 values and the elevation indicates the development of refractory sHPT. The restriction of phosphate leads to a decline of FGF23, a decreased renal phosphate reabsorption and, due to increase of 1,25 (OH)2D the intestinal phosphate absorption is enhanced /27/.

Approximately 90% of patients with stage 3 and 4 of chronic kidney disease have increased concentrations of FGF23 without hyperphosphatemia and a study over 53 months has shown that patients with higher FGF23 values have a greater progrediens in renal insufficiency than those with lower values /28/. Increased FGF23 levels at the start of dialysis are also associated with higher mortality in the first year of dialysis. According to a study /29/ patients in the highest of 4 quartiles had a 6-times greater risk of mortality than those in the first quartile of the FGF23 values.


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6.4 Parathyroid hormone (PTH)

Lothar Thomas

6.4.1 Introduction

PTH works as an interface between bone , calcium metabolism and vitamin D-metabolism. PTH regulates normal bone and mineral ion homeostasis and is central in the pathogenesis of bone disease, in primary and secondary hyperparathyroidism, and especially in advanced renal insufficiency /1/.

PTH is a 84-amino acid polypeptide [PTH (1–84)] produced by the parathyroid glands and its partial degradation within these glands results in the secretion of intact hormone and various fragments into the peripheral blood. It exerts its classical biological effects on bone and kidney through its first 34 amino acids, that is, its N-terminal structure /2/. The PTH activity is mediated via interaction with the PTH receptor PTH1.

PTH exerts the calcium concentration in plasma through:

  • Release of calcium from the bones
  • Increase of the renal-tubular calcium reabsorption.
  • Stimulation of the renal 1,25(OH)2D (calcitriol) formation which increases the calcium absorption in the small intestine. The resulting increase of calcium in peripheral blood inhibits the excretion of PTH via feedback inhibition.
  • Phosphaturic effect due to suppression of the sodium-phosphate co transporter in the proximal tubular cells of the kidney.

In addition to the intact PTH (1–84), PTH fragments formed in the parathyroids or produced by the liver circulate in plasma. Following release PTH (1–84) undergoes proteolytic degradation in Kupffer cells in the liver. The resulting carboxy-terminal (C-terminal) PTH fragments but not the N-terminal fragments re-enter the circulation. The PTH fragments and intact PTH are excreted renally. C-terminal fragments have a 5–10 times longer half-life than the intact PTH. As a consequence of the different half-lives, the following proportions of immunoreactive PTH are measured in normocalcemic patients:

  • Approximately 5–30% intact PTH (1–84)
  • Approximately 70–95% C-terminal fragments
  • Approximately 4–8% N-terminal fragments. Circulating immunoreactive PTH forms

PTH (1–84)

According to the biological effects PTH (1–84) is differentiated in the following structures /2/ (Fig. 6.4-1 – Parathyroid hormone 1–84):

  • Aminoterminal structure of 34 amino acids which exerts its biological effects through its first 34 amino acids (N-terminus). It binds to the type 1 PTH/PTHrP receptor and activates the Gs protein-mediated signal transmission in the target organs (see Fig. 6.2-4 – Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor (PTHSR) or of the calcium-sensitive receptor (CaSR)). The first 6 amino acids of the N terminus are important for binding to the type 1 PTH/PTHrP receptor. Most immunoassays for the determination of PTH 1–84 (intact PTH assays) have their main epitope in region 13–34 of the PTH (1–34) structure, early epitopes like 12–18 and 13–24 being more frequent than distal epitopes 26–32 /2/.
  • Carboxy-terminal structure of the last 50 amino acids, which has no direct influence on the type 1 PTH/PTHrP receptor.

PTH fragments

C-PTH fragments: these fragments lack a smaller or larger part of the N terminus. In healthy individuals the proportion is approximately 80% and in chronic kidney disease as much as 95%. C-PTH fragments are acutely regulated in the circulation by ionized calcium. This regulation operates within the reference interval of calcium. Hypocalcemia favors the secretion of PTH (1–84) over the C-PTH fragments /2/.

N-PTH fragments: such fragments are no proteolytic degradation products of PTH (1–84) in the liver, but are secreted by the parathyroids and make up 4–8% of the immunoreactive PTH. In the final stage of chronic kidney disease, the proportion can increase to 15%. N-PTH fragments are also increasingly secreted in patients with primary hyperparathyroidism.

Non-(1–84) (PTH) fragments

The non-(1–84) PTH fragments or amino-terminal (N)- truncated PTH fragments are large circulating carboxy-terminal ()C) fragments with a partially preserved N structure. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1–84) PTH fragments. The non-(1–84) PTH fragments differ from other circulating C-PTH fragments by their capacity to react with the 13–34 structure in intact PTH assays /3/. Their proportion of the C-terminal fragments is approximately 10% and, in healthy individuals, approximately 20% of the PTH measured with intact PTH assays. In chronic kidney disease, the proportion of non-(1–84) PTH fragments can be up to 45%.

One important non-(1–84) PTH fragment is PTH 7–84. It does not bind to PTH/PTHrP 1, is biologically inactive, and has no calcium regulating effect. However, PTH 7–84 binds to receptors, which react with the C-terminal part of PTH (C-PTH receptors). These receptors are located on osteolytic cells. PTH 7–84 suppresses bone resorption, which is triggered by osteoclast-activating substances such as 1,25(OH)2D, prostaglandin E2 and IL-11. PTH 7–84 is also supposed to have hypocalcemic properties and counteract the calcium increasing effects of PTH.

6.4.2 Indication

  • Differentiation between hyperparathyroidism-related hypercalcemia and other forms of hypercalcemia
  • Assessing the bone metabolism and therapeutic monitoring in patients with chronic kidney disease
  • In patients with vitamin D deficiency
  • In patients malabsorption syndrome
  • During adenectomy for checking the residual PTH secretion or during surgery of a large goiter.

6.4.3 Method of determination

There are three generations of PTH assays, which measure different circulating forms of PTH /4/.

First generation assays

PTH was measured by radio immunoassay (RIA), using different polyclonal antibodies directed against epitopes within the mid- or carboxy-terminal portion (e.g., 53–84 PTH or 44–68 PTH) of the PTH molecule. In addition to PTH 1–84, RIAs primarily measured PTH fragments. Because of low detection limit and cross-reactivity with PTH-fragments such assays are largely replaced by the second generation assays.

Second generation assays (intact PTH assays) /4/

These are immunometric assays (IMAs). They use two different antibodies that are directed against distinct epitopes within PTH (1–84). The capture antibody is directed against an epitope within the C-terminal structure (e.g., epitope region 39–84). The detection antibody (second antibody) is directed against an aminoterminal epitope. Most detection antibodies have their main epitope in region 13–34 of the PTH (1–34) structure, early epitopes like 12–18 and 13–24 being more frequent than distal epitopes 26–32. The detection antibody also binds fragments with a truncated N-terminus such as PTH 7–84. Second generation assays do not detect mid-regional C-PTH fragments such as 53–84 and 44–68, which are detected by first generation assays. The characterization of the second generation assays show that the detection antibody detects not only intact PTH (epitope region PTH 1–34, PTH 2–34) but also C-PTH fragments, non-(1–84) PTH fragments and PTH 7–84.

Third generation assays (bio active PTH 1–84 assay)

These immunometric two-site assays use a similar capture antibody as the second generation assays. The detection antibody is directed against epitopes at the extreme aminoterminal end (region 1–4) of the PTH molecule. These assays do not cross-react with PTH 7–84 and other amino-terminally truncated PTH fragments. In samples from healthy individuals, they measure approximately half of the values as second generation assays, but they only measure the same values as second generation assays if only PTH (1–84) is in the sample /5/.

If a patient’s sample is measured with the second and third-generation assay, the ratio of the results from the bio active PTH assay/intact PTH assay allows the proportion of amino-terminally truncated PTH fragments such as PTH 7–84 to be calculated. These fragments play an important role for CKD patients.

The KDIGO Guidelines recognize a potential benefit of the third-generation assays for patients with CKD, but they recommend that the second-generation assay continue to be used until more exhaustive results are available.

6.4.4 Specimen

Serum, plasma, in the morning in fasting state: 1 mL

For dialysis patients, the blood sample should be drawn before the dialysis.

6.4.5 Reference interval

Refer to Tab. 6.4-1 – Reference intervals for PTH.

6.4.6 Clinical assessment

Laboratories measure PTH using second-generation assays and specify the results as intact PTH, knowing that not only PTH (1–84) is measured. In the following the abbreviation PTH is used for intact PTH that stimulates the PTH/PTHrP receptor.

PTH is secreted by the parathyroid glands primarily in response to changes in blood ionized calcium (iCa). In cases of hypocalcemia, the normalization of iCa is achieved by the stimulated secretion of PTH via the PTH/PTHrP receptor of the parathyroids. In cases of normocalcemia, the regulation of iCa is mainly regulated by C-PTH fragments. With hypercalcemia, PTH secretion is suppressed and serum PTH levels are decreased. The ratio C-PTH fragments/PTH is increased, because PTH fragments such as PTH (7–84) are elevated.

C-PTH fragments with or without partially contained N-terminus do not stimulate the PTH/PTHrP receptor. If more PTH is required due to increasing hypocalcemia, as is the case with secondary hyperparathyroidism due to chronic kidney disease, vitamin D deficiency or the partial removal of parathyroids, more PTH is secreted and the ratio C-PTH fragments/PTH decreases. The reverse of this is the case with tumor hypercalcemia and vitamin D treatment.

Refer to: PTH and chronic kidney disease (CKD)

With the use of second-generation PTH assays, besides PTH (1–84) fragmented or truncated PTH structures [e.g., PTH (7–84)] are also measured to a different extent and indicate the finding of an increased PTH concentration. PTH fragments are formed in different amounts in primary and secondary hyperparathyroidism /67/. This particularly plays a role in CKD, when the PTH level is used as a surrogate marker of bone mineral disease (BMD). Due to a cross reaction of PTH with fragmented PTH structures in the second-generation PTH assays, a false impression concerning the BMD status can be mediated /8/. PTH and hypercalcemia

Hypercalcemia is one of the most common indications for determining PTH. The PTH level allows the differentiation of primary hyperparathyroidism (pHPT) from other causes of the hypercalcemia such as tumor hypercalcemia, vitamin D intoxication, sarcoidosis, and familial hypocalciuric hypercalcemia. Whereas PTH is increased or near the upper reference interval value in 95% of pHPT patients it is within the reference interval for the remaining hypercalcemia. PTH and normocalcemia

Usually, the determination of PTH is not required for normocalcemic patients. In older patients with bone complaints and normal calcium levels, mild increase in PTH can be an early sign of pHPT or of secondary hyperparathyroidism (sHPT) due to vitamin D deficiency or CKD. PTH and hypocalcemia

Hypocalcemia is associated with hyperparathyroidism and pseudohypoparathyroidism. In both cases PTH is increased and calcium is decreased.

Diseases with hyper- and hypoparathyroidism are shown in Tab. 6.4-2 – Diseases and conditions with hyperparathyroidism and hypoparathyroidism.

6.4.7 Comments and problems

Blood sampling

The blood should be drawn in the morning before 10 a.m. because of mild pulsatility and a circadian rhythm, with higher PTH levels in the evening.

Reference interval

The tests of various manufacturers do not yield comparable values for PTH. Thus, depending on the manufacturer of the kit, the difference for PTH can be up to 3.4 times in the individual sample /28/. According to the KDIGO guidelines an adynamic bone disease exists in CKD patients with PTH levels two times the upper reference interval value. The treatment of these patients with active vitamin D analogues is prohibited. Therefore it is important to always measure PTH using the assay of the same manufacturer.

Stability /29/

At room temperature up to 6 hours, at 4 °C up to 24 hours in serum /30/. Depending on the PTH assay, the values are 10–30% higher in EDTA blood or EDTA plasma than in serum. The cause is unknown.

Interference factors

The contamination of the sample with tissue plasminogen activator such as alteplase, in dialysis patients, leads to a decrease in the concentration of PTH by 2.5–33.5% within 24 hours /31/.

6.4.8 Biochemistry and physiology

PTH, a peptide of 84 amino acids, is excreted by the parathyroid glands in the form of PTH (1–84), but truncated PTH fragments such as PTH (7–84) are also released. PTH (1–84) is removed from circulation by the liver after being present for 2–4 minutes, cleaved into fragments and excreted through glomerular filtration. For binding the PTH to its receptors, only the first 34 amino acids are required. Only the first 6 amino acids of the N terminus are biologically active.

The ionized calcium (iCa) concentration in the blood is registered by the iCa-sensitive receptor of the parathyroid cells. The high sensitivity of these cells allows them to centrally regulate the calcium content in the circulation. If a hypocalcemia is registered, the parathyroids respond with an enhanced secretion of PTH, which causes the transfer of iCa into the extracellular space due to the following PTH-mediated actions:

  • Release of iCa and phosphate from the bone
  • Induction of the renal 1,25(OH)2D synthesis, which induces the increased intestinal calcium absorption
  • Increase of the renal distal-tubular iCa reabsorption.

The resulting increase of calcium in peripheral blood inhibits the excretion of PTH via feedback inhibition.

The effect of PTH on the tissues is mediated via the PTH/PTHrP receptor /32/ a protein of 585–593 amino acids. The receptor binds PTH (1–34) and PTHrP. After binding the receptor stimulates specific G proteins and mediates its signal via two different paths, the cyclic AMP and iCa to the cell nucleus. Refer to Fig. 6.2-4 – Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor (PTHSR) or of the calcium-sensitive receptor (CaSR).

The cyclic AMP mediated PTH effects on the kidneys are the stimulation of the 25-OHD-1α-hydroxylase and the suppression of the sodium-phosphate co transporters Refer to Fig. 6.3-3 – Renal reabsorption of phosphate.

In PTH stimulated calcium resorption of the bone, PTH does not have a direct effect on mature osteoclasts, but binds to osteoblastic PTH/PTHrP receptors. The osteoblastic cells are activated and secrete cytokines, which stimulate in a paracrine way the differentiation of preosteoclastic cells into active, multi nuclear osteoclasts. PTH also promotes the cell proliferation of osteoblasts and their type-I collagen production and thus also has an osteoanabolic effect in addition to the bone resorption effect.


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16. Elaraj DM, Remaley AT, Simonds WF, Skarulis MC, Libutti SK, Bartlett DL, et al. Utility of rapid intraoperative parathyroid hormone assay to predict severe postoperative hypocalcemia after reoperation for hyperparathyroidism. Surgery 2002; 132 (6): 1028–33.

17. Cesarim ALM, Arcadipane FAMC, Martins AS, Del Negro A, Rodrigues AAN, Ticani AJ, Marchi E. Pattern of intraoperative parathyroid hormone and calcium in the treatment of tertiary hyperparathyroidism. Otolaryngol Head Neck Surg 2019; https://journals.sagepub.com/doi/10.1177/0194599819866819.

18. Yamashita A, Gao P, Cantor T, Naguchi S, Uchino S, Watanabe S, et al. Comparison of PTH levels from the intact and whole PTH assays after parathyroidectomy for primary and secondary hyperparathyroidism. Surgery 2004; 135: 149–56.

19. Stenner E, Sandic S, Dobrinja C, Ruscio M, Bernardi S. The total testing process of intra-operative parathyroid hormone. A narrative review. Clin Lab 2020; 66: 229–37.

20. Goodman WG, Quarles LD. Development and progression of secondary hyperparathyroidism in chronic kidney disease: lessons from molecular genetics. Kidney Int 2008; 74: 276–88.

21. KDIGO 2017 clinical practice guideline update for the diagnosis, evaluation, prevention, and treatment of chronic kidney disease – mineral bone disorder (CKD-MBD). Kidney Int Supplements 2017; 7: 1–59.

22. Thomas MK, Demay MB. Vitamin D deficiency and disorders of vitamin D metabolism. Endocrinol Metab North Am 2000; 29: 611–27.

23. Sanchez-Regana M, Martin-Ezquerra G, Umbert-Millet P. Serum levels of parathyroid hormone and parathyroid-related peptide in psoriasis. Acta Derm Venereol 2005; 85: 420–3.

24. Shoback D. Hypoparathyroidism. N Engl J Med 2008; 359: 391–403.

25. Goswami R, Mohapatra T, Gupta N, Rani R, Tomar N, Dikshit A, Sharma K. Parathyroid gene polymorphism and sporadic idiopathic hypoparathyroidism. J Clin Endocrinol Metab 2004; 89: 4840–5.

26. Bastepe M, Jüppner H. Pseudohypoparathyroidism. New insights in an old disease. Endocrinol Metab North Am 2000; 29: 569–89.

27. Weinhaeusel A, Thiele S, Hofner M, Hiot O, Noehammer C. PCR-based analysis of differentially methylated regions of GNAS enables convenient diagnostic testing of pseudohypoparathyroidism type Ib. Clin Chem 2008; 54: 1537–45.

28. Almond A, Ellis AR, Walker SW, on behalf of the Scottish Clinical Biochemistry Managed Diagnostic Network. Current parathyroid hormone immunoassays do not adequately meet the needs of patients with chronic kidney disease. Ann Clin Biochem 2012; 49: 63–7.

29. Ercan M, Akbulut ED, Avci E, Yücel C, Oguz EF, Turhan T, Serdar M. Determining biological variation of serum parathyroid hormone in healthy adults. Biochem Med (Zagreb) 2019; 29 (3): 030702.

30. Souberbielle JCP, Roth H, Fouque DP. Parathyroid measurement in CKD. Kidney Int 2010; 77: 93–100.

31. Schiller B, Wong A, Blair M, Moran J. False low parathyroid hormone values secondary to sample contamination with tissue plasminogen activator. Nephrol Dial Transplant 2009; 24: 2240–3.

32. Brown EM, Segre GV, Goldring SR. Serpentine receptors for parathyroid hormone, calcitonin and extracellular calcium ions. Bailliere’s Clinical Endocrinology and Metabolism 1996; 10: 123–161.

6.5 Parathyroid hormone-related peptide (PTHrP)

Lothar Thomas

The N-terminal sequence of parathyroid hormone-related peptide (PTHrP) has close homology with that of parathyroid hormone (PTH). Some of the physiological functions of PTHrP are similar to PTH because both hormones bind to the PTH/PTHrP-1 receptor. PTHrP acts as an autocrine, paracrine, and endocrine hormone and is able to simulate most of the actions of PTH, including regulation of calcium ion homeostasis, bone resorption, distal tubular calcium reabsorption, and inhibition of proximal tubular phosphate transport /1/.

6.5.1 Indication

  • Differential diagnosis of hypercalcemia in the case of known or suspected tumor (relative indication)
  • Prognostic factor for the development of bone metastases, e.g. breast cancer, small cell lung cancer, renal carcinoma
  • Monitoring the course of tumor hypercalcemia
  • During bisphosphonate treatment (relative indication).

6.5.2 Method of determination

Competitive immunoassays

Radio immunoassay or ELISA using antibodies directed against the aminoterminal (amino acids 1–34) or mid-regional (amino acids 44–68 or 53–84) structures.

Immunometric assays (IRMA)

The two-site IRMA has anti-PTHrP (1–34) as the capture antibody and anti-PTHrP (37–67) as signal antibody. The assay detects PTHrP (1–84). PTHrP (18–34), PTHrP (9–34), PTHrP (1–34), and PTHrP (37–67) do not cross react in the assay /2/.

LC-MS/MS measurement

Principle: PTHrP is enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP is digested with trypsin, and PTHrP-specific tryptic peptide is analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode /3/.

6.5.3 Specimen

Heparin or EDTA plasma: 1 ml

6.5.4 Reference interval

Refer to Tab. 6.5-1 – Reference intervals for PTHrP.

6.5.5 Clinical assessment

Hypercalcemia is as a concentration above 10.2 mg/dL (2.55 mmol/L). In 80–90% of the cases, primary hyperparathyroidism (pHPT) or malignant tumors are the underlying cause. Tumor hypercalcemia is characterized by hypercalcemia and hypophosphatemia, two laboratory diagnostic findings which also indicate pHPT. Whereas for pHPT the calcium level is often borderline and is mostly below 11 mg/dL (2.55 mmol/L), the increase with tumor hypercalcemia is significant and is often above 13 mg/dL (3.25 mmol/L) /7/.

Tumor hypercalcemia is characterized by increased bone resorption with release of calcium and is related to the following disorders:

  • Osteolytic tumor metastases through the local release of cytokines with cancers of the breast, bladder, lung, uterus, and skin
  • The secretion of 1,25 (OH)2D by the tumor
  • The formation of PTHrP by the tumor.

In addition to the determination of calcium and phosphorus, PTH is often and PTHrP is occasionally determined. Usually, PTHrP determination is not required and PTH is sufficient, because /8/:

  • Calcium and PTH are increased with pHPT
  • In cases of tumor hypercalcemia, calcium is increased and PTH is low or low normal.

Therefore, from a diagnostic standpoint, the determination of PTHrP is only required if the hypercalcemia cannot be sufficiently clarified based on clinical findings, and calcium, phosphorus and PTH levels in serum/plasma (Tab. 6.5-2 – Diseases and conditions with increased PTHrP values).

6.5.6 Comments and problems

Method of determination

The PTHrP values of different assays are not comparable because they measure different structures. Tests which determine mid-regional structures have values that are 10 times higher than the values measured using aminoterminal assays. Larger structures which encompass the N-terminal and mid-regional structures are recorded using the immunometric assays. PTHrP 1–74, 1–84 or 1–86 are used for calibrating the tests /2/.

Reference interval

PTHrP is only determined in 50% of normal individuals (detection limit < 0.2 pmol/L) /2/.

Influence factor

Carboxy-terminal structures and, in a smaller percentage, mid-regional structures of PTHrP are excreted renally. In stage 4 and 5 of renal disease, these increase in the plasma.


Since PTHrP is degraded quicker in whole blood than in plasma, the sample must be centrifuged immediately and the plasma must then be measured or deep-frozen /23/.

6.5.7 Biochemistry and physiology

PTHrP is a protein of 139 amino acids (Fig. 6.5-1 – PTHrP protein: its structures and functions). PTHrP and PTH are products of two different genes, but are capable of carrying out the same actions on the PTH/PTHrP receptor 1. This is due to similar primary and secondary structures, which are necessary for binding to the receptor. Eight of the first 13 amino acids of the aminoterminal structure are identical. Although the primary structure of PTHrP and PTH between amino acids 18 and 34 is not identical, they have a comparable secondary structure, which allows binding to the receptor. In contrast to PTH, which is only formed by the parathyroids, PTHrP is synthesized in many tissues such as the epithelial, mesenchymal connective tissue, endocrine glands, and the central nervous system. Using the calcium content as a reference, PTHrP does not stimulate the enteral absorption of vitamin D to the same extent as PTH and its effect on the bone is also less. Its physiological endocrine effect is limited to an increase of renal-tubular calcium reabsorption and of the placental calcium transport /10/.

In addition to the above-mentioned endocrine effect, PTHrP also has paracrine/autocrine and intracrine effects /11/. The local autocrine/paracrine effect of PTHrP is that it mediates signals via the PTH/PTHrP receptor like an epithelial factor. In health PTHrP regulates bone development by maintaining the endochondral growth plate. It also plays a role during tooth eruption, development of mammary and regulates the vascular tone via the smooth musculature. The smooth muscles of the uterus and the β-cells of the pancreas also secrete and respond to PTHrP. The intracrine effects relate to the PTHrP influencing the cell cycle via effects on the cell nucleus.


1. Frieling JS, Lynch CC. Proteolytic regulation of parathyroid hormone-related protein: functional implications for skeletal malignancy. Int J Molecular Sciences 2019; 20: https://doi.org/10.3390/ijms20112814.

2. Ratcliffe WA, Norbury S, Heath DA, Ratcliffe JG. Development and validation of an immunoradiometric assay of parathyrin-related protein in unextracted plasma. Clin Chem 1991; 37: 678–85.

3. Kushnir MM, Rockwood AL, Strathmann FG, Frank EL, Straseski JA, Meikle AW. LC-MS/MS measurement of parathyroid hormone-related peptide. Clin Chem 2016; 62: 218–26.

4. Burtis WJ, Brady TG, Orloff JJ, Ersbak JB, Warrell RJ, Olson BR, et al. Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. N Engl J Med 1990; 322: 1106–12.

5. Pandian MR, Morgan CH, Carlton E, Segre GV. Modified immunoradiometric assay of parathyroid hormone-related protein: clinical application in the differential diagnosis of hypercalcemia. Clin Chem 1992; 38: 282–8.

6. Dumon JC, Jensen T, Lueddecke B, Spring J, Barle J, Body JJ. Technical and clinical validation of an immunometric assay for circulating parathyroid hormone-related protein. Clin Chem 2000; 46: 416–8.

7. Fritchie K, Zedek D, Grenache DG. The clinical utility of parathyroid hormone-related peptide in the assessment of hypercalcemia. Clin Chim Acta 2009; 402: 146–9.

8. Hiraki A, Ueoka H, Bessho A, et al. Parathyroid hormone-related protein measured at the time of first visit is an indicator of bone metastases and survival in lung carcinoma patients with hypercalcemia. Cancer 2002; 95: 1706–13.

9. Wada S, Kitamura H, Matsuura Y, Katayama Y, Ohkawa H, Kugai N, et al. Parathyroid hormone related protein as a cause of hypercalcemia in a B-cell type malignant lymphoma. Internal Med 1992; 31: 968–72.

10. Strewler GJ. The physiology of parathyroid hormone related protein. N Engl J Med 2000; 342: 177–85.

11. Wysolmerski JJ. Parathyroid hormone-related protein: an update. J Clin Endocrinol Metab 2012; 97 (9): 2947–56.

6.6 Vitamin D

Lothar Thomas

Vitamin D plays an essential role in bone health and in the regulation of calcium and phosphorus metabolism. However, the effects of vitamin D are not limited to mineral homeostasis and maintenance of bone health. Vitamin D physiology extends far above these functions as documented by the presence of vitamin D receptors in many organs and tissues. In addition the 25(OH)D-1α-hydroxylase, the enzyme responsible for the conversion of 25(OH)D (calcidiol) to its biologically active form 1,25 (OH)2D (calcitriol) has been found in other tissues aside from kidneys. The synthesis of calcitriol may be important in regulating cell growth and differentiation via paracrine or autocrine regulatory mechanisms. The action of calcitriol involves a nuclear vitamin D receptor that regulates the transcription of genes in vitamin D target cells that are involved in cell differentiation and calcium homeostasis /12/. The nomenclature of vitamin D precursors and metabolites is shown in Tab. 6.6-1 – Nomenclature of vitamin D precursors and metabolites.

6.6.1 Indication

25-hydroxy vitamin D [25(OH)D]

Anamnestic and clinical advices of vitamin D deficiency are:

  • Chronic kidney failure stage ≥ 2 in children and stage ≥ 3 in adults
  • Lack of sunlight (seniors)
  • Reduced intestinal intake of vitamin D due to fat malabsorption
  • Increased metabolism of vitamin D (barbiturates or anti epileptic drugs)
  • Increased loss of vitamin D binding protein (nephrotic syndrome, dialysis)
  • Hypocalcemia, hypophosphatemia, hyperparathyroidism, increased alkaline phosphatase
  • Radiological signs (pseudo fractures, Looser’s reconstruction zones, reduced bone mineral content).

Suspicion of vitamin D overdose or intoxication; increased 25(OH)D concentration.

1,25-hydroxy vitamin D [1,25 (OH)2D]

The plasma concentration of the active vitamin D reflects the calcium content and the renal function. Indications are:

  • Chronic renal disease in stages ≥ 3 (not generally recommended by the KDIGO).

Differentiation of hypercalcemias:

  • Sarcoidosis, tuberculosis, other granulomatous disease
  • Monitoring after medication of 1α-hydroxy vitamin D3 or of 1,25 dihydroxy vitamin D3
  • Suspicion of intoxication with hypercalcemia-generating plants, e.g. solanum malacoxylon
  • Hypercalciuria of unclear genesis.

Differentiation of hypocalcemia:

  • 25(OH)D-1α-hydroxylase deficiency (vitamin D-dependent rickets, VDDR), low calcitriol concentration
  • Vitamin D receptor defect (resistance to calcitriol, VDRR, high calcitriol concentration).

6.6.2 Method of determination

To overview methods of determination refer to reference /3/. 25(OH)D (calcidiol)

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) /4/

Principle: at first the specimen is prepared by precipitation of serum proteins and dissociation of vitamin D from the binding protein. After centrifugation the supernatant is transferred to a solid phase extraction plate and the retained analytes transferred to the auto sampler for chromatographic separation on an ultra-performance liquid chromatography (UPLC) system. The eluent of the UPLC system is introduced in a mass spectrometer set in positive electro spray mode for quantification of 25(OH)D.

Sequentially competitive immunoassay /5/

Principle: sequential competitive immunoassay format. The specimen is added to a reaction cuvette followed by displacement buffer for removal of vitamin D from the binding protein. Monoclonal antibody conjugated with acridinium ester is added and allows to bind 25(OH)D in the specimen. A 25(OH)D analog conjugated to bovine serum albumin and fluorescein is added along with anti-fluorescein coated paramagnetic particles. The reaction cuvette is washed, and acid and base reagents are added to initiate the chemiluminescent reaction. An inverse relation exists between the amount of 25(OH)D in the specimen and the amount of relative light units detected by the system.

