Hepatitis C is a major disease that is prevalent worldwide. According to the WHO, there are 130–170 million individuals infected with the hepatitis C virus (HCV). There are considerable regional differences: Egypt has the highest prevalence of HCV infection worldwide (the prevalence is as high as 22%) 1–4. The prevalence in Egypt increases markedly with age, with anti-HCV antibodies detected in 2–7% of children younger than 10 years of age, 10% in those 10–20 years of age, and in more than half the individuals between 40 and 50 years of age in rural areas in the Nile Delta region 5–8.
Complications of hepatitis C occur almost exclusively in patients who have developed cirrhosis. The risk for decompensation is estimated to be close to 5% per year in cirrhotics. Once decompensation has developed, the 5-year survival rate is roughly 50%. Liver transplantation is then the only effective therapy. Hepatocellular carcinoma also develops solely in patients with cirrhosis (in contrast to chronic hepatitis B) 9.
Recently, the role of vitamin D in patients with chronic liver disease (CLD) has received considerable attention, given its inherent activation process by the liver and the high prevalence of vitamin D deficiency in this patient group 10. Evidence is also beginning to indicate possible direct therapeutic benefits of vitamin D therapy. Although clear evidence of an association between vitamin D and liver disease exists, it remains unknown whether vitamin D deficiency confers an enhanced risk of liver disease or whether liver disease causes vitamin D deficiency 11.
Vitamin D is synthesized predominantly in the liver and functions as an important secosteroid hormone with pleiotropic effects. Although its key regulatory role in calcium and bone homeostasis is well established, recently, there has been increasing recognition that vitamin D also regulates cell proliferation and differentiation, and has immunomodulatory, anti-inflammatory, and antifibrotic properties. These nonskeletal effects are relevant in the pathogenesis and treatment of many causes of CLD 12. Higher vitamin D levels are associated with less liver fibrosis and less inflammation in HCV patients 13.
Hepatic fibrosis results from wound healing following acute and chronic liver injury. In response to chronic hepatic inflammation, parenchymal cells release extracellular matrix proteins including type I collagen, resulting in the progressive deposition and accumulation of fibrosis. Ultimately, fibrotic tissue can replace hepatocytes and disrupt lobular architecture, the hallmark of cirrhosis, which, in turn, results eventually in hepatic dysfunction 14. Emerging data suggest that vitamin D is an important modulator of both the inflammatory response and wound healing 15. Vitamin D may modulate the inflammatory response and subsequent fibrosis by inhibition of tumor necrosis factor-α, a cytokine that plays a central role in the regulation of the immune response 16,17, and by inhibiting the development of fibrosis directly through suppression of transforming growth factor-β, a multifunctional cytokine that may influence progression of fibrosis 18,19.
In recent years, the role of vitamin D has been studied in adult patients with chronic HCV infection. To the best of our knowledge, childhood studies are very rare in this field. We aimed to assess vitamin D in hepatitis C patients and its association with the degree of fibrosis in a group of Egyptian children and to also assess whether vitamin D could work as a reliable noninvasive marker of liver fibrosis.
Patients and methods
Sixty patients with a confirmed diagnosis of HCV infection-related CLD were recruited from the National Liver Institute, Minufiya governorate. Their age ranged from 8 to 14 years, mean 11.0±2.1. There were 34 male and 26 females. Another 60 age-matched and sex-matched healthy controls participants were included in the study from among those attending the Pediatric Clinic of the National Research Center (for follow-up of growth and development). Study inclusion criteria for the patients were age range 8–14 years, presence of a chronic HCV infection, and seronegative for hepatitis B virus and human immunodeficiency virus infections. Exclusion criteria were decompensated liver disease (cirrhosis with a Child–Pugh score>9), another cause of clinically significant liver disease, presence of hepatocellular carcinoma, and history of chronic disorders associated with changes in mineral metabolism (thyroid disorders, parathyroid disorders, Cushing’s syndrome, diabetes, or renal failure). None of the patients received calcium, vitamin D, or any medication that may have influenced bone metabolism (corticosteroids, calcitonin, cytotoxics, antimetabolites, anticoagulants, anticonvulsants, thyroxine). The controls had normal liver functions and negligible HCV antibody levels.
