Introduction

Alanine aminotransferase (ALT) is an enzyme found predominantly in the liver but also in other tissues such as the kidneys, heart, and muscle cells. An increase in ALT serum levels indicates definite liver cell injury due to many causes. An ALT blood test is often included in a liver panel and comprehensive metabolic panel to assess for damage to the liver. 

The liver has a central and critical biochemical role in the metabolism, digestion, detoxification, and elimination of substances from the body.[1] All blood from the intestinal tract initially passes through the liver, where products derived from the digestion of food are processed, transformed, and stored.[2] These include amino acids, carbohydrates, fatty acids, cholesterol, lipids, vitamins, and minerals.

Most major plasma proteins (except immunoglobulins and the von Willebrand factor) are mainly or exclusively synthesized in the liver. The liver responds to multiple hormonal and neural stimuli to regulate blood glucose concentrations.[3] Not only does the organ extract glucose from the blood to generate energy, but it also stores dietary glucose as glycogen for later use. The liver is also the major site for gluconeogenesis, which is critical for maintaining blood glucose concentration in the fasting state.[4] The liver is central in lipid metabolism; it extracts and processes dietary lipids and is the principal site of cholesterol, triglyceride, and lipoprotein synthesis.[5] Another major liver function is the synthesis of bile acids from cholesterol, with the secretion of these compounds into the bile, which facilitates the absorption of dietary fat and fat-soluble vitamins.[6]

The liver is also the primary site of metabolism of both endogenous substances and exogenous compounds (eg, drugs and toxins). This process, known as biotransformation, converts lipophilic substances to hydrophilic ones for subsequent elimination.[5] The liver is a major site of hormone catabolism and regulates plasma hormone concentrations.[6] The liver is also involved in hormone synthesis, producing the hormones insulin-like growth factor 1 (IGF-1), angiotensinogen, hepcidin, thrombopoietin, erythropoietin, and the prohormone 25-OH vitamin D. Many of these hepatic functions can be assessed by laboratory procedures to gain insight into the integrity of the liver.[1]

As a large organ, the liver has extensive reserve capacity to perform its functions in coordination with other organs. Individuals with liver disease maintain normal function despite extensive liver damage. In such cases, liver disease may be recognized only using tests that detect injury.[2] Most commonly, this is accomplished by measuring the plasma activities of enzymes found within liver cells, which are released in specific patterns with different forms of injury.[7] Chronic liver injury involves fibrosis in the liver. Markers of the fibrotic process might indicate the degree of injury.[8] Chronic damage is often due to chronic inflammation. Cytokines can alter the liver's protein production pattern, making it possible to detect inflammation, not necessarily related to the liver.[9] Some proteins are produced in increased amounts with liver regeneration and neoplasia; the markers may be useful in detecting liver cell proliferation.[10]

This review focuses on the significance of ALT in assessing hepatic injury and malfunction. ALT is aggregated primarily in the cytosol of hepatocytes, consists of 496 amino acids, and has a half-life of approximately 47 hours.[11] ALT is detectable in serum at low concentrations (typically <30 IU/L). However, any process that leads to loss of hepatocyte membrane integrity or necrosis results in the release of ALT in high concentrations in the plasma.[2]

Therefore, the elevation of serum ALT concentration is sensitive but not specific to measure hepatocellular injury, as the degree of elevation cannot determine the exact cause.[1] The most common causes are alcohol-induced liver injury, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic hepatitis B or C, autoimmune hepatitis, and drug or herbal supplement-induced liver injury.[4] Other causes include hemochromatosis, vascular disease, acute viral hepatitis, and genetic disorders affecting the liver.[7]

Etiology and Epidemiology

The liver transaminases, mainly ALT and aspartate aminotransferase (AST), are aggregated in the cytosol of hepatocytes. These enzymes are normally detectable in the serum at low concentrations, typically <30 IU/L.[12][13] However, any process that leads to loss of hepatocyte membrane integrity or necrosis releases these enzymes into the blood, where the elevated concentrations can be measured.[14] Transaminase elevation is frequent in primary care medicine, affecting 10% to 30% of the U.S. population. Less than 5% of patients with elevated transaminases will have severe liver conditions.[15] 

ALT increases affect 8.9% of the U.S. population.[16] Several physiological and risk factors may contribute to the serum levels of these enzymes, including age, sex, body mass index, pubertal age, elevated levels of triglycerides, insulin resistance, and blood glucose level.[17][18]

