Pattern of Serum Vitamin D in Individuals with non-Alcoholic Fatty Liver and ‎its Relation to Metabolic Dysfunction

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is accumulation and storage of ‎increased amounts of fat in hepatic cells; which causes liver damage similar to ‎heavy alcoholic consumption with little or no consumption of alcohol.  NAFLD ‎has two forms: simple form of hepatic steatosis without inflammation or fibrosis, ‎and progressive form with hepatic inflammation, fibrosis, advanced cirrhosis and ‎liver failure; representing about 10-30% of NAFLD cases. NAFLD progression is ‎not always linear so it is difficult to predict when and who will progress to ‎advanced stages [1-3]‎.‎

NAFLD is a common health problem; affecting about 25-30% of people all over the world due to increased rates of obesity. The prevalence of NAFLD has doubled in the previous 20 years with hazardous health consequences [3, 5].

NAFLD could be either a result or a cause of metabolic syndrome in most cases associated with diabetes type 2 and dyslipidemia [1]. Nowadays, NAFLD is considered a consequence of metabolic syndrome, insulin resistance or lifestyle-related diseases such as diabetes mellitus (DM), dyslipidemia and hypertension rather than a primary liver disease [6, 7]. The difference in age of onset, sex of cases and severity of NAFLD is related to differences in obesity and metabolic syndrome in the general population [8, 9].

Vitamin D has an anti-inflammatory and anti-fibrotic function on the hepatic stellate cells signifying an association between vitamin D deficiency in NAFLD. However, this association is still unsettled [10].

Recent studies found that fructose metabolism produces uric acid. Both fructose and serum uric acid are associated with NAFLD; as fructose metabolized to triglycerides (TG) in the liver, and subsequently induce hepatic steatosis through mitochondrial oxidative stress and accumulation of hepatic fats [11]. Other possible mechanisms by which uric acid produce NAFLD are insulin resistance and hyperinsulinemia which decrease nitrous oxide supply to the cells [12] and increase of oxidative stress [13]; all of which promote hepatic steatosis [11-13].

Our study aimed to identify association between vitamin D deficiency and metabolic changes with non-alcoholic fatty liver disease.

PATIENTS‎ AND METHODS

A cross sectional study conducted between June 2018 and December 2020. It was carried out at the clinic of gastroenterology and hepatology in a specialized hospital. The study included 170 participants involved by convenience sampling: 85 cases of non-alcoholic fatty liver and 85 healthy controls. Sociodemographic data were collected: age, sex, residence, and smoking habits. Weight and height were measured to calculate the body mass index (BMI) by dividing weight in Kg to square height in meters.

BMI from18.5 to 24.9 was considered normal, > 25 indicated overweight and >30 indicated obesity [14]. Blood pressure was measured by sphygmomanometer and hypertension was considered as ≥140 mmHg systolic and ≥90 mmHg diastolic [15].

All the participants underwent an abdominal ultrasound (US) as well as liver function tests to be included in the study. NAFLD diagnosis was established according to the American Association for the Study of Liver Diseases (AASLD), the American College of Gastroenterology, and the American Gastroenterological Association guidelines published in 2012. The diagnosis of NAFLD required: (1) evidence of hepatic steatosis (HS) by imaging ultrasound (2) no significant alcohol consumption, (3) no competing causes of HS, and (4) no coexisting causes of chronic liver disease [16]. Ultrasound features included any 4 of the following 5 sonographic features: (1) attenuation of image quickly within 4-5 cm of depth; (2) echogenic diffuse but particularly important to note brightness within the first 2-3 cm of depth; (3) liver uniformly heterogeneous; (4) thick subcutaneous depth (> 2 cm); and (5) liver fills entire field with no visible edges [17].

Patients with chronic viral hepatitis, liver cirrhosis secondary to any cause, patients with history of primary biliary cirrhosis, primary sclerosing cholangitis, and patients with heart or respiratory or renal diseases as well as pregnant women were excluded from the study. In addition, patients on calcium or vitamin D supplementation or any patients that received vitamin D supplement in the previous 2 months as vit D has a lengthy half-life [18] were also excluded from the study.

All study populations were subjected to the following laboratory investigations:

-       Random blood glucose (RBG), HbA1c, uric acid, liver enzymes: ALT and AST, lipid profile: cholesterol, TG, LDL and HDL. They were measured using fully automated analyzer Cobas 8000 (Roche Diagnostics, Mannheim, Germany).