Electrochemoluminescence immunoassay /6/

Principle: competitive immunoassay for determining 25(OH)D3 based on the streptavidin-biotin technology. The test uses a polyclonal antibody from sheep against 25(OH)D3, which is ruthenium labeled. The vitamin D3 in the specimen competes for binding with biotinylated 25(OH)D3 antigen which is bound to the streptavidin-coated micro particles. 1,25(OH)2D (calcitriol)

Immunoassay /37/

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) /7/.

6.6.3 Specimen

Serum or plasma: 1 mL

Fasting blood sample in the morning; hyperlipoproteinemia interferes the extraction of vitamin D. For dialysis patients, the blood sample should be taken before the dialysis.

6.6.4 Recommended values and reference intervals

Refer to Tab. 6.6-2 – Recommended levels for 25(OH)D and reference interval for 1,25(OH)2D.

6.6.5 Clinical significance 25(OH)D and vitamin D state

Depending on the geography, 20–50% of the European and North American population have a vitamin D deficiency. The 25(OH)D value reflects the intake of vitamin D with the diet, and its formation in the skin. The determination of 25(OH)D is therefore the best biomarker for assessing the vitamin D status. The synthesis of the active hormone 1,25(OH)2D depends on the stored amount of 25(OH)D and multiple factors, which convert 25(OH)D into 1,25(OH)2D. One important factor is the 25-OHD-1α-hydroxylase (CYP27B1), which converts 25(OH)D into 1,25(OH)2D and its inactivation through the 24-(OH)D hydroxylase (CYP24A1) to 1,24,25 (OH)D.

The evaluation of the serum level of 25(OH)D is not problem-free because the values of a reference population are not very authoritative, since individual levels depend on /11/:

  • Ecological factors (season, local weather conditions, nutrition, sunbathing)
  • Unchangeable individual factors (ethnicity, skin pigmentation, age) /12/. These factors especially affect older people in northern latitudes. As they become older, they stay indoors more, wear less exposing clothing, change their eating habits and take in less fat in their diet. Due to increasing atrophy of the skin, fewer pre-vitamin D is converted to vitamin D. Also, due to increasingly limited renal function, the hydroxylation of 25(OH)D to 1,25(OH)2D declines /13/.

The 25(OH)D level depends on the season. The highest values are measured 4–6 weeks after the highest amount of sunlight. The lowest [< 20 μg/L (50 nmol/L)] are measured at the end of winter; from January to April in Central Europe. According to a study /14/, 57% of men and 58% of women in Germany have 25(OH)D values below 20 μg/L (50 nmol/L). For persons 65–79 years of age, the prevalence is 75%.

There is an increased demand for vitamin D in patients with chronic kidney disease, during pregnancy and lactation. A sufficient vitamin D supply for newborns is only achieved if 25(OH)D in the milk of breast-feeding mother is > 30 μg/L (75 nmol/L). 25(OH)D and calcium homeostasis

25(OH)D values below 20 μg/L (50 nmol/L) promote secondary hyperparathyroidism /15/. Therefore this concentration has generally been defined as the low threshold value for inhabitants of Central Europe. In part, however, even higher 25(OH)D values, may cause an increase of PTH within the reference interval. People with an optimal vitamin D supply have PTH values below 45 ng/L (4.5 pmol/L). If the vitamin D supply is suboptimal, the PTH level elevates to 45–65 ng/L (4.5–6.5 pmol/L) /16/.

For persons over 70 years of age, secondary hyperparathyroidism can only be reliably prevented with 25(OH)D values above 40 μg/L (100 nmol/L) /17/. Post-menopausal women with 25(OH)D values ≤ 25 μg/L (62,5 nmol/L) often have increased markers of bone resorption /18/. 25(OH)D levels below 5–4 μg/L (12–10 nmol/L) are often associated with osteomalacia. For osteoporosis prevention, 25(OH)D values above 25 μg/L (62.5 pmol/L) are the goal /18/. Pleiotropic effects of vitamin D

Vitamin D receptors are present in most tissues /1/. A deficiency of 25(OH)D can therefore not only be associated with calcium homeostasis disorders and diseases of the bone, but also with the pathogenesis of chronic diseases. Thus, among other things, vitamin D affects the cardiovascular system, the immune system and the glucose tolerance, which means that a 25(OH)D deficiency can be involved with the increased risk of a negative outcome of these diseases. Diseases and conditions with decreased levels of 25(OH)D are listed in Tab. 6.6-3 – Diseases and conditions with modified 25(OH)D or 1,25 (OH)2D concentration. Findings in rickets and osteomalacia are shown in Tab. 6.6-4 – Findings for the various forms of rickets/osteomalacia. Laboratory investigations in 25(OH)D deficiency

In cases of 25(OH)D deficiency and acute or chronic disorders of the bone further investigations are calcium, phosphate, creatinine, 1,25 (OH)2D and PTH in the serum and calcium excretion in the urine. Findings on the differentiation of 25(OH)D deficiency of rickets/osteomalacia are shown in Tab. 6.6-4 – Findings for the various forms of rickets/osteomalacia. Monitoring the vitamin D treatment 

The treatment of 25(OH)D deficiency, especially for older patients, serves the purpose of reducing the risk of bone fractures and preventing falling down by improving the neurological/psychological function. The following laboratory examinations are recommended /19/:

  • Determination of serum calcium before start of treatment with 1–2 thousand IU of vitamin D, because hypercalcemia is a contraindication
  • Measurement of 25(OH)D to control if the dose makes sense. The first measurement should take place 4–6 weeks after treatment is started. Conditions with increased concentration of 25(OH)D

Conditions with increased concentration of 25(OH)D can occur with Vitamin D treatment or corresponding vitamin D3-containing pharmaceuticals (e.g., cod liver oil) or a 25(OH)D treatment.

25(OH)D concentrations above 80 μg/L (200 nmol/L) are only measured in cases with:

  • High vitamin D dosage (> 10,000 IE daily)
  • Overdose of vitamin D in patients with hypoparathyroidism
  • Intoxications with dihydrotachysterol; the intake of these preparations is not analyzed by the assays for 25(OH)D. 1,25(OH)2D and vitamin D status

1,25(OH)2D (calcitriol) is the active vitamin D and fulfills the functions of a classic hormone /20/. The hormonal signal is transmitted by specific cell receptors of the small intestine, the bone, the kidney, and numerous other organs. In addition to genomically mediated effects, there are also non-genomic effects that act quickly, within 2–6 minutes. The task of 1,25 (OH)2D is to maintain the calcium and phosphate homeostasis in association with PTH and FGF23. In addition, 1,25(OH)2D has pleiotropic effects.

Since 95% of the 25(OH)D in serum circulates as 25(OH)D3, only the 1,25(OH)2D3 is first determined.

The concentration of 1,25(OH)2D3 is 500 to 1,000 times less than that of 25(OH)D, but the affinity to the vitamin D receptor is higher for 1,25(OH)2D.

With the determination of 1,25 (OH)2D disorders in the vitamin D metabolism are diagnosed because the concentration mirrors the activity of the 25-OHD-1α-hydroxylase in the kidney. The compliance of the vitamin D intake can also be monitored. During growth spurts and during pregnancy, high concentrations of 1,25(OH)2D are measured. Hypophosphatemia, hypocalcemia and PTH stimulate the 25-OHD-1α-hydroxylase. This explains an increased formation of 1,25(OH)2D and hyperabsorption of calcium from the intestine for conditions with hypophosphatemia (e.g., for some of the cases of primary hyperparathyroidism or the treatment of rickets with vitamin D).

25(OH)D should be measured if deficiency of vitamin D is suspected although 1,25(OH)2D is the active form of the vitamin. The reasons are /36/:

  • The 25(OH)D concentration corresponds with the vitamin D intake and activity
  • The half live of 1,25(OH)2D is 4 to 6 hours as compared with a half-life of 2 to 3 weeks for 25(OH)D.
  • 1,25(OH)2D concentration may be useful for the diagnosis of genetic rickets, in chronic kidney failure and unexplained hypercalemia, or hypercalciuria.

6.6.6 Comments and problems

Blood sampling

In fasting state in the morning or before dialysis.

Method of determination

The selection of the assay for the determination of vitamin D, either 25(OH)D or [25(OH)D3 and 25(OH)D2] is important. Since some assays only measure 25(OH)D3, the laboratory must decide whether it will determine 25(OH)D, 25(OH)D3 or 25(OH)D3 and 25(OH)D2 separately. Immunoassays only show a moderate agreement with the LC-MS/MS. Concentrations below 8 μg/L (20 nmol/L) are not reliably measured /4/.

The immunoassays for determining 1,25(OH)2D only use antibodies against 1,25(OH)2D3.

Influencing factor

After a heparin injection (e.g., under dialysis treatment) there is an increase of 25(OH)D. The blood sample should be drawn before the dialysis.


At room temperature for 72 hours, decrease of 25(OH)D by 2.3% if the sample is kept in the dark. With exposure to daylight and at room temperature, a decrease by 3.4% after 24 hours and 8.5% after 7 days /21/.

6.6.7 Biochemistry and physiology

During exposure to solar ultraviolet radiation, 7-hydroxycholesterol is photosynthesized in the skin to cholecalciferol which is converted to vitamin D3. The initial substances of vitamin D3, cholecalciferol and of vitamin D2, ergocalciferol, are lipophilic and are therefore difficult to determine in serum. Their half-life in plasma is 24 hours. Cholecalciferol is formed endogenically and ergocalciferol is taken in with vegetables and fish. Vitamin D3 and vitamin D2 from dietary sources are incorporated into chylomicrons and transported by the lymphatic system into the venous circulation.

Vitamin D in the circulation is bound to vitamin D-binding protein and transported to the liver, where it is converted by vitamin D-25 hydroxylase to 25-hydroxy vitamin D [25(OH)D]. This form of vitamin D is biologically inactive and more than 99% is bound to proteins, especially to vitamin D-binding protein. 25(OH)D is the major circulating form in of vitamin D that is used by the clinicans to determine the vitamin D status.

Approximately 99.7% of total circulating 25(OH)D in serum is bound to vitamin D binding protein and albumin, and only 0.03% is free. It is hypothyzed that only free 25(OH)D can enter cells and induce its biological function.The main proportion is 25(OH)D3 (calcidiol), whereas 25(OH)D2 (ercalcidiol) is primarily measured in a greater concentration in patients with vitamin D2 substitution. The concentration of 25(OH)D3 fluctuates seasonally because the formation of cholecalciferol depends on the effect of sunlight on the skin. The two 25(OH)D molecules differ slightly in terms of their structure, but they have the same biological effect. Therefore it can be important sometimes to measure 25(OH)D3 and 25(OH)D2 to determine the vitamin D status.

Vitamin D (D2 and D3) from the diet and the skin undergo two sequential hydroxylations: first in the liver 25(OH)D2 and 25(OH)D3 and then in the kidneys, leading to its biologically active forms 1,25(OH)2D2 and 1,25(OH)2D3.The half life time of both hormones is 4–6 hours /2/.

25(OH)D is biologically inactive and must be converted in the kidneys by 25-OHD-1α-hydroxylase in the active form 1,25(OH)2D. The main regulators of 25-OHD-1α-hydroxylase activity are the serum concentrations of calcium, phosphate and PTH. The hormones 1,25(OH)2D, PTH and FGF23 regulate by several negative feedback loops the metabolism of phosphate and calcium. The function of the loops results in the enteral absorption and renal handling of calcium and phosphate. In addition the bone mineralization is modulated /2/. Refer to (Fig. 6.6-1 – Formation and metabolism of vitamin D in three steps).

The functions of 1,25(OH)2D are /1/:

  • Enhancement of the intestinal absorption of both calcium and phosphorus. 1,25(OH)2D interacts with the vitamin D receptor (VDR). It is located in the cytoplasm of the cells and belongs to the nucleic receptors of the steroid hormones /22/. After binding of a ligand, the VDR heterodimerizes with the retinoid X receptor (RXR) to form a complex (RXR-VDR), which, after reaching the cell nucleus, binds to vitamin D responsive elements (VDRE). The latter are localized in the promoter region of different genes, whose transcription rates are changed by the binding of the complex RXR-VDR (see example in Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis). In addition the expression of the epithelial calcium channel and of calbindin 9K, a calcium binding protein, is increased.
  • Inhibition PTH synthesis by negative feedback mechanisms and by increased serum calcium levels.
  • Regulation of bone metabolism through binding to its receptor on osteoblasts causing an increase in the expression of the RANKL (see Tab. 6.1-2). RANK, the corresponding receptor on preosteoclasts binds RANKL, which induces preosteoclasts to become mature osteoclasts.
  • Enhancement of the expression of 25-OHD-24-hydroxylase to catabolize 1,25(OH)2D to the biologically inactive calcitoic acid.

To evaluate the vitamin D status, the concentration of 25(OH)D in serum is measured because it represents the content of vitamin D that is available to the body. If the concentration is normal, the determination of 1,25(OH)2D is not required to asses a relevant vitamin D deficiency if chronic kidney disease in stage ≥ 3 can be ruled out /34/.

Low concentrations of 25(OH)D induce secondary hyperparathyroidism (sHPT) and stimulate the 25-OHD-1α-hydroxylase, which keeps the concentration of 1,25 (OH)2D normal for a long time despite vitamin D deficiency /19/. Thus, a 25(OH)D deficiency primarily causes osteoporosis (decrease in bone mass) as a result of the sHPT. A decrease of 1,25(OH)2D only manifests after a longer period following severe 25(OH)D deficiency. As a consequence of this, a metabolic bone disease results in the form of osteomalacia (i.e.; the reduced synthesis of bone matrix and mineralization defects). In a low vitamin D state, the small intestine absorbs 10–15% of dietary calcium, however when adequate 30–40%.

The maximum bone mass in adolescents depends on the calcium intake, bodily activity, and genetic factors. Up to 85% of the inter individual variability of the bone mass depends on genetic factors, especially on genes that encode the VDR. The VDR gene polymorphism is associated with the mineral mass of the bone. In particular, the polymorphism of the start codon (regulated by splitting the enzyme Fok1) is associated with the bone density. Thus, the Fok1 polymorphism of the VDR receptor appears to predict the bone mineral mass in children /23/.


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3. Farrell CJ, Herrmann M. Determination of vitamin D and its metabolites. Best practice & Research Clinical Endocrinology & Metabolism 201§; 27: 675–88.

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5. Hermida FJ, Fernandez M, Laborda B, Perez A, Lorenzo MJ, Magadan C. Assessment of Advia Centaur analyzer for the measurement of 25-OH vitamin D. Clin Lab 2012; 58: 987–95.

6. Roth HJ, Schmidt-Gayk H, Weber H, Niederau C. Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography – tandem mass spectrometry as a reference. Ann Clin Biochem 2008; 45: 153–9.

7. Spanaus K, von Eckardstein A. Evaluation of two fully automated immunoassay based tests for the measurement of 1,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS. Clin Chem Lab Med 2017; 55: 1305–14.

8. Holick MF, Binkley NC, Biscoff-Ferrari HA, Gordon M, Hanley DA, Heany RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Practice Guideline. J Clin Endocrinol Metab 2011; 96: 1911–30.

9. Mark S, Gray-Donald K, Delvin EE, O’Laughlin J, Paradis G, Lambert M. Low vitamin D status in a representative sample of youth from Quebec, Canada. Clin Chem 2008; 54: 1283–9.

10. Wildermuth S, Dittrich K, Schmidt-Gayk H, Zahn I, O’Riordan JLH. Scintillation proximity assay for calcitriol in serum without high pressure liquid chromatography. Clin Chim Acta 1993; 220: 61–70.

11. Mosekilde L. Vitamin D and the elderly. Clinical Endocrinology 2005; 62: 265–81.

12. Webster C. Relationship of total 25-OH vitamin D concentrations to indices of multiple deprivation: geoanalysis of laboratory results. Ann Clin Biochem 2013; 50: 31–8.

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14. Hinzpeter B, Mensink GBM, Thierfelder W, Müller MJ, Scheidt-Nave C. Vitamin D status and health correlates among German adults. EJCN 2007; 1–11.

15. Malabanan, A, Veronikis IE, Holick MF. Redefining vitamin D insufficiency. Lancet 1998; 351: 805–6.

16. Guillemant J, Cabrol S, Allendou A, Peres G, Guillemant S. Vitamin D-dependent seasonal variation of PTH in growing male adolescents. Bone 1995; 17: 513–6.

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23. Carey DE, Golden NH. Bone health in adolescence. Adolesc Med State Art Rev 2015; 26 (2): 291–325.

24. Lewis R. Mineral and bone disorders in chronic kidney disease: new insights into management. Ann Clin Biochem 2012; 49: 432–40

25. Wolf M. Verbesserung der Überlebensrate von Dialysepatienten durch aktives Vitamin D. Nieren- und Hochdruckkrankh 2008; 37: 224–8.

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27. Ramasamy I. Inherited disorders of calcium homeostasis. Clin Chim Acta 2008; 394: 22–41.

28. Svoren BM, Volkening LK, Wood JR, Laffel LMB. Significant vitamin D deficiency in youth wit type 1 diabetes mellitus. J Pediatr 2009; 154: 132–4.

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36. Virkud YV, Fernandes ND, Lim R, Mitchell DM, Rothwell WT. Case 39-2020: a 29 month-old boy with seizure and hypocalcemia. N Engl J Med 2020; 383: 2462–70.

6.7 Biochemical bone markers

Lothar Thomas

Biochemical bone markers are a tool in the diagnosis and monitoring of metabolic bone disease /1/. Their most application in clinical practice is monitoring of osteoporosis and the efficacy of anti resorptive treatment as an adjunct to bone mineral density measurements. Other applications are the use of these markers for bone diseases other than osteoporosis, such as bone mineral disorders (chronic renal failure, vitamin D deficiency, hyperparathyroidism), hypercortisolism, hyperthyroidism, diabetes mellitus type I, metastatic bone disease, multiple myeloma, and Paget’s disease.

A distinction is made between bone formation markers and bone resorption markers /2/ (Fig. 6.7-1 – Bone markers associated with bone resorption and bone formation).

Bone formation markers

These markers are direct or indirect products of the osteoblasts, which are formed during the various phases of the life cycle of the osteoblasts /1/. Important markers are:

Bone resorption markers

These markers are direct or indirect products of the osteoclasts. The cross links of the collagen molecules of the bone matrix form special structures during the synthesis or degradation that are used for analysis /3/ (Fig. 6.7-2 – Generation of biomarkers during type 1 collagen synthesis and degration).

Because most of the metabolic bone diseases are characterized by an increase in bone resorption, these markers are of special interest. Important markers are:

6.7.1 Synthesis and degradation of collagen I

Procollagen I extension peptides /4/

Bone is a composite material of about 70% mineral matter (mainly hydroxyl apatite), 5–8% water and 22–25% organic matrix (mainly type I collagen) /2/. Collagen type I is a heterodimer made up of three polypeptide chains (triple helix) and is rich in the amino acids lysine and proline. Two polypeptide chains (α1) are identical, the third (α2) has a different amino acid sequence. After synthesis, two α1chains and one α2 chain are twisted to one triple helix, the fibril. The three α chains form a triple helix by covalent binding via hydroxylated lysine remnants of the adjacent chains (Fig. 6.7-2 – Generation of biomarkers during type 1 collagen synthesis and degradation).

The α chains are synthesized as pre procollagen and consist of a signal peptide at the N-terminal end as well as N- and C-terminal pro peptides. After the pro peptides are cleaved from the fibril, it is transported to the extracellular space via the Golgi apparatus.

The fibrils arrange as bundles in the extracellular space to produce large collagen fibers. The carboxy-terminal and aminoterminal ends of pro collagen I are enzymatically cleaved during extracellular processing and fibril formation prior to incorporation of type I collagen into the bone matrix.

The cleavage of collagen type 1 yields two relative large extension peptides termed pro collagen I carboxy-terminal pro peptide (PICP) and pro collagen I N-terminal pro peptide (PINP) (Fig. 6.7-2).

N-telopeptide (NTX), C-telopeptide (CTX) /4/

When type I collagen is degraded by osteoclasts during the resorption process, NTX and CTX fragments of type 1 collagen are released in the circulation. During the degration of collagen, NTXs and CTXs are relatively specific for bone tissue breakdown. Other tissues comprised of type 1 collagen are not actively metabolized by osteoclasts, and therefore different fragments are formed during breakdown of non skeletal tissue. Immunoassays, which use antibodies against CTX are called β-crossLap tests (CTX).

Pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) /4/

Post translational processing of lysine and hydroxylysine residues to form Pyr and D-Pyr cross links are essential for stabilizing the mature forms of collagen fibers and elastin. Both cross links are present in bone and absent from most tissues including skin. D-Pyr is more bone specific because Pyr cross links are present in cartilage and several soft tissues. Three-fold more Pyr is present in bone compared to D-Pyr.

6.7.2 Significance of bone markers

In metabolic bone diseases, the remodeling process is accelerated and, consequently, the concentration of bone turnover markers in serum and urine is increased. The bone turnover markers reflect the total rate of bone formation and bone resorption of the skeleton. Bone turnover marker allow a dynamic assessment of bone metabolism, thus supplementing the static information of the bone mass, which is determined by measuring the bone mineral density /4/.

The intraindividual variation of the bone turnover markers is relatively high, which means that bone turnover markers are only recommended to a limited extent for diagnosing bone diseases, in particular if it involves the determination in urine. The currently essential clinical application is the monitoring of metabolic bone disease, especially osteoporosis under anti resorptive treatment /5/. The biomarker should be determined before, after a 3–6 month period, after 12 months, and then annually. Bone resorption markers significantly decrease as early as 3–6 months. As a rule, a decline of 30–70% in comparison to the initial value is expected, then it will reach a plateau. An insufficient decrease is a sign of failed treatment or poor compliance of the patient. Using bone mineral density measurement changes in density are normally observed after 1–2 years /6/.

The bone turnover markers provide no information about the current bone mineral content or bone fragility, nor do they provide information about a specific site with increased bone turnover in the skeleton. This is only possible using the bone mineral density measurement, because it allows the bone mass to be assessed as normal, osteopenic, or osteoporotic. Because bone mineral density provides information of the current bone status and bone markers provide information about current bone metabolism, both parameters complement each other, and provide comprehensive information about patients bone status and bone metabolic activity /2/. Thus, patients with a high rate of bone resorption have an increased fracture risk and those with a combination of low bone mineral density and a high rate of bone resorption have the highest risk of fracture /7/.

The reference intervals of the bone turnover markers have limited authority in comparison to other biomarkers in clinical chemistry. Thus, for post-menopausal osteoporosis, the bone marker levels of most patients are within the reference intervals and only relative changes compared to a value before treatment play a role. To overcome this problem and to allow the detection of physiological changes in bone marker values in an individual the use of least significant change is used (Tab. 6.7-1 – Least significant change).

Least significant change (LSC): the LSC is the minimal change of a bone turnover marker which must be occur during treatment to make a response significant. The LSC is calculated from the intraindividual and analytical variability according to a formula /2/. A change in bone markers that is higher than the LSC would have a 95% chance of being too high in comparison to normal biological variability. Thus the LSC for β-CTX measurements is 31.2% for a change in bone mineral density of the spinal column of 3.8% /9/.

Evaluation of bone turnover markers

The bone turnover markers are not recommended for diagnosing osteoporosis and for determining the bone mass. The currently best established and most common clinical application of bone turnover markers is the monitoring of osteoporosis treatment and controlling the compliance of these patients /10/.

Bone formation markers

Bone formation markers, especially the bone ALP, have advantages over resorption markers in assessing the bone participation in cases of vitamin D deficiency, hyperparathyroidism, osteomalacia/rickets, renal osteodystrophia, osteoblastic metastases, and Paget’s disease. P1NP is intended for assessing the anabolic treatment of osteoporosis with recombinant parathyroid hormone.

Bone resorption markers

In the treatment of osteoporosis, the resorption markers, particularly pyridinolines and C-telopeptides (CTX), have become significant. Experience shows, however, that in most cases (e.g., post-menopausal women, older men) the CTX determination (EDTA plasma at 8 a.m. in fasting state) is the superior marker for bone resorption and pyridinolines move into the background. In contrast, for tumor patients (e.g., bronchial carcinoma, breast carcinoma, multiple myeloma) the pyridinolines are the superior marker, determined in the first morning urine.


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2. Vesper HW. Analytical and preanalytical issues in measurement of biochemical bone markers. Labmedicine 2005; 36: 424–9.

3. McCudden CR, Kraus VB. Biochemistry of amino acid racemization and clinical application to musculoskeletal disease. Clin Biochem 2006; 39: 1112–30.

4. McCudden CR, Kraus VB. Biochemistry of amino acid racemization and clinical application to musculoskeletal disease. Clin Biochem 2006; 39: 1112–30.

5. Camacho PM, Lopez NA. Use of biochemical markers of bone turnover in the management of postmenopausal osteoporosis. Clin Chem Lab Med 2008; 46: 1345–57.

6. Anlinker M, Bieglmayer C, Dimai HP, Gasser RW, Kudlacek S, Obermayer-Pietsch B, et al. Labordiagnostik in der Prävention, Differentialdiagnose und Verlaufskontrolle der Osteoporose. J Lab Med 2009; 33: 140–6.

7. Walker-Bone K, Walter G, Cooper C. Recent developments in the epidemiology of osteoporosis. Curr Opin Rheumatol 2002; 14: 411–5.

8. Biegelmayer C, Clodi M, Kudlacek S. Biomarker in der Osteologie: Aktueller Stand. J Mineralstoffw 2007; 13: 82–7.

9. Christgau S, Cloos PAC. Current and future applications of bone turnover markers. Clin Lab 2003; 49: 439–46.

10. Lee J, Vasikaran S. Current recommendations for laboratory testing and use of bone turnover markers in the management of osteoporosis. Ann Lab Med 2012 32: 105–12.

6.8 Bone ALP

Lothar Thomas

The bone specific ALP (BALP) is produced by the osteoblast and is deposited as buds in cell membrane vesicles. BALP plays an important role in the bone formation. Approximately 20–40% of serum ALP is BALP (see also Section 1.3 – Alkaline phosphatase (ALP)).

6.8.1 Indication

Indicator of global bone formation activity:

  • Monitoring of bone mineral disease in patients with chronic renal failure
  • Management of patients with Paget’s disease
  • Monitoring of bisphosphonate therapy for osteoporosis
  • Management of patients with bone metastases.

6.8.2 Method of determination

See Section 1.3 – Alkaline phosphatase.

6.8.3 Specimen

Serum or heparin plasma, no EDTA, citrate or oxalate plasma: 1 mL

6.8.4 Reference interval

Refer to Ref. /12/ and Tab. 6.8-1 – Reference intervals for bone ALP.

6.8.5 Clinical assessment

To evaluate discrete changes of the bone metabolism, such as post-menopausal, in cases of chronic kidney disease-mineral bone disease, or after administration of glucocorticoids (e.g., after an organ transplantation) the BALP is often determined in addition to the ALP.

In one study /3/ the ALP increased by 24% after menopause and the BALP by 77%. Expressed as Z-score (multiple of the standard deviation above the mean value for pre-menopausal women), the ALP increased by 0.9 and the BALP by 2.2. The BALP responds relatively slow as bone formation marker. Chronic kidney disease-mineral bone disease (CKD-MBD)

For monitoring CKD-MBD the KDIGO guidelines /4/ recommend the ALP or BALP be determined in stages G4 - G5D every 12 months or more frequently if PTH is increased. In comparison with a healthy control group, patients with stage G4 and G5D had the values shown in Ref. /5/ and Tab. 6.8-2 – Findings in patients with CKD stages G4 and G5D in comparison to a healthy control group for BALP and other bone markers. Regarding the response of BALP to treatment of renal osteodystrophia see Tab. 1.3-4 – Diseases of the bone associated with elevated total ALP. Vitamin D deficiency

An increase in BALP or ALP is only seen after more than 6 months of vitamin D deficiency. When the vitamin D deficiency is corrected, the BALP increases even further in the first weeks, but then returns to normal over the course of 6–12 months. Fractures and polytraumas

In the first 5 days, there is a decrease of BALP in the serum, followed by an increase, which is still not normalized for large fractures after 100 days and often only reach the initial values after one year /6/. Paget’s disease

For this disease, ALP or BALP are the superior bone markers (see Section 1.3 – Alkaline phosphatase). Malignant tumors

Carcinomas metastasizing in the bone, especially carcinomas of the breasts, lungs and prostate can cause an increase of BALP /7/. The prevalence is shown in Tab. 6.8-3 – Incidence of bone metastases in advanced malignant tumors. The metastases lead to dysregulation of the bone metabolism and cause osteolytic, osteoblastic or mixed bone metastases, depending on the malignoma. In cases with osteoblastic metastases, there is an excessive bone formation with an increase of BALP. In patients with prostate carcinoma and bone metastases, resorption markers quickly respond to treatment. The BALP, if at all, only shows signs of decline after more than 4 weeks /8/. With multiple myeloma, the function of osteoclasts is particularly increased, therefore, it does not make sense to determine bone formation markers such as BALP /9/. Therapeutic measures

Administering sodium fluoride: if sodium fluoride is administered to treat osteoporosis, an increase of the BALP occurs (NaF stimulates the osteoblasts).

