Kaplan: Clinical Chemistry, 5th Edition
Clinical References - Methods of Analysis
Alanine Aminotransferase
James J Miller i i
Name: Alanine aminotransferase, ALT, L-alanine:2-oxoglutarate
aminotransferase, serum glutamate pyruvate transaminase, SGPT Clinical significance: click here
Enzyme number:  EC 2.6.1.2
Molecular mass:  Approximately 109,000 D
Chemical class:  Enzyme, protein
Method:CLSI RS4-A
Refer to Chapter 31, Liver Function, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.
Students’ Quick Hyperlink Review
•Principles of analysis and current usage
•Reference and preferred methods
•Specimen
•Interferences
•ALT reference intervals
•Interpretation
•ALT performance goals
•References
•Methods Summary Table
•Tables and Figures
•IFCC recommended procedure
i ALT
Previous and current authors of this method:
First edition:        Robert L. Murray
Methods edition: Robert L. Murray
Second edition:    Robert L. Murray
Third edition: Steven C. Kazmierczak
Fourth edition:    Steven C. Kazmierczak
reaction massFifth edition: James J. Miller
Principles of Analysis and Current Usage
Alanine aminotransferase (ALT) catalyzes the transfer of an amino group between L-alanine and L-glutamate; the corresponding ketoacids in this process are α-ketoglutarate and pyruvate
(Figure 1). In vivo, this reaction proceeds to the right to provide a source of nitrogen for the urea cycle. The pyruvate thus generated is available for entry into the citric acid cycle, whereas the glutamate is deaminated (catalyzed by glutamate dehydrogenase), yielding ammonia and α-ketoglutarate.
The reaction is reversible, with the chemical equilibrium favoring the formation of alanine and α-ketoglutarate. Because these products are relatively difficult to assay, however, analytical techniques typically force the reverse reaction, allowing quantitation of pyruvate. Two methods of ALT analysis have enjoyed wide popularity for routine clinical use: the Reitman-Frankel method [1], which involves the measurement of ALT activity by conversion of the reaction product, pyruvate, to its hydrazone (Table 1: Method 1); and the Wroblewski method [2] (Table 1: Method 2), in which the ALT reaction is coupled to a lactate dehydrogenase (LD) reaction.  This is the most common method in use today, and the former method is of historical interest only.
In the LD coupled method, the pyruvate product of the ALT reaction is reduced to lactate by nicotinamide adenine dinucleotide (NADH). The disappearance of NADH is monitored spectrophotometrically (at 340 nm).
Preferably, the absorbance change should be monitored continuously rather than by readings at several time points or only the end-point.
Reference and Preferred Methods
There have been many minor modifications in the enzymatic technique since its introduction by Wróblewski and LaDue [3-7].In addition, the specifications listed in the reference method published by the International Federation of Clinical Chemistry (IFCC) have been modified over the years, the most recent modification in 2002 being to optimize conditions for 37°C [7,8]. The main use of the IFCC reference method is to assay calibrators for use in routine methods. This allows routine methods to be traceable to the IFCC reference method, with the goal of reducing the biases between methods.
In the most recent IFCC reference method (2002) [7], 0.20 mL of serum is preincubated for 5 minutes in 2.00 mL of a mixture that contains all reactants except α-ketoglutarate. During this preincubation period, the added lactate dehydrogenase (LD) rapidly converts the endogenous pyruvate in the serum to lactate, and the pyridoxal phosphate cofactor joins with any inactive apoenzyme to form an increased amount of active ALT. With the addition of 0.20 mL of α-ketoglutarate, the primary reaction is initiated, and the concentrations shown in Table 2 are reached, exclusive of the small increases caused by the presence of endogenous material in the serum. After steady state is reached, the rate of NADH oxidation is monitored repeatedly at 339 nm. The rate of change in absorbance is corrected for a reagent blank.
