Chronic Hepatitis B and Related Liver Diseases Are Associated with Reduced 25-Hydroxy-Vitamin D Levels: A Systematic Review and Meta-Analysis

Hepatitis B infection is a major public health problem globally leading to chronic liver disease and death, which are influenced by various environmental and host factors including serum 25-hydroxy-vitamin D levels. There is no comprehensive systematic review reporting the association of serum 25-hydroxy-vitamin D levels and different stages of chronic hepatitis B. This study aimed to analyze the association of 25-hydroxy-vitamin D levels in chronic hepatitis B with various determinants and outcomes. A bibliographic search in PubMed, Google Scholar, and Scopus was conducted using the search terms “Vitamin D”, “cholecalciferol”, “calcitriol”, “Hepatitis B”, and “HBV”, which were published until September 2022. Meta-analysis using the “metafor” package in R was conducted with a random effect model. This analysis included 33 studies with 6360 chronic hepatitis B patients. The pooled estimates of serum 25-hydroxy-vitamin D level among CHB cases was 21.05 ng/mL and was significantly lower compared to healthy controls. (p < 0.005). Reduced serum 25-hydroxy-vitamin D level was significantly associated with the severity of liver fibrosis as well as HBe positivity. This analysis suggests that serum 25-hydroxy-vitamin D levels are associated with disease activity and pathobiology, although the exact nature of the cause–effect relationship cannot be discerned from this study.


Introduction
Hepatitis B infection is a public health problem affecting nearly one third of the population worldwide. In some of the infected individuals, hepatitis B infection persists as chronic infection, which later leads to complications such as hepatic fibrosis, decompensated cirrhosis, and hepatocellular carcinoma [1]. The progression of hepatitis B infection to chronic infection and liver decomposition is regulated by various host and environmental factors [1,2]. The host immune factors are controlled by nutrition, endocrine, and other determinants [1].Vitamin D is one such molecule with multiple effects on immunity, inflammation, and fibrosis. Vitamin D, a fat-soluble vitamin is crucial for a plethora of biological and physiological functions in the body. The biologically active form of vitamin D is Calcitriol (1, 25-dihydroxy vitamin D); however, the serum level of vitamin D is determined by the level of 25-hydroxy-vitamin D, which is an indicator of vitamin D sufficiency. While various classifications of vitamin D deficiency have been proposed, the widely accepted classification of vitamin D deficiency is as follows: Normal/optimum: >30 ng/mL; Insufficiency: >20 but <30 ng/mL; mild deficiency: >10 but <20 ng/mL; severe 1.
We excluded articles that were in a foreign language, or which did not contain the relevant information as per the inclusion criteria; 2.
Studies including pregnant women and children were not included; 3.
We also excluded literature reviews, editorial reviews, and systematic reviews; 4.
Editorials, brief communications, and conference proceedings were also excluded; 5.
Articles estimating the values of only 1, 25-dihydroxy vitamin D were also excluded.

Evaluation of the Methodological Quality of the Studies Included
The methodological quality was independently assessed by two reviewers (SA and NK) using Rayyan Software [14]. Discussion with a third reviewer (AB) was performed to resolve the disagreements and conflicts. The quality assessment was performed using the Downs and Black checklist for quality assessment [15]. The tool considers studyrelated characteristics of quality, external validity, biases such as study and selection bias, confounding variable consideration and power to assess the overall quality. One point (yes) or zero (no) was scored for each item, excluding the power question. The power was scored on a 6-point scale, and the post-hoc power calculations were performed using G*power software [16]. The total score determined an overall quality index, which was used to classify the studies as excellent (>25), good (18)(19)(20)(21)(22)(23)(24)(25), fair (13)(14)(15)(16)(17) and poor (<13).

Data Extraction
SA and NK extracted the data using a standardized data format from studies that gave the number of cases according to 25-hydroxy-vitamin D levels and hepatitis B infection/outcomes. Research articles with inadequate or unclear results were excluded from quantitative analysis. Any discrepancies were resolved by through detailed discussion and consensus between the two authors (SA and NK) and an independent review by the third reviewer (AB). An electronic spreadsheet was created in which the following information was recorded: authors, year of publication, country where the study was conducted, type of publication, study design, sample size, gender, age, and 25-hydroxy-vitamin D levels (in cases and controls, in case of case-control studies).

