| BackgroundThe important role of Vitamin D in regulating calcium and bone homeostasis has been well recognized. The discovery of its ligand-vitamin D receptors (VDR) in many cells and tissues and their effect on regulating the transcription of over 200 genes involved in immunomodulation, inflammation, fibrogenesis, cell proliferation and differentiation, have attracted great interest of researchers to explore its extra-skeletal effects. These extra-skeletal diseases include diabetes, pulmonary tuberculosis, cardiovascular disease, and cancers. A meta-analysis of 18 randomized trials showed that vitamin D supplementation substantially reduced total mortality.Vitamin D is a secosteroid hormone obtained through the diet or through endogenous synthesis in sun-exposed skin, then the active metabolite is obtained through two successive hydroxylations:the first hydroxylation occurs in the liver to form the intermediate 25-hydroxyvitamin D [25(OH)D],25(OH)D is transported to the kidneys and undergoes a second hydroxylation in the kidneys by the 1-alpha-hydroxylase to its most active form,1,25-dihydroxyvitamin D [1,25(OH)D], circulating in lower concentrations than 25(OH)D but has much greater affinity for VDR and is biologically more potent. However, assays for evaluating 1,25 (OH)D levels cannot be used for diagnosing vitamin D deficiency for the generally poor reliability. Therefore, the primary circulating form of vitamin D,25(OH)D, a stable vitamin D metabolite with relatively long half-life (2-3 weeks), was widely accepted as the most appropriate barometer of overall vitamin D status.In tune with the contribution liver makes in the activation of vitamin D by 25-alpha-hydroxylation, the relationship between chronic liver disease and vitamin D status has been increasingly recognized. In the past several years, vitamin D deficiency is reported to be associated with liver fibrosis and low rate of sustained virologic response (SVR) to interferon (IFN)-based therapy in genotype 1 chronic hepatitis C (CHC) patients. However, the data linking vitamin D deficiency and clinical outcomes have been conflicting in later studies in patients with different genotype HCV infection. A meta-analysis recently showed that low vitamin D status in chronic hepatitis C (CHC) patients is associated with a higher likelihood of having advanced liver fibrosis and lower odds of achieving sustained virologic response.Chronic hepatitis B virus (HBV) infection is a global health burden affecting approximately 400 million people worldwide and is the main risk factor for the development of hepatocellular carcinoma (HCC) in China. Profound and long-lasting suppression of serum HBV DNA to low or undetectable levels has been identified as a key determinant to reduce liver damage, to prevent liver disease progression and to minimize HCC risk. According to previous analysis, patients with high serum ALT, low HBV DNA levels before treatment have higher chance to obtain virological response. However, these well-characterised factors for predicting better outcome are unmodifiable, therefore, searching for new modifiable cofactors to perform optimal patient profiling before treatment is crucial to improve response rates.The relationship between vitamin D status and liver histology or antiviral response in chronic hepatitis B (CHB) is even less well characterized. We therefore performed this study to assess the relationship between serum vitamin D levels and clinical characteristics including liver histology of CHB in Chinese patients living in subtropical regions and to investigate the performance of baseline vitamin D level in influencing the rate of antiviral responses in a large well-controlled cohort of HBeAg positive CHB patients received telbivudine based therapy.Materials and MethodsPatientsIn the cross-sectional study, a total of 242 treatment-naive chronic hepatitis B patients were recruited from Nanfang hospital, Guangzhou, China. Serum samples were collected at the patients’ first presentation in our outpatient clinic between August 2009 and October 2011, then routinely stored and used for the present, retrospective study. Liver biopsy was performed in selected patients (134/242) within 6 months before enrollment to confirm the diagnosis or assess the activity grade and fibrosis stage. Inclusion criteria for the present study were chronic infection with hepatitis B virus (HBV), defined as detectable hepatitis B surface antigen (HBsAg)≥6 months, age≥ 18 years, treatment-naive for both nucleos(t)ide analogs (NAs) or interferon. Exclusion criteria were:(1) advanced liver