In physiological body fluids ammonia/ammonium has a pKa of 9.05 at 37 °C. At physiological pH approximately 99% of ammonia is present in blood as NH4+ cation in the circulation. An alkaline blood pH favours the formation non-dissociated NH3 gas, more acidic pH the formation of NH4+. The NH4+ cation diffuses or is transported more efficiently than NH3 into the tissues . Ammonia is derived mainly from deamination of the α-amino nitrogen of amino acids and is toxic at elevated concentrations. Most of the daily ammonia load comes from the digestion of dietary proteins and the bacterial synthesis of ammonia in the intestine. Under normal conditions, deamination of glutamine to glutamate in the liver releases ammonia, which then is converted to the non-toxic nitrogen-rich compound urea, which is subsequently excreted into the urine.
Neurological manifestations in patients:
- With liver cirrhosis
- With liver failure of different origins (alcohol-toxic, infectious)
- On aggressive chemotherapy
- On treatment with valproic acid
- With severe gastrointestinal hemorrhage
- With a portocaval/portosystemic shunt.
Suspected congenital metabolic disorder in newborns, children and, less commonly, adults.
Principle: in the presence of NADPH2, ammonia is transferred to 2-oxoglutarate by glutamate dehydrogenase (GLD), forming glutamate and NADP. The decrease in NADPH2 is measured. The decrease in absorbance at 334 or 340 nm is proportional to the ammonia concentration in the reaction mixture.
Principle: NH4+ cations of the sample are converted to NH3 (gas) in an alkaline buffer. The released gas diffuses through the pores of a membrane into the inner NH4Cl containing solution of a pH electrode. Ammonia is directly measured via the pH increase of the inner electrode.
In adults, hyperammonemias are usually associated with liver failure, while in children they mostly result from hereditary defects of enzymes of the urea cycle . Hyperammonemias cause neurological manifestations, which are characterized by hepatic encephalopathy and cerebral edemas of varying severity. The edemas are usually localized in the cerebral cortex, but also affect the white matter to a certain extent. In these patients, diminished urea synthetic capacity causes impairment of the physiological route of ammonia detoxification, leading to elevated ammonia levels with neurotoxic effects to the central nervous system. Hepatic encephalopathy is, however, not caused by the diminished urea synthetic capacity, but acts synergistically with systemic inflammation, hypoxia due to reduced cerebral blood circulation, and metabolic disorders that are not associated with ammonia genesis. Initial clinical symptoms are loss of appetite, vomiting and hyperventilation, later followed by lethargy, encephalopathy, and seizures. A progressive increase in ammonia to 5–6 times the upper reference interval value leads to cerebral edema, coma, and possibly death. In patients with hyperammonemia and hepatic encephalopathy, the duration of the hyperammonemia determines the reversibility of the cerebral pathology as well as the prognosis.
The rate of ammonia synthesis and the concentration in arterial blood are determined by two factors:
- The balance of protein anabolism and catabolism
- The integrity of the urea cycle.
The ammonia derived from the deamination of amino acids is detoxified through the synthesis of glutamine and urea. Glutamine is produced in all tissues, in particular muscle, by the transfer of ammonia to the α-ketoglutarate formed in the citric acid cycle. The α-ketoglutarate transports ammonia from the periphery to the liver by the incorporation of potentially two ammonia molecules. In the liver, the ammonia molecules are involved in the synthesis of urea or amino acids. Due to the large capacity of the urea cycle, liver diseases usually have no impact on this process.
Hereditary hyperammonemias are caused by disorders classified as neurometabolic diseases, a large group of heterogeneous diseases which primarily affect the central nervous system () . Their cumulative incidence is 1 in 500 individuals. Neurometabolic diseases can be caused by an enzyme defect of the urea cycle or can occur secondary to organic aciduria or fatty acid oxidation disorders. In children aged up to 2 years, clinical symptoms of hereditary hyperammonemias often appear as early as in the first weeks of life. A second peak of incidence occurs at age 12 to 15 years, at the end of puberty, when the growth rate declines.
Suspected manifest encephalopathy in hereditary hyperammonemia is diagnosed based on clinical symptoms. In children these are nausea, vomiting, disorientation, seizures, and lethargy. Newborns refuse food, and infants and older children have anorexia and ataxia. It must be noted that genetic urea cycle disorders can occur at any age.
There are also documented cases of neurometabolic diseases in older age groups. The classification of hereditary hyperammonemias is based on the biochemical substances produced or metabolized, or the enzyme defect that is present.
- Urea cycle disorders (cumulative incidence 1 : 20,000; the most common defect is ornithine transcarbamylase deficiency) .
- Amino acid transport disorders such as hyper ornithinemia-hyperammonemia-hypercitrullinemia (HHH) syndrome and lysin uric protein intolerance (LPI)
- Organic acidurias (cumulative incidence 1 : 10,000) such as propionic aciduria and methyl malonic aciduria
- Fatty acid oxidation disorders (e.g., acyl-CoA dehydrogenase deficiency)
- Mitochondriocytopathies with disorders of energy metabolism (e.g., respiratory chain defects, pyruvate carboxylase deficiency)
- Hyperinsulinism-hyperammonemia (HI/HA) syndrome.
Before considering a diagnosis of a hereditary disorder in newborns or infants, acquired hyperammonemias must be excluded (e.g., liver insufficiency, portosystemic shunt, liver bypass, Reye’s syndrome, valproic acid therapy, hypovolemic shock, transient neonatal hyperammonemia, neonatal Herpes simplex virus infection, perinatal asphyxia, cardiac insufficiency, sepsis, infection with urea-splitting bacteria).
It is important to determine several biomarkers, since ammonia levels can be below 100 μg/dL (60 μmol/L) or only slightly elevated during the asymptomatic interval of neurometabolic disorders. Increased protein intake or extreme physical stress can also lead to a physiologically elevated concentration of ammonia. The following assays are recommended:
- Blood gas analysis; hyperammonemia with metabolic acidosis is more likely to be due to organic aciduria than an urea cycle disorder, although acidosis can also develop in the latter. However, urea cycle disorders are much more frequently associated with alkalosis than with acidosis.
- Blood glucose; if levels are normal, HI/HA syndrome can be excluded
- Ketone bodies in urine; ketonuria is more suggestive of organic aciduria
- Serum urea; although the serum urea concentration is often low in urea cycle disorders, it is not a sensitive and specific parameter
- ALT, prothrombin time and albumin are important for excluding acute liver failure.
Special investigations in suspicion of hyperammonemia and neurometabolic disorders
In many cases, a general differentiation of disorders is possible by measuring amino acids and organic acids in urine and/or, if necessary, plasma (). For final differentiation, the activity of the enzyme suspected to be deficient is measured and molecular genetic assays are carried out.
Genetic urea cycle defects can occur in every age. In all defects, alanine and glutamine are elevated.
The incidence of ornithine transcarbamylase (OTC) deficiency with an estimated incidence of approximately 1:40,000 is the most common disorder in urea genesis. The plasma ammonia concentration frequently exceeds 1,700 μg/dL (1,000 μmol/l). The chromatogram of organic acids shows a large peak of orotic acid. Children with severe OTC deficiency typically present after 24 h with irritability, poor feeding, progressive lethargy, and seizures progressing to hyperammonemic coma .
In citrullinemia (argininosuccinate synthetase deficiency), the citrulline concentration is above 1,500 μmol/L. In ornithine transcarbamoylase (OTC) and carbamoyl phosphate synthetase (CPS) deficiency, the citrulline and arginine concentrations are reduced.
Argininosuccinase deficiency is characterized by high urinary clearance of argininosuccinate with only slightly elevated levels in plasma.
In arginase deficiency, the concentration of arginine in plasma and urine is significantly elevated.
It is not possible to differentiate OTC, CPS and N-acetyl glutamate synthetase (NAGS) deficiencies from other urea cycle defects based on the amino acid profile. It can, however, be done using the allopurinol loading test, because in all urea cycle enzyme defects, except CPS and NAGS deficiency, there is increased formation of orotic acid from carbamoyl phosphate due to impaired metabolism of the latter in the urea cycle (). Allopurinol, or its metabolite oxipurinol ribonucleotide, inhibit orotidine mono phosphate decarboxylase, resulting in increased orotic acid in urea cycle enzyme defects. This is, however, not the case in NAGS or CPS deficiency. The allopurinol loading test is especially useful for differentiating between OTC, CPS and NAGS deficiency .
Allopurinol loading test
Allopurinol is administered as a single dose (adults 300 mg), and after 6 hours urine is collected over 24 hours. Increased excretion is indicative of OTC deficiency and rules out CPS- or NAGS deficiency. The test has a high specificity for urea cycle defects and is also used for identifying asymptomatic heterozygous carriers of OTC deficiency.
Increased clearance of the respective amino acids is found in HHH and LPI syndrome (). Excretion of orotic acid may also be increased. In HHH syndrome, hyperornithinemia may be absent if protein intake is low. In LPI syndrome, the plasma concentration of the dibasic amino acids, except cysteine, may be only slightly elevated or even normal. Therefore, measurements in spontaneous morning urine are important in the diagnosis of these disorders .
The measurement of organic acids in urine and plasma is performed using gas chromatography mass spectrometry. Relatively common organic acidurias/acidemias include methylmalonic aciduria, propionic acidemia, and isovaleric acidemia.
In disorders of fatty acid oxidation, β-oxidation is activated in the catabolism of fatty acids, producing dicarboxylic acids such as adipic acid, sebacic acid and suberic acid, which are excreted in urine . High excretion of dicarboxylic acids in combination with diminished or absent excretion of ketone bodies indicates a disorder of fatty acid oxidation. The dicarboxylic acid pattern also provides information as to whether the acyl-CoA dehydrogenases which metabolize short-chain or long-chain fatty acids are affected. For example, the presence of hydroxy dicarboxylic acids indicates a deficiency of 3-hydroxy-acyl-CoA dehydrogenase, which metabolizes long-chain fatty acids.
Acquired hyperammonemias are predominantly of hepatic origin and caused by severe liver disease, usually liver cirrhosis. The latter leads to reduced synthesis of urea and glutamine, which is further compromised by the increased production of ammonia induced by the catabolic state. In liver cirrhosis, there is a combination of metabolic (reduction of the liver parenchyma) and hemodynamic components (portocaval/portosystemic shunts). Ammonia-containing blood reaches the systemic circulation directly from the intestine, bypassing the liver.
Acquired hyperammonemias can cause hepatic encephalopathy. About 5–6 fold elevations of blood ammonia [concentrations above 300 μg/dL (176 μmol/L)] will produce coma, however the brain will not develop tolerance for repeated exposure to ammonia. The persistent or episodic hyperammonemia can be expected to exact a permanent toll on brain function . Hyperammonemic encephalopathy is a clinical syndrome characterized by abnormal mental status and neuromuscular manifestations, such as asteriks, tremor, opthalmoplegia, incoordination, and incontinence. Latent encephalopathy is diagnosed by psychomotor tests such as linking numbers and arithmetic exercises and measuring reaction time . Other causes of acquired hyperammonemia are rare. They are listed in .
The term hepatic encephalopathy (HE) covers a multitude of potentially reversible symptoms ranging from discrete neuropsychiatric abnormalities to coma . HE is a secondary, metabolic, cerebral and neuromuscular disorder which is caused by chronic liver disease or acute, severe failure of the liver parenchyma. To differentiate HE from other encephalopathies, the following conditions must be present :
- Fulminant hepatic failure, also known as acute HE or type A HE. In this acute type of HE, a cerebral edema develops, with the associated symptom of intracranial pressure.
- Surgical or spontaneous portocaval shunt without underlying liver disease (type B HE).
- Liver cirrhosis with the signs of functional impairment or portal hypertension (type C HE). This type of HE is subcategorized into an episodic (triggered by precipitating factors), a persistent, and a minimal form.
The hyperammonemic HE results from the reduced capacity of the liver to synthesize urea and glutamine. Ammonia from endogenous protein metabolism and from enteral bacterial breakdown of protein is insufficiently metabolized by the liver. This occurs in liver diseases with extensive reduction of functional liver parenchyma such as liver cirrhosis and acute liver failure with severe impaired function of parenchymal tissue.
With a portocaval shunt, intestinal blood bypasses the liver and reaches directly the systemic circulation. In shunt-operated patients, this occurs via portocaval or splenorenal shunts. In acute liver failure intrahepatic shunts allow blood to circulate unchanged from the portal vein to the hepatic vein.
Triggering factors of HE in patients with liver cirrhosis and portal hypertension include:
- Gastrointestinal hemorrhages (esophageal varices)
- Alkalosis, hypophosphatemia
- Dietary problems (high protein intake, alcohol, obstipation, vomiting, diarrhea, surgery)
- Sedatives, hypnotics, diuretics
- Acute and chronic infections, in particular in conjunction with long-term treatment with corticosteroids
- Insufficiently controlled use of diuretics (carbonic anhydrase inhibitors).
Causes of acute liver failure with HE include:
- Acute viral hepatitides
- Toxic hepatitides (e.g., from amanita phalloides, acetaminophen, industrial solvents, other hepatotoxic substances)
- Acute drug-induced toxic hepatitis (tricyclic antidepressants, NSAID, INH, halothane)
- Hypoxic liver failure (acute right heart failure)
- Other causes such as Budd-Chiari syndrome, acute gestational fatty liver, severe malignant liver infiltration, autoimmune hepatitis. Acquired hyperammonemias are listed in .
Increased GGT activity can increase the rate of ammonia formation in plasma manyfold the mean value of healthy individuals. In samples with elevated GGT the glutamate is cleaved, producing NH3. With a GGT level of 1,000 U/L, glutamate cleavage activity is 35 times higher compared to activity in the reference range . Blood specimens anticoagulated with dipotassium EDTA should contain 5 μL of sodium borate (0.4 mol/L) (pH 7) and 50 μL of L-serine solution (0.1 mol/L) per 1 mL blood, for prevention of glutamate cleavage.
Hemolysis causes falsely elevated levels, since the ammonia concentration is approx. 3 times higher in the erythrocytes than in plasma. Approximately 75% of the ammonia in whole blood is found in erythrocytes.
Levels in capillary blood are higher than those in arterial blood. No differences are observed in arterial and venous plasma of resting patients without liver disease. Blood collection after physical work leads to elevated levels in venous plasma .
For transportation to the laboratory blood samples must be cooled in ice-water and transported within 15 minutes. If it can be ensured that the transport temperature will not exceed 20 °C, the specimen may be transported without ice after being briefly cooled in ice beforehand. A temperature of –30 °C allows long-term storage of the plasma without an increase in ammonia levels .
Oxidation of fat and carbohydrates yields as the only end products CO2 and H2O, both of which are eliminated via the lungs and kidneys. Oxidation of proteins additionally leads to a formation of HCO3– and NH4+ . Complete oxidation of protein, however, yields HCO3– and NH4+ in stoichiometric amounts. In man, ingestion of an average of 100 g protein per day results in the daily formation of about 1 mole each of HCO3– and NH4+. Such high amount of HCO3– cannot be excreted via the kidneys in view of the limited urine volume. The major pathway for metabolically generated HCO3– is hepatic urea synthesis in the liver consuming HCO3– and NH4+ in the same stoichiometry as they are produced during protein breakdown:
Thus, the formation of approx. 30 g of urea per day leads to the elimination of 1 mole of the strong base HCO3– (pK 6.1) and 1 mole of the weak acid NH4+ (pK 8.9). This mechanism contributes significantly to stabilizing the body’s pH level. NH4+ can also be disposed by glutamine formation that occurs in the liver and other organs, and NH4+ can be excreted into urine after renal hydrolysis of glutamine . Therefore, the route of nitrogen disposal by either urea or glutamine synthesis determines the rate of HCO3– removal and the liver becomes an important organ in acid-base homeostasis.
Ammonia is formed in all tissues and is toxic in particular for the nerve tissue. Physiologically, most of it is eliminated via the liver and a small amount of it via the kidneys . The ammonia released in the tissues is detoxified by converting it to glutamine for transport to the liver. Glutamine is produced in the tissues from glutamate and ammonia in a reaction catalyzed by the enzyme glutamine synthetase.
The plasma concentration of glutamine is approximately 700 μmol/L, which is high compared to the other amino acids. Another form of transport for ammonia is alanine. In this process alanine transaminase and glutamate transaminase catalyze the transfer of amino groups from most amino acids to form L-alanine from pyruvate or L-glutamate from α-ketoglutarate (see ). Since L-alanine is also the substrate for glutamate transaminase, all the amino nitrogen from amino acids that undergo transamination can be concentrated in glutamate.
The glutaminase- and GLD-mediated reactions occur in the mitochondria of the hepatocytes. The intramitochondrial concentration of glutamate determines the rate of formation of N-acetyl glutamate and urea. The first step in urea synthesis, the formation of carbamoyl phosphate, takes place close to the oxidative deamination of glutamate and the citric acid cycle. Both generate the substrates for the synthesis of carbamoyl phosphate (). The formation of urea, which starts with the formation of carbamoyl phosphate, occurs in a four-step cycle which ends with the cleavage of arginine into ornithine and urea. Urea is water-soluble, non-toxic, well permeable and is eliminated via the kidneys.
The two major ammonia detoxifying systems, urea and glutamine synthesis are heterogeneously distributed in the liver acinus. Whereas periportal hepatocytes contain urea cycle enzymes, only a small perivenous cell population at the acinar outflow is able to eliminate ammonia by glutamine synthesis. Thus, the two major ammonia detoxification systems, urea and glutamine synthesis, are anatomically organized in sequence. Perivenous glutamine synthetase acts as a high affinity scavenger for NH4+ ions not extracted by upstream urea synthesis and the latter pathway is a periportal low affinity system for ammonia detoxification .
