Leakage of Enzymes from Cells

Enzymes are retained within their cells of origin by the plasma membrane surrounding the cell. The plasma membrane is a metabolically active part of the cell, and its integrity depends on the cell’s production of ATP. Any process that impairs ATP production by depriving the cell of oxidizable substrates or by reducing the efficiency of energy production by restricting the access of oxygen (ischemia or anoxia) promotes deterioration of the cell membrane. The earliest sign of impaired energy metabolism is the efflux of potassium with influx of sodium; water thus accumulates within the cell, causing it to swell. The next and most serious stage is the entry of Ca²⁺, which stimulates intracellular enzymes, leading to both cell damage and disruption of the cell membrane. Finally, free radicals formed during these processes may cause further damage. The membrane becomes leaky; if cellular injury becomes irreversible, the cell will die, although enzyme loss may also occur without the occurrence of irreversible injury. Small molecules are the first to leak from damaged or dying cells, followed by larger molecules, such as enzymes and other proteins. Cytosolic proteins appear early on in the plasma, followed much later by mitochondrial and membrane-bound enzymes. It appears that ATP must decline to below a certain level before substantial enzyme release occurs. Ultimately, the complete content of the necrotic cells is discharged.

Because of very high concentrations of enzymes within the cells—thousands or even tens of thousands times greater than concentrations in extracellular fluid—and because extremely small amounts of enzyme can be detected by their catalytic activity, increased enzyme activity in the extracellular fluid or plasma is an extremely sensitive indicator of even minor cellular damage, some causes of which are listed in Table 22-1.

A reduction in the supply of oxygenated blood perfusing any tissue will promote enzyme release, such as occurs in myocardial infarction. Cells of the affected region rapidly begin to deteriorate and die, releasing their protein and enzyme contents to the systemic circulation, which accounts for the rapid rise in serum biomarkers that is characteristic of this condition. The liver is also very sensitive to hypoxia, which results from diminished cardiac output (heart failure). Direct attack on the cell membranes by such agents as viruses or organic chemicals also causes enzyme release, which is particularly important in the case of the liver. Skeletal muscles also contribute enzymes to blood. Again, the cause may be poor perfusion, hypothermia, or direct trauma to the muscles (crush injuries). Infection, inflammation (polymyositis), degenerative changes (dystrophies), drugs, and alcohol (alcoholic myopathy) will cause enzyme leakage from myocytes.

Efflux of Enzymes from Damaged Cells

Once conditions for leakage of enzymes from cells have become established, the speed and extent to which the process is reflected in enzyme changes in the blood depend on several factors.

The driving force of enzyme release is the steep concentration gradient that exists between the interior and the exterior of the cells. The rate of escape of enzyme molecules is presumably controlled to some extent by diffusion; therefore, smaller enzyme molecules might be expected to appear in the extracellular fluid earlier than larger ones.

The way in which released enzyme molecules are transferred from the interstitial fluid to the blood varies from one tissue to another; they may pass directly through the capillary wall, or lymphatic transfer may occur. Direct transfer occurs to a large extent in the liver, which is a highly vascular tissue with many permeable capillaries, although evidence suggests that liver enzymes may also be subject to lymphatic transfer. On the other hand, the capillaries of skeletal muscle are relatively impermeable, and in this tissue it is probable that released enzymes mainly reach the circulatory system by way of drainage from the lymphatic system. Lymph drainage is also important in transporting enzymes released from damaged intestinal, pancreatic, and myocardial cells to the circulation, although, following myocardial infarction, a minor proportion of myocardial enzymes reaches the circulation by direct capillary transfer.

The intracellular location of the leaking enzymes affects the rates at which they appear in the circulation. As would be expected, the most sensitive indicators of cell damage are the molecules that are present in the soluble fraction of the cell. Release of structurally bound membrane proteins requires both a leaky cell membrane and a dissociation or degradation, which is a slower process.³⁹ Enzymes associated with subcellular structures, such as mitochondria, are less readily released into the circulation and often indicate irreversible cellular injury. This fact has been used in attempts to distinguish reversible leakage, presumed to reflect damage only to the cell membrane, from necrotic lesions, in which intracellular structures are destroyed.

