Vitamin D in Preclinical Models of Fatty Liver Disease.

Simple steatosis in non-alcoholic fatty liver disease (NAFLD) progresses to non-alcoholic steatohepatitis (NASH) when excessive fat accumulation is accompanied by ballooning, inflammation, and progressive hepatocellular injury. Due to the increasing global incidence of NAFLD/NASH and the lack of effective drugs, current treatment options are currently dominated by lifestyle interventions, including dietary and physical activity modifications. In this regard, vitamin D has received widespread attention in recent years. In line with its pleiotropic physiological effects, preclinical animal models and patient cohorts have demonstrated anti-inflammatory, anti-fibrotic and anti-proliferative effects of vitamin D on NAFLD and NASH. Several animal models have confirmed the association of vitamin D deficiency and NALFD/NASH severity in humans and revealed potential benefits of dietary vitamin D supplementation. These preclinical models also provide critical guidance to define the roles and therapeutic potential of vitamin D as well as its downstream functional mechanisms in the pathogenesis of fatty liver disease. This review summarizes vitamin D research in currently available animal models of fatty liver disease.

Four-Week Omega-3 Supplementation in Carriers of the Prosteatotic PNPLA3 p.I148M Genetic Variant: An Open-Label Study.

BACKGROUND/AIMS: The PNPLA3 loss-of-function variant p.I148M is a strong genetic determinant of nonalcoholic fatty liver disease. The PNPLA3 protein functions as an intracellular lipase in the liver, with a greater activity on unsaturated fatty acids. This study aimed to determine whether short-term supplementation with omega-3 fatty acids impacts hepatic steatosis differently in PNPLA3 p.148I wild-type individuals as compared to homozygous carriers of the PNPLA3 p.148M variant. METHODS: Twenty subjects with hepatic steatosis (50% women, age 18-77 years) were included. Ten subjects homozygous for the PNPLA3 148M variant were matched to 10 wild-type individuals. The subjects received 4 g omega-3 fatty acids (1,840 mg eicosapentaenoic acid and 1,520 mg docosahexaenoic acid) a day for 4 weeks. Transient elastography with a controlled attenuation parameter (CAP) was used to quantify liver fat before and after the intervention. Body composition, fibrosis, liver function tests, serum free fatty acids (FFA) and glucose markers were compared. RESULTS: Patients homozygous for the PNPLA3 p.148M variant (risk group) demonstrated no significant changes in CAP compared to baseline (284 ± 55 vs. 287 ± 65 dB/m) as did the control group (256 ± 56 vs. 262 ± 55 dB/m). While serum liver enzyme activities remained unchanged in both groups, the risk group displayed significantly (p = 0.02) lower baseline FFA concentrations (334.5 [range 281.0-431.0] vs. 564.5 [range 509.0-682.0] µmol/L), which markedly increased by 9.1% after the intervention. In contrast, FFA concentrations decreased significantly (p = 0.01) by 28.3% in the wild-type group. CONCLUSIONS: Short-term omega-3 fatty acid supplementation did not significantly alter hepatic steatosis. The nutrigenomic and metabolic effects of omega-3 fatty acids should be investigated further in carriers of the PNPLA3 148M risk variant.

Short-term Dietary Interventions for the Management of Nonalcoholic Fatty Liver.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects millions of individuals on a global scale and currently no gold standard treatment exists. The risk of developing NAFLD is considerably higher with increasing body mass index. Consequently, weight loss should be recommended to all overweight patients with fatty liver. However, lifestyle interventions, irrespective of weight status, may also influence the condition. The aim herein is to present examples of short-term interventions which assess direct effects of dietary-related components on hepatic steatosis. METHODS: This review includes studies with short-term dietary-related interventions of up to 16 weeks that evaluate their efficacy in reducing intrahepatic lipid contents (hepatic steatosis). This review primarily focuses on the three main macronutrients: dietary carbohydrates, fats and proteins. RESULTS: High saturated fat intake and high consumption of carbohydrates, particularly from simple sugars such as fructose are reported as risk factors for hepatic steatosis. Overall, shortterm hypocaloric diets have shown beneficial effects in reducing intrahepatic lipid contents. Macronutrient manipulations such as carbohydrate restriction as well as the consumption of unsaturated fatty acids are also reported to have efficacious effects. CONCLUSION: This review highlights the different dietary interventions that can influence hepatic steatosis in the short term, illustrating both pro and anti-steatotic effects.