Glucocorticoids: they lead to a decrease of osteocalcin within 24 hours (by approximately 50% after 1 g of methylprednisolone i.v.) /10/, but a decrease of BALP only occurs after a few weeks.

Bisphosphonate treatment: after alendronate is administered, for example, a new, lower plateau of the BALP is reached after 6 months /11/.

Hormone substitution: after substitution with estrogen or estrogen/gestagen, a lower BALP plateau is reached after 12 months /12/.


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3. Garneo P, Delmas PD. Assessment of serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. J Clin Endocrin Metab 1993; 77: 1046–53.

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6. Herrmann W, Quast S, Hagenbourger O, Laurer H, Marzi I. The role of bone alkaline phosphatase (BAP) as a diagnostic marker of fracture repair. Clin Lab 1998; 44: 855–8.

7. Hannon RA, Eastell R. Bone markers and current laboratory assays. Cancer Treatment Reviews 2006; 32, Suppl 1: 7–14.

8. Berruti A, Dogliotti L, Tucci M, et al. Metabolic effect of single dose pamidronate administration in prostate cancer patients with bone metastases. Int J Biol Markers 2002; 17: 244–52.

9. Coleman RE. The role of bone markers in metastatic bone disease. Cancer Treatment Reviews 2006; 32, Suppl 1: 1–2.

10. Peretz A, Moris M, Willems D, Bergmann P. Is bone alkaline phosphatase an adequate marker of bone metabolism during acute corticosteroid treatment? Clin Chem 1996; 42: 102–3.

11. Kress BC, Mizrahi IA, Armour KW, Marcus R, Emkey RD, Santora AC II. Use of bone alkaline phosphatase to monitor alendronate therapy in individual postmenopausal osteoporotic women. Clin Chem 1999; 45: 1009–17.

12. Overgaard K, Alexandersen P, Riis BJ, Christiansen C. Evaluation of a new commercial IRMA for bone-specific alkaline phosphatase during treatment with hormone replacement therapy and calcitonin. Clin Chem 1996; 42: 973–4.

6.9 Osteocalcin (OC)

Hilmar Stracke, Lothar Thomas

OC (bone gamma-carboxylglutamic acid-containing protein; GLA) is synthesized by the osteoblasts during the matrix mineralization phase and is incorporated into the organic bone matrix /1/. A small fraction of newly synthesized OC is secreted in the circulation and, in individuals with normal renal function, excreted in the urine. During bone resorption OC is degraded, however up to 70% also enter the circulation. Because OC in circulation may be both newly synthesized during bone formation and released during resorption, there is some question whether OC should be considered a marker of osteoblast activity or an indicator of bone matrix metabolism or turnover /2/. Its synthesis is regulated by 1,25(OH)2D.

6.9.1 Indication

Monitoring bone remodeling in:

  • Osteoporosis (assessment of the bone turnover)
  • Carcinoma with bone metastases
  • Primary hyperparathyroidism
  • Renal osteopathy.

6.9.2 Method of determination

Immunometric assay, enzyme-linked immunosorbent assay (ELISA). Most commercially available assays use antibodies which detect N-terminal and mid-regional fragments of the OC (N-MID osteocalcin) /3/. There is no OC reference preparation. The patient values obtained using various test kits are not comparable /3/.

6.9.3 Specimen

Serum, EDTA or lithium heparin plasma; blood sampling at 8–9 a.m. in fasting state: 1 mL

6.9.4 Reference interval

Approximately 2–10 (11), (15), (17), (34) μg/L* /3/

* Depending on the test kit

6.9.5 Clinical significance

In clinical diagnostics OC is used a bone formation marker and the serum level reflects 10–40% of the OC that is not incorporated in the bone matrix, but is released into the circulation as intact 1–49 OC. Released OC is rapidly metabolized, one third is retained as an intact molecule, one third is a large N-terminal fragment of the OC (amino acids 1–43), and one third exists as a mid-regional fragment and in the form of smaller fragments /3/.

An increased serum concentration of OC is the result of an enhanced bone remodeling. Quantitative histomorphometric examinations and examinations of the kinetics and balance of calcium have shown that OC is more a biomarker of bone formation than of bone resorption /4/.

The OC fragments are eliminated by metalloproteinases of the liver and kidney. Since OC fragments are excreted renally, an increased OC concentration can also be caused by limited renal clearance.

Increases of OC in serum are mostly related to an enhanced bone turnover, as with primary hyperparathyroidism, Paget’s disease, and high-turnover osteoporosis.

The OC concentration in the serum is, like all bone metabolism markers, higher in children than in adults. In children, OC correlates to the growth rate, therefore there is a peak during puberty. Whereas, for example, CTX are relatively consistently higher in post menopausal than in premenopausal women, there is a temporary increase for OC in the time following menopause. In premenopausal women, there are significantly higher OC levels during the luteal phase /3/.

One advantage of OC in comparison to the bone-specific ALP is in the stronger and faster reaction when glucocorticoids are administered (suppression of osteoblast activity). Disadvantages are dependency on the glomerular filtration rate, weaker response with Paget’s disease, and limited bone formation specificity. The clinical significance of OC in comparison to ALP is shown in

Since the OC gene expression is regulated directly by 1,25(OH)2D and corticosteroids, the OC values of patients who are treated with these medications should be interpreted with caution.

6.9.6 Comments and problems

Sample collection

Blood collection is recommended in the morning in fasting state. There is a circadian rhythm of the OC with high values in the early morning and a nadir in the afternoon and early evening. Then an increase follows, which reaches its maximum value from midnight to 4 a.m. /12/.


Anticoagulated blood samples have excessively low OC values in comparison to the serum. In one study /13/ the level was only 83.6% of the level in serum when Li-heparin was used, 65.1% for EDTA, 70.4% for sodium citrate (0.13 mol/L) and 37.3% for oxalate/fluoride.

Method of determination

In addition to intact OC the N-terminal and mid-regional fragments (N-MID-OC) are also measured.

Influence factors

OC formation is stimulated by physiological levels of 1,25(OH)2D. Parathyroid hormone and the glucocorticoids prednisolone and deflazacort suppress the 1,25(OH)2D stimulated OC synthesis. OC is important for the synthesis of clotting factors of the prothrombin complex and other vitamin K-dependent proteins. Dicumarol suppresses the OC synthesis.

Seasonal rhythms

Since higher bone resorption rates are observed in winter with lower supplies of vitamin D and a vitamin D deficiency also stimulates the osteoblasts, the highest OC values are observed in February and the lowest in July.


N-MID-OC: in whole blood, at room temperature (RT) for 8 hours, at RT for 24 hours in serum, for 48 hours at RT 1 week at 4–8 °C, and for 1 year at –20 °C in EDTA plasma.

Intact OC: the serum should be separated and stored within 1 hour /14/.

6.9.7 Biochemistry and physiology

OC is a protein composed of 49 amino acids and has a MW of 5.8 kDa; it is formed from a larger 11 kDa precursor by proteolytic cleavage and contains up to three γ-carboxyglutamic acid residues (bone gla protein).

The precursor consists of three structures:

  • A signal peptide made up of 23 amino acids, which is cleaved after translation
  • The target peptide of 26 amino acids, which directs the peptide to the γ-carboxylation
  • The OC protein of 49 amino acids. The OC is one of the three vitamin K-dependent proteins, which are formed from the osteoblasts. The others are the matrix Gla protein and protein C. Vitamin K is an essential cofactor of the post-translational γ-carboxylation of OC. Through carboxylation, a second carboxyl group is introduced to the glutamyl residues in the positions 17, 21 and 24 during the formation of γ-carboxy glutamyl residues (Gla) (Fig. 6.9-1 – γ-carboxylation of glutamyl residues). This modification leads to a conformational change of the protein with an increase of the affinity for calcium and hydroxyl apatite /4/.

OC is the largest portion of the non collagenous proteins of the organic bone matrix. It is considered to be a specific marker of osteoblast function and of osteoid mineralization, since it is exclusively synthesized by osteoblasts. The synthesis is stimulated by vitamin D.

The overwhelming portion of the OC is incorporated into the bone matrix after its release from the osteoblasts. A small proportion enters the circulation. Circulating OC has a half-life of 4 min. and is primarily eliminated via the kidney. In the presence of a GFR below 30 [mL × min–1 × (1.73 m2)–1], the plasma OC level exceeds the reference interval. Similar interactions are observed, when the OC concentration is set in relation to the renal plasma flow.

The OC concentration rises with age. Women have higher serum levels than men. OC is not only present in bone and serum but is also found in extraosseous calcifications during the early mineralization phase and in atherosclerotic plaques.


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6.10 N-terminal propeptide (PINP)

Lothar Thomas

The bone matrix is comprised up to 90% of collagen type I, which is synthesized by osteoblasts. The cleavage of collagen type I yields two relative large extension peptides termed pro collagen I carboxy-terminal pro peptide (PICP) and pro collagen I aminoterminal pro peptide (PINP) (see Section 6.7.1 – Synthesis and degradation of collagen I and Fig. 6.7-2 – Generation of biomarkers during type 1 collagen synthesis and degration). The PINP has a MW of 100 kDa and the P1CP of 35 kDa. Both pro collagen extension peptides circulate in the blood and are indicators of bone formation /1/. The P1NP is preferred for determination.

6.10.1 Indication

  • Evaluation of progress and therapeutic monitoring of the post menopausal osteoporosis
  • Vitamin D deficiency-caused secondary hyperparathyroidism
  • Assessment of the mineral bone disease in chronic kidney disease (CKD-MBD).

6.10.2 Method of determination

Two-site immunoassay using two monoclonal antibodies against intact P1NP. In addition to the intact P1NP, dimer and trimer forms are also detected /23/.

Principle: the sample is incubated with a biotinylated antibody and then a second ruthenium labeled antibody is added to the preparation, together with streptavidin coated micro particles. A sandwich complex is formed, which binds to the micro particles through biotin-streptavidin. The micro particles are magnetically bound to the surface of an electrode. Applying a voltage to the electrode induces a chemiluminescent emission, which is measured using a photomultiplier and is transformed into a concentration value via a calibration curve.

6.10.3 Specimen

EDTA plasma, heparin plasma, serum: 1 ml

6.10.4 Reference interval

35 years and older /3/: 13.8–60.9 μg/L
 /3/: 13.9–85.5 μg/L

6.10.5 Clinical assessment

A distinction is made between intact P1NP (trimeric form) and the monomer form. The natively formed trimeric form with a half-life of 10 hours is converted into the monomer form in vivo. Diagnosis of post menopausal osteoporosis

Post menopausal women without osteoporosis have serum P1NP levels of 19–100 μg/L (central 95% range), the mean value and the standard deviation are 47.7 ± 19,9 μg/L /4/. For women with post menopausal osteoporosis, the concentrations are in a comparable magnitude (50.5 ± 18,6 μg/L).

In a further study /3/ 74% of the post menopausal women over 35 years of age with osteoporosis had somewhat higher mean P1NP values than premenopausal women without osteoporosis and 32.5% had concentrations above the upper reference interval value. Both studies show that the diagnosis of the post menopausal osteoporosis with P1NP is only possible in a few cases. Monitoring the treatment for osteoporosis

The essential indication of the P1NP determination is the monitoring of osteoporosis treatment, particularly under anabolic treatment with recombinant parathyroid hormone (rPTH) /5/.

Thus, in one study /3/ under 3-month treatment with rPTH (teriparatide):

  • The concentrations of P1NP increased for 83% of the patients treated with rPTH and
  • 88% of the patients treated with bisphosphonate (alendronate) showed a decrease in comparison to the initial value
  • A least significant change (LSC, see Section 6.7.2 – Significance of bone markers) of 20% served as a change criterion.

A further study on treating post menopausal women with rPTH (teriparatide) has shown that an increase of P1NP in the first 3 months after the start of treatment has the highest diagnostic sensitivity for predicting an improvement of bone mineral density (BMD) of the spine in the following 18 months. The improvement was defined as an increase of the bone mineral density above 3% /6/.

6.10.6 Comments and problems


EDTA plasma or heparin plasma should be preferred.

Method of determination

Tests which only measure the trimeric form yield lower values than those that also detect monomer P1NP.


At room temperature 24 hours, at up to 8 °C 5 days, 6 months at –20 °C.


1. Bieglmayer C, Dimai HP, Gasser RW, Kudlacek S, Obermayer-Piertsch B, Woloszcuk W, et al. Biomarkers of bone turnover in diagnosis and therapy of osteoporosis. Wien Klin Wschr 2012; 162: 464–77.

2. Brandt J, Krogh TN, Jensen CH, Fredriksen J, Teisner B. Thermal instability of the trimeric structure of the N-terminal propeptide of human procollagen type 1 in relation to assay technology. Clin Chem 1999; 45: 47–53.

3. Garneo P, Vergnaud P, Hoyle N. Evaluation of a fully automated serum assay for total N-terminal propeptide of type 1 collagen in postmenopausal osteoporosis. Clin Chem 2008; 54: 188–96.

4. Martinez J, Olmos JM, Hernandez JL, Pinedo G, Llorca J, Obregon E, et al. Bone turnover markers in Spanish postmenopausal women. The Camargo cohort study. Clin Chim Acta 2009; 409: 70–4.

5. Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, et al. The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 2003; 349: 1207–15.

6. Chen P, Satterwhite JH, Licata AA, Lewiecki EM, Sipos AA, Misurski DM, et al. Early changes in biochemical markers of bone formation predict BMD response to teripartide in postmenopausal women with osteoporosis. J Bone Mineral Res 2005; 20: 962–70.

6.11 Pyridinoline and Deoxypyridinoline

Lothar Thomas

Post translational processing of lysine and hydroxylysine residues to form the pyridinium cross links pyridinoline (PYD; hydroxylysylpyridinoline) and deoxypyridinoline (DPD; lysylpyridinoline) are essential for stabilizing the mature forms of collagen and elastin. Both cross links are absent from most tissues and are found in bone. PYD and DPD are liberated from bone matrix by osteoclasts and about 60% is bound to protein and the remaining 40% is free /1/. Because most metabolic bone diseases are characterized by an increase in bone resorption, these biomarkers are of special interest.

6.11.1 Indication

Detection of increased rate of bone resorption associated with the following bone diseases /2/:

  • Post menopausal osteoporosis
  • Primary hyperparathyroidism
  • Assessment of mineral bone disease in cases of chronic kidney disease (CKD-MBD)
  • Paget’s disease
  • Tumor-associated hypercalcemia
  • Carcinoma with bone metastases

Evidence of a treatment effect:

  • 6 months after the start of hormone substitution
  • 3 months after the start of bisphosphonate treatment
  • 1 month after the start of parenteral bisphosphonate and/or parathyroid hormone treatment

6.11.2 Method of determination

A distinction is made between the assays of total (t) and free (f) pyridinolines in the urine. The former are determined with the HPLC, the latter with HPLC or immunoassays.

PYD and DPD by means of HPLC

The determination consists of /3/:

  • Acid hydrolysis in order to release the protein-bound cross links
  • Partition chromatography using cellulose
  • HPLC separation using reversed phase principle. The measurement is undertaken fluorometrically with PYD and DPD being separately determined in a single run (Fig. 6.11-1 – Structure and separation of the pyridinolines). Omitting acid hydrolysis allows the free fraction of cross links to be determined.

Immunoreactive deoxypyridinoline 

This method measures the free fraction of DPD. The determination is preformed by competitive enzyme immunoassay using solid-phase immobilized monoclonal antibodies and enzyme-conjugated DPD. By acid hydrolysis of the sample before starting the immunoassay the sum of the free and peptide-bound fraction of PYD and DPD can also be determined by immunoassay /4/.

Immunoreactive pyridinoline

This method measures the free and protein-bound fraction of PYD. DPD shows 100% cross reactivity. The determination is performed using a competitive enzyme immunoassay with solid-phase immobilized PYD and enzyme-conjugated polyclonal antibodies.

6.11.3 Specimen

Spot morning urine without additives: 5 mL

6.11.4 Reference interval

Refer to References /34/ and Tab. 6.11-1 – Reference intervals for pyridinolines.

6.11.5 Clinical significance

Since PYD and DPD are only present in mature collagen type 1 and not in the collagen of the skin, the renal secretion of PYD and particularly of DPD can serve as a specific biomarker for the quantitative assessment of bone resorption. In healthy individuals, 15–85% of total PYD (tPYD) and total DPD (tDPD) are present in the urine in free form as fPYD and fDPD. The remainder is bound to peptides from the bone collagen.

The excretion of the pyridinolines in the urine correlates significantly to the histologically measured bone resorption (osteoclast surface) /5/.

The premenopausal range is seen as a reference interval of the pyridinolines. Post menopausal DPD values above 60 μg/g creatinine are interpreted as increased bone resorption. Approximately 30–40% of the post menopausal women have values above this threshold. Post menopausal osteoporosis

Since the osteoclasts mainly release crosslinked telopeptides and other peptides from the bone collagen, tPYD and tDPD increase post menopausally more rapidly than fPYD or fDPD.

tPYD and tDPD also decrease more rapidly than fPYD and fDPD during hormone substitution after menopause /6/. The more enhanced the bone resorption, the less the proportion of fDPD, and as bone resorption increases, the proportion of fDPD becomes less. It is true that tDPD and fDPD correlate /7/, but the evidence of fDPD becomes less important in comparison to the total excretion due to the decreasing proportion of the fDPD with increasing total excretion. The same applies to the measurement of tPYD and fPYD. Therefore the measurement of tPYD and tDPD is favored /7/.

In the first morning urine, post menopausal women have higher excretion than during the day, which is even more accentuated in post menopausal women with osteoporosis (Fig. 6.11-2 – Examination of tDPD in pre- and post menopausal women/8/. In women with osteoporosis, bone resorption increases especially at night. Apparently, the bodily activity during the day leads to bone resorption approaching normal.

In addition to the daily rhythm, there is a monthly and annual rhythm of tDPD, at least in premenopausal women (Fig. 6.11-3 – Seasonal rhythms of 25-hydroxy vitamin, parathyroid hormone and total deoxypyridinoline in pre menopausal women/9/. This figure also shows that, for a low 25(OH)D in February, mild regulatory hyperparathyroidism and mild increased bone resorption occurs.

The bone resorption in winter in premenopausal women hardly increases with low 25(OH)D values, but it shows a partial sharp increase in post menopausal women in February with low 25(OH)D values (Fig. 6.11-3 – Seasonal rhythms of 25-hydroxy vitamin, parathyroid hormone and total deoxypyridinoline in premenopausal women). Pyridinoline (tPYD and tDPD) in tumor patients

For patients with lung carcinoma and radiologically proven bone metastases, pyridinolines (tPYD and tDPD) are sensitive markers /10/. However, patients without bone metastases and patients with benign lung diseases also show some signs of increased excretion. Thus, the diagnostic sensitivity is high, but the specificity is low. In patients with lung carcinoma or breast carcinoma without bone metastases, increased values of tPYD and tDPD are often found because, presumably, the tumors produces substances such as PTHrP that cause increased bone resorption.

In tumor patients, the bone metastases are shown with more sensitivity by tPYD and tDPD than by NTX and CTX. This may be due to the fact that, in tumor patients, the degradation of collagen no longer occurs regularly as in post menopausal women through cathepsin K, but irregularly through, for example, matrix metalloproteinases, so that structures other than the telopeptide of collage type 1 occur.

Due to the seasonal rhythm with the vitamin D deficiency phase during the winter in Central and Northern Europe, the pyridinolines, like all markers, lose their diagnostic effect. Due to vitamin D substitution for tumor patients during the winter, the pyridinolines and other bone resorption markers can gain in importance for the detection of bone metastases /1011/. Disadvantages of pyridinolines

The disadvantage of tPYD and tDPD in comparison to serum markers is the relation to creatinine excretion if spot urine is investigated.


A reason as to why the tDPD excretion relative to creatinine can be increased in post menopausal women is that they are less physically active than younger premenopausal women. This causes muscle mass and urine creatinine to decrease more sharply than the bone mass, which results in an increased ratio of tDPD/creatinine. tDPD/creatinine is disproportionately increased with hyperthyroidisms as well /6/. One cause is the increase of muscle metabolism.

Hormone replacement therapy (HRT)

During HRT, the serum level of the sexual hormone-binding globulin (SHBG) increases /12/; this is a result of the passage of estradiol through the liver. SHBG binds estradiol and testosterone equally, therefore the concentration of free testosterone in women decreases under oral HRT. The reduced free testosterone leads to a decline in muscle mass, the creatinine excretion is therefore lower and the ratio of tDPD/creatinine turns out higher. Since the β-CTX (see Section 6.12 – N-telopeptide and C-telopeptide) are measured in the EDTA plasma, they indicate the bone resorption in women under HRT more correctly than the ratio of tDPD/creatinine. Thus, in all of the post menopausal women with HRT there was a decrease of the β-CTX in relation to the values of premenopausal women. This was not the case, however, for all HRT women when the ratio tDPD/creatinine was measured.

In another series of women under various forms of HRT, it was seen that low-dose transdermal patches (25 μg estradiol administered per day) led to normal tDPD/creatinine ratio in the first morning urine. It is documented that this form of HRT does not lead to an increase in SHBG. Therefore, the muscle mass will not decrease and the creatinine excretion in the urine also does not decrease.


These patients with well functioning kidneys excrete little creatinine in the urine, which is why the ratio of tDPD/creatinine turns out too high.

Bisphosphonate treatment

With Paget’s disease and osteoporosis, bisphosphonate treatment leads to a sharp decrease of tPYD and tDPD, while fPYD and fDPD do not decrease /5/. Vitamin D treatment in older women with vitamin D deficiency leads to a sharp decrease of CTX and a less pronounced decrease of tPYD and tDPD, while fPYD and fDPD do not decrease /13/. The fDPD is thus not suitable for either the monitoring of the bisphosphonate treatment or the vitamin D treatment.

Tab. 6.11-2 – Pyridinolines in diseases with increased bone resorption and during treatment shows diseases with changed bone resorption and the behavior of the pyridinolines in the monitoring of the treatment.

6.11.6 Comments and problems

Method of determination

tDPD are part of an extensive laboratory standardization program at the Centers of Disease Control of the United states /14/.

Urine sample

Most of the experiences are present in the first morning urine. It provides the following advantages:

  • The urine is more concentrated than in later samples
  • The peaks of the pyridinolines are significantly higher due to the higher degradation rates at night
  • The sample must not be exposed to direct sunlight, because pyridinolines are fluorescing structures that can be destroyed by long-term exposure to UV light.

Influencing factors /14/

  • Intraindividual variation: tPYD 71 (57–78)%, tDPD 67 (53–75)%
  • Day-to-day variation: tPYD 16 (12–21)%, tDPD 16 (5–24)%
  • Inter individual variation of premenopausal women: tPYD 26 (12–63)%, tDPD 34 (8–98)%
  • Inter individual variation of post-menopausal women: tPYD 36 (22–61)%, tDPD 40 (27–54)%.


1. Christenson RH. Biochemical markers of bone metabolism: An overview. Clin Biochem 1997; 30: 573–91.

2. Withold W. Monitoring of bone turnover: Biological preanalytical and technical criteria in the assessment of biochemical markers. Eur J Clin Chem Clin Biochem 1996; 34: 785–99.S

3. Uebelhart D, Gineyts E, Chapuy MC, Delmas PD. Urinary excretionof pyridinium crosslinks: a new marker of bone resorption in metabolic bone disease. Bone Mineral 1990; 8: 87–96.

4. Kraenzlin ME, Kraenzlin CA, Meier C, Giunta C, Steinmann B. Automated HPLC assay for urinary collage crosslinks: effect of age, menopause, and metabolic bone diseases. Clin Chem 2008; 54: 1546–53.

5. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinium crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 1991; 6: 639–44.

6. Garnero P, Gineyts E, Arbault P, Christiansen C, Delmas PD. Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion. J Bone Miner Res 1995; 10: 641–9.

7. Meyer-Lüerssen B, Traber L, Knörzer Th, Schmidt-Gayk H. Bone resorption marker in pre- and postmenopausal females. Clin Lab 2000; 46: 285–90.

8. Aoshima H, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, and Inoue T. Circadian variation of urinary type I collagen cross-linked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks. Bone 1998; 22: 73–8.

9. Woitge HW, Knothe A, Witte K, Schmidt-Gayk H, Ziegler R, Lemmer B, Seibel MJ. Circannual rhythms and interactions of vitamin D metabolites, parathyroid hormone, and biochemical markers of skeletal homeostasis: a prospective study. J Bone Miner Res 2000; 15: 2443–50.

10. Muley Th, Herb KP, Tuengerthal S, Schmidt-Gayk H, Ebert W. Markers of bone metabolism do not prove bone metastases in lung cancer patients. CECHTUMA 2003; 4th Central European Conference on human tumor markers. Karlovy Vary, Czech Republic, Abstract, 2003.

11. Grant WB. An estimate of premature cancer mortality in the U.S. due to inadequate doses of solar ultraviolet-B radiation. Cancer 2002; 94: 1867–75.

12. Gower BA, Nyman L. Associations among oral estrogen use, free testosterone concentration, and lean body mass among postmenopausal women. J Clin Endocrinol Metab 2000; 85: 4476–80.

13. Kamel S, Brazier M, Rogez JC, Vincent O, Maamer M, Desmet G, Sebert JL. Different responses of free and peptide-bound cross-links to vitamin D and calcium supplementation in elderly women with vitamin D insufficiency. J Clin Endocrinol Metab 1996; 81: 3717–21.

14. Vesper HW, Demers LM, Eastell R, Garnero P, Kleereoper M, Robins SP, et al. Assessment and recommendations on factors contributing to preanalytical variability of urinary pyridinoline and deoxypyridinoline. Clin Chem 2002; 48: 220–35.

15. Rauch F, Schönau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994; 102: 94–7.

16. Schönau E, Rauch F. Biochemical measurements of bone metabolism in childhood and adolescence. J Lab Med 2003; 27: 32–42.

17. Garton M, Martin J, Stewart A, Krukowski Z, Matheson N, Loveridge N, et al. Changes in bone mass and metabolism after surgery for primary hyperparathyroidism. Clin Endocrinol 1995; 42: 493–500.

18. Ureña P, Ferreira A, Kung VT, et al. Serum pyridinoline as a specific marker of collagen breakdown and bone metabolism in hemodialysis patients. J Bone Miner Res 1995; 10: 932–9.

6.12 N-telopeptide and C-telopeptide

Lothar Thomas

During bone resorption, N-telopeptide (NTX) and C-telopeptide (CTX) of type 1 collagen are released into circulation (Fig. 6.7-2 – Generation of biomarkers during type 1 collagen synthesis and degradation). NTX and CTX are relative specific biomarkers for the degradation of bone. None-bone tissues comprised of type 1 collagen form different fragments during degradation /1/. Therefore NTX and CTX have an optimal bone specificity under these preconditions. In addition both telopeptides are important bone resorption markers and are the largest fraction indicating increased bone resorption and lead to significant elevations in serum and urine in comparison to PYD and DPD.

The CTX are preferred in comparison to DPD in clinical laboratory diagnostics because:

  • CTX occur as α-CTX (no isomerization of asparagine acid) and β-CTX (isomerized asparagine acid). Isomerization is a spontaneous process which occurs slowly in the post-translational period. Therefore, the β-form is only present in mature tissues such as bones and β-CTX tests only detect collagen fragments from mature type 1 collagen. Newly formed collagen is not determined, which is why the β-CTX test is not a bone formation marker /2/.
  • The β-CTX test is also called β-CrossLaps assay. β-CTX can be determined using immunoassays on automated platforms with good precision in the serum and urine.

6.12.1 Indication

Determination of the bone resorption:

  • Estimation of an increased regular bone turnover (e.g., post menopausal)
  • Monitoring the treatment for osteoporosis
  • Monitoring the bone turnover of dialysis patients
  • Monitoring the treatment of malign bone disease.

6.12.2 Method of determination

Enzyme immunoassay

Two-site immunoassay specifically for the crosslinked β-isomerized type-1 collagen telopeptide fragments. Two monoclonal antibodies are used, each of which records the antigen Glu-Lys-Ala-His-βAsp-Gly-Gly-Arg (CrossLap-antigen) /3/.

Principle: the specimen is incubated in a tube with a biotinylated antibody and then a second ruthenium-labeled antibody is added, together with streptavidin-coated micro particles. A sandwich complex is formed, which binds to the micro particles through biotin-streptavidin. The micro particles are magnetically bound to the surface of an electrode. Applying a voltage to the electrode induces a chemiluminescent emission, which is measured using a photomultiplier. The emission is transformed into a concentration via a calibration curve.

6.12.3 Specimen

EDTA blood, urine: 2 mL

Preparation of the patient, see Section 6.12.6 – Comments and problems.

6.12.4 Reference interval

Refer to Ref./3/ and Tab. 6.12-1 – Reference interval for β-CTX.

6.12.5 Clinical assessment

In clinical practice, measurements of either serum or urine β-CTX are used in the diagnosis and management of a spectrum of metabolic and malignant bone diseases. β-CTX is released in the circulation in remodeling of the bone in high amounts and in remodeling of the skin to a small proportion. Therefore β-CTX has developed as a sensitive and specific biomarker of bone turnover (Tab. 6.12-2 – β-CTX in diseases with increased bone turnover). Because of its high sensitivity for the degradation of type 1 collagen, the possibility for the determination in serum and the adaption to automated analyzers has favored β-CTX as a widely used bone resorption marker /18/. Notes for the assessment of β-CTX concentration /4/

Day to day variability

Serum β-CTX levels show low variability.

