Abstract
Background/Objectives
To determine associations between serum 25-hydroxyvitamin D (25(OH)-D) concentrations and histologic nonalcoholic fatty liver disease (NAFLD) severity.
Subjects/Methods
Clinical, laboratory, and histology data were collected retrospectively in a pediatric cohort with biopsy-confirmed NAFLD. Serum 25(OH)-D concentrations were used to define vitamin D deficiency (≤20 ng/ml), insufficiency (21–30 ng/ml), and sufficiency (≥31 ng/ml).
Results
In all, 234 patients (78% non-Hispanic, median age 14 years) were included. The majority (n = 193) were either vitamin D insufficient (50%) or deficient (32%). Eighty-four patients (36%) reported taking vitamin D supplements at the time of biopsy; serum 25(OH)-D concentrations were not higher in those supplemented. There were no differences in the demographic, clinical, and laboratory characteristics of the three vitamin D status groups. Severity of steatosis, ballooning, lobular/portal inflammation, and NAFLD activity score were also not different between the groups. The proportion of patients with significant fibrosis (stage ≥ 2) was higher in those with insufficiency (29%) compared to those who were sufficient (17%) or deficient (15%, p = 0.04). After controlling for important covariates selected from age, body mass index, ethnicity, vitamin D supplementation, and season, the insufficient group had increased odds of a higher fibrosis score compared to the sufficient group (adjusted OR, 2.04; 95%CI, 1.02–4.08).
Conclusions
Vitamin D deficiency and insufficiency are common in children with NAFLD, but not consistently related with histologic disease severity. Prospective longitudinal studies are needed to determine optimal dosing strategies to achieve sufficiency and to determine whether adequate supplementation has an impact on histology.
Introduction
Nonalcoholic fatty liver disease (NAFLD) affects approximately one third of adults and one in ten children [1]. NAFLD has become a major public health concern, and an increasingly common indication for liver transplantation in adults [2] and in children [3]. Already the most prevalent pediatric liver disease [4], NAFLD is increasingly recognized even in preschool-aged children [5]. While NAFLD is closely related to obesity and metabolic syndrome in both adults and children [1], many children have distinct histological patterns. Specifically, children may manifest with an increased periportal distribution of steatosis, inflammation, hepatocellular injury, and fibrosis. This suggests the potential for unique pathophysiological or developmental differences in the onset and progression of NAFLD between adults and children [6]. Identifying the underlying mechanisms that contribute to the progression of disease in children is essential to develop targeted and effective therapies.
Vitamin D deficiency is highly prevalent among adults with NAFLD [7, 8]. In the context of nonalcoholic steatohepatitis (NASH), the expression of hepatic vitamin D receptors (VDR) is inversely proportional to the severity of steatosis and lobular inflammation [9]. Vitamin D has both anti-inflammatory and antifibrotic properties [10, 11], which would support a potential mechanism for its use in the treatment of NAFLD. While vitamin D deficiency has been linked to several biomarkers of systemic inflammation in this age group, independently of obesity [12], a recent meta-analysis of 937 subjects from six studies did not find an association between hypovitaminosis D and histologic liver disease severity in adults with NAFLD [13]. Randomized controlled trials (RCTs) investigating the effects of vitamin D supplementation in adults with NAFLD have shown inconsistent results [14, 15]. Notably, the optimal frequency, formulation and dose of vitamin D supplementation necessary to achieve sufficiency has not been determined in this population. A recent prospective study found that supplementation with 2000 IU of cholecalci-ferol daily for 6 months failed to correct hypovitaminosis D in the majority of patients with NASH [15]. Absence of normalization of plasma 25-hydroxyvitamin D (25 (OH)-D) concentrations in this context was accompanied by lack of normalization of serum aminotransferase levels [15].
