With a global prevalence of NAFLD at 20–30% and a similar estimated prevalence in the German population 1 it has become more and more important to enhance diagnostic efficiency for this disease. Liver biopsy is still considered gold standard for confirmation of NAFLD, for risk assessment of liver cirrhosis and HCC, and the only method to detect and diagnose NASH 15. Given the limitations of this method and the very low proportion of patients, who actually undergo liver biopsy, it is no surprise that NAFLD and in particular asymptomatic progressive liver disease (i.e. NASH with compensated cirrhosis) remain severely underdiagnosed 7. As elevations of serum liver enzyme concentrations are still the most common symptom resulting in further clinical workup and referral to hepatologists 20,21, in the present study utility of serum liver enzymes for detection or diagnosis of NAFLD or NASH was tested. Despite significant differences of ALT, AST and γGT serum concentrations between NAFL and NASH and significant correlations of these factors with NAS and SAF, performance to separate NAFL and NASH was insufficient for clinical application.
The central finding of this retrospective data analysis is that even in morbidly obese individual serum liver enzyme concentrations cannot efficiently separate NAFL and NASH. All AUROCs calculated to discern NAFL from NASH demonstrated only poor to mediocre performance of liver serum enzymes as classifiers with a maximum AUROC of 0.7073. For clinical use, this value should be at least above 0.85. Additionally 55% of the whole cohort did not exhibit elevations of any of the three tested liver enzymes and 63% of those without elevations had NASH. This confirms previous findings that normal serum concentrations of liver transaminases cannot reliable to rule out NAFLD or the need for a liver biopsy .In a study analyzing histological and clinical features of NAFLD it has been shown that 59% of patients with normal serum liver transaminases had NASH and 35% had advanced fibrosis or cirrhosis 22. Another study aimed at serum based identification of NASH found that in patients with normal serum transaminases 46% had NASH 23. In a direct comparison between 63 NAFLD patients with normal transaminase and 395 NAFLD patients with elevated transaminases, no difference in the distribution of fibrosis grades was found in 59% of patients with normal and in 74% of patients with elevated transaminases NASH was present. In a large population based study with more than 4900 participants (Heinz Nixdorf Recall Study) we identified the proportion of diabetics as 13.7% 16. In that study elevated transaminases were found in only 2% of the participants, although 90% of patients with type 2 diabetes mellitus have NAFLD and approximately 38% NASH 24. The authors of some of these works suggest that including measures of glucose metabolism and IR (i.e. HbA1c) should be included into detection algorithms 23. This is in line with a serum based score to separate NAFL and NASH, recently introduced by us 25. The included parameters age, γGT, HbA1c, M30, and adiponectin were selected from all available parameters by a nonbiased ensemble feature selection, including machine learning algorithms 26. Of note, with adiponectin and HbA1c two factors associated to glucose metabolism and IR (and adipose tissue status) were selected. To develop an efficient, clinically applicable serum based diagnostic algorithm sufficiently powered prospective studies assessing clinical outcome and development of various markers over the disease course are required. Measures of IR and glucose metabolism should be included in such an analysis.
It has been argued that adapting the current limits of normal for serum liver enzymes would improve their diagnostic accuracy. However, when calculating optimized cut-offs for either 95% sensitivity or specificity from our data between 62% and 91% of patients would remain unclassified with a lower boundary of 15 to 20 U/l. In the past, various studies aimed to lower the upper limits of normal for ALT in different patient populations. Studies from China (22.15 U/l for men, 22.40 U/l for women), USA (30 U/l and 19 U/l), Taiwan (22 U/l and 17 U/l) and India (27 U/l and 17 U/l) resulted in dramatically lower thresholds, in a similar range as those identified by us 27–30. Based on these and further studies, current AASLD guidelines recommend a hepatitis B-screening at ALT serum concentrations above 35 U/l for men and 25 U/l for women 31. However, to achieve a 95% sensitivity for the detection of NASH in our cohort, the cut-off would have to be even lower (AST: 15.5 U/l; γGT: 19.5 U/l). Lowering of the upper limit of normal values to the point where adequate sensitivity is reached, would lead to a very high rate of false positive results for NAFLD/NASH when used as screening in the general population. In our opinion these findings of other groups and our own rule out any applicability of the liver serum values for diagnostic or screening purposes in NAFLD.
Another result of this study is that histological evaluation by NAS and by SAF differed in a substantial proportion (20%) of the study population. NAS and SAF were designed for different purposes and the NAS classification is usually broken down into three groups with 1–2 points as definitely no NASH, 3–4 as borderline NASH (requiring further examination or follow up) and ≥ 5 as definite NASH. To reduce ambiguity and allow more robust statistical evaluation we applied a simplified classification, interpreting NAS up to 4 as NAFL. This might be one cause, why 64 patients more were classified as NASH by SAF than by NAS. However, 10 patients were classified as NAFL by SAF and NASH by NAS. A study with a more diverse patient population, different outcome scenarios, and rigorous histological work-up would be required to investigate if one scoring system is superior in identifying patients at higher health risk. With our current understanding 32–34 the observed differences between NAS and SAF classification might be a matter of interpretation in borderline NASH cases.
A direct comparison of serum liver enzyme concentrations yielded a significant difference between NAFL and NASH, independent of classification by NAS or SAF. The effect size was small and the overlap of the range of values was quite broad. Furthermore, all three serum parameters correlated significantly positively with the NAS and the SAF score, with slightly stronger correlations with the SAF score. Overall the rank correlation coefficients indicate a modest correlation between the histological assessed liver injury and serum concentrations of enzymes. However, due to this positive correlation and the small but significant difference between NAFL and NASH serum liver enzymes might support surveillance. When the diagnosis NAFLD is established (i.e. histologically or by ultrasound) an increase of serum liver enzymes, even within the current normal range, would indicate disease progression. Though, this has to be tested in an appropriately designed study. Our finding that the serum parameters exhibited slightly stronger correlations with the SAF score than with NAS can only be attributed to the addition of the fibrosis grade to the SAF score. This clearly indicates that serum liver enzymes reflect not only injury of the hepatic parenchyma but also fibrogenesis. It has been shown multiple times that fibrogenesis and progression to advanced fibrosis occurs in a similar proportion of NAFL and NASH patients 10,35−37, albeit possibly with different pace. The hypothesis that serum liver parameters reflect liver injury and fibrogenesis in parallel would explain the limited clinical benefit for screening or diagnostic purposes in NAFLD, as progression of fibrosis is rather rare in this disease.
In summary, our data fortify findings of the past 15 years, that elevation of serum liver enzymes ALT, AST and γGT is no reliable sign for NASH or progressive NAFLD. Conversely, serum concentrations of these factors in normal range do not exclude NASH. An adaptation of normal ranges would probably dramatically increase false positive results without enhancing clinical risk assessment. Serum liver enzymes might still have use in disease surveillance, when NAFLD or NASH will be established by other diagnostic measures. It is time to move on from serum liver enzyme elevations in NAFLD detection or risk assessment and focus studies on other markers, in particular those related to adipose tissue dysfunction, glucose metabolism, and IR.