Alia S Dadabhai, Behnam Saberi, Katie Lobner, Gerard E Mullin, Division of Gastroenterology and Hepatology, the Johns Hopkins University School of Medicine, Baltimore, MD 21224, United States

Russell T Shinohara, Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States

Author contributions: Dadabhai AS, Saberi B and Mullin GE contributed equally to the data evaluation, manuscript preparation, editing, figures, and final submission; Shinohara RT provided biostatistics methodology oversight, data extractions and meta-analysis with pooled data figure preparation and manuscript preparation and editing; Lobner K provided informatics methodology support, conducted the literature search, and reviewed and edited the manuscript.

Conflict-of-interest statement: All the authors declare that they have no competing interests.

Data sharing statement: Technical appendix, statistical code, and dataset are available from the corresponding author at adadabh1@jhmi.edu.

Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

Correspondence to: Alia S Dadabhai, MD, Assistant Professor, Division of Gastroenterology and Hepatology, the Johns Hopkins University School of Medicine, 4940 Eastern Ave A504, Baltimore, MD 21224, United States. adadabh1@jhmi.edu

Telephone: +1-410-5507857 Fax: +1-410-5507861

Received: November 16, 2016
Peer-review started: November 21, 2016
First decision: December 1, 2016
Revised: December 14, 2016
Accepted: January 2, 2017
Article in press: January 3, 2017
Published online: February 18, 2017

To investigate the relationship between vitamin D and liver fibrosis in hepatitis C-monoinfected or hepatitis C virus (HCV)-human immunodeficiency virus (HIV) co-infected patients.

Pertinent studies were located by a library literature search in PubMed/Embase/Cochrane/Scopus/LILACS by two individual reviewers. Inclusion criteria: (1) studies with patients with HCV or co-infected HCV/HIV; (2) studies with patients ≥ 18 years old; (3) studies that evaluated liver fibrosis stage, only based on liver biopsy; and (4) studies that reported serum or plasma 25(OH)D levels. Studies that included pediatric patients, other etiologies of liver disease, or did not use liver biopsy for fibrosis evaluation, or studies with inadequate data were excluded. Estimated measures of association reported in the literature, as well as corresponding measures of uncertainty, were recorded and corresponding odds ratios with 95%CI were included in a meta-analysis.

The pooled data of this systematic review showed that 9 of the 12 studies correlated advanced liver disease defined as a Metavir value of F3/4 with 25(OH) D level insufficiency. The meta-analysis indicated a significant association across studies.

Low vitamin D status is common in chronic Hepatitis C patients and is associated with advanced liver fibrosis.

Hepatitis C virus (HCV) infection remains one of the most common etiologies of liver disease worldwide. A number of epidemiological papers have addressed the global prevalence of Hepatitis C. Lanini et al[1] reported that 100 million people globally have serological evidence of current or past HCV infection causing 700000 deaths annually while others suggest that the actual occurrence is double[2]. HCV remains the most common indication for liver transplantation in the United States[3]. Chronic infection with HCV can lead to liver inflammation, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Liver fibrosis is a result of excessive accumulation of extracellular matrix proteins, as part of the wound healing response to chronic injury and chronic inflammation[4]. Various factors have been associated with progression of fibrosis including duration of infection, age, male sex, diabetes, alcohol consumption and human immunodeficiency virus (HIV) co-infection[5].

