Association between Liver Enzymes and Dyslipidemia in Yemeni Patients with Type Two Diabetes Mellitus

Type two diabetes mellitus (T2DM) is characterized by chronic hyperglycemia and is associated with dyslipidemia and liver function disorders. This study was conducted to assess the association between liver enzymes and elevated lipid prole in a sample of Yemeni patients with T2DM. A case-control study comprising 142 T2D patients and 142 healthy control subjects was carried out at the outpatient clinics of Ibn-Sina hospital, Mukalla, during the period from January to May 2020. Serum fasting blood glucose (FBG), total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) were analyzed using the Cobas Integra Plus 400 autoanalyzer. Also, anthropometric and blood pressure measurements were taken from each participant. Independent sample T-test and Pearson correlation coecient were used. T2D patients had signicantly higher FBG (P= <0.0001), total cholesterol (P= <0.0001), LDL-C (P= <0.0001), and GGT (P= <0.0001) while, HDL-C was signicantly lower in T2D patients (P= 0.021). In correlation analysis, serum GGT was positively associated with FBG (r= 0.216; P= <0.0001), total cholesterol (r= 0.196; P= 0.0001), triglyceride (r= 0.123; P= 0.038), and LDL-C (r= 0.209; P= <0.0001). Also, serum ALT was positively associated with FBG (r= 0.145, P= 0.014) and triglyceride (r= 0.172, P= 0.004). In conclusion, higher levels of ALT and GGT could be used as the predictive markers for NAFLD in T2D patients with hyperlipidemia. Thus, routine screening of liver enzymes Data presented as mean±SD for normal continuous variables and median (interquartile range) for continuous non-normal variables. Independent sample T-test for normally distributed continuous variables and Mann-Whitney U test for skewed continuous variables. P-value <0.05 was considered statistically signicant.


Introduction
Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia which results from defective insulin action and secretion or both [1]. World Health Organization projects that the number of diabetic patients will exceed 350 million by 2030 [1]. Previous data have documented liver disease is a major cause of morbidity and mortality of type 2 diabetes patients [2][3]. It is well known that the liver is a vital organ in the metabolism of carbohydrates and in maintaining glucose homeostasis during fasting and postprandial period [2,4].
Non-alcoholic fatty liver disease (NAFLD) is the scope of chronic liver disease in T2D patients [5], which is characterized by excess deposition of fat in the liver and associated with hepatic insulin resistance [3] and T2D risk [5]. Serum alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) has been shown to be good biomarkers of NAFLD. ALT has been considered the speci c marker of liver injury, as found in high concentrations in hepatocytes [6], while GGT is present on the surface of most cell types and highly active in the liver, kidneys, and pancreas [7]. Also, GGT is responsible for extracellular glutathione catabolism and may be linked to oxidative stress [8] and chronic in ammation [9]; both oxidative stress and chronic in ammation are important pathways for hepatic insulin resistance (IR) and subsequently T2D development [10].
Hyperinsulinemia and IR play an important role in lipid abnormalities for T2D patients [2,11]. Also, altered lipoprotein patterns and liver enzymes have been identi ed as indepen dent risk factors for the development of cardio vascular disease (CVD) [10][11][12]. Moreover, higher levels of triglycerides, LDL-C, total cholesterol, and lower levels of HDL-C were reported in T2D patients than normal healthy subjects [13].
However, few studies reported the correlation between liver enzymes and lipid pro le in T2D patients, hence this case-control study was conducted to assess the association between liver enzymes and elevated lipid pro le in a sample of Yemeni patients with T2D.