The relation between tissue injury and the appearance of enzymes in the circulation is most clearly seen in myocardial infarction, in which a relatively short episode of damage is followed by rapid transfer of enzymes to the circulatory system. About 24 hours after a myocardial infarction, the pattern of relative activity of various enzymes in the circulatory system closely resembles that in myocardial tissue. These relationships are less clearly recognized in other conditions, such as chronic liver disease, in which enzyme release is a process that continues over a period of time. The pattern of relative enzyme activities in serum in chronic disease may also become distorted by differential rates of removal of enzymes from the circulation and possibly by differential changes in rates of enzyme synthesis in affected tissue.

Release of enzymes from damaged or dying cells and changes in the rate of enzyme production constitute the most important mechanisms by which changes in enzyme activity in the serum or plasma are produced. However, other possibilities exist and appear to account for some changes of diagnostic importance. For example, much of the γ-glutamyltransferase activity of liver cells is located on their exterior surfaces. It is possible that ectoenzymes such as this may be eluted from the surfaces, especially where detergent action of the blood is increased through accumulation of bile salts. This process does not involve cell damage in the sense of increased membrane permeability, as evidenced by lack of correlation between activities in the serum of γ-glutamyltransferase and the aminotransferases in liver disease of different types.

Altered Enzyme Production

Small amounts of intracellular enzymes physiologically present in the plasma can be assumed to result from wear and tear of cells or leakage of enzyme from healthy cells. This contribution of enzymes to the circulating blood may decrease as the result of a genetic deficiency of enzyme production (e.g., as is the case for alkaline phosphatase in hypophosphatasia or in individuals homozygous for the “silent” gene for serum cholinesterase), or when enzyme production is depressed as a result of disease (e.g., cholinesterase in liver disease). However, cases in which enzyme production is increased are of more general interest in diagnostic enzymology. For example, an increase in the number and activity of alkaline phosphatase–producing osteoblasts of bone is responsible for the increased concentration of alkaline phosphatase in the serum of normally growing children. Increased osteoblastic activity also accounts for increased concentrations of this enzyme in the serum in various types of bone disease.

The process of enzyme induction also increases enzyme production. An example of such induction is the increased activity of γ-glutamyltransferase in serum, which results from administration of drugs such as barbiturates or phenytoin, and from intake of ethanol.

Clearance of Enzymes

Significant evidence is available about the way in which enzymes are cleared from the circulation. Few enzyme molecules are small enough to pass through the glomerulus of the kidney; therefore urinary excretion is not a major route for elimination of enzymes from the circulation. An exception to this is α-amylase; increased concentrations of this enzyme in the blood (e.g., after acute pancreatitis) are accompanied by increased excretion in the urine.

Evidence now suggests that many enzymes are not inactivated in the plasma but are rapidly removed, probably by the reticuloendothelial system, such as the bone marrow, spleen, and liver (Kupffer cells), or, to a lesser extent, by nearly all cells in the body. The mechanism appears to consist of receptor-mediated endocytosis (the process of recognition, specific accumulation, and uptake of protein by specific cell surface receptors followed by fusion with lysosomes, digestion of ingested protein, and recycling of the receptor back to the cell membrane). For example, hepatic Kupffer cells have been shown to take up several tissue-derived enzymes—such as creatine kinase, adenylate kinase, cytoplasmic and mitochondrial aspartate aminotransferase, and malate and alcohol dehydrogenases—by receptor-mediated endocytosis, which may have affinity for lysine residues on these enzymes. The adult isoform of intestinal alkaline phosphatase is a galactosyl-terminal glycoprotein that reacts with a galactosyl-specific receptor on the hepatocyte membrane and undergoes subsequent endocytosis. This process is rapid, accounting for the extremely short plasma half-life of this isoform. However, in hepatic cirrhosis, in which considerable reduction in parenchymal cell mass often occurs, the plasma concentration and half-life of the isoform increase. Other alkaline phosphatase isoenzymes and isoforms are sialoglycoproteins that do not react with the galactosyl receptor and therefore are protected from rapid uptake from blood. Indeed, examples are known of excessive sialylation of alkaline phosphatases produced by malignant cells, prolonging their plasma half-lives and facilitating their detection. This example illustrates the importance of understanding the processes by which enzymes are cleared from plasma.

