Kaplan: Clinical Chemistry, 5th Edition

Clinical References - Methods of Analysis

Alanine Aminotransferase
James J Milleri 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

iALT
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
Fifth edition: James J. Miller

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

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.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

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.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

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 level. 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

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

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 suggest 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

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995; 33: 231-
238.
10 DiMagno EP, Corle D, O'Brien JF, Masnyk IJ, Go VL, Aamodt R. Effect of long-term
freezer storage, thawing, and refreezing on selected constituents of serum. Mayo Clin
Proc 1989; 64: 1226-1234.
11 Ono T, Kitaguchi K, Takehara M, Shiiba M, Hayami K. Serum-constituents analyses:
effect of duration and temperature of storage of clotted blood. Clin Chem 1981; 27: 35-
38.
12 McEnroe RJ, Burritt MF, Powers DM, Rheinheimer DW, Wallace BH. Eds. CLSI:
Interference Testing in Clinical Chemistry, Approved Guidelines –Second Edition CLSI
document EP7-A2, 2005 Accessed 19 Jan 2009
13 Karlsen RL, Kristiansen G, Solberg JH. Effects of metronidazole (Flagyl) on the
determination of serum ASAT on the SMA 12/60 Auto Analyser. Scand J Clin Lab
Invest 1983; 43: 175-177.
14 Stromme JH, Rustad P, Steensland H, Theodorsen L, Urdal P. Reference intervals for
eight enzymes in blood of adult females and males measured in accordance with the
International Federation of Clinical Chemistry reference system at 37˚C: part of the
Nordic Reference Interval Project. Scand J Clin Lab Invest 2004; 64: 371-384.
15 Soldin SJ, Hicks JM, editors. Pediatric reference ranges. Washington, D.C.: AACC Press:
1995.
16 Cordoba J, O'Riordan K, Dupuis J, Borensztajin J, Blei AT. Diurnal variation of serum
alanine transaminase activity in chronic liver disease. Hepatol 1998; 28: 1724-1725.
17 Dufour DR. Laboratory guidelines for screening, diagnosis and monitoring of hepatic
injury. Washington, D.C.: National Academy of Clinical Biochemistry; 2000.
18 Price CP, Alberti KGMM. Biochemical Assessment of Liver Function. In: Wright R,
Alberti KGMM, Karran S, Millward-Sadler GH, editors. Liver and biliary disease—
pathophysiology, diagnosis, management. London: W.B. Saunders; 1979. p. 381-416.
19 Majhi S, Baral N, Lamsal M, Mehta KD. De Ritis ratio as diagnostic marker of alcoholic
liver disease. Nepal Med Coll J 2006; 8: 40-42.
20 Giannini E, Risso D, Botta F, Chiarbonello B, Fasoli A, Malfatti F, et al. Validity and
clinical utility of the aspartate aminotransferase-alanine aminotransferase ratio in
assessing disease severity and prognosis in patients with hepatitis C virus-related chronic
liver disease. Arch Intern Med 2003; 163: 218-224.
21 Giannini EG, Zaman A, Ceppa P, Mastracci L, Risso D, Testa R. A simple approach to
noninvasively identifying significant fibrosis in chronic hepatitis C patients in clinical
practice. J Clin Gastroenterol 2006; 40: 521-527.
22 Fraser CG. Biological variation: from principles to practice. Washington, DC: AACC
Press; 2001, p. 140.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Tables
Table 1: Methods of Alanine Aminotransferase (ALT) Analysis
Method 1: Dinitrophenylhydrazine coupling (colorimetric) (Reitman and Frankel [1]);
quantitative
Principle of analysis: click here
Comments: Serum
Method 2: Enzymatic (ultraviolet monitoring) (Wróblewski and LaDue [2]); quantitative
Principle of analysis*: UV monitoring of NADH disappearance at 340 nm:

Usage: Serum; most frequently employed procedure

*Ala, Alanine; α-KG, α-ketoglutarate; Gl, glutamate; Lac, lactate; NAD , nicotinamide adenine dinucleotide;
+

NADH, reduced nicotinamide adenine dinucleotide; Pyr, pyruvate.

Table 2: Conditions of the 2002 IFCC ALT Reference Method

Component/Condition Concentration/Value
L-Alanine (mmol/L) 500
2-Oxoglutarate (mmol/L) 15
Buffer & concentration (mmol/L) Tris 100
pH 7.15
Pyridoxal phosphate (mmol/L) 0.1
NADH (mmol/L) 0.18
LDH (U/L) 1700
Volume fraction (v/v) 0.0833
Temperature (°C) 37.0
Wave length (nm) 339
Band width (nm) ≤2
Light path (mm) 10
Incubation time (s) 300
Delay time (s) 90
Measurement interval (s) 180
Readings (measurement points) ≥6

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 Table 3: Tissue Distribution of ALT in Normal Human Adult Tissues
Tissue U × 10-3 g of Wet Ratio of Activity in
Tissue Homogenate Tissue to That in Serum
Heart 7.1 444
Liver 44 2750
Skeletal muscle 4.8 300
Kidney 19 1188
Pancreas 2 125
Spleen 1.2 75
Lung 0.7 44
Serum 0.016 1

From Wróblewski F: Adv Clin Chem 1:313-351, 1958.