The literature linking serum 25(OH)-D concentrations with liver disease severity in children with NAFLD is also inconclusive. While some cohort studies in children with presumed or confirmed NAFLD have reported significant associations between 25(OH)-D levels and presence or severity of NAFLD [16, 17], others found no associations [18, 19]. Studies with biopsy-confirmed cases of NAFLD have included smaller cohorts to date (n = 73–103), which may be contributing to variability in findings. Notably, it is not known whether vitamin D supplementation, which is commonly recommended in routine clinical practice or even self-administered by patients, could potentially influence associations. Therefore, further validation in larger cohorts is needed to determine if there are consistent associations between vitamin D status and pediatric NAFLD severity.
The objective of this study was to determine the prevalence of vitamin D insufficiency/deficiency in a large pediatric cohort with histologically confirmed NAFLD and to investigate whether serum 25(OH)-D concentrations were associated with features of liver disease severity.
Subjects and methods
Subjects and study design
This was a retrospective study performed at Cincinnati Children’s Hospital Medical Center with Institutional Review Board approval and a waiver of informed consent. Inclusion criteria were histologically confirmed NAFLD in children and adolescents evaluated at the Steatohepatitis Center at Cincinnati Children’ s Hospital Medical Center from April 1, 2009 to July 30, 2018 and body mass index (BMI) at or above the 85th percentile for age and sex. Exclusion criteria were secondary causes of hepatic steatosis (e.g. medication related), evidence of other concurrent liver diseases (e.g. autoimmune hepatitis, viral hepatitis), and history of weight loss surgery.
Clinical records were reviewed to collect information regarding the patients’ age, sex and ethnicity (defined and grouped as per the National Institutes of Health categories) [20], type 2 diabetes (T2DM) status, liver histology, as well as their anthropometrics within 3 months of and closest to the 25(OH)-D measurement. The formulation (ergocalci-ferol vs. cholecalciferol), frequency and dosage of vitamin D supplementation prescribed in relation to serum evaluation and liver biopsy were also determined. Laboratory data obtained within 3 months of the serum 25(OH)-D measurement, including serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), and glycated hemoglobin (HbA1C), were also documented.
Serum 25(OH)-D was measured using a chemiluminescent immunoassay. With respect to quality control, our institution used a vendor (Quantimetrix) who provided two external controls, representing both a high and a low 25(OH)-D concentration. In addition to the two external controls, our lab ran an internal control of human pool plasma with a 25(OH)-D concentration within the known established range. Thus, three separate controls were used to validate the assay.
Clinical and histological variables
Patients were classified according to serum (25(OH)-D concentrations as deficient (≤20ng/ml), insufficient (21–30 ng/ ml), and sufficient (≥31ng/ml) [21]. To categorize obesity severity, patients were defined as overweight (BMI: 85th to <95th percentile for age and sex based on CDC growth charts), obese class I (BMI: 95th percentile to <120% of the 95th percentile), obese class II (BMI: 120 to <140% of the 95th percentile), or obese class III (BMI ≥ 140% of the 95th percentile) [22]. Diagnosis of type 2 diagnosis mellitus (T2DM) was defined as HbA1c>6.4%, oral glucose tolerance test (OGTT) with plasma glucose >200 mg/dl at 2 h or confirmation of T2DM diagnosis by an endocrinologist.
NAFLD was defined histologically as the presence of steatosis involving at least 5% of the hepatocytes in the absence of evidence of other etiologies of chronic liver disease. Each biopsy was assigned a score for steatosis (0–3), lobular inflammation (0–3), hepatocyte ballooning (0–2) and fibrosis (0–4), and the NAFLD activity score (NAS) was calculated, as per Kleiner et al. [23]. For the purposes of the analyses, an NAS of ≥5 was used as a cutoff to distinguish those with mild versus severe liver disease. Significant fibrosis was defined as fibrosis ≥ stage 2.