The half-lives of enzymes in plasma vary from a few hours to several days, but in most cases, the average half-life (t_1/2) is 6 to 48 hours. Rates of decay may also be expressed as k_d values—the fractional disappearance rate—and the relationship to t_1/2 values is as follows:

Typical disappearance rates from human blood for several clinically relevant enzymes are shown in Figure 22-1.

Clinical Significance

Serum CK is increased in nearly all patients when injury, inflammation, or necrosis of skeletal or heart muscle occurs.

Elevation of serum CK activity may be the only sign of subclinical neuromuscular disorders.⁴³ In case series, 30 to 44% of asymptomatic subjects with persistent hyperCKemia up to fivefold the upper reference limit (URL) have myopathy. Serum CK activity is greatly elevated in all types of muscular dystrophy. In progressive muscular dystrophy (particularly Duchenne sex-linked muscular dystrophy), enzyme activity in serum is highest in infancy and childhood (7 to 10 years of age) and may be increased long before the disease is clinically apparent. Serum CK activity characteristically falls as patients get older and as the mass of functioning muscle diminishes with progression of the disease. About 50 to 80% of asymptomatic female carriers of Duchenne dystrophy show threefold to sixfold increases in CK activity. High values of CK are noted in viral myositis, polymyositis, and similar muscle diseases. However, in neurogenic muscle diseases, such as myasthenia gravis, multiple sclerosis, poliomyelitis, and parkinsonism, serum enzyme activity is not increased. Very high activity is also encountered in malignant hyperthermia, an inherited life-threatening condition characterized by high fever and brought on by administration of inhalation anesthesia (usually halothane) to the affected individual.

Skeletal muscle that is diseased or damaged (such as by extreme exercise) may contain significant proportions of CK-MB owing to the phenomenon of “fetal reversion,” in which fetal patterns of protein synthesis reappear. Thus serum CK-MB isoenzyme may increase in such circumstances. This explanation may also account for the elevated CK-MB values sometimes observed in chronic renal failure (uremic myopathy).

In acute rhabdomyolysis due to crush injury, with severe muscle destruction, serum CK activities exceeding 200 times the URL may be found. If the CK remains below 5000 U/L (about 30 times the URL) during the first 3 days after the insult, the probability of developing acute renal failure appears to be low.⁵ Serum CK can also be increased by other direct trauma to muscle, including intramuscular injection and surgical intervention. Finally, a number of drugs when given at pharmacologic doses can increase serum CK activities. The drugs principally responsible are statins, fibrates, antiretrovirals, and angiotensin II receptor antagonists. Varying degrees of myopathy may occur with statin use, ranging from mild myalgic syndrome alone to rhabdomyolysis (0.02%).⁸⁶

Changes in serum CK and its MB isoenzyme following acute myocardial infarction have been the mainstay of diagnosis for many years.¹⁴ However, it is now more advantageous to use more cardiac-specific nonenzymatic markers, such as cardiac troponin I or T. CK-MB determination can still be used with some success to estimate the extent of myocardial necrosis to assist with assessment of infarct prognosis. When peak CK-MB is compared with estimates of infarct size, good correlations can be obtained (Table 22-4). A problem with using CK-MB for this purpose is the requirement for frequent sampling to ensure that peak CK-MB values are correctly identified.

