Vitamin D and histologic severity of nonalcoholic fatty liver disease: A systematic review and meta-analysis

Accepted Manuscript Title: Vitamin D and histologic severity of nonalcoholic fatty liver disease: a systematic review and meta-analysis Authors: Veeravich Jaruvongvanich, Wasin Ahuja, Anawin Sanguankeo, Karn Wijarnpreecha, Sikarin Upala PII: DOI: Reference:

S1590-8658(17)30204-9 http://dx.doi.org/doi:10.1016/j.dld.2017.02.003 YDLD 3375

To appear in:

Digestive and Liver Disease

Received date: Revised date: Accepted date:

2-12-2016 2-2-2017 5-2-2017

Please cite this article as: Jaruvongvanich Veeravich, Ahuja Wasin, Sanguankeo Anawin, Wijarnpreecha Karn, Upala Sikarin.Vitamin D and histologic severity of nonalcoholic fatty liver disease: a systematic review and meta-analysis.Digestive and Liver Disease http://dx.doi.org/10.1016/j.dld.2017.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 Vitamin D and histologic severity of nonalcoholic fatty liver disease: a systematic review and meta-analysis Running title: Vitamin D and NAFLD histologic severity

Veeravich Jaruvongvanich MD 1,2, Wasin Ahuja 3, Anawin Sanguankeo, MD 4,5, Karn Wijarnpreecha 4, Sikarin Upala, MD, MS 4,5

1

Department of Internal Medicine, University of Hawaii, Honolulu, HI, USA

2

Department of Internal Medicine, King Chulalongkorn Memorial Hospital, Bangkok, Thailand

3

Department of Internal Medicine, Norwalk Hospital, Norwalk, CT, USA

4

Department of Internal Medicine, Bassett Medical Center and Columbia University College of

Physicians and Surgeons, Cooperstown, NY, USA 5

Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol

University, Thailand

Veeravich jaruvongvanich: [email protected] Wasin Ahuja: [email protected] Karn Wijarnpreecha: [email protected] Sikarin Upala: [email protected] Anawin Sanguankeo: [email protected]

Corresponding author Sikarin Upala, MD, Department of Internal Medicine, Bassett Medical Center and Columbia University College of Physicians and Surgeons, Cooperstown, NY 13326, United states. [email protected], Office: 607-547-4805, Fax: 607-547-6612

2 Abstract Background: NAFLD and vitamin D deficiency often coexist and epidemiologic evidence has shown that both of these conditions share several risk factors. Recent studies investigating the relationship between vitamin D levels and severity of NAFLD showed conflicting results. Thus we conducted a systematic review and meta-analysis to evaluate association between vitamin D and NAFLD histologic severity. Methods: A comprehensive search of the databases of the MEDLINE and EMBASE was performed from inception through November 2016. Observational studies compared serum vitamin D levels among NAFLD patients with high and low histologic severity, which was determined by NAFLD activity score (NAS) and fibrosis score. We calculated pooled mean difference (MD) of 25hydroxyvitamin D levels with 95% confidence intervals (CI) using random-effects model. Results: Data were extracted from 6 studies involving 974 NAFLD patients. There was no difference in 25-hydroxyvitamin D levels among NAFLD patients with high NAS (score of ≥ 5) versus low NAS (pooled MD = -0.93, 95%CI -2.45 to 0.58, I2 = 0%) and also high fibrosis score (score of ≥ 3) versus low fibrosis score (pooled MD = 0.88, 95%CI -2.65 to 4.42, I2 = 64%). Conclusions: Despite evidence implicating vitamin D in NAFLD pathogenesis, serum 25hydroxyvitamin D may not be associated with NAFLD histologic severity.