Written informed consents were obtained from all the parents of pediatric patients and the controls after the nature of the procedure had been fully explained. The study was approved by the medical ethics committee of the National Research Center.
All children were subjected to the following:
- Detailed assessment of history.
- Full clinical examination.
- Serum samples were taken and stored frozen at −80°C for further determination of the selected parameters. All the experiments were conducted in duplicate.
- HCV diagnosis was made on the basis of the presence of quantitative and qualitative PCR for HCV performed by TaqMan technology.
- Anti-HCV antibodies in serum were detected by radioimmunoassay (Lumipulse II HBsAg; Fujirebio Co. Inc., Tokyo, Japan). The detection threshold was 615 IU (3200 copies/ml).
- Serum aspartate aminotransferase and alanine aminotransferase (AST and ALT) levels (U/l) were determined using the method of Gella et al.20, where the transfer of the amino group from aspartate or alanine formed oxalacetate or pyruvate, respectively, and the color developed was measured at 520 nm.
- Serum albumin was detected using the human serum EIA kit (Cayman Chemical Co., Ann Arbor, Michigan, USA). Concentrations of albumin (mg/dl) were obtained from a calibration curve.
- Total bilirubin (mg/dl) was measured using the method of Doumas et al.21, where it reacted with diazotized sulfanilic acid in the presence of caffeine with the final azo-pigment product. The color developed was read at 546 nm.
- 25-hydroxy vitamin D [25(OH) vitamin D] was assayed in serum using a quantitative enzyme immunoassay 22. Serum 25-hydroxyvitamin D is the major circulating form of vitamin D and a standard indicator of vitamin D status 23. In the present study, two categories were considered: category 1: less than 30 ng/ml (vitamin D deficient) and category 2: at least 30 ng/ml (normal levels) 24.
Histopathological examination (for patients)
Formalin-fixed paraffin-embedded liver biopsies were used for histological analysis. Histological sections were evaluated by two independent pathologists in a blinded manner. Fibrosis staging was assessed semiquantitatively according to the METAVIR system 25,26.
Statistical analysis was carried out using the statistical package for social sciences, version 16 for windows (SPSS Inc., USA). Continuous data were expressed as mean±SD and were compared using Student’s t-test. Pearson’s correlation analysis was carried out to evaluate the association between continuous exposure and continuous covariates. Multiple linear regression analysis was carried out to determine the influence of multiple variables (serum 25-hydroxy vitamin D, age, sex, BMI) on a dependent variable (liver fibrosis). Categorical data were expressed as frequencies and percentages, and were analyzed using the two-tailed χ2-test. We evaluated serum 25-OH-vitamin D levels as categorical variables (binary: insufficient and normal value) using the χ2-test to calculate an odds ratio (OR) for the progression of fibrosis on the basis of vitamin D levels. We carried out multiple logistic regression analysis to evaluate the impact of vitamin D categories as well as other known variables (age, sex, BMI) that influence fibrosis progression. P value less than 0.05 was considered as statistically significant.
The current study included 60 chronic HCV patients (mean age 10.97 years±2.09, 56.7% males and 43.3% females) and another 60 healthy controls. Another 60 age-matched and sex-matched healthy participants were included as the control group. Fig. 1 shows the methods of transmission of HCV in the patient group. In 47% of the patients, transmission was through the vertical route (maternal), in 30%, it was through transfusion or injection, whereas in 23%, it was unknown. In 50% of the HCV patients, serum 25-hydroxy vitamin D less than 30 ng/ml (insufficient) was found, whereas the remaining 50% were found to have serum 25-vitamin D of at least 30 ng/ml (adequate) (Fig. 2). The stages of liver fibrosis in the patient group are shown in Fig. 3. The F1 stage was found in 30%, F2 in 23%, F3 in 34%, and F4 in 13% of the patients.