Statistical analyses
Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC). Descriptive statistics of patients’ demographics, clinical characteristics, laboratory values, and disease severity were summarized in the overall cohort and by vitamin D status groups. N and percentages were reported for categorical variables, and medians with quartiles and means with standard deviations for continuous variables. Univariate analysis for marginal relationship between each of these characteristic variables/outcomes within the vitamin D status groups was done using chi-square tests for nominal categorical variables, Kruskal-Wallis tests for lab variables, and analysis of variance (ANOVA) for the ordinal histology outcomes. In addition, the conditional relationship between the outcomes and vitamin D status groups after controlling for potential covariates was modeled through multivariable logistic regressions. A backward model selection procedure was used to identify the final model for each outcome. BMI group variable was always put in the model regardless of being selected or not. Other covariates being considered in the model selection are age, sex, ethnicity, season, diabetes diagnosis, vitamin D supplementation. Odds ratios and 95% confidence intervals with p values were reported. Statistical significance was claimed at the 0.05 level.
Results
Of the 249 patients who had undergone a liver biopsy for the investigation of fatty liver disease during the study period, 237 (95%) had serum 25(OH)-D concentrations measured within 3 months of the biopsy. Of these 237 patients, only three had a BMI less than the 85th percentile and as such were excluded from further analyses. The demographic and baseline clinical, laboratory, and histological characteristics of the final cohort of 234 patients that met inclusion criteria are summarized in Table 1. About a third (35%) had an NAS ≥ 5. No patients had cirrhosis, but 13% had stage 2 and 9% had stage 3 fibrosis.
Table 1.
Demographics, and baseline clinical, laboratory, and histological characteristics of the study cohort
| Variable | All children, n = 234 |
|---|---|
| Vitamin D status; n (%) | |
| Deficient | 76 (32%) |
| Insufficient | 117 (50%) |
| Sufficient | 41 (18%) |
| Age (years) | 14 (12–16) |
| Male sex; n (%) | 155 (66%) |
| Ethnicity | |
| N (%) Hispanic | 51 (22%) |
| N (%) non-Hispanic | 183 (78%) |
| BMI (z-score) | 2.5 (2.2-2.7) |
| Obesity; n (%) | |
| Overweight | 5 (2%) |
| Obese Class I | 45 (19%) |
| Severe obesity Class II | 80 (34%) |
| Severe obesity Class III | 104 (45%) |
| Type 2 diabetes mellitus (T2DM); n (%) | 37 (16%) |
| Patients prescribed vitamin D supplementation; n (%) | 84 (36%) |
| Laboratory data at time of liver biopsy | |
| ALT (U/L) | 83 (54-124) |
| AST (U/L) | 48 (33-70) |
| GGT (U/L) | 46 (31-65) |
| ALP (U/L) | 201 (122 -290) |
| HbA1c (%) | 5.2 (5.0-5.5) |
| Histology data | |
| Steatosis score (mean, SD) | 1.9 (0.9) |
| Lobular inflammation score (mean, SD) | 1.2 (0.7) |
| Hepatocellular ballooning score (mean, SD) | 0.7 (0.6) |
| Portal inflammation score (mean, SD) | 0.8 (0.6) |
| Fibrosis stage (mean, SD) | 0.9 (0.9) |
| Fibrosis stage (n (%) in each category) | |
| 0 | 95 (42%) |
| 1 | 83 (36%) |
| 2 | 29 (13%) |
| 3 | 21 (9%) |
| 4 | 0 (0%) |
| NAFLD activity score (mean, SD) | 3.9 (1.5) |
| NAS score (n (%) in each category) | |
| 1 | 13 (6%) |
| 2 | 32 (14%) |
| 3 | 48 (20%) |
| 4 | 59 (25%) |
| 5 | 47 (20%) |
| 6 | 28 (12%) |
| 7 | 4 (2%) |
| 8 | 2 (1%) |
| NAS ≥ 5, n (%) | 81 (35%) |
Data are presented as N (%) for categorical variables, and medians and interquartile ranges or mean (SD)
ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, BMI body mass index, GGT gamma glutamyl transpeptidase, HbA1c hemoglobin A1c, NAFLD nonalcoholic fatty liver disease, NAS NAFLD activity score, T2DM Type 2 diabetes mellitus
The majority of patients (n = 193; 82%) were either vitamin D insufficient (50%) or deficient (32%) at the time of liver biopsy. Approximately one third of the study cohort had been prescribed vitamin D supplements (Table 1). There was seasonal variation in 25(OH)-D concentrations, with the highest proportion of vitamin D deficiency in the winter (December to February) (p < 0.01). Baseline demographics and anthropometrics of the patients were not significantly different among the groups based on vitamin D deficiency status (Table 2).