Keywords: Fibrosis; Meta-analysis; NAFLD activity score; Nonalcoholic fatty liver disease; Liver histology, Vitamin D

3 Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. It affects approximately 30% of the US population, or between 75 million and 100 million individuals [1]. It has been shown to have a 34% increased risk of all-cause mortality [2]. Low vitamin D level affects approximately 25-50% of the US population [3]. Up to 92% of patients with chronic liver disease have low vitamin D levels [4]. Therefore, unsurprisingly, NAFLD and low vitamin D levels are often found together. This was initially thought to be from the fact that both conditions share many common risk factors. However, several epidemiological studies demonstrated that there is an independent association between vitamin D levels and the risk of NAFLD [5, 6]. Since vitamin D has been shown to have significant functions in immune modulation, cell differentiation and proliferation, and regulation of inflammatory response [7], low vitamin D level is thought to play a role in the complex pathogenesis of NAFLD by up-regulating hepatic inflammatory response through adipocytokines such as adiponectin, leptin, resistin [7]. Subsequent studies have investigated the relationship between vitamin D level and NAFLD histologic severity. However, the data is limited and conflicting [8-14]. Thus, to further investigate this association, we conducted a systematic review and meta-analysis of observational studies to evaluate association between vitamin D level and NAFLD histologic severity.

Methods This systematic review and meta-analysis was conducted and reported according to the Metaanalysis Of Observational Studies in Epidemiology statement [15] and was registered in PROSPERO (registration number: CRD42016051458).

Search Strategy Two authors (SU and VJ) independently searched published studies indexed in PubMed/MEDLINE and EMBASE databases from inception to November 2016 using the search strategy that comprised terms for NAFLD and vitamin D as detailed in Item S1 in supplementary material without language restriction. References of selected retrieved articles were also manually reviewed.

4 Eligibility criteria Our inclusion criteria were as follows: (i) Case-control, cross-sectional or cohort studies evaluating the association between vitamin D level and histologic severity, (ii) Participants were adult patients with NAFLD, (iii) NAFLD was diagnosed based on the detection of hepatic steatosis by biopsy or by imaging studies (ultrasonography, computed tomography or magnetic resonance imaging) with the exclusion of other causes of hepatic steatosis, (iv) Serum vitamin D was a measure of 25hydroxyvitamin D, (v) Outcomes were histologic features including NAFLD activity score (NAS) and fibrosis score. The NAS is the scoring system of histologic evaluation using an 8-point scale. It comprises of three variables including steatosis (0-3), ballooning (0-2) and lobular inflammation (0-3). The score of ≥ 5 is largely considered diagnostic of nonalcoholic steatohepatitis (NASH) [16]. Currently, NAS is used to grade the activity of NASH, rather than to make a histological diagnosis of NASH [17]. We used the cut-off of 5 in this meta-analysis (NAS 0-4 versus 5-8). Fibrosis score is determined with the staging from F0 to F4 [18]. Advanced fibrosis is defined as F3-4. We used the cut off of 3 (F0-2 versus F3-4). We further subcategorized fibrosis score into 3 subgroups as followed: F0 versus F1-2 versus F3-4. Two authors (SU and VJ) independently reviewed and evaluated the eligibility of all identified articles. Quality of each study was also independently evaluated by the two authors using the Newcastle-Ottawa quality assessment scale which assessed each study in three areas including (i) the selection of the study participants, (ii) the comparability of the groups and (iii) the ascertainment of the outcome of interest [19]. Difference in determination of the eligibility of each study by the two authors was resolved by discussion with all authors. We contacted the authors of the primary reports to request any unpublished data. If the authors did not reply, we used the available data for our analyses.

Data Extraction The following data were abstracted from each study using standardized case record form: first author name, year of publication, study design, study location, number of participants, participant baseline characteristics, method used to identify and verify NAFLD as well as vitamin D level. This data extraction was independently extracted by the two authors.