Hypothyroidism is a common cause of endocrine myopathy. About 60% of hypothyroid subjects show an average elevation of CK activity fivefold greater than the URL. The major isoenzyme present is CK-MM, suggesting muscular involvement.

During normal childbirth, a sixfold elevation in maternal total serum CK activity occurs. Surgical intervention during labor further increases the activity of CK in serum. CK-BB may be elevated in neonates, particularly in brain-damaged or very low birth weight newborns. The presence of CK-BB in blood, usually at low concentrations, may however represent a physiologic finding in the first days of life.

Methods for Determination of Creatine Kinase Activity

Numerous photometric, fluorometric, and coupled enzyme methods have been developed for the assay of CK activity, using the forward (Cr → CrP) or the reverse (Cr ← CrP) reaction. Currently, all commercial assays for total CK are based on the reverse reaction, which proceeds about six times faster than the forward reaction.

CK catalyzes the conversion of CrP to Cr with concomitant phosphorylation of ADP to ATP. The ATP produced is measured by hexokinase (HK)/glucose-6-phosphate dehydrogenase (G6PD) coupled reactions that ultimately convert NADP⁺ to NADPH, which is monitored spectrophotometrically at 340 nm. Szasz and colleagues optimized the assay by adding N-acetylcysteine to activate CK, EDTA to bind Ca²⁺ and to increase the stability of the reaction mixture, and adenosine pentaphosphate (Ap₅A) in addition to AMP to inhibit adenylate kinase (AK).⁸⁵ A reference method based on this previous experience was developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) for the measurement of CK at 37 °C.⁷⁶

Specimens for CK analysis include serum and plasma heparin. Anticoagulants other than heparin should not be used in collection tubes because they inhibit CK activity. CK activity in serum is relatively unstable and is rapidly lost during storage. Average stabilities are less than 8 hours at room temperature, 48 hours at 4 °C, and 1 month at −20 °C. Therefore the serum specimen should be chilled to 4 °C if the serum is not analyzed immediately, and stored at −80 °C if analysis is delayed for longer than 30 days. It is not necessary to add any thiol agent for storage because the optimized assay formulation containing EDTA, 2 mmol/L, and N-acetylcysteine, 20 mmol/L, reactivates CK in serum to the extent of 99% after it has been stored for 1 week at 4 °C. A moderate degree of hemolysis is tolerated because erythrocytes contain no CK activity. However, severely hemolyzed specimens are unsatisfactory because enzymes and intermediates (AK, ATP, and G6P) liberated from the erythrocytes may affect the lag phase and side reactions occurring in the assay system.

Reference Intervals

Serum CK activity is subject to a number of physiologic variations. It is influenced by sex, age, race, muscle mass, and physical activity. The distributions of CK activity are notably skewed toward higher values in reference populations. Men have higher values than women, and blacks have higher values than nonblacks. In white subjects, the reference interval was found to be 46 to 171 U/L for males and 34 to 145 U/L for females, when measured with an assay traceable to the IFCC 37 °C reference procedure.⁸¹ Newborns generally have higher CK activity resulting from skeletal muscle trauma during birth. Serum CK in infants decreases to the adult reference interval by 6 to 10 weeks.

CK activity in the serum of healthy people is due almost exclusively to CK-MM activity (although small amounts of CK-MB may be present) and is the result of physiologic turnover of muscle tissue. Exercise and muscle trauma can increase serum CK.⁷ Sustained exercise, such as that performed by well-trained long-distance runners, increases the CK-MB content of skeletal muscle, which may produce abnormal serum CK-MB concentrations.

Methods for Separation and Quantification of Creatine Kinase Isoenzymes

Electrophoretic methods are useful for separation of all CK isoenzymes. The isoenzyme bands are visualized by incubating the support (e.g., agarose, cellulose acetate) with a concentrated CK assay mixture using the reverse reaction. NADPH formed in this reaction is then detected by observing the bluish-white fluorescence after excitation by long-wave (360 nm) ultraviolet light. NADPH may be quantified by fluorescent densitometry, which is capable of detecting bands of 2 to 5 U/L. The mobility of CK isoenzymes at pH 8.6 toward the anode is BB > MB > MM, with the MM remaining cathodic to the point of application. The discriminating power of electrophoresis also allows the detection of abnormal bands (Figure 22-3). Disadvantages of electrophoresis include that the turnaround time is relatively long, the procedure is highly labor intensive and is not adaptable to clinical chemistry analyzers, and interpretative skills are required.