Analytical Methods for Quantification of Vitamin D and Implications for Research and Clinical Practice.

A plethora of contradictory research surrounds vitamin D and its influence on health and disease. This may, in part, result from analytical difficulties with regard to measuring vitamin D metabolites in serum. Indeed, variation exists between analytical techniques and assays used for the determination of serum 25-hydroxyvitamin D. Research studies into the effects of vitamin D on clinical endpoints rely heavily on the accurate assessment of vitamin D status. This has important implications, as findings from vitamin D-related studies to date may potentially have been hampered by the quantification techniques used. Likewise, healthcare professionals are increasingly incorporating vitamin D testing and supplementation regimens into their practice, and measurement errors may be also confounding the clinical decisions. Importantly, the Vitamin D Standardisation Programme is an initiative that aims to standardise the measurement of vitamin D metabolites. Such a programme is anticipated to eliminate the inaccuracies surrounding vitamin D quantification.

Assessment of 3-epi-25-hydroxyvitamin D levels during cholecalciferol supplementation in adults with chronic liver diseases.

Recently, hepatic immaturity was cited as a possible reason for high levels of the C-3 epimer of 25-hydroxyvitamin (25(OH)D) in premature infants: however what role, if any, the liver plays in controlling epimer concentrations is unknown. This study assesses 3-epi-25-hydroxyvitamin D (3-epi-25(OH)D) levels during the course of cholecalciferol supplementation in adults with chronic liver diseases (CLD). Vitamin D metabolites were analyzed in 65 CLD patients with 25(OH)D <30 ng/mL who received 20 000 IU cholecalciferol/week for 6 months. The primary outcome assessed serum 25(OH)D and 3-epi-25(OH)D in response to supplementation. Corresponding values from 16 CLD patients with sufficient vitamin D levels receiving no supplementation were compared. The epimer was detected in all samples and at lower relative concentrations with lower vitamin D baseline status, i.e., severe vitamin D deficiency (<10 ng/mL) as compared with deficient (10-19.9 ng/mL), insufficient (20-29.9 ng/mL), or sufficient (≥30 ng/mL) vitamin D levels (2.4% vs. 4.8%, 5.2%, 5.8%, respectively; P < 0.001). Similar relative concentrations for 3-epi-25(OH)D, ranging from 4.3%-7.1% (absolute concentrations: 1.1-4.0 ng/mL; all P < 0.001), were obtained in response to cholecalciferol in all supplemented patients, regardless of inadequacy threshold. Epimer levels significantly decreased (P = 0.007) in unsupplemented patients, coinciding with decreasing serum 25(OH)D concentrations over time. No epimer differences between patients with (n = 17) or without (n = 48) cirrhosis were demonstrated. The 3-epi-25(OH)D was present in serum of all patients at comparable levels to those reported by others. Epimer levels increased linearly with increasing 25(OH)D levels after supplementation. However, no effect of cirrhosis on epimer concentrations was observed.

Vitamin D supplementation: less controversy, more guidance needed.

Vitamin D is a secosteroid hormone with multiple functions that extend beyond the regulation of intestinal calcium absorption. In recent years, the publication of research articles investigating associations between vitamin D status and health has reached an all-time high, and an increase in supplementation studies has followed. Given the pleiotropic effects of vitamin D, the scientific focus has gone beyond its known classic benefits on skeletal health to include diabetes and cardiovascular, neurological, respiratory, renal, and liver diseases, yet numerous conflicting findings continue to emerge. This review presents some examples of recent work within the context of controversies surrounding vitamin D and highlights key factors that should be considered when designing vitamin D supplementation regimens.

Effect of Short-Term Vitamin D Correction on Hepatic Steatosis as Quantified by Controlled Attenuation Parameter (CAP).