Table 2.
Comparison of the clinical, laboratory and histology findings among patients grouped by vitamin D status
| Variable | Vitamin D deficiency (N = 76) | Vitamin D insufficiency (N = 117) | Vitamin D sufficiency (N = 41) | p value |
|---|---|---|---|---|
| Age | 15 (12, 17) | 14 (12, 16) | 15 (12, 17) | 0.53 |
| Male sex; n (%) | 51 (67.1%) | 74 (63.2%) | 30 (73.2%) | 0.51 |
| Hispanic ethnicity; n (%) | 22 (29%) | 25 (21 %) | 4 (10%) | 0.06 |
| BMI z-score | 2.5 (2.2, 2.7) | 2.5 (2.3, 2.7) | 2.45 (2.0, 2.7) | 0.58 |
| T2DM; n (%) | 13 (17%) | 18 (15%) | 6 (15%) | 0.94 |
| Season at the time of vitamin D measurement | <0.01 | |||
| Spring | 18 (24%) | 27 (23%) | 6 (15%) | |
| Summer | 13 (17%) | 31 (27%) | 21 (51%) | |
| Autumn | 18 (24%) | 36 (31%) | 9 (22%) | |
| Winter | 27 (36%) | 23 (20%) | 5 (12%) | |
| Prescribed vitamin D supplementation (overall) | 29 (38%) | 40 (34%) | 15 (37%) | 0.86 |
| Weekly supplementation | 21 (28%) | 15 (13%) | 6 (15%) | 0.05 |
| Daily supplementation | 8 (10%) | 25 (21%) | 9 (22%) | |
| Laboratory investigations | ||||
| ALT (U/L) | 90 (63, 125) | 77 (53, 114) | 97 (60, 126) | 0.36 |
| AST (U/L) | 47 (34, 76) | 48 (33, 67) | 50 (30, 70) | 0.54 |
| GGT (U/L) | 46 (31, 63) | 46 (30, 65) | 47 (33, 72) | 0.80 |
| ALP (U/L) | 179 (120, 290) | 206 (130, 290) | 212 (121, 259) | 0.76 |
| HbA1c (%) | 5.3 (5.0, 5.5) | 5.1 (4.9, 5.5) | 5.2 (5.0, 5.4) | 0.18 |
| Histology data | ||||
| Steatosis | 1.9 (0.9) | 2.0 (0.8) | 2.0 (1.1) | 0.72 |
| Lobular inflammation | 1.3 (0.7) | 1.2 (0.7) | 1.2 (0.7) | 0.89 |
| Ballooning | 0.7 (0.6) | 0.7 (0.6) | 0.7 (0.6) | 0.84 |
| NAFLD activity score (NAS) | 3.8 (1.5) | 3.9 (1.4) | 3.9 (1.6) | 0.89 |
| Patients with NAS ≥ 5, n (%) | 23 (30.3%) | 42 (35.9%) | 16 (39.0%) | 0.59 |
| Portal inflammation | 0.7 (0.6) | 0.8 (0.7) | 0.9 (0.6) | 0.54 |
| Fibrosis stage | 0.7 (0.9) | (1.0) | 0.8 (1.0) | 0.07 |
| Patients with fibrosis ≥ 2, n (%) | 11 (14.5%) | 34 (29.1%) | 7 (17.1%) | 0.04 |
Data are presented as N (%) for categorical variables, and medians and interquartile ranges or mean (SD)
25(OH)-D 25-hydroxyvitamin D, ALT alanine aminotransferase, AST aspartate aminotransferase, ALP alkaline phosphatase, BMI body mass index, GGT gamma glutamyl transpeptidase, HbAlc hemoglobin A1c, NAFLD nonalcoholic fatty liver disease, NAS NAFLD activity score, T2DM Type 2 diabetes mellitus
Association between 25(OH)-D concentrations and laboratory variable data
Overall, there was no significant difference in median ALT, AST, GGT, ALP, and HbA1c levels among the vitamin D status groups (Table 2). The results were similar even after excluding those who had been prescribed vitamin D supplements at the time of liver biopsy (Table 3).