5 Statistical Analysis The outcome of the meta-analysis is the net difference of serum 25-hydroxyvitamin D level between NAFLD patients with higher and lower histologic severity as determined by NAS (< 5 versus ≥ 5) and fibrosis stage (< 3 versus ≥ 3). Subgroup analysis was performed to compare the difference of serum 25-hydroxyvitamin D level between NAFLD patients with F3-4 vs. F0 and F1-2 vs. F0. The effect size was estimated as unstandardized mean differences (MDs) with 95% confidence interval (CI). In light of the high likelihood of between-study variance, a random-effects model was used. The heterogeneity of effect size estimates across these studies was quantified using the Q statistic, its pvalue, and I2 (P<0.10 was considered significant). A value of I2 of 0–25% indicates insignificant heterogeneity, 26–50% low heterogeneity, 51–75% moderate heterogeneity and 76–100% high heterogeneity [20]. Publication bias was assessed using funnel plot and Egger’s regression test [21]. Data analysis was performed using Comprehensive Meta-Analysis 3.3 software from Biostat, Inc.

Results The initial search yielded 155 articles; 135 articles were excluded based on title and abstract review (33 articles did not study in human, 86 articles were not observational studies, 16 articles performed in children). A total of 20 articles underwent full-length review. Fourteen articles were excluded (3 articles were not observational studies, 3 articles performed in children, 4 articles had not adequate data to combine, and 4 articles did not report outcome of interest). Six observational studies (all cross-sectional [8, 9, 12-14, 22]) involving 974 NAFLD patients were included in the meta-analysis. Item S2 outlines the search methodology and selection process. Table 1 describes the detailed characteristics and quality assessment of included studies.

Vitamin D and NAS Six studies [8, 9, 12-14, 22] assessed the association between serum 25-hydroxyvitamin D and NAS. We found no significant difference in serum 25-hydroxyvitamin D among NAFLD patients with high NAS (score of ≥ 5) compared with those with low NAS with the pooled MD of -0.93 (95% CI 2.45 to 0.58) ng/ml. The forest plot of this meta-analysis is shown in Figure 1. The statistical heterogeneity was low with I2 of 0% (Pheterogeneity = 0.64). To investigate potential publication bias, we examined the contour-enhanced funnel plot of the included studies that assessed MD of serum vitamin

6 D (Item S3). Publication bias is presence as there are more studies that favor positive results and the Pvalue for Egger’s regression test is significant (P < 0.01).

Vitamin D and fibrosis score Six studies [8, 9, 12-14, 22] assessed the association between serum 25-hydroxyvitamin D and fibrosis score. We found no significant difference in serum 25-hydroxyvitamin D among NAFLD patients with high fibrosis score (score of ≥ 3) compared with those with low fibrosis score with the pooled MD of 0.88 (95%CI -2.65 to 4.42) ng/ml. The forest plot of this meta-analysis is shown in Figure 2. The statistical heterogeneity was moderate with I2 of 64% (Pheterogeneity = 0.62). Funnel plot was symmetric and, thus, did not suggest publication bias (Item S4). As we further categorized patients into 3 subgroups based on fibrosis score, there were no differences in serum 25-hydroxyvitamin D in those with F1-2 (pooled MD = -0.25, 95%CI -3.43 to 2.93 ng/ml), or in those with F3-4 (pooled MD = -0.33, 95%CI -6.00 to 5.33 ng/ml) compared with F0 as shown in Item S5.

Discussion This is the first meta-analysis on the relationship between serum vitamin D and NAFLD histologic severity. Our study found no association between serum 25-hydroxyvitamin D levels and disease severity as determined by NAS and fibrosis score among patients with NAFLD. Insulin resistance is a key risk factor of NAFLD that links to development of oxidative stress and lipotoxicity [23]. Vitamin D deficiency is independently associated with insulin resistance [24-26] and supplementation of vitamin D has shown to improve insulin sensitivity [27, 28]. Adipose tissue inflammation has been recognized as another key component of NAFLD [29]. Vitamin D has an active role in adipose tissue by reducing inflammation and inhibiting adipogenesis [30]. Moreover, vitamin D was also found to significantly modulate the liver inflammation and liver fibrosis in in vitro and in vivo models [31-34]. Thus we hypothesized that low serum vitamin D might associate with worsening histologic severity of NAFLD given its metabolic, anti-inflammatory, and anti-fibrotic properties that could impact on various steps of disease progression and severity of NAFLD [35]. Vitamin D deficiency is highly prevalent in patients with cirrhosis [36]. Low vitamin D levels are associated with advanced liver disease and poor outcomes in cirrhotic patients [37, 38]. Few studies