Diurnal variation /15/

The highest values are in the early morning hours and lowest values during the afternoon and evening (Fig. 6.12-1 – Circadian variation of the βCTX concentration in serum).

Seasonal variation

Because of moderate vitamin D deficiency the β-CTX serum level is higher in winter than in summer (Fig. 6.12-2 – β-CTX and 25 (OH)D in post menopausal women in January and February with and without hormone replacement treatment).


A single meal within 60–120 min. before blood collection can decrease the serum β-CTX concentration by up to 50%.

Hormone replacement therapy

The β-CTX level declines as shown in Fig. 6.12-3 – β-CTX levels in women with hormone replacement therapy.

6.12.6 Comments and problems

Preparing the patient for blood sampling

  • 12 hours of abstinence from food, before blood collection. After waking up, only water is allowed until the blood sample is taken /15/
  • Blood collection between 7 a.m. and 9 a.m. in fasting state /16/
  • If it is not possible to draw blood before 9 a.m., the recommended alternative to β-CTX determination is to determine pyridinolines in morning spot urine
  • For dialysis patients, the blood sample should be taken before the dialysis.


EDTA plasma is better suited than serum, because the stability of β-CTX is greater.

Stability of β-CTX

In the EDTA whole blood 6 hours at room temperature (RT). In EDTA plasma 24 hours at RT, 48 hours at 4 °C, and 1 month at –20 °C. In serum at RT 12–24 hours.


1. Bieglmayer C, Dimai HP, Gasser RW, Kudlacek S, Obermayer-Piertsch B, Woloszcuk W, et al. Biomarkers of bone turnover in diagnosis and therapy of osteoporosis. Wien Klin Wschr 2012; 162: 464–77.

2. Garnero P, Borel O, Delmas PD. Evaluation of a fully automated serum assay for C-terminal cross-linking telopeptide of type 1 collagen in osteoporosis. Clin Chem 2001; 47: 694–702.

3. McCudden CR, Kraus VB. Biochemistry of amino acid racemization and clinical application to musculoskeletal disease. Clin Biochem 2006; 39: 1112–30.

4. Herrmann M, Seibel M. The amino- and carboxyterminal cross-linked telopeptides of collagen type I, NTX-1 and CTX-1: a comparative review. Clin Chim Acta 2008; 393; 57–75.

5. Camacho PM, Lopez NA. Use of biochemical markers of bone turnover in the management of postmenopausal osteoporosis. Clin Chem Lab Med 2008; 46: 1345–57.

6. Reginster JY, Henrotin Y, Christiansen C, et al. Bone resorption in post-menopausal women with normal and low BMD assessed with biochemical markers specific for telopeptide derived degradation products of collagen type 1. Calcif Tissue Int 2001; 69: 130–7.

7. Iki M, Morita A, Ikeda Y, et al. Biochemical markers of bone turnover predict bone loss in perimenopausal women – the Japanese Population-based Osteoporosis (JPOS) Cohort Study. Osteoporos Int 2006; 17: 1086–95.

8. Chubb SAP. Measurement of C-terminal telopeptide of type 1 collagen (CTX) in serum. Clin Biochem 2012: 45 (12) 928-35.

9. Garnero P, Sornay-Rendu E, Claustrat P, Delmas PD. Biochemical markers of bone turnover, endogenous hormones and the risk of fractures in postmenopausal women: the OFELY study. J Bone Miner Res 2000; 15: 1526–36.

10. Garnero P, Hausherr E, Chapuy MC, et al. Markers of bone resorption predict hip fracture in elderly women: the EPIDOS study. J Bone Miner Res 1996; 11: 1531–8.

11. Rosen CJ, Chesnut III CH, Mallinak NJ. The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab 1997; 82: 1904–10.

12. Guillaume J, Lafage-Proust MH, Souberbielle JC, Lechevallier S, Deleaval P, Lorriaux C, et al. Severe secondary hyperparathyroidism in patients on haemodialysis is associated with a high initial serum parathyroid hormone and beta CrossLaps level: Results from an incident cohort. PLOS One 2018; 13 (6): e0199140

13. Hannon RA, Eastell R. Bone markers and current laboratory assays. Cancer Treatment Reviews 2006; 32, Suppl 1: 7–14.

14. Ryan CW, Huo D, Bylow K, et al. Suppression of bone density loss and bone turnover in patients with hormone-sensitive prostate cancer and receiving zoledronic acid. BJU Int 2007; 100: 70–5.

15. Qvist P, Christgau S, Pedersen BJ, Schlemmer A, Christiansen C. Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting. Bone 2002; 31: 57–61.

16. Schmidt-Gayk H, Spanuth E, Kötting J, Bartl R, Felsenberg D, Pfeilschifter J, Raue F, Roth HJ. Performance evaluation of automated assays for α-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone (Biorose multicenter study). Clin Chem Lab Med 2004; 42: 90–5.

17. Elenaei MO, Musto R, Alagband-Zadeh J, Moniz C, Le Roux CW. Postprandial bone turnover is independent of calories above 250 kcal. Ann Clin Biochem 2010; 47: 318–20.

18. Schmidt-Gayk H. Knochenstoffwechsel. In Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books: 311–30.

Table 6.1-1 Significant factors in the regulation of osteoblasts /1/

Regulation factors

Epidermal growth factor (EGF)

factor (EGF)EGF promotes the renewal of mesenchymal stem cells, from which the osteoblasts are created. The self renewal is critically dependent on the expression of genes such as the stem cell gene Sca-1/Ly-6A.

Fibroblast growth factor 23 (FGF23) /6/

The FGF23/klotho system is important in the renal excretion of phosphate. Failure of this system results in reduced excretion of phosphate and causes hyperphosphatemia and vascular calcification. FGF23 is a 251 amino acid peptide synthesized by osteocytes and osteoblasts in response to high phosphate intake, hyperphosphatemia and increase in 1,25 (OH)2D3 (calcitriol). The central target organ of FGF23 is the kidney. High FGF23 induces a rapid and marked inhibition of renal phosphate reabsorption resulting in severe hypophosphatemia, bone demineralization and low calcitriol. FGF23 inhibits 1a-hydroxylase expression in the renal proximal tubule and stimulates the 24-hydroxylase which converts calcitriol and 25 OHD into active metabolites. See also Section 6.3 – Phosphate.

OsteoBlast-stimulating factor (OSF-1)

This factor, also known as pleio-trophin, has a chemotactic effect on the osteoprogenitor cells and stimulates the activity of mature osteoblasts.

Parathyroid hormone, growth hormone, insulin-like growth factor-1, prostaglandins

These hormones and chemokines have a stimulating effect on the new formation of stem cells. They also stimulate the osteogenic differentiation of bone morphogenetic protein primed cell populations.

Bone morphogenetic protein (BMP)

There are at least 30 BMPs. The BMPs are the largest group of the transforming growth factor (TGF-β)-super family. The BMPs have osteoinductive properties and regulate the differentiation of mesenchymal cells in components of the bone, cartilage, or fat tissue.

Runt homology domain transcription factor-2 (Runx2)

Runx2 is the key osteoblast differentiation transcription factor. It is essential for the differentiation of the mesenchymal stem cell (MSC) into the osteoblast line and simultaneously suppresses the transition of MSC into the chondrocyte and adipocyte line. The TGF-β/BMP and Wnt/β-catenin signal paths promote the expression of Runx2 to induce the osteogenic cell phenotype. In an early stage, Runx2 triggers the expression of the bone matrix protein genes, the proteins osteocalcin, osteopontin and Col1a1. Several proteins interact and modulate Runx2. Runx2-transgenic mice do not experience any osteoblast differentiation and therefore do not show any bone formation.

Wingless-ints (Wnts)

Wnts are secreted lipid-modified glycoproteins, which activate different intracellular signaling pathways including the Wnt/β-catenin signal pathway. Together with the TGF-β/BMP and Runx2, the Wnts play an important role in skeletal development, osteoblast differentiation, and bone formation.

The Wnt/β-catenin signal pathway stimulates bone formation in the following way:

  • Activation and differentiation of osteoblasts and suppression of the differentiation of mesenchymal cells into chondrocytes and adipocytes
  • Increase of the growth rate of osteoblasts and suppression of their apoptosis
  • Suppression of the formation of osteoclasts.

DKK1 is a soluble inhibitor of the Wnt/β-catenin signal pathway. It contains two cysteine-rich domains, from where the aminoterminal bind to the low-density lipoprotein receptor-related protein (LRP5/LRP6) component of the Wnt receptor complex. Over expression of DKK1 in osteoblasts inhibits the Wnt signaling pathway and causes osteopenia and disruption of the hematopoietic stem cell niche. In a study /3/ DKK1 was determined in post menopausal women with osteoporosis (2.737 ± 148 ng/l) and without osteoporosis (2.219 ± 207 ng/l.)

Table 6.1-2 Significant factors in the regulation of osteoclasts /1/

Regulation factors


The monocyte-colony stimulation factor (M-CSF) is essential for the development of osteoclast predecessor cells.

Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL)

RANKL is synthesized by osteoblasts and controls the differentiation of pre-osteoclasts to osteoclasts via the Receptor Activator of Nuclear Factor Kappa B (RANK). RANK is localized on pre-osteoclasts and cooperates directly with other receptors such as the osteoclast-associated receptor (OSCAR) and a triggering receptor expressed on myeloid cells (TREM-2) (Fig. 6.1-3 – Mechanisms of osteoclast activation).

Osteoclast-associated receptor (OSCAR)

OSCAR is an immunoglobulin-like receptor and, like RANKL is involved in the osteoblast-osteoclast interaction. OSCAR reacts via the adapter molecule FCRγ which has an immunoreceptor tyrosine-based activation motif (ITAM) that is critical for th activation of calcium signalling. In the event of low plasma calcium, RANK, OSCAR and TREM-2 cooperate, phosphorylation of ITAM occurs, osteoclasts are activated, and the concentration of calcium increases.


Activated osteoclasts express integrins, which detect matrix proteins such as osteopontin (OSP) and bone sialo protein (BSP). The binding of the integrins to these proteins is connected to intracellular signaling of the osteoclasts, increased intracellular calcium concentration and increased synthesis of free radicals, which overall leads to the activation of osteoclasts. Actin-based adhesion structures, also called podocytes, are the result. The bone-resorbing osteoclast shows polarity; degradation products of collagen and matrix proteins are transported to the basolateral cell membrane by means of transcytosis. The release of calcium and phosphate, which are located in a resorptive hemi vacuole, is less clear. They should selectively and actively make their way to the extracellular spaces via ion channels.

Bone-specific alkaline phosphatase (bone ALP)

The bone ALP releases phosphate from the bone minerals, increases the extracellular phosphate concentration, and regulates the expression of genes which are associated with osteoblast differentiation. In the case of hypophosphatemia osteoclasts are activated.

Table 6.1-3 Effect of estrogens and androgens on bone metabolism

Estradiol (E2)


  • Effect on osteoblast, osteocyte, and osteoclast
  • Effect on osteoblasts and osteoclasts
  • E2 receptors are on osteoblasts and osteoclasts
  • Androgen receptors are on osteoblasts and osteoclasts
  • Suppresses bone resorption by suppressing the osteoclasts
  • Testosterone can be converted in the bone into the biologically active metabolite dihydrotestosterone (5α-reductase-catalyzed) and E2 (aromatase-catalyzed)
  • Promotes apoptosis of osteoclasts
  • Androgen deficiency (orchiectomy) leads to increased bone loss
  • Suppresses the production of bone-resorbing cytokines (IL-1, TNF-α, M-CSF, IL-6, PG) in osteoblasts and mononuclear cells in the bone marrow; this, in turn, influences the osteoclasts paracrinally
  • Suppress IL-6 production in stromal cells
  • Adrenal androgens (DHEA) also protect against post menopausal bone loss; DHT is, however, more potent than DHEA in its effect on osteoblasts (in vitro)
  • Increases the production of IGF-1 and type 1-collagen synthesis in osteoblasts and stimulates the secretion of calcitonin
  • Testosterone increases the production of IGF-1 and growth hormone synthesis
  • Stimulates the intestinal calcium absorption, partially mediated via calcitriol
  • Stimulates the intestinal Ca2+absorption, partially mediated via calcitriol
  • Promotes the renal-tubular calcium reabsorption, renal tubuli have E2 receptors
  • Promotes the renal-tubular Ca2+reabsorption, renal tubuli have androgen receptors

DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; IL-1, interleukin-1; IL-6, interleukin-6; M-CSF, macrophage-colony stimulating factor; PG, prostaglandin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α

Table 6.1-4 Biomarkers for the diagnosis and monitoring of metabolic bone disease and osteoporosis



Calcium, phosphate, in serum

Hyperparathyroidism, vitamin D deficiency, malignant tumor with bone metastases.

Ionized calcium (Ca2+)

Determination of functional calcium

Total protein

Adjustment of serum calcium for total protein in hypo- or hyperproteinemia.


Diagnosis of renal phosphate loss or phosphate elevation in renal insufficiency.

Alkaline phosphatase (ALP)

ALP, which is expressed in many tissues, is found in higher concentrations in active osteoblasts and is released by osteoblasts into the circulation during osteogenesis. In healthy individuals, osteoblasts are the origin of approximately half of the circulating ALP.


Estimation of glomerular filtration rate.

Serum protein electrophoresis

Suspicion of multiple myeloma.

Complete blood count

Leukocytosis and eosinophilia are indicative of glucocorticoid-induced osteoporosis.


Indicative of an alcohol or medication-dependent disorder of bone metabolism.


Indicative of a thyroid hormone dependent disorder of bone metabolism.


Indicative of an acute or chronic systemic inflammation.

25(OH)D* in serum

Suspicion of deficient vitamin D intake or of reduced enteral absorption. In cases of reduced 25(OH)D, possibly additional 1,25 (OH)2D3* determination, in order to establish disturbed renal conversion of 25(OH)D into 1,25 (OH)2D3.

Calcium in urine

With normal renal function and a balanced diet,renal calcium excretion in 24-hour urine is a quantitative measure of bone resorption, particularly when associated with a marked increase of bone resorption, as may occur in primary hyperparathyroidism. Since renal calcium excretion is, in many cases, strongly dependent upon intestinal calcium absorption, a 2-hour fasting urine sample can provide better information concerning osseous calcium release than a 24-hour sample Refer to Section 6.2.1 – Calcium and ionized calcium in blood.

Parathyroid hormone*

Suspicion of primary and secondary hyperparathyroidism and vitamin D deficiency.

Sex hormones

FSH, LH, estradiol, sex hormone-binding globulin, prolactin. In women (men) with increased bone resorption for evaluation of the etiology.


Suspicion of hypercortisolism-dependent disorder of bone metabolism.

Vitamin B12, folic acid

If deficiency, disturbance of cellular functions.

Tissue transglutaminase antibodies

In young persons with reduced 25(OH)D and suspicion of deficient vitamin D absorption e.g., with celiac disease.

Bone turnover markers /18/ (see also Sections

Markers of bone formation: bone ALP /19/, osteocalcin, N-terminal pro peptide of type 1 collagen (P1NP) /20/ with the following indications:

  • Bone ALP: osteomalacia/rickets, renal osteopathy, vitamin D deficiency, hyperparathyroidism, Paget’s disease, bone metastases
  • Osteocalcin: osteoporosis, renal osteopathy, bone metastases
  • P1NP: control of osteoporosis therapy by means of rPTH.

Bone resorption markers: pyridinolines /21/, amino- and carboxy-terminal cross-linked collagen type 1 telopeptides such as NTX and β-CTX (β-crosslaps) /22/, tartrate-resistant acid phosphatase (TRAP-5b) /23/. Indications: for osteoporosis (post-menopausal women, older men), the determination of β-crosslaps (blood sampling in EDTA tubes around 8 AM) is the better marker in comparison to pyridinolines. In tumor patients with bone metastases, e.g. lung carcinoma, breast cancer, multiple myeloma, the determination of pyridinolines in the first morning urine is the superior marker. There is still too little data available for the clinical assessment of TRAP-5b.

* The repetition of these tests during the winter months (January to April) is recommended. Fasting morning (7:30 - 8:30 AM) blood sampling is best. For urine tests such as pyridinolines approximately 10 mL of first morning urine.

Table 6.1-5 Bone metabolism in puberty, pregnancy, and with osteoporosis

Clinical and laboratory findings

Normal growth

The growth of the skeletal system starts with the proliferation and differentiation of cartilage cells in the epiphyseal plates. The cartilage tissue is replaced by bone tissue after chondroclastic resorption. The GH required for the growth process stimulates the IGF-1 production of the osteoblasts, particularly however in the liver, which leads to significantly increased IGF-1 concentrations in adolescence. This promotes the growth of all organs, including the bones. In contrast, fetal growth is not or hardly dependent on IGF-1.

Within the scope of net growth of bone mass in adulthood, e.g. by adapting to bio mechanical stress of the skeleton, sexual steroids play a dominant role. Due to their osteoanabolic effects, sexual steroids cause a shift of the equilibrium between bone formation and bone resorption in favor of formation. Regular bone remodeling needs optimal nutrition, healthy lifestyle, and is only possible with physiological concentrations of sexual steroids.

During adolescence, the bone mass is built up independently of sexual steroids and at the end of the adolescence and puberty it reaches its maximum, now under the influence of sexual steroids. The maximum bone mass of an individual is determined by genetics, nutrition and lifestyle habits (movement, exposure to sunlight) and possible damaging influences on bone mass development (smoking, alcohol, drugs).

After age 35, the bone mass continuously decreases at a rate of approximately 0.5% per year in both sexes. For the first decade after menopause, the loss of bone mass increases in women to 3–4% per year and then typically normalizes again to a decrease of 0.5% every year. In men, the bone mass continuously decreases after age 35 at about 0.5% per year.

The changes in bone metabolism during adolescence and puberty lead to an increase in the plasma concentration of the bone markers ALP, bone ALP and osteocalcin. The influencing factors are:

  • Thyroid hormones, which stimulate growth; hypothyroidism in childhood results in stunted growth
  • Testosterone, because it seems to accelerate growth during puberty. In contrast, estradiol accelerates the closure of the epiphyseal growth plates after initial stimulation of the growth
  • Treatment with estradiol: the hormone is used to reduce the final size of girls who are predicted to experience unusual macrosomia. Men form estradiol from testosterone so that epiphyseal growth plate closure takes place according to estradiol increase. Men with an aromatase deficiency or a lack of estradiol receptors have a delayed epiphyseal growth plate closure and therefore experience macrosomia /24/.

Pregnancy /25/

The mineral balance of pregnant women must adapt to the needs of the fetus and the placenta. Such hormone-induced adaptations temporarily lower the calcium content of the mother’s skeleton, but have no long-term effects, unless the mineral content of the skeleton was already reduced due to malnutrition or a vitamin D deficiency. During pregnancy, the following changes of biomarkers of the bone metabolism are recorded:

  • Parathyroid hormone (PTH): decrease in the first trimester to 10–30% of the values before pregnancy, then an increase until the end of the pregnancy to values in the mid-range of the reference interval
  • PTH-related peptide (PTHrP): increased during the entire pregnancy
  • 1,25 (OH)2D3: values double early in the pregnancy and remain high until childbirth. The increase is independent of PTH because 1,25(OH)2D3 increases and PTH decreases.
  • 25-OHD-1α hydroxylase: the enzyme is up-regulated as a response to the increase in estradiol, prolactin and human placental lactogen
  • Calcitonin; the concentration increases because the C-cells of the thyroid, breast and placenta contribute to the increase.

Osteoporosis – Generalized /26/

Osteoporosis is a systemic bone disease, which predisposes to an increased risk of fracture due to reduced bone mineral density and micro-architectural damage of the bone tissue. There is a reduction in bone mass without a specific defect of bone formation. The balance between bone formation and resorption is disrupted. The prevalence of osteoporosis increases from 7% to 19% in women aged 55 to 70. Morphometrically verifiable vertebral crush fractures and peripheral fractures increase exponentially. This also effects approximately 10% of men over 60. A distinction is made between primary osteoporoses (80–90%) and secondary osteoporoses (10–20%). The latter are related to different diseases (endocrine, gastrointestinal, congenital, myelogenous, medicinal cause) and are often of complex nature, based on known or unknown comorbidities. For the primary osteoporoses, a distinction is made between type 1 (post-menopausal osteoporosis) and type 2 (age-associated osteoporosis).

Diseases for which the suspicion of secondary osteoporosis must be examined are /27/:

  • Endocrine diseases: hypogonadism in men and women, hyperthyroidism, Cushing’s syndrome, osteomalacia. Assays: TSH, dexamethasone suppression test, testosterone, estradiol, sexual hormone-binding globulin.
  • Gastroenterological diseases: colitis ulcerosa, Crohn’s disease, anorexia nervosa, primary biliary cirrhosis. Assays: CRP, antimitochondrial antibodies, perinuclear antineutrophil cytoplasmic antibodies (p-ANCA), anti-saccharomyces cerevisiae antibodies (ASCA), tissue transglutaminase antibodies.
  • Malignant tumors: systemic hematological diseases, multiple myeloma, lymphoma, solid tumors, mastocytosis. Assays: complete blood count and differential, serum protein electrophoresis.
  • Chronic inflammatory diseases: rheumatic arthropathies, autoimmune diseases. Assays: CRP, rheumatic factors, antibodies against citrullinated peptides (anti-CCP), antinuclear antibodies.
  • Chronic kidney disease. Assays: creatinine and estimated GFR, albumin excretion.
  • Addiction. Assays: alcohol, mean corpuscular volume, GGT, CDT (see Section 18.6 – Carbohydrate-deficient transferrin (CDT) and alcohol abuse).

A loss of bone mass of ≥ 1 standard deviation from the norm for a given age is called osteopenia, whereas, by definition, one only speaks of osteoporosis upon the presence of osteopenia and one or more non-traumatic fractures /28/. Osteoporosis is thus a loss of structure, mass and function of the bone, mostly associated with vertebral, femoral neck or radius fractures. According to WHO guidelines, osteoporosis already exists in women with osteopenia and a loss of bone mass of ≥ 2.5 standard deviations below the maximum bone mass of a young adult (approx. 35 years old).

Osteoporosis is a combination of bone fragility and reduced bone mass. The latter is caused by a combination of various disorders such as gonadal hormone deficiency, inadequate absorption of vitamin D and calcium, physical inactivity, comorbidity, an inadequate protein/calories ratio, and by medication /26/.

Decreasing estrogen and androgen concentrations result in a serious disruption of the bone metabolism with consecutively occurring osteoporotic loss of bone mass in both sexes. Malfunctions of the bone metabolism (premature epiphyseal closure and suspended bodily growth) due to increased concentrations of sexual hormones outside of adolescence are unknown.

The diagnosis of osteoporosis is made on the basis of clinical, radiological and bone mineral density findings, case history information (family history, nutrition, medication, stimulants, comorbidities) and, if possible, osteohistological findings. Clinically the osteoporosis is either asymptomatic or involves back pain, loss of body size, spine deformations, and bone fractures with little or no significant trauma.

A very important factor for all types of osteoporosis is the supply of vitamin D. Since this is often insufficient during the winter months, it is important to examine the concentration of 25(OH)D, PTH and bone ALP and C-terminal cross links or pyrodinolines between January and April. At this interval the lowest vitamin D concentrations and the most cases of acute secondary hyperparathyroidism are diagnosed. A one-time examination, for example, during the summer half of the year, is insufficient for patients with osteoporosis.

Laboratory findings /2930/: biomarkers are determined for the etiological clarification of the loss of bone mass and to assess the bone metabolism. Orientating examinations are:

  • Carboxy-terminal collagen cross links (CTX) a marker of increased bone resorption
  • Vitamin D, for determining a sub-optimal supply
  • Parathyroid hormone to rule out secondary hyperparathyroidism.

– Post-menopausal osteoporosis, osteoporosis type 1, estrogen deficiency-associated osteoporosis

With post-menopausal osteoporosis, reduced estrogen concentrations in the plasma are associated with enhanced bone resorption. The coupled bone formation is delayed and cannot compensate for the resorption of substance due to the high number of activated osteoclasts. One cause is that the reduced positive influence of the estrogens on differentiated osteoblastic cell functions (collagen type 1, growth factor formation) reduces bone formation. The increased bone resorption causes the post-menopausal accelerated loss of bone mass and bone structure, especially of the trabecular bone. This leads to increased concentrations of bone turnover markers (markers of bone formation and bone resorption)in the plasma. This condition of loss of bone mass with increased bone metabolism is called high turnover osteoporosis.

The initially increased bone metabolism normalizes in the post menopause after 10–15 years. After the post menopausal high turnover phase, the dominating processes within the scope of age-associated osteoporosis determine the further development of the bone mass. It is possible that the increase in the static and functional stress on the remaining bone mass releases the decisive signals for suppressing the increased bone resorption and remodeling at a low level occurs.

– Androgen deficiency-mediated osteoporosis (AMO) in men

AMO is a bone metabolism dysfunction, mostly seen in patients with a low maximum initial bone mass and a lifestyle that stresses the bone metabolism (lack of mobility, calcium and vitamin D-deficient nutrition, consumption of alcohol, cigarettes). To what extent a discrete lack of gonadal and adrenal androgens contribute to the occurrence of masculine osteoporosis is uncertain. At an advanced age, osteoporosis in men becomes more acute due to age-associated loss of bone mass /31/.

The bone forming effect of gonadal and adrenal androgens (testosterone, dihydrotestosterone, dehydroepiandrosterone) results from a stronger effect, which stimulates osteogenesis and a weaker effect, which suppresses the bone resorption /32/. Consequently, hypogonadism leads to decreased osteogenesis, which causes loss of bone mass with only slightly increased bone resorption. Only the turnover markers for bone formation tend to be low normal, whereas the findings for bone resorption show no signs of consistent changes in masculine hypogonadism.

Since estradiol is formed in men from testosterone stimulated by aromatase, and men also have estradiol receptors on the bones, an estradiol deficiency can play an important role. In one third of men with osteoporosis, estradiol values under 13 ng/L are measured. Below this cutoff, increased bone absorption occurs in women as well /33/. A low estradiol concentration in men correlates to low bone density in the lumbar spine and the femoral neck /34/. Another indicator of the estradiol effect are the casuistics of men with low bone density and an estrogen receptor defect /35/ or an aromatase deficiency /36/.

– Glucocorticoid excess-associated osteoporosis

Exogen (glucocorticoid treatment) and endogen-produced (Cushing’s syndrome) supra physiological glucocorticoid concentrations cause a loss of bone mass in patients of all ages and genders, particularly of the trabecular bone with significant, measurable changes in the bone metabolism markers in serum and urine. The cause of the rapid loss of bone mass under glucocorticoids is the combination of indirect stimulation of the bone resorption, direct suppression of the osteoblast proliferation, and inhibition of osteoblastic cell functions (effects of osteocalcin, type I collagen and growth factor). The remodeling is almost completely suppressed.

The stimulation of bone resorption results from:

  • Suppression of the intestinal (calcitriol-mediated) calcium absorption
  • Inhibition of the renal calcium reabsorption
  • Development of secondary hyperparathyroidism
  • Reduction of the gonadal and adrenal steroid hormone production, by which the anti resorptive effect of estrogens and androgens also diminishes /37/.

For renal calcium reabsorption, it must be noted that reabsorption is influenced by sodium secretion in the presence of excess of glucocorticoids; a high salt intake leads to high calcium secretions. Furthermore, regarding glucocorticoid excess, hyperthyroidism and vitamin D deficiency, the effects in post-menopause are more serious than in pre-menopause.

– Juvenile osteoporosis (JO)

The bone mass that is accumulated in childhood and adolescence is essential to the health of bones in adults. One important criterion is the maximum bone mass, which is reached before age 18 in the lumbar spine and in femur, whereas it can increase up to age 50 for the skull and the radius, through continuous, periosteal growth. A small number of children, particularly those suffering from malnutrition, have an exceedingly low bone mass in relation to the age of the skeleton and the stage of sexual maturity. Already at age 10–14, they are predisposed to fractures of the distal end of the radius or ulna and they are predisposed to osteoporosis in adulthood. In addition to the constitutional delay of puberty, malnutrition or malnourishment also play an important role in genetically associated diseases /38/. The bone metabolism biomarkers in serum and urine yield no characteristic findings.

– Senile osteoporosis, type II osteoporosis /39/

Histomorphometric techniques demonstrate a reduction in bone formation (mean wall thickness) in both sexes that probably contributes to the decline in bone mass with aging. Senile osteoporosis in men and women is due, at least in part, to alterations in calcium economy. To maintain mineral balance in young men the calcium requirements are 400–600 mg/day. However, the range of demand is higher in older individuals. Mechanical forces exert major effects on bone and is one of the important variables for the dimorphism in bone mass and structure. Because of the clear decline in physical activity and muscle strength with aging and mechanical force, senile bone loss in men may in part relate to a diminution of the trophic effects of mechanical force on skeletal tissues.

