Alanine aminotransferase (ALT, formerly SGPT) determination methods are fundamental laboratory techniques for assessing hepatocellular injury and monitoring liver function. This technical article explains the analytical principles, sample requirements, reagent systems, and operating procedures of the major ALT assay methods, including the IFCC-recommended kinetic UV method and the Reitman-Frankel colorimetric method. It also covers reference intervals, interference control, quality assurance, and the clinical, pharmaceutical, and industrial applications of ALT measurement.
Technical Overview
Alanine aminotransferase (ALT, EC 2.6.1.2), historically known as serum glutamate pyruvate transaminase (SGPT) or glutamate pyruvate transaminase (GPT), is a pyridoxal phosphate-dependent cytosolic enzyme that catalyzes the reversible transfer of an amino group from L-alanine to alpha-ketoglutarate, producing pyruvate and L-glutamate. The enzyme is present at its highest concentration in liver tissue, with lower amounts in kidney, heart, and skeletal muscle, which makes serum ALT activity a relatively specific biochemical marker of hepatocellular damage.
ALT determination methods quantify the catalytic activity of the enzyme rather than its mass. One unit (U) of ALT activity is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under specified conditions, and results are reported as U/L. Modern ALT assays are standardized against the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference measurement procedure at 37 degrees Celsius, which specifies buffer composition, substrate concentrations, pH, and optional pyridoxal-5-phosphate activation to ensure comparability of results between laboratories and instrument platforms.
- Enzyme commission number: ALT, EC 2.6.1.2
- Catalyzed reaction: L-alanine + alpha-ketoglutarate to pyruvate + L-glutamate
- Primary clinical role: detection and monitoring of hepatocellular injury
- Standard reference procedure: IFCC method at 37 degrees Celsius
- Common reporting units: U/L (microkatal/L in some regions)
Testing Samples
Serum
Serum is the preferred and most widely used sample matrix for ALT determination. Venous blood is collected into a plain tube or a serum-separator tube, allowed to clot for 20 to 30 minutes at room temperature, and then centrifuged to obtain a clear, cell-free serum specimen. Serum should be visually inspected and grossly hemolyzed, icteric, or severely lipemic specimens should be documented or rejected according to laboratory policy, because these conditions can interfere with photometric measurement.
Plasma
Heparinized plasma is an acceptable alternative to serum on most automated chemistry analyzers and provides faster turnaround because no clotting time is required. Anticoagulants such as EDTA, citrate, oxalate, and fluoride are generally not recommended because they can dilute the sample, chelate essential metal ions, or chemically interfere with the coupled indicator reaction. If a laboratory wishes to accept plasma collected in these anticoagulants, the method must first be validated against serum through a method-comparison study.
Sample Handling and Stability
ALT is a relatively stable enzyme compared with many other clinical enzymes. Under typical conditions, serum ALT activity remains acceptable for at least 24 hours at 20 to 25 degrees Celsius, for approximately 3 to 7 days at 2 to 8 degrees Celsius, and for several months when frozen at minus 20 degrees Celsius or below. Samples should be tightly sealed to prevent evaporation, protected from repeated freeze-thaw cycles, and transported promptly to the laboratory with minimal mechanical stress that could cause hemolysis.
Other Sample Types
Beyond clinical specimens, ALT determination methods are adapted to non-clinical matrices in research and industry. These include liver tissue homogenates and hepatocyte culture lysates in toxicology studies, plasma or serum from animals in veterinary diagnostics, and aqueous extracts of meat or fish used as freshness indicators in food quality research. Because enzyme activity in these matrices can be far higher than in human serum, samples usually require dilution and preliminary validation to confirm matrix compatibility.
Testing Items
The core measured quantity is the catalytic activity concentration of ALT in serum or plasma, expressed in U/L. Typical reference intervals for healthy adults, when the IFCC 37 degrees Celsius method without pyridoxal-5-phosphate is used, are approximately 9 to 50 U/L for men and 7 to 35 U/L for women, although each laboratory must verify or establish intervals appropriate to its own method, instrument, and patient population.
- ALT catalytic activity concentration in serum or plasma (U/L)
- Interpretive flags such as critical high values, often above 10 times the upper reference limit
- Paired AST results and the AST/ALT ratio (De Ritis ratio) for differential diagnosis when requested
- Serial ALT monitoring for patients on hepatotoxic drug therapy or following acute hepatitis
Clinical decision limits may differ from statistical reference intervals. For example, donor screening and drug-safety monitoring programs frequently apply fixed cutoff values defined by regulatory or clinical guidelines, and interpretation should always consider sex, age, body mass index, and recent physical activity or alcohol intake.
Testing Methods
Several analytical strategies exist for ALT determination. The two most important are the continuous-monitoring kinetic UV method based on the LDH/NADH coupled indicator reaction, which is the current international reference and routine method, and the classic Reitman-Frankel colorimetric endpoint method, which is still used in laboratories with basic equipment. Additional approaches include dry-film chemistry, pyruvate oxidase-based colorimetric assays, and point-of-care testing systems.