Study Design and Subjects Selection
This is a case-control study was carried out at the College of Medicine and Health Sciences, Hadhramout University, and the subjects were selected from the diabetic outpatient clinic of Ibn-Sina hospital, Mukalla during the period from 1 st January to 30 th May 2020. A total of 284 Yemeni adult subjects, randomly selected, and recruited into this study. At recruitment, an in-person interview was conducted using a structured questionnaire to collect health-related information. The study group was subdivided into two groups: 142 healthy control subjects composed of 51 males and 91 females (age: 46.0±7.94 yr.), and 142 T2D patients composed of 64 males and 78 females (age: 54.0±8.29 yr.). T2D patients were those who reported being diagnosed with T2D. Healthy control subjects were selected from the remaining participants who were free of T2D and were matched for age, sex, and dialect group with cases on a 1:1 ratio. Moreover, the selected healthy control subjects were screened for the presence of undiagnosed T2D at the time of blood donation by measuring fasting blood glucose (FBG). Healthy control subjects with FBG ≥7.0mmol/L were excluded from the study. Written consent was obtained from each participant entered into the study. The study was approved by the Ethics Committee of the Medicine and Health Sciences College, Hadhramout University, Mukalla, Yemen. Patients with co-morbidities such as chronic liver disease, chronic renal disease, cardiovascular disease, and malignancy were excluded.

Data Collection
A questionnaire form focusing on demographic information and diabetes history was given to all subjects. The patient's demographic information, clinical presentation, medical history, and physical ndings were taken from each subject. This included the patient's age, sex, smoking status (never, current or past), hypertension status (yes or no), diabetes status (yes or no) diabetes duration (years), diabetes medication, and diabetes complications. Patients were diagnosed with diabetes based on medical history, present intake of diabetes medications, or according to the American Diabetes Association (ADA) criteria [14]. Classi cation of Body Mass Index (BMI) was based on the World Health [15].

Anthropometric and Blood Pressure Measurements
Weight and height were measured following measured according to WHO guidelines [15]. Body mass index (BMI) was calculated as weight/height 2 (Kg/m 2 ). Obese subjects were de ned as BMI ≥30 kg/m 2 and normal-weight subjects having a BMI of 18-25 according to WHO guidelines [15]. Patients who had a blood pressure of ≥140/90 mmHg or were taking antihypertensive medications were diagnosed with hypertension [16]. A true healthy normal ALT level ranges from 29 to 33 IU/l for males, and 19 to 25 IU/l for females and levels above this should be assessed as described by the American College of Gastroenterology (ACG) [17].

Biochemical Investigations
Ten milliliters of the venous blood sample was obtained from consenting subjects. The blood samples were collected by vein puncture in tubes without anticoagulant. The blood samples were then transported to the laboratory immediately. The serum was separated and stored at −20°C freezers till analyses. The serum samples of matched case-control pairs were randomly placed next to each other with the case/control status blinded to the laboratory personnel and were processed, and tested in the same batch. All laboratory equipment was calibrated. Thawing freezing was avoided by dividing the samples into aliquots. Plasma fasting blood glucose (FBG), total cholesterol, triglycerides, and HDL-cholesterol (HDL-C) were determined enzymatically using a chemical autoanalyzer (Cobas Integra 400 Plus, Roche diagnostic GmbH, Mannheim, Switzerland), following the standard procedures as described by the manufacturer. Concentrations of LDL-cholesterol (LDL-C) were calculated using Friedwald's formula [18].
All biochemical investigations were analyzed in the National Center for Public Health Labs-Mukalla, Yemen.

Statistical Analysis
Data were analyzed using the Statistical Package for the Social Sciences for Windows (version 24) and are expressed by mean ± standard deviation (SD) for continuous variables (normally distributed). Noncontinuous variables are expressed by median (inter-quartile range) and n (percentage) for categorical variables. Independent Student's t-test used for normally distributed continuous variables and Wilcoxon signed-rank test for skewed continuous variables. The Pearson correlation test was performed with ALT, AST, and GGT as the dependent variables. The statistical analysis was conducted at a 95% con dence level and a P -value <0.05 was considered statistically signi cant.