Most reference methods, including the current IFCC procedure [7], have included pyridoxal phosphate. The need for addition of this cofactor has been widely debated. The essential nature of the cofactor has long been recognized, but because it is usually present in human serum in adequate amounts, many investigators do not add this component to the reaction mixture. In the occasional patient with severe vitamin B deficiency, this could lead to a serious underestimation of AST activity.
The presence of aminotransferases in the reagents is possible if the LD is not carefully prepared. Good-quality enzymes will not pose a problem; in any event, a blank determination will identify the problem. The presence of pyruvate is a potential source of error, since in the presence of endogenous LD, pyruvate will be converted to lactate, with simultaneous consumption of NADH. This problem is circumvented by the addition of a large excess of LD, so that endogenous pyruvate is converted during the preincubation period, eliminating interference during the measurement period.
Some older reference methods based on the Wróblewski coupled enzymatic method used phosphate buffer, which retards the recombination of added pyridoxal phosphate with the apoenzyme. If a large amount of the inactive enzyme is present, a falsely low activity will be observed. Activation of apoenzyme is more efficient in Tris buffer. However, NADH is somewhat less stable in Tris buffer than it is in phosphate buffer. For this reason, the Tris concentration is kept relatively low at 100 mmol/L.
Most routine methods today use the Wróblewski coupled enzymatic method and may be traceable to the current IFCC reference method.
The American Association for Clinical Chemistry proposed a method [3] for the small clinical chemistry laboratory that differs from the IFCC in that (1) a single reagent is used to avoid a two-step addition procedure, (2) the reaction is read after 150 seconds for the following 180 seconds, and (3) pyridoxal phosphate is not added.
The drawbacks of the dinitrophenylhydrazine method are that (1) the pyruvate produced by the reaction results in feedback inhibition of ALT, and thus specimens exhibiting high activity are spuriously lowered; and (2) any ketone in serum can react, though most do not result in an absorbance change in the region measured. However, acetoacetic acid and hydroxybutyric acid, both components of ketosis, do cause false elevations.
The U.S. National Institute of Standards and Technology (NIST) Standard Reference Material No. 909b is a lyophilized human serum preparation intended for use in evaluating the accuracy of routine methods. It is available to manufacturers and laboratories for the validation of ALT methods.
Specimen
Serum is the preferred specimen. Oxalate, heparin, and citrate do not inhibit the enzymatic activity but may introduce slight turbidity. Hemolyzed specimens should be avoided, since erythrocytes contain three to five times more ALT activity than is found in serum. ALT is stable in serum for 3 days at room temperature or 1 week at 4°C [9].A marked decrease in ALT activity is seen following freeze/thaw cycles 10]. ALT has been found to be stable in whole blood for up to 24 hours [11]. Urine has little or no activity and is not recommended for analysis.  Interferences
There is significant ALT activity in erythrocytes and significant hemolysis (>300 mg/dL hemoglobin) may artifactually increase apparent ALT activity. Icteric (bilirubin <40 mg/dL) and lipemic (triglycerides <3000 mg/dL) specimens generally do not interfere with measurement of ALT [12]. Metronidazole (Flagyl) may interfere with ALT methods because of its relatively high concentration and absorbance near 340 nm [13].
Alanine Aminotransferase Reference Interval
When analyzed at 37°C by methods employing activation with PP, the normal adult reference interval is approximately 8 to 47 U/L [14].However, it must be noted that ALT activities are age dependent. Healthy newborns have been reported to show an upper reference interval of up to double the adult le
vel. These values decline to adult levels by approximately 3 months of age. This increased activity has been attributed to seepage from the neonate’s immature hepatocytes, which have more permeable membranes. Men have been reported to show higher ALT values than women. Upper reference limits in individuals 10 years of age are approximately half of those seen in individuals at 40 years of age [15]. ALT activity peaks at approximately the fourth to fifth decade of life and then gradually declines.