Vitamin D is a hormone that has numerous biological properties that influence host physiology by regulating epigenetic regulation of more than 2000 genes throughout the body. Vitamin D is best known for its role in maintaining bone mineralization but has diverse and profound influences which can determine disease development and outcome. Although referred to as a vitamin, this steroid hormone is synthesized in the body by a series of hydroxylation reactions that occur in skin (7-hydroxylation), the liver (25-hydroxylation) and the kidney (1-hydroxylation)[6] (Figure 1). Reduction of the enzymatic conversion of 7-dehydrocholesterol to 1.25 hydroxy vitamin D at any of the three conversion steps can result in suboptimal vitamin D status[7]. Vitamin D has a number of influences on innate and adaptive immunity which are pertinent to study in conditions that are driven by chronic inflammation and maladaptive tissue injury[8,9]. Given the ubiquitous distribution of vitamin D receptors in virtually every cell in the body-suboptimal vitamin D status has been studied for its relationship to numerous diseases[10]. For example, there is substantial evidence that vitamin D benefits rheumatoid arthritis, due to its immunomodulatory effect[11]. The role of vitamin D in various cancers has been established linked to its antiproliferative action mediated through vitamin D nuclear receptor[12]. There have been numerous reports on lower serum vitamin D levels in patients with chronic liver disease from various etiologies[13]. In chronic HCV, Low vitamin D levels have been reported in 46% to 92% of patients[10] raising suspicion of its potential contribution to disease pathogenesis. There is growing evidence from various groups, that vitamin D levels are inversely correlated with liver inflammation and stage of liver fibrosis in patients with HCV; however, the studies are heterogeneous with occasionally the results are conflicting. Additionally, the methods of reporting liver fibrosis were variable.

The aim of this study was to evaluate the relationship between vitamin D status and hepatic fibrosis based on histopathological staging in patients with chronic HCV mono-infection or co-infected HIV-HCV infection, by performing a systematic review of the scientific literature followed by a meta-analysis.

The initial protocol established a series of mesh terms used to identify articles that would evaluate the severity of liver fibrosis in chronic hepatitis C patients with vitamin D levels. Eighteen hundred and twelve articles were found using PubMed (n = 468)/EMBASE (n = 1269)/Cochrane (n = 23)/Scopus (n = 42)/LILACS (n = 10) search engines. Mesh terms used were liver fibrosis/vitamin d/cirrhosis/Ergocalciferols/25 hydroxyvitamin/25 hydroxy d/25(OH) D. Detailed evaluation of the articles by at least two independent reviewers (total of three) assessed the sufficiency of data, method of fibrosis qualification, relevance to the topic to narrow the studies to twelve. The data extraction algorithm is summarized in Figure 3. Table 1 reflects the characteristics of the studies relating fibrosis to chronic hepatitis C and vitamin D level. When patients were stratified according to vitamin D status, we found substantial differences between the levels of severity of liver fibrosis. The sensitivity analysis with different cutoffs for the Monte Carlo simulations showed robustness of the result to the choice of cutoff, with significant effects for all thresholds employed.

The results of our systematic analysis of the literature demonstrated an association between advanced liver fibrosis (defined as Metavir F3/F4) in chronic hepatitis C (CHC) with vitamin D status as reflected by 25-hydroxyvitamin D [25(OH)D] serum levels. In nine[21-29] of twelve studies (75%) that qualified for data extraction (Tables 1 and 2) the final analysis demonstrated an overall association between low vitamin D status as defined as serum 25(OH)D < 15 ng/mL with advanced liver fibrosis (F3/F4 stage disease) in CHC as proven by biopsy analysis for fibrosis stage. These data are highly consistent with prior reports, and the expected pathophysiological interference of 25-hydroxylation of vitamin D as liver fibrosis increases and functional hepatic capacity decreases over the course of hepatitis C disease progression[6].

Inclusion criteria

Age ≥ 18 yr

Studies including mono-infected HCV or co-infected HCV/HIV

Studies that evaluated liver fibrosis stage, only based on liver histology

Studies that reported serum or plasma 25(OH)D levels

Exclusion criteria

Age < 18 yr

Other etiologies of liver disease, other than hepatitis C

Studies that used non-invasive methods in evaluating liver fibrosis

Inadequate data

HCV: Hepatitis C virus; HIV: Human immunodeficiency virus.