Physiological Factors

1. Extreme physical exertion can induce a reversible elevation in serum ALT, approximately 2 to 2.2-fold higher than the normal limit.[19] While the source of elevated ALT in these individuals could be non-hepatic, it is likely released from the exercising skeletal muscles.
2. ALT activity has a diurnal variation; the nadir value is at 4:00 hr, and the peak value is at 16:00 hr.[20]
3. ALT is higher in males compared to females. These gender-based differences in ALT levels are possibly related to hormonal differences between males and females.[21]
4. Ethnicity affects ALT levels. Research shows that Mexican Americans have a higher ALT elevation prevalence than other ethnicities.[22] This finding could be related to higher incidences of metabolic syndrome, a major cause of elevated ALT in Mexican Americans.
5. Research has found no association between the hypoxia-inducible factor 3 alpha subunit (HIF3A) rs3826795 polymorphism and ALT. However, a significant interaction between obesity and rs3826795 concerning ALT was found. Rs382695 G-allele number elevation of ALT is significant only in obese children but not non-obese children.[23]
6. In another study, Bekkelund and Jorde found that serum ALT was associated with body fat mass index in men. ALT was associated with lean mass index in men and women in the overweight and obese population.[24] They found body mass index to be the most determining factor for ALT, and gender was the most influencing factor for AST.[25]

Pathological Causes

1. Depending on the region, alcoholic hepatitis and non-alcoholic fatty liver disease (NAFLD) are considered the most common causes of abnormally high ALT levels.
2. Non-alcoholic steatohepatitis (NASH)
3. Chronic hepatitis B or C
4. Autoimmune hepatitis
5. Alpha-1 antitrypsin deficiency
6. Drug-associated, occupational exposure, or herbal supplement-induced liver injury
7. Hemochromatosis
8. Wilson disease
9. Celiac disease
10. Ischemic hepatitis
11. Budd-Chiari syndrome
12. Vascular disease
13. Genetically-related conditions affecting the liver[26]

Pathophysiology

ALT catalyzes the transfer of amino groups from the L-alanine to alpha-ketoglutarate, and the conversion products are L-glutamate and pyruvate. The process is critical in the liver in the tricarboxylic acid (TCA) cycle. Pyruvate can be used in the citric acid cycle to produce cellular energy. The coenzyme needed for this reaction is pyridoxal 5'-phosphate, also known as vitamin B6.[27] The pyridoxal 5'-phosphate (P-5′-P) is bound to the inactive apoenzyme and is a true prosthetic group. The pyridoxal 5'-phosphate attached to the apoenzyme accepts the amino group from the first substrate, aspartate or alanine, to form enzyme-bound pyridoxamine-5′-phosphate and the first reaction product, oxaloacetate or pyruvate, respectively.[28] The coenzyme in the amino form then transfers its amino group to the second substrate, 2-oxoglutarate, to form the second product, glutamate. The pyridoxal 5'-phosphate (P-5′-P) is regenerated.[29]

Both coenzyme-deficient apoenzymes and holoenzymes may be present in serum.[30] Therefore, adding P-5′-P under measurement conditions that facilitate recombination with the enzymes usually increases aminotransferase activity. For clinical assays, following the principle that all factors affecting the reaction rate must be optimized and controlled, adding P-5′-P in aminotransferase methods is recommended to ensure that all enzymatic activity is measured.[31]

ALT is found ubiquitously throughout the human body, in the kidney, myocardium, skeletal muscle, brain, pancreas, spleen, and lungs. More specifically, the highest tissue concentration of ALT activity is in the cytosol of hepatocytes. The activity of ALT in hepatocytes is approximately 3000 times higher than that of serum ALT activity.[32] Therefore, in patients with acute or chronic hepatocellular injury, the release of ALT from dying or damaged hepatocytes increases serum ALT levels. The half-life of ALT is approximately 47 hours in circulation.[33]

Specimen Requirements and Procedure

According to the International Standard ISO 6710, the light green cap contains lithium-heparin and is used for the hepatic function panel.[34] A trained clinician will disinfect the skin and wrap an elastic strap around the arm to visualize a vein. A blood sample is collected. The sample must be placed in the correctly colored cap, as the various color caps have a particular additive.[35] After the specimen is collected and fulfills the requirements, it is sent for testing. Hepatic function panels evaluate for ALT through blood samples. The quality of blood specimens is vital to decrease laboratory errors, prevent diagnosis delay, and ensure a proper diagnosis.[36] All specimens should undergo measurement of the hemolysis index, as hemolyzed blood is considered unsuitable for testing. The inappropriate quality or sample volume contributes to approximately 80% to 90% of laboratory errors.[37]