7 investigating the association between serum vitamin D and NAFLD histology demonstrated conflicting results. It should be noted that these studies had relatively small sample sizes [14, 22, 39]. Recently, a study from the NASH Clinical Research Network involving 190 biopsy-proven NAFLD adults found an independent association between vitamin D and increased NAS and the presence of fibrosis [40]. However, it is notable that factors affecting NAFLD severity including body mass index, obesity, diabetes mellitus, and metabolic syndrome were relatively enriched in the vitamin D deficient group. Our meta-analysis comprehensively assessed all available studies and showed no association between serum 25-hydroxyvitamin D levels and histologic severity of NAFLD. Randomized controlled trials performed in NAFLD patients also found no change in serum liver enzymes, liver steatosis and metabolic or cardiovascular parameters in the vitamin D supplement group [41, 42]. However, the results have to be interpreted with caution given the limited number of participants and relatively shortterm follow up. Our result is also supported by a recent study [13] evaluating the relationship between hepatic expression of several vitamin D metabolizing genes and downstream mediators of vitamin D signaling, which include cytochrome complexes, vitamin D binding protein, and group specific component gene, and NAFLD severity. This study demonstrated a lack of association between these genes and disease severity. A few limitations in this study should be acknowledged. First, many of the included studies did not adjust for potentially important confounders including body mass index, insulin resistance, metabolic syndrome and diabetes. This could affect the validity of the results. Second, statistical heterogeneity is present in this study. This might be explained by the differences in population enrolled and methods of serum 25-hydroxyvitamin D measurement. Third, this is a meta-analysis of crosssectional studies, which can only demonstrate an association, not causality. In summary, despite evidence implicating vitamin D in NAFLD pathogenesis, our study suggests that serum 25-hydroxyvitamin D may not be associated with NAFLD histologic severity as determined by NAS and fibrosis score. However, our results should be interpreted with caution given low numbers of observed subjects and lack of confounder adjustments. Further study into whether vitamin D may play a role in the progression of NAFLD is warranted.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

8 Informed consent: No informed consent. Disclosure statement: The authors have nothing to disclose Author contributions: VJ conceived of the study, assessed the quality of the studies, and drafted the manuscript. SU and WA searched the literature, assessed the quality of the studies, performed the statistical analysis, and drafted the manuscript. AS and KW participated in the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.

Conflict of interest We declare no conflict of interest. Potential competing interests: None. Acknowledgement: We thank Matthew Roslund for validation of the search.