A recent systematic review of the literature by Abbasi et al[30] studied the relationship between low vitamin D status [< 20 ng/mL 25 OH(D)] and the severity of the CLD. A comparatively abridged search strategy yielded 641 articles for consideration and ultimately 19 articles and 4895 study patients with CLD for data extraction showing that almost 80% of patients with chronic liver disease had severe vitamin D deficiency. García-Álvarez et al[31] conducted a systematic review evaluating the relationship of vitamin D status to advanced liver fibrosis in CHC-naïve patients and sustained virological response (SVR) to therapy using pegylated interferon/ribavirin (Peg-IFN/RBV). Seven of fourteen papers utilized for their extraction evaluated advanced liver fibrosis (1083 patients) and eleven for SVR (2672 patients). Approximately 70% of CHC patients had low 25(OH)D whereby the definition of insufficiency varied (20 or 30 ng/mL), and 50% of the HCV-infected patients had 25(OH)D levels < 10 or 20 ng/mL. Overall, low vitamin D status was related to a diagnosis of advanced stage of liver disease. Luo et al utilized a search methodology restricted to PubMed and Embase databases before October 2013 included studies that analyzed the association between serum vitamin D status and the severity of liver fibrosis in 8231 CHC patients without other restrictions yielding six global studies for data extraction[13]. One study recruited 6567 participants as part of the Swiss Hepatitis C Cohort Study[23] raising concerns for skewing of the extracted data. The mean data from extracted studies suggested that lower serum vitamin D is a risk factor for progressive liver fibrosis in CHC patients. However, there was a high heterogeneity and inconsistencies depending upon data set utilized (OR data studies vs mean data extracted). Our search methodology instead included 2521 patients which incorporated the 2012 study by Lange et al[32] which evaluated 468 HCV patients treated with alpha interferon-based regimens for vitamin D status and advanced disease demonstrating that fibrosis stages F2-F4 vs F0-F1 associated with low 25(OH)D.

The nine studies showing a positive association between low vitamin D with an advanced stage of fibrosis had variations in their definition of vitamin D status which challenged our ability to Meta-analyze the data. Low vitamin D was stratified according to by either insufficient (I) or deficient (D) (Table 1) in eight[21-27,29] of the nine studies. Gerova et al[28] used three categories; mild insufficiency, profound insufficiency, and deficiency. Overall, of the twelve papers in our final analysis, two[28,33] utilized nmol/L to measure serum 25(OH) vitamin D status. Insufficiency was defined as < 30 ng/mL in seven with another two using equivalent levels in nmol/L[28,34], < 20 ng/mL in two[23,25] while El-Maouche studied only deficient patients (< 15 ng/mL)[20]. The definition of “deficiency” was utilized by all but two[20,34] of the studies as < 10 ng/mL 25(OH) vitamin D. The prevalence of vitamin D deficiency in a population depends on upon the definition used [< 20 or < 30 ng/mL (50 or 75 nmol/L)]. In the National Health and Nutrition Examination Survey (NHANES), 41.6 percent of United States adults had (25[OH]D) levels < 20 ng/mL (50 nmol/L)[35]. The Institute of Medicine recommends the attainment of the serum 25(OH)D levels of > 20 < 40 ng/mL (50 to 100 nmol/L), however, many define sufficient vitamin D status as 25(OH)D > 30 and < 50 ng/mL (75 to 125 nmol/L)[36,37].

Hepatitis C genotype (1-6) did not change the outcome of analyses between advanced fibrosis in CHC with vitamin D status[20,33,34]. The geographical latitudes of study site and variable seasonal fluctuations have provided challenges to vitamin D status, but did not appear to influence the outcome of the negative outcome studies[20,33,34] Esmat et al[34] conducted a open-labelled RCT of 101 HVC4 Egyptian patients undergoing standard of care (SOC) Peg-IFN/RBV plus/minus 15000 IU vitamin D₃ (cholecalciferol). The fibrosis stage (F1-F3) at baseline was not different according to 25(OH) vitamin D levels. El-Maouche et al[20] evaluated HIV-HCV co-infected patients for histological fibrosis using the Metavir system [0 (no fibrosis) to 4 (cirrhosis)] and used banked serum as a source for vitamin D determination. Similar to Esmat et al[34], the prevalence of significant fibrosis (F2 ≥ 2) was similar among those with and without low vitamin D while low 25(OH)D status was not associated with significant fibrosis after adjusting for other confounders. Finally, Kitson et al[38] from Australia evaluated pre-treatment 25-hydroxyvitamin D [25(OH)D] level in a cohort of 274 treatment-naive patients with HCV-1 to evaluate the association between vitamin D status, virological response, and liver histology after 48 wk of pegylated interferon alfa-2a plus ribavirin therapy. Baseline 25(OH)D level did not vary with fibrosis stage (F3/4 vs F0-2).