Immunochemical methods are applicable to the direct measurement of CK-MB. Immunoinhibition techniques measuring the catalytic activity of the B subunit of CK dimer were first introduced. T

Although a modification was developed to avoid these interferences, interferences from CK-BB, macro-CKs, or CK-Mt led to low specificity of most immunoinhibitin methods. Furthermore, because the CK-B subunit accounts for one half of CK-MB activity, the change in absorbance should be doubled to obtain CK-MB activity. This resulted in a significant decrease in the analytical sensitivity of the method.

Currently, the most common approach is to measure concentrations of the CK-MB protein (“mass”) by using immunoassays with monoclonal antibodies.⁵⁸ Measurements use the “sandwich” technique, in which one antibody specifically recognizes only the MB dimer. The sandwich technique ensures that only CK-MB is estimated because neither CK-MM nor CK-BB reacts with both antibodies. Mass assays are more sensitive than activity-based methods with a limit of detection for CK-MB usually less than 1 µg/L. Other advantages include sample stability, noninterference with hemolysis, anticoagulants or other catalytic activity inhibitors, full automation, and fast turnaround time. With CM-MB mass assays, the upper reference limit for males is 5.0 µg/L, with values for females being less than male values, although many laboratories use a single reference limit (male).

Clinical Significance

Serum ALD determinations have been of some clinical interest in primary diseases of skeletal muscle. Some researchers believe that increased ALD activity is useful in distinguishing neuromuscular atrophies from myopathies in combination with the CK/AST ratio.²⁷ In general, however, measurement of ALD activity in the serum of subjects with suspected muscle disease does not add information to that available more readily from measurement of other enzymes, especially CK.²² At the time of this book’s writing, ALD measurement has largely been discontinued within clinical laboratories and is not routinely available.

Methods for Measurement of Aldolase Activity

All assay methods are based on the forward ALD-catalyzed reaction. In the analytical approach on which all commonly used procedures and kits are based, the ALD reaction is coupled with two other enzyme reactions. Triosephosphate isomerase (EC 5.3.1.1) is added to ensure rapid conversion of all GLAP to DAP. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is added to reduce DAP to glycerol-3-phosphate, with NADH acting as hydrogen donor. The decrease in NADH concentration is then measured.

ALD activity in serum is quite stable. Activity is unchanged at ambient temperatures for up to 48 hours and at 4 °C for several days. Hemolyzed specimens should not be used; plasma is preferred over serum because of the possible release of platelet enzyme during the clotting process.

Reference Intervals

The reference interval for the activity of ALD in adults is 2.5 to 10 U/L, measured at 37 °C. However, a definite sex difference has been noted, with men having higher values. Serum ALD activity in the neonate is fourfold that seen in adult, and in children is twice that in the adult. Adult values are attained by the time the child reaches puberty.⁹¹

Clinical Significance

In preliminary studies, GP-BB was significantly more sensitive than CK-MB and myoglobin for the diagnosis of acute myocardial infarction within the first 4 hours after onset of chest pain.² However, GP-BB is not a heart-specific protein, and its specificity as a marker for myocardial damage is limited.

Methods for Measurement of Glycogen Phosphorylase Isoenzyme BB

Manual ELISA assays are available for measurement of the isoenzyme GP-BB. The calculated upper reference limit is 10 µg/L.