- Aminotransferase reactions. In protein metabolism, aminotransferases transfer amino groups to and from glutamate for the breakdown and resynthesis of amino acids. The glutamate produced in the breakdown of amino acids is converted to α-ketoglutarate by GLD. The α-ketoglutarate is then metabolized in the citric acid cycle.
- Completion of amino acid degradation. Glutamate completes amino acid degradation through oxidative deamination by GLD to α-ketoglutarate and NH4+.
- Urea genesis. In the liver the intramitochondrial concentration of glutamate determines the production of N-acetyl glutamate by N-acetyl glutamate synthetase and, thus, governs the rate of urea genesis.
- Glutamine synthetase which adds ammonia to glutamate to form glutamine.
- Production of glutathione. Glutamate, cysteine and glycine are synthesized into glutathione, which protects the cells from oxidation.
- The central nervous system (CNS). In the CNS a glutamate-glutamine shuttle is essential for the non-toxic recycling of glutamate from astrocytes to neurons. Glutamate is the major excitatory neurotransmitter of the CNS. Astrocyte uptake of glutamate is crucial for preventing toxic extracellular uptake of glutamate. Glutamate taken up by astrocytes is converted to glutamine. This glutamine can then be released for re-uptake by neurons and used to regenerate glutamate for neurotransmission. In hyperammonemia, increased formation of glutamine and glutamate may act as a sump for ammonia, leading to accumulation of glutamine in the brain and causing brain swelling through shifts in intracellular osmoles.
Renal ammoniogenesis is the direct elimination of ammonia via the kidneys . When ammonia is produced in the kidneys, glomerular filtered luminal glutamine as well as contra luminal plasma glutamine is metabolized to glutamate and ammonia by GLD of the tubular cells. NH3 is released into the tubular lumen and binds with H+ to form NH4+. Ammoniogenesis thus serves the elimination of protons and is excreted at a rate of approximately 35 mmol/24 h. About one third of the ammonia formed in the kidneys is released into the urine and two thirds into the renal veins.
The detoxification of ammonia via the kidneys and liver is influenced by the acid-base balance. For example, a decrease in the extracellular pH from 7.4 to 7.3 causes a 70% decrease in GLD activity in the periportal hepatocytes. As a result, in acidosis, urea synthesis in the periportal hepatocytes is reduced in favor of increased glutamine production in the perivenous cells. The urea cycle is down regulated in order to conserve HCO3– which would otherwise be used up in the formation of carbamoyl phosphate. The increased amounts of glutamine produced are transported to the kidneys and help compensate the acidosis. By formation of ammonia, which binds with H+ to form NH4+ , the latter are eliminated and not reabsorbed by the tubules.
The NH4+ and HCO3– homeostasis is also regulated by the concerted action of the liver and kidneys. In renal insufficiency with reduced renal elimination of NH4+, the urea cycle is stimulated, since the increase in NH4+ in the periportal hepatocytes activates GLD. This leads to increased consumption of HCO3– with consecutive compensation of the alkalosis by metabolic acidosis.
In patients with liver cirrhosis, urea synthesis is reduced by approx. 80% due to the reduced liver parenchyma. This leads to deficient consumption of HCO3– and consequently metabolic alkalosis . The alkalosis activates hepatic glutamine synthetase and stimulates the production of glutamine approx. 5-fold. Thus, the flux of ammonia through the urea cycle is increased despite decreased capacity of the cycle, and the cirrhotic patient excretes near-normal amounts of urea. If acidosis develops due to sepsis, cardiac insufficiency or drugs, this compensatory stimulation is lost due to inhibition of glutamine synthetase.
The ammonia concentration in portal vein blood is approximately 154 μg/dL (90 μmol/L). The ammonia is produced from amino acids and urea by bacteria in the intestinal tract. Ammonia transported into the liver is normally eliminated as follows:
- 70% via the urea cycle
- 30% via the formation of glutamine with subsequent recirculation and introduction into the urea cycle. In liver cirrhosis, the number of glutamine-producing cells around the central vein is diminished to such an extent that the capacity for synthesis decreases significantly so that the NH4+ can no longer be taken up, resulting in hyperammonemia.
In newborns, the enzymes of the urea cycle have approximately 50% activity and reach adult capacities within 6 months. Genetic defects of enzymes 1–4 of the urea cycle lead to inhibition of the formation of urea, resulting in hyperammonemia. Deficiencies of the 5th enzyme, arginase, are less commonly associated with hyperammonemia.
- Unconjugated bilirubin (Bu), the bilirubin fraction that is present in the first few days of life. Bu is extremely apolar and practically insoluble in water at physiological pH and normal body temperature. In plasma it is present in a folded structure, the so-called ZZ conformation, loosely bound to albumin. This type of bilirubin is also referred to as ZZ bilirubin. There are three metabolic and excretory pathways for the elimination of ZZ bilirubin: conjugation with glucuronic acid, photo isomerization, and oxidation.
- Conjugated bilirubin (Bc) bound to sugar; the glucuronidation products are bilirubin mono glucuronide (C-8), bilirubin mono glucuronide (C-12), and bilirubin diglucuronide. The conjugates are water-soluble and are secreted into bile by hepatocytes against a concentration gradient. In icteric sera with a high proportion of conjugated bilirubin, the main fraction consists of bilirubin mono glucuronide.
- δ-bilirubin (Bδ); bilirubin is covalently bound to albumin via an amide bond between its propionic acid side chain and the ε-amino group of a lysine residue on albumin.
- Unbound unconjugated bilirubin (BF) also referred to as free bilirubin (BF).
Due to the different reactions of Bu, Bc and Bδ with diazo reagent, the following bilirubin fractions are differentiated in routine clinical diagnostics:
- Total bilirubin: diazo reagent reacts with Bu, Bc and Bδ in the presence of an accelerator
- Direct bilirubin: diazo reagent reacts immediately without the presence of an accelerator. The main fraction of Bc and Bδ as well as a small but variable fraction of Bu are measured. The serum concentration of direct-reacting bilirubin is therefore only a limited indicator of the concentration of Bc.
- Indirect bilirubin: is the difference of total bilirubin minus direct bilirubin.
Since Bc is a better criterion for the differential diagnosis of jaundice than direct bilirubin, the latter should no longer be assayed.
Diagnosis, differential diagnosis and monitoring of jaundice.
Bu, Bc and Bδ are measured.
Principle: in the presence of caffeine reagent, Bu, Bc and Bδ react with diazotized sulfanilic acid producing azobilirubin of red color in neutral solution. Addition of ascorbic acid, alkaline tartrate and dilute HCl changes the color of the azodye to blue and shifts the absorption maximum from 530 to 598 nm. Bc and Bδ react rapidly with diazotized sulfanilic acid, Bu does so slowly, but quickly after displacement from albumin by caffeine reagent the reaction proceeds rapidly. For the sample blank, the diazotized sulfanilic acid is replaced by sulfanilic acid.
Principle: Bu, Bc and Bδ react with 2.5-dichlorobenzene- diazonium salt in 0.1 mol/L HCl to form azodyes, which are measured quantitatively at 540–560 nm. For the determination of BT, Bu is released from albumin by the detergent Triton X-100. The reaction mixture of the sample blank contains only 0.1 mol/L HCl; therefore no color reaction occurs.
Used for the bilirubin determination in neonatal plasma containing mainly Bu.
Principle: the absorbance of the plasma within a capillary tube is measured spectrophotometrically at approximately 460 nm. The spectral interference by hemoglobin is compensated by an additional measurement at 550 nm. The absorbance values at both wavelengths represent the sum of absorbencies of hemoglobin and bilirubin present in the specimen. The difference of absorbance A460–A550 represents the absorbance of bilirubin only, because the absorbance of hemoglobin is identical at both wavelengths.
Principle: the film slide contains three layers:
- The upper spreading layer containing caffeine and sodium benzoate to separate Bu from albumin
- The second layer retains proteins
- The third layer is the reaction zone. Bu and Bc interact with a specific charged polymer called a mordant. The concentrations of Bu and Bc are calculated from the measured reflection densities and the predetermined molar reflectivities of the two bilirubin species at two wavelengths (400, 460 nm) and use of simultaneous equations.
Principle: BT, in the presence of bilirubin oxidase, is oxidized to biliverdin by molecular oxygen.
The purple compound is measured at 450 nm. Bu, Bc and Bδ are measured at pH 8.2 in the presence of sodium dodecyl sulfate and sodium cholate.
The method is used to determine total bilirubin in newborns.
Principle: the meters work by directing light (380–760 nm) into the skin of the neonate and measuring the intensity of specific wavelength that is returned. The meter analyzes the spectrum of optical signal reflected from subcutaneous tissues. The optical signals are converted to electrical signals by a photocell. These are analyzed by a microprocessor to generate a serum bilirubin value. The light absorption of interfering factors, such as hemoglobin, melanin, and dermal thickness, is mathematically subtracted to estimate the bilirubin concentration in the capillary beds and subcutaneous tissue.
Direct reacting bilirubin
Bilirubin immediately reacts with diazo reagent in the absence of an accelerator such as caffeine reagent. Determination is of Bc, Bδ and partially of Bu. This method is still used as part of many mechanized analysis systems, but should be replaced by the specific determination of Bc.
Unconjugated bilirubin (Bu)
Is calculated based on the difference of total bilirubin minus direct bilirubin. The goal is the determination of Bu. The calculation is meaningful only in the case of hemolytic jaundice and hereditary hyperbilirubinemia since little Bδ accumulates, but not in obstructive hepatobiliary jaundice, because of the associated almost proportional increase in both Bc and Bδ. Bδ enters into the determination of direct reacting bilirubin and leads to a falsely low value for Bu.
Diazo method: keeping Bu from reacting requires dilution of the serum with HCl and incubating for at last 5 min. before adding diazo reagent. Bδ enters the reaction only to a limited extent.
Multilayer film-slide technology: different methods based on film slide technology are available allowing separate determination of Bu, Bc and Bδ.
Unbound unconjugated bilirubin (free bilirubin; BF)
Peroxidase method: non albumin bound bilirubin is oxidized to a color-less product by ethyl hydrogen peroxide in a reaction catalyzed by horseradish peroxidase, while albumin-bound bilirubin is protected from oxidation . The rate of decrease in absorbance of bilirubin is measured at 440 nm.
- Serum, plasma: 1 mL
- Capillary plasma (Heparin, EDTA): 0.05 mL
Hyperbilirubinemia is a symptom and clinically causes jaundice if bilirubin levels are ≥ 4 mg/dL (68 μmol/L) in neonates and infants and ≥ 3 mg/dL (51 μmol/L) in older children and adults.
There is overproduction of bilirubin, which is most commonly caused by hemolytic anemias, neonatal jaundice, ineffective erythropoiesis, infections such as malaria, transfusion reactions, burns, resorption of large hematomas, and hereditary hyperbilirubinemia.
The most frequent causes are infectious or toxic injury of the liver parenchyma. Generally, possible causes include acute and chronic viral hepatitides, bacterial and parasitic liver diseases, liver metastases, drug-induced parenchymatous and cholestatic liver injury as well as involvement of the liver in other underlying diseases. Another significant group are hereditary hyperbilirubinemias.
Post hepatic jaundice
This is caused by mechanical obstruction of the bile ducts (bile duct stones, carcinoma of the pancreatic head, biliary atresia, primary sclerosing cholangitis).
Differential diagnostic information is provided by:
- Total bilirubin (BT = Bu + Bc + Bδ)
- The concentration of conjugated bilirubin (Bc)
- The Bc to BT ratio
- The LD to AST ratio
- The levels of ALT, GGT and ALP activities.
Prehepatic jaundice due to hemolysis or ineffective erythropoiesis can be ruled out if BT is above 6 mg/dL (103 μmol/L). Significantly elevated bilirubin levels are only seen in transfusion incidents in the AB0 system, hemolytic crises (e.g., in sickle-cell anemia, neonatal jaundice, and certain hereditary hyperbilirubinemias).
The determination of direct reacting bilirubin and the direct reacting bilirubin/total bilirubin ratio are useful in the differentiation of hemolytic from hepatobiliary jaundice only up to a BT concentration of 3 mg/dL (51 μmol/L) . Using a ratio of 0.33 as a discriminator, values below predict hemolytic jaundice with a diagnostic sensitivity of 80% and values above predict hepatobiliary jaundice with a sensitivity of 86%.
In differentiation to hepatic jaundice, an LD/AST ratio of ≥ 5 is suggestive of hemolytic jaundice. Other findings pointing to hemolytic jaundice include:
- In urine: bilirubin negative and urobilinogen positive
- Decrease in serum haptoglobin, and reticulocytosis.
If hyperbilirubinemia is accompanied by elevated liver enzymes, then the jaundice is likely to be hepatic. In this case, differentiation from post hepatic jaundice is required.
Hyperbilirubinemia has low diagnostic sensitivity for liver diseases and therefore is not a screening parameter. More than 40% of patients with clinical liver disease have a BT concentration below 1.2 mg/dL (20 μmol/L) and another 25% have subicteric levels i.e., bilirubin concentrations in the range of 1.2–2.9 mg/dL (20–50 μmol/L). Many liver patients present with sub icterus as the main symptom of concern .
In hepatic jaundice, Bu, Bc and Bδ are elevated, with Bc accounting for over 50%. The main causes are viral hepatitides and conditions with reduced bilirubin excretion due to lack of energy, such as sepsis, total parenteral nutrition, or serious surgical interventions. The proportions of Bc and Bδ of BT are of prognostic value. For example, a decrease in Bc is a sensitive indicator of improvement while an increase in Bδ predicts prolonged illness. In the case of decreasing or slightly to moderately elevated BT levels, direct bilirubin can account for 80% of total bilirubin, since Bδ, which enters into the determination of direct reacting bilirubin, has a half-life of 18 days.
In newborns and infants, Bδ is low in non-hepatic jaundice (e.g., neonatal jaundice, septic shock, hemolysis). An increase of Bδ over 10% of BT, suggests a hepatogenic cause (e.g., Cytomegalovirus infection, biliary atresia, hepatitis B infection) .
Other findings are:
- In urine: bilirubin positive, urobilinogen positive.
In obstructive jaundice, ALT activities are rarely higher than 10-fold the upper reference interval value. The elevation in BT is due to the parallel increase in Bc + Bδ, with Bc accounting for the largest proportion (). In acute disorders with jaundice, fresh obstructive jaundice can be ruled out if ALT is normal or elevated more than 25-fold .
The success of an invasive procedure for the correction of cholestasis can be assessed quickly by monitoring Bc. Due to its short half-life, Bc decreases faster than BT . The determination of Bc however, may not be performed as direct bilirubin, because Bδ, which still remains elevated for several weeks post obstruction, is measured as part of it .
Other findings in post hepatic jaundice:
- In urine: bilirubin positive, urobilinogen negative.
Hyperbilirubinemia in neonates, infants, older children and young adults presents a differential diagnostic problem. In neonates, the condition can be benign, as in breast milk jaundice, or potentially fatal, as in hereditary fructose intolerance. The jaundice can originate primarily in the liver, as in acute viral hepatitis, or outside the liver, as in biliary atresia, or can be secondary to a non-hepatic cause, such as hemolysis (e.g., due to glucose-6-phosphate dehydrogenase (G6-PD) deficiency, or sepsis). Hereditary hyperbilirubinemias must always be included in the differential diagnosis .
Hereditary hyperbilirubinemia is characterized by liver dysfunction without hepatocellular damage (). One of the first steps in the diagnosis is the measurement of BT and the quantitative determination of Bc and Bu. Conjugated hyperbilirubinemias are always caused by a hepatobiliary disorder, and conjugated bilirubin is harmless. Chronic, more severe unconjugated hyperbilirubinemias can cause bilirubin encephalopathy, while mildly elevated concentrations of Bu have an anti oxidative effect and counteract the development of oxidative stress.
- Disorders of bilirubin conjugation and elimination. Non-conjugated hyperbilirubinemias include type I and type II Crigler-Najjar syndrome (Arias syndrome) and Gilbert’s syndrome.
- Hereditary cholestasis with predominantly conjugated hyperbilirubinemia. These include Dubin-Johnson syndrome, Rotor syndrome, benign recurrent intrahepatic cholestasis (BRIC), progressive familial intrahepatic cholestasis (PFIC), and Alagille syndrome.
In theory, the measurement of Bc is a good indicator for differentiating between the two hereditary types of hyperbilirubinemia. In practice, however, this is not the case, since most mechanized analytical systems measure direct bilirubin but not Bc. This is too unspecific, because direct bilirubin is also measured in healthy individuals where physiologically it should not be detectable.
Neonates have a reduced erythrocyte life span compared to adults. The conversion of hemoglobin (Hb) to unconjugated bilirubin and the conjugation of the latter are reduced in the postnatal period, leading to increased serum levels of unconjugated bilirubin. In addition, the Hb from hematomas also has to be metabolized in the postnatal period. Moreover, an acute-phase reaction activates hem oxygenase-1, leading to further accumulation of Hb.
Approximately 60% of term and 85% of pre-term newborns will develop clinically apparent jaundice because of an increase in unconjugated bilirubin . This physiological jaundice becomes clinically apparent on day 3, peaks on day 5–7 when most newborns are already at home, and resolves by day 14. Physiological jaundice is usually benign. However, if unconjugated bilirubin levels get too high, bilirubin can cross the blood brain barrier where it is neurotoxic. Therefore it is useful to classify newborn jaundice according to the age when the baby becomes visibly jaundiced.