Table 1 compares different variables between HCV patients and controls. In terms of mean age and BMI, no significant differences were detected between both groups (P>0.05). The mean serum AST, ALT, and bilirubin were significantly higher in the patient group than in the controls (P<0.05), whereas the mean serum albumin and 25-hydroxy vitamin D levels were significantly lower in the patient group than in the controls (P<0.05).
In HCV patients, serum hydroxy vitamin D was not significantly associated with BMI, any of the liver enzymes (AST and ALT), serum albumin, and serum bilirubin (P>0.05). However, a significant negative association was found between serum 25-hydroxy vitamin D and stages of liver fibrosis in the patient group (P<0.005) (Table 2). Multiple linear regression analysis was carried out to confirm the previous association after adjustment for age sex and BMI and it showed the same significant inverse association between serum hydroxyl-vitamin D and the stages of liver fibrosis in the patient group (P<0.05) (Table 3).
Table 4 shows that HCV patients with low serum 25-hydroxy vitamin D levels are more at risk of developing moderate to severe fibrosis compared with those with adequate levels of serum 25-hydroxyl vitamin D [OR=11 and 95% confidence interval (CI)=3.292–36.751 before adjustment for age, sex, and BMI]. After adjustment for age, sex, and BMI, the association was the same (OR=18.233 and 95% CI=4.253–78.165).
Our study showed that vitamin D insufficiency [serum level of 25 (OH) vitamin D<30 ng/ml] was prevalent in 50% of the patients in the patient group. A significantly lower serum level of 25-hydroxy vitamin D was found in the patient group compared with the controls, whereas a significant inverse association was found between 25-hydroxyl vitamin D and stages of liver fibrosis. Moreover, those HCV patients with insufficient vitamin D levels were more at risk of developing severe fibrosis compared with those with adequate vitamin D levels.
The prevalence of vitamin D deficiency in the general population affects all age groups and ranges from 20 to 100% in terms of serum 25(OH) vitamin D concentrations less than 20 ng/ml 27. Arteh et al.10 found vitamin D less than 32 ng/ml (i.e. 80 nmol/l) in 92% of 118 patients with CLD (HCV with cirrhosis, n=43; HCV without cirrhosis, n=57; non-HCV-related cirrhosis, n=18). Similarly, Fisher and Fisher 28 found inadequate 25(OH) vitamin D levels (<32 ng/ml) in 91% of patients with noncholestatic CLD, and the majority (68%) were vitamin D deficient (<20 ng/ml). Both studies showed a higher prevalence than in our study and that may be because of the differences in the age groups of the patients; our study included children whereas their study included adults.
Previously, vitamin D deficiency was considered to be found predominantly in cholestatic liver disorders because of impaired intestinal absorption commonly observed in such patients 29. Accumulating evidence, however, supports its widespread presence in CLD, irrespective of etiology. Putz-Bankuti et al.30 reported an inverse association (r=−0.21, P=0.08) between serum 25(OH) vitamin D levels and the severity of liver disease in a cohort of 75 patients with cirrhosis. This study is in agreement with the current study, which showed a significant inverse association between serum 25(OH) vitamin D and stages of liver fibrosis (r=−0.381, P=0.003). A low vitamin D status is also evident in patients with other CLDs, particularly in the presence of cirrhosis. For instance, 50 patients with nonalcoholic steatohepatitis and 10 patients with simple steatosis were observed by Targher et al.31 to have lower 25(OH) vitamin D concentrations compared with 60 controls matched for age, sex, and BMI. Overall, this Italian-based study found lower vitamin D status in patients with nonalcoholic fatty liver disease (NAFLD), which was also associated with histopathological NAFLD features. These findings were corroborated recently by the same group 32 in a larger study (n=262), in which low 25(OH) vitamin D levels were associated independently with NAFLD (n=162) and serum 25(OH) vitamin D was significantly (P<0.001) lower than that in patients who were free from NAFLD and other liver diseases (15±9 vs. 21±9 ng/ml). It is noteworthy that when compared with the lowest quartile, patients in the highest quartile of serum 25(OH) vitamin D levels showed an OR of 4.7 (95% CI, 2.2–10.3, P<0.001) for NAFLD.