Figures
Figure 1: Amino transfer catalyzed by ALT.

Figure 2: Reaction of pyruvate with dinitrophenylhydrazine, as in Method 1.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Figure 3: Conversion of pyruvate to lactate with lactate dehydrogenase, as in Method 2.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Procedure: Kinetic Analysis of ALT [3]
Principle
The rate of the reaction in which an amino group is transferred from L-alanine to 2-
oxoglutarate to form pyruvate and L-glutamate catalyzed by the action of ALT is monitored by
use of an indicator reaction. The pyruvate formed is converted to lactate, and monitoring the
absorbance change at 339 nm of the consumed NADH follows the reaction.
Reagents

1. Tris, L-alanine buffer (121.1 mmol/L Tris, 630 mmol/L L-alanine, pH 7.15). Dissolve
1.47 g of tris(hydroxymethyl)aminomethane and 5.61 g of L-alanine (free acid) in 80 mL of
distilled water. Adjust to pH 7.15 at 37°C with 1 mol/L HCl (approximately 8.0 mL). Allow the
solution to cool to the calibration temperature, and bring to a volume of 100 mL in a volumetric
flask. This is Solution 1, stable for 3 months at 2°C to 8°C. Check for bacterial growth.

2. Tris/hydrochloric acid buffer (121.2 mmol/L Tris, pH 7.15). Dissolve 1.47 g of

tris(hydroxymethyl)aminomethane in 80 mL of distilled water. Adjust to pH 7.15 at 37°C with 1
mol/L HCl. Allow the solution to cool to the calibration temperature, and bring to volume of 100
mL in a volumetric flask. This is Solution 2, stable for 3 months at 2°C to 8°C. Check weekly for
bacterial growth.

3. Pyridoxal phosphate solution (6.3 mmol/L pyridoxal phosphate). Dissolve 16.7 mg of

pyridoxal phosphate in Solution 2, and bring to 10 mL volume. This is Solution 3. Stable for 1
week at 2°C to 8°C when stored in a dark bottle.

4. Reduced nicotinamide adenine dinucleotide (11.34 mmol/L NADH). Dissolve 16.1

mg of the disodium salt of NADH (or an equivalent amount correcting for water of hydration) in
2.0 mL of Solution 2. This is Solution 4, stable for 1 week at 2°C to 8°C when stored in a dark
bottle.

Diluent for reagent enzymes. Dissolve 1.2 g bovine serum albumin and 0.9 g NaCl in 100 mL
of water. Stable at least 1 month at 2°C to 8° C.

5. Lactate dehydrogenase (3.57 mkat/L or 214,000 U/L). Dilute the enzyme in Diluent
for Reagent Enzymes. This is Solution 5, stable for at least 2 days at 4°C.

6. Reaction solution. Mix 10 mL of Tris-aspartate (Solution 1) with 0.2 mL of pyridoxal

phosphate (Solution 3), 0.2 mL of NADH (solution 4), and 0.1 mL of the enzymes (Solution 5).
Mix thoroughly, and store in a dark bottle. Stable for 1 day at 2°C to 8°C.

7. Start reagent solution. Dissolve 407 mg of 2-oxoglutaric acid, disodium salt in 10.0 mL
of distilled water. Stable for 1 week at 2° to 8° C.
Assay
Equipment: Spectrophotometer with ≤ 2nm band pass at 339 nm with a constant
temperature cuvette capable of maintaining a constant temperature with less than 0.1°C
fluctuation. A recording spectrophotometer is preferable.
1. Add 2 mL of reaction solution and 0.2 mL of serum to the cuvette.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Clinical References - Methods of Analysis 3-11

2. Mix and allow to stand for 5 minutes.

3. Add 0.2 mL of start reagent solution.
5. Mix, wait 90 s, and record the change in absorbance for an additional 180 s. If no
recorder is available, record at least every 30 s.
6. A reagent blank is run by replacing the sample with 9 g/L sodium chloride in step 1.
7. A sample blank should be checked by replacing the start reagent in step 3 with 9 g/L
sodium chloride. The sample blank is not included in the calculation of AST activity, but
a rate > 1% of the ALT activity indicates that the material is not suitable as a calibrator.
For the reagent blank of the sample blank, replace both the start reagent and the sample
with 9 g/L sodium chloride.
Calculations
If the change in absorbance per second (ΔA/Δt) is greater than 0.0025 per second, dilute
the sample 5- to 10-fold with 9 g/L NaCl, and repeat the measurement.
Correct for the blank reaction using the following schema:

(ΔA/Δt)A = Measured reaction rate with serum in step 1

(ΔA/Δt)B = Measured reaction rate with NaCl in place of serum in step 1

ΔA/Δt ALT = (ΔA/Δt)A − (ΔA/Δt)B and

ALT activity = 1905 x ΔA/Δt ALT μkat/L

ALT activity in μkat/L can be converted to U/L by multiplying by 60.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.