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HbA1C as a potential Biomarker of Non-Alcoholic Fatty Liver Disease: comparison with BMI, WHR and WC

Background: Nonalcoholic fatty liver disease (NAFLD) is a slow progressing disease common in obese and diabetic individuals. Present study attempts to establish association of glycosylated hemoglobin (HbA1C) with NAFLD and its severity, compared with body mass index (BMI), waist to hip ratio (WHR) and waist circumference (WC). Methods: This case control study was conducted on a sample of 450 individuals including 150 cases and 300 age and gender matched controls recruited from Dow Radiology Institute through convenient sampling. Severity of the disease among cases was determined upon ultrasonic evidence. Detailed history and physical examination was recorded. HbA1C was determined by TINIA method. The study was part of a project funded by Higher education of Pakistan. Results: Among the cases 66% whereas 32% of controls had HbA1C levels more than normal value (5.7%). HbA1C, type II DM and BMI were significantly associated with NAFLD (cOR= 4.12, 2.88, 2.25 (overweigh) and 4.32 (obese) respectively)..WC was found significantly associated with NAFLD in both genders (cOR in males= 5.50 and females= 5.79, p<0.01). After adjustment with BMI and Type II DM, HbA1C was found significantly associated with NAFLD (aOR=3.40, p<0.001) along with BMI more than 30Kg/m2 (aOR= 3.30, p< 0.001). After stratification of data on the basis of gender HbA1C and WC remained significant (aOR= 2.08 and 2.91, p<0.05 respectively) while BMI became insignificant (aOR= 1.65 and 1.73, p>0.05 for overweight and obese) in males. In females, HbA1C, WC and BMI (only in obese individuals) were found significantly associated (aOR=5.20 p<0.001, 4.28 p=0.035 and 2.90 p=0.029) respectively. AUC for HbA1C was valid for total

adjustment with BMI, WHR and WC. HbA1C may be presented as a potential biomarker for NAFLD examined with WC in adult population. Evaluation of HbA1C for potential biomarker for NAFLD merits more studies in other settings.

Background
Non Alcoholic Fatty Liver Disease (NAFLD) refers to a condition where excess fat accumulates in the liver of the people with no history of alcohol consumption. Fat molecules are deposited in the form of triacylglycerol (TAG) in hepatocytes. NAFLD is alarmingly increasing round the globe. It is known to progress into Non-Alcoholic Steatohepatitis (NASH), followed by cirrhosis of liver and eventually failure or development of carcinoma of liver [i]. The exact mechanism of the development of both NAFLD and subsequently NASH needs to be further explored. Type II Diabetes Mellitus (Type II DM), insulin resistance, obesity and dyslipidemias are the some of the known risk factors and comorbidities [ii]. In Type II DM, insulin resistance results in high blood glucose levels with an increased tendency to glycosylate the plasma proteins non-enzymatically resulting in formation of glycosylated hemoglobin (HbA 1C ) and other advanced glycation end products (AGEs). As this reaction is irreversible, HbA 1C once formed, persists till the survival of red blood cells (RBC) (Mean 116 and 106 days in males and females respectively).
The estimated global prevalence of NAFLD ranges from 6.3 -33% among general population [iii]with an increasing trend worldwide [iv]. It is the most common chronic liver disease in USA (25-30%) and Asia Pacific region (13-60%). The reported figures from Asian countries are from India 5 -28%, China 5 -24%, Japan 9 -14%, Hong Kong 16%, Taiwan 11 -41% and Indonesia 30% [v]. In  It is therefore tempting to speculate that simultaneous non-enzymatic glycosylation of enzymes involved in glucose and lipid metabolism may trigger lipid accumulation in liver even in the absence of diagnosed diabetes mellitus. Hence raised HbA 1C may be a useful biomarker of NAFLD. Moreover the various indices of body weight measurements as known risk factors of NAFLD may also be compared with the association of HbA 1C with NAFLD.
This case control study was designed (1) to measure association of HbA 1C with its severity and (2) Compare the associations of HbA 1C with BMI, WHR and WC in the study population.

Methods
This case control study was conducted at Dow University of Health Sciences (DUHS).

Individuals undergoing upper abdominal ultrasonography (U/S) at Department of Radiology
DUHS were recruited for the study. Those having fatty liver disease on ultrasound were identified as cases, while people showing no fatty infiltration were included as control.
Informed consent was obtained after explaining the study procedures and outcomes, those who refused to be included were dropped out. Considering the prevalence of the condition in Pakistan 6 , sample size was calculated by OpenEpi as 104 (52 each in case and control group). However to improve strength of study, the total sample size was increased to 450 with case to control ratio of 1:2. The participants were recruited through convenient For the purpose of standardization, subjects undergoing ultrasonography by two trained sonologists were included in the study. Patients with chronic liver disease, tumors, acute hepatitis, and those having history of alcohol consumption were excluded. Patients with type 1 diabetes mellitus and individuals with known NAFLD were also excluded. History regarding presenting complaints, comorbidities, lifestyle, dietary intake and medication were recorded on structured performa. Detailed physical examination was carried out.
Height in meters and weight in Kg was recorded for BMI calculation as Kg/m 2 , waist circumference (WC) and waist-hip ratio (WHR) were measured by standard method 11 .
Blood samples were collected in fasting state in appropriate containers with bar code, for estimation of blood glucose (FBS) by enzymatic method expressed in mg/dl and HbA 1C by turbidimetric inhibition immunoassay (TINIA) expressed in percentages (%). The value 5.7% and below was taken as normal [ii]. The study was approved by institutional review board DUHS (IRB-447/DUHS/-14) and funded by Higher Education Commission Pakistan.

