Prevalence of asymptomatic nonalcoholic fatty liver disease in nondiabetic participants: a study from South India

Background Nonalcoholic fatty liver disease (NAFLD) is a common chronic disease that is rapidly increasing worldwide. Aim The aim of this study was to investigate the risk factors associated with NAFLD in nondiabetics from South India. Participants and methods We recruited 345 asymptomatic participants consecutively, and the study period was between January 2014 and December 2017. All participants underwent risk factor evaluation, fasting serum lipid profile, C-reactive protein (CRP), hemoglobin A1c, liver function test, and abdominal ultrasound. Results Of 345 participants, men represented 213 (71%). The mean age of the participants was 58.4±11.1 years, with age range from 24–68 years. Prevalence of NAFLD was seen in 22%. On risk factor evaluation, 107 (31%) were hypertensive, 89 (25.7%) smoked, 52 (15%) were overweight, 110 (24.9%) were obese, and 113 (32.7%) had dyslipidemia. The mean CRP was 12.7±9.5 mg/l. High γ-glutamyl transferase levels, elevated CRP levels, obesity (45, 59.2%), high total cholesterol (49, 64.4%), low levels of high-density lipoprotein (28, 36.8%), high levels of low-density lipoprotein (27, 35.5%), and high triglycerides (31, 40.7%) were significantly associated with NALFD compared with non-NAFLD. After adjustment using multiple regression analysis, obesity (odds ratio: 3.5; 95% CI: 2.18–6.16), high total cholesterol [odds ratio: 4.9; 95% confidence interval (CI): 2.91–9.43], low high-density lipoprotein (odds ratio: 2.3; 95% CI: 1.20–4.47), high low-density lipoprotein (odds ratio: 2.9; 95% CI: 1.58–6.17), high triglycerides (odds ratio: 2.4; 95% CI: 1.33–4.60), and elevated CRP levels (odds ratio: 2.0; 95% CI: 1.21–3.39) were significantly associated with NAFLD. Conclusion Our study established obesity, CRP positivity, and dyslipidemia as independently associated with NAFLD in South Indian patients.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a separate hepatic condition distinguished by abnormal fat deposits in liver cells causing chronic liver disease [1]. NAFLD is now recognized as one of the major chronic liver diseases in industrialized countries [2]. At present, NAFLD is an increasing major health problem worldwide [3]. At the time of diagnosis, most patients with NAFLD have minimal signs and symptoms of liver disease, even though some patients have discomfort or sensation of fullness on the right side of the upper abdomen; there is generalized fatigue with hepatomegaly in most of the patients. Prevalence of NAFLD is 15-40% in Western countries and 9-40% in Asia [4]. Recent studies from the Indian subcontinent recorded a NAFLD prevalence of 9-32% in the general population [5,6]. Studies have established hypertension, type II diabetes, smoking, obesity, and dyslipidemia to be significantly associated with NAFLD [5,6]. The aim of this study was to investigate NAFLD risk factors among nondiabetics in general population. Limited studies are available on this topic from Indian subcontinent.
Yashoda Hospital and Nizam's Institution of Medical Sciences, Hyderabad. The study period was from January 2014 to December 2017, and the study was approved by Institutional Ethics Committee.
Detailed medical history (present and past) was collected from all participants. Standardized questions were adapted from the behavioral risk factor surveillance system prescribed by the Centers for Disease Control and Prevention. All participants underwent glycosylated hemoglobin A1c (HbA1c), lipid profile, C-reactive protein (CRP), ultrasound abdominal and liver function test [alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), and alkaline phosphatase], and viral screening (HIV and hepatitis B and C). NAFLD was diagnosed based on criteria defined by American Gastroenterology Association [7], and based on the ultrasonography, NAFLD was classified into grade I, grade II, and grade III [8].

Including and excluding criteria
Participants older than 18 years were included in the study. If participants having any present or past history of NAFLD; type I or type II diabetes; cardiovascular disease; cerebrovascular disease; other liver diseases like viral hepatitis, primary biliary cirrhosis, druginduced liver damage, autoimmune hepatitis, biliary obstruction, alcoholic liver disease, and Wilson's disease; α 1 -antitrypsin deficiency; HIV infection; alcohol intake more than 20 g/day; severe end organ damage; malignancy; previous gastrointestinal tract surgery; ingestion of drugs known to produce hepatic steatosis; patients taking corticosteroids, high-dose estrogens, methotrexate, tetracycline hydrochloride, amiodarone, or tamoxifen citrate in the previous 6 months; those who had received any prior therapies that may have been beneficial for NAFLD, such as vitamin E, pentoxifylline, and pioglitazone; and those prescribed diet and exercise for weight loss were excluded from the study.