A hemolysis index assesses the sample at specific wavelengths to determine the potential concentration of cell-free hemoglobin and ensure quality. If the hemolysis index is unavailable, a visual inspection should be conducted.[38] Blood specimens with fibrin strands or clots should not be used for testing. Blood tubes filled at less than 90% of their nominal volume should not be used for testing to maintain specimen integrity.[39] Only in emergent situations that require evaluating prothrombin time and fibrinogen assay are blood coagulation tubes filled to 70% of their nominal volume usable.[40]

Diagnostic Tests

A panel of laboratory tests to assess liver functions, also known as the liver function test, is commonly used in clinical practice. The liver function test comprises the following:[40]

1. Serum bilirubin
2. Serum alanine aminotransferase (ALT)
3. Serum aspartate transaminase (AST)
4. Serum alkaline phosphatase (ALP)
5. Serum gamma-glutamyltransferase (GGT)
6. Prothrombin time or an International Normalized ratio (INR)
7. Serum albumin

Testing Procedures

The liver function tests are performed on semi-automatic or fully automated analyzers, which are based on the principle of photometry. Photometry is the measurement of light absorbed in the ultraviolet (UV) to visible (VIS) to infrared (IR) range. This measurement determines the amount of an analyte in a solution or liquid. Photometers utilize a specific light source and detectors that convert light through a sample solution into a proportional electrical signal. These detectors may be photodiodes, photoresistors, or photomultipliers.[41] Photometry uses Beer–Lambert’s law to calculate coefficients obtained from the transmittance measurement. A test-specific calibration function establishes a correlation between absorbance and analyte concentration to achieve highly accurate measurements.[42]

Interfering Factors

Patients are instructed to avoid certain medications and foods before a hepatic function panel to ensure the integrity of blood specimens.[43] Drug hepatotoxicity can be non-idiosyncratic or idiosyncratic. Also, drug-associated hepatotoxicity can be classified as immune-mediated and non-immune-mediated. The incidence of drug-induced liver injury is 19 cases per 100,000 persons. The most common drug causing drug-induced liver injury is amoxicillin or clavulanate.[44][45] Hepatitis E infection can masquerade drug-induced liver injury in 3% to 13% of the cases.[46] Tacrine, a medication for Alzheimer disease, was withdrawn from the market because of significant liver injury. This medication caused elevations of ALT levels that trended as high as 20 times the normal reference level.[47] 

Up to 5% of patients on statin medications developed elevations in ALT.[48] Ceftriaxone, phenytoin, carbamazepine, cotrimoxazole, and allopurinol have been reported to cause liver injury. Also, tricyclic antidepressants, imipramine, and amitriptyline have links to elevations in ALT.[49] Elevation of serum ALT and AST has been reported in patients taking these medications: isoniazid, pyrazinamide, rifampicin, ibuprofen, or dapsone.[50] The website maintained by the National Institute of Diabetes and Digestive Diseases (NIDDK) is a valuable resource for clinicians and researchers interested in liver hepatotoxicity and other drugs that can increase serum ALT. As stated earlier, periods of intense exercise should be avoided before testing, as this may increase ALT levels.[51]

Results, Reporting, and Critical Findings

The results of a hepatitis panel should correlate with the initial findings in a complete history and physical examination. A thorough review should include essential questions regarding the patient’s age, past medical history (diabetes, obesity, hyperlipidemia, inflammatory bowel disease, celiac sprue, thyroid disorders, autoimmune hepatitis, acquired muscle disorders, alcohol consumption, medication use, toxin exposure), and family history of genetic liver conditions (Wilson disease, alpha-1-antitrypsin deficiency, hereditary hemochromatosis).[32]

A review of systems should also include signs and symptoms of chronic liver disease such as jaundice, ascites, peripheral edema, hepatosplenomegaly, gynecomastia, testicular hypotrophy, muscle wasting, encephalopathy, pruritus, and gastrointestinal bleeding.[52] Other tests that help determine the cause of elevated transaminase levels found on a hepatitis panel include fasting lipid levels, hemoglobin A1c level, fasting glucose, complete blood count with platelets, a complete metabolic panel, iron studies, hepatitis C antibody, and hepatitis B surface antigen testing.[53] A hepatitis panel reference range can fluctuate amongst different laboratories. Reported values also vary depending on gender, body mass index, and past medical history. Repeated liver enzymes are typically unnecessary in the workup for elevated transaminase levels.[54]

Clinical Significance

An increase in ALT serum levels indicates definite liver cell injury due to many causes.[55] Although specific hepatic diseases are associated with an elevation in ALT levels, no correlation exists between the absolute peak of the ALT elevation and the magnitude of hepatic injury.[54] An increase in both AST and ALT serum levels is common. ALT levels greater than 1000 U/L should be considered acute ischemic liver injury, severe drug-induced liver injury, or acute viral hepatitis. Other causes include common bile duct stones and hepatitis E infection.[56]