Table 3.
Comparison of the clinical, laboratory and histology characteristics of patients not prescribed vitamin D supplementation among patients grouped by vitamin D status (n = 150)
| Variable | Vitamin D deficiency (N = 47) | Vitamin D insufficiency (N = 77) | Vitamin D sufficiency (N = 26) | p value |
|---|---|---|---|---|
| Age | 14 (12, 16) | 13 (10, 15) | 14.5 (11, 17) | 0.29 |
| Male sex; n (%) | 32 (68%) | 54 (70%) | 18 (69%) | 0.97 |
| Hispanic ethnicity; n (%) | 13 (28%) | 16 (21%) | 3 (12%) | 0.29 |
| Laboratory | ||||
| ALT (U/L) | 73 (46, 121) | 74 (59, 119) | 92.5 (63, 160) | 0.69 |
| AST (U/L) | 54 (34, 72) | 48 (33, 67) | 52.5 (38, 83) | 0.44 |
| GGT (U/L) | 46 (31, 63) | 43 (28, 64) | 49.5 (37, 75) | 0.44 |
| ALP | 175 (121, 320) | 217.5 (134, 290) | 196 (133, 252) | 0.73 |
| HbA1c (%) | 5.3 (5.1, 5.6) | 5.1 (4.8, 5.4) | 5.2 (5.0, 5.4) | 0.12 |
| Histology | ||||
| Steatosis | 1.9 (0.9) | 2.0 (0.9) | 1.9 (1.1) | 0.89 |
| Lobular inflammation | 1.3 (0.8) | 1.2 (0.7) | 1.2 (0.7) | 0.92 |
| Ballooning | 0.7 (0.5) | 0.8 (0.6) | 0.8 (0.7) | 0.52 |
| NAFLD activity score | 4.0 (1.6) | 4.1 (1.4) | 3.9 (1.6) | 0.87 |
| Patients with NAS ≥ 5; n (%) | 16 (34%) | 28 (36%) | 10 (38%) | 0.95 |
| Portal inflammation | 0.7 (0.6) | 0.7 (0.6) | 0.9 (0.5) | 0.46 |
| Fibrosis stage | 0.9 (1.0) | 1.1 (1.0) | 0.7 (0.9) | 0.12 |
| Patients with fibrosis ≥ 2, n (%) | 8 (17%) | 24 (31%) | 4 (15%) | 0.14 |
Data are presented as medians and interquartile ranges or mean (SD)
25(OH)-D 25-hydroxyvitamin D, ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, BMI body mass index, GGT gamma glutamyl transpeptidase, HbA1c hemoglobin A1c, NAFLD nonalcoholic fatty liver disease, NAS NAFLD activity score, T2DM Type 2 diabetes mellitus
Association between 25(OH)-D concentrations and histologic severity of NAFLD
There was no statistically significant difference in the severity of steatosis, ballooning, lobular or portal inflammation and NAS among the groups (Tables 2 and 4). This was observed even after excluding those who had been prescribed vitamin D supplementation at the time of biopsy (Table 3). While there was no significant difference in mean fibrosis score across vitamin D status groups, the proportion of patients with significant fibrosis (fibrosis score ≥ 2) was significantly higher in those with vitamin D insufficiency when compared to those who were vitamin D sufficient or deficient (Table 2, Fig. 1). This relationship did not persist after excluding the patients prescribed vitamin D supplementation (Table 3). In addition, there were differences of laboratory and histological data among patients without vitamin D supplementation (Supplementary Table 1). After controlling for age, vitamin D supplementation and season (the confounders automatically selected from a list of possible confounders (which originally also included ethnicity, BMI ɀ-score and T2DM) by the statistical model based on their significance), the vitamin D insufficiency group had two times the odds of having higher stage fibrosis compared to the vitamin D sufficiency group (adjusted OR, 2.04; 95%CI, 1.02–4.08) (Table 4). Similarly, after controlling for the same confounders, the vitamin D insufficiency group had more than two times the odds of having significant fibrosis (stage ≥ 2) compared to the vitamin D sufficiency group (adjusted OR, 2.22; 95%CI, 0.86–5.70).