- Uncommon on days 1–2. The underlying disease for jaundice is antibody mediated hemolysis e.g., Rhesus, ABO, and others. Newborns of Rh(D) negative mothers, or newborns of mothers with a positive antibody screen, should routinely have cord blood sent for blood group and direct antibody test (Coomb's test). Any newborn who is Coomb's test positive should have a total bilirubin determination in the first 24 hours. Any newborn who is clinically jaundiced within the first 24 hours requires urgent testing to exclude hemolysis.
- Normal incidence on days 3–10, mostly uncomplicated, in rare cases complicated (e.g., G6PDH deficiency) or increased incidence of premature newborns
- Prolonged jaundice is defined as a duration of more than 14 days in full-term infants and more than 21 days in premature infants. It is clinically useful to classify (i) Predominantly unconjugated prolonged jaundice usually found in breast milk nutrition. Breast fed neonates are four times more likely to have hyperbilirubinemia with levels above 10 mg/dL (172 μmol/L) than those on a formula diet. However, they do not have higher bilirubin peak levels . (ii) Predominantly conjugated prolonged jaundice usually always pathological. The newborn should be investigated for intra-hepatic (e.g., hepatitis) and obstructive (e.g., biliary atresia) causes of prolonged jaundice. See .
Physiological transient unconjugated jaundice in the first week of life must be differentiated from hyperbilirubinemia which exceeds a time-dependent physiological upper limit on the bilirubin nomogram and can lead to bilirubin-induced neurologic dysfunction (BIND). Refer to .
In most cases, total bilirubin is determined in neonates. If the upper limit in the bilirubin nomogram is exceeded or if there is prolonged jaundice, unconjugated and conjugated bilirubin has to be determined in addition to total bilirubin.
Hereditary disorders of bilirubin clearance (Gilbert’s syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome), G6-PD deficiency and hereditary spherocytosis alone do not cause neonatal hyperbilirubinemia. Frequently they are not diagnosed until jaundice is provoked by an extrinsic event.
Discharge before 72 hours is a risk factor for the development of severe hyperbilirubinemia, mainly because outpatient surveillance cannot be as close as that provided in the postnatal ward.
Total bilirubin concentrations increase with perinatal age and higher bilirubin levels are tolerated at older age. However, studies on bilirubin induced neurological damage only provide limited evidence on harmful total serum bilirubin levels, because most factors that increase the risk of neurodevelopmental delay (e.g., asphyxia, intracranial hemorrhage, prematurity) also increase total serum bilirubin levels and bilirubin induced neurotoxicity (BIND), especially in pre-term infants .
Most pre-term infants less than 35 weeks gestational age have elevated total serum bilirubin concentrations which often present as jaundice. When left unmonitored or untreated in these infants, an elevated total bilirubin level can progress to silent symptomatic neurologic manifestations. Acute bilirubin encephalopathy is acute progressive, and often reversible with aggressive intervention, whereas kernicterus (or chronic bilirubin encephalopathy) is the syndrome of chronic, post-icteric and permanent neurologic sequelae that is associated with more serious and usually irreversible manifestations . Dysmyelination and degeneration in the globus pallidus, subthalamic nucleus, and cerebellum are neuropathologic findings of kernicterus in neonates, albeit characteristically late (> 10 days) in onset .
Low bilirubin kernicterus in pre-term neonates, though rare, remains an unpredictable and refractory form of brain injury. Low bilirubin kernicterus is defined as the occurrence of kernicterus at total bilirubin levels below commonly recommended exchange transfusion thresholds (). The exchange transfusion treatment thresholds reflect both the total bilirubin level and the presence of neurotoxicity risk factors.
- Hypoalbuminemia < 2.5 g/dL. Mean serum albumin levels reported for pre-term infant below 30 weeks` gestation are approximately 1.9 g/dL (90% CI 1.2–2.8 g/dL) and do not approach 2.5 g/dL until 36–37 weeks’ gestation
- Co-morbid CNS findings of intraventricular hemorrhage and periventricular white matter injury
- Perinatal and early postnatal infection/inflammation is an important contributer to brain structural and functional abnormalities later in life. Notable contributers are chorioamnionitis, sepsis and necrotizing enterocolitis
- Chronic bilirubin-induced neuroinflammation. Persistent inflammation is recognized as an important risk factor for central nervous system injury in pre-term neonates.
- Total bilirubin to gauge the size of the neonate's bilirubin
- Unconjugated unbound bilirubin (no commercial assay is available)
- Bilirubin/albumin ratio can serve as a proxy for unbound bilirubin but has a limited and conflicting track record in predicting adverse neurodevelopmental outcome. Japanese investigators /, / found that a bilirubin-albumin ratio of ≥ 0.50 μmol/L/μmol/L (≥ 4.25 mg/dL/g/dL) for infants of 30–34 weeks gestation and ≥ 0.40 μmol/L/μmol/L (≥ 3.4 mg/dL/g/dL) for infants of below 30 weeks' gestation predicted putative neurotoxic unbound bilirubin levels of ≥ 1.0 μg/dL.
- Unconjugated unbound bilirubin/total bilirubin ratio (Bf/TBC; μg/mg). In a study the ratio was tested in neonates who were admitted to the neonatal intensive care unit. One group had normal and the other had abnormal auditory brainstem response. In normal newborns the Bf/TBC was mean + SD = 0.062 ± 0.034 (range 0.009–0.200) in the abnormal group mean + SD = 0.109 ± 0.039 (range 0.034–0.167).
The presence of conjugated bilirubin indicates a pathologic process and usually prolonged jaundice. Conjugated hyperbilirubinemia can be of infectious, endocrine or genetic etiology. The most common surgically correctable cause is extrahepatic biliary atresia.
If conjugated hyperbilirubinemia is present, the following laboratory tests must be performed within 24 h according to the suspected diagnosis:
- Sodium, potassium, creatinine, urea
- Blood count including reticulocytes
- Blood group typing (ABO, Rh)
- Blood gas analysis
- ALT, AST, GGT, LD
- Glucose, lactate, ammonia
- Cholesterol, triglycerides
- Blood and urine culture tests for pathogenic bacteria
- Stool color analysis
- PT and aPTT
- Serology for hepatitis A, B, C
- IgM antibodies for rubella, toxoplasmosis, Herpesvirus, Cytomegalovirus.
- TSH, free T4, cortisol and α1-antitrypsin
- Organic acids and amino acids in urine
- Enzyme tests in red blood cells for galactosemia.
Tables describing scores and staging of liver diseases:
Pre analytic factors
- Use of “adult-size” lancets that are too long for blood collection in neonates. There is a risk of puncture wound osteomyelitis.
- In vitro hemolysis, in particular when using micro containers for blood collection. Hemolysis occurs in 0.2% of specimens and is the cause of specimen rejection by the laboratory in 39.7% of cases.
- Exposure of the specimen to light.
Calibration: the recommended standard reference material for the calibration of bilirubin assays is the SRM 916a preparation by the National Institute for Standards and Technology (NIST).
Total bilirubin: so far it is not known whether the reference method recommended by the NCCLS measures Bδ correctly. The linear range of the assay is up to 27 mg/dL (462 μmol/L). Interference by hemoglobin is found only at concentrations ≥ 2 g/L.
Direct reacting bilirubin: the extent to which Bu is simultaneously measured depends on the pH of the reaction mixture: the lower the pH, the less Bu is measured. Bδ is always included in the measurement. Laboratories should ensure that the assays they use do not measure direct reacting bilirubin above 0.1 mg/dL (1.7 μmol/L) in healthy individuals .
Conjugated bilirubin: the method used for the determination of Bc does not detect Bu. However it has interference by the presence of hemoglobin, since during preincubation of the specimen with HCl hemoglobin is oxidized to methemoglobin, producing H2O2. The latter causes destruction of the azobilirubin formed in the main reaction resulting in falsely low results.
Enzymatic method for BT: at concentrations below 1.1 mg/dL (19 μmol/L), the values are on average 0.1 mg/dL (1.7 μmol/L) lower than those measured with the reference method. Direct bilirubin concentrations in the sera of newborns with non-conjugated hyperbilirubinemia are higher when measured using the bilirubin oxidase method than with the diazo method. This is also the case in neonates after light therapy . Hemoglobin concentrations ≥ 2 g/L lead to a 5–18% decrease in BT.
Spectrophotometric determination of bilirubin: Bilirubinometers are used to measure neonatal bilirubin. Measurements are carried out at two wavelengths i.e., of 460 nm and 550 nm. Although hemoglobin is compensated by the measurement at two wavelengths, a free hemoglobin concentration of 4 g/L causes a reduction in bilirubin within the threshold range for phototherapy from 16.3 mg/dL (279 μmol/L) to 15.6 mg/dL (265 μmol/L) . Carotenes which often can cause interference are only in very small quantities in the serum of neonates. The most common interference mimicking falsely high values is the plasma turbidity (lipemia), because the light dispersion it causes is greater at 460 nm than at 550 nm. The results obtained with bilirubinometers are highly dependent on the protein matrix. The calibration should therefore be performed with commercial sera specifically designed for this type of assay, or adult sera whose concentrations were determined with the reference method. Levels above 17.5 mg/dL (300 μmol/L) should not be measured spectrophotometrically, since absorbance values are obtained that deviate from the theoretical linearity of Lambert-Beer’s law. For the decision on whether to proceed with exchange transfusion, the direct spectrophotometric bilirubin determination must not be used as the sole method of analysis. Additional bilirubin determination based on the diazo reaction (Jendrassik/Gróf, DPD) is recommended .
- Day-to-day variation: BT 30%.
- Fasting: BT elevation by 100 to 200% after 48 h.
- Physical stress: 30% increase in BT.
- Pregnancy: 33% decrease in BT during the last trimester.
- Oral contraceptives: 15% decrease in BT .
Exposure of the specimen to light: up to 30% decrease in BT after 1 h.
During phototherapy in hyperbilirubinemic neonates non-conjugated bilirubin is converted to photo isomers which are more water-soluble. The diazo method measures higher levels in neonates receiving phototherapy than the bilirubin oxidase method . Neonatal bilirubin levels measured with the multi layer film-slide technique are higher than the determination based on the diazo reaction .
Approximately 80–85% of the bilirubin produced daily originates from the degradation of hemoglobin released by the breakdown of senescent erythrocytes. At the end of their life span of approximately 120 days, the erythrocytes are taken up by macrophages of the reticuloendothelial system, in particular in the spleen. The degradation of hemoglobin occurs in two steps. The Fe3+ heme binds to the membrane-bound enzyme hem oxygenase and is reduced to Fe2+ heme in a reaction catalyzed by NADPH-cytochrome P450 reductase (). The Fe2+ heme is then broken down by oxidation into equimolar amounts of CO and biliverdin. The hemoglobin molecule degrades, globin is metabolized, iron binds to transferrin, and the biliverdin is reduced to bilirubin by cytosolic biliverdin reductase. The CO produced binds to hemoglobin to form carboxyhemoglobin and is then eliminated via the pulmonary gas exchange.
The breakdown of 1 g of hemoglobin yields 34 mg of bilirubin. Approx. 250 mg of bilirubin is produced daily in the physiological breakdown of hemoglobin. The remaining 15–20% of the bilirubin produced daily is derived from the breakdown of heme-containing proteins such as myoglobin, cytochromes and catalases. Bilirubin accumulates in the bone marrow in the case of ineffective erythropoiesis that is associated with impaired maturation processes, such as in megaloblastic anemia, thalassemia, porphyria, and myelodysplastic syndrome. In these conditions, up to 80% of the bilirubin can result from ineffective erythropoiesis.
Bu binds to albumin in the blood for transport to the liver. Upon reaching the liver, bilirubin dissociates from albumin in the space of Disse and is taken up by the hepatocyte across the sinusoidal part of the cell’s plasma membrane. The uptake is mediated by a transport system and occurs actively against a concentration gradient. The sinusoidal membrane’s transport system for organic ions and possibly bilirubin, but not for bile acids, is believed to consist of bilitranslocase, bromosulfophthalein/bilirubin binding protein (BBBP), and organic anion-binding protein (OABP). Organic anion transport peptides (OATPs) may also play a role. The transport of unconjugated bilirubin across the sinusoidal part of the plasma membrane is, however, not yet well understood .
In the cytosol of the hepatocyte, Bu binds to ligandin and Z-protein. Ligandin has a higher affinity and transports Bu to the endoplasmic reticulum where it is glucuronidated by the action of uridine-5’-diphosphate glucuronyltransferases (UGTs) . Essential cosubstrates of the UGTs are uridine diphosphate glucuronic acid or other UDP sugars. Bu is glucuronidated as follows ():
- At the propionic acid side chain located at C8 of the two central pyrole rings, forming bilirubin mono glucuronide (C8)
- At the propionic acid side chain located at C12 of the two central pyrole rings, forming bilirubin mono glucuronide (C12)
- Both isomers can then be further glucuronidated to diglucuronides.
By its conversion to glucuronide bilirubin becomes water-soluble. The transport of the glucuronide across the canalicular domain of the hepatocyte’s plasma membrane is mediated by multi drug resistance proteins (MRPs), of which six are known.
Approximately 80% of the bilirubin secreted into bile is bilirubin diglucuronide, 15% is bilirubin mono glucuronide, and 5% is mixed conjugates with sugars such as glucose and xylose. About 1–2% of the bilirubin is secreted into bile non-conjugated.
The excretion of the Bc from the hepatocyte into the bile capillaries depends on the energy available and is the slowest process in bilirubin metabolism. A healthy liver can eliminate approximately 1 g of conjugated bilirubin per day i.e., 2–5 times the amount of bilirubin produced by the body. Inhibition of the excretion mechanism by drugs such as digoxin or by bromosulfophthalein causes Bc to accumulate in the hepatocyte from where it is then released into plasma.
Bc reaches the intestines via the biliary tract. Due to the impact of intestinal bacteria urobilinogen is formed. Approximately 70% of the urobilinogen is reabsorbed in the intestine, transported to the liver via the portal vein, and excreted again via the biliary tract (enterohepatic circulation). Approximately 2–4 mg of urobilinogen is excreted in urine daily. This amount is 2–3 times higher if bilirubin levels are increased (e.g., due to hemolytic anemia) and 4–10 higher if the liver parenchyma is damaged (e.g., in acute viral hepatitis or in the presence of a portocaval shunt). If the bile ducts are obstructed, the enterohepatic urobilinogen circulation is interrupted, and urobilinogen is not detectable when bilirubinuria is present.
In conjugated hyperbilirubinemias, in particular obstructive jaundice, a rapid decrease in Bc (half-time of only hours) is the most sensitive indicator for complete removal of the occlusion. Assays measuring BT are, however, unsuitable for monitoring the decline of conjugated hyperbilirubinemia, since they also measure Bδ with a half-time of 18 days. Bc can, however, be monitored using methods that specifically measure Bc.
In acute hepatitides, parenchymal cell damage leads to jaundice. It results from impaired excretion of biliary excreted substances into the bile capillaries and regurgation of Bc back into the bloodstream.
UDP-glucuronyltransferase (UGT1A1) glucuronidates bilirubin and is encoded by the UGT1A1 gene. A polymorphism in the promoter of the gene, which contains repeats of thymidine and adenine (TA) (TATA box), is responsible for the amount of UGT1A1 in the cell. If the TATA box is
- homozygous for 7 TA repeats (genotype 7/7), the cell contains low UGT1A1 activity
- homozygous for 6 TA repeats (genotype 6/6, wild type), the cell contains high UGT1A1 activity
- heterozygous (genotype 6/7), the cell contains moderate UGT1A1 activity.
In Europe, 24% of individuals have genotype 6/6, 39% genotype 6/7, and 8% genotype 7/7. A variation in nucleotide 211 (G to A, heterozygous and homozygous) causes hyperbilirubinemia in neonates.
Bu is highly apolar and practically insoluble in water. This is due to its folded configuration, which is stabilized by six bridging hydrogen bonds yielding the so-called ZZ conformation (). Under the effect of phototherapy in the neonates with unconjugated hyperbilirubinemia, the bridging hydrogen bonds rupture, resulting in isomers with a partially open (ZE or EZ) or completely open (EE) conformation. These conformations are more water soluble and can therefore be better eliminated.
The cause of bilirubin encephalopathy is free bilirubin, whose concentration increases with a BT concentration above 17 mg/dL (291 μmol/L). Free bilirubin dissolves readily in fat and is neurotoxic in that it damages the mitochondria in the astrocytes. Since the binding of Bu to albumin decreases in acidosis as well as being impaired by certain drugs such as salicylic acid, both factors also promote an increase in BF.
Bδ has a half-life of 18 days due to its covalent linkage with albumin. It is not detected in healthy individuals, in Gilbert’s syndrome, and in neonatal hyperbilirubinemia. In diseases with conjugated hyperbilirubinemia (e.g., parenchymatous and obstructive jaundice, Bδ can account for 20–50% of BT in the acute phase). As the clinical condition improves and BT decreases, the Bδ fraction can increase to 50–90% so that hyperbilirubinemia can persist for a relatively long period of time.
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Hermann Seim, Lothar Thomas
Carnitine (3-hydroxy-4-N-trimethylammonium butyrate) occurs in nature as the L-(–) stereoisomer, either in free form or esterified through its hydroxyl group with fatty acids. A range of endogenous carnitine can be measured in plasma :
- Total L-carnitine: the sum of free carnitine and esterified carnitine
- Free L-carnitine: the proportion of the unesterified carnitine in plasma is about 90% of total carnitine.