IFCC Continuous-Monitoring (Kinetic UV) Method
The kinetic UV method is the internationally recommended procedure for ALT and is implemented on virtually all automated biochemistry analyzers. Its principle is a two-step coupled enzyme reaction. First, ALT converts L-alanine and alpha-ketoglutarate into pyruvate and L-glutamate. Second, the indicator enzyme lactate dehydrogenase (LDH) reduces the newly formed pyruvate to lactate while oxidizing NADH to NAD. The oxidation of NADH causes a decrease in absorbance at 340 nanometers that is directly proportional to ALT activity, and the absorbance decline is monitored continuously over a defined linear interval.
- Primary reaction: L-alanine + alpha-ketoglutarate to pyruvate + L-glutamate (ALT)
- Indicator reaction: pyruvate + NADH + hydrogen ion to lactate + NAD (LDH)
- Monitoring wavelength: 340 nanometers (334 or 365 nanometers on mercury-line instruments)
- Molar absorptivity of NADH at 340 nm: approximately 6.22 x 10^3 L per mol per cm
Typical reagent composition according to the IFCC 37 degrees Celsius formulation includes 100 mmol/L Tris buffer at pH 7.15, L-alanine at approximately 500 mmol/L, alpha-ketoglutarate at approximately 15 mmol/L, NADH at approximately 0.18 mmol/L, LDH at approximately 1700 U/L, and, in formulations that follow the full IFCC 2002 reference procedure, pyridoxal-5-phosphate at approximately 0.16 mmol/L to ensure full apoenzyme activation. Commercial kits are usually supplied as a two-reagent liquid system in which reagent 1 contains buffer, alanine, NADH, LDH, and activator, while reagent 2 contains alpha-ketoglutarate as the reaction-starting component.
The measurement sequence is designed to eliminate interference from endogenous pyruvate. Serum is first mixed with reagent 1 and pre-incubated so that LDH consumes any native pyruvate in the sample. Reagent 2 is then added to initiate the ALT reaction, and after a short lag phase the analyzer records the absorbance decrease every 30 to 60 seconds for 1 to 3 minutes. Activity is calculated from the linear change in absorbance per minute using the following general relationship: ALT activity in U/L equals the change in absorbance per minute multiplied by total reaction volume, divided by the product of the NADH molar absorptivity, the optical path length, and the sample volume, with appropriate unit conversion. Samples exceeding the linear range, typically above 400 to 1000 U/L depending on the kit, must be diluted with isotonic saline and re-assayed.
Reitman-Frankel Colorimetric (Dinitrophenylhydrazine) Method
The Reitman-Frankel method is a classic endpoint colorimetric procedure suitable for laboratories equipped only with a water bath and a visible-light spectrophotometer. Serum is incubated at 37 degrees Celsius for 30 minutes with a substrate solution containing L-alanine and alpha-ketoglutarate in phosphate buffer at pH 7.4. During incubation, ALT generates pyruvate in proportion to its activity. The reaction is stopped by adding 2,4-dinitrophenylhydrazine (DNPH) in hydrochloric acid, which reacts with pyruvate to form pyruvate dinitrophenylhydrazone. After a fixed development period, sodium hydroxide solution is added, producing a reddish-brown chromophore that is measured at approximately 505 to 520 nanometers against reagent and sample blanks.
Quantification is achieved by comparing the absorbance of the test sample with a calibration curve prepared from pyruvate standards, and results are traditionally expressed in Karmen units or converted to U/L. The method is inexpensive and technically simple, but it suffers from limited specificity because DNPH also reacts with residual alpha-ketoglutarate and other carbonyl compounds, requiring careful blanking. Sensitivity and precision are inferior to the kinetic UV method, which is why the Reitman-Frankel method has been largely replaced in modern clinical laboratories and is now mainly of historical, educational, or resource-limited settings value.
Dry-Film Chemistry and Point-of-Care Testing
Dry chemistry systems use multilayer analytical slides or test strips that contain all necessary reagents in a dried format. A small drop of serum, plasma, or whole blood is applied to the slide, and the ALT reaction proceeds within the film layers; the quantified signal is read by reflectance densitometry rather than transmission photometry. These systems require no liquid reagent preparation, generate minimal waste, and deliver results within roughly 10 to 15 minutes, making them well suited to blood donor screening, outpatient clinics, field surveys, and emergency settings. Analytical performance is generally acceptable for screening purposes, but results should be confirmed with the reference kinetic method when clinically significant abnormalities are detected.
Pyruvate Oxidase Colorimetric Method and Other Approaches
An alternative colorimetric strategy couples the ALT reaction to pyruvate oxidase. The pyruvate generated by ALT is oxidized by pyruvate oxidase to produce hydrogen peroxide, which is then quantified through a peroxidase-catalyzed Trinder reaction using 4-aminoantipyrine and a phenol or aniline derivative to form a colored dye measured at 500 to 550 nanometers. This approach is useful for open-channel analyzers that lack a reliable 340 nanometer measurement channel. Additional techniques described in the literature include immobilized-enzyme biosensors, flow-injection analysis, and microfluidic chip assays; these remain largely confined to research or specialized instrumentation but demonstrate the continuing methodological evolution of ALT determination.
