Serum vitamin D concentrations are associated with obese but not lean NAFLD: a cross-sectional study

Background: Low serum vitamin D concentrations have been reported to be associated with an increased risk of non-alcoholic fatty liver disease (NAFLD). However, whether slim or obese people show a similar association between vitamin D and NAFLD remains speculative. This study aimed to explore the relationship between serum vitamin D concentrations and NAFLD in lean and obese Chinese adults. Methods: This cross-sectional study included 2538 participants (1360 men and 1178 women) who underwent their health checkups at the First Aliated Hospital of Zhejiang University School of Medicine in 2019. NAFLD was diagnosed by liver ultrasound excluding other causes. The association of serum vitamin D concentrations with NAFLD was analyzed in lean and obese participants. Results: The overall prevalence of NAFLD was 33.61% (13.10% in lean and 53.32% in obese) in this study population. The serum vitamin D levels of obese NAFLD patients were lower than that of the obese NAFLD-free controls. However, the serum vitamin D levels of lean NAFLD patients were comparable to that of the lean NAFLD-free controls. Serum vitamin D levels were negatively correlated with the prevalence of NAFLD in obese but not lean participants. Serum vitamin D levels were independently associated with the risk of NAFLD in obese participants, with an adjusted odd ratio (95% CI) of 0.986 (0.979–0.992). However, the serum vitamin D levels were not related to the risk of NAFLD in lean participants. Conclusions: Low serum vitamin D levels are associated with NAFLD in obese but not lean participants. VD suciency, ≥ 100 nmol/L; VD insuciency, 50–100 nmol/L; and VD deciency,


Background
Non-alcoholic fatty liver disease (NAFLD) is hepatic steatosis when liver lipid deposition is not secondarily caused by heavy drinking or other known etiologies with or without in ammation and brosis (1). NAFLD is currently one of the world's highest prevalence of chronic liver diseases, affecting approximately 29.2% of adults in China (2). NAFLD includes simple steatosis, steatohepatitis, cirrhosis, and even hepatocellular carcinoma (3,4). Patients with NAFLD have increased risks of cardiovascular disease, stroke, type 2 diabetes, and extrahepatic malignancies (5)(6)(7)(8). The high prevalence and serious clinical harms of NAFLD make it a global research hotspot in recent years (9).
Obesity is closely related to NAFLD, and the prevalence and risk of NAFLD in obese individuals are higher than those in lean individuals (10). But recently, many studies have shown that non-obese people also have a high prevalence of NAFLD (11,12).
We have previously reported that in China, the prevalence of NAFLD in the non-obese population is 7.3%, and 8.9% of the nonobese adults developed NAFLD during a 5-year follow-up (13). Compared with obese NAFLD patients, lean NAFLD patients are usually asymptomatic and di cult to diagnose, but in fact, they also have severe liver histological necrotizing in ammation and high mortality (14). NAFLD in the non-obese population may also cause signi cant health problems (15). Therefore, the identi cation, diagnosis, and treatment of non-obese NAFLD are very important.
The risk factors for non-obese NAFLD remain unclear. Previous cross-sectional studies have shown that vitamin D de ciency was associated with an increased risk of NAFLD, and vitamin D levels were negatively associated with the severity of NAFLD (16,17).
Several prospective studies have pointed out that serum vitamin D de ciency was accompanied by an increased risk of incident NAFLD (18)(19)(20)(21). Our recent study showed that the serum vitamin D levels in high-fat diet-fed mice were signi cantly decreased, and vitamin D supplementation ameliorated high-fat diet-induced hepatic steatosis in mice (22). Vitamin D supplementation could also improve hepatic steatosis in patients with NAFLD (23,24). However, it is unclear whether obesity affects the correlation between vitamin D concentrations and NAFLD, and whether serum vitamin D concentrations are related to NAFLD in lean individuals.
In this study, we aimed to explore the correlation between serum vitamin D concentrations and NAFLD in obese and lean Chinese adults.