- Total acylcarnitine: L-carnitine is esterified through its hydroxyl group with long-chain (C12–C18) or shorter-chain fatty acyl groups (C2–C10). The proportion of total acylcarnitine in plasma is about 5% of total carnitine. Acyl compounds contain the acyl group (R-CO-)
- Acetylcarnitine: the proportion of L-carnitine trans esterified to acetyl carnitine is about 5% of total carnitine.
Carnitines are low molecular weight endogenous compounds present in all mammalian species. The biological activities of carnitines are:
- To function as an essential carrier of acyl groups from the cytoplasma into the mitochondria and hence to the site of fatty acid β-oxidation and consequently to produce energy
- To maintain inner mitochondrial concentrations of free coenzyme A by accepting short-chain acyl groups for the corresponding acyl-CoA and transporting them out of the mitochondria.
Mitochondrial fatty acid oxidation is a fundamental source of cellular energy, particularly in cardiac and skeletal muscle. Approximately 98% of the total carnitine pool within the body is located in these tissues. Despite the fact that only 1% of the body’s carnitine pools is present in blood, the plasma concentration is used as an indicator of the total body carnitine content.
L-Carnitine body pool is achieved by absorption from dietary source, in particular red meat, and by endogenous biosynthesis requiring lysine and methionine.
Suspected carnitine deficiency in conjunction with:
- Symptoms such as myasthenia, myalgia, cardiomyopathy, hypo ketotic hypoglycemia, failure to thrive in neonates and infants
- Malnutrition including kwashiorkor and cachexia
- Long-term carnitine-free parenteral nutrition
- Drug-induced deficiency caused by treatment with (e.g., valproic acid or pivalic acid)
- Congenital enzyme defects (e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD))
- Organic acidurias (e.g., propionic aciduria)
- Lipid storage myopathies
- Pregnancy, hemodialysis (situational).
There are two main principles for the determination of free carnitine and total carnitine which is often determined concomitantly. One is a colorimetric assay the other is a radioisotopic assay.
L-Carnitine reacts with acetyl CoA catalyzed by carnitine acetyl transferase (CAT, EC 126.96.36.199) to form acetyl L-carnitine and CoASH. CoASH reacts nonenzymatically with 5,5’-dithiobis-2-nitro benzoate (DTNB) to form 5-thio-2-nitro benzoate (TNB). The concentration of TNB is measured spectrophotometrically. Two sets of test tubes are used. In the first set free carnitine is determined. To the second set 2 mol/L KOH is added used for hydrolyzing the sample for total carnitine determination. The values thus obtained have to be multiplied by factors corresponding to dilution of the samples and other variables. The CAT is highly specific for L-(–) carnitine.
The radioisotopic assay is based on the stoichiometric acetyl-transfer from 14C-labelled acetyl coenzyme A to carnitine catalyzed by CAT, and measurement of isotope content of the 14C acetyl carnitine formed.
The following methods are used for determining L-carnitine and its esters: gas chromatography and high-pressure liquid chromatography (HPLC), often coupled with tandem mass spectrometry or HPLC electrospray mass spectrometry .
- Serum (EDTA plasma), urine: 1 mL
- Biopsy material, seminal plasma
L-carnitine is synthesized in the liver from methionine and protein-bound lysine. However, most of the carnitine required daily comes from dietary sources, in particular red meat. No hereditary defect of carnitine synthesis is currently known, although the synthetic pathway is not yet fully developed in premature infants .
L-carnitine deficiency is a condition in which free L-carnitine is reduced. Often, the L-carnitine/acylcarnitine ratio is decreased due to the latter being elevated. Cellular L-carnitine deficiencies are of clinical significance. They cannot always be detected by measuring the serum L-carnitine concentration.
L-carnitine deficiencies are classified as follows based on clinical and etiopathogenic criteria:
- Muscular; only the muscles are affected
- Systemic; L-carnitine is reduced in all tissues
- Primary; this type of deficiency is a disorder of L-carnitine metabolism, which results in a low concentration of free L-carnitine. It is associated with cardiomyopathy, encephalopathy and myasthenia and can lead to premature death if untreated. The disorder is due to a defect of L-carnitine uptake into the cell or a defect of L-carnitine transport to the mitochondrion.
- Secondary; this type of L-carnitine deficiency is milder than the primary type and results from diminished renal or hepatic function, extreme malnutrition, or use of medications, such as valproic acid or pivampicillin.
Primary L-carnitine deficiency is a rare disease, whereas the secondary type is clinically common. The deficiency can be efficiently treated by supplementation with L-carnitine (approximately 10–100 mg/kg per day). The response to L-carnitine application in muscular L-carnitine deficiency varies, in the systemic form it is vital.
The L-carnitine concentration in the tissues is 10–100-fold higher than that in blood or extracellular fluid. What is important clinically is that:
- Reduced tissue L-carnitine levels indicate L-carnitine deficiency syndrome
- A persistently reduced serum L-carnitine concentration over weeks or months suggests L-carnitine deficiency in the tissues
- A normal serum L-carnitine concentration does not rule out L-carnitine deficiency in individual organs, in particular the heart and skeletal muscles, since there may be isolated dysfunction of the L-carnitine transport system of the cell membrane (e.g., muscular L-carnitine deficiency).
Myogenous primary L-carnitine deficiency
The deficiency is confined to the muscles . The diagnosis can be made by determining L-carnitine in muscle tissue, since the serum L-carnitine concentration is within the reference range. Even though 99% of the L-carnitine pool resides in the skeletal muscle, nearly 90% of this pool must be depleted before the serum level falls below the reference interval . Clinical symptoms include episodes of myasthenia and myalgia, in particular of the extremities and neck muscles; fasting or a high-fat diet induce an adjusted form of ketogenesis. Histochemically, a form of lipid storage myopathy is present, in which lipid containing vacuoles are stored in type I muscle fibers. This form of the deficiency can manifest from infancy to adulthood and is less progressive than the systemic form. Muscular L-carnitine deficiency is inherited in an autosomal recessive pattern, or acquired.
Myogenous secondary L-carnitine deficiency
Secondary deficiency is confined to the muscles, but associated with other muscular diseases (e.g., Duchenne muscular dystrophy or metabolic myopathies (mitochondriopathies).
- Defect of the L-carnitine transporter of the cell membrane (L-carnitine uptake defect, CUD). In CUD, the resorption of L-carnitine from the intestine and the reabsorption of L-carnitine in the proximal renal tubules are inhibited, and in other tissues the accumulation of L-carnitine within the cell is reduced.
- Defects of enzymes which transport long-chain fatty acids to the mitochondria i.e., carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), and the carnitine carrier, also known as carnitine-acylcarnitine translocase.
Clinical findings: there are two forms of CUD: a cardiomyopathic form which begins in early childhood, and a hepatic form with recurrent symptoms similar to Reye’s syndrome.
Laboratory findings: free and total L-carnitine are very low, acylcarnitine is normal. Fasting glucose and ketone bodies are reduced. Further results: ammonia elevated, metabolic acidosis, creatine kinase elevated, myoglobin elevated, aminotransferases elevated.
Clinical findings: only the liver is affected. Episodic liver dysfunction, often accompanied by hypoglycemia. Some patients develop renal tubular acidosis. The mothers of these patients may have a history of acute hepatic steatosis in pregnancy.
Laboratory findings: total L-carnitine reduced, acylcarnitine reduced, free L-carnitine normal or elevated. Fasting hypoglycemia and hypo ketonemia. Metabolic acidosis, ammonia normal, aminotransferases and creatine kinase normal.
Clinical findings: this deficiency can result in sudden infant death or can be of a milder nature. Both cases are characterized by cardiomyopathic and hepatic dysfunction of different severity.
Laboratory findings: acylcarnitine elevated, free L-carnitine reduced. Fasting hypoglycemia and hypo ketonemia. Metabolic acidosis, ammonia, lactate, uric acid, aminotransferases and creatine kinase elevated.
Clinical findings: there are two forms:
- Neonatal form, which is usually lethal and may be accompanied by cystic kidneys
- Childhood-onset form that manifests in adulthood. This deficiency is one of the most common biochemically defined causes of myoglobinuria in adults . CPT-2 deficiency becomes evident in particular after intense physical stress. It is associated with myalgias with myoglobinuria and rhabdomyolysis. Other precipitating factors include fasting with and without physical exercise, cold exposure, and recurrent infections, all of which ultimately are conditions of increased fatty acid oxidation. There is little or no lipid storage in the skeletal muscle /, , /.
Laboratory findings: total L-carnitine normal or elevated, acylcarnitine elevated, free L-carnitine reduced. Fasting glucose and ketone bodies reduced. Liver enzymes, creatine kinase and myoglobin elevated.
This group includes L-carnitine deficiencies that are associated with defined genetic defects outside the carnitine system, complex diseases, or extreme malnutrition. Genetic defects include organic acidurias, enzyme defects of mitochondrial β-oxidation and of the respiratory chain (). In serum there is a marked deficiency of free L-carnitine (below 18 μmol/L).
Secondary L-carnitine deficiency often presents as a mere functional deficiency in which free L-carnitine is reduced, but total L-carnitine is still within the reference range due to elevated acylcarnitine . Thus L-carnitine is lacking as a transfer recipient for excessive pathogenic acyl groups from coenzyme A.
Since in colorimetric assays the sulfhydryl group of free coenzyme A is determined using dithiobisnitrobenzoate, endogenous SH groups interfere with the assay. This can be prevented by prior oxidation with H2O2 and subsequent decomposition of the latter by catalase .
The concentration of total L-carnitine in erythrocytes is comparable to that in serum, but with a different degree of acylation /, /. The concentration of L-carnitine in leukocytes is higher than that in serum and dependent on the degree of activation .
Total L-carnitine can be stored for up to 1 week at 4–6 °C. Changes in the free L-carnitine and acylcarnitine fractions can occur proportionally to the duration of storage due to hydrolysis, in particular of the short-chain acylcarnitines. For long-term stability of total L-carnitine and its fractions, deep-frozen storage is required .
Before a fatty acid (FA) liberated from triglycerides can enter the metabolic pathway it must be activated to form an acyl-CoA. This activation step is catalyzed by acyl-CoA synthetase via a two-step reaction :
1) the formation of an intermediate fatty acyl-AMP with the release of pyrophosphate
2) the formation of a fatty acyl-CoA with the release of AMP
Activated fatty acids are unable to cross the inner mitochondrial membrane in a free form. An L-carnitine acyl transferase (CPT I) that is located at the outer face of the inner mitochondrial membrane catalyzes the formation of acyl carnitine esters and free coenzyme A from L-carnitine and acyl CoA esters. The free coenzyme is then available to the cell for further use and the carnitine ester is passed to the inner surface of the membrane by a carnitine-acylcarnitine translocase that is located within the membrane. At the inner face, a second acyl transferase (CPT II) acts on the acylcarnitine and coenzyme A within the mitochondrion, to release L-carnitine and to reform the fatty acid CoA ester, the substrate of β-oxidation. The L-carnitine, in turn, is recycled back to the outer surface of the membrane by the translocase, where it can participate in another round of the trans esterification/translocation system () .
The total L-carnitine pool in adults is 15–20 g, 95% of which resides in the skeletal muscle and is replaced relatively slowly.
Most of the L-carnitine required daily comes from dietary sources. L-carnitine is absorbed in the duodenum and jejunum by a Na+-dependent active transport mechanism or by diffusion and comes from animal products (meat, milk) (). Plant sources of L-carnitine are limited. The bioavailability of orally applied L-carnitine is approximately 5–20%.
The smaller proportion of the L-carnitine supply is synthesized in the body from the essential amino acids lysine and methionine (). In addition, vitamins C, B6 and niacin as well as Fe2+ are required. The last step in the biosynthesis, the hydroxylation of γ-butyrobetaine, occurs in the liver and kidneys.
The liver and kidneys play a key role in the homeostasis of L-carnitine metabolism. Following its intestinal absorption, L-carnitine travels to the hepatocytes via the portal vein blood and is partially esterified with fatty acids to form acylcarnitines. Free L-carnitine and the acylcarnitines are distributed to the organs via the blood circulation. In the kidneys, 95% of the filtered L-carnitine is reabsorbed. Clearance of acylcarnitine is significantly higher than that of free L-carnitine.
Fatty acids are metabolized to CO2 and H2O in all organs, except the brain and the erythrocytes. This process occurs in the mitochondria in the intermediate vicinity of the citric acid cycle and the respiratory chain. The hydrogen produced in the breakdown of fatty acids is transferred to FAD and NAD and metabolized for energy.
- Fatty acids are activated in the cytoplasm of the cells by linking of their carboxyl group with acetyl-CoA. This step is catalyzed by thiokinases (acyl-CoA synthetases).
- For transport to the mitochondrion, the fatty acids must be converted to L-carnitine ester (acylcarnitine). This reaction is catalyzed by carnitine acyltransferases (CAT), the most important ones being the carnitine palmitoyltransferases (CPT), which catalyze the transport of long-chain fatty acids. The CoA residue is exchanged for L-carnitine by the action of CPT I on the outer mitochondrial membrane (). The fatty acid residue is transferred back to CoA by the action of CPT II on the inner mitochondrial membrane.
- Fatty acid metabolism begins in the mitochondrial matrix, where, catalyzed by acyl-CoA dehydrogenase, the fatty acids undergo oxidization to form unsaturated compounds.
Three types of acyl-CoA synthetases, carnitine acyltransferases and acyl-CoA dehydrogenases are known:
- With a substrate specificity for long-chain fatty acids (C14 to C20; palmitoyl)
- With a specificity for medium-chain fatty acids (C8 and C10; octanyl)
- and with a specificity for acetate and propionate only.
Carnitine acyltransferase (CAT, EC 188.8.131.52)
The CAT resides in the mitochondria and peroxisomes and catalyzes the reversible transfer of short-chain acyl groups from coenzyme A to carnitine. In the numerous secondary L-carnitine deficiencies with elevated acylcarnitine, the CAT is responsible for removing toxic acyl groups from the mitochondria. The activity of this enzyme determines the availability of free CoA.
Carnitine octanoyltransferase (EC 184.108.40.206)
This enzyme catalyzes the reversible transfer of medium-chain acyl residues (C8 and C10 with highest affinity) from coenzyme A to L-carnitine. Due to the localization in the mitochondria and peroxisomes, this enzyme accounts for the transport of medium-chain acyl residues from the peroxisomes to the mitochondria as the site of energy production (carnitine shuttle).
Carnitine palmitoyltransferase (CPT, EC 220.127.116.11)
This enzyme is required for transporting the long-chain fatty acids into the mitochondria. CPT consists of two enzymes, CPT I and CPT II, which differ in their location (CPT I is located in the outer mitochondrial membrane, CPT II on the matrix side of the inner mitochondrial membrane), specific regulation, and physiological function (). The physiological inhibitor of CPT I is malonyl-CoA, the first intermediate in the synthesis of fatty acids (). Malonyl-CoA regulates fatty acid oxidation by inhibiting CPT I.
- When carbohydrates are consumed (high insulin/glucagon ratio), hepatic lipogenesis is increased, malonyl-CoA increases, CPT I is inhibited, and newly formed long-chain acyl-CoA fatty acids are converted to triglycerides instead of being oxidized. The triglycerides are released from the liver as very-low-density lipoproteins (VLDL) and stored in adipose tissue.
- In the fasting state (low insulin/glucagon ratio), the concentration of malonyl-CoA is low due to the low substrate flow through glycolysis, CPT I is active, and the free fatty acids supplied by adipose tissue undergo β-oxidation, producing ketone bodies in the process.
The translocase is located in the inner mitochondrial membrane and catalyzes the transport of acylcarnitines of any chain length and L-carnitine across the mitochondrial membrane (both directions) in stoichiometric exchange for the relevant substance according to concentration gradient. The long-chain acylcarnitines are transported into the mitochondria for β-oxidation in exchange for mitochondrial L-carnitine ().
17. Duran M. Disorders of mitochondrial fatty acid oxidation and ketone body handling. In: Blau M, Duran M, Blaskovics ME, Gibson KM, eds. Physician’s guide to the laboratory diagnosis of metabolic diseases. Berlin; Springer 2002: 309–334.
21. Campos Y, Huertas R, Lorenzo G, Bautista J, Gutierrez E, Aparicio M, Alesso L, Arenas J. Plasma carnitine insufficiency and effectiveness of L-carnitine therapy in patients with mitochondrial myopathy. Muscle Nerve 1993; 16: 150–3.
23. Bennett MJ, Hale DE. Defects of mitochondrial-oxidation enzymes. In: Ferrari R, DiMauro S, Sherwood G, eds. L-Carnitine and its role in medicine: from function to therapy. London: Academic Press, 1992: 187–206.
24. Richter T, Müller DM, Rotzsch C, Seim H. Carnitinmangel bei Kindern nach langzeitiger Sondenernährung über eine perkutane endoskopisch kontrollierte Gastrostomie (PEG). Monatsschr Kinderheilkd 1996; 144: 716–21.
27. Reuter SE, Evans AM, Fauli RJ, Chace DH, Fornasini G. Impact of haemodialysis on individual endogenous plasma acylcarnitine concentrations in end-stage renal disease. Ann Clin Biochem 2005; 42: 387–93.
