ABSTRACT

Objectives:

We aimed to identify the potential role of vitamin D₃ among patients with chronic hepatitis B (CHB) in Birjand, Iran.

Materials and Methods:

In this case-control study, 292 patients were selected with CHB and 304 healthy subjects as control groups in the outpatient clinic of the infectious diseases department between January 2018 and December 2019. We quantified the levels of total vitamin DD₃ in serum samples of them. Logistic statistical analysis was applied at the significance level of 5%.

Results:

The mean age and serum vitamin DD₃ level of the study and control groups were; 39.9±12.3 years, 43.0±9.3 years and 17.76±5.53 ng/mL, 22.07±2.41 ng/mL, respectively. So, a significant difference between means of vitamin DD₃ serum in the two groups was observed (p>0.05). The prevalence of vitamin DD₃ deficiency was higher among patients with hepatitis B virus (63.0%) than the healthy group (32.9%). Frequency distribution of serum vitamin DD₃ levels showed a significant difference between the two groups (p=0.001). The risk of vitamin DD₃ deficiency was significantly more than the healthy group (odds ratio: 3.17, p<0.001).

Conclusions:

According to the results; a high risk of vitamin DD₃ deficiency related to CHB was found in this city. Future studies are warranted to consider the impact of vitamin D supplementation in CHB.

Keywords:

Vitamin D 3 level, liver disease, chronic HBV infection

Introduction

The liver is the main place for vitamin D₃ synthesis, where 25-hydroxylation occurs (1). Vitamin D₃ plays an appearing role in metabolic liver diseases. There is evidence about the interrelationship between vitamin D₃ and different chronic liver diseases owing to its immunomodulatory role (2,3). About 240 million individuals are infected with hepatitis B virus (HBV) chronically all over the world (4).

It has been shown that vitamin D₃ has very important biologic effects (5,6). Vitamin D₃ levels can affect the immune system and host response to HBV infection.

But, the association between vitamin D₃ metabolism and chronic hepatitis B (CHB) is less well characterized yet (7).

Different studies suggested low levels of vitamin D₃ are associated with high levels of HBV replication in CHB patients recently. Although, a study found a positive relationship between hepatitis B surface antigen (HBsAg) seroclearance and vitamin D₃ levels (8).

Also, another one showed a significant relationship between higher levels of HBV replication and low levels of vitamin D₃ in CHB infection (9). In addition, the role of vitamin D₃ may also affect disease progression in patients with HBV infection.

According to the mentioned contents, it was assumed that vitamin D₃ level may be one of the responsible agents for the very low serum levels of CHB patients. So, the purpose of this study was to specify the risk, associated factors, and symptoms related to vitamin D₃deficiency among CHB patients compared to healthy individuals in Birjand.

Materials and Methods

This case-control study was carried out in Khorasan Jonoobi province of Iran in 2019 in the outpatient clinic of the infectious diseases department.

Sampling

In this study, 292 patients with CHB (HBsAg positive, anti-HBs negative), were randomly selected according to the calculated sample size by the following formula with a power of 90%. In addition, 304 natural immunized persons (HBsAg negative, anti-HBs has normal liver enzymes who have not received antiviral treatment were included. the healthy group was selected from collected samples of the master plan of the province (26).

Vitamin D₃ Level Classification

Total vitamin D₃ levels were measured in the serum samples. Based on the World Health Organization, a level of 30 ng/mL or above is considered as vitamin D₃ sufficiency (10). Then, vitamin D₃ status was classified as normal (≥30 ng/mL), insufficient (20-29.9 ng/mL), and deficient (<20 ng/mL).

Laboratory Tests

For laboratory tests, 10 ccs of venous blood were taken from patients and healthy controls. The serum levels of vitamin D₃ were measured using a COBAS e411 analyzer, manufactured by Mannheim Roch diagnostic Gmbh in Germany, with the Elecsys kit (REF0589413). Other tests were performed on patients and healthy controls according to laboratory routines. Levels of alanine transaminase (ALT), aspartate transaminase (AST), (glycated hemoglobin) hemoglobin A1C (HbA1C) and bilirubin levels of liver enzymes were measured by the ARCHITECT I system biochemical auto-analyzer. Levels of total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), fasting blood sugar (FBS), and body mass index (BMI) were also measured.