– Tumor-associated osteoporosis/osteopathy

Imaging diagnostics, findings of bone scintigraphy and hypercalcemia with suppressed PTH often point to bone metastases and tumor-associated osteoporosis. Four tumors that most frequently metastasize in the bones (prostate, mama, lung, renal and thyroidal carcinomas) contribute significantly to the disability and pain in later tumor stages. The determination of organ-related tumor markers and bone turnover markers can be a valuable to diagnose progressive disease. Most tumors that metastasize in the bone secrete cytokines or the PTH-related peptide, which are responsible for stimulating bone resorption and hypercalcemia.

Laboratory findings: Bone turnover marker results are dependent on the tumor state:

  • Breast carcinoma and lung carcinoma /40/: as a rule, the bone resorption markers are increased more frequently than the markers of bone formation. Determination of pyrodinolines in early morning urine is recommended.
  • Prostate cancer /41/: bone metastases can be associated with increases of ALP, bone ALP and osteocalcin, increased urinary pyrodinolines, and normal calcium excretion.
  • Multiple myeloma: osteocalcin normal or low, pyridinolines may be increased.

Drug-induced metabolic bone disease /42/

Glucocorticoids, cyclosporine: Cause loss of bone mass, cyclosporine alone increases the osteocalcin concentration.

Cholesterol-bonding resins (cholestyramine): Long-term use can cause low vitamin D values due to resorption suppression of fat-soluble vitamins.

Anticonvulsants: chronic administration of diphenylhydantoin, phenobarbital and carbamazepine can produce changes of biomarkers such as hypocalcemia, elevated ALP, decreased 25(OH)D, increased PTH and CTX. Drug-induced increases in hepatic P450 enzyme activity results in increased catabolism of vitamin D. This leads to decreased intestinal calcium absorption, hypophosphatemia, increase in PTH and alterations in bone remodeling. Dilantin has direct effects on bone and mineral metabolism at the cellular level. The spectrum of disorders ranges from decreased bone mass, radiologic changes of rickets, osteopenia and osteomalacia.

Heparin: Long-term treatment (over a period of 4 months) with heparin doses > 15,000 IU/day can cause fractures /43/.

Smoking and alcohol: The harmful effects of smoking and alcohol on the bone metabolism are based on direct toxic effects on osteoblastic cells. Hypophosphatemia frequently found in alcoholics are the result of malnourishment, the ingestion of antacids with an aluminum content and loss of phosphates through vomiting.

Table 6.1-6 Diseases involving disorders of the bone mineralization

Clinical and laboratory findings

Rickets and osteomalacia /44/

Osteomalacia is a disease of the adult skeleton in which the mineralization of newly formed bones is disrupted. In children, this disease manifests itself as rickets. A lack of calcitriol [1,25 (OH)2D3], calcium (calcipenic form of osteomalacia), or phosphate (phosphopenic form of osteomalacia) causes mineralization disorders of the osteoid and an increase in the production of osteoblastic osteoid. The reduced mineralization of osteoid that is increasingly produced, together with the typical clinical symptoms, is called rickets in a growing skeletal system and osteomalacia in mature skeletons. In rickets softness of the metaphyses of the long bones, together with continued cartilaginous expansion of the growth plates (physes), can result in bowing or knobby deformities of the long bones /45/.

The cause of vitamin D, calcium and/or phosphate deficiency is most often:

Malnutrition, particularly due to long lactation periods for newborn (mother’s milk is deficient in calcium, phosphate, and vitamin D)

Calcium and phosphate deficiency in the diet or due to intraluminal calcium precipitation in diets rich in phytate.

Aluminium. Children with parenteral nutrition can develop rickets despite the fact that the parenteral solutions contain adequate amounts of calcium, phosphate and vitamin D.

Little exposure to sunlight (cutaneous metabolization of 7-dehydrocholesterol into cholecalciferol through sunlight is inhibited) in older patients or due to protective clothing.

In chronic kidney disease with reduced renal phosphate clearance, an increase in phosphate causes suppression of the 25-OHD-1α-hydroxylase, and in end stage renal failure the production of this enzyme, that is essential for the vitamin D metabolism, ceases. Decreasing concentrations of 1,25(OH)2D3 combined with phosphate-induced hypocalcemia can lead to renal osteodystrophy.

Laboratory findings: for the findings for osteomalacia/rickets, see Tab. 6.1-8 – KDIGO classification of chronic kidney disease – mineral bone disorder (CKD-MBD) and renal osteodystrophy.

Calcipenic osteomalacia

Calcipenic osteomalacia can occur in cases of:

  • Calcium deficiency in the diet
  • Vitamin D deficiency (lack of sunlight)
  • Malabsorption syndrome due to insufficient absorption of fat-soluble vitamin D precursors, e.g. in cases of celiac disease, postgastrectomy syndrome, particularly after a Billroth-II resection, short bowel syndrome and, more rarely, non-operated Crohn’s disease
  • e.g., in cases of celiac disease, postgastrectomy syndrome, particularly after a Billroth-II-resection, short bowel syndrome and, more rarely, non-operated Crohn’s disease
  • Liver function disorders (hepatic osteodystrophy) which can cause stimulated catabolism of vitamin D and its metabolites (anti-epileptic drugs). Further loss of vitamin D and its metabolites may be due to gallic acid-binding cholestyramine, due to decreased activity of hepatic 25-OHD 1α hydroxylase or direct osteoblast-damaging effect of ammonia-containing hepatotoxins in preterminal liver failure. Hepatic osteodystrophy often worsens after liver transplantation. A complete lack of 25-OHD-1α-hydroxylase is not compatible with life.
  • Hereditary pseudo-vitamin D deficiency rickets type I (PDDR) an autosomally recessive defect of the renal 25-OHD-1α hydroxylase in the absence of kidney failure
  • Vitamin D-resistant rickets (VDRR) a functional defect of the vitamin D receptor with consecutive end organ resistance and alopecia.

Laboratory findings: for the findings for calcipenic osteomalacia, see Tab. 6.1-9 – Laboratory findings in osteomalacia.

Phosphopenic osteomalacia

Phosphopenic osteomalacia is much rarer than the calcipenic forms /46/. The clinical symptoms are similar to those of the calcipenic form. The causes of this osteomalacia are:

  • Malnutrition, e.g. continuous parenteral nutrition, many years of antacid treatment, phosphate binders, premature births, newborn infants who are nursed for a long period of time
  • X-chromosomal hypophosphatasia (XLH); familial rickets, particularly in males with dentition problems in childhood and deformed lower extremities at the start of the static load due to renal phosphate loss, discrete defect of the 25-OHD-1α hydroxylase and osteoblast dysfunction. Sporadic forms (new mutations) and autosomally dominant cases (HBD, hypophosphatemic bone disease) also occur.
  • Increased renal loss with Fanconi syndrome and other tubular phosphate loss syndromes; increased renal loss also with tumor-induced osteomalacia; neoplastic production of a peptide (phosphatonin, FGF-23) /47/. FGF-23 induces hypophosphatemia and hyperphosphaturia, intestinal phosphate malabsorption and decreased 1,25(OH)2D3 with normal 25(OH)D.
  • Hypophosphatasia: This is a rare hypophosphatasia involving metatarsal stress fractures, diffuse bone pain and osteopenia with low activity of the liver-bone-kidney isoform of the ALP; the intestinal and placental ALP isoforms have normal activity. There is an autosomally recessive inheritance /48/. The concentrations for calcium, 25(OH)D and PTH are normal with often increased concentrations for phosphate, ethanolaminephosphot, pyridoxal phosphate and inorganic pyrophosphate, where the latter could be responsible for the mineralization disorder in this disease.

Laboratory findings: for the findings for phosphopenic osteomalacia, see Tab. 6.1-10 – Laboratory test results for calcipenic osteomalacia.

Hyperparathyroidism – Generalized

The four parathyroid glands regulate serum calcium concentration and bone metabolism through the secretion of parathyroid hormone (PTH). In turn, serum calcium concentrations regulate PTH secretion. High calcium concentrations inhibit PTH secretion by the parathyroid glands and low concentrations stimulate PTH /49/. PTH regulates calcium fluxes across bone, kidneys and intestines. Malignant tumors and pHPT are the most common causes of hypercalcemia (more than 90%).

Laboratory findings: for diseases with increased PTH excretion see Tab. 6.1-11 – Phosphopenic osteomalacia (OM).

– Primary hyperparathyroidism (pHPT)

Solitary parathyroid adenomas or multiglandular hyperparathyroidism cause increase in PTH. In HPT the PTH level is above or near the upper reference interval value /49/. See also Section 6.4 – Parathyroid hormone (PTH).

Due to the routine determination of calcium in the serum, the diagnosis of pHPT is often made and is the third most common endocrinological diagnosis after diabetes mellitus and thyroid dysfunctions, with a prevalence of 30 per 100,000. Women (women/men, 3/1) in their 50s and 60s are especially affected by this disease.

If pHPT is diagnosed in young people and in the case of familial accumulation, the following should always be looked for:

  • Multiple endocrine neoplasia (MEN)
  • pHPT with hypophyseal tumor, gastrinoma or insulinoma
  • pHPT with medullary thyroid carcinoma and pheochromocytoma.

In 80% of the cases, the pHPT involves a solitary adenoma with normal remaining parathyroids, in approx. 20% of the cases all four or more epithelial bodies are adenomatously altered. The prevalence of the parathyroid carcinoma in pHPT is 0.5%.

Due to the early diagnosis, the majority of pHPT cases are clinically asymptomatic. The classic changes to the skeleton, nephro calcinosis, nephro lithiasis and hypercalciuria, are rarely found or only found to a discrete extent. More commonly, limited performance and fatigue are specified as the neuromuscular symptoms. Refer also to Section 6.4 – Parathyroid hormone.

– Secondary hyperparathyroidism (sHPT)

sHPT is a common sequence of chronic kidney disease or malabsorption syndrome. A continuously decreased calcium concentration reactively causes a constant increase of PTH secretion. Several abnomalitis in parathyroid gland function contribute to the persistent PTH concentration in serum /50/: secretion by individual parathyroid cells, increases in PTH production per cell owing to enhanced gene expression and cellular enlargement or hypertrophy, and increases in the number of parathyroid cells because of tissue hyperplasia. See also paragraphs 1.2, 6.2, 6.3, 6.4, and 6.7.

– Tertiary hyperparathyroidism

Tertiary HPT can develop in patients with secondary HPT over years, because the parathyroid glands are autonomous and PTH secretion is not linked to the serum calcium concentration. In patients with renal osteodystrophy, the long-term existence of sHPT leads to an adenomatous transformation of the parathyroid glands with a loss of calcium mediated suppressibility of the PTH secretion. Such discrepancies are the result of:

  • Stimulation of the PTH secretion over many years due to low calcium
  • A lack of suppression of PTH production due to decreased calcitriol formation.

Increased PTH secretion leads to increased serum calcium and thus to a laboratory configuration (increased calcium and PTH) which is comparable to the pHPT.

Laboratory findings: see Tab. 6.1-11 – Phosphopenic osteomalacia (OM).

Familial hypocalciuric hypercalcemia (FHH)

The three forms of FHH (FHH 1, FHH 2 and FHH 3) are heterogeneous autosomally dominant diseases of the extracellular calcium homeostasis. The calcium concentration in the serum is increased and excretion in the urine is inadequately decreased. FHH 1 and FHH 2 have normal PTH values, slightly increased magnesium, and are clinically asymptomatic. FHH3 is associated with increased PTH values, hypophosphatemia, and osteomalacia. Approx. 65% of the patients with FHH have FHH1, due to loss-of-function mutations in the gene CASR, which encodes the G-protein-coupled receptors. Gα1 mutants with loss-of-function cause FHH2 and mis-sense mutations in the adapter protein 2 cause FHH 3 /51/. See also Section 6.3 – Phosphate.

Chronic kidney disease – Mineral bone disorder (CKD-MBD) /1752/

The bone complaints associated with CKD-MBD are pain and fractures. Many patients with post menopausal or age-related osteoporosis are in stages 1–3 of CKD. Patients with advanced CKD (stages 4–5 and dialysis), for whom abnormal biochemical examinations show that the criteria of CKD-MBD are relevant, have renal osteodystrophy (ROD). Whereas in cases of osteoporosis, decreased bone mineral density (BMD) is a factor, abnormal bone quality can exist in cases of CKD-MBD with normal or even increased BMD (Tab. 6.1-7 – Prevalence of bone disease in patients with CKD-MBD).

The KDIGO definitions of CKD-MBD and renal osteodystrophy are shown in Tab. 6.1-8 – KDIGO classification of Chronic kidney disease – Mineral bone disorder (CKD-MBD) and renal osteodystrophy.

Osteoporosis and adynamic bone disease (ABD) are disorders of CKD-MBD. Both are associated with low bone mass, bone fractures, vascular calcifications, and increased mortality. Whereas with osteoporosis, bone degradation exceeds bone formation, there is low to no bone formation in cases of ABD. Although PTH values ≤ 150 ng/L (15 pmol/L) have a good predictive value for low bone turnover, values up to 450 ng/L (45 pmol/L) can also be associated with ABD, which is why a bone biopsy and histomorphology investigations are required.

Hypoparathyroidism /53/

Hypoparathyroidism occurs due to PTH deficiency or lack of PTH effect. PTH secretion is inadequate to mobilize calcium from bone, reabsorb calcium from distal nephron and stimulate 1α-hydroxylase activity. Hypoparathyroidism can first occur as just asymptomatic hypocalcemia or cause mild neuromuscular irritability Chvostek’s- and Trousseau’s signs (Tab. 6.1-13 – Causes of hypoparathyroidism).

Laboratory findings: the findings for hypoparathyroidism are listed in Tab. 6.1-13.

Paget disease of bone (PDB)

PDB is a chronic disease of the adult skeleton with one or more localities of aggressive osteoclastic bone resorption, followed by an incomplete osteoblastic repair. The disease begins at one point of the bone and can continuously progresses over the entire bone. The bone resorbing function of the osteoclast is greatly magnified, owing to an increase in its number, size and increasing activity. The abnormal remodeling leads to the expansion and softening of the bone, which sometimes involves pain, fractures and deformities of the bone, but no signs of neoplastic transformation. PDB affects individuals > 50 years of age, men somewhat more frequently than women and with a prevalence of 1.5% in Germany.

PDB is a local, monostotic or polyostotic increase of bone remodeling, which often affects the pelvis, femur, spine, skull and tibia and generally does not spread to other skeletal regions outside of the localities found in the initial diagnosis. No bio mechanically reliable, compact lamellar bone tissue is formed instead, woven bone rich with blood vessels and connective tissue.

Genetic factors are important PDB and mutations in only a single SQSTM1 gene predispose to classical PDB. Mutations show incomplete penetrance, emphasizing the role of environmental factors such as infection with paramyxoviruses /54/. The mutations cause changes in the ubiquitin domain result in either mis-sense or truncing mutations within the C-terminus of the p62 protein. This protein includes an ubiquitin-associated domain, one class of ubiquitin binding domains, that interact with the protein modifier ubiquitin. Ubiquitin modification of proteins serves as a scaffold to establish signal-induced protein-protein interactions that are essential within signaling pathways, leading to activation of transcription factors, including NF-kB /54/.

The MPB diagnosis is based on the characteristic radiological findings. The bone scintigraphy is a valid procedure for finding even small Paget lesions in the skeleton. MPD is mostly asymptomatic and is incidentally diagnosed based on X-ray images or increased ALP.

Laboratory findings: the local high turnover status of a Paget lesion is reflected in the findings of biomarkers. ALP and bone ALP are increased, but not for small, local lesions. Bone resorption markers yield few valid results. Determination of PTH and vitamin D is recommended for differentiation from other bone diseases.

Fibrous dysplasia (FD)

FD occurs in both genders. It is a rare, monostotically and polyostotically occurring fibrous destruction of bone, involving fractures and mutilating deformations of the skeleton. The monostotic form only manifests itself after age 30, whereas the polyostotic form is already manifested during childhood. The findings of bone metabolism markers are often not very conclusive.

In cases of McCune-Albright syndrome, FD involves hyper pigmentations (café-au-lait spots) and increased activity of endocrine organs (hyperthyroidism, Cushing’s syndrome, acromegaly, pubertas praecox). Etiologically, the underlying cause of FD is an activating mutation of the Gs-alpha subunit of the receptor adenylate cyclase-coupling G protein /55/.

Celiac disease (CD) /56/

CD is often associated with an abnormal bone metabolism, disorder involving both mineralization, leading to osteomalacia and bone mass reduction, resulting in osteoporosis. The prevalence of osteoporosis is 35–85% in untreated children and the prevalence of secondary hyperparathyroidism is 30–35%. Two mechanisms are considered to be responsible: malabsorption and local inflammation. Following a gluten-free diet in children leads to normal bone mass within one year, but not in adults.

Laboratory findings: in one study /56/ 41% of children with CD had a calcium concentration below 9.2 mg/dL (2.3 mmol/L), 54% had PTH values > 95 ng/L, and the average concentration of 25(OH)D was 22 ± 11 μg/L (control group 54 ± 26 μg/L). At the time of diagnosis, all were positive for anti-transglutaminase IgA and 65% of these were also positive for anti-transglutaminase IgG. Anti-transglutaminase antibody values normalized in 20 of 34 children with gluten-free diet after a mean period of 6 months.

Osteitis Fibrosa Cystica /57/

Osteitis fibrosa cystica is a skeletal disorder that is caused by a sustained hyperparathyroidism, often due to chronic renal failure, resulting in formation of cyst-like tumors in the bone.

Laboratory findings: in a study /57/ the patient showed a decreased calcium concentration of 7.6 mg/dL (1.9 mmol/), a phosphate concentration in the normal range, a decreased 25-(OH) D concentration of 5.2 ng/mL (13 nmol/l) and an increased PTH level of 4,081 ng/L (normal range 11.5 to 78.4 ng/L).

Table 6.1-7 Prevalence of bone diseases in patients with CKD-MBD determined by bone biopsy

Bone disease



CKD 3–5







Mild BD

Slightly increased




Osteitis fibrosa










Adynamic BD





Mixed BD





BD, bone disease; reduced, amount of non-mineralized osteoid increased

Table 6.1-8 KDIGO classification of Chronic Kidney Disease – Mineral Bone Disorder (CKD-MBD) and renal osteo­dystrophy /17/

Definition of CKD-MBD

A systemic disorder of mineral and bone metabolism due to CKD manifested either one or a combination of the following:

  • Abnormalities of calcium, phosphorus, PTH, or vitamin D metabolism
  • Abnormalities in bone turnover, mineralization, volume, linear growth, or strength
  • Vascular or soft tissue vascularization.

Definition of renal osteodystrophy

  • Renal osteodystrophia is an alteration of bone morphology in patients with CKD
  • It is one measure of the skeletal component of the systemic disorder of CKD-MBD that is quantifiable by histo morphometry or bone biopsy

Diagnosis of CKD-MBD see Tab. 6.4-4 – Diagnosis and monitoring of chronic kidney disease mineral bone disease.

Table 6.1-9 Laboratory findings in osteomalacia

Laboratory examination

Rickets and osteomalacia

Calcium i. S.


Phosphate i. S.


Calcium excretion

Phosphate excretion

2/3 of the phosphate intake







Intact PTH

Calcidiol (for vit. D deficiency)


Normal to

* Deoxypyridinoline or other resorption markers

** In cases of primary calcium deficiency within the framework of initial renal osteodystrophia.

Table 6.1-10 Laboratory test results for calcipenic osteomalacia



Liver function



Calcium i. S.

Phosphate i. S.









Intact PTH

Total ALP


Bone ALP






Calcidiol, 25(OH)D; VDDR, vitamin D-dependent rickets, disturbed 1α-hydroxylase; VDRR, vitamin D-resistant rickets, disturbed calcitriol receptor; VDDR and VDRR are also called PVDR = pseudo-vitamin D deficiency rickets type I and II. DPD, deoxypyridinoline; N, normal; NI, not indicated

Table 6.1-11 Phosphopenic osteomalacia (OM)


Phosphopenic OM
due to malnutrition

Phosphopenic OM
due to XLH and TIO

Calcium i. S.



Phosphate i. S.


Phosphate excretion

(2/3 of the intake)

Normal (2/3 of the intake)

Intact PTH







Calcitriol (1,25(OH)2D3)



Bone ALP




DPD, deoxypyridinoline; OM, osteomalacia; TIO, tumor-induced osteomalacia; XLH, X-chromosomal hypophosphatemia.

Table 6.1-12 Diseases with increased PTH secretion


Primary HPT

Secondary HPT

Tertiary HPT


Increased PTH secretion due to parathyroid adenoma or carcinoma

Reactively increased PTH secretion due to renal or intestinal-related calcium deficiency

Autonomic hyperplasia of the parathyroids with increased PTH after many years of secondary HPT

Laboratory findings


Calcium i. S.

/N (< 2.4 mmol/L)

/N (> 2.4 mmol/L)

Phosphate i. S.

/N (renal)

/N (dialysis)

/N (intestinal)

/N (intestinal)

Calcium 24 h* urine



or zero (dialysis)

/N/ (intestinal)

Phosphate 24 h* urine

2/3 of the intake


or zero (dialysis)








/N (intestinal)

/N (intestinal)






Bone ALP

Resorption marker*

* Excretion in the 24-hour urine; ** resorption marker: pyridinoline (PYb), deoxypyridinoline (DPD), β-CrossLaps (CTX), NTX, tartrate-resistant acid phosphatase (TRAP-5b). Comment: The calcitriol values are only elevated for primary HPT if there is a sufficient concentration of 25(OH)D (calcidiol).

Table 6.1-13 Causes of hypoparathyroidism

Removal or destruction of the parathyroid glands

  • Postoperatively, e.g. after a thyroid operation
  • Autoimmune disease, e.g. associated with a polyglandular insufficiency:
  • Schmidt’s syndrome
  • Post radiatio
  • Neoplastic or granulomatous destruction of the parathyroid glands with consecutive PTH deficiency

Dysfunction of the parathyroid glands

  • Mutations in PTH or calcium receptor genes
  • Hypomagnesemia
  • Hyperparathyroidism of the mother with suppressed parathyroid gland function of the newborn

Lack of parathyroid gland development in newborns

  • DiGeorge’s syndrome with cardiac and facial anomalies, cleft palate
  • Thymic aplasia and hypocalcemia
  • Kearns-Sayre’s syndrome with mitochondrial myopathy, retinal degradation, arrhythmia, heart failure, deafness, and hypocalcemia

Disrupted PTH effectiveness

  • Hypomagnesemia
  • Disrupted post-PTH receptor mechanisms within the scope of pseudohypoparathyroidism (PHP) type la (mutation of the Gs-alpha protein): target organ resistance to PTH, TSH, gonadotropins and glucagon together with Albright’s osteodystrophia (small body size, round face, adiposity, brachydactyly, subcutaneous calcifications). Albright’s osteodystrophia without PTH resistance is also called pseudo PHP.
  • PHP type Ib (disrupted PTH receptor expression): renal and bone PTH resistance with normal activity of the Gs-alpha protein
  • PHP type Ic: like PHP type Ia without detected mutation of the Gs-alpha protein
  • Pseudohypoparathyroidism (PHP) type II: PTH resistance of bone and partially of the kidneys (cause as yet unclear)

Table 6.1-14 Changes of bone markers in hypo- and pseudohypoparathyroidism



type I

type II


Calcium i. S.


Phosphate i. S.







Intact PTH











Calcium excretion


cAMP in the urine


Urine phosphate in the EH test



Decreased or reduced; Elevated or increased; PTH, parathormone; GFR, e.g., determined by creatinine clearance or cystatin C; EH test, Ellesworth-Howard test, PTH injection; Hypo P, hypoparathyroidismd; PHP, pseudohypoparathyroidism: pseudo PHP, pseudo-pseudo hypoparathyroidism; N, normal; Ni, no increase; Sn, subnormal

Table 6.2-1 Reference intervals for calcium in serum and plasma

Total calcium (Adults) /5/

Calcium (spectrophotometry)

8.6–10.3 (2.15–2.58)

Calcium (atomic absorption)

8.8–10.2 (2.20–2.54)

Ionized calcium /7/

4.5–5.3 (1.12–1.32)

Total calcium (Newborns) /8/

Full-term delivery

8.0–11.0 (2.0–2.75)

Premature delivery

7.0–11.0 (1.75–2.75)

Total calcium (Infants/children) /9/

0–5 days

7.9–10.7 (1.96–2.66)

1–3 years

8.7–9.8 (2.17–2.44)

4–6 years

8.8–10.1 (2.19–2.51)

7–9 years

8.8–10.1 (2.19–2.51)


8.9–10.1 (2.22–2.51)

12–13 years

8.8–10.6 (2.19–2.64)

14–15 years

9.2–10.7 (2.29–2.66)

16–19 years

8.9–10.7 (2.22–2.66)

Ionized calcium/10/

Cord blood

5.20 ± 0.24 (1.30 ± 0.061)

1. day

4.40 ± 0.24 (1.10 ± 0.059)

3. day

4.52 ± 0.20 (1.13 ± 0.051)

5. day

4.86 ± 0.21 (1.22 ± 0.053)

After age 1 and adults

4.49–5.21 (1.12–1.30)

Data expressed in mg/dL (mmol/L), * Values expressed as x ± s for a collective of only 7–19 children in each case, other references express: for 0–1 month 3.9–6.0 mg/dL (1.0–1.5 mmol/L) and for 1–6 months, 3.7–5.9 mg/dL (0.95–1.5 mmol/L). Conversion: mg/dL × 0.2495 = mmol/L

Table 6.2-2 Protein correction of total serum calcium

Correction of calcium (Ca) to protein 7.76 g/dL /14/

Corrected calcium (mg/dL) = Measured calcium (mg/dL) 0.6 + Protein (g/dL) 19.4

Correction of calcium to albumin 4 g/dL according to Payne

Corrected Ca (mg/dL) =

Measured Ca (mg/dL) – albumin (g/dL) + 4.0

Corrected Ca (mmol/L) =

Measured Ca (mmol/L) – 0.25 × Albumin (g/dL) + 1

The Payne formula is based on the observation that each 1 g/dL reduction in albumin < 4.0 g/dl results in an approximately 0.8 mg/dL (0.2 mmol/l) reduction in total calcium. Payne RB, Carver ME, Morgan DB. Interpretation of total calcium: effects of adjustment for albumin concentration on frequency of abnormal values and on detection of change in the individual. J Clin Pathol 1979; 32: 56–60.

Table 6.2-3 Diseases that can cause hypercalcemia /18/



Clinical and laboratory findings






, n

Neoplastic disease /21/– Breast cancer, lung carcinom, multiple myeloma, hypernephroid carcinoma, pancreatic cancer, ovarian cancer

For non-clinical, unselected patients, the estimated frequency of tumor-related hypercalcemia is 15 per 100,000. Approximately 10–20% of tumor patients, mostly in the advanced stage, develop hypercalcemia. It is most commonly encountered in patients with breast cancer, squamous cell lung cancer, epidermoid tumors of the ENT (ear, nose and throat) region and in cases of multiple myeloma. While breast cancer and multiple myeloma are associated with osteolytic hypercalcemia, in other cancers the osseous calcium release is humorally induced. Adenocarcinomas of the lung, the carcinomas of the head and neck area, urogenital tract, pancreas, ovaries and with T-cell leukemia in adults synthesize parathyroid-related peptide (PTHrP) which is homologous to PTH in the first 15 amino acids and, like PTH, causes stimulation of the osteoclasts and the renal-tubular calcium reabsorption /22/.

Rarely hypercalcemia involves acute and chronic leukemia, prostate cancer, gastric and small cell lung carcinoma. The cause of hypercalcemia with multiple myeloma are mediators of bone resorption formed by the myeloma cells. In cases of Waldenstroem’s macroglobulinemia, pseudo hypercalcemia can occur due to increased binding of calcium to monoclonal IgM /23/. Clinical symptoms such as confusion, polyuria, polydipsia, nausea, vomiting, constipation, and bone pain only occur if calcium values are above 12 mg/dL (3.0 mmol/L) /24/.

, n

, n


Hyperparathyroidism – Primary hyperparathyroidism (pHPT)

The estimated frequency of pHPT in the population is 28/100,000; in the clinic, 20–30% of hypercalcemias are due to pHPT. Hyperparathyroidism is often diagnosed during screening due to an increased calcium concentration. The complications of hypercalcemia, such as peptic ulcers, pancreatitis, kidney stones, are no longer common diagnostic criteria. In 85% of the cases, there is a singular adenoma of the parathyroids, primary hyperplasia, which affects all four epithelial bodies, is found in about 10%. In half of these cases familial hypocalciuric hypercalcemia or the multiple endocrine neoplasia (MEN) type 1 or 2a is present. The cause of hypercalcemia is increased intestinal calcium absorption due to PTH-induced 1,25(OH)2D synthesis in the vascular endothelium. The kidneys support hypercalcemia through increased PTH-induced calcium reabsorption.

Laboratory findings: serum calcium is generally above 10.4 mg/dL (2.6 mmol/L) and PTH is more than 30% above the upper reference interval value of 65 ng/L (6.5 pmol/L). The phosphorus is often subnormal. Hypercalciuria is common, but not as common as would be expected due to increased serum calcium. If PTH is normal or only slightly elevated and there is hypercalciuria, but 1,25(OH)2D is increased, then this can be an indicator of pHPT. See also Section 6.4 – Parathyroid hormone (PTH).