cirrhosis (Child-Pugh score> 6); (2) presence of hepatocellular carcinoma or AFP≥ 50 ng/ml; (3) other causes of liver disease or mixed causes (excessive alcohol consumption, hepatitis C, hepatitis D, autoimmune liver disease, Wilson disease, hemochromatosis); (4) human immunodeficiency virus co-infection; (5) malignant disease or severe chronic disease.In the longitudinal study,606 treatment-naive adults from Effort study aged 18-65 years, with hepatitis B surface antigen (HBsAg)-positive for at least 6 months, hepatitis B e antigen (HBeAg)-positive and hepatitis B e antibody (HBeAb)-negative were included, patients with HBV DNA >5 logio copies/mL and ALT≥ 2 and< 10 × ULN were eligible to be randomized at a 1:1 ratio to the telbivudine-based optimized group or telbivudine monotherapy group. Five hundred and sixty patients completed the 104-week follow up. Only 554 patients have sufficicent serum for 25(OH)D assays. This multicenter, open-label, randomized, controlled,2-year study (NCT00962533) was conducted at 24 centers in China from August 2009 to March 2012. For more inforation about the Effort study, please refer to the paper published on Hepatlogy 2014.Serological assays and HBV-DNA assaysThe normal range for serum ALT level is 0 to 40 U/L. HBV-DNA was quantified using the Roche Diagnostics Cobas Taqman 48 (Meylan, France). Serological assays including HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc, were determined using commercial Architect 12000 platform (Abbott Laboratories, North Chicago, IL).HBV genotypingHBV genotypes were determined using the PCR based restriction fragment length polymorphism (RFLP) as described previously.Vitamin D assaySerum levels of 25(OH)D were measured using an automated electrochemiluminescense-based assay, Elecsys Vitamin D Total (Roche Diagnostics, Mannheim, Germany). The data are expressed as nanograms per milliliter.25(OH)D concentrations<20 and <30 ng/ml were defined as deficiency and insufficiency, respectively.Histological assessmentLiver biopsy was evaluated to assess the inflammation and fibrosis by an experienced pathologist according to the Knodell histologic activity index (HAI, score of 3 or less indicate mild or no necroinflammation,10 or above indicate pronounced necroinflammation) and the Ishak system (0 indicates the absence of fibrosis,3 or more indicate significant fibrosis), respectively.Statistical analysisStatistical analysis of the data was performed using the statistical package SPSS (version 18.0; SPSS, Inc., Chicago, IL, USA). Continuous variables are presented as the mean ± SD and categorical variables are presented as frequencies (%). Levels of HBV-DNA and HBsAg were transferred to logio copies/mL. Chi-square was used for categorical variables. Mann-Whitney or Kruskal-Wallis test was used for similar comparison of nonparametric data. After univariate analysis, only variables associated with the dependent variable were included in the multivariate logistic analysis for significant associations. Only patients with complete data for the remaining covariates were included in multivariate analysis.Results1. Cross-sectional studyPatient CharacteristicsTwo hundred and forty two patients were included in the present study according to the above-described criteria. All patients lived in Guangdong Province (about 23° north). One hundred and eighty nine patients (78.1%) were HBeAg-positive and 53 patients (21.9%) were HBeAg-negative. There were more men than women in the study population, with 82% of patients in HBeAg-positive group and 85% in HBeAg-negative group being male. Mean alanine transferase (ALT) levels were significantly higher in HBeAg-positive patients than that in HBeAg-negative patients. HBV genotype B was predominant in both HBeAg-positive and HBeAg-negative groups.Serum 25(OH)D levelsThe mean value of serum 25(OH)D level was 33.90±10.67 ng/ml.59.9% patients had normal serum 25(OH)D level (>30ng/ml); 31.4% had a vitamin D level at 20-30ng/ml; and 8.7% had vitamin D deficiency (<20ng/ml), respectively..Characteristics of patients with different vitamin D stratificationWe stratified the patients into 3 groups according to the above defined cutoff values of vitamin D (<20 ng/ml,20-30 ng/ml,>30 ng/ml, respectively), and their characteristics were compared. As shown in Table 3, patients with vitamin D deficiency (<20 ng/ml) are younger and had significantly lower levels of creatinine and hemoglobin, more females and higher percentages of patients with genotype B HBV infection, and having blood samples mostly drawn in winter/spring months in comparison to