Clinical Significance

Liver disease is the most important cause of increased transaminase activity in serum. In most types of liver disease, ALT activity is higher than that of AST; exceptions may be seen in alcoholic hepatitis, hepatic cirrhosis, and liver neoplasia. In viral hepatitis and other forms of liver disease associated with acute hepatic necrosis, serum AST and ALT activities are elevated even before the clinical signs and symptoms of disease (such as jaundice) appear. Activities for both enzymes may reach values as high as 100 times the upper reference limit, although 10-fold to 40-fold elevations are most frequently encountered. The most efficient aminotransferase threshold for diagnosing acute liver injury lies at seven times the upper reference limit (sensitivity and specificity >95%). Peak values of transaminase activity occur between the 7th and 12th days; activities then gradually decrease, reaching normal levels by the 3rd to 5th week if recovery is uneventful. Peak activities bear no relationship to prognosis and may fall with worsening of the patient’s condition.

Persistence of increased ALT for longer than 6 months after an episode of acute hepatitis is used to diagnose chronic hepatitis. Most patients with chronic hepatitis have maximum ALT less than seven times the upper reference limit. ALT may be persistently normal in 15 to 50% of patients with chronic hepatitis C, but the likelihood of continuously normal ALT decreases with an increasing number of measurements. In patients with acute hepatitis C, ALT should be measured periodically over the next 1 to 2 years to determine if it becomes and stays normal.¹⁵

The picture in toxic hepatitis is different from that in infectious hepatitis. In acetaminophen-induced hepatic injury, the transaminase peak is more than 85 times the upper reference limit in 90% of cases—a value rarely seen with acute viral hepatitis. Furthermore, AST and ALT activities typically peak early and fall rapidly.¹⁵

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of aminotransferase increases other than viral and alcoholic hepatitis. NAFLD includes a spectrum of liver pathology, from simple steatosis to nonalcoholic steatohepatitis (NASH), in which inflammatory changes and focal necrosis may progress to liver fibrosis, cirrhosis, and hepatic failure. NAFLD is now considered to be an additional feature of the “metabolic syndrome.” Indeed, serum aminotransferase elevation in NAFLD is associated with higher body mass index, waist circumference, serum triglycerides, and fasting insulin and lower HDL cholesterol—all features characteristic of this syndrome.

Aminotransferase activities observed in cirrhosis vary with the status of the cirrhotic process and range from the upper reference limit to four to five times higher, with an AST/ALT ratio (AAR) greater than 1. This appears to be attributable to a reduction in ALT production in a damaged liver, associated with reduced clearance of AST in advancing liver fibrosis. An AAR ≥1 has ≈90% positive predictive value for diagnosing the presence of advanced fibrosis in patients with chronic liver disease. Furthermore, the amount of elevation in the AAR can reflect the grade of fibrosis in these patients.

Twofold to fivefold elevations of both enzymes occur in patients with primary or metastatic carcinoma of the liver, with AST usually being higher than ALT, but activities are often normal in the early stages of malignant infiltration of the liver. Slight or moderate elevations of AST and ALT activities have been observed after administration of various medications, such as nonsteroidal anti-inflammatory drugs, antibiotics, antiepileptic drugs, statins, or opiates. Over-the-counter medications and herbal preparations are also implicated. In patients with increased transaminases, negative viral markers, and a negative history for drugs or alcohol ingestion, the work-up should include less common causes of chronic hepatic injury (e.g., hemochromatosis, Wilson’s disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, celiac disease, α₁-antitrypsin deficiency).⁶⁷

Although serum activities of both AST and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver-specific enzyme. Serum elevations of ALT activity are rarely observed in conditions other than parenchymal liver disease.³⁴ Moreover, elevations of ALT activity persist longer than do those of AST activity. Thus the incremental benefit of determination of AST, in addition to ALT, may be limited.

After acute myocardial infarction, increased AST activity appears in serum, as might be expected from the high AST concentration in heart muscle. AST activity also is increased in progressive muscular dystrophy and dermatomyositis, reaching concentrations up to eight times normal; they are usually normal in other types of muscle disease, especially in those of neurogenic origin. Pulmonary emboli can increase AST to two to three times normal, and slight to moderate elevations are noted in acute pancreatitis, crushed muscle injury, and hemolytic disease.