The inclusion criteria for CHB patients were: patients who were admitted to the infectious diseases outpatient clinic with the diagnosis of CHB with the approval of the infectious specialist according to clinical and serological signs, willingness to participate in the study, didn’t receive supplementation or injection of calcium and vitamin D₃ in the last six months and also with age ≥18 years. In addition, 304 healthy subjects without a history of hepatitis B disease were selected as healthy control group 26.

Patients’ and healthy controls exclusion criteria include severe renal disease, history of cardiovascular disease, co-infection with cancer, pregnancy, diabetic disease, thyroid disorders, other viral hepatitis (hepatitis C virus, hepatitis D virus, human immunodeficiency virus), and other causes of liver disease such as alcohol consumption.

Variables of interest were: age, sex, BMI, and clinical symptoms such as FBS, glycated HbA1C, blood pressure, and HBsAg.

Ethics Approval

This study was approved by the Birjand University of Medical Sciences (BUMS) Ethics Board Committee (approval number: IR.BUMS.REC.1398.324).

Statistical Analysis

Descriptive statistics were used to describe the data, chi-square test was applied to determine the difference of symptoms related to vitamin D₃ deficiency between the study groups. Also, logistic regression was done to specify the relationship between considered variables and vitamin D₃ deficiency in two groups. All analysis was done by SPSS version 22.0. The significance in all of these tests was two-tailed with a 5% significance level.

Results

Out of the all subjects who participated in the study, 48.6% were male in the case group; with a mean age 29±5.3; and 52.2% female with mean age (31.5±7.8); also, of the 304 healthy subjects, 22.1% male; mean age 23.1±7.7, and 77.8% female with mean age 29.1±11.7. So; the distribution of gender was similar in the patients’ group (Table 1). Healthy controls were younger than patients but no significant difference between them was observed (p>0.05). So that, the mean age of the CHB and control groups were, 32.9±12.3 years and 28.18±11.21 years, respectively. There was no significant difference in HbA1C level between genders, 5.71% vs 5.43% (p=0.343) (Table 1).

The mean of vitamin D₃ serum levels in the CHB and control groups were; 17.76±5.53 ng/mL, 22.07±2.41 ng/mL, respectively with significant differences (p=0.031). When categorized as deficient, insufficient, and sufficient. Then among the healthy subjects, vitamin D₃ levels were classified as 32.9%, 25.96%, and 41.15%, respectively.

Of 292 patients, 184 (63.1%), 56 (19.2%), and 52 (17.8%) had vitamin D₃ deficiency, insufficiency, and sufficient vitamin D₃serum levels, respectively. The prevalence of vitamin D₃ deficiency was high among CHB patients (63.1%) as well as in healthy individuals (32.9%). Vitamin D₃levels frequency distribution showed a significant difference in the two groups (p=0.001) (Table 2).

There is a negative correlation between vitamin D₃levels and BMI and HbA1C in patients. Also, a positive correlation was noticed between age and vitamin D₃levels, but none of these have significant values in the control group (p>0.05) (Table 3).

The results of the comparison of laboratory characteristics of CHB patients and healthy controls showed that the mean of ALT in patients was 32.82 IU/mL (8-117) and AST was 32.21 IU/ML (10-167). Of the all patients in total (21.04%) had ALT and (17.54%) had AST higher than 40 IU/ML while in the healthy group, only 15% had ALT and 5% had AST higher than 40 IU/mL. Also, there was a significant difference in ALT and AST between the two groups (p=0.001). Based on this result, between variables such as LDL, HDL, and BMI, age, sex, and vitamin D₃levels; was observed no significant difference.

According to the logistic regression results, the risk of vitamin D₃deficiency in men is 45% higher than in women which is not statistically significant [odds ratio (OR): 1.54, p=0.114). Among the patients, 75.3% were urban and 24.7% were rural residents. The distribution of vitamin D₃ deficiency in rural people is more than in urban population, which is significant in both groups (OR: 2.321, p=0.004). There was no significant difference in the BMI distribution between the two groups (p=0.13). In general, 47.3% of patients had a desirable weight, 35.5% had overweight and 8.2% had a BMI of more than 30 (Table 4).