Table 4.
Logistic regression results for NAFLD severity outcomes (n = 234)
| Characteristics | Odds of vitamin D status classification |
|||||
|---|---|---|---|---|---|---|
| Unadjusted |
Adjusteda |
|||||
| OR (95% CI) |
p | OR (95% CI) |
p | |||
| Deficiency vs. sufficiency | Insufficiency vs. sufficiency | Deficiency vs. sufficiency | Insufficiency vs. sufficiency | |||
| NAS | 0.91 (0.47, 1.78) | 1.02 (0.54, 1.91) | 0.91 | 1.15 (0.57, 2.31) | 1.25 (0.66, 2.38) | 0.79 |
| Proportion of patients with NAS ≥ 5 | 0.68 (0.31, 1.50) | 0.88 (0.42, 1.82) | 0.59 | 0.70 (0.3, 1.63) | 0.89 (0.42, 1.92) | 0.65 |
| Fibrosis stage | 0.93 (0.46, 1.90) | 1.77 (0.91, 3.44) | 0.04 | 1.10 (0.52, 2.35) | 2.04 (1.02, 4.08) | 0.03 |
| Proportion of patients with fibrosis ≥ 2 | 0.82 (0.29, 2.31) | 1.99 (0.80, 4.92) | 0.04 | 0.98 (0.33, 2.91) | 2.22 (0.86, 5.70) | 0.06 |
OR odds ratio, 95% CI 95% confidence interval, NAS NAFLD activity score, NAFLD nonalcoholic fatty liver disease
Adjusted by age, season, and/or vitamin D supplementation based on model selection results
Fig. 1.
The proportion of patients with fibrosis severity: mild (F 0–1) and advanced (F 2–4)
Use of vitamin D supplementation and association with 25(OH)-D concentrations
Various oral vitamin D supplementation regimens had been prescribed to patients (Table 2): ergocalciferol either 50,000 IU weekly or 2000–5000 IU daily, or cholecalciferol 50,000 IU weekly or 800–4000 IU daily. Of the 84 patients who had been prescribed vitamin D supplements, 42 (50%) were on weekly (50,000 IU) and 42 (50%) on daily vitamin D supplementation. Those with vitamin D deficiency were more likely to be prescribed weekly high-dose vitamin D supplementation (p = 0.05). Overall, supplementation (any vs. none) was not associated with higher 25(OH)-D concentrations: 23.8 ± 0.7 in the nonsupplemented vs. 24.1 ± 0.9 in the supplemented group (p = 0.79), even after adjustments for season (p = 0.18). Furthermore, no significant differences in 25 (OH)-D concentrations were found between those prescribed daily vs. weekly supplementation.
Discussion
This is the largest pediatric study investigating the relationship between serum 25(OH)-D concentrations and histologic liver disease severity in children with NAFLD to date. In this study, we found that 82% of children with NAFLD were vitamin D insufficient or deficient within 3 months of their liver biopsy. There was no association between serum 25 (OH)-D concentrations and serum aminotransferases or histologic scores. While the proportion of patients with significant fibrosis stage ≥ 2 was significantly higher among those who were vitamin D insufficient, this relationship did not exist among those who were deficient in 25(OH)-D or when supplemented patients were excluded from the analyses, suggesting that a pathophysiologic relationship is less likely. Vitamin D supplementation was equal among all three groups; however, those who were deficient were more likely to be prescribed weekly high-dose vitamin D supplementation. Overall, supplementation was not associated with significantly higher serum vitamin 25(OH)-D concentrations.
