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Association of serum alanine aminotransferase and γ-glutamyltransferase levels within the reference range with metabolic syndrome and nonalcoholic fatty liver disease

Background/Aims Nonalcoholic fatty liver disease (NAFLD) has recently been found to be a novel component of metabolic syndrome (MS), which is one of the leading causes of chronic liver disease. The serum alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) levels are suggested to affect liver fat accumulation and insulin resistance. We assessed the associations of serum ALT and GGT concentrations within the reference ranges with MS and NAFLD. Methods In total, 1,069 subjects enrolled at the health promotion center of Wonkwang University Hospital were divided into 4 groups according to serum ALT and GGT concentrations levels within the reference ranges. We performed biochemical tests, including liver function tests and lipid profiles, and diagnosed fatty liver by ultrasonography. Associations of ALT and GGT concentrationgrading within the reference range with fatty liver and/or MS were investigated. Results The presence of MS, its components, and the number of metabolic abnormalities [except for high-density lipoprotein-cholesterol (HDL-C) and fasting blood glucose] increased with the ALT level, while the presence of MS, its components, and the number of metabolic abnormalities (except for HDL-C) increased with the GGT level. The odds ratios for fatty liver and MS increased with the ALT level (P<0.001 and P=0.049, respectively) and the GGT level (P=0.044 and P=0.039, respectively). Conclusions Serum ALT and GGT concentrations within the reference ranges correlated with the incidence of NAFLD and MS in a dose-dependent manner. There associations need to be confirmed in large, prospective studies.

marker of hepatic steatosis or hepatic dysfunction. Also, as a gluconeogenic enzyme, the ALT is associated with hepatic IR. 7 A number of MS components, obesity, IR, and high sensitivity-C-reactive protein (hs-CRP), are strong predictors of increased ALT activity in NAFLD. 8 Elevated serum ALT levels have a positive association with MS-related diseases, such as type 2 DM and cardiovascular disease. 9,10 In recent data, both normal and increased levels of ALT are associated with long-term development of multiple metabolic disorders. These results indicate the potential for ALT values within the reference interval as biomarkers for the risk of MS. 11 Several prospective epidemiologic studies have demonstrated that increased concentrations of hepatic enzyme in serum, even within the reference interval, may be related to an increased risk of type 2 DM and the MS, as well as death. 12 In addition, ALT is a predictor of mortality due to unrecognized liver diseases, but also due to other causes of ALT elevation, linked to non-liver health risks, such as atherosclerosis, hypertension (HTN), and type 2 DM. 7 Even within the normal range, there is a positive association between the aminotransferase concentration and mortality from liver disease. 13 As GGT is a sensitive indicator of liver damage, 14 excess deposition of fat in the liver is associated with an elevated serum GGT and IR. 15,16 GGT is located on the external surface of most cells and mediates the uptake of glutathione, 17 an important component of intracellular oxidative defenses. 18 GGT is also considered a marker of oxidative stress and it may be directly involved in the generation of reactive oxygen species. The serum GGT level has been associated with many cardiovascular disease risk factors, DM, MS, and IR. 18,19 In Korean adults, serum GGT is closely correlated with IR and the increased number of components of MS, 20 as a surrogate marker of IR, inflammation, and MS. 21 Some studies suggest that within its reference interval, the serum GGT concentration has been closely related to the presence of components of the MS. 7 Recent reports suggest that an increased GGT level is a risk factor for advanced fibrosis in NAFLD 21 and, with weight loss, a decrease in GGT activity is predictive of improved lobular inflammation and fibrosis of liver. 20 Thus, measurement of ALT and GGT may identify subjects in the general population with a risk of NAFLD and MS. 7 However, limited data exist on the significance of GGT or ALT activities within reference interval in subjects with NAFLD and MS.
Therefore, we assessed the association of serum ALT and GGT activities within reference range with MS and NAFLD.

Subjects
The medical records of 2,024 subjects who attended our

Assessment of hepatic steatosis
We used ultrasonography as a non-invasive method to diagnose fatty liver disease. All ultrasound scans were performed by two skilled operators using a high resolution B-mode scanner (SSD-5500; Aloka, Tokyo, Japan). Liver steatosis was assessed on the basis of abnormally intense, high-level echoes arising from the hepatic parenchyma, the liver-kidney difference in echo amplitude, the echo penetration into the deep portion of the liver, and the clarity of the blood vessel structure in the liver. 22

Subject assessment and anthropometric measurements
A self-administered questionnaire also served to measure medical history of the examinee and family, medication history, and lifestyle (cigarette smoking, drinking alcohol, and exercise). Anthropometric measurements of height, body weight, and WC were made on the same day. The height was measured to the nearest 0.5 cm. The body weight was measured in light clothing and without shoes to the nearest 0.1 kg. The WC was measured to the nearest 0.1 cm with flexible tape at the midpoint between the lower rib margin and the iliac crest in the horizontal position.