Risk factor assessment
According to Joint National Committee VI-VII, hypertension was defined as a systolic blood pressure more than 140 mmHg and/or a diastolic blood pressure more than 90 mmHg based on the average of the two blood pressure measurements, or a patient's selfreported history of hypertension or antihypertensive use, supported by documents. Diabetes was diagnosed if the patient was on antidiabetic medications. Smokers were defined as those reporting daily smoking, and exsmokers and occasional smokers were classified as nonsmokers. BMI value of 25.1-30 kg/m 2 was considered overweight and more than 30 kg/m 2 was measured as obese. Dyslipidemia was defined (ATP III) as one or more of the following: total cholesterol more than 200 mg/dl, low-density lipoprotein (LDL) cholesterol more than 130 mg/dl, high-density lipoprotein (HDL) cholesterol below 40 mg/dl, very low-density lipoprotein (VLDL) cholesterol more than 30 mg/dl, and triglycerides more than 150 mg/dl. The CRP was assessed by quantitative analysis, and more than 10.1 mg/l was considered positive [9,10].

Discussion
In our study, we found a prevalence of 22% of NAFLD in asymptomatic participants from South India; other studies have found similar findings from 9.3 to 29.5% in Asian countries [11,12], 17.1% in Mexico [13], 30% in Israel [14], 20% in Romania [15], and 22.6% in Italy [16]. Ethnicity and racial differences possibly account for the small changes in the prevalence of NAFLD and less prevalence of NAFLD in African Americans [17].
The difference may be owing to less visceral fat and a different lipoprotein metabolism in African Americans [18].
Recent studies have established increasing prevalence of NAFLD with age [1]. We found our study the mean age (59.9 years) had borderline significant association with NAFLD; other studies have noted slightly lower mean age (49.14+9.65) years [8] and 55.4 years [19].
Fatty liver occurs in all age groups. The liver regulates alcohol metabolism, and as the body ages, toxicity elevates owing to increased organ damage. These developments are related to a mitochondrial transport flaw rising with age and decrease in the smooth endoplasmic reticulum and metabolism of CYP2E1-dependent microsomal ethanol oxidation functions [20].
In our study, we noted that among patients with NAFLD, 43 (56.5%) were men, whereas other studies have noted in 53.3% of men [21]. Our study established no significant association between men and women with NAFLD. However, some studies have noted NAFLD being more predominant in women [1].
We found hypertension in 22 (28.9%) patients with NAFLD, with no significant association with NAFLD. However, some studies have found a significant association with NAFLD [22,23].
This study found smoking was seen in 21 (27.6%) of patients with NAFLD, with no significant association with NAFLD. In contrast, other studies have noted smoking was association with NAFLD [24].
This study found obesity was present in 45 (59.2%) of patients with NAFLD, with a significant association with NAFLD. Our findings were advocated by other studies, which showed the prevalence ranges from 22-74% in obese children and adults [25,26]. De Sousa et al. [26] found in a population-based study, central obesity was seen in 50.5% of men and 38.9% of women, and it had an independent association with NAFLD. Cotrim  Asian participants have a higher ratio of visceral fat and a minor lean body mass than Caucasian participants with the same BMI. Central obesity is an important factor for insulin resistance, and an increased visceral adiposity may cause pathogenesis of NAFLD. Visceral adipose tissue is strongly associated with insulin resistant and produces more free fatty acid than accomplish adipose tissue in other sites. In liver deposited of undeveloped lipogenesis causing to NAFLD [27].
Studies have reported that obesity is a major risk factor for NAFLD [27]. In our study, after adjustment of logistic regression analysis, obesity was an independent risk factor for NAFLD (odds ratio: 3.5; 95% CI: 2.18-6.16). Our findings were advocated by de Sousa and colleagues [26][27][28].   Differences in body fat distribution of patients with NAFLD may have a genetic predisposition. Deposits of lipids within hepatocytes, in the form of triglycerides, can lead to development of NAFLD. The primary metabolic abnormalities leading to lipid accumulation are not proper understood, but they may consist of alterations in the pathways of uptake, synthesis, degradation or secretion in hepatic lipid metabolism and finally from insulin resistance. Insulin resistance is one of the factor for development of NAFLD [8]. The pathophysiology of NAFLD is still incompletely understood. Storage of triglycerides in hepatocytes due to oxidative stress, lipid peroxidation, and proinflammatory cytokines [e.g. tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)]. Animal studies have shown elevated fatty acids in the liver, and it may cause higher levels of TNF-α. When hepatocytes get damaged, liver-specific macrophages (Kupffer cells) are activated and produce more TNF-α and IL-6 into the blood stream, which leads to the production of the acutephase protein high-sensitivity CRP [35].
In our study, we established that elevated total cholesterol (odds ratio: 4.9; 95% CI: 2.91-9.43), LDL (odds ratio: 3.0; 95% CI: 1.55-6.07) triglycerides (odds ratio: 2.4; 95% CI: 1.33-4.60), and low HDL (odds ratio: 2.3; 95% CI: 1.20-4.47) are independent association with NAFLD; our findings are supported by Amirkalali et al. [36].In our study, we found elevated CRP levels were significant associated with NAFLD compared without NAFLD, and our findings are supported by Nigam et al. [35]. Very few studies have found elevated CRP levels are a useful diagnostic marker for NAFLD [37,38]. Studies have noted CRP levels were higher in Asians Indians than Caucasians [39]. Park et al. [40] showed in their study that elevated high-sensitivity CRP level was associated with NAFLD in nonobese healthy men. The proposed mechanism between CRP and NAFLD is the rise in acute-phase cytokines. IL-6 is a strong booster and elevates CRP levels.
Our study found that elevated ALT (72.8±14.8 IU/l), AST (47.4±11.7 IU/l), and alkaline phosphatase (128.1 ±13.8 IU/l) are significantly associated with NAFLD compared with without NAFLD. A similar finding was noted by Agrawal et al. [32], who noted in their study ALT had a mean value of 97.0±56.0 IU/l, AST had a mean value of 75.0±50 IU/l, and alkaline phosphatase had a mean value of 216.0±77 IU/l. Jali et al. [31] noted elevated levels of AST (30%) and ALT (22%) in NAFLD. Amirkalali et al. [36] showed in their study very low prevalence of AST (3.9%) and ALT (10.4%) in NAFLD. However, some studies have found no significant association AST and ALT with NAFLD [42,43]. Studies have established GGT levels were significantly associated with NAFLD [44,45].
Our study showed elevated GGT levels were significantly associated with NAFLD when compared with non-NAFLD; our findings are advocated by Saxena and colleagues [44,45].