In contrast to the current study, Corey et al.33 evaluated the impact of vitamin D levels on the progression of CLD in a longitudinal case–control study as part of the HALT-C trial. The sample included well-defined patients with HCV who had not achieved a sustained virological response. This study failed to find a difference in vitamin D levels in patients with and in those without progressive CLD during a 4-year period. Both groups showed a decrease in vitamin D levels; however, 75% of the patients had normal vitamin D levels at baseline. The authors speculate that this specific observation may have resulted from supplementation as detectable vitamin D2 levels were identified in 55% of the cohort. Another study carried out by Duarte et al.34 evaluated 100 patients with chronic hepatitis C, and found no difference in the mean vitamin D levels in those with and without cirrhosis. Their vitamin D levels were 46.6 ng/ml in noncirrhotic individuals and 45.6 ng/ml in cirrhotic patients.
In a population with genotype 1 chronic hepatitis C, a significant (P<0.0001) inverse correlation between 25(OH) vitamin D serum concentrations and stages of fibrosis was reported 35,36. Moreover, multivariate logistic regression analysis showed an independent association between low 25(OH) vitamin D and increased necroinflammatory activity (OR 2.23, 95% CI 1.01–4.93, P=0.04), and more severe fibrosis (OR 0.94, 95% CI 0.89–0.99, P=0.02); thus, the lower the vitamin D, the higher the grade of inflammation and the stage of fibrosis 37. These findings are in agreement with those of the current study, which showed an inverse association between 25(OH) vitamin D and stages of liver fibrosis in HCV patients and a significant association between low 25(OH) vitamin D and severe fibrosis (OR=18.233, 95% CI=4.253–78.165).
In Egypt, Schaalan et al.38 and El Husseiny et al. 39 studied vitamin D deficiency in HCV-4-infected patients. They found a significantly negative correlation between vitamin D and interleukin-17 (IL-17), IL-23 PIIINP, and MCP-1. This negative correlation appears to highlight, at least in part, how these cytokines are involved with vitamin D in immune responses in HCV-4-related liver disease, and could explain how vitamin D deficiency plays a role in liver fibrosis as detected in our study.
The current study has several important limitations. First, it is a cross sectional case–control study capturing the relationship between vitamin D and liver disease at only a single point in time and therefore could not assess changes in vitamin D temporally with the progression of liver disease. Second, the number of patients was limited to only 60. In addition, we evaluated only patients with hepatitis C-induced liver disease and did not study the role of vitamin D in other forms of CLD. Although clear evidence of an association between vitamin D and liver disease exists in our study, it remains unknown whether vitamin D deficiency confers an increased risk of liver disease or whether liver disease causes vitamin D deficiency.
Vitamin D deficiency is highly prevalent in young patients with chronic HCV infection and is directly associated with disease severity. Chronic HCV patients with insufficient vitamin D levels have significantly increased ORs for severe fibrosis compared with patients with adequate vitamin D levels. Vitamin D deficiency and CLD may represent cause or effect as multiple endogenous and environmental factors affect vitamin D metabolism. Benefits of normalized vitamin D status are emerging in patients with CLD. We recommend the inclusion of vitamin D assessment and replacement in the management of chronic HCV patients. We also recommend the use of serum 25-hydroxy vitamin D as a reliable noninvasive biomarker of liver fibrosis in those patients. However, further studies in children are needed to evaluate the role of vitamin D and its supplementation in patients with chronic HCV infection.
Conflicts of interest
There are no conflicts of interest.
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