Statistical Analysis
Data analysis was performed using the 'meta' and 'metafor' package in RStudio 2022.07.2 Build 576 with R for Windows version 4.2.1. The packages contain functions to estimate the common effect and random effects, generate meta-analytical plots such as forest plots, funnel plots, as well as sub-group and meta-regression analysis. A mean difference with a 95% confidence interval was used to determine the difference between the serum 25-hydroxy-vitamin D levels between chronic hepatitis B patients and controls. Cochran Q test and I 2 statistics were used to estimate the heterogeneity between the studies. The percentage of variation across studies leading to heterogeneity rather than chance was defined as low, moderate, or high for values of 25%, 50%, and 75%, respectively. The publication bias was assessed using Funnel plots and Egger's test.

Search Results and Study Selection
A total of 6458 articles were identified through the search strategy, out of which 3297 were removed as duplicates and 3120 were excluded after screening titles and abstracts. Full-text review of 40 articles was performed, out of which 7 articles were excluded for not matching the eligibility criteria. A total of 33 articles were included in the meta-analysis. The details of the screening process are described in Figure 1. conducted, type of publication, study design, sample size, gender, age, and 25-hydroxyvitamin D levels (in cases and controls, in case of case-control studies).

Statistical Analysis
Data analysis was performed using the 'meta' and 'metafor' package in RStudio 2022.07.2 Build 576 with R for Windows version 4.2.1. The packages contain functions to estimate the common effect and random effects, generate meta-analytical plots such as forest plots, funnel plots, as well as sub-group and meta-regression analysis. A mean difference with a 95% confidence interval was used to determine the difference between the serum 25-hydroxy-vitamin D levels between chronic hepatitis B patients and controls. Cochran Q test and I 2 statistics were used to estimate the heterogeneity between the studies. The percentage of variation across studies leading to heterogeneity rather than chance was defined as low, moderate, or high for values of 25%, 50%, and 75%, respectively. The publication bias was assessed using Funnel plots and Egger's test.

Search Results and Study Selection
A total of 6458 articles were identified through the search strategy, out of which 3297 were removed as duplicates and 3120 were excluded after screening titles and abstracts. Full-text review of 40 articles was performed, out of which 7 articles were excluded for not matching the eligibility criteria. A total of 33 articles were included in the meta-analysis. The details of the screening process are described in Figure 1.
Out of the studies selected for review, 18 were cross-sectional studies, 10 were casecontrol studies, 2 were cohort studies, and 3 were randomized controlled trials. All the studies had cases with CHB or inactive HBV carriers as the study population. One study involved patients with CTP-A cirrhosis and one study had patients with HBsAg seroclearance. Only the relevant and suitable data of CHB patients in each study were included in the meta-analysis.
As per the Downs and Black quality assessment, 8 studies were graded as good quality, 23 as fair and 2 studies were graded as poor. The agreement between the reviewers was calculated using the Cohen's kappa statistic, and showed almost perfect agreement (99.7%) with Cohen's k value 0.927 [17]. The study characteristics of the studies included in the review are described in Table 2.

Meta-Analysis
Meta-analysis was performed to study the difference between 25-hydroxy-vitamin D levels among cases with CHB and healthy controls. The units of 25-hydroxy-vitamin D levels were transformed from nmol/L to ng/mL to maintain uniformity in the results. As described in the forest plot ( Figure 2a) The average serum 25-hydroxy-vitamin D levels were significantly lower in CHB patients than in healthy controls and the pooled mean difference was −0.59 ng/mL (−0.82, −0.35) (Cochran Q test p < 0.001, I2 = 91.1%). As seen in Figure 2b, the pooled mean difference of vitamin D levels between HBV carriers and healthy controls was −1.32 ng/mL (−2.84, −0.20) (Cochran Q test p < 0.01, I2 = 96%).   inactive HBV carriers and healthy controls [18][19][20], (c) treatment-naïve and on-treatment Hepatitis B patients [20,22,25,41].
The funnel plot for 25-hydroxy-vitamin D levels in CHB versus healthy controls is asymmetrical, as shown in Figure 3a. The Egger's test, however, suggested no evidence of potential publication bias (p = 0.4143).

Sensitivity Analysis
After excluding four studies that were lying outside the funnel plot, the standardized mean difference between CHB cases and controls was 0.3274 ng/mL [0.2104, 0.4443], which was statistically significant with a p value < 0.001 (Cochran Q test p = 0.1238, I 2 = 38.4%)

Meta-Regression
We performed meta-regression analysis to see if latitude, age, male-to-female ratio among cases and control, and type of assay used for detection had any effect on the standardized mean difference of 25-hydroxy-vitamin D levels among CHB and healthy controls. As shown in Table 3, we could not find any significant association between these variables and the standardized mean difference, except that the method of detection had

Sensitivity Analysis
After excluding four studies that were lying outside the funnel plot, the standardized mean difference between CHB cases and controls was 0.3274 ng/mL [0.2104, 0.4443], which was statistically significant with a p value < 0.001 (Cochran Q test p = 0.1238, I 2 = 38.4%)