Discussion

This study was conducted for the first time in this province (a region in the East of Iran) regarding vitamin D₃ pattern in patients with CHB and also to investigate factors associated with vitamin D₃ deficiency in CHB in comparison with the healthy group. In a period (1990-2010), the prevalence of vitamin D₃ deficiency was studied in Iranian society, and according to the results, in all regions; both sexes had moderate and significant vitamin D₃deficiency (11).

Nghiem et al. showed that vitamin D₃ deficiency existed in many CHB patients and this deficiency had a relationship with the complications and outcome of the disease. Decreased liver function due to HBV-induced injuries to liver cells can be one of the causes of vitamin D₃ deficiency in CHB (12,13).

The results of this study showed that different degrees of the prevalence of vitamin D₃deficiency existed among patients and healthy controls. The duration of exposure to sunlight exposure is an important factor in the changes in vitamin D₃levels (14).

In some studies, vitamin D₃levels were found to be inversely proportional to HBV-DNA viral load and a sufficient level of vitamin D₃ (15).

Previous studies showed the association between D₃ level and CHB (16,17,18), this study was in line with these studies too (19).

In this study, vitamin D₃ insufficiency/deficiency accounted for 82.1% of patients, which was similar to the reported prevalence from Japan and Germany (20,21). However, these results indicate that the relatively high prevalence of vitamin D₃deficiency is similar to another study (22). It might be because sunshine hours differ among various latitudes as sunlight-related ultraviolet rays are the substantial factor for vitamin D₃synthesis (22). However, this study showed that there was no association between the serum vitamin D₃ level and biological factors in both study groups. This failure could be due to variations in the subject’s age, HBV genotype, and racial background.

BMI higher than normal is considered to be an effective factor in the level of vitamin D₃stores (23). In this study, however, no significant relationship was found between serum levels of vitamin D₃and BMI. This result was different from other studies (21,22).

The current study, showed no association between the biochemical parameters and the serum vitamin D₃ level by sex. It is obscurant whether vitamin D₃deficiency is effective in CHB. We assume that sunlight exposure time might be short in the healthy subjects since they might go out less frequently than patients with CHB.

A high prevalence of vitamin D₃insufficiency in healthy individuals, as well as CHB patients, can be associated with consuming poor foods in vitamin D₃ and lower sun exposure. Also, the results of Tabrizi et al. (21) as a systematic review study showed a high prevalence of vitamin D₃deficiency among the Iranian population. The main reason for the higher vitamin D₃ deficiency prevalence in these people may be due to spending more time at home, and the clothing that may result in reduced vitamin D₃ synthesis. Also, the vitamin D₃ content is low in the Iranian diet (24).

In the published records on the prevalence of vitamin D₃deficiency showed that vitamin D₃deficiency prevalence was significantly different based on geographical regions in the Iranian population (10). In this study, however, vitamin D₃deficiency was not correlated with liver function parameters significantly, probably due to that vitamin D₃serum levels are affected by multiple factors. Roughly, one billion people worldwide apparently are vitamin D₃deficient (11,25). In line with a previous study (3), our results confirm an inverse correlation between BMI and HbA1C loads and vitamin D₃levels in the patients’ group.

Study Limitations

Some limitations of this study were: influencing of several factors on serum vitamin D₃ levels; such as seasonal variation, diet, and geographical habitation. However, any information about these affecting factors for study subjects was not available. Another limitation is that vitamin D₃ may be an additional factor for the clinical outcomes of HBV infection and its interaction with vitamin D₃ receptor on the pathogenesis of HBV infection needs to be explored further. However, this study could not demonstrate the relevance between assessed variables and serum level vitamin D₃, partially due to having only a few patients with advanced liver fibrosis.

Conclusion

Our study findings reveal that probably this studied population suffers from an insufficiency of vitamin D₃. This indicates the need to consume foods rich in vitamin D₃, require higher sun exposure, or vitamin D₃supplementation should be recommended in this area. It is noteworthy that although vitamin D₃deficiency is apparent in patients, this deficiency is also a noticeable difference with healthy people. Therefore, supplementation of vitamin D₃ with the initial dose should be recommended and initiated.