- Vitamin D level in addition was estimated   using Abbott Architect i-2000SR (Abbott Park, IL, USA). Architect 25-OH Vitamin D assay is a quantitative chemiluminescent microparticle immunoassay permitted by FDA to measure 25-hydroxyvitamin D in serum. Principle of the test is depending on that the sample and pre-treatment reagents are mixed with assay diluent then combine with paramagnetic anti-vitamin D coated microparticles to make a reaction mixture. Vitamin D in the sample then bind to anti-vitamin D coated microparticles. After incubation anti-Biotin acridinium-labeled conjugate complex is added to the reaction mixture and fixes to unoccupied binding sites of the anti-vitamin D coated microparticles. Pre-trigger and trigger solutions are added after washing. The reaction then is measured as relative light units (RLUs). An indirect relationship presents between the quantity of vitamin D in the serum and the RLUs sensed by the Architect i System optics.

Vitamin D level was classified into three categories as following: sufficient if more than 30 ng/ml, insufficient from 20 to 30 ng/ml and deficient less than 20 ng/ml [19].

Statistical analysis:

Data were analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Kolmogorov- Smirnov test normality distribution of variables. Student t-test was used to compare two groups in normally distributed quantitative variables, while Mann Whitney test was used for non-normal distributed quantitative variables. Chi-square test was done in categorical variables. Results Significance was judged at 5% level.

Odds ratio was used to estimate risk factors led to fatty liver. Also, the effect of vitamin D on blood sugar, blood lipids and uric acid assessed by regression analysis. Sensitivity and specificity for different parameters affecting NAFLD were estimated.

Ethical consideration: The study had ethics approval from the associated institutions and was done according to Helsinki Declaration. All patients signed a written informed consent before inclusion into this study. Privacy and confidentiality of patients were kept.  Approval from the manager of the private hospital and Ethical Committee of Faculty of Medicine, Suez University was obtained.

RESULTS:

Demographics, medical history and laboratory findings were compared between both groups. NAFLD group was younger in age and most of them were males. That group had higher BMI, liver enzymes, uric acid, RBS, HBAIC, and more impaired lipid profile. All these variables were significantly different between both groups, except for residence, smoking, hypertension, and mean LDL and HDL levels (Table 1).

Three fourths of NAFLD group had vitamin D deficiency in comparison to only 58% of control group. Only 7.1% of NAFLD group had sufficient vitamin D level; in contrast to 22.4% in the other group. This difference was statistically significant (P = 0.01)).

Univariate analysis showed that important risk factors of fatty liver were vitamin D deficiency, male sex, overweight, hypercholesteremia, hyperglycemia and elevated liver enzymes meanwhile multivariate analysis shows that only male sex and overweight were risk factors of NAFLD (Table 2).

Subgroup analysis of patients with NAFLD based on vitamin D level status revealed that cholesterol level, triglycerides, random blood sugar (RBS), hypertension (HTN) and overweight were higher in the deficient group, but the difference was not statistically significant (Table 3).

Meanwhile, when we compared vitamin D deficient cases with those with insufficient or normal level among all participants, we found that BMI, RBS, hypercholesterolemia, hypertriglyceridemia and LDL were significantly higher in the deficient group. Although HbA1C, ALT and AST, uric acid and HTN were higher in the deficient groups than those with insufficient or normal level, the difference was statistically insignificant.

Using receiver operating characteristic (ROC) statistics; to show the predictors of NAFLD, showed that sensitivity and specificity of vitamin D deficiency, at a cutoff value of ≤18.5 ng/mL, were 68.24% and 52.92%, respectively. Sensitivity and specificity of hypertriglyceridemia, at a cutoff value of >113, were 77.65% and 48.24%, respectively. Sensitivity and specificity of HBA1C, at a cutoff value of >5.2mg/L, were 69.41% and 56.47%, respectively. While, sensitivity and specificity of hypercholesterolemia, at a cutoff value of >190, were 61.18% and 50.59%, respectively. This indicated that hypertriglyceridemia, HbA1c and Vitamin D deficiency (77.6, 69.4, and 68.2%) respectively were the most sensitive indicator and predictive of NAFLD (Table 4; Figure 1, 2).