patients with 25(OH)D concentrations at 20-30 ng/ml or≥ 30 ng/ml. In contrast, the mean values of body mass index (BMI), white blood cell count, platelet count, ALT, bilirubin, albumin, prothrombin time, HBsAg and the composition of genotypes, liver histology stage showed no significant differences among the three groups.Uni-and multivariate logistic regression analysis of factors associated with vitamin D statusTo further identify the independent predictors of insufficient vitamin D levels (<30ng/ml), we therefore performed uni-and multivariate logistic regression analysis. We selected age, gender, season, BMI, hemoglobin, white blood cells, platelet, ALT, bilirubin, albumin, creatinine, prothrombin time, HBV genotype, HBeAg status, HBV DNA, HBsAg, necroinflammation grade and fibrosis stage as covariates. In univariate analysis, gender (male vs. female, p= 0.002), age (p= 0.009), season of blood sampling (winter/spring vs. summer/autumn, p= 0.007), creatinine (p= 0.013), genotype (B vs. C, p= 0.016) and HBeAg status (positive vs. negative, p= 0.024) were independently associated with 25(OH)D insufficiency (<30 ng/ml). Only gender (p= 0.007), age (p= 0.001), season of blood sampling (p= 0.040) and genotype (p =0.004) were independent factors in multivariate logistic regression analyses. As an association between HBV genotype and vitamin D status was demonstrated above, we further stratified patients into two groups (genotype B and C). The mean 25(OH)D was significantly lower in patients with genotype B HBV infection than those with genotype C (32.51±11.06 vs.35.77±10.16 ng/ml,p= 0.023, Figure 2). Furthermore, patients with genotype B HBV infection had a higher prevalence of vitamin D insufficiency than those with genotype C (46.3% vs.30.6%, p= 0.021).Relationship between vitamin D status and HBV DNA levelTo further investigate the inverse correlation between serum 25(OH)D concentration and HBV viral load demonstrated in the Germany cohort, we validated the correlation in our cohort. However, no significant association was found between HBV DNA and vitamin D status (r=-0.025,p= 0.700). When we stratified patients according to HBV DNA<6 (n= 44) and≥ 6 log10 copies/mL (n= 198), the mean 25(OH)D levels (34.14±10.52 vs.33.73±10.74 ng/ml, p= 0.920) and the prevalence of vitamin D insufficiency (40.9% vs.39.9%, p= 0.902) in these two groups are comparable. To further explore the association between HBV DNA and vitamin D level, we analyzed the data after stratification by HBV genotypes, similarly, no significant association was found between HBV DNA and vitamin D status in patients with genotype B(r= 0.008,p= 0.928) or genotype C HBV infection(r=-0.052,p= 0.612).Relationship between vitamin D status and liver histologyLiver biopsy was available in 134 out of 242 patients to confirm the diagnosis or assess the activity grade and fibrosis stage. Percentage of patients with different necroinflammation and fibrosis stage are as following:HAI 0～36.0%(n= 8), HAI 4～69.0%(n= 12), HAI 7～944.0%(n= 59), HAI≥1041.0%(n= 55); F1 6.0%(n= 8), F2 28.4%(n= 38), F3 35.8%(n= 48), F4 20.1%(n= 27), F5 7.5%(n= 10) and F6 2.20%(n= 3), respectively. Mean 25(OH)D concentration did not significantly vary between patients with minimal and significant necroinflammation (HAI 0-9 34.21±11.27 vs. HAI 10～1831.94±10.98 ng/ml, p= 0.219), patients with different fibrosis stage (F1 34.85±14.31, F2 32.37±10.82, F3 32±9.93, F4 34.49±12.29, F5 35.99±10.02, F6 37.33±22.77 ng/ml, p= 0.748), mild or significant fibrosis (F1～2 33.04±11.41 vs. F3～633.40±11.10 ng/ml, p= 0.907). Moreover, the prevalence of vitamin D insufficiency did not significantly vary in patients with minimal or significant necroinflammation (41.8% vs.50.9%, p= 0.296), mild or significant fibrosis (45.7% vs.45.5%,p= 0.983).2. Longitudinal studyPatient characteristicsFive hundred and fifty four patients from muti-centers in China from August 2009 to March 2010 including 449 men (81.0%) with a mean age of 30.19 ± 8.97 years were recruited underwent 25(OH)D testing. The descriptive characteristics of the participants are presented. The mean value of circulating 25(OH)D was 29.82 ± 11.20 ng/ml. In all,302 (54.5%) patients had vitamin D insufficiency (< 30 ng/ml), whereas the remaining 252 (45.5%) patients had normal (> 30 ng/ml) 25(OH)D serum levels.Latitude and vitamin D statusAssays of mean total 25(OH)D and prevalence of vitamin D insufficiency in different areas are presented in Table 2. The primary contrast across groups was gradient change in 25(OH)D according to distance from the equator (latitude), with the following