Diurnal variations in ALT have been observed in both healthy individuals and those with cirrhosis. Up to 45% variation may be seen, with higher values being observed in the afternoon [16]. Other factors that have been reported to affect ALT include African-American race (15% higher than Caucasians), body mass index (40 to 50% higher with high body mass index), and exercise (20% lower in those who exercise) [17]. Ingestion of food causes no changes in measured ALT activity.
Interpretation
In contrast to aspartate aminotransferase (AST), which is found in both the cytoplasm and mitochondria, ALT is found exclusively in the cytoplasm. The tissue distribution of ALT and the ratio of ALT tissue activity to ALT plasma activity are presented in Table 3. Based on activity per gram of wet tissue, liver has the greatest amount of enzyme activity, with kidney being the next most active tissue.
Liver disease, in particular hepatocyte necrosis, is the most important cause of increased ALT activity. Because serum activities of ALT are unusually sensitive to liver damage, increases in ALT readily occur following moderate to excessive use of alcohol or following exposure to a variety of hepatotoxic agents. ALT is often used as part of a battery of enzymes to establish the presence and extent of liver damage. The half-life of ALT is
approximately 47 ± 10 hours [18].
ALT is usually higher than AST in most types of liver disease in which the activity of both enzymes is predominantly from the hepatocyte cytosol. When liver necrosis is substantial, as in individuals with alcoholic and viral hepatitis, mitochondrial AST is also released into the blood, and AST activity is usually higher than ALT. The ratio of AST to alanine aminotransferase (ALT), sometimes called the De Ritis Ratio, is often used to evaluate alcoholic liver disease [19] and severity of liver disease in viral hepatitis [20,21]. This ratio is only pertinent in isolated liver disease when comorbidities that increase AST are not present.
Alanine Aminotransferase Performance Goals
The current target for total error in ALT measurements is < 20% (CLIA). Biological variation data sugge
st that in patients with stable ALT activities, total error of < 30% is required for optimum use [22]. The interlaboratory coefficients of variation listed for various ALT methods in the 2007 College of American Pathologists Participant Summary Report (C-B) are all < 5%, indicating that this target is easily met using current ALT methods.
References
1 Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic
oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56-63.
2 Wróblewski F, LaDue JS. Serum glutamic-pyruvic transaminase in cardiac and hepatic
disease. Proc Soc Exp Biol Med 1956; 91: 569-571.
3 Butler TJ, Klotzsch SG, Osberg IM. Alanine aminotransferase, ALT provisional.  In
Faulkner WR, Meites, S, editors:  Selected methods of clinical chemistry. Washington
D.C.: American Association for Clinical Chemistry; 1982. p. 69-73.
4 Wilkinson JH, Baron DN, Moss DW, Walker PG. Standardization of clinical enzyme
assays:  a reference method for aspartate and alanine transaminases. J Clin Pathol 1972;
25: 940-944.
5 Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical
Pathology. Recommended methods for the determination of four enzymes in blood.
Scand J Clin Lab Invest 1974; 33: 291-305.
6 Enzyme Commission of the German Society for Clinical Chemistry. Recommendations
of the German Society for Clinical Chemistry. Z Klin Chem Klin Biochem 1972; 10:
281-91.
7 Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PFH, et al. IFCC
primary reference procedures for measurement of catalytic activity concentrations of
enzymes at 37°C. International Federation of Clinical Chemistry and Laboratory
Medicine. Part 4. Reference procedure for the measurement of catalytic concentrations of alanine aminotransferase. Clin Chem Lab Med 2002; 40: 718-24.
8 Bergmeyer HU, Horder M, Rej R. International Federation of Clinical Chemistry (IFCC).
Approved recommendation on IFCC methods for the measurement of catalytic
concentrations of enzymes. Part 3. IFCC method for alanine aminotransferase. J Clin
Chem Clin Biochem 1986; 24: 481-95.
9 Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time

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