Participants
We enrolled adults who underwent health checkups at the First A liated Hospital of Zhejiang University School of Medicine in 2019 as participants in our cross-sectional study. The analysis included participants with complete anthropometric, biochemical data records (including serum vitamin D concentrations) and liver ultrasound results. We excluded the following participants: (1) participants with incomplete anthropometric and biochemical data; (2) men with alcohol consumption > 210 g/week and women with alcohol consumption > 70 g/week; (3) participants with other chronic liver diseases caused by autoimmune hepatitis or viral hepatitis; and (4) participants who use hepatotoxic drugs (such as sulfonamide and azithromycin). The nal analysis included 2538 participants (1360 men and 1178 women).
The personal information of all participants was anonymous. The study was approved by the Ethical Committee of the First A liated Hospital of Zhejiang University School of Medicine.

Clinical examinations
Clinical examinations included questionnaires, medical history, anthropometry, and biochemical measurements. Through the examination, the physician recorded the medical history (including previous diseases and drug prescriptions) and drinking frequency and amount. The smoking history was also recorded and distinguished as yes and no.
The anthropometric measurements were performed as previously described, including body weight, standing height, waist circumference, and blood pressure (25,26). Weight and height were measured with light clothing and no shoes. Waist circumference was measured when the patient exhales with the tape measure placed between the lowest rib and the top edge of the top. Blood pressure was measured after resting for 5 minutes. Body mass index (BMI) was calculated as the weight (kg) divided by the height (m) squared.
Fasting blood samples were taken from the anterior cubital vein and were used for biochemical analysis. Measurements include liver enzymes, blood lipids, glucose, and uric acid. All biochemical values were measured by a Hitachi 7600 clinical analyzer (Hitachi, Tokyo, Japan) using standard methods. Serum 25-hydroxyvitamin D levels were measured with electrochemiluminescence immunoassay (ECLIA) platform using the Roche cobas e602 analyzer (Roche Diagnostics GmbH, Germany).

Diagnostic criteria and de nitions
Lean was de ned as BMI < 24 kg/m 2 , and obesity was de ned as BMI ≥ 24 kg/m 2 (27). The quartiles of serum vitamin D levels

Diagnosis of NAFLD
An abdominal ultrasound examination was performed by experienced ultrasonographers, using the Toshiba Nemio 20 ultrasound system (Toshiba, Tokyo, Japan) with a probe of 3.5 MHz. The ultrasonographers were unaware of the study's purpose and laboratory values. Fatty liver disease was diagnosed according to the standards of the Chinese Liver Disease Association (29).

Statistical analysis
The statistical analysis was performed by SPSS (SPSS, Chicago, IL) for Mac version 18.0. Continuous variables were presented as mean ± SD or median and interquartile range (IQR). Student's t-test was applied to compare continuous data, and χ 2 test was applied to compare categorical variables. Cochran-Armitage trend test showed the trend of prevalence. Stepwise multiple regression analysis was used to identify possible risk factors for NAFLD (Backward LR; Entry: 0.05, Removal: 0.10). P < 0.05 (twotailed test) was considered to be statistically signi cant.

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A total of 2538 participants (1360 men and 1178 women) were included in this study, and 853 (33.61%) had NAFLD. The prevalence of NAFLD was 13.10% in lean participants (BMI < 24 kg/m 2 ), and was 53.32% in obese participants (BMI ≥ 24 kg/m 2 ). We compared clinical characteristics based on NAFLD status (Table 1). We found that both lean and obese NAFLD patients were older, had higher BMI, larger waist circumference, higher systolic and diastolic blood pressure, and had elevated serum levels of alanine aminotransferase, γ-glutamyl transpeptidase, triglyceride, LDL-cholesterol, uric acid and fasting glucose, but lower serum HDL-cholesterol levels than corresponding controls. Besides, vitamin D levels of obese NAFLD patients were lower than those of obese NAFLD-free controls (59.03 ± 19.46 versus 63.56 ± 22.09 nmol/L, P < 0.001), but this was not observed in lean participants (Table 1). Data are expressed as mean (SD).