Generally, mitochondrial AST (m-AST) activity in serum shows a marked increase in patients with extensive liver cell degeneration and necrosis. Of particular interest is the usefulness of the ratio between m-AST and total AST activities for diagnosing alcoholic hepatitis. The ratio seems to identify the liver cell “necrotic type” condition (i.e., slight enzyme increase concomitant with relatively high activities of mitochondrial enzymes) typical of alcoholic hepatitis.⁵⁶

Several authors have described AST linked to immunoglobulins, or macro-AST. Typical findings include a persistent increase in serum AST activity in an asymptomatic subject, with absence of any demonstrable pathology in organs rich in AST. Increased AST activity might reflect decreased clearance of the abnormal complex from plasma. Macro-AST has no known clinical relevance. However, identification is important to avoid unnecessary diagnostic procedures in these subjects. Laboratory procedures for the demonstration of macro-AST include electrophoresis with specific enzyme stain (atypical origin band) and differential precipitation with polyethylene glycol (PEG) 6000 (see “Amylase” section later in this chapter).

Methods for Measurement of Transaminase Activity

The assay system for measuring transaminase activity contains two amino acids and two oxo-acids. Because no convenient method is available for assaying amino acids, formation or consumption of the oxo-acids is measured. Continuous-monitoring methods are commonly used to measure transaminase activity by coupling transaminase reactions to specific dehydrogenase reactions. The oxo-acids formed in the transaminase reaction are measured indirectly by enzymatic reduction to corresponding hydroxy acids, and the accompanying change in NADH concentration is monitored spectrophotometrically. Thus oxaloacetate, formed in the AST reaction, is reduced to malate in the presence of malate dehydrogenase (MD).

Pyruvate formed in the ALT reaction is reduced to lactate by lactate dehydrogenase (LD). The substrate, NADH, and an auxiliary enzyme, MD or LD, must be present in sufficient quantity so that the reaction rate is limited only by the amounts of AST and ALT, respectively. As the reactions proceed, NADH is oxidized to NAD⁺. The disappearance of NADH is followed by measuring the decrease in absorbance at 340 nm for several minutes, either continuously or at frequent intervals. The change in absorbance per minute (ΔA/min) is proportional to the micromoles of NADH oxidized and in turn to micromoles of substrate transformed per minute. A preliminary incubation period is necessary to ensure that NADH-dependent reduction of endogenous oxo-acids in the sample is completed before 2-oxoglutarate is added to start the transaminase reaction. After a brief lag phase, the change in absorbance (ΔA) is monitored. As already mentioned, supplementation with P-5′-P ensures that all transaminase activity of the sample is measured.

Because of the large numbers of AST and ALT activity measurements performed daily in clinical laboratories throughout the world, standardization of transaminase measurements is a priority need for patient care. As discussed in Chapter 8, the reference system approach, based on the concepts of metrologic traceability and the hierarchy of analytical measurement procedures, gives clinical laboratories and the medical community universal means of creating and ensuring the comparability of results. In this system, the IFCC reference measurement procedure forms the highest metrologic level and thereby constitutes the definition of the respective measurable enzyme quantity.³⁰ Primary IFCC procedures for the measurement of catalytic activity concentrations of AST and ALT at 37 °C have been published.^79,80 Values assigned to the manufacturer’s product calibrators and measurement results of lower metrologic levels, including those used in daily routine practice, should be traceable to these top-level reference measurement procedures, thus improving the accuracy and comparability of transaminase results. It should be remembered that the concept of the reference system is valid only if the reference procedure and corresponding routine procedures have identical, or at least very similar, specificities for the measured enzyme. Thus it will not be possible to calibrate procedures for aminotransferases that do not incorporate P-5′-P using a procedure that does, such as the IFCC reference procedure, because the ratio of pre-formed holoenzyme to apoenzyme differs among specimens.