, n

, n


– Tertiary hyperparathyroidism

Stimulation of PTH secretion over several years due to low serum calcium and no suppression of PTH synthesis due to decreased renal synthesis of 1,25(OH)2D lead to hypercalcemia and biochemical findings like that for pHPT. The causes are chronic real disease and, in rare cases, several years of vitamin D deficiency, high-dosage phosphate treatment for phosphate diabetes or primary biliary cirrhosis.



Vitamin D – Vitamin D-induced hypercalcemia

In cases of granulomatous diseases such as sarcoidosis, tuberculosis, silicosis, berylliosis, histoplasmosis, leprosy, disseminated candidiasis and Crohn’s disease, there is an unregulated, increased conversion of 25(OH)D to 1,25(OH)2D due increased activity of 25-OHD-1α-hydroxylase in the macrophages of the granuloma. Biochemical findings are an increase in 1,25(OH)2D and low PTH level.




– Vitamin D overdose

The cause of vitamin D-induced hypercalcemia include dosage errors during high-dosage medication of vitamin D3 (cholecalciferol) above 50,000 units per week or of active vitamin D metabolites such as 1α-hydroxy cholecalciferol (alfacalcidol), 1,25-dihydroxy vitamin D (calcitriol) and dihydrotachysterol. The indication for the intake of these substances is usually the treatment of rickets, hypoparathyroidism, renal bone mineral disease or malabsorption-related calcium deficiency. These preparations are also used for treating osteoporosis /25/.

In osteoporosis patients, hypercalcemia can occur with a twice daily intake of 0.5 μg calcitriol with a simultaneous intake of 0.5 g calcium over several months . Hypercalcemia can also occur during treatment of psoriasis patients with calcipotriol /26/.

Treatment in cases of kidney failure: these patients must be treated if there is secondary hyperparathyroidism (sHPT) with PTH values more than 2–4 times the upper reference interval value because hyperphosphatemia must be prevented. The PTH level is reduced through continuous treatment with calcitriol, because this prevents the transcription and secretion of PTH via the vitamin D receptor. The treatment range of calcitriol is limited, however, particularly if it is administered together with calcium-containing phosphate binders. An incorrect dosage and improper monitoring of the treatment increases the risk of hypercalcemia. The half-life of the hypercalcemia for a moderate overdose is approximately 3 weeks. A long-term overdose leads to calcification of tendons, ligaments, joints, vessels and inner organs. Therefore, the strict monitoring of the treatment with cholecalciferol and/or calcitriol by determining calcium, phosphate, ALP and PTH is recommended /27/.



Vitamin A overdose

Hypercalcemia occurs in patients receiving high doses of vitamin A, as treatment for acne, Kaposi syndrome, promyelocytic leukemia and other neoplasias. Hypercalcemia results due to increased bone resorption. PTH and 1,25(OH)2D are normal /28/.




Milk-alkali syndrome

The milk-alkali syndrome is a complication in patients with peptic ulcers and who drink quantities of milk and take in 30–40 g of calcium carbonate per day. Ulcer treatment with these antacids requires monitoring of the calcium level. The symptoms of the milk-alkali syndrome are hypercalcemia, metabolic alkalosis, renal failure, and the excretion of alkaline urine /29/.



Thiazide medication

The renal excretion of calcium is suppressed. Thiazides lead to a temporary increase of calcium in some patients. If the hypercalcemia does not normalize 2 weeks after discontinuation the treatment, pHPT is often present /15/. The thiazide test may be of further help, if borderline hypercalcemia is present in combination with elevated PTH and underlying pHPT is suspected. Administering thiazide reduces the calciuria and the serum concentration of calcium increases. Normal parathyroids respond with a decrease of PTH. This is not the case for pHPT.





Increased bone resorption is the cause of the rarely occurring hyperthyreote hypercalcemia. PTH and 1,25(OH)2D are normal /30/.



Addison’s disease

Due to the deficiency of glucocorticoids, the intestinal calcium absorption is increased and the renal calcium excretion is decreased. This also occurs after discontinuation of treatment with glucocorticoids.



Familial hypocalciuric hypercalcemia (FHH) /31/

FHH results from disorders in the signaling pathway of the calcium sensitive receptor (CaSR). A distinction is made between 3 types of FHH (see Tab. 6.1-6). Patients with FHH 1 and FHH 2 are generally clinically asymptomatic, although some adults with FHH 1 suffer from pancreatitis, chondrocalcinosis, or pHPT. In comparison to pHPT the FHH is likely if the calcium/creatinine ratio (both in mmol) in the urine is below 0.01. The ratio has a diagnostic sensitivity of 81% for confirming the FHH with a specificity of 88% to rule out the pHPT. The second morning spontaneous urine is used for determination of the calcium/creatinine ratio. Medications that cause a change of the calcium excretion (diuretics, lithium) should be stopped 2 months before the calcium/creatinine ratio is determined.

Laboratory findings: it is important to distinguish between FHH and pHPT. In FHH, the serum calcium is rarely > 12.8 mg/dL (3.2 mmol/L) and PTH is < 30 ng/L (3.0 pmol/L). In FHH3 PTH is increased and phosphorus is decreased.



Neonatal heavy hyperparathyroidism (NSHPT)  /31/

NSHPT is a very rare disease with hyperparathyroidism-related bone damage due to a lack of mineralization, which occurs in children as early as age 1–6 months. There is a homozygote loss of function in the gene of the calcium-sensitizing receptor.

Laboratory findings: serum calcium 11–28.4 mg/dL (2.73–7.1 mmol/L), very high PTH, occasionally also in the reference interval, serum phosphorus below 5.6 mg/dL (1.8 mmol/L), ALP more than 4 times the age-dependent upper reference interval value.

Idiopathic infantile Hyper­calcemia /32/

The CYP24A1 gene encodes the 25-(OHD) 24-(OH)D-1α-hydroxylase, the enzyme for the degradation of 1,25 (OH)2D. Mutations in CYP24A1 cause an increase of 1,25 (OH)2D and increase of the intestinal calcium absorption. At age 6–8 months, particularly with vitamin D supplementation, an increase of calcium to approximately 4 mmol/L results, PTH is decreased to below 1 ng/L (0.1 pmol/L).





Hypercalcemia can occur a few days or weeks after immobilization. It especially occurs in chronic immobilization (tetraplegia) or if there is another illness without stress of the bones. PTH and 1,25(OH)2D are normal.

Table 6.2-4 Diseases that may cause hypocalcemia /1117/



Clinical and laboratory findings






Malabsorption e.g., vitamin D deficiency, malabsorption syndrome /21/

Diseases which cause enteral calcium and vitamin D malabsorption over a long period of time are associated with the development of secondary hyperparathyroidism and osteomalacia. Such diseases include for example celiac disease and chronic pancreatitis.

Laboratory findings: serum calcium is commonly at the lower reference interval value, 25(OH)D is decreased, 1,25(OH)2D is slightly decreased or normal, PTH is elevated, and the ALP is increased.


Hypoparathyroidism (surgical, idiopathic, miscellaneous)

Postoperative hypoparathyroidism occurs after thyroid surgery as well as neck surgery, if there are malignant tumors, as a result of parathyroid gland removal. The control is achieved intraoperatively by determining the PTH level. Hypocalcemia tends to occur during the early postoperative period, but in rare cases may not occur until years later.

Idiopathic hypoparathyroidism is an autoimmune disorder and usually occurs at an early age. It often occurs together with other autoimmune disorders such as Addison’s disease, Hashimoto’s thyroiditis, pernicious anemia and hypogonadism.

Some rare causes of hypoparathyroidism are hemosiderosis, Wilson’s disease, and metastatic disease.

Laboratory findings: calcium < 8.0 mg/dL (2.0 mmol/L), phosphorus > 5 mg/dL (1.6 mmol/L), intraoperative decrease of PTH, postoperative PTH is low to low-normal.



Pseudohypoparathyroidism represents a group of states with end organ resistance to PTH, and elevated PTH.

Type 1 is characterized by a defect of the Gs-protein-mediated signal transmission before the formation of cAMP and type 2 is associated with a defect at a locus beyond the synthesis of cAMP (Fig. 6.2-4 – Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor or of the calcium-sensitive receptor.

For further information see Tab. 6.4-5 – Biochemical and clinical findings in pseudo hypoparathyroidism.

Chronic kidney disease (CKD)

CKD is characterized by glomerular and tubular sclerosis with a resulting decrease of the GFR and reduction of the parenchymal tissue. From stage 3 of CKD, there is a reduced calcitriol formation as a result of direct retention of phosphorus or the stimulation of FGF-23. The decreased calcitriol leads to reduced absorption of calcium from the intestines and a decreased reabsorption in the proximal tubuli of the kidneys. The result is a decline of serum calcium level, which is compensated by increased PTH secretion. The result is secondary hyperparathyroidism, which intensifies the hyperphosphatemia. According to the KDIGO guidelines, biomarkers are recommended for monitoring of CKD mineral bone disease (CKD-MBD), see Tab. 6.4-4 – Diagnosis and monitoring of Chronic Kidney Disease-Mineral Bone Disease (CKD-MBD) according to KDIGO.

↓, n

Hungry bone syndrome /33/

This syndrome can occur in dialysis patients with severe secondary or tertiary hyperparathyroidism after removal of the parathyroids or during treatment with cinacalcet (calcimimetic). The reason is due to remineralization of the skeleton, which occurs when the effect of the stongly increased PTH is no longer a factor. Bone formation increases dramatically, osteoclast-related bone degradation is sharply reduced.

Laboratory findings: long-term hypocalcemia, a slight decrease of serum phosphate, and a further increase of ALP. The ALP increases on the 4th postoperative day and reaches its peak value after 7–14 days. The higher the preoperative values of PTH, ALP and calcium, the greater the postoperative risk of hungry bone syndrome.



Autosomal dominant hypocalcemia with hypercalciuria (ADHH) /31/

There is an activating mutation of the calcium-sensitive receptor gene. More than 25 mutations have been described to date. The prevalence is 1 : 70,000 and is somewhat lower than for FHH. The clinical symptoms of hypocalcemia are neuromuscular irritability, paresthesias, muscle cramps, and carpopedal spasms.

Laboratory findings: the clinical symptoms are severe in the calcium range of 4.8–7.0 mg/dL (1.20–1.75 mmol/L) and slight in the range of 6.0–7.8 mg/dL (1.50–1.95 mmol/L). The lowest serum calcium value is 4.8 mg/dL (1.20 mmol/L), two thirds of the patients have PTH values over 10 ng/L (1.0 pmol/L), the calcium/creatinine ratio (mmol/mmol) is 0.37 (0.03–0.82), magnesium is below 17 mg/dL (0.70 mmol/L).



Nephrotic syndrome

Due to proteinuria, there is a loss of vitamin D-binding protein, which leads to a decreased concentration of 25(OH)D in serum. The low amount of unbound 25(OH)D appears to be sufficient, because in the case of nephrotic syndrome, iCa and also the PTH concentration usually remain normal.


n, ↓


Liver cirrhosis

If the reduction in albumin synthesis is associated with values blow 30 g/L, there is a decrease of calcium, iCa remains relatively unaffected. Calcium excretion declines if vitamin D deficiency is concomitantly present.

, n



Tumors with osteoblastic metastases

Tumors associated with osteoblastic metastases such as breast carcinoma, prostate carcinoma, lung carcinoma, thyroid carcinoma cause the hungry bones syndrome. The skeleton system takes up a great amount of calcium, phosphate and magnesium, thus causing a decrease in their blood concentrations.




Acute pancreatitis

Sapofonication of calcium by fatty acids within the necrotic tissue is the reason for a decrease in calcium concentration. Hypocalcemia is common when amylase and lipase are normal again. In addition, hypoalbuminemia and hypomagnesemia can be measured.


Adrenal hyperplasia, glucocorticoid therapy

Cortisol suppresses intestinal calcium absorption and increases renal elimination. Excess cortisol leads to severe osteoporosis.



Diuretics (thiazides)

Thiazides reduce the renal calcium excretion, probably due to decreased calcium mobilization from the bone. This is therapeutically applied in patients with idiopathic hypercalciuria.



Furosemide, ethacrynic acid

The renal excretion of calcium is increased by these diuretics and the result is hypercalciuria.




Antiepileptic drugs (phenytoin, phenobarbital, primidone, carbamazepine)

Anti convulsants cause a deficiency of 25(OH)D due to activation of intrahepatic oxidases. Diphenylhydantoin also inhibits calcium absorption in the intestines. Because of this, osteomalacia is occasionally found to occur in patients with seizures.

Status post surgical treatment of pHPT

After a successful surgery, a massive influx of calcium and phosphate into the bone can occur.

Leukemia therapy

In the event of successful leukemia treatment (e.g., treatment of Burkitt lymphoma) the massive release of phosphates can cause hypocalcemia.

Table 6.2-5 Adult calcium excretion on regular diet /37/

24-hour urine

2-hour urine

< 250 mg (6.2 mmol)

+ ≤ 0.2 g/g creatinine (0.57 mmol/mmol creatinine)

< 300 mg (7.5 mmol)

+ ≤ 4 mg (0.1 mmol)/kg body weight

Conversion: mg × 0.02495 = mmol

Table 6.2-6 Calcium/creatinine ratio (mmol/mmol) in the urine of children on regular diet /38/



0–< 1 yr


1–< 2 yrs


2–< 5 yrs


5–< 10 yrs


10–18 yrs


The ratios apply to 2-hour urine or time-independent urine. The calcium excretion in the 24-hour urine (pre-pubescent boys) is 0.332 ± 0.122 mg/kg per 24 h /39/.

Table 6.2-7 Diseases associated with increased urinary calcium excretion /42/

Clinical and laboratory findings

Malignant tumor, metastases

Hypercalciuria is caused by increased calcium mobilization from the bone. It is encountered mainly in:

  • Tumors with PTHrP production: renal, bronchial, ovarian carcinoma (resorptive hypercalciuria)
  • Tumors with metastatic spread in the skeleton (resorptive hypercalciuria)
  • Multiple myeloma (resorptive hypercalciuria)

Primary tumors of the skeleton are less common cause of hypercalciuria.

Kidney stones

A calcium excretion of more than 0.10 mmol/kg body weight in 24-hours is found in approximately 50% of patients with calcium oxalate or calcium apatite nephro lithiasis and is one of the risk factors for stone formation. Hypercalciuria is supposed to be caused by the inhibition of the tubular calcium reabsorption. Although both an increased intake of NaCl as well as a decreased potassium intake cause a rise in renal calcium excretion, they are not the reason for the hypercalciuria observed in patients with nephro lithiasis.

Primary hyperparathyroidism

An increase in calcium excretion does not occur until hypercalcemia is present in combination with an increased glomerular filtration of calcium. In these cases the renal tubules are not capable of reabsorbing the calcium load, despite an elevated re-absorptive rate (absorptive and resorptive hypercalciuria).

Renal-tubular acidosis

Hypercalciuria due to increased accumulation of calcium in the primary urine because acidosis is associated with an increased dissociation of protein-bound calcium. In addition, the renal tubular reabsorption of calcium is decreased.

Hyperthyroidism, Cushing’s syndrome

Increased glomerular filtration and reduced tubular calcium reabsorption. Glucocorticoids reduce the enteral calcium absorption.

Immobilization, absence of gravity

Immobilization and absence of gravity lead to release of calcium from the bone. The concentration of PTH is low-normal.

Boeck’s disease (sarcoidosis)

The enteral absorption of calcium is increased; the underlying cause is thought to be the increased synthesis of 1,25(OH)2D in the granuloma cells. As a result of this, the urinary calcium excretion is increased (absorptive hypercalciuria).

Milk-alkali syndrome

Occurs during treatment of gastroduodenal ulcers with large quantities of calcium salts or due to daily consumption of several liters of milk (absorptive hypercalciuria).

Condition after ovari ectomy, estrogen deficiency

Estrogen deficiency promotes the mobilization of calcium from the bone, as part of an osteoporotic process. The concentration of PTH is low-normal.

Table 6.2-8 Causes of secondary hypercalciuria /40/


  • Increased intake of calcium, sodium, albumin, carbohydrates, alcohol
  • Decreased intake or reduced absorption of phosphate and potassium

Secondary increase of intestinal calcium absorption

  • Vitamin D therapy
  • Endogenic over-production of 1,25(OH)2D (primary hyperparathyroidism, granulomatous diseases, lymphomas, severe hypophosphatemic diseases)

Increased osteoclastic bone resorption

  • Bone metastases
  • Multiple myeloma
  • Primary hyperparathyroidism
  • Paget’s disease
  • Hypothyroidism
  • Long-term immobility

Reduced renal-tubular calcium reabsorption

  • Loop diuretics
  • Bartter syndrome
  • Sponge kidney
  • Primary renal-tubular defects
  • Endogenic or exogenic glucocorticoid excess
  • Genetic disorders (calcium-sensitive receptors, chloride channels)

Table 6.3-1 Reference intervals for phosphate

Adults /5/

2.6–4.5 (0.84–1.45)

Children /6/

1–30 days

3.9–7.7 (1.25–2.50)

1–12 mos

3.5–6.6 (1.15–2.15)

1–3 yrs

3.1–6.0 (1.00–1.95)

4–6 yrs

3.3–5.6 (1.05–1.80)

7–9 yrs

3.0–5.4 (0.95–1.75)

10–12 yrs

3.2–5.7 (1.05–1.85)

13–15 yrs

2.9–5.1 (0.95–1.65)

16–18 yrs

2.7–4.9 (0.85–1.60)

Data expressed in mg/dL (mmol/L).

Conversion: mg/dL × 0.3229 = mmol/L

Table 6.3-2 Causes of hypophosphatemia

Acute phosphate shift from the extracellular to the intracellular compartment

  • Recovery from diabetic ketoacidosis
  • Dextrose infusion
  • Resumption of food intake after a period of fasting
  • Respiratory alkalosis
  • Recovery from acute respiratory acidosis
  • Very strenous physical activity (bodybuilding)
  • Leukemias and lymphomas

Inadequate phosphate intake

  • Parenteral nutrition without phosphate

Impaired enteral phosphate absorption

  • Phosphate binders, antacids

Renal tubular leak

  • Sporadic hypophosphatemia (late onset)
  • Oncogenic hypophosphatemia

Undetermined mechanisms

  • Alcoholism

Table 6.3-3 Diseases and conditions associated with hypophosphatemia /17/

Clinical and laboratory findings

Competitive athletes, bodybuilding

A few days prior to a competitive event, athletes initiate carbohydrate loading as well as dehydrating. The glycogen stores that are empty because of dieting and persistent training are replenished by an excessive intake of carbohydrates. During this phase, phosphate shifts from extracellular to intracellular space. In bodybuilders, the development of large muscle mass leads to phosphate requirements. If the intake of phosphate is inadequate low, hypophosphatemia and muscle weakness ensue /22/.

Primary Hyperparathyroidism

Hypophosphatemia or levels below 3.5 mg/dL (1.13 mmol/L) are, like hypercalcemia, an important essential finding in suspicion of hyperparathyroidism. It is important to make repeated determinations and the age as well as the gender dependence of the phosphate concentration needs to be kept in mind /1/.

Intestinal malabsorption

Vitamin D and calcium malabsorption cause hypophatemia as a result of secondary hyperparathyroidism /1/.

Vitamin D deficiency rickets

All types of this disorder are associated with an increase in ALP. Rapid normalization of phosphate occurs after vitamin D is administered. The product of calcium × phosphate is below 24 [mg/dL]2.


Due to loss of fluids and the administering of glucose infusions with a shift of phosphate from the extracellular to intracellular space, most cases show a mild hypophosphatemia on post-operative day 2–3. The decrease of phosphate concentration is, on average, 0.5 mg/dl (0.16 mmol/L) /23/. In cases of surgical complications and long-standing acute-phase reaction, hypophosphatemias are common and lead to deficient generation of ATP with serious consequences.

Severe burns

A few days after severe burns, hypophosphatemia occurs depending on the extent of the burns /1/.

Lupus erythematosus

In 20% of the patients with juvenile systemic lupus erythematosus, decreased phosphate concentrations were measured /24/.

Diabetic ketoacidosis (DKA)

Patients with DKA are more likely to be admitted to a clinic with hyperphosphatemia, resulting from osmotic diuresis. Rehydration and administering of insulin lead to a phosphate shift to the intracellular space. The result is the formation of hypophosphatemia, usually below 2 mg/dl (0.65 mmol/L) 16–24 hours after the start of treatment /1/.


The development of hypophosphatemia is due to decreased food intake, vomiting, diarrhea, liver disease, and malnutrition. Hypomagnesemia, hypokalemia, vitamin D deficiency, ketoacidosis or lactate acidosis and respiratory alkalosis also commonly occur /1/. Due to the shift of phosphate to the intracellular space, the infusion of dextrose solution can lead to phosphate concentrations in the serum below 1 mg/dL (0.32 mmol/L). The combination of rhabdomyolysis and hypophosphatemia is found exclusively in patients with severe alcoholism /25/.

Antacids treatment

Aluminum hydroxide containing antacids bind phosphate in the intestine. Even dosages of 3 × 30 mL daily can lead to hypophosphatemia after 2–4 weeks. The excretion of phosphate in the stool increases. The excretion of phosphate in urine decreases to below 50 mg (1.6 mmol)/24 hours. The renal secretion of phosphate which normally compensates for fluctuations in the dietary intake of phosphate, is not capable of compensating.

In the presence of hypophosphatemia, the calcium concentration in serum is normal. Also normal are PTH and 1,25(OH)2D. Osteomalacia can develop after more than two years intake of antacids. Patients who take antacids for a long period of time should have an excretion of phosphate in the urine above 300 mg (9.7 mmol)/24 hours. This is an indication that adequate intestinal absorption of phosphate still takes place /1/.

Oncogenic osteomalacia

Oncogenic hypophosphatemic osteomalacia is mainly acquired in adulthood. This disease often involves bone pain and muscle weakness. The leading biochemical finding is hypophosphatemia. According to a series of 71 case reports, the tumor was discovered prior to detection of hypophosphatemia in 8 cases. It was found in 17 cases during the first year and in the remaining cases within a period of up to 15 years after the detection of hypophosphatemia /12/. The tumor is usually benign and involves for example, soft tissue tumors such as hemangioma, fibroangioma, mesenchymal tumors or bone tumors such as fibroma and osteoblastoma /26/. Tumors such as lung carcinoma can be associated with the secretion hormones like ACTH and corticotropin.

Laboratory findings: hypophosphatemia due to decreased renal-tubular phosphate reabsorption, 25(OH)D normal, 1,25 (OH)2D is decreased.

Hereditary hypophosphatemia – Generalized /27/

Hypophosphatemia due to isolated renal phosphate wasting is defined by three well defined Mendelian disorders: X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR) and hereditary hypophosphatemic rickets with hypercalciuria (HHRH).

– X-linked hypophosphatemia /27/

X-linked hypophosphatemia (XLH) occurs in 1 out of 25,000 newborns and is the most common form of hereditary rickets. Patients present with short stature, lower extreme deformity from rickets, bone pain, joint pain and dental abscesses. Inactivating mutations in the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) are responsible for XLH.

Laboratory findings: the excretion of phosphate is increased because the TmP/GFR is decreased; the activity of ALP is increased. PTH is normal or slightly increased, 1,25 (OH)2D is normal, but inadequately declined relative to hypophosphatemia; calcium excretion is normal /10/.

Autosomal dominant hypophosphatemic rickets (ADHR) /27/

Two groups of patients were identified. The first group consisted of patients who presented with renal phosphate wasting as adolescents or adults. They presented with bone pain, weakness and insufficiency fractures, but did not manifest lower extremity deformity. The second group presented with phosphate wasting, rickets, and lower extremity deforming as children. ADHR results from mutations in R 176 or R179 in the protein FGF23. These arginines make up the RXXR cleaving sites and mutations in either of these arginines protect FGF23 from degradation.

Autosomal recessive hypophosphatemic rickets (ARHR) /27/

These patients present with rickets and osteomalacia with isolated renal phosphate wasting and normocalciuria. There is a homozygote mutation in the gene for Dentin matrix protein 1 (DMP1). DMP1 is a non-collagen bone matrix protein.

Laboratory findings: hyperphosphaturia in cases of normocalciuria, the concentration of FGF23 in the plasma is increased.

Hereditary hypophosphatemic rickets with hypercalciuria

A very rare disease with growth delay, radiological signs of rickets, nephrocalcinosis in the absence of glucosuria, proteinuria and acidosis. Nucleotide sequence revealed a single-nucleotide deletion in the gene SLC34A3.

Laboratory findings: hyperphosphemia, hypercalciuria, hypophosphatemia, elevated 1,25(OH)2D.

Kidney damage

Due to the nephrotoxicity of medications, particularly of cytostatics such as ifosfamid, tubular kidney damage can occur up to fully developed Fanconi syndrome. This is particularly described in cases involving children. In a case report of a child with myopathy, the serum phosphate level was 1.4 mg/dl (0.45 mmol/L), the phosphate clearance which was corrected to the body’s surface was 24.7 mL/min. (normal below 16 mL/min.), PTH and 1,25(OH)2D were normal. There was also lactaturia, glucosuria and aminoaciduria /28/.

Hepatic resections, liver transplantation /29/

Hypophosphatemia is a post-operative complication after liver resection, and liver cancer. Thus, after resection, the phosphate level decreases to 1 mg/dL (0.32 mmol/L) with complications such as cardiac arrhythmia, infection, liver failure and respiratory distress. Low phosphate values are also measured after liver transplantation, especially in patients with parenteral nutrition.

Hyperventilation /30/

Hyperventilation causes a respiratory alkalosis, which in turn mediates an increase in intracellular pH. This stimulates phosphofructokinase activity in the glycolytic pathway with a subsequent increase in demand for phosphate, which is shifted intracellularly to form glycolytic intermediate metabolites. A similar phenomenon is observed with an elevation in intracellular pH, which occurs in metabolic alkalosis. In a case report, the patient with hyperventilation had severe hypophosphatemia (0.23 mmol/L), blood pH 7.53, PCO2 3.6 kPa, PO2 16.9 kPa, actual bicarbonate 12 mmol/L.

Table 6.3-4 Diseases and conditions associated with hyperphosphatemia

Clinical and laboratory findings

Chronic kidney disease (CKD) /13/

The association of elevated serum phosphate levels with increased mortality was documented in many studies in patients with CKD and with kidney transplant.

The concentration of phosphate in serum is an indicator for monitoring the phosphate retention as a consequence of a decreased glomerular filtration rate (GFR). Increased phosphate values are a risk factor for the progression of CKD with complications such as mineral bone disease (CKD-MBD), vascular calcification, left ventricular hypertrophy and increased mortality. Prospective studies have shown that even patients who do not have CKD, but do have borderline or actual hyperphosphatemia, have increased mortality and increased cardiovascular risk. In a 2-year study of patients undergoing chronic hemodialysis, the mortality was 39% for phosphate values above 7.9 mg/dL (2.55 mmol/L) and 18% for above 6.6 mg/dL (2.13 mmol/L) /31/. In one study /32/ of CKD-predialysis patients, the increase of the hazard ratio for mortality was 1.07 for each phosphate increase of 0.3 mg/dL (0.10 mmol/L).

With a GFR decline below 60 [mL × min–1 × (1.73 m2)–1], renal-related elevation of phosphate start, however, levels above the upper reference interval value only occur with an estimated GFR below 30 [mL × min–1 × (1.73 m2)–1]. In the GFR range of 30–60 [mL × min–1 × (1.73 m2)–1] a larger percentage of patients have phosphate values above 4.6 mg/dL (1.5 mmol/L) /33/.

The kidneys are capable of detecting changes in the phosphate level in plasma and of regulating the concentration by means of active reabsorption. This takes place by means of sodium-phosphate cotransporters of the proximal tubular cells, a process which is independent of hormones. The phosphate concentration of the extracellular fluid (ECF) is also regulated by humoral factors. The main factors are PTH and the fibroblast growth factor 23 (FGF23). The increase of phosphate in the ECF causes the increase of PTH and FGF23. Both induce phosphaturia due to decreased phosphate reabsorption in the kidneys. These effects are independent of the calcium and 1,25(OH)2D levels which both have a regulating effect on PTH and FGF23, but in different ways. A high concentration of calcium or 1,25(OH)2D suppresses the secretion of PTH and stimulates that of FGF23 /7/.

With a GFR of around 30 [mL × min–1 × (1.73 m2)–1] the clearance of phosphate decreases and the phosphate concentration in serum increases. In the event of a further decline in GFR with an increase of phosphate, the hyperphosphatemia becomes a central problem because increasing FGF23 and PTH concentrations are required in order to keep the serum phosphate concentration in the reference interval and secondary hyperparathyroidism (sHPT) develops. In this situation, the following exist: hyperphosphatemia with accompanying hypocalcemia, decrease in 1,25(OH)2D and the increase of PTH and FGF23. A restriction of phosphate can correct the sHPT independently of the increase of serum calcium and of 1,25(OH)2/34/. According to recent suggestions /33/ decreasing GFR at first causes a decrease of 1,25 (OH)2D and then of PTH. With decline of GFR, the increase of PTH precedes the increase of phosphate and the decrease of calcium. Serum FGF23 is elevated in stages 4 and 5 of CKD and increases with an elevation of phosphate.