Association of serum vitamin D levels with the prevalence of NAFLD
We classi ed all participants into quartiles by their serum vitamin D levels and analyzed the association of vitamin D quartiles with the prevalence of NAFLD. We found that serum vitamin D quartiles were negatively associated with the prevalence of NAFLD in obese participants ( Table 2). The prevalence of NAFLD was 56.54%, 59.77%, 52.15%, and 44.51% in the rst, second, third, and fourth quartiles of serum vitamin D in obese participants (P for trend < 0.001; Table 2). However, serum vitamin D quartiles were not associated with the prevalence of NAFLD in lean participants (Table 2). We also divided all participants into three groups according to their vitamin D adequacy status and analyzed the association of vitamin D adequacy status with the prevalence of NAFLD in lean and obese participants, respectively. We found that participants with vitamin D de ciency had the highest prevalence of NAFLD (57.38%), followed by those with vitamin D insu ciency (52.06%), and vitamin D su ciency (41.82%) in the obese group (P for trend = 0.045; Table 3). However, the prevalence of NAFLD was comparable among lean participants with different vitamin D adequacy status (Table 3). Participants were classi ed into three groups according to their serum vitamin D levels: VD su ciency, ≥ 100 nmol/L; VD insu ciency, 50-100 nmol/L; and VD de ciency, < 50 nmol/L.

Association of serum vitamin D levels with risk of NAFLD
Next, multiple logistic regression analyses were conducted to explore the risk factors of NAFLD in lean and obese participants.
We found that male gender, high BMI and waist circumference, high serum levels of albumin, alanine aminotransferase, aspartate aminotransferase, uric acid, and fasting blood glucose, and low serum levels of HDL-cholesterol were correlated with increased risks of NAFLD in both lean and obese participants (Table 4). We also found that serum vitamin D concentrations were another factor associated with the risk of NAFLD in obese participants, with an adjusted OR (95% CI) of 0.986 (0.979-0.992). However, serum vitamin D concentrations were not associated with the risk of NAFLD in lean participants (Table 4). Backward stepwise regression was used in multivariate logistic regression analyses (probability to enter = 0.05 and probability to remove = 0.10).
We further analyzed the correlation between vitamin D quartiles and the risk of NAFLD (  (Table 5). Similarly, we analyzed the correlation between vitamin D adequacy status and risk of NAFLD (Table 6).
Among obese participants, vitamin D de ciency showed an increased risk of NAFLD compared with those with vitamin D su ciency, with an adjusted OR (95% CI) of 1.906 (1.005-3.614). However, vitamin D de ciency was not associated with an increased risk of NAFLD in lean participants (Table 6). These results showed that decreased serum vitamin D concentrations were associated with an increased risk of NAFLD in obese but not lean participants.   Model 3 was further adjusted for waist circumference, systolic blood pressure, albumin, alanine aminotransferase, aspartate aminotransferase, total cholesterol, LDL-cholesterol, fasting blood glucose, serum uric acid and smoking history.

