Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation
Phase: NA
Conditions: Vitamin D and Hepatitis B Virus
Sponsor: Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation

Title:  
The	   causal	   relationship	   between	   serum	   vitamin	   D	   level	   and	  
hepatitis	  B	  virus	  replication	  in	  patients	  with	  chronic	  hepatitis	  B	  
virus	  infection	    
 
 
NCT 03068767	  
	  
	  
Date	  of	  document:	  June	  1,	  2017	   	  
	  
	  
IRB	  approval	  letter	  (as	  attached)	   	  
	  
	  
 

Abstract 
Background and Aims: Vitamin D deficiency is common in patients with chronic 
hepatitis B virus (HBV) infection. Previous study showed an inverse association 
between vitamin D and HBV DNA levels. However, the causal relationship between 
vitamin D and HBV replication remains inconclusive. 
Methods: HBV carriers receiving regular 6-month surveillance without current 
antiviral treatment or cirrhosis were invited to participate into this trial. The markers 
of HBV replication included serum HBV DNA and quantitative HBsAg (qHBsAg) 
levels. Those with undetectable HBV DNA or sufficient vitamin D levels, cancer or 
electrolyte imbalance were excluded. The eligible subjects were randomized to 
receive either vitamin D supplement 2000 IU per day for 2 months (vitamin D group) 
or none (control group). Levels of vitamin D, HBV DNA and qHBsAg before and 
after 2 months of treatment or follow-up were compared between these two groups. 
The primary endpoint was to identify any difference between two groups. 
 
 

Introduction 
Hepatitis B virus (HBV) infection is a global health problem, especially in Taiwan. 
The clinical manifestations of HBV infection include acute/fulminant hepatitis or 
various forms of chronic infection. Chronic HBV infection may lead to inactive 
carrier, chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC).1-5 Low 
vitamin D is frequently found in patients with chronic hepatitis B (CHB) than healthy 
controls.6 Furthermore, several cross-sectional studies showed an inverse association 
between serum vitamin D level and HBV DNA level.7-10 However, the causal 
relationship cannot be confirmed from these studies. 	  
Cholecalciferol is the precursor of the bioactive vitamin D. It is rare in natural 
foods and the major source is synthesized in the skin during exposure to sunlight.11 It 
is hydroxylated in liver to calcidiol, which is released into the plasma and exerts its 
biological functions by signaling through a nuclear vitamin D receptor. Therefore, 
liver has a close relationship with the synthesis of vitamin D. Vitamin D plays a 
crucial role in the bone metabolism and affect immune function not only in innate but 
also in adaptive immunity.12,13 In clinical practice, low vitamin D is associated with 
autoimmune, infective disease, and the development of malignancy.14  

 
Vitamin D has been reported to be an important immune modulator of hepatitis 
C virus (HCV) infection and metabolic liver disease.15-20 Regarding HBV-infected 
patients, the relationship among vitamin D level, HBV viral load and liver 
dysfunction remains largely unclear. To clarify the causal relationship, we conducted 
a randomized controlled trial of providing vitamin D supplement to HBV patients 
with insufficient vitamin D (<30 ng/mL) level to investigate the effect of vitamin D 
level on HBV replication in terms of HBV DNA and qHBsAg levels. 
 
	  
 

Materials and Methods 
Patients and study design 
This was a randomized controlled trial. The sample size was determined using two 
samples t-test power calculation. The 1 log increase of serum HBV DNA level was 
defined as a significant difference between two samples. Effect size (Cohen’d=0.39) 
was obtained using two parameters including difference and standard deviation. The 
power and a significant level were set as 0.8 and 0.05, respectively. The sample size 
was calculated out as 82 in each group. The ratio of exclusion was estimated as 20 %. 
Therefore, the sample size was set as 196 in this clinical trial. In our clinical 
experience, the majority of vitamin D deficiency patients can achieve adequate serum 
vitamin D levels after two months vitamin D supplement. Furthermore, the liver 
dysfunction due to hepatitis flare or cirrhosis could be a confounding factor for the 
low serum vitamin D levels in HBV-infected patients. According to the above 
consideration, our study was designed to enroll 196 non-cirrhotic, HBV-infected 
patients with normal or minimally elevated serum ALT levels. Thus, chronic HBV 
patients receiving regular 6-month surveillance and without current antiviral treatment 
in the outpatient clinic of Taipei Tzu Chi Hospital were invited to join the study from 