28. Schoderbeck M, Auer B, Legenstein E, Genger H, Sevelda P, Salzer H, Marz R, Lohninger A. Pregnancy-related changes of carnitine and acylcarnitine concentrations of plasma and erythrocytes. J Perinat Med 1995; 23: 477–85.
33. Demirkol M, Sewell AC, Böhles H. The variation of carnitine content in human blood cells during disease – a study in bacterial infection and inflammatory bowel disease. Eur J Pediatr 1994; 153: 565–8.
Uric acid and its ionized form, monosodium urate, are the end products of purine metabolism in humans. The size of the body’s uric acid pool is the result of production and elimination. Hyper- and hypouricemia are not diseases. While hyperuricemia is primarily a symptom of gout, it also occurs secondarily in association with risk factors of cardiovascular disease and in association with metabolic syndrome and conditions with increased cell turnover. Hypouricemia is of limited pathological significance. Urinary uric acid excretion can be a useful marker in the workup of hyper- and hypouricemia.
- Together with physical examination in internistic medical check-up
- Family history of gout or history of renal calculi
- Clinical symptoms indicative of an acute gout attack
- Monitoring of gout therapy
- In patients with hypertension, hyperlipidemia, overweight, prediabetes, diabetes mellitus, chronic kidney disease
- Cardiovascular disease and stroke
- Suspected gestosis
- Diseases, conditions and therapies possibly causing secondary hyperuricemia (e.g., polycythemia vera, starvation diets, alcohol consumption, cytostatic therapy and tumor radiotherapy, cyclosporine therapy in transplant recipients)
- In children with hypo ketonemic hypoglycemia.
Detection of increased endogenous uric acid synthesis:
- Gout in childhood and adolescence
- Nephrolithiasis associated with the formation of uric acid- or calcium-containing kidney stones with normal or borderline serum uric acid levels
- Conditions associated with hypouricemia.
Principle: uric acid is degraded by uricase to allantoin and H2O2. The resulting H2O2 is quantitated by use of catalase and aldehyde dehydrogenase (ADH). The increase in NADPH concentration, measured by change in absorbance at 340 or Hg 334 nm is proportional to the amount of uric acid ().
In the first step uric acid is oxidized by oxygen and uricase form allantoin, carbon dioxide and H2O2. For routine diagnostic purposes most assays involve a peroxidase or catalase system coupled with an oxigen acceptor to produce a chromogen.
- Trinder’s reaction: in the presence of peroxidase, H2O2 reacts with a chromogen system consisting of phenol and 4-aminophenazon to form a red chinonimine whose absorption is followed at approximately 500 nm. There are a number of modifications for this method which all involve the phenol component. Either chlorinated phenols or chlorinated benzene sulfonic acids are used .
- Kageyama’s reaction: catalase, in the presence of H2O2, converts methanol to formaldehyde. The latter reacts with acetyl acetone in the presence of ammonium ions, forming a yellow dye whose absorbance is measured at 410 nm .
- Serum, plasma (no EDTA, citrate, oxalate): 1 mL
- Urine: 24 h-collection without additive
- Uric acid/creatinine ratio: spontaneously voided urine sample without additives.
By definition, hyperuricemia is a condition when the plasma uric acid concentration exceeds the solubility limit of monosodium urate at 37 °C of 6.8 mg/dL (400 μmol/L). At higher concentrations the plasma is supersaturated, leading to the precipitation of monosodium urate under certain physical conditions. Hypouricemia is defined as a uric acid level ≤ 2 mg/dL (119 μmol/L).
In the period around the 1920s, uric acid levels in the population of industrial nations were below 3.5 mg/dL (210 μmol/L), increasing continuously up to 2-fold over the next 50 years. Levels in women are about 0.5 to 1 mg/dL (30–60 μmol/L) lower than those in men due to the uricosuric effect of estrogens. After menopause, uric acid levels in women increase and eventually approximate those in men (). The intraindividual and inter individual variation in serum uric acid is multifactorial and influenced by genetic and environmental factors. Approximately two-thirds of the uric acid produced daily is excreted via the kidneys, the remainder via the intestine in stool.
Hyperuricemia has a high prevalence in the population. The Framingham study found that 9.2% of men and 0.4% of women had hyperuricemia, and 19% of those with hyperuricemia had gout . A German study found that 2.6% of the female blood donors and 28.6% of the male donors were hyperuricemic. In women hyperuricemia often does not occur until after menopause.
Hyperuricemia is associated with other metabolic disorders, such as insulin resistance, diabetes mellitus, metabolic syndrome, obesity, hyperlipoproteinemia, excessive alcohol consumption and diseases such as hypertension and chronic renal insufficiency, and is an independent risk factor for cardiovascular disease /, /.
- Reduced renal uric acid excretion due to genetic defects (URAT1 transporter) or multiple causes (congestive heart failure, volume depletion, diuretics) that lead to increased renal tubular reabsorption of uric acid. 90% of individuals with gout-related hyperuricemia have reduced uric acid excretion. The extent of hyperuricemia is also influenced by environmental factors.
- Overproduction of uric acid due to a diet high in meat and fish, or excessive alcohol consumption.
The differentiation between uric acid overproduction and reduced renal elimination is evaluated by determining the uric acid excretion in a urine sample collected over a period of 24 h or calculating the uric acid/creatinine ratio in a spontaneously voided urine sample.
- Primary forms. These are most commonly caused by genetic variations of the urate transport molecules in the renal tubules which, in combination with a protein-rich diet, lead to increased renal reabsorption of uric acid (underexcretors). These patients require a 1–2 mg/dL (59–119 μmol/L) higher plasma uric acid concentration compared to healthy individuals to excrete the same amount of uric acid. In rare cases, the condition may be caused by hereditary enzyme defects which lead to increased uric acid production (over producers). Both inherited disorders often lead to gout, which is then classified as primary.
- Secondary forms. These include all cases in which hyperuricemia or gout is due to diseases that do not primarily affect purine metabolism. Causes include increased production of uric acid from exogenous or endogenous purines, reduced renal excretion of uric acid, or physiologically unspecific causes () .
At a pH of 7.4, approximately 90% of uric acid exists in the form of monosodium urate. Concentrations of 8 mg/dL (476 μmol/L) and higher can cause the urate to precipitate in the tissues. Gout occurs in 0.5–7% of men and 0.1% of women. There is no clear correlation between the serum uric acid concentration and the precipitation of a gout attack. According to one study , the annual incidence of gout is 0.5% for uric acid levels of 7.0–8.9 mg/dL (416–529 μmol/L) and 4.9% for levels above 9.0 mg/dL (535 μmol/L), with gout attacks usually only occurring after 20–40 years of persistent hyperuricemia. In patients with a congenital enzyme defect, the first gout attack usually occurs as early as in adolescence.
Hyperuricemia is diagnosed if uric acid levels in morning fasting serum are elevated two to three times on different days to concentrations above 6.0 mg/dL (357 μmol/L) in women and above 7.0 mg/dL (416 μmol/L) in men. To allow a general assessment, patients should remain on their usual diet, medication and alcohol intake as in the weeks/months prior to the test . For influences of drugs refer to .
Measurement of uric acid in urine
Once gout has been diagnosed, its possible causes must be investigated. The determination of urinary uric acid allows to answer the following questions:
- If hyperuricemia is present, is it of endogenous origin i.e., caused by disease, or of exogenous origin i.e., caused by diet?
- If nephrolithiasis is present, is the patient an over producer or underexcretor?
- If uric acid levels are borderline or elevated, is renal function impaired?
Uric acid excretion in the 24 h-urine sample and/or the uric acid/creatine ratio in spot urine allow the following diagnostic conclusions:
- A uric acid excretion up to 600 mg (3.57 mmol) on a low-purine diet and up to 800 mg (4.76 mmol) on a normal diet in hyperuricemic patients indicate that the hyperuricemia is due to impaired tubular uric acid secretion and is therefore primary. In these underexcretors, the fractional excretion of uric acid (FEUA) is less than 4.0% .
- An increased FEUA or uric acid/creatinine ratio is indicative of uric acid overproduction, regardless of whether serum uric acid is elevated. Normalization of uric acid excretion on a low-purine diet indicates that the uric acid is synthesized from exogenous purines. If this is not the case, then the uric acid is mainly a breakdown product of endogenous purines.
The following should be taken into account when interpreting uric acid excretion:
- Is the glomerular filtration rate (GFR) reduced? If this is the case, overproduction may be missed in the presence of normal uric acid excretion.
- In patients with impaired GFR, elevated uric acid excretion confirms overproduction of uric acid
- The uric acid/creatinine ratio in spot urine which, if above 0.80, is used an indicator for uric acid overproduction, correlates only moderately with uric acid excretion in 24 h-urine due to considerable diurnal fluctuation in uric acid excretion
- A free uric acid clearance rate above 10% indicates increased excretion, a rate below 4% indicates decreased excretion (see calculation in .
- Has the patient been taking any drugs that can influence the production and elimination of uric acid ()?
Although hyperuricemia has a high prevalence, most people affected by it do not develop any clinical symptoms. However, the incidence of complications correlates with the level of serum uric acid () and the level of uric acid excretion.
- Acute gouty arthritis
- Inter critical gout
- Tophaceous, often primary chronic gout, accompanied by soft tissue and bone tophi
- Renal involvement in gout (e.g., nephrolithiasis, interstitial urate nephropathy (gouty kidney) with hypertension, and obstructive uric acid nephropathy
- Association with hypertension, metabolic syndrome and cardiovascular disease.
Acute gouty arthritis
Acute gouty arthritis clinically presents as severely painful mono arthritis, predominantly of the first metatarsal phalangeal joint (podagra). The pain often lasts for a week and is self-limiting. Uric acid levels can be normal during an acute attack and are therefore best measured 2–3 weeks after the attack. Since uricemias are often detected early during a checkup, timely medically prescribed treatment has led to a decline in the incidence of acute gouty arthritis.
Inter critical gout
This is the condition that occurs after the acute gout attack has resolved and the patient has become asymptomatic. If no prophylactic therapy is initiated, the intervals between attacks can become shorter and the attacks longer. More joints can become involved in the disease and tophaceous gout can develop. Clinically, inter critical gout rarely presents with tophi, but radiographic examinations often reveal bone destruction.
Tophi are nodular masses of monosodium urate crystals which deposit in bones, usually near joints, in cartilage, bursae and synovial tendon sheaths. This form also occurs as primary chronic gout i.e., without previous acute gouty arthritis. It is associated with older age of onset, manifests as polyarticular gout, especially in the lower joints, and commonly also affects women .
About 30–40% of patients with an acute gout attack have a history of renal calculi. Approximately 40% of patients with myeloproliferative diseases also develop renal calculi. However, not all kidney stones in hyperuricemic patients are urate calculi. In gout patients, the prevalence of nephrolithiasis correlates with the level of serum uric acid and urinary uric acid excretion (). Approximately 85% of renal calculi in hyperuricemic patients contain uric acid.
Urate calculi form when urine becomes oversaturated with undissociated uric acid. Although hyperuricosuria may be present, the main causes of the formation of urate calculi are low urine pH and low urine volume, not hyperuricosuria . About 90% of patients with urate calculi have a first morning urine pH below 5.7, and many even have an average pH of 5.5 . All patients with gouty diathesis have an increased filtered uric acid load and acidic urine, but only 28% develop urate calculi on a low-purine diet . Besides gout, there are other causes associated with the formation of acidic urine, such as physical exercise and dehydration, which lead to the formation of urate calculi. In patients with an ileostomy or Crohn’s disease, dehydration is thought to be the main factor .
This nephropathy, which is also known as gouty kidney, is a manifestation of chronic gout. Here, precipitation of mono urate crystals in the medullary interstitium and renal pyramids has led to inflammatory changes. Urate nephropathy is associated with a limited glomerular filtration rate, proteinuria and hypertension.
Acute uric acid nephropathy is acute post renal failure caused by the precipitation of urate crystals in the tubules and collecting ducts due to acute severe overproduction of uric acid. It is promoted by dehydration and acidosis. Findings include hyperuricemia above 12 mg/dL (714 μmol/L) and a random spot urine uric acid/creatinine ratio above 1.0. In other forms of acute renal failure the ratio is below 1.0.
Acute uric acid nephropathy occurs in blast crises in leukemias before or during cytostatic therapy. It can also occur at the beginning of uricosuric therapy, if therapy concepts such as sufficient fluid intake, neutralization of urine, and up titration of uricosuric medication are disregarded.
- Resolve the acute gout attack. Most patients with acute attacks, in whom a diagnosis of crystal arthropathy was confirmed by joint puncture, are treated with non-steroidal anti-inflammatory drugs (NSAID), which have fewer side effects and a longer-lasting effect than colchicine. Alternatives are corticosteroids or ACTH. While all these drugs help eliminate the acute pain, they do not correct the cause of the hyperuricemia nor the deposition of urate crystals in the tissues. Colchicine is the drug of choice in patients in whom the diagnosis of crystal arthropathy is not confirmed.
- Prevent further gout attacks and reverse the complications of gout. The likelihood of a recurrent gout attack within a year is 78%, and within 5 years 89%. During this period, the gouty tophi continuously become larger, trigger a destructive inflammatory response in the tissues and lead to the destruction of cartilage and bones. The body’s uric acid pool increases continuously. To correct these complications, the uric acid pool must be normalized again. This requires serum uric acid levels to be reduced to below 6.8 mg/dL (404 μmol/L), or better, to below 5.0 mg/dL (297 μmol/L). Reducing the concentration only to 8 mg/dL (476 μmol/L), for example, is insufficient, because although the rate at which gouty tophi develop is slowed down, there is no reduction in the uric acid pool. Therapy with specific uric acid-lowering drugs containing xanthine oxidase inhibitors, such as allopurinol, begins 2–3 weeks after the acute gout attack. Patients treated with uricosuric drugs, such as sulfinpyrazone and probenecid, are typically under 60 years of age, have a creatinine clearance above 80 [ml × min.–1 × (1.73 m2)–1], uric acid excretion below 800 mg (4.76 mmol)/24 h, are on a specific diet (avoidance of offal, seafood, and fructose-containing drinks) and have no renal calculi.
- Eliminate associated conditions that contribute to the hyperuricemia and gout. These include criteria of the metabolic syndrome, such as obesity, hypertension, hypertriglyceridemia, and insulin resistance. In addition, excessive alcohol consumption must be avoided.
Hypouricemia is defined as a condition with serum uric acid concentration ≤ 2 mg/dL (119 μmol/L). It has a prevalence of 0.2–0.5% in outpatients and of about 1% in clinical patients. Hypouricemias usually occur without clinical symptoms and are coincidental findings . Hypouricemias can be caused by:
- Reduced uric acid formation. This form of metabolic hypouricemia is found in hereditary xanthinuria, hereditary purine nucleoside phosphorylase deficiency, and allopurinol therapy.
- Increased renal uric acid excretion. Causes include uricosuric drugs, syndrome of inappropriate secretion of antidiuretic hormone (SIADH), Fanconi syndrome, malignant diseases, AIDS, severe liver injury, severe burns, diabetes mellitus, and hyper eosinophilic syndrome.
- A combination of metabolic and renal hypouricemia.
In most cases, hypouricemia is caused by drugs that interfere with renal tubular uric acid transport . These include acetohexamide, allopurinol, azathioprine, bishydroxycoumarin, clofibrate, contrast agent, fenofibrate, fenoprofen, guaifenesin, halogenates, losartan, phenylbutazone, probenecid, salicylates, and tienilic acid. Once these drugs are discontinued, uric acid levels generally normalize within 14 days.
Blood should preferably be collected in the morning and after fasting, not after intense physical work or after excessive exposure to the sun, which can lead to considerable increases. A normal continental breakfast does not significantly increase uric acid levels. Uric acid is not subject to circadian variation, but undergoes daily fluctuations.
Anticoagulants and stabilizers
EDTA, citrate, oxalate, sodium fluoride, cyanide, formaldehyde and oxonic acid cause reduced levels by inhibiting uricase.
Method of determination
Methods that measure the H2O2 produced in a coupled reaction, may generally experience interference from light scattering due to turbidity of the sample, spectral interference by bilirubin and hemoglobin, or interference by reducing substances such as ascorbic acid or phenol-type substances that are similar to those of the O2 acceptor . Trinder’s reaction produces falsely low readings in the presence of calcium dobelisate and α-methyldopa. Homogentisic acid at levels above 50 mg/L leads to elevated values in the aldehyde dehydrogenase method and causes pseudo hypouricosuria in the uricase-peroxidase reaction .
Increased excretion of homogentisic acid occurs in alkaptonuria, a hereditary disease caused by a deficiency of the enzyme homogentisic acid oxidase (EC 18.104.22.168), which results in insufficient conversion of homogentisic acid to 4-maleylacetoacetate.
In men, uric acid levels reach a plateau at 20–24 years of age and subsequently remain constant through life provided that weight remains the same. In women, levels increase between the ages of 15 to 19 years, then plateau until menopause before rising again .
The lower uric acid concentration in women compared to men is due to higher uric acid clearance. A metabolic balance study of 19- to 32-year-old individuals on an isoenergetic formula diet showed uric acid levels of 3.0 ± 0.5 mg/dL (178 ± 30 μmol/L) for women and 4.1 ± 0.7 mg/dL (244 ± 42 μmol/L) for men . Women who use oral contraceptives have lower uric acid levels than women of the same age group who do not . Caucasians have higher uric acid levels than blacks .