Discussion
In this study, we explored the correlation between serum vitamin D concentrations and NAFLD in Chinese adults. We found that serum vitamin D concentrations of obese NAFLD patients were lower than those of obese controls without NAFLD. We also found that serum vitamin D concentrations were negatively correlated with the prevalence of NAFLD in obese but not lean participants.
Our further analysis showed that decreased serum vitamin D concentrations or vitamin D de ciency were associated with an increased risk of NAFLD in obese but not lean participants. These ndings suggested a signi cant correlation between serum vitamin D concentrations and NAFLD in obese but not lean participants.
Several studies have reported that there is a signi cant correlation between serum vitamin D concentrations and NAFLD (30,31), and this was con rmed by a meta-analysis including 12794 participants of 17 studies (32). Moreover, low serum vitamin D concentrations are related to greater severity of hepatic steatosis and necrotizing in ammation both in children and in adults (33,34). Preclinical investigations found that vitamin D supplementation signi cantly improved liver steatosis in high-fat diet-fed mice (35). Besides, we and others found that vitamin D receptor (VDR) is upregulated in the steatotic livers, and maybe a therapeutic target for NAFLD (35,36). As we know, low serum vitamin D levels are more commonly observed in obese than in lean individuals (37). However, whether lean or obese individuals showed a similar association of vitamin D with NAFLD remains speculative and should be investigated. In this study, we provided evidence that low serum vitamin D levels were associated with obese but not lean populations.
The explanations for why obese and lean individuals have inconsistent correlations between vitamin D and NAFLD remains unclear, although several possibilities exist. First, obesity is closely associated with low vitamin D levels itself and maybe a major factor causing this result (38,39). With fewer outdoor activities and low exposure to sunlight, obese individuals may have decreased vitamin D synthesized in the liver or percutaneously (8). A genetic study showed that each increase in BMI will reduce serum vitamin D concentration by 1.15% (40). Second, patients with vitamin D de ciency have higher serum levels of proin ammatory cytokines and promote the development of NAFLD (41). In the non-alcoholic steatohepatitis (NASH) stage, vitamin D de ciency can also actively regulate the synthesis of endogenous fatty acids in the liver by weakening the enterohepatic circulation (42). Third, vitamin D can increase the expression of peroxisome proliferator-activated receptor γ (PPAR-γ), thereby promoting the secretion of serum triglycerides and the accumulation of lipid droplets in hepatocytes (43). Therefore, under vitamin D de ciency conditions, the ow of free fatty acids (FFAs) in the blood increases, and fat deposition is accelerated into hepatocytes, contributing to the progress of NAFLD (44). Further researches are needed to clarify these possibilities.
Recently, researchers have subdivided NAFLD into obese and lean subtypes according to their obesity status, and many studies have focused on lean NAFLD (45)(46)(47). Vitamin D concentrations are closely related to NAFLD, and vitamin D de ciency is considered a risk factor for NAFLD (31,44). However, it was not clear whether vitamin D concentrations were also associated with lean NAFLD. In this study, we found that low vitamin D concentrations are associated with obese but not lean NAFLD. Our results suggest that the vitamin D concentration may be an important predictor of NAFLD screening in obese but not lean population.
In this study, some limitations are acknowledged. First, our NAFLD was diagnosed based on ultrasound. Although ultrasound NAFLD diagnosis has been widely used clinically as a screening method for hepatic steatosis, it is still insu cient to detect mild steatosis and cannot replace the gold standard for liver biopsy. The correlation between vitamin D levels and NAFLD histological severity was not explored in this study. Second, this is a single-center cross-sectional study. Our sample size may be insu cient to represent the entire Chinese adult population, and further multi-center cohort studies are needed. Third, this study classi ed lean and obese participants by the BMI but did not include waist circumference or waist-to-hip ratio. It may mix some central obese patients with lean participants.

Conclusions
Our cross-sectional study provided evidence that there was a signi cant correlation between serum vitamin D concentrations and NAFLD in obese but not lean participants. Further research is needed to explore the complicated relationships and possible mechanisms between obesity, vitamin D levels, and NAFLD.

Declarations
Ethics approval and consent to participate The study was approved by the Ethical Committee of the First A liated Hospital of Zhejiang University School of Medicine.

Consent for publication
Not applicable.

Availability of data and materials
The data that support the ndings of this study are available from the First A liated Hospital, Zhejiang University School of Medicine but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the First A liated Hospital, Zhejiang University School of Medicine.

Competing interests
The authors declare that they have no competing interests. Authors' contributions QQW and XYS collected and analyzed participant data, and completed the manuscript writing. JHW and JWZ did data collection and interpretation. CFX did the study design and implementation, manuscript drafting, and critical revision of the manuscript for important intellectual content. All authors read and approved the nal manuscript. QQW and XYS contributed equally to this study.