Meta-Regression
We performed meta-regression analysis to see if latitude, age, male-to-female ratio among cases and control, and type of assay used for detection had any effect on the standardized mean difference of 25-hydroxy-vitamin D levels among CHB and healthy controls. As shown in Table 3, we could not find any significant association between these variables and the standardized mean difference, except that the method of detection had

Sensitivity Analysis
After excluding four studies that were lying outside the funnel plot, the standardized mean difference between CHB cases and controls was 0.3274 ng/mL [0.2104, 0.4443], which was statistically significant with a p value < 0.001 (Cochran Q test p = 0.1238, I 2 = 38.4%)

Meta-Regression
We performed meta-regression analysis to see if latitude, age, male-to-female ratio among cases and control, and type of assay used for detection had any effect on the standardized mean difference of 25-hydroxy-vitamin D levels among CHB and healthy controls. As shown in Table 3, we could not find any significant association between these variables and the standardized mean difference, except that the method of detection had a significant impact in CHB cases (p = 0.0306). The scatterplot of latitude, methods, and gender ratio in cases versus controls was plotted as shown in Figures 6-8.

Discussion
According to WHO, the current global burden of chronic hepatitis B-infected people is around 296 million people, with around 1.5 million new infections added every year [49]. A total of 33 studies were included in the present analysis, covering a total population of 6360, with 6037 for chronic hepatitis B, and 240 inactive carriers. The majority of the studies used 25-hydroxy-vitamin D to assess serum vitamin D levels in chronic hepatitis B patients. Our results indicate that chronic hepatitis B infection was associated with reduced 25-hydroxy-vitamin D levels. The role of 25-hydroxy-vitamin D in hepatitis B has been implicated by various studies, though its association with disease status has not been analyzed. This meta-analysis has explained the association of serum 25-hydroxy vitamin D levels in different stages of chronic hepatitis B patients. Our study has shown that significantly low levels of 25-hydroxy-vitamin D are associated with chronic hepatitis B patients compared to healthy controls. Furthermore, we found that this association was also found among inactive carriers of hepatitis B infection. In addition, our results also pointed out that serum levels of 25-hydroxy-vitamin D were further low in treatment-naïve patients compared to those on antiviral treatment. Thus, it would be plausible to suggest that lower 25-hydroxy-vitamin D levels can influence the viral outcome, and vice versa, pointing toward a multifaceted crosstalk among host and viral factors. As a causal factor, low serum 25hydroxy-vitamin D levels might influence the viral outcome by affecting appropriate immune response; thus, leading to chronicity. On the other hand, liver inflammation and pathology in hepatitis can compromise the 25 hydroxylation of cholecalciferol in the liver, the key step in vitamin D activation.