August 2017 to September 2018. All had positive hepatitis B surface antigen (HBsAg) 
for more than 6 months. Those with positive anti-hepatitis C virus (HCV) antibody, 
alcoholic liver disease, other known causes of chronic hepatitis or hepatocellular 
carcinoma were excluded. Cirrhosis was diagnosed based on histological findings or 
ultrasonographic evidence of nodular liver surface with coarse echotexture and 
splenomegaly. After obtaining written informed consent, levels of serum vitamin D, 
HBV DNA and quantitative HBsAg (qHBsAg) levels were measured. Those who had 
HBsAg loss, undetectable HBV DNA and adequate vitamin D level (≥30 ng/mL) 
were excluded. The eligible subjects were randomized into two groups by using the 
computer random distribution. Patients in the vitamin D group received vitamin D 
supplement (D3, 2000 IU/day; Metagenics, Aliso Viejo, CA 92656) for 2 months and 
untreated patients were used as the control group. Levels of vitamin D, HBV DNA 
and qHBsAg before and after 2 months of treatment or follow-up were compared 
between these two groups. The primary endpoint was to identify any difference 
between two groups. Patients lost to follow-up or poor drug compliance (vitamin D 
intake amount < 80%) were excluded for per-protocol analysis. Both intention-to-treat 
and per-protocol analyses were conducted.  

 
Laboratory testing 
Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 
measured using routine automated methods (Dimension Flex®reagent cartridge, USA; 
SIEMENS, Dimension, NY, USA). The upper limit of normal of ALT was 63 U/L in 
men and 59 U/L in women. The HBeAg and Anti-HCV were assayed using Abbott 
ARCHITECT, Wiesbaden, Germany. The Fibrosis 4 index (FIB-4) was calculated 
base on the formula: FIB-4 = Age (years)×AST (U/L)/[PLT(109/L)×ALT1/2(U/L)]. 
Based on our previous study, FIB-4 >= 0.87 was defined as the presence of significant 
fibrosis (>=F2).21 
 
Quantification of serum vitamin D level 
Serum vitamin D level was measured using LIAISON 25OH vitamin D Total assay 
(DiaSorin Inc, USA) based on an automated chemiluminescent immunoassay (CLIA) 
technology. Serum vitamin D concentration ＜20 ng/ml was defined as deficiency, < 
30 and ≥ 20 ng/ml as insufficiency, whereas concentrations ≥30 ng/ml were 
considered as sufficiency.  

 
Quantification of HBV DNA and HBsAg levels 
Serum HBV DNA level was quantified using Roche COBAS AmpliPrep/COBAS 
TaqMan HBV Test (Roche Molecular Systems, Inc, USA). The lower limit of 
quantification was 20 IU/ml. Serum qHBsAg level was assayed using the Architect 
HBsAg QT (Abbott Laboratories) according to the manufacturer’s instruction. The 
detection range is from 0.05 to 250 IU/ml. If the qHBsAg level was found to be 
higher than 250 IU/ml, the samples were 1:100 or 1:1000 diluted to obtain a reading 
within the range of the calibration curve. 
 
Ethical considerations 
The study was performed in accordance with the principles of the 1975 Declaration of 
Helsinki and approved by the Ethical Committee of Taipei Tzu Chi Hospital, 
Buddhist Tzu Chi Medical Foundation. Each participant provided written informed 
consent at the time of enrollment. The register number of clinical trial is NCT 
03068767.  
	  

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