Statistical Analysis:
Data was analyzed using the software SPSS version 21.0 and STATA 14. Chi-square, ANOVA and binary logistic regression were used for analysis. Frequencies and proportions were generated for all categorical variables, study participants' characteristics and body weight measurements with NAFLD. These were compared using Chi-square (x2) test, while mean differences for anthropometric measures with NAFLD severity grades were assessed using ANOVA. Binary logistic regression (univariate and multivariate) were used to analyse the factors associated with the occurrence of NAFLD. Results of regression were reported as crude and adjusted odds ratio (OR) and 95% confidence interval (CI). ROC curve was plotted to compare each variable with NAFLD. P value < 0.05 was taken as significant.

Results
Baseline characteristics are given in Table-1. Females dominated the sample with 56% in cases and 60.3% in controls (Table-1). 43.96 ± 11.06 years was recorded as the mean age of study sample. Odds for HbA 1C was significantly high (cOR= 4.12 (2.72-6.25)) and it was found to be consistently high after adjusting with history of Type II DM and the indices of body measurements (BMI, WHR and WC) 3.40 (2.19-5.26), in males (2.08 (1.06-4.11)) and females 5.20 (2.79-9.68) ( Table-3). BMI was significantly associated with NAFLD, however after adjusted with Type II DM and HbA 1C , the odds of BMI were found significant only in obese individuals, further after stratification of data on the basis of gender it became insignificant in males. In both genders WHR was found not significant. Odds for HbA 1C and WC were found consistently significant in total study sample as well as in both genders (Table-3 Present study reports significantly higher HbA 1C levels in individuals with NAFLD (p<0.001, Table-1) that increased with grades of severity (Table-2). Chronic hyperglycemia results in non-enzymatic glycosylation of various proteins that may trigger the immune response with consequent subclinical inflammation of soft tissues including liver [xi]. Glycosylated Hemoglobin reflecting long term glycemic control has been studied for its association with NAFLD. A study has reported conflicting results that "increased pancreatic echogenicity is associated with deteriorating glycemic parameters and higher risk of glycemic progression and incident diabetes, independent of HbA 1C concentration and NAFLD" [xii]. Recently it has been claimed that hemoglobin glycation index (difference between observed and predicted HbA 1C levels based on plasma glucose levels) can identify the non-diabetic individuals at higher risk of developing fatty liver [xiii]. Present study reports a significant association of HbA 1C with NAFLD .This association was positive both in diabetic and nondiabetic, obese and lean persons (cOR=4.12, p<0.001). This indicates that those who have HbA 1C higher than 5.7% are 4 time more prone to develop fatty liver disease. Similarly a Chinese study has also reported HbA 1C as an independent risk factor for development of NAFLD in elderly people 16 . Moreover 11.55% (52 individuals) of the study sample were diagnosed as Type II diabetic at the time of recruitment and in concordance with others 7 more than 90 % of individuals with Type II DM had NAFLD. While 99 (66%) had more than normal HbA 1C (Table-1). This indicates that more than half of the individuals did not know about their high HbA 1C which may have been in pre-dibetic 17  Excess abnormal fat predisposes to obesity related disease regardless of total body fat.
Present study found both BMI and WC significantly different (p<0.001) with presence of NAFLD in both genders (Table-1) while WHR was significantly different only in males. All of these indices were significantly associated with NAFLD (Table-3 This study also demonstrated significant increases in various severity grades of fatty liver with the rise in HbA 1C levels, BMI and WC (Table-2). Increasing insulin resistance has been reported with higher grades of NAFLD in diabetic and pre-diabetic individuals[xx] while other have claimed that glyacated albumin/glycated hemoglobin is inversely proportional to the severity grades of NAFLD [xxi]. With this data it is tempting to speculate that investigation of HbA 1C and central obesity may give an insight to the presence of NAFLD well before it is diagnosed.

Conclusions
Present study provides substantial evidence that high HbA 1C level is significantly associated with presence of NAFLD in both diabetic and non-diabetic individuals. HbA 1C is the single risk factor that is strongly associated with NAFLD and its severity. Among the indices of body measurements WC was found a strong risk factor in both genders, BMI was only significant in obese females while association of WHR with NAFLD was not found significant in both genders. HbA 1C may be presented as a potential biomarker for NAFLD examined with WC in adult population. Evaluation of HbA1C for potential biomarker for NAFLD merits more studies in other settings.

Limitations
Liver biopsy was not performed owing to its invasive nature with no justification for the test in controls. Secondly Ultrasonography of liver may not identify cases of NAFLD with early changes. And therefore some of the potential cases may have been grouped as controls.

Declarations
Ex-Professor of Medicine, Dow University of Health Sciences