Discussion
According to WHO, the current global burden of chronic hepatitis B-infected people is around 296 million people, with around 1.5 million new infections added every year [49]. A total of 33 studies were included in the present analysis, covering a total population of 6360, with 6037 for chronic hepatitis B, and 240 inactive carriers. The majority of the studies used 25-hydroxy-vitamin D to assess serum vitamin D levels in chronic hepatitis B patients. Our results indicate that chronic hepatitis B infection was associated with reduced 25-hydroxy-vitamin D levels. The role of 25-hydroxy-vitamin D in hepatitis B has been implicated by various studies, though its association with disease status has not been analyzed. This meta-analysis has explained the association of serum 25-hydroxy vitamin D levels in different stages of chronic hepatitis B patients. Our study has shown that significantly low levels of 25-hydroxy-vitamin D are associated with chronic hepatitis B patients compared to healthy controls. Furthermore, we found that this association was also found among inactive carriers of hepatitis B infection. In addition, our results also pointed out that serum levels of 25-hydroxy-vitamin D were further low in treatment-naïve patients compared to those on antiviral treatment. Thus, it would be plausible to suggest that lower 25-hydroxy-vitamin D levels can influence the viral outcome, and vice versa, pointing toward a multifaceted crosstalk among host and viral factors. As a causal factor, low serum 25hydroxy-vitamin D levels might influence the viral outcome by affecting appropriate immune response; thus, leading to chronicity. On the other hand, liver inflammation and pathology in hepatitis can compromise the 25 hydroxylation of cholecalciferol in the liver, the key step in vitamin D activation.
A study explored the effect of Vitamin D supplementation on HBV replication but did not find any significant change in HBV DNA levels before and after supplementation. Furthermore, this study studied the effect after supplementation of 2 months, and the longterm effect of supplementation on viral activity remains to be explored [48]. Another study has suggested that impaired liver function in HBV-related cirrhosis could be responsible for insufficient vitamin D hydroxylation and subsequent activation leading to reduced levels in the blood [9]. In line with this, one prospective study has found that serum 25-hydroxy-vitamin D levels negatively correlated with the severity of cirrhosis, with the lowest levels found in decompensated end-stage liver disease [50]. Moreover, as the liver is the principal organ for different transport protein synthesis, reduced production of 25hydroxy-vitamin D binding protein in chronic hepatitis could further promote insufficient 25-hydroxy-vitamin D levels in circulation. Lastly, ethnic and geo-environmental factors such as location of residence, skin tone, seasonal variations, exposure to sunlight, and nutritional intake can influence the serum 25-hydroxy-vitamin D3 levels Our next key observation was reduced levels of 25-hydroxy-vitamin D levels in HBe antigen-positive patients compared to HBeAg-negative ones. It highlights that 25-hydroxyvitamin D levels affect the E antigen secretion viral proliferation in the host. In agreement with this, another study has found that 25-hydroxy-vitamin D levels inversely correlated with HBV DNA load in chronic hepatitis B patients [12].
Although a previous meta-analysis has shown the inverse correlation between 25hydroxy-vitamin D levels and HBV DNA in chronic hepatitis B patients, no study has ever explored the detailed analysis of all studies estimating 25-hydroxy-vitamin D levels in hepatitis B patients with a different disease or treatment status. Our study has shown that 25-hydroxy-vitamin D levels varied significantly in treatment-naïve CHB patients compared to those on antiviral treatment. This again strengthens the fact that HBV infection in the liver impairs 25-hydroxy-vitamin D metabolism and its levels. Host immunological response is also governed by 25-hydroxy-vitamin D levels. A study on hepatitis B patients on interferon therapy has found that HBV DNA levels decreased more rapidly in patients with a higher level of serum 25-hydroxy-vitamin D. Thus, it signifies that 25-hydroxy-vitamin D influences the cellular immune response to a great extent [51].
Furthermore, results from subgroup analysis among CHB patients with fibrosis and cirrhosis indicated that 25-hydroxy-vitamin D levels positively correlated with fibrosis severity by the common effects model [p < 0.01]. A study administering ergo/cholecalciferol in alcoholic liver disease patients has demonstrated that the biological availability of 25-hydroxy-vitamin D positively correlated with patients with mild to moderate liver fibrosis compared with patients with severe liver disease (Child Pugh-C) [52]. The negative correlation between the severity of fibrosis/cirrhosis and 25-hydroxy-vitamin D levels could also be due to the fact that vitamin D, through the activation (VDR) and Calciumsensing receptor (CaSR), imparts portal hypotensive effect as evident from a study in a rat model [53].
Meta-regression was performed to analyze how common variables such as age, sex, latitude, and the method of 25-hydroxy-vitamin D estimation influenced or showed any association with the serum 25-hydroxy-vitamin D level (intervention effect) in the metaanalysis. The analysis showed no significant association between these variables with the estimation of serum Vitamin D levels in cases vs. healthy control, suggesting these factors were adjusted and nullified.
However, the study has some limitations which should be considered while interpreting the results. The heterogeneity among studies was quite high due to different study types, sample size, and different disease states related to chronic HBV. There was also significant publication bias as evident from our findings. However, sensitivity analysis was performed to check the robustness of the results and models and it was found that after excluding four studies the mean difference remained statistically significant. Lastly, although we tried to incorporate all eligible published studies, there are still chances of missing a few studies from the grey literature.

Conclusions
The meta-analysis has covered the most updated and pooled estimate of serum 25hydroxy-vitamin D levels in altogether and different disease states of chronic hepatitis B patients. Presumably, this is the first systematic review and meta-analysis which has identified major differences in serum 25-hydroxy-vitamin D levels correlating with disease activity such as HBeAg status and severity in terms of fibrosis and cirrhosis. Notwithstanding the wide heterogeneity among the included studies, our analysis strongly suggests that serum 25-hydroxy-vitamin D levels are associated with disease activity and pathobiology, although the exact nature of the cause-effect relationship cannot be discerned from this study. Future research is necessary to conduct in this area to validate the therapeutic and preventive role of vitamin D against chronic hepatitis B and related liver diseases.
Thus, this detailed meta-analysis provides corroborative evidence about the role of vitamin D in hepatitis B-related diseases and suggests that monitoring 25-hydroxy-vitamin D status and supplementation of vitamin D in chronic hepatitis B patients to prevent long-term complications should be addressed in future well-designed study cohorts.