The manner by which vitamin D may influence the course of CHC may be due to effects on viral clearance, immune modulation, cell differentiation and proliferation and inflammation regulation. Vitamin D is not only involved in calcium homeostasis but has also has been associated with the mechanism of cellular proliferation, and immunomodulation[39]. Several studies have shown that vitamin D levels are inversely correlated with stage of liver fibrosis in patients with CHC. Nine[21-29] of the twelve studies that we included for data extraction reported the inverse correlation of vitamin D levels with the stage of liver fibrosis in patients with CHC. Vitamin D has anti-inflammatory, anti-proliferative and anti-fibrotic effects that dampen inflammatory cell recruitment to the liver and mitigate hepatic fibrosis progression[40]. HCV may also have its own direct actions that impair vitamin D activity and status. It has been hypothesized that HCV affects 25-hydroxylation of vitamin D through cytokine induction or oxidative stress or through disruption in lipid metabolism where HCV suppress 25(OH)D levels due to a decrease in the production of vitamin D precursor, 7-dehydrocholesterol[10].

The profound relationship of vitamin D to immunity and inflammation, and our findings raise questions about how vitamin D status may impact the outcome of the many non-HCV chronic liver diseases. Individuals with chronic liver disease have significant global prevalence, morbidity, poor quality of life and mortality. Prior works have demonstrated adverse survival outcomes in patients with lowered vitamin D status[41,42]. In our analyses, we excluded papers reporting the analysis of vitamin D in chronic liver diseases other than HCV including chronic hepatitis B (CHB) which has a higher global prevalence of approximately 300 million infected individuals. Yu et al[43] evaluated the potential association between serum vitamin D level and liver histology or virological parameters in treatment-naïve patients with chronic hepatitis B infection in Southern China. They reported that patients infected with genotype B had a higher prevalence of vitamin D insufficiency than individuals with CHC. Furthermore, in chronic hepatitis B patients, serum 25(OH) D was not correlated with viral load or fibrosis. Mi et al[44] reported that vitamin D status was not different among Asians with non-cirrhotic CHB and CHC.

Low vitamin D status is associated with the risk of progression and the severity of hepatic inflammation in patients with non-alcoholic fatty liver disease[45,46]. Primary biliary cirrhosis has been extensively analyzed for correlations of vitamin D status predicting the outcome to ursodeoxycholic acid (UCDA) therapy and the influence of vitamin D supplementation to UCDA intervention[47-49]. Autoimmune hepatitis (AIH) has also been studied for the potential influence of vitamin D given the epidemiological association of this hormone with a number of diseases with autoimmunity[50,51]. However, there are not sufficient studies to draw meaningful conclusions of serum 25(OH)D and AIH at this time.

Altered vitamin D physiology via resistance from genetic polymorphisms of the vitamin D receptor (VDR) could also influence the outcome of CHC. Baur et al[25] demonstrated that low 25(OH)D plasma levels and VDR bAt[CCA]haplotype were associated with rapid fibrosis progression in CHC, separately and synergistic when co-present. Petta et al[52] reported that low hepatic VDR expression was inversely related to the severity of advanced liver fibrosis in patients with genotype 1 cCHC patients. Grunhage reported that a single nucleotide polymorphism (SNP) linked to the DHCR7 gene coding vitamin D precursor dehydrocholesterol was related to altered serum 25(OH)D in chronic liver disease patients with no or mild fibrosis[53].

CHC with severely low vitamin D status is accompanied by advanced liver fibrosis. Interventional trials aimed to normalize vitamin D status in early stages of CHC may shed light on whether correction of vitamin D status in this patient population should become the standard of care.