Strategies for controlling the phosphate increase include the prevention of sHPT and CKD-MBD, dietary reduction by means of adequate protein intake, treatment with phosphate binders and 1,25 (OH)2D. For the diagnosis of CKD-MBD, see Tab. 6.4-4 – Diagnosis und monitoring of Chronic Kidney Disease-Mineral Bone Disease (CKD-MBD) according to KDIGO.

Laboratory findings: for chronic dialysis patients, the KDIGO guidelines recommend the following target values /35/:

Calcium 8.4–9.5 mg/dL (2.1–2.4 mmol/L)

Phosphate 3.5–5.5 mg/dL (1.13–1.78 mmol/L)

Ca × phosphate below 55 [mg/dL]2

PTH 150–300 ng/L (15–30 pmo/L).

Hypoparathyroidism, pseudohypoparathyroidism type I and II

The increased tubular reabsorption of phosphate can cause hyperphosphatemia. PTH suppresses the renal tubular reabsorption of phosphate. The deficiency of PTH due to surgical or traumatic hypoparathyroidism or a tubular resistance of PTH receptor in the case of pseudohypoparathyroidism reduce the proximal-tubular secretion phosphate.

Increased phosphate intake (orally, intravenously)

Taking phosphate tablets or laxatives containing phosphate can lead to hyperphosphatemia, particularly if vitamin D therapy is being carried out simultaneously. A common cause is also enemas with solutions containing phosphates. In one case report /36/, a phosphate nephropathy developed with an increase of phosphate of 4.1 mg/dL (1.32 mmol/L), which decreased to the reference interval within 24 hours.

Acute tumor lysis syndrome (TLS)

Laboratory findings in acute TLS are marked hyperuricemia, hyperphosphatemia, hypocalcemia and hyperkalemia. TLS results from the destruction of a large number of tumor cells during chemotherapy. Since lymphoblasts have a 4-times higher concentration of phosphate than lymphocytes, TLS often occurs during treatment of the lymphoblastic lymphoma, lymphoblastic leukemia, and Burkitt lymphoma /37/. See also Section 5.4 – Uric acid.

Crush syndrome

The laboratory findings are similar to those of tumor lysis syndrome /25/.

Acute acidosis

In acute acidosis the hyperphosphatemia results from the shift of phosphate from the intracellular to the extracellular compartment. Such conditions exist in cases with metabolic acidosis (e.g., diabetic ketoacidosis, lactic acidosis) respiratory acidosis, and tissue hypoxia /7/.

Table 6.3-5 Diseases with Cp-increase


Clinics and laboratory findings

Hyperparathyroidism, malabsorption syndrome

Due to increased PTH secretion, tubular phosphate reabsorption is decreased.

Phosphate diabetes, renal-tubular acidosis

Decrease of tubular phosphate reabsorption due to congenital or acquired damage of renal tubules.

Table 6.3-6 TRP(%) with diseases



Primary hyperparathyroidism


Phosphate diabetes

< 80

Renal-tubular acidosis

< 80

Table 6.3-7 Reference intervals of the tubular maximum for phosphate reabsorption (TmP/GFR)

Age/gender /18/






3 months



6 months



2–15 yrs



25–35 yrs



25–35 yrs



Table 6.4-1 Reference intervals for PTH


Reference interval

Intact PTH

15–65 ng/L (1.5–6.5 pmol/L) /6/

Bioactive PTH

Depending on the manufacturer, the upper reference interval value is approximately 50–60% of second generation PTH assays.

Conversion: ng/L × 0,106 = pmol/L

Table 6.4-2 Diseases and conditions with hyperparathyroidism and hypoparathyroidism

Clinical and laboratory findings

Primary hyperparathyroidism (pHPT) – Generalized /9/

The cause of pHPT is often an adenoma of a single parathyroid gland, seldom hyperplasia of all parathyroid glands, and only rarely parathyroid carcinoma, familial pHPT or multiple endocrine neoplasia (MEN). Solitary adenomas are monoclonal or oligoclonal tumors, and this is the case in polyglandular pHTP as well. Parathyroid adenomas reflect an overgrowth from somatic or germ-line mutations in parathyroid progenitor cells. Solitary adenomas account for approximately 85% of pHPT. Hyperfunction in multiple parathyroid glands (hyperplasia, multiple adenomas and polyclonal hyperfunction) occurs for most of the remainder. Less than 1% of patients have parathyroid carcinoma. Approximately 75% of patients with sporadic pHPT are women with an average age of 55 years. The annual incidence is 10 in 100,000 individuals. About 80% of the cases of pHPT are sporadic in nature and in 20% the disease is inherited. Among the inherited forms are:

  • Multiple endocrine neoplasia type 1 (MEN type 1): these patients have various combinations of tumors (e.g., of parathyroid glands, enteropancreatic, anterior pituitary, and other tumors). By the age of 40 years patients with MEN type 1 have endocrine disorders with the following frequencies: hyperparathyroidism 85%, Zollinger-Ellison syndrome 35%, prolactinoma 25%. The cause is the presence of a germ cell mutation which inhibits the tumor suppressor gene MEN1.
  • Familial hypocalciuric hypercalcemia. This disorder is based on an inactivating mutation in the gene that codes the calcium-sensing receptor, which result in an insensitivity of the parathyroid cells to inhibition by plasma calcium.
  • Neonatal severe pHPT. The newborns have marked enlargement of all parathyroid glands, very high PTH serum levels, and hypercalcemia above 16 mg/dL (4 mmol/L). Pathophysiologically homozygous inactivating germ-line mutations of the Calcium-sensing receptor gene are responsible for the disease.

Some 5% of patients with pHPT have autoantibodies against the calcium-sensing receptor. The pathophysiological role of autoantibodies is unknown /10/.

Manifestations of pHPT: the PTH excess manifests as abnormal fluxes of calcium and phosphate in bone, in the kidneys, and in the gastrointestinal tract:

  • Persistently elevated PTH levels have a catabolic effect on bones and lead to bone mineral disease (BMD) with osteopenia and an increased risk of bone fracture, particularly of the vertebra. The PTH-induced increase in bone resorption results in hypercalciuria. On bone density measurement, approximately 15% of patients have a lumbar Z-score of less than minus 1.5. Nonetheless, no significant changes are found in a large proportion of patients with mild pHPT. In parathyroid ectomized patients with bone involvement, an increase in bone density of 6–14% is observed after 1 year /11/.
  • About 20% of pHPT patients have nephro lithiasis. Kidney stones no longer occur following parathyroid ectomy in over 90% of patients with nephro lithiasis.
  • In pHPT the hypercalcemia leads to hypercalciuria and the PTH stimulated enhanced synthesis of 1,25(OH)2D results in absorptive hypercalciuria.

Clinical symptoms /12/: the majority of patients with pHPT have no symptoms that can be related to pHPT or to hypercalcemia, but up to 50% have mild symptoms such as fatigue or muscle weakness. Among asymptomatic patients only 25% have progressive disease. Osteopenia generally occurs after 10 years.

Laboratory findings: some pHPT patients manifest elevated calcium of only less than 1 mg/dL (0.25 mmol/L). Patients < 45 years of age with pHPT and hypercalcemia often do not exceed the upper reference interval value of PHT [65 ng/L (6.5 pmol/L)]. This is because younger persons have lower PTH values than older individuals. In these cases the threshold for suspecting pHPT should be around 45 ng/L (4.5 pmol/L) /13/. Occasionally pHPT patients have persistently normal calcium levels, but elevated PTH concentrations. If all causes of sHPT have been ruled out, this represents an early manifestation of pHPT. In older patients with hypercalcemia values of greater than 65 ng/L (6.5 pmol/L) are indicative of pHPT. In a study /14/ out of 56 patients with surgically confirmed pHPT, only 1 had a lower value; the median in all patients was 165 ng/L (16.1 pmol/L), and the highest level was 1200 ng/L (113 pmol/L). The ruling out of a vitamin D deficiency is important (25 OHD < 20 μg/L). Hypophosphatemia, or values lower than 3.5 mg/dL (1.13 mmol/L), are only found in 40% of asymptomatic patients, and this holds true for hypercalciuria as well. Depending upon the extent of the pHPT, biochemical markers of bone remodeling, such as ALP, bone ALP and pyridinolines, may be elevated (Tab. 6.4-3 – Calcium (Ca), phosphate (P) and PTH in hyperparathyroidism). In many cases both ALP and the bone resorption markers are normal. 25(OH)D in serum is low-normal, and 1,25 (OH)2D is near the upper reference interval value or slightly elevated.

Indication for surgery in asymptomatic patients: according to the Consensus of the National Institutes of Health of the United States, the criteria are /15/: serum calcium above 12 mg/dL (3.0 mmol/L), calcium excretion higher than 400 mg (10 mmol)/24 hours, reduced bone density (Z-score less than minus 2), reduced eGFR in the absence of other causes, age younger than 50 years.

– Neonatal severe hyperparathyroidism (NSHPT)

Mean PTH values are on average 10-fold above than the upper reference interval value, moderate [12–13 mg/dL (3.0–3.25 mmol/L)] to severe [30 mg/dL (7.5 mmol/L)] elevations in calcium. Relative hypocalciuria is present in spite of the hypercalcemia.

– Intraoperative PTH measurement

The most common complication following total thyroidectomy is hypoparathyroidism and, following parathyroid surgery, a persistent hyperparathyroidism. PTH concentrations are generally measured pre-operatively as well as 10 minutes after the thyroid has been removed or after parathyroidectomy. In patients who developed symptomatic hypocalcemia following thyroidectomy, PTH concentrations were 7.6 ± 12.0 ng/L (0.77 ± 1.2 pmol/L), in comparison with 55.7 ± 31.8 ng/L (5.8 ± 3.2 pmol/L) in asymptomatic patients. An intraoperative level below 10 ng/L (1 pmol/L) had a positive predictive value for hypoparathyroidism of 100%, and a negative predictive value of 91% /16/. In parathyroid surgery, a remaining hyperparathyroidism is unlikely if PTH concentrations in the 10-minute post-ablation sample decreases by more than 50% in comparison with the pre-resection value /17/. With the use of a third generation PTH assay, a decrease of 50% occurs within 5 minutes /18/. The onset of hypocalcemia and its associated symptoms will usually occur within 6–24 hours after the operation. Critical points of intraoperative PTH testing process are described in Ref. /19/.

Secondary hyperparathyroidism (sHPT) – Generalized

The pathogenesis of sHPT is complex and driven by several factors, including vitamin D deficiency, hypocalcemia, and hyperphosphatemia. Elevated FGF23 concentrations exacerbate sHPT through further reductions in 1,25(OH)2 D (calcitriol) levels. Calcitriol deficiency results in decreased intestinal absorption of calcium and may lead to hypocalcemia, a major stimulus for PTH secretion. This leads to parathyroid cell proliferation, contributing to sHPT. The incidence and severity of sHPT increases as kidney function declines and can lead to significant abnormalities in bone mineralization and turnover /21/.

– Chronic kidney disease /2021/

sHPT develops in CKD if the GFR is moderately to severely decreased; decline below 44 [mL × min–1 × (1.73 m2)–1] (e.g., G3b to G5 CKD stages). An increase in PTH inactivates the sodium-phosphate co transporter of the proximal renal tubular cells and causes reduced phosphate reabsorption. Hyperphosphatemia, hypocalcemia and a diminished synthesis of 1,25(OH)2D induce increased PTH secretion. The result is parathyroid gland diffuse or nodular hyperplasia. The hyperplasia leads not only to increased parathyroid gland volume but also to downregulation of the calcium-sensing receptor and vitamin the D receptor.

A decrease of ionized calcium (iCa) level in blood is believed to be the triggering cause of sHPT in CKD, since iCa dependent signaling influences three parathyroid gland functions, namely, PTH gene expression, PTH secretion, and parathyroid cell proliferation. A different theory emphasizes the role of phosphate. In CKD, phosphate is retained and the resulting hyperphosphatemia reduces the activity of 25-OHD-1α-hydroxylase so that less calcium is absorbed by the intestine. The increase in phosphate leads to elevated secretion of fibroblast growth factor 23 (FGF23). In this situation, the following exist: hyperphosphatemia with accompanying hypocalcemia, decreased 1,25(OH)2D and elevated PTH and FGF23. According to more recent notions, decreasing GFR at first causes a decline of 1,25 (OH)2D and then of PTH. The increase in PTH precedes that of phosphate and the decrease in calcium. According to other theories, the hyperphosphatemia does not trigger the sHPT but rather, only reinforces it (see Section 6.3 – Phosphorous). The consequences of CKD induced sHPT are metabolic bone disease (CKD-MBD), with disturbances of calcium and phosphate metabolism that lead to calcification of blood vessels and of soft tissue.

The sHPT in CKD increases bone turnover by stimulating osteoblast activity and osteoclast proliferation. The combination of sHPT and bone mineralization deficiencies leads to various forms of renal osteodystrophia (ROD) (see Section 1.3 – Alkaline phosphatase (ALP)).

Laboratory findings /21/: the concentration of PTH is an important criterion with regard to the treatment of CKD-MBD. Thus, in stage G5 CKD, PTH values should be maintained 2 to 9-fold above the upper reference interval value of the assay by the administration of vitamin D, calcitriol or vitamin D analogs.

In one study /5/ in dialysis patients, second generation PTH values were 223 ng/L (range 5–2844) while those of third generation PTH were 138 ng/L (range 4–1580). Patients with PTH levels between 500 and 900 ng/l should visit a surgeon.

With PTH values below 65 ng/L (6.5 pmol/L), adynamic bone disease (ABD) was present in 48–75% of cases. ABD is very likely to occur with PTH values of below 150 ng/L (15 pmol/L) and bone ALP of less than 7 μg/L. See also Tab. 6.4-3 – Calcium (Ca), phosphate (P) and PTH in hyperparathyroidism.

Target values are 3.5–5.5 mg/dL (1.13–1.78 mmol/L) for phosphate and 8.4–9.6 mg/dL (2.1–2.4 mmol/L) for calcium. Recommendations of the KDIGO /21/ for biochemical diagnosis of (CKD-MBD) are shown in Tab. 6.4-4 – Diagnosis und monitoring of Chronic Kidney Disease-Mineral Bone Disease (CKD-MBD) according to KDIGO.

– Vitamin D deficiency /22/

1,25 (OH)2D (calcitriol) is the most important steroid hormone with regard to bone mineral metabolism. Its precursor, 25(OH)D (calcidiol), is absorbed enterally from diet or is synthesized in the skin. It is found in the circulation, bound to vitamin D-binding protein. The first step is 25-hydroxylation in the liver, followed by 1α-hydroxylation in the kidneys. The active hormone exerts its effect via vitamin D receptors. When 25(OH)D concentrations declines below 15 μg/L, PTH rises, in order that calcium concentrations can be maintained.

– Psoriasis

Patients with psoriasis have significantly higher PTH values than healthy individuals. In one study PTH was 42.3±1.8 ng/L in psoriatic patients compared with 23.4 ± 9 ng/L in the control group /23/.

Hypoparathyroidism – Generalized /24/

Hypoparathyroidism causes hypocalcemia because PTH secretion is inadequate to mobilize calcium from bone, to reabsorb calcium from the distal nephron, and to stimulate renal 25-OHD-1α-hydroxylase. In consequence little 1,25(OH)2D is generated resulting in decreased intestinal vitamin D absorption. Hypoparathyroidism can be congenital or acquired. The decrease in serum calcium level has to be evaluated and this is usually accomplished by the additional determination of phosphate, PTH and creatinine.

Laboratory findings: PTH is inappropriately low in patients with low iCa or albumin-corrected calcium (see Section 6.2 – Calcium). Phosphate levels are elevated or at the upper reference interval value. The determination of 25(OH)D is important in order to exclude vitamin D deficiency. The concentration of magnesium should also be determined because both hypermagnesemia and hypomagnesemia can cause hypocalcemia by inducing functional hypoparathyroidism. In hypomagnesemia PTH concentration is inappropriately low or near the lower reference interval value, in the presence of mild hypocalcemia. The reason is that magnesium is essential for both PTH secretion and for the function of the calcium-sensing receptor. In hypermagnesemia the parathyroid is unable to secrete sufficient PTH because the secretion is inhibited caused by a magnesium overdose or due to renal insufficiency.

– Acquired hypoparathyroidism

Acquired hypoparathyroidism is most commonly the result of thyroid or parathyroid gland surgery or neck dissection. It is present, by definition, if 6 months following the operation PTH secretion is not capable of maintaining normal plasma calcium concentration. The level of PTH is either undetectable or below 7 ng/L (0.7 pmol/L). Serum calcium is usually below 9.0 mg/dL (2.25 mmol/L) and phosphate above 4.5 mg/dL (1.45 mmol/L). Alcohol intoxication, aluminum toxicity and hypomagnesemia can lead to transient hypoparathyroidism.

– Immune-mediated hypoparathyroidism /25/

In idiopathic hypoparathyroidism autoantbodies against the calcium-sensing receptor (CaSR) play a role. The presence of CaSR autoantibodies in association with specific human leukocyte antigen DR haplotypes indicate organ-specific autoimmunity. Clinical symptoms are tonic-clonic seizures, calcification of the basal ganglia and cataract.

Laboratory findings: transient hypocalcemia, hyperphosphatemia, reduced or borderline low PTH values, normal 25(OH)D and magnesium, elevated ALP.

– Autosomal dominant hypoparathyroidism, familial hypercalciuric hypocalcemia, familial hypocalcemia

  • Familial hypercalciuric hypocalcemia: mutations in the CaSR or GNA11 genes lead to over activity in the respective protein. The altered CaSR protein is more sensitive to calcium, meaning even low levels of Ca can trigger it to stimulate Gα11 signalling. Mild hypocalcemia and hypomagnesemia, hypercalciuria, reduced or low-normal PTH.
  • Familial isolated hypoparathyroidism: mutations in the signal peptide of the CaSR which lead to lack of secretion of PTH.
  • X-linked hypoparathyroidism: deletions in chromosomes 2p25.3 and Xq27.1 lead to aplasia of the parathyroid glands.
  • Further disorders: DiGeorge syndrome, syndrome of hypoparathyroidism, deafness and renal anomalies, syndrome of hypoparathyroidism growth and mental retardation and dysmorphism.

Pseudohypoparathyroidism type I (PHPI) /26/

The PHPI is defined as PTH resistance that produces a compensatory increase of the PTH level, hypocalcemia, hyperphosphatemia. The disease is divided into the three groups PHPIa, PHPIb and PHPIc. In contrast to PHPIa and PHPIc, the defect in PHPIb patients is limited with PTH resistance to the proximal renal tubulus. Thus, these patients do not manifest the clinical picture of Albright’s hereditary osteodystrophia (AHO).

PHPI is caused by mutations in regulatory regions that lead to tissue-specific diminished transcription of the GNAS gene encoding the GNAS complex [stimulatory G protein α-subunit (Gsα)]. In addition alternative splice variants of GNAS gene seem to be involved in transcription regulation of and are transcribed from different promoter regions and alternative first exons. The Gsα is located downstream of the type 1 PTH/PTHrP receptor (see Section 6.2.3 – Biochemistry and physiology). The PTH-dependent regulation of calcium and phosphate homeostasis via the type 1 PTH/PTHrP receptor, which mediates its effect by cyclic AMP, is disturbed in PHPIa and PHPIc.

Pseudohypoparathyroidism type Ia (PHPIa)

Clinical findings: PHPIa, also called Albright’s hereditary osteodystrophia (AHO), is characterized by short stature, obesity, round face, brachydactyly, heterotopic calcification, mental retardation.

Laboratory findings: PTH resistance affects the proximal tubulus to the greatest extent. Phosphate secretion in the proximal tubules is decreased and hyperphosphatemia and hypophosphaturia result; the excretion of cyclic AMP is also reduced (see Section 6.6 – Vitamin D). Due to a reduced stimulation of 25-OHD-1α-hydroxylase, the synthesis of which is partially dependent upon the formation of cyclic AMP, serum concentrations of 1,25(OH)2D are also decreased (Tab. 6.4-5 – Biochemical and clinical findings in pseudohypoparathyroidism).

Pseudohypoparathyroidism type Ib (PHPIb)

This type is characterized by PTH-resistant hypocalcemia and hyperphosphatemia, without the clinical symptoms of AHO which are present in PHPIa. The hypocalcemia develops during the first decade of life. The changes in calcium and phosphate concentrations are dependent upon the magnitude of the PTH resistance. PTH-dependent mobilization of calcium and phosphate from bone appears to be normal, but is at times so marked that bone disease similar to that seen in hyperparathyroidism occurs; in these cases, therefore, the term pseudo-hypohyperparathyroidism (PHP-HPT) is used.

Laboratory findings: the diagnosis of PHPIb can be difficult based upon the clinical and laboratory findings. This is because the hypocalcemia is not yet present at birth or in early childhood and may only become apparent later in the form of convulsions. Furthermore, PHPIb and type II PHP have the same GNAS protein activity so that these two types cannot be distinguished by the measurement of cyclic AMP excretion. Molecular genetic diagnostics is therefore recommended /27/.

Pseudohypoparathyroidism type Ic (PHPIc) and type II PHP

These two variants of pseudohypoparathyroidism are characterized to a lesser degree than the types mentioned above. Patients with type II PHP, like those with PHPIb, do not manifest AHO. In the parathyroid hormone test they show normal cyclic AMP excretion and no phosphaturia.

Pseudo-pseudohypoparathyroidism (PPHP)

In PPHP the phenotypic features correspond to those of PHPIa. There are, however, no comparable disturbances of calcium and phosphate metabolism, and PTH is normal.

Table 6.4-3 Calcium (Ca), phosphate (P) and PTH in hyperparathyroidism




Clinical and laboratory findings

Primary hyperparathyroidism (pHPT)

P mostly below 3.5 mg/dL (1.13 mmol/L). Ca corrected on serum protein (for formula, see Tab. 6.2-2 – Protein correction of serum calcium); in more than 98% of cases, greater than 10.2 mg/dL (2.55 mmol/L).

Secondary hyperparathyroidism (sHPT) – Renal insufficiency

PTH should be regularly monitored when there is a decrease of the estimated GFR in stages G3b to G5 of CKD such as GFR below 44 [mL × min–1 × (1.73 m2)–1].

, n

– Malabsorption syndrome

25(OH)D resorption is decreased in cases with fat malabsorption and the formation of 1,25 (OH)2D is reduced.


Differentiation of the types is possible using molecular genetic investigations.

Table 6.4-4 Diagnosis and monitoring of Chronic Kidney Disease-Mineral Bone Disease (CKD-MBD) according to KDIGO /21/


Time interval

CKD stage G3a, in children stage G2: calcium, phosphorus, PTH, ALP

Calcium und phosphorus every 6–12 months, PTH based on baseline level and CKD progression.

CKD stage G4: calcium, phosphorus, PTH

Every 6–12 months, PTH every 6–12 months

CKD G5, including G5D: calcium, phosphorus, PTH

Every 1–3 months, PTH every 3–6 months

CKD stages 4–5D: ALP every 12 months, or more frequently in the presence of elevated PTH

Table 6.4-5 Biochemical and clinical findings in pseudohypoparathyroidism /24/























AHO, Albright’s hereditary osteodystrophy ; PHP Ia, pseudohypoparathyroidism type Ia; PPHP, pseudo-pseudohypoparathyroidism; PHP Ib, pseudohypoparathyroidism type Ib; Gsα, subunit of the guanyl nucleotide-binding protein; , decreased; , elevated; +, present; –, absent; N, normal

Table 6.5-1 Reference intervals for PTHrP


PTHrP (pmol/L)

Anti-PTHrP (1–34)

< 2,5 /2/



0.6–2.2 /3/

Two-site immunometric assay

Anti-PTHrP (1–74)

< 5,1 /4/

Anti-PTHrP (1–74)

< 1,5 /5/

Anti-PTHrP (1–84)

< 1,0 /6/

Anti-PTHrP (1–86)

< 0,23 /2/

Table 6.5-2 Diseases and conditions with increased parathyroid-related protein (PTHrP)

Clinical and laboratory findings

Malignant tumor

Solid tumors, which are often associated with elevated PTHrP concentrations are metastasized cancers of the breast, lung, kidney, bladder and esophagus. In one study /7/ PTH and PTHrP was measured in 123 hypercalcemic patients. 47 patients had tumor hypercalcemia and, of these, 15 had an elevated PTHrP concentration. The diagnostic sensitivity of PTHrP was 33% with a diagnostic specificity of 95%. The PTH level was the lowest in patients with elevated PTHrP. A PTH level above 26 ng/L (2.6 pmol/L) predicted a normal PTHrP concentration with a probability of 95% in the pathological PTHrP group and 100% in the overall hypercalcemia collective. The authors only give a low priority to the PTHrP measurement. In older studies /46/ the diagnostic sensitivity of PTHrP was 60–80%, depending on the test used. Many patients whose tumor is histologically a squamous cell carcinoma have elevated PTHrP values with hypercalcemia /4/. The PTHrP increase does not precede the hypercalcemia.

Malignant hematological systemic disease

PTHrP only plays a subordinate role with these hypercalcemias. The hypercalcemia with multiple myeloma is mostly triggered by local factors. According to a study /9/ PTHrP was increased in B-cell lymphoma when generalized lymphadenopathy had developed. PTH had a low normal concentration and 1,25(OH)2D was normal.

Primary hyperparathyroidism (pHPT)

In case of pHPT, PTHrP is normal. PTH is conclusive and its determination is always a priority in the event of hypercalcemia. PTHrP should only be determined if hypercalcemia is confirmed and PTH does not show the expected value.

Table 6.6-1 Nomenclature of vitamin D precursors and metabolites, modified according to Ref. /2/

Chemical name

Clinical name



Pro-vitamin D3

Cell membrane lipid component


Pre-vitamin D3

Present in diet or photosynthesized in skin


Pre-vitamin D2

Equivalent to vitamin D3 as precursor for active vitamin D

Calcidiol (25 (OH)D)

25-Hydroxyvitamin D

Reflects vitamin D status

Calcitriol (1,25 (OH)2D)

1,25 Hydroxyvitamin D

Active form of vitamin D

Table 6.6-2 Recommended cutoffs of serum 25(OH)D and reference values for 1,25(OH)2D3


Adults /8/
nmol/L (ng/ml)

Children /9/
nmol/L (ng/ml)


≥ 75 (30)

≥ 75 (30)


≥ 50 (20)


25–49 (10–19)

< 37.5 (15)


< 25 (10)

< 27.5 (11)


≥ 325 (150)

1,25 (OH)2D3

Reference interval

Reference interval

Less than 50 years



50 years and older



Pregnant women






Conversion 25 (OH)D: ng/ml × 2.5 = nmol/L

Conversion 1,25 (OH)2D: ng/L × 2.6 = pmol/L

Table 6.6-3 Diseases and conditions with modified 25(OH)D or 1,25 (OH)2D3 concentration

Clinical and laboratory findings

Healthy individuals /11/

Children up to 1 year of age who have experienced a second winter, women and children of immigrants with dark skin (melanin absorbs UV radiation), individuals who are confined indoors or who have been deprived of sunlight for longer than 8–12 weeks, often have a 25(OH)D deficiency. This also applies to many healthy individuals older than 50 in Central and Northern Europe in the months from January to April. The average 25(OH)D concentrations for elderly Europeans are around 8.4–22 μg/L (21–55 nmol/L) and in the USA around 28–34 μg/L (71–86 nmol/L). Europeans living in senior homes have values between 3.6 and 14.8 μg/L (9–37 nmol/L) and in the USA between 21.2 and 26 μg/L (53–65 nmol/L). In Germany, 57% of adults have deficient or severe deficient 25(OH)D levels and this also applies to as many as 75% of elderly people over 65 years of age. Deficiency is defined as ranging between 10 and 20 μg/L (25–50 nmol/L) and severe deficiency as ranging between 5 and 10 μg/L (12.5–25 nmol/L) /14/.

cHildhood: Vitamin D deficiency can be divided into genetic, prenatal or perinatal, and childhood causes. Genetic disorders alter the metabolism of vitamin D (e.g., deficiency in enzymes 25-hydroxylase or 1α hydroxylase). Prenatal and perinatal causes include maternal vitamin D deficiency, prematurity, and exclusive breast-feeding without vitamin D. Childhood cause include obesity, malabsorption, and low sun exposure, as well as decreased nutritional intake, which seems to be the mostly likely explanation /36/.

Risk of fracture in older individuals /11/

25(OH)D deficiency and calcium deficiency result in secondary hyperparathyroidism (sHPT) with increased bone turnover, accelerated bone loss and, especially in older adults, the deficiency leads to fractures, due to senile (type 2) osteoporosis. The bone turnover is increased with 25(OH)D concentrations of < 20 μg/L (50 nmol/L) and the density of the hip bones decreases with values starting below 12.5 μg/L (30 nmol/L). Only a 2-year vitamin D treatment can reverse such a condition. In cases of pseudo-fractures, Looser’s reconstruction zones or reduced mineral content of the bones, there is often a deficiency of 25(OH)D as well.

In a study low plasma 25(OH)D concentrations were associated with osteoporotic fractures; however Mendelian randomization analysis provided no evidence supporting a causal role for vitamin D in the risk of osteoporotic fractures /35/.