To measure uric acid in urine, refrigerated samples must be warmed up for 60 min. at 37 °C or for 10 min. at 60 °C. Otherwise readings will be falsely low by 20%, since part of the precipitated sodium mono urate does not dissolve .
Approximately 3 days at room temperature.
The total amount of uric acid (C5H4N4O3) present in the body is referred to as uric acid pool and is approximately 1 g. Although purines are synthesized and metabolized in all tissues, uric acid synthesis only occurs in tissues that contain xanthine oxidase, mainly the liver and the small intestine ().
Approximately 80% of uric acid is eliminated through the kidneys and less than 20% through the intestine. The physiological renal excretion is up to 800 mg (4.76 mmol) per day. Uric acid reaches the large intestine through saliva, bile, gastric and pancreatic juices and is broken down into CO2 and NH3.
At pH 7.4, most uric acid circulates in plasma in an ionized form as monosodium urate and mono- potassium urate, only a small amount of it is present in the form of free acid. The solubility product of the salts is 8.4 mg/dL (500 μmol/L) and that of the free acid 6.8 mg/dL (400 μmol/L). Precipitation of urates, in particular in the less perfused tissues, occurs as a result of an increase in uric acid, cooling, or a pH shift towards acidosis.
The presence of hyperuricemia is the indicator of an increased uric acid pool, which can result from uric acid overproduction, reduced excretion of uric acid, or a combination of both. The uric acid pool can be elevated up to 30 g .
In 99% of cases, primary hyperuricemia is due to selective impairment of renal uric acid elimination. In healthy individuals, uric acid clearance is 8–10 mL/min., with approximately 6–12% of the filtered uric acid appearing in urine. The filtered uric acid is almost completely reabsorbed in the proximal tubule, subsequently secreted in the distal tubule, and partly reabsorbed. It is believed that, in hyperuricemia, tubular secretion of uric acid is impaired.
In less than 1% of cases, primary hyperuricemia is due to endogenous overproduction of uric acid. Possible causes include deficiencies of enzymes in uric acid metabolism or increased activity of enzymes (e.g., PRPP synthetase) ().
Lesch-Nyhan syndrome, an X-chromosomal disorder with a recessive inheritance pattern, is caused by deficient activity of hypoxanthine-guanine phospho-ribosyl transferase (HGPRT). This enzyme is responsible for the resynthesis of adenosine mono phosphate (AMP) and guanosine mono phosphate (GMP) from purine bases released in the endogenous degradation of DNA and RNA. HGPRT deficiency is associated with a reduced intracellular concentration of AMP and GMP. As a result, adenosine phospho ribosyl transferase (APRT), the enzyme which catalyzes the de novo synthesis of nucleotides, is not feedback-inhibited by the two nucleotides, thus promoting the de novo synthesis of purines from 5-phospho ribosyl-1-pyrophosphate. As a result, the de novo synthesis can be increased up to 20-fold, which leads to a significant increase in the pool of uric acid and to a 3–4 fold higher excretion of uric acid .
- The result of the urate crystal-dependent mechanism is tubular obstruction by uric acid precipitation. Uric acid has a pKa of 5.75 and is thus a weak acid. At pH 5.0, urine is saturated with uric acid at a concentration as low as 15 mg/dL (892 μmol/L), while at pH 7.0 saturation is reached at a concentration of 200 mg/dL (11.9 mmol/L). In the presence of acidic urine, uric acid can precipitate in the distal tubule and the collecting duct, causing tubular obstruction. Granulocytes migrate into the tissue and interstitial nephritis develops as a late effect of sterile inflammation. Depending on the interstitial uric acid concentration, which mirrors the serum concentration, needle-shaped monosodium urate crystals are deposited in the interstitium, resulting in the formation of urate micro tophi.
- The urate crystal-independent mechanisms are activated by acute changes in the autoregulation of renal blood flow. Reduced renal perfusion leads to tissue hypoxia, and the subsequent re perfusion injury triggers an inflammatory response. The damaged cells release cytokines and chemokines, and adhesion molecules are expressed, causing granulocytes and monocytes to accumulate in the peri tubular capillaries. This further reduces renal perfusion, leading to vascular and tubular injuries and the development of renal insufficiency.
Increased intake of dietary purines is not a significant factor in enzyme deficiency-induced hyperuricemia, but it does promote the manifestation of gout.
Secondary hyperuricemia is the result of purine overload. They can be due to endogenous or exogenous causes.
It is not always possible to clearly differentiate between primary and secondary hyperuricemia, because often an hereditary disposition to gout becomes manifest only through other influences (e.g., high intake of purines, or in the course of other underlying diseases).
Increasing nutritional purine intake leads to an increase in serum uric acid levels, where the magnitude of the increase depends on the type of purines consumed. AMP and GMP lead to a greater increase in uric acid when consumed as nucleotides than when consumed in the form of RNA and DNA. Due to its higher susceptibility to hydrolysis, RNA leads to a two-fold increase in uric acid compared to the same intake of DNA. Approximately 60% of RNA compared to only 30% of DNA is reabsorbed by the intestine.
Administration of sugars, such as fructose, sorbitol and xylitol, leads to an increase in serum uric acid levels. This is thought to be caused by an increase in the de novo synthesis of purine and increased formation of uric acid from preformed purines.
Fasting causes a rapid increase in uric acid as a result of increased endogenous production of purines due to the metabolism of endogenous substances on the one hand and reduced renal uric acid secretion due to acidosis on the other hand.
All conditions associated with acidosis lead to reduced renal uric acid excretion.
Urate has antioxidant properties in vivo and inhibits the destructive effect of reactive oxygen species (ROS) on proteins, lipids and deoxyribonucleic acids. An increased uric acid concentration is also thought to have an oxidative effect under certain conditions.
The enzyme xanthine oxidase, which catalyzes the conversion of the purines xanthine and hypoxanthine to uric acid, has two isoforms:
- The dehydrogenase form, which forms uric acid and NADH2
- The oxidase form, which generates uric acid and H2O2.
Uric acid excretion requires special transporters, which are located in the proximal tubular cells of the kidneys, in the intestinal enterocytes and the vascular smooth muscle cells. Their role in the homeostasis of uric acid is not yet well understood .
11. LI M, Hu X, Fan Y, Li K, Zhang X, Hou E, Tang Z. Hyperuricemia and the risk for coronary heart disease morbidity and mortality: a systematic review and dose-response meta-analysis. www.nature.com/scientific reports 2016: .
17. Richette P, Doherty M, Pascual E, Barskova V, Becce F, Castaneda J, et al. 2018 updated European Leage Against Rheumatism evidence-based recommendations for the diagnosis of gout. Ann Rheum Dis 2019; .
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38. Park JT, Kim DK, Chang TI, Kim HW, Chang JH, Park SY, et al. Uric acid is associated with the rate of residual renal function decline in peritoneal dialysis patients. Nephrol Dial Transplant 2009; 24: 3520–5.
42. Niskanen L, Laaksonen DE, Lindström J, Eriksson JG, Keinänen-Kiukaanniemi S, Llanne-Parikka P, Aunola S, et al. Serum uric acid as a harbinger of metabolic outcome in subjects with impaired glucose tolerance. Diabetes Care 2006; 29: 709–11.
43. Jalal DI, Rivard CJ, Johnson RJ, Maahs DM, McFann K, Rewers M, Snell-Bergeon JK. Serum uric acid levels predict the development of albuminuria over 6 years in patients with type 1 diabetes: findings from the Coronary Artery Calcification in Type 1 Diabetes study. Nephrol Dial Transplant 2010; 25: 1865–9.
45. Strasak A, Ruttmann E, Brant L, Kelleher C, Klenk J, Concin H, et al. Serum uric acid and risk of cardiovascular mortality: a prospective long-term study of 83683 Austrian men. Clin Chem 2008; 54: 273–84.
48. Moriwaki Y, Yamamoto T, Nasako Y, Ohata H, Takahashi S, Tsutsumi Z, et al. Pseudohypouricosuria in alcaptonuria: homogentisic acid interference in the measurement of urinary uric acid with the uricase-peroxidase reaction. Ann Clin Biochem 1999; 36: 501–3.
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Acetoacetate (AcAc), β-hydroxy butyrate (β-HB) and acetone are ketone bodies. They are produced by ketogenesis, a mitochondrial process in which acetyl-CoA from the β-oxidation of fatty acids is converted to AcAc. A small amount of AcAc is converted to acetone by spontaneous decarboxylation. The greater portion is converted to β-HB by the enzyme β-hydroxy butyrate dehydrogenase (β-HBD) (). The ratio of ketone bodies in the blood depends on the redox state of the cells. In healthy individuals on a normal diet, AcAc and β-HB are present in approximately equimolar amounts. Acetone accounts for less than 5%.
Ketone bodies can be measured in blood and urine. In individuals on a normal diet, the capacity of the tissues to oxidize ketone bodies is sufficient to metabolize the amounts released by the liver. This is not the case in conditions of insufficient carbohydrate intake, fasting, and poorly controlled diabetes mellitus.
Differentiation of hyperglycemias with and without metabolic acidosis from other acidoses with a high anion gap such as:
- Diabetic ketoacidosis (diabetic coma)
- Hyperglycemic hyper osmolar non-ketotic syndrome
- Alcoholic ketoacidosis
- In combination with lactate measurement, differentiation from lactic acidosis and acidoses associated with sepsis, shock, intoxication (CO, cyanide, salicylates), severe hypoxemias, convulsions, malignancies (lymphomas)
- Intoxication with ethyl substitutes (glycols, methanol)
In combination with glucose and lactate, as a screening test for suspected congenital metabolic disorder in neonates and infants.
Although all three ketone bodies can be quantified in serum and urine, the following methods have become established for practical reasons:
- Quantitative measurement of β-HB in serum
- Combined measurement of β-HB and AcAc, also sequentially to differentiate between the two
- Rapid assays for determining ketone bodies in urine, which can also be used in diluted serum.
Quantitative determination of β-hydroxy butyrate (β-HB)
Principle: initially, the serum sample is deproteinized thus avoiding the spontaneous decarboxylation of AcAc into acetone. β-HB is determined by its quantitative conversion into AcAc and NADH catalyzed by the enzyme β-HBD. AcAc can also be determined according this principle. Since the reaction equilibrium is on the side of the β-HB, the AcAc is bound to hydrazone (e.g., by using hydrazine) thus removing it from the reaction. The increase of NADH2 is measured spectrophotometrically .
In a commercial card test, diaphorase catalyzes the reduction of nitro blue tetrazolium (NBT) by NADH2 to form a blue product whose absorbance is measured.
Quantitative measurement of total ketone bodies
Principle: β-HB and AcAc are determined as total ketone bodies in a recycling reaction. The serum/plasma sample is incubated with thio-NAD and β-HBD. AcAc is converted to β-HB and NADH2 to NAD (). Then β-HB is converted to AcAc again and thio-NADH is formed from thio-NAD. The increase in thio-NADH is measured spectrophotometrically and depends on the concentration of total ketone bodies in the sample . Separate measurement of β-HB is also possible. For this, AcAc is converted to acetone in a prior reaction.
Separate measurement of β-hydroxy butyrate and acetoacetate
Principle: AcAc and β-HB are measured in separate assay samples in a reaction catalyzed by β-HBDH. In the first assay sample, AcAc is converted to β-HB and the total amount of ketone bodies is determined, while in a second sample only β-HB is measured. The concentration of AcAc is determined from the difference in absorbance in assay sample 1–2. In an indicator reaction catalyzed by NADH oxidase, the resulting NADH is converted to H2O2. The latter oxidizes a chromogen in a reaction catalyzed by peroxidase, and the absorbance of this chromogen is measured photometrically .
Semi quantitative determination of ketone bodies in urine or serum (rapid assay)
Principle: in an alkaline environment AcAc and acetone react with sodium nitroprusside and glycine producing a purple-colored complex. Only AcAc and acetone are measured in the reaction, but not β-HB. The analytical sensitivity for AcAc is 50 mg/L and for acetone 500 mg/L.
Test strips allow a distinction to be made between several ketone body concentration ranges. The test may also be used for semi quantitative measurements in serum and plasma. The sample should be diluted with physiological saline solution at a ratio of 1 : 2 .
- Whole blood: 0.1–1 mL
- Serum, plasma (if only β-HB is measured): 1 mL
Calculation of the anion gap
- Anion gap (mmol/L) = [Na+] – ([Cl–] + [HCO3–])
- Reference interval 8–16 mmol/L.
Normal anion gap metabolic acidoses
These acidoses are associated with hyperchloremia and can be caused by systemic infections, renal tubular acidosis, medication of carbo anhydrase inhibitors, and hyperkalemic acidosis.
These types of acidoses are associated with normochloremia or occasionally with hypochloremia and may result from:
- Ketoacidosis; AcAc and β-HB are elevated (e.g., in diabetic and alcoholic ketoacidosis and congenital metabolic disorders such as organic acidurias)
- Lactic acidosis; lactate is elevated (e.g., in impaired tissue perfusion and congenital lactic acidoses) (see )
- Uremia; reduced excretion of acids such as phosphates and sulfates
- Rhabdomyolysis; increased release of sulfur-containing amino acids
- Intoxication with salicylates and ethyl substitutes such as ethylene glycol, formaldehyde, toluol, methanol.
At physiologic pH, the ketone bodies β-HB and AcAc circulate in plasma as anions. The H+-ions of the ketone bodies are buffered by HCO3–, and reduce the HCO3– concentration resulting in metabolic acidosis. Since HCO3– as a measured anion is replaced by the not-measured keto anions, the anion gap increases. The retention of keto anions, which results in an increase in anion gap is quantitatively similar to the decrease in plasma HCO3–-concentration. The fractional reabsorption of AcAc and β-HB in the kidney is only 75–85%. Therefore, ketonuria occurs during periods of enhanced keto acid production because of the difference between the quantity of keto anions filtered compared with the amount reabsorbed. The absolute quantity of glucose and keto anions in diabetic ketoacidosis excreted in the urine is directly related to the glomerular filtration rate .
In patients with intact kidney function, there is a quantitative relationship between ketonemia and ketonuria. In the presence of ketonemia (β-HB and AcAc), urine assays show a positive 1+ positive result for levels ≥ 8 mg/dL (0.8 mmol/L), and a 3 + positive result for levels ≥ 13 mg/dL (1.3 mmol/L) . Using rapid tests for detecting ketosis can be problematic. The tests only respond to AcAc and acetone, but not to β-HB. The ratio of ketone bodies in the blood depends on the redox state of the cell. In severe ketoacidosis, for example, the ratio of β-HB to AcAc is shifted towards β-HB (e.g., 6 : 1) due to an enormous excess of NADH.
The concentration of AcAc in urine may be only just above the detection limit, even though the patient has severe clinical symptoms. Under treatment, clinical symptoms improve, the formation of excess NADH is reduced, and less AcAc is converted to β-HB, leading to increased AcAc in urine. This results in the paradoxical situation that the improvement of clinical symptoms is accompanied by apparently worsened ketonuria. Therefore, only the quantitative measurement of serum β-HB, or better β-HB and AcAc, reflects the progress of ketosis.
The main ketoacidoses are:
DKA is a metabolic derangement consisting of three concurrent abnormalities: high blood glucose, high ketone bodies, and metabolic acidosis /, /. Approximately 25–40% of children with type 1 diabetes present with DKA at initial manifestation, while in adult diabetics DKA develops as a result of a comorbidity due to poor compliance or incorrect insulin administration (insulin pump). Another type of diabetes associated with the production of ketone bodies besides type 1 is ketosis prone diabetes, which is an intermediate form between type 1 and type 2. About half of all type 1 patients have no autoantibodies at initial manifestation, but a lack of β-cell reserve and ketosis .
DKA is differentiated from hyperglycemic hyper osmolar non-ketotic syndrome (HHNS). The hallmark of HHNS is severe hyperglycemia, elevated serum osmolality, and extensive dehydration with absence of ketoacidosis. Approximately 20% of patients with DKA and HHNS are admitted to hospital in a comatose state .
Both HHNS and DKA are characterized by insulinopenia and clinically they differ in:
- Age of onset (DKA in adolescent type 1 diabetics, HHNS in older type 2 diabetics)
- Extent of dehydration (mild to moderate in DKA, severe in HHNS)
- Severity of ketosis (moderate to severe in DKA, lack of ketogenesis in HHNS).
In glycogen storage diseases, in particular type I glycogenosis (von Gierke disease), there is reduced hepatic synthesis of glucose, leading to hypoglycemia, lactic acidosis and ketonemia even after short periods of fasting. Hypoglycemia and ketonemia occur rarely in type IV and occasionally in type VI, in which they are milder in nature .
Detection limit of rapid assays
Rapid urine assays are based on the sodium nitroprusside method. The detection limit is 50 mg/L for AcAc and 500 mg/L for acetone; β-HB is not detected.
AcAc is unstable and quickly decarboxylated to β-HB and acetone. At a storage temperature of –20 °C the concentration decreases by about 40% and at a temperature of –80 °C it decreases by 15% within 40 days . Therefore, the serum proteins must be precipitated with perchloric acid immediately after collection if AcAc is to be measured. β-HB is stable for up to 4 h at 4 °C in whole blood and for up to 48 h in serum and plasma .
- Two acetyl-CoA molecules generated from the β-oxidation of fatty acids are condensed by the enzyme acetoacetyl-CoA thiolase to form the intermediate acetoacetyl-CoA
- A third acetyl-CoA molecule is subsequently condensed by HMG-CoA synthase to form the key intermediate β-hydroxy-β-methylglutary-CoA (HMG-CoA)
- HMG-CoA is then split into acetoacetate and acetyl-CoA by the enzyme HMG-CoA lyase
- Part of the acetoacetate is reduced to β-HB by β-HBD in the presence of NADH. The ratio of AcAc to β-HB depends on the intramitochondrial NADH/NAD ratio.