Acknowledgment

We would like to thank all the study subjects for their participation. Dentistry Clinical Research Development Center, Birjand University of Medical Sciences, Birjand, Iran for consulting.

Ethics

Ethics Committee Approval: This study was approved by the Birjand University of Medical Sciences (BUMS) Ethics Board Committee (approval number: IR.BUMS.REC.1398.324).

Informed Consent: Written consent was obtained from the all of patients.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: F.O., M.Z., Concept: F.O., M.Z., Desing: F.O., M.Z., Data Collection or Processing: M.Z., Analysis or İnterpretation: F.O., M.Z., Literature Search: F.O., M.Z., Writing: F.O., M.Z.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The financial support of the study was provided by the researchers.

References

Arteh J, Narra S, Nair S. Prevalence of vitamin D deficiency in chronic liver disease. Dig Dis Sci 2010;55:2624-2628.

Osmani F, Azarkar G. Fitting logistic regression models to assess vitamin D deficiency with clinical parameters in chronic hepatitis B patients. Infectious Disease Modelling. 2021;6:612-617.

Coussens AK, Wilkinson RJ, Hanifa Y, Nikolayevskyy V, Elkington PT, Islam K, Timms PM, Venton TR, Bothamley GH, Packe GE, Darmalingam M, Davidson RN, Milburn HJ, Baker LV, Barker RD, Mein CA, Bhaw-Rosun L, Nuamah R, Young DB, Drobniewski FA, Griffiths CJ, Martineau AR. Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment. Proc Natl Acad Sci U S A 2012;109:15449-15454.

Demir C, Demir M. Vitamin D levels in patients with chronic hepatitis B virus infection and naturally immunized individuals. Internal Medicine Inside 2013;1:1-4.

Azarkar G, Osmani F. Clinical characteristics and risk factors for mortality in COVID-19 inpatients in Birjand, Iran: a single-center retrospective study. Eur J Med Res 2021;26:79.

Efe C, Kav T, Aydin C, Cengiz M, Imga NN, Purnak T, Smyk DS, Torgutalp M, Turhan T, Ozenirler S, Ozaslan E, Bogdanos DP. Low serum vitamin D levels are associated with severe histological features and poor response to therapy in patients with autoimmune hepatitis. Dig Dis Sci 2014;59:3035-3042.

Fedirko V, Duarte-Salles T, Bamia C, Trichopoulou A, Aleksandrova K, Trichopoulos D, Aleksandrova K, Trichopoulos D, Trepo E, Tjønneland A, Olsen A, Overvad K, Boutron-Ruault MC, Clavel-Chapelon F, Kvaskoff M, Kühn T, Lukanova A, Boeing H, Buijsse B, Klinaki E, Tsimakidi C, Naccarati A, Tagliabue G, Panico S, Tumino R, Palli D, Bueno-de-Mesquita HB, Siersema PD, Peters PH, Lund E, Brustad M, Olsen KS, Weiderpass E, Zamora-Ros R, Sánchez MJ, Ardanaz E, Amiano P, Navarro C, Quirós JR, Werner M, Sund M, Lindkvist B, Malm J, Travis RC, Khaw KT, Stepien M, Scalbert A, Romieu I, Lagiou P, Riboli E, Jenab M. Prediagnostic circulating vitamin D levels and risk of hepatocellular carcinoma in European populations: a nested case-control study. Hepatology 2014;60:1222-1230.

Fujii H, Nakai K, Yonekura Y, Kono K, Goto S, Hirata M, Shinohara M, Nishi S, Fukagawa M. The vitamin D receptor activator maxacalcitol provides cardioprotective effects in diabetes mellitus. Cardiovasc Drugs Ther 2015;29:499-507.