Viral hepatitis is liver inflammation from hepatitis A, B, C, D, and E. Acute hepatitis A, compared to hepatitis C and B, is associated with increased serum ALT and AST levels, reaching 3000 to 4000 IU/L. The diagnosis of chronic hepatitis will have elevations in ALT levels for greater than 6 months.[54] Common clinical signs of viral hepatitis include jaundice, anorexia, fatigue, vomiting, fever, nausea, and hepatomegaly. The risk factors for viral hepatitis include travel to areas where hepatitis is endemic, multiple sexual partners, occupational exposure to chemicals and hepatotoxicants, and intravenous drug use.[57] Hepatitis serology labs should also be ordered to confirm the diagnosis and the type of viral hepatitis.[58]

The most efficient aminotransferase threshold for diagnosing acute liver injury is 7 times the upper limit (sensitivity and specificity >95%).[59] In acute viral hepatitis, peak values of transaminase activity occur between the 7th and 12th days; activities then gradually decrease, reaching physiologic concentrations by the 3rd to 5th week if recovery is uneventful.[57] Peak activities bear no relationship to prognosis and may fall with the worsening of the patient’s condition, perhaps due to a lack of further functional hepatocytes to continue enzyme release.

The persistence of increased ALT for longer than 6 months after an episode of acute hepatitis is used to diagnose chronic hepatitis.[60] Most patients with chronic hepatitis have a maximum ALT of fewer than seven times the upper 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 increasing measurements.[61] In patients with acute hepatitis C, ALT should be measured periodically over the next 1 to 2 years to determine if it stays normal.[62]

Ischemic liver injury, also known as ischemic hepatitis, occurs when there is an acute reduction in blood perfusion to the liver, leading to necrosis of hepatic centrilobular cells on histology.[63] Hepatic damage is higher in septic shock, where a decrease in blood perfusion to the liver is due to infection. A recent study revealed the incidence of abnormally elevated ALT was more sensitive to the diagnosis of ischemic hepatitis due to septic or hypovolemic shock.[64] In evaluating septic shock as a potential cause for ischemic liver injury, serum lactate, serum CRP, blood counts, D-dimer levels, and blood cultures should be measured. 

Medications can account for an elevation in ALT. Paracetamol toxicity (also known as acetaminophen) has been shown in a recent study to account for almost half of drug-induced liver injuries. In paracetamol toxicity, the levels of serum ALT are usually higher than 1,000 U/L.[56] Therefore, paracetamol toxicity should be among the differential diagnoses of patients with acute liver failure. A review of hepatotoxic medicines is vital in ensuring the proper diagnosis. Drugs associated with an elevation of transaminases include tacrine, imipramine, amitriptyline, isoniazid, pyrazinamide, rifampicin, ibuprofen, nimesulide, cotrimoxazole, phenytoin, dapsone.[45][43]

Non-alcoholic fatty liver disease (NAFLD) should merit consideration among the most common causes of abnormally elevated ALT levels in asymptomatic patients.[65] NAFLD is the fat accumulation within the liver in patients who do not consume alcohol. NAFLD has the potential to progress into hepatic fibrosis and cirrhosis, increasing liver-related morbidity and mortality.[66] NAFLD is usually associated with higher ALT and GGT levels in patients with impaired glucose tolerance or type 2 diabetes mellitus. NAFLD risk factors include morbid obesity, hyperglycemia, hypertriglyceridemia, hypertension, and decreased insulin sensitivity.[67] A NAFLD fibrosis score and radiological imaging such as CT or MRI of the liver should be considered to assess the severity and progression of NAFLD. The diagnosis of NAFLD is made with steatosis in 5% or greater of hepatocytes.[68]

In 1957, DeRitis described in a publication the ratio between AST and ALT in the diagnosis of viral hepatitis, where ALT is usually higher than AST.[69] Later, the usefulness of this ratio was highlighted in alcoholic hepatitis, where AST is mostly more elevated than ALT. Therefore, the ratio between AST and ALT is >2.0 for alcoholic hepatitis, 1.5 to <2.0 in acute viral hepatitis, and >1.0 in fibrosis and cirrhosis.[70] Many laboratories do not include this ratio in their reports because it is not specific, and hemolysis can affect AST.[71] The ratio is affected by the number of days post-exposure and the severity of the disease. Additional critical factors are the relatively short half-life of AST (18 hours) compared to ALT (47 hours), gender, and intra-individual variation of both AST and ALT.[72]