Ketosis develops as a result of excess production of acetyl-CoA. For example, β-oxidation of 1 mol of C16 fatty acid is converted to 8 moles of acetyl-CoA. This is enzymatically condensed with oxaloacetate derived from carbohydrate metabolism, to give citrate, which is the major component of the tricarboxylic acid cycle. An increase in the glucagon/insulin ratio increases fatty acid oxidation and thus the formation of ketone bodies. The increased formation of ketone bodies results from a reduction in the:
- Availability of carbohydrates (e.g., due to fasting, frequent vomiting, alcoholism, glycogen storage disease)
- Reduced glucose uptake in the cells due to insulin deficiency (e.g., in diabetic ketoacidosis).
DKA is the result of absolute or relative insulin deficiency in combination with increased activity of counter regulatory hormones (glucagon, catecholamines, cortisol, growth hormone). Absolute insulin deficiency is often present in children with undiagnosed diabetes type 1 or in insulin-dependent diabetics who fail to inject insulin. Relative insulin deficiency is due to elevated levels of counter regulatory hormones such as occur with vomiting, gastrointestinal disease with diarrhea, trauma, or sepsis.
- Increased production of glucose in the liver and kidneys by glycogenolysis and gluconeogenesis
- Reduced glucose uptake in muscle and fat tissues due to insulin deficiency-induced reduced translocation of (GLUT)4-glucose transporters of the cell membrane, resulting in hypoglycemia and hyper osmolality. Hyperglycemia above the renal glucose threshold of 180 mg/dL (10 mmol/L) in combination with hyper ketonemia results in osmotic diuresis with dehydration and electrolyte loss, which are often exacerbated by vomiting.
- Increased lipolysis and ketogenesis, resulting in ketonemia and metabolic acidosis
- Inhibition of hepatic glycolysis in which glucose is converted to pyruvate, which then is used for the synthesis of amino acids and lipids, the generation of ATP in the citric acid cycle, and the formation of NAD by conversion of pyruvate to lactate
- A low concentration of insulin triggers the mobilization of fatty acids from adipose tissue by stimulating hormone-sensitive lipase. The free fatty acids are transported to the liver where they are metabolized by β-oxidation, thus producing ketone bodies.
The main factor in the development of ketoacidosis in DKA is the excess of glucagon and its effect on the hepatocyte, since glucagon inhibits lipogenesis and stimulates fatty acid oxidation. The first step in the synthesis of free fatty acids is the conversion of acetyl-CoA to malonyl-CoA by the action of acetyl-CoA carboxylase. The free fatty acids are either used for lipogenesis or undergo mitochondrial fatty acid oxidation and ketogenesis ().
Glucagon inhibits acetyl-CoA carboxylase, resulting in less malonyl-CoA. Malonyl-CoA is a strong inhibitor of fatty acid oxidation since it inhibits carnitine-palmitoyl transferase I (CPT I), which mediates the transport of free fatty acids to the mitochondrion (). Due to the decrease in malonyl-CoA in diabetic ketoacidosis there is increased activity of CPT I, leading to increased fatty acid oxidation. The resulting excess acetyl-CoA is not fed into the mitochondrial citric acid cyle, but takes the alternative pathway, forming AcAc and β-HB ().
The pathway of ketone body production can be inhibited by activation of carbohydrate metabolism. In ketoacidosis induced by fasting this occurs by consuming an adequate amount of carbohydrates, in DKA by administering insulin, which promotes glucose uptake into the cells. Both cases lead to increased production of oxaloacetate, an acceptor for acetyl-CoA which feeds the latter into the citric acid cycle.
The insulin-antagonistic effect of catecholamines, cortisol and growth hormone in DKA leads to reduced peripheral glucose uptake. In the absence of insulin, catecholamines also promote the breakdown of triglycerides in adipocytes and increase the release of fatty acids.
- Stage I: lipoprotein lipase of the vascular endothelium fails to be activated by insulin. Triglycerides are not cleaved off by very-low-density lipoproteins (VLDL) and transported into the fat cells, resulting in the hypertriglyceridemia in DKA. In the tissue cells, lipase is elevated. As a result, increasing amounts of fatty acids are released into the circulation.
- Stage II: the counter regulatory hormones glucagon, catecholamines and cortisol stimulate hepatic gluconeogenesis and glycogenolysis in the skeletal muscle and inhibit glucose uptake and oxidation by cells. The result is excess glucose production relative to glucose use and hyperglycemia.
- Stage III: osmotic diuresis and dehydration. Glucosuria results in an osmotic diuresis with resultant polyuria and polydipsia. If fluid intake is maintained dehydration is minimal, and blood glucose will stabilize at about 300–400 mg/dL (16.7–22.2 mmol/L). If fluid intake cannot be maintained, as would occur during severe DKA or illness associated with vomiting, dehydration results. With severe dehydration the glomerular filtration rate (GFR) and the filtered glucose decrease, resulting in marked hyperglycemia. The GFR is reduced by approximately 25%. Blood glucose levels are near 600 mg/dL (33.3 mmol/L). Blood glucose concentrations above 800 mg/dL (44.4 mmol/l) usually indicate a GFR that is reduced by about 50% .
- Stage IV: the accumulation of glucose in the extracellular space causes an osmotic shift of cellular water to the extracellular compartment. This results in a dilutional hyponatremia. In the early phase of DKA, serum potassium and phosphate levels are normal or elevated, since the acidosis causes the potassium to shift to the extracellular compartment. Potassium losses are caused by urinary excretion of potassium along with keto acids and the effect of the increased aldosterone secretion as response of dehydration. As a result there is a total body potassium deficiency of 5–10 mmol/kg of body weight. Acidosis and hyperglycemia also cause a loss of phosphate.
- Brain edema
Brain edema is the most serious complication of childhood DKA . The incidence is about 1% with mortality rates between 21–50%. Osmotic activity of particles in brain cells during consistent hyperglycemia prevent cellular dehydration. As glycemia rapidly diminishes with onset of therapy, osmolytes remain within the brain cells causing an osmotic gradient that drives water from the extracellular compartment into the cytoplasm, causing intracellular swelling. This is due to the fact that osmolytes are eliminated from within the cell through osmolyte channels at a slow rate. Therefore water flow may be an issue of timing and cell membrane osmolyte channel number of the cell, rather than just an issue of osmotic gradient.
Hyperglycemic hyper osmolar non-ketotic syndrome (HHNS)
HHNS is characterized by hyperglycemia, increased serum osmolality, and prolonged dehydration in the absence of ketoacidosis (). Glucosuria reduces the renal concentration capacity, thus increasing the loss of water. The reduction of the intravascular volume in combination with the possible presence of renal insufficiency reduce the GFR and lead to a rise in blood glucose. Since patients with HHNS have a higher concentration of insulin in portal vein blood than those with DKA, the liver is able to metabolize fatty acids via a non-ketogenic pathway. Hyper osmolality and dehydration inhibit lipolysis, resulting in a reduced fatty acid supply for the liver. Patients with HHNS therefore do not have significant ketosis or acidosis. However, due to the higher glucose levels, they have greater volume depletion than DKA patients, leading to the development of pre renal azotemia .
Alcoholic ketoacidosis results when mobilization of fatty acids occurs in conjunction with a ketogenic state in the liver. This condition is caused by a decreased ratio of insulin to glucagon. Reduced insulin levels result from glycogen depletion from starvation, decreased gluconeogenesis, and suppression of insulin release due to activation of sympathetic nerves . Activation of the sympathetic nervous system and increased levels of growth hormone, cortisol, and ethanol account for the increased magnitude of fatty acid mobilization, as compared with simple starvation. Ethanol metabolism leads to an increased ratio of NAD/NADH that contributes to decreased gluconeogenesis and facilitates production of ketone bodies, specifically β-hydroxybutyric acid () /, /.
When keto acids enter the extracellular fluid, the dissociated H+ reacts with bicarbonate to generate CO2 and water. As a consequence, the bicarbonate level increases; this accounts for the increase in the anion gap .
26. Toledo JD, Modesto V, Peinador M, Alvarez P, Lopez-Prats JL, Sanchis R, Vento M. Sodium concentrations in rehydration fluids for children with ketoacidotic diabetes: effect on sodium concentration. J Pediatr 2009; 154: 895–900.
Lactate is the final product of anaerobic glucose metabolism. It is oxidized in the citric acid cycle in the presence of oxygen, or undergoes gluconeogenesis within the Cori cycle. Lactate is one of the intermediates of metabolism, whose concentration increases as metabolism is altered during hypoxia. Lactate is therefore considered a marker of tissue hypoxia.
Plasma, whole blood
- Prognosis and monitoring of circulatory shock and intoxications
- Diagnosis of occult tissue hypoxias with a normal arterial PO2 and monitoring of treatment results
- Evaluation of unclear cases of metabolic acidoses, especially those associated with increased anion gap and comatose patients
- Diagnosis of acute intestinal vascular occlusion
- Fetal distress during labor and delivery
- Primary test in children with suspected congenital metabolic disorders (e.g., in combination with glucose, ketone bodies and ammonia).
Acute inflammation in the central nervous system.
The measurement of L-lactate is based on the conversion to pyruvate. This reaction is either catalyzed by lactate dehydrogenase (LD) to form NADH + H+ or by lactate oxidase (LOD) to form H2O2.
Principle: lactate is oxidized to pyruvate by LD in the presence of NAD . The NADH formed is measured spectrophotometrically at 340 or 366 nm as a measure of lactate. The balance of the reaction is such that lactate formation is greatly favored. In order that quantitative turnover of lactate occurs, certain reaction conditions have to be met:
- Alkaline reaction environment; this causes the reaction equilibrium to shift towards pyruvate
- Pyruvate must be removed at equilibrium; accomplished by the transamination reaction
- The H+ ions must be captured; achieved by the presence of alkaline buffer environment.
Lactate oxidase dehydrogenase (LOD) catalyzed reaction
Principle of amperometric measurement: this method uses a lactate-sensitive electrode on which the enzyme LOD is immobilized by attachment to a membrane surrounding an amperometric electrode. LOD generates H2O2 from lactate and O2. The H2O2 diffuses towards a platinum electrode, which is maintained at a certain potential relative to a silver reference cathode. At the platinum electrode, H2O2 is oxidized to O2, resulting in a change of potential, which is directly related to the lactate concentration.
- Capillary blood: mix 1 volume part of capillary blood into 1 volume part of 0.6 mol/L perchloric acid (approximately 7%)
- Arterial or venous whole blood: collect in 5 mL tube containing 12.5 mg of sodium fluoride and 10 mg of potassium oxalate
- Arterial or venous plasma, obtained from stabilized whole blood (see above)
- Cerebrospinal fluid, centrifuged.
The lactate concentration in blood reflects the ratio of lactate production to lactate consumption by the different organs. Lactate is produced by muscle, in particular during intense physical exercise, as well as by the brain, intestine, and red cells. It is metabolized by the liver, the kidneys, and the heart. Lactate assays therefore do not measure the lactate turnover of a specific organ, but the measured concentration is the net result of the production and consumption of the whole organism. The baseline lactate concentration is kept constant within a narrow range. The quantitative contribution of an organ to the entry into or removal of lactate from the blood depends on influencing factors such as rest, exercise, hypoxia, diet, alcohol, and drugs. Critically ill and malnourished patients may have severe tissue hypo perfusion, but only slightly elevated lactate levels due to the lack of availability of metabolizable glucose .
Elevated blood lactate levels can be due to increased production, reduced clearance, or a combination of both, depending on clinical circumstances. Severe hyper lactatemia only develops in the setting of increased peripheral production together with reduced hepatic metabolic capacity due to a liver disease .
Lactate is the product of glycolysis. The reversible reaction expressed by the equation promotes lactate synthesis at a normal lactat/pyruvate ratio of 10 : 1.
If blood lactate is elevated, a differential diagnosis is required to distinguish between hyper lactatemia and lactic acidosis. Lactate levels of 18–45 mg/dL (2–5 mmol/L) are defined as hyper lactatemia, higher levels as lactic acidosis.
To assess the pathological quality of elevated lactate concentrations, the following biomarkers allow the evaluation of pathology:
- Blood pH, HCO3–, PCO2, PO2
- Calculation of the anion gap
- Ketone body concentration in serum or urine
- Monitoring of the lactate concentration
- Creatinine and urea
- Toxicological investigations
- Organic acids in urine.
- Moderately elevated lactate levels (18–45 mg/dL; 2.0–5.0 mmol/L)
- Absence of metabolic acidosis (pH > 7.30)
- Absence of major perfusion defects of organs.
Hyper lactatemia without acidosis occurs in the setting of increased glycolysis of glucose to lactate (e.g., intense physical work, catecholamine infusion, or alkalosis). Causes and diseases associated with hyper lactatemia without acidosis are shown in .
In contrast to hyper lactatemia, in lactic acidosis the homeostatic regulation of lactate metabolism has failed. This can be due to excessive stress on the existing regulation or due to mitochondrial dysfunction.
- Type A is caused by an imbalance of the requirement and supply of organs with oxygen. Due to hypo perfusion induced tissue hypoxia, glucose metabolism switches from aerobic mitochondrial to anaerobic cytoplasmic glycolysis. This results in reduced oxidation of pyruvate in the citric acid cycle, accumulation of lactate, and reduced synthesis of ATP.
- Type B is caused by an existing organic or systemic disease without primary indication of hypo perfusion and hypoxia of organs (e.g., sepsis, diabetes mellitus, acute or chronic liver disease, renal failure, malignant tumor, medication, drugs and toxins, as well as congenital metabolic disorders).
Hyper lactatemias with progression to lactic acidosis are listed in . Lactic acidosis is the most common type of metabolic acidosis. Like diabetic and alcoholic acidosis, it is associated with an increased anion gap. In contrast to ketoacidosis, which is also associated with elevated lactate, although usually at levels below 45 mg/dL (5.0 mmol/L), ketone bodies are not elevated in lactic acidosis.
In lactic acidosis, it initially seems easier to determine the severity of metabolic acidosis by measuring the pH rather than lactate, since the formation of lactate and H+ ions is a stoichiometric process. The H+ ions are formed during the hydrolysis of ATP and immediately used for oxidative phosphorylation under aerobic conditions. Under anaerobic conditions this step is inhibited, and for each molecule of lactate one H+ ion is produced. In pure lactic acidoses, such as hemorrhagic shock, there is thus a calculable relationship between the blood pH, the measured PCO2 and the concentration of HCO3– ().
This does not apply for complex conditions, and these can be predicted. They are:
- Concomitant renal insufficiency
- Preexisting disorders of acid-base-balance such as metabolic alkalosis in the presence of chronic obstructive pulmonary disease.
Even the increased anion gap that is present in hyper lactatemia does not provide any further information, since it is influenced by ketone bodies and other non-measured anions. This is the case (e.g., with organic acidurias and fatty acid oxidation defects) where the anion gap is not so much due to the lactate but rather due to the organic acids, and is often above 25 mmol/L. This is also the case with exogenously supplied organic acids such as salicylates.
Patients with lactic acidosis do not present with a clear clinical picture. Common symptoms are tachypnea, hypotension, and impaired mental state. In many cases, a combination of type A and type B lactic acidosis is present such as increased synthesis and decreased elimination of lactate and H+ ions .
The etiology of hereditary lactic acidoses can be of primary or secondary nature (). They are a primary phenomenon in disorders of pyruvate and hepatic glycogen metabolism, gluconeogenesis defects, disorders in the citric acid cycle, and respiratory chain disorders. They occur as a secondary phenomenon in disorders of acetyl-CoA metabolism, if pyruvate cannot enter the citric acid cycle.
Lactic acidosis in children primarily suggests a hereditary metabolic disorder, although it is much more frequently due to other causes such as shock, sepsis, cardiopulmonary diseases or drug poisoning.
Forearm ischemic work test
The test is used for the detection of some metabolic myopathies. When performed under ischemic conditions, the forearm ischemic work test according to McArdle () may induce exercise intolerance (muscle cramps, pain, rhabdomyolysis and compartment syndrome) in patients with glycogenosis .
A new non-ischemic forearm work test () avoids the exercise intolerance associated with McArdle’s test. The new test measures lactate and ammonia during grip exercise . shows the behavior of lactate and ammonia during the test for different myopathies.
Many inflammatory, vascular, metabolic and neoplastic diseases of the brain and meninges present with an elevated cerebrospinal fluid (CSF) lactate. The determination of lactate is of diagnostic importance in differentiating :
- Between bacterial and viral meningitis
- Between transitory ischemic attacks and generalized seizures in cases lacking history and clinical findings
- Between artificial blood contamination of CSF and cerebral or subarachnoidal hemorrhage.
The lactate concentration is of prognostic value in vascular and traumatic brain diseases and in intoxications, but not in inflammatory diseases. The behavior of CSF lactate in meningeal and cerebral diseases is shown in .
Arterial or capillary blood should be collected. Venous specimens should be obtained without the use of a tourniquet. The lactate concentration is generally 4.5–9.0 mg/dL (0.5–1.0 mmol/L) higher in venous blood than in arterial blood. Influencing factors such as venous stasis, problems with vascular puncture, or crying in children also cause elevations.