In our cohort, the majority (82%) of children with NAFLD were vitamin D insufficient or deficient. According to population-based data, approximately 15% of children aged 12–19 years across the BMI spectrum are vitamin D insufficient or deficient [24]. In the context of pediatric NAFLD, vitamin D insufficiency ranges from 47 to 80 % in countries such as Italy, UK, and the USA [16, 18, 19]. Previous studies have attributed the vitamin D insufficiency/ deficiency seen in patients with NAFLD to obesity [12]. Factors such as reduced dietary intakes of vitamin D and the distribution of 25(OH)-D into large volume adipose stores have been postulated to contribute to obesity-related hypovitaminosis D [25–27]. The reported prevalence of 25(OH)-D insufficiency and deficiency in predominantly Caucasian obese children, aged 6–18 years, in the USA is similar to that found in our cohort at 78% (overweight), 87% (obese class I), 91% (obese class II and class III) [28]. Given the retrospective design of our study and the fact that all our patients were overweight or obese, we could not distinguish an obesity-independent effect of vitamin D deficiency on NAFLD. Nevertheless, it is clear that vitamin D insufficiency and deficiency are highly prevalent in obese children with NAFLD.
In our study, there was a significantly higher proportion of patients with severe fibrosis in the vitamin D insufficient group, but not in the deficiency group. In contrast, Nobili et al. reported an association between fibrosis severity and vitamin D status [16]. In that study, a cutoff of 20 ng/ml was used to divide the NAFLD cohort of 73 Italian children (with those falling <20 ng/ml classified as vitamin D deficient, and those with 25(OH)-D >20 ng/ml classified as normal). While the age, median BMI ɀ-score and mean NAS of that study cohort were similar to ours, only 19 patients in the Italian cohort had stage 2 fibrosis (and none had stage 3–4). In addition, the number of patients with insufficient 25(OH)-D concentrations was not reported. In our study, 14.5% of deficient and 29.1% of insufficient patients had stage 2 fibrosis or worse (Table 2). The results of our study are consistent with those reported by Hourigan et al. who also did not observe an association between vitamin D levels and NAFLD severity, including fibrosis, in a multicenter North American cohort of 102 well-characterized children with biopsy-confirmed NAFLD. Patients included in that study had similar baseline BMI ɀ-score as our cohort [19], but were more likely to be of Hispanic ethnicity (70% of total cohort). Nearly half of the cohort in the Hourigan et al. study lived in a Southern latitude (56%), though no relationship with vitamin D status and Northern or Southern latitude was identified. By comparison, our cohort was largely non-Hispanic and was recruited from a Northern latitude, where average ultraviolet (UV) light exposure index between 2015 and 2016 was 4.7 in Fig. 2 (Global Solar UV Index using a scale of 1 (or “Low”) to 11 and higher (or “Extreme”)).
Fig. 2.
The UV light dataset as estimated by UV Index (UVI) in Cincinnati (CIN), downloaded from 2015–2016
A plausible reason that could explain the discrepancies in the published pediatric studies on the association of pediatric NAFLD with serum vitamin 25(OH)-D concentrations is the variability in the vitamin D supplementation. Both prescription and over-the-counter supplements could potentially impact the findings if a biological role for vitamin should exist in the pathogenesis of NAFLD. In addition, it should be noted that long-term supplementation may lead to 25(OH)-D concentrations that do not necessarily reflect liver disease severity. Over one third of our cohort reported vitamin supplementation at time of biopsy, but the proportion reporting vitamin D supplementation was similar across all three groups. The Endocrine Society recommends supplementing obese children with 600–1000 IU per day [29]. In our study, even though the minimum dose of vitamin D prescribed was 800 IU per day, vitamin D supplementation was not associated with higher serum 25(OH)-D values. This may be reflective of heterogeneity in the duration of treatment, poor compliance, or the fact that higher doses of vitamin D supplementation are required to bring serum 25(OH)-D values to a normal range in adolescents with severe obesity and NAFLD. In our cohort, patients with vitamin D deficiency were more likely to be prescribed weekly supplementation. However, previous studies have suggested that daily administration may be superior to intermittent dosing [30, 31]. Evidence-based guidelines regarding the optimal dosing strategy for vitamin D are needed for children.