mean concentrations:36.09 ± 10.99 ng/ml in Sourth China (N23°-28°); 29.26 ± 10.49 ng/mL in Central China (N29°-36°); 24.61 ± 9.57 ng/mL in North China (N37°-43°), p< 0.001. Prevalences of vitamin D insufficiency across countries also resulted in statistical differences (32.4%,56.6% and 72.7%, respectively, p< 0.001).Predictors of vitamin D statusTo further identify the independent predictors of vitamin D insufficiency (< 30 ng/mL), we therefore performed uni- and multivariate logistic regression analysis. We selected age, gender, season, latitude, BMI, hemoglobin, white blood cells, platelet, ALT, bilirubin, albumin, creatinine, HBV genotype, HBV DNA, necroinflammation grade and fibrosis stage as covariates. As biochemical and blood tests were assessed at local laboratories, we have adjusted the data for different ULN in different local laboratories. In univariate analysis, gender (male vs. female, p-0.001), season of blood sampling (winter/spring vs summer/autumn, p=0.011), lattitude (p< 0.001), and bilirubin (p=0.008) were independently associated with Vitamin D insufficiency. Only gender (p=0.001), season (p=0.009), and latitude (p< 0.001) were independent factors in multivariate logistic regression analyses.Baseline predictors of virologic responseUnivariate analysis looking for an influence of age (years), gender (M/F), season, latitude, BMI, ALT, HBV DNA level, HBsAg level and vitamin D status on the probability of virologic response is summarized in Table 4. Variables that were significant in the univariate analysis were ALT, HBV DNA level, HBsAg level and vitamin D status. In multivariate analysis, vitamin D status remained the independent factor associated with virologic response besides ALT and HBV DNA level.Performance of baseline vitamin D level for virologic responseThe receiver operating characteristic (ROC) curves were generated from univariate and multivariate analyses of ALT, HBV DNA levels and vitamin D levels in order to evaluate the performance of these variables in predicting the presence of virologic response in CHB patients. The ability of ALT, HBV DNA levels and serum vitamin D to predict virologic response give an area under the curve (AUC) of 0.589 (p= 0.001,95% CI:0.536-0.642),0.668(p< 0.001,95% CI:0.631-0.731),0.576(p-0.006,95% CI:0.525-0.627), respectively.Conclusions1. In the cross-sectional study, the major findings are:(1) The prevalence of vitamin D deficiency in our cohort was significantly lower than the recent data from European studies (46.4%-81%) but comparable with the Australian study (16%); (2) gender (OR= 0.936, p=0.007), age (OR= 2.886, p=0.001), season of blood sampling (OR= 1.809, p=0.040) and genotype (OR= 0.407, p=0.004) were independent factors associated with 25(OH)D insufficiency; (3) no significant association was found between HBV DNA and vitamin D status (r=-0.025, p=0.700), the prevalence of vitamin D insufficiency did not significantly vary in patients with minimal or significant necroinflammation (41.8% vs.50.9%, p=0.296), mild or significant fibrosis (45.7% vs.45.5%, p=0.983); (4) the mean 25(OH)D was significantly lower in patients with genotype B HBV infection than those with genotype C (32.51±11.06 vs.35.77±10.16ng/ml, p=0.023). Furthermore, patients with genotype B HBV infection had a higher prevalence of vitamin D insufficiency than those with genotype C (46.3% vs.30.6%, p=0.021).2. In the longitudinal study, we found:(1) 302 (54.5%) patients had vitamin D insufficiency (< 30 ng/ml); (2) there was a gradient change in 25(OH)D levels according to distance from the equator (latitude):36.09 ± 10.99 ng/ml in Sourth China (N23°-28°); 29.26 ± 10.49 ng/mL in Central China (N29°-36°); 24.61 ± 9.57 ng/mL in North China (N37°-43°), p< 0.001; (3) gender (OR= 2.192, p= 0.001), season (OR=0.646, p= 0.011), and latitude (OR= 0.181, p< 0.001) were independent factors associated with 25(OH)D insufficiency; (4) baseline ALT (OR= 1.134, p= 0.021), HBV DNA (OR= 0.421,p< 0.001), treatment group (OR= 0.436, p< 0.001) and 25(OH)D level (OR= 0.404, p< 0.003) were independent factors associated with virologic response; (5) baseline vitamin D status predicts virologic response in patients with chronic hepatitis B treated with Telbivudine, the virologic response rate was lower in patients with Vitamin D deficiency compared with those in whom vitamin D level is normal, The ability of serum vitamin D to predict virologic response give an area under the curve (AUC) of 0.576 (p= 0.006,95% CI: 0.525-0.627), the best cut-off value is 29.91 ng/ml. |
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