Quality Control and Lab Safety

The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations require a laboratory to have quality control (QC) procedures to monitor the accuracy and precision of the complete testing process.[73] For non-waived tests, laboratory regulations require, at the minimum, analysis of at least 2 levels of control materials every 24 hours.[74] To ensure accurate results, laboratories can assay QC samples more frequently. The procedure is stable if the QC material result is within limits and patient samples are suitable. If not, the procedure is inaccurate, and patient samples may not be suitable. Corrective action is needed.[75]

The Levey-Jennings plot is the most common presentation for evaluating QC results. This representation displays sequential QC results over time, enabling a swift visual performance evaluation. Under stable conditions aligning with the procedure's specifications, the mean value signifies the anticipated QC result, while the standard deviation lines depict anticipated imprecision. Presuming a Gaussian (normal) distribution of imprecision, the results ideally demonstrate a uniform spread around the mean, with a higher frequency of observations closer to the mean compared to the extremes of the distribution.[76]

Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[77] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month), the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[78]

External quality control, also known as external quality assessment (EQA) or proficiency testing (PT), is a vital process for ensuring the accuracy and reliability of laboratory testing.[79] In this assessment, surrogate samples are provided by an independent external organization, and the laboratory is unaware of the expected values for these samples. The results obtained from analyzing these EQA/PT samples are compared with target values assigned to the samples. This comparison verifies that the laboratory's measurement procedures align with the expected performance standards. Participation in EQA/PT programs allows laboratories to evaluate and manage the quality of their testing routines with the support of an independent party. It provides valuable insights into individual laboratories' performance and helps standardize measurement procedures across different laboratories.[80] By enrolling in EQA/PT programs, laboratories can demonstrate their commitment to quality improvement and gain confidence in the accuracy of their testing processes.

Westgard rules represent a comprehensive set of statistical criteria employed in quality control practices within laboratories. These rules serve as analytical tools to detect errors or inconsistencies that might occur during the testing process.[81] Each Westgard rule targets specific deviations within quality control (QC) data, highlighting potential issues such as random errors, systematic shifts in means, trends in consecutive measurements, or extreme outliers.[82] In case of a rule violation, proper corrective and preventive action should be implemented before patient testing.[83] 

Biological variation data suggest that an imprecision of less than 9.7%, a bias of ±11.5%, and a total error of ±27.5% for ALT is required for clinical use of its determinations. In general, the imprecision target is easily met using current aminotransferase methods. However, it is important to note that while imprecision targets may be consistently met by current methods, ensuring compliance with bias and total error criteria is equally essential.[84] Focusing solely on meeting imprecision targets might overshadow the necessity of addressing bias and overall total error, which are critical factors influencing the accuracy and reliability of clinical interpretations.

Collecting blood samples is a crucial yet potentially hazardous task, bearing risks for clinicians and patients if not executed properly.[85] Structured educational programs are essential to mitigate these risks. These programs aim to standardize blood collection, educate individuals on associated risks, and address common mistakes, enhancing laboratory safety standards.[86]

Clinicians should demonstrate proficiency before engaging in patient care involving blood collection. This involves understanding and adhering to protocols that prioritize safety measures. Personal protective equipment (PPE), such as single-use nonsterile gloves, eye protection, and masks in scenarios where exposure to blood is possible, serves as a frontline defense against accidental contamination or infection.[87] Additionally, using trays or tube holders during sample collection requires careful consideration. Protocols should be in place to prevent cross-contamination, especially when these items are used for multiple patients.[88]

Proper disposal of needles minimizes the risk of needle-stick injuries, and utilizing designated sharps containers ensures safe containment of used needles, significantly reducing the potential for accidental injuries.[89] Overall, comprehensive education, strict adherence to safety protocols, and the utilization of appropriate protective gear and disposal methods are fundamental in minimizing risks associated with blood sample collection for both healthcare personnel and patients.

Enhancing Healthcare Team Outcomes

Multi-disciplinary and interprofessional rounds are when medical and other health providers collaborate to enhance patient outcomes. Clinicians may review the significance of the hepatic function test panel in correlation with each patient's medical history and physical examination to detect any drug-induced hepatic injury and build a differential diagnosis. Changes in the patient's patterns of ALT and AST over time or other liver function tests may necessitate a referral to gastroenterologists. Clinical pharmacists can also advise about potential contraindications or hepatotoxic medication interactions. The charge nurse of each floor can communicate updates on a patient's response to treatment, other laboratory orders, and current disposition. A multi-disciplinary approach can ensure a higher quality of care and enhance outcomes.[90]