Ghaziasadi A, Ziaee M, Norouzi M, Malekzadeh R, Alavian SM, Saberfar E, Judaki MA, Ghamari S, Khedive A, Namazi A, Rahimnia R, Jazayeri SM. The prevalence of hepatitis B virus surface antigen (HBsAg) variations and correlation with the clinical and serologic pictures in chronic carriers from Khorasan Province, North-East of Iran. Acta Med Iran 2012;50:265-272.

Heshmat R, Mohammad K, Majdzadeh S, Forouzanfar M, Bahrami A, Ranjbar Omrani G, Nabipour I, Rajabian R, Hossein-Nezhad A, Rezaei Hemami M, Keshtkar AA, Pajouji M. Vitamin D deficiency in Iran: A multi-center study among different urban areas. Iran J Public Health 2008;37:72-78.

Hoan NX, Van Tong H, Le Huu Song CGM, Velavan TP. Vitamin D deficiency and hepatitis viruses-associated liver diseases: A literature review. World J Gastroenterol 2018;24:445-460.

Hu YC, Wang WW, Jiang WY, Li CQ, Guo JC, Xun YH. Low vitamin D levels are associated with high viral loads in patients with chronic hepatitis B: A systematic review and meta-analysis. BMC gastroenterol 2019;19:84.

Ma Y, Zhang P, Wang F, Yang J, Liu Z, Qin H. Association between vitamin D and risk of colorectal cancer: a systematic review of prospective studies. J Clin Oncol 2011;29:3775-3782.

Osmani F, Ziaee M. Assessment of the risk factors for vitamin D3 deﬁciency in chronic hepatitis B patient using the decision tree learning algorithm in Birjand. Informatics in Medicine Unlocked. 2021;23:100519.

Manco M, Ciampalini P, Nobili V. Low levels of 25-hydroxyvitamin D3 in children with biopsy-proven nonalcoholic fatty liver disease. Hepatology 2010;51:2229-2230.

Osmani F, Hajizadeh E, Rasekhi A, Akbari ME. Prognostic factors associated with locoronal relapses, metastatic relapses, and death among women with breast cancer. Population-based cohort study. Breast. 2019;48:82-88.

Osmani F, Hajizadeh E, Rasekhi A. Association between multiple recurrent events with multivariate modeling: a retrospective cohort study. J Res Health Sci 2018;18:e00433.

Plum LA, DeLuca HF. Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov 2010;9:941-955.

Rode A, Fourlanos S, Nicoll A. Oral vitamin D replacement is effective in chronic liver disease. Gastroenterol Clin Biol 2010;34:618-620.

Schillie S, Xing J, Murphy T, Hu D. Prevalence of hepatitis B virus infection among persons with diagnosed diabetes mellitus in the United States, 1999-2010. J Viral Hepat 2012;19:674-676.

Tabrizi R, Moosazadeh M, Akbari M, Dabbaghmanesh MH, Mohamadkhani M, Asemi Z, Heydari ST, Akbari M, Lankarani KB. High prevalence of vitamin D deficiency among Iranian population: a systematic review and meta-analysis. Iran J Med Sci 2018;43:125-139.

Wong GL, Chan HL, Chan HY, Tse CH, Chim AM, Lo AO, Wong VW. Adverse effects of vitamin D deficiency on outcomes of patients with chronic hepatitis B. Clin Gastroenterol Hepatol 2015;13:783-790.e1.

Yu R, Sun J, Zheng Z, Chen J, Fan R, Liang X, Zhu Y, Liu Y, Shen S, Hou J. Association between vitamin D level and viral load or fibrosis stage in chronic hepatitis B patients from Southern China. J Gastroenterol Hepatol 2015;30:566-574.

Talaei A, Yadegari N, Rafee M, Rezvanfar M. Vitamin D Deficiency and Its Cut-off Point among young teenagers. Birjand Univ Med Sci 2011;18:210-216.

Hoan NX, Khuyen N, Binh MT, Giang DP, Van Tong H, Hoan PQ, Trung NT, Anh DT, Toan NL, Meyer CG, Kremsner PG, Velavan TP, Song LH. Association of vitamin D deficiency with hepatitis B virus-related liver diseases. BMC Infect Dis 2016;16:507.

The Importance of Vitamin D Deficiency as a Potential Marker Among Chronic Hepatitis B Patients