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11 (32) Ning C, Liu L, Lv G, et al. Lipid metabolism and inflammation modulated by Vitamin D in liver of diabetic rats. Lipids in health and disease. 2015;14:31. (33) Wang H, Zhang Q, Chai Y, et al. 1,25(OH)2D3 downregulates the Toll-like receptor 4-mediated inflammatory pathway and ameliorates liver injury in diabetic rats. Journal of endocrinological investigation. 2015;38:1083-91. (34) Duran A, Hernandez ED, Reina-Campos M, et al. p62/SQSTM1 by Binding to Vitamin D Receptor Inhibits Hepatic Stellate Cell Activity, Fibrosis, and Liver Cancer. Cancer cell. 2016;30:595609. (35) Eliades M, Spyrou E. Vitamin D: a new player in non-alcoholic fatty liver disease? World journal of gastroenterology. 2015;21:1718-27. (36) Stokes CS, Volmer DA, Grunhage F, et al. Vitamin D in chronic liver disease. Liver international : official journal of the International Association for the Study of the Liver. 2013;33:338-52. (37) Finkelmeier F, Kronenberger B, Zeuzem S, et al. Low 25-Hydroxyvitamin D Levels Are Associated with Infections and Mortality in Patients with Cirrhosis. PloS one. 2015;10:e0132119. (38) Paternostro R, Wagner D, Reiberger T, et al. Low 25-OH-vitamin D levels reflect hepatic dysfunction and are associated with mortality in patients with liver cirrhosis. Wiener klinische Wochenschrift. 2016. (39) Manco M, Ciampalini P, Nobili V. Low levels of 25-hydroxyvitamin D(3) in children with biopsy-proven nonalcoholic fatty liver disease. Hepatology (Baltimore, Md). 2010;51:2229; author reply 30. (40) Nelson JE, Roth CL, Wilson LA, et al. Vitamin D Deficiency Is Associated With Increased Risk of Non-alcoholic Steatohepatitis in Adults With Non-alcoholic Fatty Liver Disease: Possible Role for MAPK and NF-kappaB? The American journal of gastroenterology. 2016;111:852-63. (41) Sharifi N, Amani R, Hajiani E, et al. Does vitamin D improve liver enzymes, oxidative stress, and inflammatory biomarkers in adults with non-alcoholic fatty liver disease? A randomized clinical trial. Endocrine. 2014;47:70-80. (42) Barchetta I, Del Ben M, Angelico F, et al. No effects of oral vitamin D supplementation on nonalcoholic fatty liver disease in patients with type 2 diabetes: a randomized, double-blind, placebocontrolled trial. BMC medicine. 2016;14:92.

12

13 Figure legends Figure 1 Random effects pooled mean difference of 25-hydroxyvitamin D levels in NAFLD patients with high and low NAS, NAS; nonalcoholic fatty liver disease activity score

Figure 2 Random effects pooled mean difference of 25-hydroxyvitamin D levels in NAFLD patients with high and low fibrosis score

14

Table 1 Characteristics of included studies Targher et al. [14] Barchetta et al.

Bril et al. [9]

Anty et al. [8]

Luger et al. [12]

Patel et al. [13]

[22] Country

Italy

Italy

United States

France

Austria

United States

Year

2007

2012

2015

2016

2016

2016

Study design

CS

CS

CS

CS

CS

CS

Total number

60

25

239

360

46

244

Study sample

Adults with

Patients who

General

Morbidly obese

Adults planned to

Adults who

NAFLD who

underwent liver

population who

patients, referred

undergo gastric

underwent

attended

biopsy for clinical

volunteered or

for bariatric

bypass

evaluation for the

outpatient clinic

purposes at

from outpatient

surgery, between

suspected

Campus Bio-

clinic

2006 and 2012

NAFLD

Medico Hospital Diagnosis of

Liver biopsy

Liver biopsy

Liver biopsy

Liver biopsy

Liver biopsy

Liver biopsy

CLIA

Calorimetric

CLIA

CLIA

N/A

CLIA

NAFLD Vitamin D measurement Mean 25-

method 20.4 ± 8.8

21.9 ± 12.3

21.8 ± 1.0

18.8 ± 1.9

15.6 ± 5.8

27.6 ± 11.8

Mean ALT

105 ± 42

87.5 ± 46.6

64 ± 4

N/A

36.4 ± 20.8

N/A

Diabetes (%)

N/A

N/A

76

N/A

N/A

47.5

Mean BMI

26.3

30.5

34.3 ± 0.4

42.2

43.8

36.3

Mean age

47.0 ± 3.0

48.7 ± 13.3

53 ± 1

40 ± 3.2

42.0 ± 13.0

49.9 ± 11.0

Female (%)

33.3

48

14.6

83.9

80

35.7

Quality of

4,2,3

4,2,3

5,2,3

5,2,3

4,2,3

5,2,3

hydroxyvitam in D levels (ng/ml)

(years)

15 study (Selection, comparability, outcome)

Abbreviation: ALT: alanine aminotransferase; BMI: body mass index; CS: cross-sectional study; N/A: not applicable; NAFLD: nonalcoholic fatty liver disease; US: United States