Limitations of study
The main drawback of the study was that we were unable to examine insulin resistance in our participants. The second drawback was that we were unable to analyze grade I to grade III with multiple logistic regression owing to small number. In our study, all asymptomatic participants underwent for one laboratory examination at the time of recruitment. Some studies have found liver biopsy as a gold standard for NAFLD diagnosis, but it is an invasiveness and painful procedure with complications, and sometimes, mortality also occurs. In this study, we performed ultrasonography, which is a noninvasiveness procedure and a promising tool for NAFLD in asymptomatic participants, and it also cost-effective. Ultrasonography has a sensitivity of 60-94% and specificity of 88-95% [46]. In our study, we assessed the relationship between CRP and NAFLD and analyzed multiple logistic regressions for potential risk factors to NAFLD.

Conclusion
In this study, we established dyslipidemia, obesity, and CRP positivity as independent risk factors for NAFLD in asymptomatic nondiabetic South Indian participants. We need long-term follow-up studies from India on patients with NAFLD to understand the temporal evolution of NAFLD, metabolic syndrome, and coronary artery disease in our population. From our results, we suggest that every asymptomatic participant older than 40 years should undergo ultrasonography screening. Further studies should explore these findings.
VCS Srinivasarao Bandaru was involved in data collection and manuscript writing; Jaydip R. Chaudhury in manuscript review and data analysis; Palle L. Reddy in manuscript writing and interpretation of data; Somala N. Reddy in data collection and statistical analysis; Pradeep K. Misra in manuscript review and data collection; Banda Balaraju in manuscript review and interpretation of data; and Kandadai R. Mridula in study design, manuscript preparation, data review, manuscript review, and statistical analysis. All authors have read the manuscript and approved it for submission. This study was funded by Yashoda Research Foundation, Hyderabad, India.

Financial support and sponsorship
Nil.

Conflicts of interest
There are no conflicts of interest.