Method Selection and Performance Requirements
For routine clinical work, the IFCC-traceable kinetic UV method at 37 degrees Celsius is strongly preferred because of its specificity, precision, and international standardization. Regardless of the platform used, the method should demonstrate within-run and between-run coefficients of variation typically below 5 percent, linearity covering the clinically important range, calibration traceability to the IFCC reference measurement procedure, and documented interference thresholds for hemolysis, icterus, and lipemia. Two-level internal quality control materials should be run at least daily and results should be accepted according to standard statistical quality control rules, with regular participation in external quality assessment schemes.
Applications
Clinical Diagnostics
ALT determination is a cornerstone of liver function testing. Elevated serum ALT activity indicates hepatocellular injury and is central to the diagnosis and follow-up of acute and chronic viral hepatitis, alcoholic and non-alcoholic fatty liver disease, autoimmune hepatitis, cirrhosis, ischemic hepatopathy, and liver involvement in systemic illness. Serial ALT measurement guides treatment decisions in patients receiving potentially hepatotoxic medications such as antituberculosis agents, methotrexate, statins, anticonvulsants, and certain herbal preparations, and it is routinely included in general health examinations, preoperative assessments, and the workup of jaundice and abnormal liver imaging.
Blood Donor Screening
Serum ALT testing was historically introduced in transfusion medicine as a surrogate marker for non-A, non-B hepatitis before specific hepatitis C virus serology became available. Many blood establishments continue to include ALT screening as part of donor selection algorithms to reduce the residual risk of transfusion-transmitted infections and to identify donors with asymptomatic liver disease who require further medical evaluation.
Pharmaceutical, Biotechnology, and Research Use
In drug development, ALT activity in serum is a key safety biomonitoring parameter in preclinical toxicity studies and in clinical trials, where elevations above defined thresholds trigger investigation for drug-induced liver injury. Cell-based platforms use ALT released into culture medium as a quantitative marker of hepatocyte damage in in vitro cytotoxicity assays. Research applications also include exercise physiology, nutrition studies, and epidemiological investigations of metabolic liver disease in population cohorts.
Veterinary and Food Industry Applications
Adapted ALT assays are used in veterinary medicine to evaluate hepatic disorders in companion animals and livestock. In food science, declining ALT activity in post-mortem muscle tissue has been investigated as one component of multi-index freshness assessment for meat and fish products, demonstrating the versatility of transaminase measurement beyond clinical chemistry.
FAQ
What is the normal reference range for ALT?
With the IFCC 37 degrees Celsius method, typical adult reference intervals are approximately 9 to 50 U/L for men and 7 to 35 U/L for women. Exact limits vary with the reagent system, instrument, and population studied, so laboratories must verify their own intervals, and interpretation should account for individual factors such as body mass index and recent alcohol use.
Which ALT determination method is the most accurate?
The IFCC continuous-monitoring kinetic UV method with LDH/NADH coupling at 37 degrees Celsius is the internationally recognized reference and routine method. It offers high specificity, excellent precision, and standardization across laboratories, and results are traceable to the IFCC reference measurement procedure.
Is fasting required before an ALT test?
Strict fasting is not mandatory because ALT activity is not acutely affected by a normal meal, but an 8 to 12 hour fast is often recommended to avoid lipemia-related photometric interference and to harmonize results with other liver tests. Patients should also avoid strenuous exercise and alcohol consumption for at least 24 hours before sampling, as both can transiently elevate ALT activity.
How does hemolysis affect ALT results?
Red blood cells contain ALT at concentrations several times higher than serum, so hemolysis releases enzyme into the sample and falsely elevates results, while cell fragments also scatter light at 340 nanometers. Mild hemolysis may be tolerated with caution, but moderately or grossly hemolyzed specimens should be rejected and recollected.
What is the difference between ALT and AST testing?
ALT is located mainly in the liver cytoplasm and is the more liver-specific enzyme, whereas aspartate aminotransferase (AST) is abundant in heart, skeletal muscle, and liver, including mitochondria. The simultaneous determination of both enzymes and calculation of the AST/ALT ratio helps distinguish hepatocellular from cholestatic or extrahepatic causes of enzyme elevation.
How should ALT samples be stored if testing is delayed?
Serum or plasma can be held at room temperature for up to about 24 hours or refrigerated at 2 to 8 degrees Celsius for several days with minimal loss of activity. For longer storage, specimens should be frozen at minus 20 degrees Celsius or below, avoiding repeated freeze-thaw cycles that can degrade enzyme activity and damage the sample matrix.
Why do very high ALT samples sometimes require dilution and repeat testing?
In the kinetic UV method, extremely elevated ALT activity can exhaust NADH and deplete substrates before or during the reading window, causing a non-linear absorbance change and falsely low results. Laboratories therefore dilute samples that exceed the validated linear range with saline and re-assay, multiplying the result by the dilution factor.