AST activity in serum is stable for up to 48 hours at 4 °C. Specimens have to be stored frozen if they are to be kept longer. ALT activity should be assayed on the day of sample collection because activity is lost at room temperature, 4 °C, and −25 °C. ALT stability is better maintained at −70 °C. Hemolyzed specimens should not be used, especially when AST is measured, because of the large amount of this enzyme present in red cells.

Reference Intervals25,29

Using methods traceable to the IFCC reference system, the AST upper reference limit for adults, calculated as the 97.5-percentile of the reference distribution, is 35 U/L, with no significant sex-related differences. Conversely, a clear difference in ALT activities has been noted between adult males and females. Corresponding ALT upper reference limits are 60 U/L and 42 U/L respectively. ALT does not reveal a distinct age dependency during childhood, whereas serum AST activity in neonates and in children younger than 3 years old is twice that in adults. Adult values are attained by the time the child reaches puberty.

Methods for Separation and Quantification of AST Isoenzymes

AST isoenzymes can be separated into anionic (cytoplasmic AST) and cationic bands (m-AST) by electrophoresis. However, the low concentration of m-AST in normal sera is usually below the limit of detection of this method. Immunoprecipitation assays using antibodies directed against both mitochondrial and cytosolic isoenzymes allow instead measurement of low concentrations of the m-AST isoenzyme present in serum. A homogeneous inhibition assay using proteinase K (EC 3.4.21.14) for selective proteolysis of cytosolic AST has been described and made amenable to automation, permitting m-AST to be measured with convenience that approaches that of the total AST assay.⁶³

About 5 to 10% of the activity of total AST in serum from healthy individuals is of mitochondrial origin. The upper reference limit for m-AST activity measured at 37 °C is 3.0 U/L.

Clinical Significance⁷⁴

GLD is increased in the serum of patients with hepatocellular damage. Fourfold or fivefold elevations are seen in chronic hepatitis; in cirrhosis, increases are only up to twofold. Very large rises in serum GLD occur in halothane toxicity, and notable increases are seen in response to some other hepatotoxic agents.

GLD potentially offers differential diagnostic potential in the investigation of liver disease, particularly when interpreted in conjunction with other enzyme test results. The key to this differential diagnostic potential is to be found in the intraorgan and intracellular distribution of the enzyme as discussed earlier in this chapter. As an exclusively mitochondrial enzyme, GLD is released from necrotic cells; therefore, when compared with hepatic disorders with extensive necrosis, release is less in diffuse inflammatory processes, and in these conditions, the release of cytoplasmic enzymes, such as ALT, is quantitatively more pronounced. Together with m-AST, GLD is of value in estimating the severity of liver cell damage.

GLD is more concentrated in the central areas of the liver lobules than in the periportal zones. This pattern of distribution is the reverse of that of ALT. Pronounced release of GLD therefore is to be expected in conditions in which centrilobular necrosis occurs (e.g., as a result of ischemia, in halothane toxicity).

Methods for Determination of Glutamate Dehydrogenase Activity

Continuous-monitoring methods have been developed for determination of GLD using both forward and reverse reactions. The equilibrium favors the formation of glutamate, and higher reaction rates are observed when 2-oxoglutarate is used as a substrate. Serum is added to a solution of NADH, an ammonium salt, and ADP in buffer at pH 7.5, and the reaction is initiated by the addition of the substrate, 2-oxoglutarate. The rate of decrease in absorbance at 340 nm is measured. The German Society for Clinical Chemistry has published optimum reaction conditions for 37 °C.¹³ Oxamate is incorporated into the reaction mixture because this acid inhibits LD activity, avoiding the critical consumption of NADH by this enzyme in serum.

GLD activity in serum is stable at 4 °C for 48 hours and at −20 °C for several weeks.

Reference Intervals

The GLD upper reference limits are 6 U/L (women) and 8 U/L (men) when a method optimized at 37 °C is used.