Animal and in vitro data suggest an anti-inflammatory and an antifibrotic potential of vitamin D [10, 11]. For instance, in different mouse models, vitamin D supplementation has been shown to be beneficial in preventing pulmonary [32] and renal interstitial fibrosis [33] by suppressing ultrastructural changes, as well as inhibiting myofibroblastic proliferation. In addition, in a prospective study of 53 presumably healthy British Bangladeshi adults, circulating Matrix Metalloproteinase 2 and 9 (MMP 2,9) levels were inversely related to serum 25(OH)-D concentrations, and furthermore, MMP2,9 levels dropped with vitamin D supplementation [34]. MMPs are involved in tissue remodeling and fibrosis, and thus may serve as one pathophysiologic link between vitamin D status and risk of development of hepatic fibrosis in NAFLD. The serum 25 (OH)-D concentration associated with optimum antifibrotic potential is not known. An RCT of vitamin D supplementation (50,000 IU, orally administered, weekly dose for 12 weeks) in adult NAFLD patients failed to show a histological benefit [14]. The only pediatric RCT assessing the efficacy of vitamin D supplementation on liver histology in children is by Della Corte et al. In that study, 41 patients ages 4–16 years with a mean BMI ɀ-score of 2.2 and histologically confirmed NASH were randomized to a combination of cholecalciferol (800 IU/day) and docosahexaenoic acid (DHA) 500mg/day (n = 18; fibrosis stage 2: 6 patients and stage 3: 1 patient) or placebo (n = 23; fibrosis stage 2: 5 patients). Repeat histology at 12 months did not reveal histological improvement in the supplemented group [35]. It remains unknown whether higher doses of vitamin D supplementation has an impact on the severity of fibrosis seen in pediatric NAFLD. Further, to have the power to show a possible effect of vitamin D supplementation on fibrosis, an adequate number of patients with severe fibrosis would need to be included.
The major strengths of our study include the large sample size of children and adolescents with biopsy-confirmed NAFLD, documentation of vitamin D prescription, and season of measurement of serum 25(OH)-D. The study was limited by the retrospective and crosssectional design, which precluded assessing patients’ adherence to the prescribed dose of vitamin D, or impact of duration and amount of vitamin D supplementation on liver histology. In addition, we did not have dietary information to control for vitamin D intake as foods can contain D2 and/or D3. Given the exploratory retrospective observational nature of this study, we aimed to capture every available signal from the data and therefore did not do multiple testing adjustment. The study of this cohort may have introduced selection bias because we only perform liver biopsies in those who have more concerning biochemical or imaging findings. Given that all our patients were overweight or obese, and the absence of a control arm of BMI-matched, non-NAFLD patients, we were unable to evaluate the effect of obesity on the 25 (OH)-D concentrations of this NAFLD cohort. However, the main objectives of the present study were to determine if vitamin D deficiency is common in pediatric NAFLD and if it is associated with liver disease severity, both of which were addressed in our study.
In summary, in this large cohort of children with histologically confirmed NAFLD, the prevalence of vitamin D insufficiency and deficiency was high. While vitamin D insufficiency was associated with the severity of hepatic fibrosis in this cohort, the lack of association with vitamin D deficiency does not support a strong pathophysiological link between vitamin D status and histologic NAFLD severity in children. Well-powered pediatric RCTs are needed to determine the impact of adequate vitamin D supplementation (that achieves normalization of vitamin D status) on the severity and outcomes of NAFLD.
Supplementary Material
Acknowledgments
Funding
SD was funded in part by NIH R21 AA022742; RO1 DK 113196; RO1 GM119174; P50 AA024333; UO1 AA021890, UO1AA026975, UO1 DK061732 and the Mikati Foundation Grant. SO was funded by NIH T32 DK007727. SAX was funded in part by R01 DK100429.
Footnotes
Supplementary information The online version of this article (https://doi.org/10.1038/s41430-019-0493-y) contains supplementary material, which is available to authorized users.
Conflict of interest The authors declare that they have no conflict of interest.
References
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