Fat malabsorption, celiac disease /19/

Reduced intestinal absorption, biliary cirrhosis, short bowel syndrome, or exocrine pancreas insufficiency can lead to a 25(OH)D deficiency. The resistance of such patients must be substituted. Young adults with 25(OH)D deficiency should undergo a screening investigation for celiac disease (e.g., with the determination of antibodies against tissue transglutaminase) (see Table 25.10-1 – Tissue transglutaminase antibodies).

Increased metabolism

The activation of microsomal P450 enzymes of the liver by barbiturates and anti epileptic drugs leads to a 25(OH)D deficiency. In particular, when anti epileptic drugs are taken, investigation should be made in winter to ensure that the concentration of 25(OH)D does not decline below 20 μg/L (50 nmol/L). An increased turnover of 25(OH)D also exists with primary hyperparathyroidism. In this case, an increased formation of 1,25(OH)2D and 24,25(OH)2D can cause a decrease in 25(OH)D.

Liver disease

In serious damage of liver parenchyma, the synthesis of 25(OH)D can be impaired. However, this is seldom the case because the 25-hydroxylation is scarcely restricted even in late stages of liver cirrhosis.

Chronic kidney disease (CKD)

CKD is characterized by glomerular and tubular sclerosis. The result is a reduction of the glomerular filtration rate (GFR) and reduction in renal parenchymal mass. These changes have the following effects on the homeostasis of calcium and phosphate /24/:

  • Reduced glomerular filtration of phosphate and impaired tubular responsiveness to hormonal influences leads to phosphate retention
  • Reduction of renal parenchyma leads to impaired synthesis of 25-OHD-1α-hydroxylase and thus failure to increase 1,25(OH)2D when required. The concentration of 1,25(OH)2D begins to fall at stage 3 CKD either as a direct result of phosphate retention or as a secondary effect of FGF23. Usually a decrease in 1,25(OH)2D becomes clinically evident at stage 4–5 CKD.
  • Reduced expression and responsiveness of the vitamin D receptor on the parathyroid glands which results in a compensatory increase of PTH in serum. The circulating concentration of 1,25(OH)2D can be maintained, but at the expense of increased parathyroid tissue activity. In late stage CKD, the concentration of 1,25(OH)2D is reduced if the patients are not treated with vitamin D.
  • Reduced expression of calcium sensing receptor on the parathyroid glands, which stimulates parathyroid hyperplasia and affects the normal feedback mechanisms for PTH release
  • Enhanced parathyroid cell proliferation under direct influence of hyperphosphatemia.

The deficiency of 1,25(OH)2D is an early complication in patients with CKD, because its concentration is continuously reduced with a decrease of GFR. The calcitriol deficiency is caused by insufficient renal-tubular reabsorption of 25(OH)D, inhibition of the 25-OHD-1α-hydroxylase due to FGF23 and hyperphosphatemia. For some patients, a 25(OH)D deficiency will cause an increase of 1,25(OH)2D deficiency. Due to 1,25(OH)2D deficiency, the feedback mechanism for PTH release is reduced, because the parathyroids have their vitamin D receptors regulated downward. This leads to increased secretion of PTH, a rise in serum PTH, and the formation of a polyglandular hyperplasia of the parathyroids. Through treatment with vitamin D, the PTH gene transcription is reduced and an increased expression of the vitamin D receptor and the calcium-sensing receptor of the parathyroids is stimulated. The parathyroids are thus sensitized for calcium and 1,25(OH)2D, which results in suppression of the parathyroidal hyperplasia and decline of the PTH secretion.

Laboratory findings: the stimuli of PTH production are hypocalcemia, hyperphosphatemia and the 25(OH)2D deficiency. Therefore, the KDIGO /13/ recommends regularly measuring 25(OH)D in stages G3a–G5D of CKD and treatment at stage GD5 requiring PTH-lowering therapy. Treatment with calcitriol, vitamin D analogues or calcimimetics is recommended in the event of rising PTH values in order to reduce them.

Dialysis patients /25/

Research on the survival rates of dialysis patients has shown that the mortality rate was significantly increased for patients with 1,25(OH)2D and 25(OH)D deficiency and no treatment with calcitriol, vitamin D analogues or calcimimetics, independently of calcium, phosphate and PTH.

Kidney transplantation

After renal transplantation, an increase in 1,25(OH)2D is often detected, with a well-functioning transplanted organ and normal 25(OH)D. The causes for this are increased PTH and hypophosphatemia (the result of persistent hyperparathyroidism). Increased 1,25(OH)2D can lead to remission of the polyglandular hyperplasia (desired) and to hypercalcemia (not desired).

Nephrotic syndrome

During nephrotic syndrome, transcalciferin bound 25(OH)D is excreted into the urine due to the molecular weight of 55 kDa. During peritoneal dialysis, transcalciferin trespasses into the dialysate together with vitamin D metabolites; in addition, 1,25(OH)2D, bound to albumin, is also lost.


In cases of sarcoidosis, other granulomatous diseases and lymphomas, the 1,25(OH)2D concentration may be increased. The reason for this is extrarenal 25-OHD-1α-hydroxylase synthesized in the granulomas (e.g., of the lungs). Depending on the available 25(OH)D the granulomas produce 1,25(OH)2D. Sarcoidosis patients are especially hypercalcemic during the summer.

Idiopathic infantile hypercalcemia /26/ (see also Section 6.2 – Calcium)

A genetic mutation in the CYP24A1 enzyme which is responsible for the degradation of 1,25 (OH)2D to 1,24,25(OH)2D causes the disease. The following values were found for one patient: fractional calcium absorption 37.4% (normally 22–27%), 25(OH)D 15–50 μg/L, 1,24,25 (OH)2D 0.62 μg/L (normally 3.49 ± 1,5 μg/L), calcium in serum 11.8 mg/dL (2.95 mmol/L).

Rickets/osteomalacia – Generally

In addition to the acquired vitamin D deficiency, congenital disorders of the vitamin D metabolism such as vitamin D-dependent rickets (VDDR) type 1 or type 2 can be the cause of osteomalacia.

If typical laboratory findings of VDDR are present (calcium and phosphate in serum decreased, urinary excretion of calcium and phosphate decreased, ALP increased) and 25(OH)D is normal, the 1,25(OH)2D should be determined /19/:

  • If 1,25(OH)2D is low and PTH elevated, the VDDR 1 (defect in 25-OHD-1α-hydroxylase) should be considered (determine creatinine in order to rule out any renal bone disease)
  • If 1,25(OH)2D is increased and PTH elevated, the VDDR 2 (end organ resistance, vitamin D receptor defect) must be considered.

– Vitamin-dependent rickets type 1 (VDDR 1) /27/

This disease, also known as hereditary pseudo vitamin D deficiency rickets type 1 (PDDR), is rare and of autosomal recessive inheritance. From a clinical standpoint, there is nanism and motor impairment.

– Vitamin-dependent rickets type 2 (VDDR 2) /27/

This disease, also known as hereditary hypocalcemic vitamin D-resistant rickets (HVDDR) is rare and the product of an individual genetic disorder, which is localized on chromosome 12 at 12q13–14. There is autosomal recessive inheritance. Alopecia is an important characteristic.

– Vitamin D-deficiency-rickets/osteomalacia /27/

In the case of vitamin D deficiency rickets, decreased, normal or increased concentrations of 1,25(OH)2D are measured, depending on the stage or exposure to UV light or vitamin D supply of the past few days.

Laboratory findings: in the case of serious vitamin D deficiency, 25(OH)D and 1,25(OH)2D are decreased. A supply of small quantities of vitamin D (e.g., 1,000 IU per day) for several days or a little UV light immediately lead [even with subnormal 25(OH)D] to increased formation of 1,25(OH)2D. The high 1,25(OH)2D concentration continues until the rickets is cured (e.g., more than 6 months). A vitamin D deficiency thus cannot be diagnosed from the measurement of 1,25(OH)2D. The determination of 25(OH)D must be requested for the diagnosis of vitamin D deficiency.

Diabetes mellitus (DM)

Patients with DM have a 25(OH)D deficiency more often than healthy individuals. This also applies to the metabolic syndrome. The β-cells of the pancreas have a vitamin D receptor and also activity of 25-OHD-1α-hydroxylase. Depending on the study, children with diabetes type 1 have a reduced 25(OH)D concentration in up to 43% of the cases. Since these patients have more fractures or bone fragility, a normal 25(OH)D concentration is important /28/.

High blood pressure /29/

Low concentrations of 25(OH)D and 1,25(OH)2D are independently associated with an up regulated renin-angiotensin-aldosterone system. Increased plasma renin in the setting of low 1,25(OH)2D may increase sympathetic activity and the intra-glomerular pressure, predisposing to arterial hypertension, a decline of GFR and subsequent cardiovascular disease.

Cardiovascular events

Both the Framingham offspring study and the Ludwigshafen risk and cardiovascular health study showed that low 25(OH)D concentrations are an independent predictor of cardiovascular mortality and fatal strokes.

Total mortality

1,25(OH)2D is an independent predictor of total mortality in patients admitted to a heart center. In a prospective cohort study /30/, patients with circulating 1,25(OH)2D concentrations below 17 ng/L (42.5 nmol/L) had approximately 4 times higher 1-year mortality risk than patients with 1,25(OH)2D concentration higher than 44 ng/L (110 nmol/L).

Colorectal carcinoma (CRC) /31/

Normal 25(OH)D values should reduce the risk of CRC and low values should increase the risk. As a preventive measure for older people in whom CRC and vitamin D deficiency are often associated, a 25(OH)D concentration > 32 μg/L (80 nmol/L) is considered.

Tobacco-related cancer

Chemicals in tobacco smoke affect the vitamin D metabolism and vitamin D can modify the carcinogenicity of these chemicals. Multi variable hazard ratios for tobacco-related cancers (lung cancer, head and neck cancer, bladder cancer, pancreatic cancer, stomach cancer, renal cancer, liver cancer) were, on average, 1.75 for 25(OH)D values < 5 μg/L in comparison to ≥ 20 μg/L /32/.

Total parenteral nutrition (TPN) /33/

To maintain a 25(OH)D concentration of 30–100 μg/L (75–250 nmol/L), TPN patients require a daily dosage of 3,000–4,000 IU of vitamin D3.

Poisoning by Vitamin D

Vitamin D poisonings are detected by the measurement of 25(OH)D. Vitamin D poisoning is caused by an increased concentration of 25(OH)D, which overrides the specificity of the 1,25(OH)2D receptor. When the 25(OH)D is increased, the value of 1,25(OH)2D is mostly normal, because hypercalcemia, hyperphosphatemia and suppressed PTH down regulate the 25-OHD-1α-hydroxylase, and thus, the formation of the 1,25(OH)2D is decreased.

Table 6.6-4 Findings for the various forms of rickets/osteomalacia









Vitamin D deficiency

, n,

e.g., UV deficiency







Renal P transport







Renal P transport

VDDR type I3

, ↓ ↓

, ↑↑




VDDR type II4

, n

, n



Vit. D receptor

Fanconi syndrome


, n




Renal P transport

1 Gender-related hypophosphatemia; 2 Autosomal dominant hypophosphatemic bone disease; 3 Autosomal recessive vitamin D-dependent rickets type I; 4 Vitamin D-dependent rickets type II (hereditary resistance to calcitriol). PTH, parathyroid hormone; P, phosphate; * 25-OHD-1α-hydroxylase.

n, normal; increased; ↑↑ greatly increased; decreased; ↓↓ greatly decreased.

Table 6.7-1 Least significant change (LSC) /8/


LSC (%)

N-terminal propeptide (P1NP)


Bone ALP




β-CTX (β-CrossLaps)


Tartrate-resistant phosphatase


Table 6.8-1 Reference intervals for bone ALP

ELISA (U/L) /1/



Immunometric assay (μg/L) /2/



Table 6.8-2 Findings in patients with CKD stages G4 to G5D in comparison to healthy controls /5/




Albumin (g/dL)

3.6 ± 0.6

4.2 ± 0.3

Calcium (mg/dL)

8.4 ± 1.1

9.8 ± 0.5

Phosphate (mg/dL)

6.3 ± 2.5

3.2 ± 0,8


0.34 ± 0.06

0.41 ± 0.08

PTH (ng/L)

324 (261–369)

53 (45–56)

tDPD (nmol/L)

62 (55–74)

35 (27–40)


75 (63–88)

49 (42–56)


46 (42–51)

26 (24–29)

25(OH)D (μg/L)

8.0 (5.3–11)

8.1 (5.8–11)

Values expressed as mean value ± SD or median and 95% CI, BMD, bone mineral density

Table 6.8-3 Incidence of bone metastases in advanced malignant tumors /11/

Carcinoma type

Incidence (%)

Multiple myeloma


Breast carcinoma


Prostate carcinoma


Thyroid carcinoma


Bladder carcinoma


Lung carcinoma


Renal cell carcinoma




Table 6.9-1 Comparison of osteocalcin with ALP




Primary HPT

Secondary HPT

Bone metastases

n or


  • high turnover

n or

  • low turnover

n or


Paget’s disease

n or

Rheumatoid arthritis

, (N), ()

Hepatobiliary disease


HPT, hyperparathyroidism; N, within the reference interval; , significantly increased; , significantly decreased

Table 6.9-2 Diseases with increased osteocalcin (OC) concentration

Clinical and laboratory findings

Primary hyperparathyroidism (pHPT)

Increased levels of OC are found in almost all patients with pHPT. Usually there is a positive correlation with the ALP. The OC concentration also correlates with the concentration of serum calcium, parathyroid hormone, and the adenoma weight of the parathyroids /5/.

Secondary hyperparathyroidism (sHPT)

Markedly elevated OC values, which are in part due to a reduced renal elimination of OC if the glomerular filtration rate is < 30 [mL × min–1 × (1.73 m2)–1/6/. If changes in renal elimination are taken in consideration, the OC determination may be useful in patients with renal osteodystrophia (chronic hemodialysis patients) as well as for the assessment of the bone turnover in kidney transplant recipients /7/.

Cancer with bone metastases

OC always indicates metastatic bone involvement by increased concentrations in tumor patients. The activity of the ALP often shows a parallel course to OC. In cancer patients with soft tissue metastases, the values for OC and ALP are within the reference interval.


The usefulness of the OC determination in patients with osteoporosis is differently assessed by various authors. In osteoporotic patients with normal bone turnover, there is no difference to a control group of the same age with healthy bone. A significantly decreased OC level in the serum is found in patients with low turnover osteoporosis in whom reduced bone formation is histomorphometrically detected /89/. Patients with high turnover osteoporosis have OC values in the upper reference interval or increased values.


Both the values for OC as well as ALP are increased in cases of osteomalacia (adults) or rickets (children). After vitamin D is administered, the serum concentration declines usually following a transient increase.

Paget’s disease

For Paget’s disease, OC only has limited diagnostic value /10/. The values are usually elevated. In comparison, ALP increase is considerably higher. Treatment with calcitonin or bisphosphonates also results in a decline of ALP, and in most cases, of OC as well.

Rheumatoid arthritis (RA)

Patients with RA often have significantly lower levels than patients in control groups. The administering of glucocorticoids makes the assessment of the OC concentration more difficult. In contrast, the ALP is mostly only moderately increased. Increased OC values in patients with RA have also been described, however /11/.

Hepatobiliary disease

Since the OC is synthesized in the osteoblasts, these diseases are always associated with OC levels in the reference interval whereas the ALP activity is increased.

Table 6.11-1 Reference intervals for pyridinolines

Women /3/
(μg/g creatinine)



  • premenopausal



  • postmenopausal



Women /4/
(nmol/mmol creatinine)

  • premenopausal

45 ± 13

9.6 ± 2.6

Men /4/
(nmol/mmol creatinine)

  • 20–60 years

45.2 ± 17.1

9.5 ± 3.6

Values are 5th and 95th percentiles in Ref. /3/ and x ± s in Ref. /4/. Determination with HPLC after acid hydrolysis. The conversion factor of PYD μg/g creatinine in μmol/mol creatinine is 0.263. The conversion factor of DPD μg/g creatinine in μmol/mol creatinine is 0.278.

Table 6.11-2 Pyridinolines in diseases with increased bone resorption and during treatment


Clinical and laboratory findings

Postmenopausal /6/

Detection of increased bone resorption

Increased in 40% of post menopausal women

Children /1516/

Enhanced growth rate or growth hormone therapy; Ehlers-Danlos syndrome type VI

Depending on the growth rate, the tPYD/tDPD ratio (normal 4 : 19 is reversed (1 : 4)

Primary hyperpara­thyroidism /17/

Estimation of bone involvement

Significant increase in tPYD and tDPD. Within 2 days following parathyroid ectomy. The excretion of tDPD decreases on average 21–15 μmol/mol creatinine

Secondary hyperparathyroidism /18/

β-CTX and TRAP 5b and ALP may be superior to pyridinolines

Excretion of tPYD and tDPD in urine is not recommended

Osteomalacia /13/

After vitamin D treatment, a sharp decrease of β-CTX, less of tDPD, no decrease of fDPD

Patients with vitamin D deficiency and increased PTH show an increase in tPYD and tDPD

Bone metastases /10/

Increased excretion in cases of breast and lung carcinoma in approximately 90% of cases

tPYD and tDPD excretion depends on the severity of the disease

Tumor patients without radiologically verified bone metastases /10/

Increased values for breast and lung carcinoma in approximately 70% of cases (e.g., due to PTHrP)

tPYD and tDPD excretion depends on the extent of the metastasis

Paget’s disease

Determination of pyrodinolines not required, ALP is the leading marker

tPYD and tDPD excretion depends on the severity of the disease

Transplantation patients

DPD and ALP are important, because glucocorticoids slow the bone formation (low bone ALP) and increase the degradation

tDPD increased or near the upper reference interval value

Hyperthyroidism /6/

Elevated tDPD/creatinine ratio, because of decreased urine creatinine

tDPD and tPYR increased

Rheumatic diseases

tPYD excretion correlates with biochemical variables of inflammatory activity and is increased as a marker for cartilage remodeling; tPYD/DPD ratio above 6 : 1; tDPD can also be elevated with increased bone degradation

tPYD and tDPD correlate with the activity of the imflammation markers

Estrogens (treatment monitoring)

Treatment monitoring of estrogens

Determination after 6 months of treatment; β-CTX shows an even more marked decrease than tDPD

Already 25 μg transdermal estrogen daily normalize the tDPD excretion

Bisphosphonate (treatment monitoring) /6/

Treatment monitoring of bisphosphonate /6/

Determination after 1 month after start of intravenous administration and 3 months after oral administration; β-CTX shows an even more marked decrease than tDPD

Suppression of bone resorption

Calcitonin (treatment monitoring)

Treatment monitoring of calcitonin

Decrease of tDPD after 1 day

Only a slight decrease of tDPD

Table 6.12-1 Reference intervals for β-CTX

β-CTX (β-CrossLaps) in the EDTA plasma


0.1–0.6 μg/L /3/


0.299 ± 0.137 μg/L /3/


0.556 ± 0.226 μg/L /3/

Table 6.12-2 β-CTX in diseases with increased bone turnover /45/

Clinical and laboratory findings

Increased regular bone degradation

In women from age 25, there is uniform bone loss, with a β-CTX upper reference interval value of 0.6 μg/L. After menopause, more than a third of the women exhibit increased bone loss and the β-CTX values continuously increase, which means that the concentration in the serum is 50–100% higher in comparison to pre-menopausal levels. But the β-CTX levels of women with hormone substitution are below 0.6 μg/L (Fig. 6.12–3 – Age of the women and β-CTX level in the EDTA plasma).

The values are the highest in men aged 20–30 and continuously decrease until age 60 and then behave indifferently there after. The seasonal variations in the concentration for both genders is 20–30%, with a higher serum level in winter time.


Since osteoporosis is a heterogeneous disease, inter individual bone turnover can vary greatly. There is usually a large overlap of the values between normal, osteopenic and osteoporotic individuals. In a study involving 800 elderly women, serum β-CTX was the best discriminator for differentiating these three groups of people /6/. Pre-menopausal women over 45 years of age with increased β-CTX levels showed significantly greater bone loss in the later stage of life than those with lower values /7/. Generally speaking, high or increased β-CTX levels are associated with increased bone loss.

Bone turnover and risk of fracture

Bone mass, rate of bone loss, and the risk of osteoporotic fracture are interrelated, and both low bone mass and rapid bone loss are independent predictors of future fracture risk. Fractures are the main complications of osteoporosis and the β-CTX level can be an indicator. This not only applies to patients with post menopausal osteoporosis, but generally to patients of any age, regardless of their bone mineral density and previous fractures /8/. In the prospective OFELY study /9/, post menopausal women in the highest quartile of the β-CTX concentration, had a 2-times greater risk of fracture within 5 years than those in the lower quartiles.

In the EPIDOS study, women with a femur bone density below 2.5 SD and increased β-CTX had an increased risk of hip fracture (odds ratio 4.8) /10/. In general, older women with increased concentrations of β-CTX had an increased fracture risk.

Pre-treatment bone turnover and therapeutic effect

The β-CTX level prior to intervention can predict the improvement of bone mineral density (BMD) under treatment. In a study /11/ the β-CTX concentrations of early post menopausal women were divided into quartiles. Patients under hormone replacement treatment with values in the highest quartile showed a significantly sharper increase of BMD than those in the lower quartiles.

For patients who were treated with hPTH (1–34), more fractures were prevented with higher β-CTX concentrations /12/.

Monitoring of the anti-osteoporosis treatment /8/

Changes in the β-CTX level under anti-resorptive treatment or hormone replacement therapy give information about whether patients benefit from the treatment and whether there is compliance. They also reflect the mechanism of the effect of the medication, help in finding the effective dosage and make a statement about whether the treatment is effective on the bone. For example, enteral reabsorption can be problematic during oral bisphosphonate treatment. A decrease of the β-CTX level provides assurance that the substance was resorbed and is effective on the bones. Thus, anti-resorptive treatment with bisphosphonate (alendronate) with a reduction of the β-CTX level by 20% indicates an increase of bone mineral density (BMD) in the hips and spine. Patients undergoing hormone replacement treatment, whose β-CTX levels are below 60% of the initial value 3 months after the start of the treatment, are likely to experience an BMD increase of 2–3% within 3 years. But up to 50% of post menopausal women undergoing hormone replacement treatment do not carry this out correctly, as seen in studies over a period of 1–5 years. If the treatment is carried out correctly, the β-CTX concentration decreases within 3–6 months.

Paget’s disease

The concentration of β-CTX is increased. The ALP is more cost-effective for assessing the progress and treatment of this disease.

Renal osteodystrophy

The concentration of β-CTX is increased, the increase correlates to the serum creatinine value.

Bone metastasis /13/

Bone scintigraphy is the gold standard for diagnosing metastatic bone disease. The diagnostic sensitivity and specificity of the resorption markers, and thus, of β-CTX is insufficient and there is a considerable overlap of the values of patients with and without bone metastasis. With the presence of metastases, the β-CTX convey prognostic information about expected, negative skeletal events such as fractures, however. β-CTX also provides information about the reduction of such a risk under bisphosphonate treatment. The β-CTX is generally increased 2–7 times in cases of metastatic bone disease and decreases 60–80% under bisphosphonate treatment. Other treatment regimes such as androgen ablation or estrogen substitution do not lead to a change of the β-CTX concentration /14/.

Figure 6.1-1 Sequence of bone remodeling, modified according to Ref. /1/. 1. Resting bone with lining cells that contain osteocytes. 2. The lining cells recede and the underlying membrane is removed by metalloproteinase. 3. Osteoclasts are recruited and activated followed by fusion to become multi nucleated osteoclasts. 4. Osteoclasts digest the underlying bone forming a resorption cavity. 5. Osteoblasts are recruited to the cavity. 6. Osteoblasts lay down new osteoid, which is then calcified.

2 3 4 5 6 Osteoclast 1 Bone Lining Cells Osteoblast

Figure 6.1-2 Osteoblast differentiation starting from the stem cell (SC), modified from Ref. /1/. The stem cell differentiates into the osteoblast (OB) via the mesenchymal stem cell (MSC) and the pre-osteoblast (PreOB). Paracrine, endocrine and autocrine factors with a stimulating effect: endothelial growth factor, EGF; insulin growth factors, IGFs; bone morphogenetic protein-2, BMP-2; transgrowth factor-β, TGF-β; vascular endothelial growth factor, VEGF; fibroblast growth factor-2, FGF-2; parathyroid hormone, PTH.

SC MSC PreOB OB EGF Insulin-IGFs BMP-2TGF-β1/2IGF1 VEGFFGF-2 Runx2Wnt/β-Katenin PTH IGF1/2 Prostaglandins IGF1 1.25 (OH) 2 D 3 DKK1

Figure 6.1-3 Mechanisms of osteoclast activation by cytokines, macrophage colony-stimulating factors (M-CSF) and Receptor Activator of Nuclear factor-kappa B Ligand (RANKL) formed by osteoblasts and stromal cells. Osteoprotegerin (OPG) is an inhibitor of osteoclast formation.

M-CSF OPG RANK RANKsignaling pathway Fusion anddifferentiation Activeosteoclast RANK-L Osteoblast/ stromal cell 00

Figure 6.1-4 Collagen synthesis from two α1-chains and one α2-chain. Markers of the collagen formation are carboxy-terminal pro collagen type I pro peptide (PICP) and N (amino)-terminal pro collagen type I pro peptide (PINP). Collagen degradation and the markers of collagen resorption (CTX, C-terminal cross links; NTX, N-terminal cross links) are shown. With kind permision from Ref. /16/.

α1(OH) α2(OH) α1(OH) Procollagen Procollagen type I N-terminal propeptide (= PINP ) N-Telo- peptide (NTX) C-Telo- peptide (CTX) Helical area Tropocollagen Procollagen type I C-terminal propeptide (= PICP ) Procollagen peptidase

Figure 6.1-5 Bone formation and bone resorption markers, modified according to Ref. /16/.

FormationOsteocalcinAP, BAPCollagenpropeptides(PICP, PINP) Osteoblast Matrix Osteoclast ResorptionOsteocalcinBSPTRAPHydroxyprolineCalcium (U)Hydroxylysine glycosidesPyridinium crosslinksCrosslinked telopeptides(NTX, CTX)ICTP

ALP, alkaline phosphatase; BALP, bone ALP isozyme; BSP, bone sialo protein; CTX, C-terminal crosslinks; ICTP, collagen type 1 C-terminal telopeptide; NTX, N-terminal crosslinks; PICP, carboxy-terminal procollagen type 1 propeptide; PINP, N-terminal procollagen type 1 propeptide; TRAP, tartrate-resistant acidic phosphatase.

Figure 6.2-1 Relationship between calcium excretion in the 24-hour urine and dietary intake of calcium /41/. The daily calcium intake is displayed on the abscissa and the calcium excretion is displayed on the ordinate. 10 mmol corresponds to 400 mg.

Resorptive + absorptivehypercalciuria Resorptive hypercalciuria Absorptive hypercalciuria Mean mmo l Dietary calcium 25.0 20. 0 15.0 10.0 7. 5 5. 0 2.5 Urinary calcium (mmol/24 h) 2. 5 5.0 7.5 10.0 12.5 15.0

Figure 6.2-2 Differentiation of hypercalciuria using the calcium/creatinine ratio. The secondary hypercalciuria must first be ruled out. With kind permission from Ref. /40/. PTH, parathyroid hormone.

Normal serum Caund Ca/Creatinine-ratio in urine (mmol/mmol) Normal PTH and serum Ca Increased PTH and normalor low serum Ca Fasting hypercalciuria undetermined pathology Renal hypercalciuria Absorptive hypercalciuria < 0.57 ≥ 0.57 Fasting-hypercalciuria

Figure 6.2-3 The maintenance of calcium homeostasis.

150–200 mg 200 mg 1,000 mg 800 mg 1.2–1.4 kg(> 99%)Bone Small intestine Plasma 200 mg Kidney Fast interchangeable pool 1200–1400 mg (< 1%)Extracellularfluid

Figure 6.2-4 Gs-protein-mediated signal transmission of the parathyroid hormone sensing receptor (PTHSR) or of the calcium-sensitive receptor (CaSR), modified according to Ref. /28/. The Gs-protein acts in the cellular signal transmission as an on-off switch.

1) The Gs-protein consists of the subunits α, β and γ. In the resting state, all three proteins are non-covalently bound to each other and the α-unit has bound GDP. The switch position is “Off”.

2) If the membrane-bound PTHSR or CaSR is activated by the binding of PTH or calcium (Ca), it interacts with the Gs-protein. This results in the dissociation of GDP from the receptor and GTP is bound.

3) The binding of GTP leads to the dissociation of the Gs protein in the α- and βγ-subunits. The α-unit binds to the effector, which possesses adenylate cyclase activity and cleaves ATP into cyclical AMP and Pi. The switch position is “On”.

4) The α-unit (a guanosin triphosphate), which is bound to the effector, hydrolyzes GTP to GDP. This inactivates the α-subunit and allows it to combine again with the βγ-subunit. Hence, the switch is in “Off” position.

Overall, by this process, PTH or Ca, the “first messengers”, release cyclic AMP, the “second messenger” and thus transmit a signal into the cell.