- Mix 1 mL of blood with 1 mL of ice-cold 7% perchloric acid immediately after collection, leave for 10 min., centrifuge and measure in the supernatant. If this cannot be done on the ward, place heparinized blood on ice and immediately transport it to the laboratory.
- Collect blood with a syringe containing 2 mg of sodium fluoride and 2 mg of potassium oxalate per mL of blood collected.
- In heparinized plasma obtained from ice-cooled whole blood, lactate increases by less than 0.9 mg/dL (0.1 mmol/L) within 120 min. at 4 °C .
- In fluoride- and oxalate-stabilized whole blood stored for 30 min. at room temperature and for 8 h at 4 °C, lactate increases by no more than 0.9 mg/dL (0.1 mmol/L) .
Assay-dependency of results
The enzymatic measurement in plasma and the amperometric analysis in whole blood produce similar results . Whole blood and plasma values measured with the enzymatic method are not comparable. The higher the hematocrit, the more they differ.
Leukocytosis increases the lactate concentration in stabilized whole blood by no more than 2.7 mg/dL (0.3 mmol/L) within 8 h . Glycolates and glyoxylic acid lead to falsely high lactate values in POCT analyzers that use aperometric measurement .
In most tissues dietary carbohydrates are metabolized to pyruvate via glycolysis in the cytoplasm of the cells. One molecule of glucose is converted into two molecules of pyruvate, generating energy for the cell in the form of two molecules of ATP without consuming O2 /, /.
- Oxidative decarboxylation. Pyruvate is decarboxylated to acetyl-CoA in the mitochondrion by pyruvate dehydrogenase, fed into the citric acid cycle and metabolized to CO2 and H2O. In combination with the oxidative phosphorylation of the respiratory chain, a total of 18 molecules of ATP are produced per decarboxylated molecule of pyruvate.
- Conversion to lactate by LD. Two molecules of ATP per molecule of pyruvate are produced. A resting person produces 1300 mmol of lactate per day, 40–60% of which is taken up by the liver and converted to glucose via gluconeogenesis (Cori cycle). Some gluconeogenesis also occurs in the skeletal muscles, heart and kidneys . Lactate can be converted back to pyruvate. This process uses four molecules of ATP. Since most pyruvate is fed into the citric acid cycle, its plasma concentration is low and the ratio of lactate to pyruvate is 10 : 1.
- Carboxylation to malate and oxalacetate.
- Transamination to alanine, catalyzed by alanine aminotransferase.
In tissues such as the red blood cells, brain, skeletal muscle, intestinal mucosa or the adrenal cortex, the pyruvate produced by glycolysis does not enter the mitochondrion, but is mostly converted to lactate. This is then transported to the liver, converted back to pyruvate and then to glucose through gluconeogenesis, or it is used in the synthesis of fatty acids.
The liver is the main organ involved in the production of glucose and clearance of lactate. In a fasting state, lactate is primarily used for gluconeogenesis due to increased activity of mitochondrial pyruvate carboxylase. In a non-fasting state, the oxidation of pyruvate to acetyl-CoA is the preferred pathway. It is stimulated by the pyruvate dehydrogenase complex in the mitochondrion.
Whether pyruvate is oxidized to acetyl-CoA or reduced to lactate depends on the NADH/NAD ratio. Adequate availability of NAD promotes the conversion to acetyl-CoA and thus the synthesis of ATP by oxidative phosphorylation. A decrease in the O2 supply to the vessels triggers a series of reactions:
- More O2 is extracted from capillary blood by expansion of the capillary bed per tissue volume. This especially affects organs that have sudden high energy consumption, such as the skeletal muscle and, less frequently, the heart muscle.
- If oxygen supply is inadequate, glycolysis is increased, causing lactate to accumulate in the cell. Lactate leaves the cell in exchange for OH– ions, mediated by a membrane-associated antiport system that is dependent on the blood pH. While alkalemia promotes the removal, acidemia increases the uptake of lactate into the cells.
Together with lactate, H+ ions leave the cell. However, hyper lactatemia does not necessarily lead to acidosis. Whether or not it develops depends on the lactate concentration, the body’s buffering capacity, and the coexistence of pathological conditions, such as sepsis or liver disease, which predispose the patient to tachypnea and alkalosis. In the presence of ischemic injury, for example, the liver produces lactate instead of utilizing it.
In hypovolemic and cardiogenic shock, blood lactate levels do not always correlate with the severity of tissue hypo perfusion. This is the case, for example, with hypo perfusion in the innervation area of the splanchnic nerve. In some cases there is no hyper lactatemia, while in other cases large amounts of lactate can be produced in the intestine, resulting in hyper lactatemia despite the absence of marked hypo perfusion. The lactate concentration is an indicator of the severity of shock and is of prognostic value only if the diagnosis of hypo perfusion is confirmed and other causes of hyper lactatemia can be excluded .
The severity of hyper lactatemia depends on the substrate availability. For example, severely ill or malnourished patients can have mild hyper lactatemia despite severe hypo perfusion, but a high mortality probability .
If anaerobic glycolysis prevails in hypo perfusion, the pH decreases. Clinical symptoms include dilatation of the smooth vascular muscles, vasodilatation and hypotension.
In sepsis, unforeseeable metabolic changes occur that lead to elevated glucose consumption and accumulation of lactate despite adequate oxygen supply to the organs. Endotoxinemia reduces the arterial glucose concentration and increases the glucose content in the muscles with normal plasma insulin levels. It has been suggested that one cause of the hyper lactatemia may be the conversion of a proportion of the enzyme activity of pyruvate dehydrogenase to an inactive isoform by endotoxin. As a result, more pyruvate is converted to lactate instead of entering the citric acid cycle. The accumulation of lactate in sepsis probably results from increased glucose turnover in combination with inadequate pyruvate metabolism in the citric acid cycle .
Hereditary hyper lactatemia
Congenital hyper lactatemia is primarily the result of disorders of energy metabolism and are due to defects in the nuclear or mitochondrial genome. The incidence is approximately 1 in 5,000 neonates, for glycogenoses it is approximately 1 in 25,000 births in Europe. Even though the individual disorders are rare, the hyper lactatemias they cause pose diagnostic problems, since hyper lactatemia is much more likely to be due to secondary causes than primary defects. Patients with primary hyper lactatemia usually have severe metabolic acidosis, hyperventilation and ataxia or an altered neurological state .
2. Toffaletti J, Hammes ME, Gray R, Lineberry B, Abrams B. Lactate measured in diluted and undiluted whole blood and plasma: comparison of methods and effect of hematocrit. Clin Chem 1992; 38: 2430–4.
14. Ariza M, Gothard JW, McNaughton P, et al. Blood lactate and mixed venous-arterial pCO2 gradient as indices of poor peripheral perfusion following cardiopulmonary bypass surgery. Intensive Care Med 1991; 17: 320–4.
16. Dettmer M, Holthaus CV, Fuller BM. The impact of serial lactate monitoring on emergency department resuscitation interventions and clinical outcomes in severe sepsis and septic shock: an observational cohort study. Shock 2015: 43: 55–61.
17. Nguyen HB, Rivers EP, Knoblich BP, Jacobsen G, Muzzin A, Ressler JA, et al. Early lactate clearance is associated with improved outcome in severe sepsis and septic shock. Crit Care Med 2004; 32: 1637–42.
18. Bortwick IA, Brunt LK, Mitchem KL, Chaloner C. Does lactate measurement performed on admission predict clinical outcome on the intensive care unit? A concise systematic review. Ann Clin Biochem 2012; 49: 391–4.
26. Lange CM, Bojunga J, Hofmann WP, Wunder K, Mihm U, et al. Severe lactic acidosis during treat-ment of chronic hepatitis B with entecavir in patients with impaired liver function. Hepatology 2009; 50: 2001–6.
30. Cheung PY, Robertson CMT, Finer NN. Plasma lactate as a predictor of early childhood neurodevelopmental outcome of neonates with severe hypoxaemia requiring extracorporal membrane oxygenation. Arch Dis Child 1996; 47: F47–F50.
Data expressed in μg/dL, values are x ± 2 s or * median ± s; values for venous plasma; conversion: μg/dL × 0.5872 = μmol/L
MCAD, medium-chain acyl-CoA dehydrogenase deficiency
Amino acids, organic acids
↑, elevated; n, normal; ↓, reduced; +, present; AS, argininosuccinate synthetase; AL, argininosuccinase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamylase; HHH, hyperornithinemia-hyperammonemia-hypercitrullinemia syndrome; LPI, lysinuric protein intolerance; HI/HA, hyperinsulinism-hyperammonemia syndrome; Kb, ketone bodies
Conversion: mg/dL × 17.104 = μmol/L, * Values expressed as 2.5 th and 97.5th percentiles
Clinical and laboratory findings
Clinical and laboratory findings
Clinical and laboratory findings
Birth weight (g)
Risk factors: Apgar < 3 at 5 min; PaO2 ≤ 40 mmHg at > 2 h, pH ≤ 7.15 at ≥ 1 h,
Birth weight < 1,000 g, hemolysis; clinical or central nervous system deterioration;
total protein ≤ 4 g/dL or albumin ≤ 2.5 g/dL
PTT, prothrombin time
The median survival time is 11 months in stage 1, three months in stage 2 and one month in stage 3. The 1-year survival rates in stages 1–3 are 39%, 12% and 3%, respectively.
Data in μmol/L
No further significant changes after 1st year of life. Data in μmol/L.
* NCP, non-collagen protein, specified as x ± 2 s
Data expressed in μmol/L
Diseases, defects, metabolic situations
Clinical and laboratory findings
Clinical and laboratory findings
Data expressed in mg/dL (μmol/L). For children values are the 2.5th and 97.5th percentiles, for adults x ± 2s. Conversion: mg/dL × 59.485 = μmol/L
Conversion of uric acid: mg × 59.48 = μmol
Reduced renal elimination
Increased uric acid clearance
Decreased/normal uric acid clearance
HGPRT, hypoxanthine-guanine phosphoribosyltransferase; PRPP, phosphoribosyl pyrophosphate
Cr, creatinine; UA, uric acid; S, serum; U, urine
* In low doses, ** In higher doses
Values expressed in mg/dL (μmol/L)
* Values expressed in mg/dL (μmol/L); ** Data in mg (mmol/L)
Clinical and laboratory findings
* Hyperglycemic hyperosmolar non-ketotic syndrome; pos., positive; neg., negative
Data expressed in mg/dL (mmol/L); values are 5th and 95th percentiles; conversion: mg/dL × 0.11 = mmol/L.
Clinical and laboratory findings
Clinical and laboratory findings
Figure 5.1-1 Structural and functional organization and regulation of the hepatic and renal ammonia metabolism, modified from Ref. /, /. NH4+ ions produced during proteolysis in the liver cell are either detoxified via the urea cycle or temporarily stored in the mitochondria in the form of glutamine. NH4+ produced in different tissues are transported to the liver in the form of glutamine and channeled into the urea cycle like endogenous glutamine. If acidosis is present, HCO3– is conserved and the urea cycle is down regulated. Under these conditions, the kidneys maintain the homeostasis of NH4+ by increased absorption of glutamine and by excretion of NH4+ in urine.
In urea cycle defects, one of the following 5 enzymes of the cycle is deficient: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (AS), argininosuccinase (AL), arginase.
The function of the urea cycle depends on the availability of acetyl-CoA from fatty acid oxidation and pyruvate metabolism. Disorders of these metabolic pathways lead to the development of secondary hyperammonemias.
NAGS, N-acetyl-glutamate synthetase; GLD, glutamate dehydrogenase; AST, aspartate aminotransferase
Figure 5.1-2 Metabolic pathways of glutamate, modified from Ref. . The figure shows the pathways of urea synthesis, of energy synthesis in the citric acid cycle, of the formation of the neurotransmitter γ-aminobutyric acid, of the buffering of protons, and of the synthesis and degradation of amino acids. NAG, N-acetyl glutamate; α-KG, α-ketoglutarate; GAD, glutamate decarboxylase; GLD, glutamate dehydrogenase; GABA, γ-aminobutyric acid.
Figure 5.2-2 Hour-specific bilirubin nomogram showing the increase in bilirubin in term or near-term neonates. The increase of the low-risk line (40th percentile) is 0.1 mg × dL–1 × h–1, that of the intermediate-risk line (75th percentile) 0.15 mg × dL–1 × h–1, and that of the line representing high risk of bilirubin encephalopathy (95th percentile) 0.20 mg × dL–1 × h–1. With kind permission from Ref. .
Figure 5.2-4 Structure of unconjugated bilirubin (Bu). Due to its two propionic acid side chains, Bu primarily appears non-apolar; simplified structure at the top. The folded structure in the ZZ conformation (bottom) is apolar, since the propionic acid chains are firmly linked to the pyrrole nitrogens via bridging hydrogen bonds.
– Its vital role in the β-oxidation of long-chain fatty acids in the mitochondria, whereby the activated fatty acids are transported across the inner mitochondrial membrane in the form of acylcarnitines. This function can be impaired due to L-carnitine deficiency or reduced carnitine palmitoyl transferase (CPT) activity.
– Its influence on the degree of acetylation, particularly of coenzyme A, one of the key substances in intermediary metabolism. Many of the pleiotropic effects of L-carnitine are attributable to the regulation of the availability of coenzyme A for essential metabolic pathways.
Figure 5.3-3 Endogenous synthesis of L-carnitine. With kind permission from Ref. . While most tissues are capable of synthesizing butyrobetaine from protein-bound trimethyllysine, most of the butyrobetaine is produced in skeletal muscle. The hydroxylation of butyrobetaine to L-carnitine only occurs in the liver and kidney. The rate-limiting step in the endogenous synthesis of L-carnitine is the hydrolysis of muscle protein.
Figure 5.3-4 Regulatory role of carnitine palmitoyl transferase I (CPT I) in the liver. With kind permission from Ref. . The enzyme activity is controlled by malonyl-CoA. A rising concentration inhibits CPT I, which promotes the synthesis of triglycerides from fatty acid acyl-CoA and glycerate 3-phosphate. A low concentration activates CPT I, leading to increased fatty acid metabolism and formation of ketone bodies. ACC, acetyl-CoA carboxylase.
Figure 5.4-1 Serum uric acid concentration as a function of age, sex, and race. Data from Bogolusa Heart Study. With kind permission from Ref. . ○ Caucasian and female; □ Caucasian and male; ● Black and female; ■ Black and male.
Figure 5.4-2 Important enzymatic steps in the regulation of purine metabolism : 1. Phosphoribosyl pyrophosphate synthetase (PRPP synthetase); 2. Glutamine phosphoribosyl pyrophosphate amidotransferase; 3. AMP deaminase, 4. Adenosine deaminase; 5. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT); 6. Adenosine phosphoribosyl transferase (APRT); 7. Xanthine oxidase; ■ Feedback inhibition.
Figure 5.5-3 Pathophysiology of diabetic ketoacidosis. In insulin deficiency, glucagon stimulates the lipolysis in adipose tissue as well as hepatic glycogenolysis and gluconeogenesis. Inhibition of acetyl-CoA carboxylase occurs, resulting in malonyl-CoA deficiency. As a result, carnitine palmitoyl transferase is no longer inhibited, allowing more fatty acids to be transported into the mitochondria for oxidation and ketogenesis. ↑ promoting effect, ↓ inhibitory effect
Figure 5.5-4 Pathophysiology of hyperglycemic hyperosmolar non-ketotic syndrome (HHNS) (left) and diabetic ketoacidosis (DKA) (right). With kind permission from Ref. . The absence of ketoacidosis in HHNS can be explained firstly by the fact that there is still sufficient endogenous insulin and, secondly, by the fact that there is little activity of the insulin counter regulatory hormones as compared with DKA.
Figure 5.5-5 Pathophysiology of alcoholic ketoacidosis. Starvation, hypovolemia and relative insulin deficiency cause fatty acids to be mobilized from adipose tissue and to be metabolized to ketonic acids and lactate. Ethanol abuse leads to increased formation of NADH2 and depletion of NAD. Consequently, the citric acid cycle, gluconeogenesis, and the formation of pyruvate from lactate are inhibited. Modified from Ref. . ↑ promoting effect, ↓ inhibitory effect
Figure 5.6-1 Mean lactate (A–D) and ammonia (E–F) levels in the non-ischemic forearm work test in different myopathies. The bright areas correspond to x ± 2 s. With kind permission from Ref. . C, healthy individuals; DC, patients with myasthenia, but without histological muscle damage; Glyc, patients with type 5 glycogen storage disease (myophosphorylase deficiency, McArdle’s disease) and type 3 glycogen storage disease (debranching enzyme deficiency); Mito, patients with mitochondrial myopathy.
Figure 5.6-2 The cell obtains its energy aerobically via the citric acid cycle by oxidation of the substrates glucose, amino acids and fatty acids, and anaerobically via glycolysis. The reducing equivalents produced in this process, such as NADH2 and FADH2 , lead to the formation of ATP and the reduction of molecular O2 to H2O within the respiratory chain.
Lactate produced by glycolysis in the muscle cell is oxidized to pyruvate within the hepatocyte and then converted to glucose by gluconeogenesis. The muscle cell and hepatocyte thus participate in a metabolic cycle known as the Cori cycle. The glycolysis relies on the supply of NAD+, which is provided under aerobic conditions through the oxidation of NADH2 in the respiratory chain and under anaerobic conditions through the synthesis of lactate. In tissue hypoxia, the aerobic production of NAD is reduced, resulting in an increase in the NADH/NAD+ ratio, which promotes the production of lactate from pyruvate and thus the development of lactic acidosis.