Clinical and metabolic characteristics according to ALT and GGT activities
Clinical characteristics of the study subjects according to serum GGT and ALT were presented in Tables 1, 2 and 3. As   (Table 3).  The factors (ALT grading and GGT grading) correlated with fatty liver or metabolic syndrome were evaluated using the multiple logistic regression analysis after adjustment for clinical and biochemical parameters. The several variables with P less than .1 in univariate analysis were included. * Adjusted for obesity, systolic blood pressure >130 mmHg, diastolic blood pressure >85 mmHg, metabolic syndrome, fasting blood sugar, GGT, † Adjusted for obesity, systolic blood pressure >130 mmHg, diastolic blood pressure >85 mmHg, metabolic syndrome, fasting blood sugar, ALT, § Adjusted for age, sex, systolic blood pressure >130 mmHg or diastolic blood pressure >85 mmHg, HDL cholesterol, fatty liver, body weight, GGT, ∥ Adjusted for age, sex, systolic blood pressure >130 mmHg or diastolic blood pressure

Association of MS and fatty liver with ALT and GGT activities
In male subjects, the presence of MS, its components, and the number of metabolic abnormalities increased with increased ALT grading, except the low HDL-C and fasting plasma glucose>100 mg/dL (Table 4). With increased ALT grading in female subjects, the presence of MS, its components, and the number of metabolic abnormalities increased, except the low HDL-C (Table 5). With increased GGT grading in total subjects, the presence of MS, its components, and the number of metabolic abnormalities increased, except the low HDL-C ( Among the hepatic enzymes, ALT is most closely related to liver fat accumulation. 24 Our results appear to agree with the findings of several previous studies that increased ALT concentration, even within the reference interval, is an independent predictor of incident NAFLD 12 and ALT is more closely associated than GGT with both hepatic IR and a later decline in hepatic insulin sensitivity. 25 This finding could be explained by the higher specificity of ALT for liver injury. 24 Also, as a gluconeogenic enzyme, 12 increased ALT has been demonstrated to be an indicator of impaired insulin signaling, which might not necessarily be associated with liver injury due to hepatic steatosis. 26,27 GGT might also be involved in the pathogenesis of NAFLD through another mechanism, such as oxidative stress. 28 Elevated ALT could be a prognostic marker for development of various MS-related diseases, including type 2 DM, cardiovascular disease, and atherothrombosis, and a minor ALT elevation is also an important feature for effective screening of MS. 7 Thus, individuals with a minor elevation in the serum ALT level that is close to the upper limits of the reference range are also of concern because elevated ALT itself is closely associated with IR, even in the absence of NAFLD and obesity. 7 Because the mean ALT activities in subjects with 2 or 3 metabolic risk factors is not particularly high, tending only to be close to the upper limit, screening for serum ALT levels may facilitate identifying the potential risk for MS-related diseases in the general population, 7 in addition to detection of liver disease, even within the reference interval.
The mechanism underlying the relationship between IR and GGT elevation has not been clarified, although hepatic steatosis or hepatic IR caused by visceral obesity may be the first mechanism. [29][30][31][32][33] Because oxidative stress can play a role in the pathophysiology of cardiovascular disease, and GGT has a pivotal role in maintaining intracellular glutathione transport into most types of cells, oxidative stress provides a second possible mechanism. [34][35][36] Increased GGT activity may be a response to oxidative stress, one which can increase the transport of glutathione precursors into cells. In addition, GGT leaks into serum possibly as a result of normal cell turnover and cellular stress. 8 Subclinical inflammation may be another possible cause. 37,38 In the present study, the white blood cell counts were also related to serum GGT and ALT concentrations. The significant associations between serum GGT and ALT activities and MS, even after adjusting for fatty liver, suggest that another mechanism governs the relationship between serum enzymes and MS, such as oxidative stress or subclinical inflammation.
In our study, the prevalence of NAFLD diagnosed by ultrasonography was 25.3%. Male gender was significantly associated with NAFLD. In addition, the increased ALT and GGT activities with the reference interval were associated with various cardiovascular disease risk factors, prevalence of fatty liver, number of components of metabolic abnormalities, and presence of MS.
In this study, using ROC curve, cut-off levels of ALT and GGT activities to predict NAFLD or MS were estimated. In male subjects, ALT activities within third grading were considered to predict NAFLD and MS, as cut-off levels. In female subjects, ALT activities within second grading were estimated as adequate cut-off levels to predict NAFLD and MS. In total subjects, GGT activities within third grading were considered as cut-off levels to NAFLD and MS. Therefore, even ALT and GGT levels within reference range may be considered to predict NAFLD and/or MS. However, this study is inadequate to clarify significance of ALT and GGT activities within reference level as predictor of NAFLD and/or MS.
In summary, even ALT and GGT activities within the reference interval correlated with incident NAFLD and MS in a dose-dependent manner, although the risks in lower quartiles were low according to the ALT or GGT grading. Therefore, increased ALT and GGT activities, despite remaining within the reference interval, might be an important preclinical marker of NAFLD and MS. Although the mechanism through which serum ALT and GGT are related to the risk for NAFLD and MS remains to be elucidated, ALT and GGT might be not only indicators of liver injury by hepatic steatosis, but also an early indicator of impaired insulin signaling, subclinical inflammation, and oxidative stress.