Discussion
Despite the incidence of diabetes is increasing worldwide and its prevalence is higher in developing countries, no studies have examined the relationship between liver enzymes with hyperlipidemia among T2D patients in Yemen. Additionally, most people aged ≥45 years in devel oping countries suffer from diabetes [19]. These ndings were convenient with our study showed that T2D patients had signi cantly higher mean age compared to healthy control subjects (Table 1). Besides, the present study observed that BMI, systolic BP, and diastolic BP were signi cantly higher in T2D patients than healthy control subjects.
T2D patients had signi cantly higher FBG, total cholesterol, and LDL-C compared with healthy control subjects. In contrast, HDL-C was signi cantly lower in T2D patients, whilst, no signi cant difference was found among both groups for triglyceride. Additionally, higher levels of GGT were revealed in T2D patients. In contrast, AST was signi cantly lower in T2D patients. No signi cant difference was found among both groups for ALT. Such a positive relationship between liver enzymes and lipid pro le has been observed in previous studies [2,[20][21][22]. Furthermore, a study by Adeniran et al. observed that ALT and AST were signi cantly increased with dyslipidemia in Nigerian patients diagnosed with T2DM [23].
Our study further revealed positive correlations between GGT with FBG, total cholesterol, triglyceride, and LDL-C across the combined group before and after adjustment for age and BMI, whilst, the association between ALT with FBG and triglyceride was no longer signi cant after adjustment for age and BMI. From the above data, our ndings agree with previous studies [24][25]. This nding supports the role of hepatic insulin resistance in the pathogenesis of NAFLD [24,26]. Moreover, Marchesinia et al. showed an association between higher ALT levels with increased IR, blood lipids [27], and fatty liver [28] in T2D patients. Besides, a positive relationship between elevated liver enzymes with fasting and postprandial glucose was observed [29]. The liver is a vital organ in maintaining glucose homeostasis during the fasting and postprandial period [2,4].
Since the liver is the important organ involved in carbohydrate and lipid metabolism and due insulin resistance is common in T2D patients, the function of the liver gets disturbed. Insulin contributes a proin ammatory effect to liver abrasion [30]. Hyperlipidemic pro le is observed due to increased transportation of fat to the liver with respect to decreased oxida tion [4]. The impairment of the normal process of syn thesis and elimination of triglycerides may progress to brosis, cirrhosis, and hepatocellular carcinoma [31][32]. One study also reported a correlation between ALT activity and increased fatty liver [28].
GGT is known as a marker of hepatobiliary dis orders and is associated with other pathologi cal conditions like diabetes. Free radicals generated by diabetes consume glutathione which induces the increased expression of GGT in hepatocytes. Various studies have suggested the association of GGT concentrations with T2D [33][34][35][36] and hyperlipidemia [37]. These ndings are in agreement with our study; GGT was signi cantly associated with the hyperglycemic and hyperlipidemic pro le. We observed ALT and GGT together were positively correlated. Moreover, some data also reported ele vated GGT levels with ALT in T2D patients with dyslipidemia [34,35,38]. Although we did not con rm the presence of fatty liver by ultrasound techniques, we showed the relationship of ALT, AST, and GGT with the pre dictors of diabetes and lipid pro le parameters, presenting hepatocellular injury. In contrast, the present study showed a weak positive correlation between AST with blood glucose and lipid pro le; being AST is a less spe ci c biomarker of hepatic injury than ALT and GGT [34].
A study of male Korean workers found that AST was independently associated with diabetes [39], while in a study of male Japanese o ce work ers AST was not associated with T2D risk [35]. Some studies also reported that ALT is a signi cant predictor of diabetes while AST is not [2,40]. These ndings are in agreement with our ndings as AST does not show considerable relation ship with the studied parameters. However, our study is limited to the standard method of liver biopsy for the prediction of NAFLD but it goes with the analysis of the Third National Health and Nutritional Examination Survey where individuals with NAFLD are known to have elevated aminotransferases; AST, and ALT. Moreover, Clark et al. sug gested that mild or chronic elevations of these aminotransferases may be due to NAFLD [41][42]. One limitation of the present study was the small sample size. Thus, further large sample size studies with more biomarkers such as insulin, hs-CRP, and adipokines are required with liver enzymes and lipid pro le. Finally, we conclude that ALT and GGT are the predictive markers for NAFLD in T2D patients with hyperlipidemia.

Conclusion
Higher levels of ALT and GGT may be used as the predictive markers for NAFLD in T2D patients with hyperlipidemia. Thus, routine screening of liver enzymes and lipid pro le in T2D patients is recommended for the early detection of liver abnormalities and diminish diabetes complications. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, lowdensity lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase.