Alanine aminotransferase: Is it healthy to be “normal”?
Authors: D. Robert Dufour
Journal: Hepatology (2009)

There are many definitions of the word “normal”. For physicians and patients, the term is typically used as a synonym for “healthy”. For most laboratory tests, however, reference intervals (commonly referred to as “normal values”) use another definiton of the word, meaning typical of the population. ALT, alanine aminotransferase; HBV, hepatitis B virus; HCV, hepatitis C virus; ULN, upper limit of the normal range. The accepted method for determining reference intervals involves selecting apparently healthy individuals, and setting the reference limits (upper and lower ends of the “normal range”) to include the central 95% of values for the test, after excluding any results considered outliers. Figure 1 shows results of such a study for alanine aminotransferase (ALT) based on data from medical students. Using this data would produce an upper reference limit (usually called upper limit of normal [ULN]) of 69 U/L in men and 56 U/L in women. As documented in the article by Dutta et al. in this issue,1 this approach often leads to significant differences in ALT ULN between laboratories. The median ULN was 63 U/L in men (with a range of 32-72) and 52 U/L in women (with a range of 31-72). Such variability has been recognized previously in a brief report on 11 laboratories involved in the Nonalcoholic Steatohepatitis Clinical Research Network; ULN ranged from 35-79 U/L in men and 31-55 U/L in women.2 Distribution of ALT results from 590 medical students (442 male, 148 female). Results show skewing toward higher results, which is typical for ALT and many laboratory tests. The upper limit of the reference interval (ULN) calculated from the central 95% of results is 69 U/L in men and 56 U/L in women. Some of this difference may be due to use of different instruments. To produce similar results, reference materials with known values must be available to instrument manufacturers. These materials are then used to “set” each individual instrument, producing results that are quite similar. Such reference materials are not available for ALT. It is also important that laboratories use standardized methods to perform the measurement. Although a recommended procedure is available from the International Federation for Clinical Chemistry, few U.S. manufacturers actually use this procedure. Are ALT values actually that different between laboratories? Both of the studies looked at results on samples sent out by the College of American Pathologists as part of proficiency testing. These unknown samples differ somewhat from actual patient samples, in that they are typically lyophilized pools of serum from a variety of individuals. Although this approach can cause differences from results obtained using fresh frozen samples,3 it is the best way currently available for comparing results. In both studies, differences in proficiency testing results were not felt to be clinically important. For example, in the study by Dutta et al. in this issue,1 using a sample with results slightly above most laboratories' upper reference limit, average ALT varied from 69-83 U/L between laboratories using different instruments. Much smaller differences were seen (for the three most commonly used instruments, a range of 4-8 U/L) between laboratories using the same instruments. Differences in laboratory procedures explains only part of the difference in reference limits between laboratories. Why then is ULN so different between laboratories? The most likely explanation is that individuals used to establish the reference limits are only “apparently healthy”, and have other reasons for increased ALT. The American Association for the Study of Liver Diseases guidelines on laboratory tests for liver disease4 summarize factors that are known to affect ALT results. The most important of these, other than known liver disease, are exercise and body mass index. Other factors known to cause liver disease include medications, herbal supplements, and excessive ethanol ingestion. In the current study, 40% of laboratories based ULN solely on manufacturer's suggestions; no information was available on exclusion criteria from most manufacturers. Of those laboratories performing their own studies to determine ULN, only half excluded patients with known liver disease, and only 12% excluded overweight or obese individuals. Only one-quarter of the laboratories excluded persons on prescription medications or who had a history of ethanol use, and none inquired about herbal supplements or exercise history. Does inclusion of persons with factors that might affect ALT results make a significant difference in determining ULN? In a study of blood donors, Prati et al.5 evaluated factors related to ALT levels. ALT levels were related to exercise, ethanol ingestion, age, and blood levels of glucose, cholesterol, and triglycerides. ULN was determined using individuals at lowest risk for liver disease, excluding persons with positive tests for hepatitis B or C, a body mass index greater than 24.9, those taking any medications, and those with abnormal levels of cholesterol, triglycerides, or glucose. In this group, ULN was 30 U/L in men and 19 U/L in women. The new ULN increased sensitivity for hepatitis C virus (HCV) viremic blood donors from 40% to 61%, with a decline in specificity from 97.6% to 95.5%. Using the old ULN (>40 U/L in men, 30 U/L in women), half of the individuals with increased ALT had no obvious cause for liver injury, but 85% of these had steatosis on liver ultrasound. It is not clear whether complete evaluation was performed in those with increased ALT using the new ULN. In the author's laboratory, ULN was determined in a similar fashion, but without evaluating laboratory results for cholesterol, triglycerides, or glucose; ULN was 33 U/L in men and 19 U/L in women. Are there any other approaches to determining “healthy” levels for laboratory tests? For several tests (lipids, glucose, hemoglobin A1c), ULN is determined from outcome studies, based on occurrence of disease manifestations in long-term follow-up. This approach is most appropriate in situations where disease is common in the population, and asymptomatic over long periods of time. It requries availability of data on outcomes in patients and correlation of those outcomes with laboratory test results. For cholesterol and glucose tests, there is a clear “threshold” level above which risk of subsequent development of complications begins to increase sharply. Although chronic liver injury (particularly nonalcoholic fatty liver disease) is clearly not as prevalent as atherosclerosis, it is probably at least as common as diabetes. One study using proton magnetic resonance spectroscopy found that about one-third of a representative population sample in Dallas, TX, had hepatic steatosis.6 A prospective study of nonalcoholic fatty liver disease in Korea showed that risk of subsequent disease development began to increase at ALT levels greater than 16 U/L.7 Thus, chronic liver disease may be a condition in which population-based reference limits will not be effective, and where outcomes studies are needed to determine appropriate ULN. What data exist on health-based outcomes for ALT? Little information actually exists on the relationship between ALT and liver disease mortality in long-term follow-up. A recent study of patients with HCV found that liver-related mortality was 11-fold higher in those with persistent elevation of ALT than in those with persistently “normal” ALT (ULN of 33 U/L).8 Another recent study, in persons with hepatitis B e antigen (HBeAg)-negative hepatitis B and initial ALT < 2 times ULN (36 U/L), found a trend to higher mortality in those with ALT > 2 times ULN on follow-up, while risk of hepatocellular carcinoma increased starting at ALT values greater than 18 U/L.9 In a population-based study from Korea, risk of death from liver disease began to rise at ALT values greater than 20 U/L.10 Although preliminary, these data suggest that risk for liver-related death increases at values well below the ULN provided by most laboratories. Increased ALT has also been linked to risk of death from all causes or cardiovascular disease,10-13 although not in all studies.14 In studies that evaluated differing cut-points lower than the ULN, risk begins to increase even at values < ULN.10, 13 These data point out the limitations of the current approach used to establish ULN for ALT. The wide variation in levels considered “abnormal” documented in published surveys1, 2 is an impediment to patient care, because it leads to differences in recognition of liver disease and decisions to treat in hepatitis B virus (HBV).15 Typical ULN are insensitive to the presence of liver disease, and do not detect limits at which risk of liver mortality seems to increase. The time has come for those involved in the care of patients with liver disease to raise this issue with the laboratories they use. At present, several steps should be taken by laboratories to evaluate their reference limits for ALT. First, they should review how they have established their reference limits. If reference limits in males are > 40 U/L and in females > 25 U/L, it is likely that a faulty approach was used to establish them. If based on manufacturer's suggestions or publications in the literature, find out how their suggested limits were developed, and what exclusion criteria were used. If (as shown in the study in this issue), patients likely to have liver disease were included, new reference limits need to be established. At a minimum, persons with known (HBV, HCV) or suspected (obese individuals, diabetics) liver disease should be excluded from studies, as should individuals taking any potentially hepatotoxic medications. Manufacturers should work to develop suggested reference limits using a similar process, and should work to reduce differences in results between methods. Further studies should be done to attempt to develop outcomes-based reference limits for ALT, as data suggest that risk may increase at levels that are even lower than would be predicted using idealized techniques to determine population-based reference limits.