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in Western countries. A meta-analysis has confirmed decreased serum 25-hydroxyvitamin D levels in NAFLD patients. This intervention study investigates whether vitamin D correction ameliorates hepatic steatosis. METHODS: We prospectively recruited 40 patients from an outpatient liver clinic with vitamin D deficiency (serum 25-hydroxyvitamin D < 20 ng/ml). Controlled attenuation parameter (CAP) during transient elastography quantified hepatic steatosis. Patients with significant liver fat accumulation were included, which was defined by a CAP value >/= 280 dB/m. Patients received 20,000 IU vitamin D/week for six months, while vitamin D status, liver function tests (LFTs), CAP and body composition were monitored. RESULTS: The cohort comprised 47.5% women (age 54.9 +/- 12.1 years; BMI 29.5 +/- 3.0 kg/m2). Mean serum vitamin D level was 11.8 +/- 4.8 ng/ml. CAP decreased significantly from baseline (330 +/- 32 vs. 307 +/- 41 dB/m) during supplementation (P = 0.007). A mean CAP reduction relative to baseline was demonstrated at four weeks and three and six months: -5.3 +/- 13.8%; -6.0 +/- 14.6% and -6.4 +/- 13.0%, respectively. During these time points, restoration of serum vitamin D levels was observed (34.6 +/- 12.9, 36.3 +/- 10.2, 34.8 +/- 9.8 ng/ml; P < 0.0001). Liver function tests and body composition remained unchanged. CONCLUSIONS: Hepatic steatosis, as assessed by CAP, significantly improves after only 4 weeks of vitamin D correction. Hepatic steatosis is a dynamic process, that can be monitored in the short-term using such non-invasive methods.

Chemotyping the distribution of vitamin D metabolites in human serum.

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the 'FDA Guidance for Industry Bioanalytical Method Validation'. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Vitamin D supplementation reduces depressive symptoms in patients with chronic liver disease.

BACKGROUND AND AIMS: Vitamin D deficiency and depression frequently occur in patients with chronic liver diseases (CLD). Depression has recently been inversely associated with vitamin D in a meta-analysis, and vitamin D receptor is expressed in brain. This pilot study investigates whether vitamin D replacement ameliorates depressive symptoms in CLD patients and consists of a cross-sectional and an interventional analysis. METHODS: Overall, 111 patients with CLD were included in the cross-sectional analysis. The Beck Depression Inventory II (BDI-II) was used to assess depression. Chemiluminescence immunoassay and LC-MS/MS quantified serum 25-hydroxyvitamin D levels. For the interventional analysis, 77 patients with inadequate vitamin D concentrations received 20,000 IU vitamin D per week for six months. The final follow-up was carried out six months post supplementation. RESULTS: In the cross-sectional analysis, 81% of patients (median age 55 years, 47% women) had inadequate baseline vitamin D levels (<30 ng/ml), and 31% presented with depressive symptoms (BDI-II score ≥14). Depression severity correlated inversely with vitamin D level in depressed patients (ß = -0.483, P = 0.004). Depression scores improved significantly from baseline in depressed patients after three and six months (P = 0.003 and P = 0.004, respectively) of supplementation, with vitamin D levels increasing to normal (P < 0.0001). Subgroup analyses revealed this anti-depressant effect of vitamin D to occur predominantly in women. The final follow-up showed increases in median BDI-II scores in the setting of decreased vitamin D levels. CONCLUSIONS: Vitamin D levels correlated with BDI-II scores, and vitamin D replacement significantly improved depressive symptoms in women with CLD. Adjuvant vitamin D may be considered in these patients. REGISTRATION NO: DRKS00007782 German Clinical Trials Registry (DRKS).

Vitamin D modulates biliary fibrosis in ABCB4-deficient mice.

PURPOSE: Impaired vitamin D receptor signaling represents an aggravating factor during liver injury, and recent studies suggest that vitamin D might exert a protective role in chronic hepatobiliary diseases. We hypothesized that vitamin D supplementation would ameliorate liver fibrosis in ATP-binding cassette transporter B4 knockout (Abcb4 (-/-)) mice as a preclinical model of sclerosing cholangitis. METHODS: Abcb4 (-/-) and wild-type mice were fed a regular chow diet (600 IU vitamin D/kg food) or diets with lower (100 IU/kg) and higher (2,400 IU/kg) vitamin D concentrations for 12 weeks. Serum 25-hydroxyvitamin D concentrations were measured by chemiluminescence immunoassays. Liver injury and biliary fibrosis were assessed by liver enzyme activities, histopathology and hepatic collagen contents. Hepatic mRNA expression of markers for fibrosis, vitamin D and bile acid metabolism were analyzed by quantitative PCR. RESULTS: Different vitamin D concentrations were observed depending on genotype and diet group, with Abcb4 (-/-) mice on the control diet showing lower vitamin D concentrations compared to wild-type mice. Abcb4 (-/-) animals on the low vitamin D diet demonstrated the most advanced liver fibrosis and highest hepatic collagen contents. Feeding Abcb4 (-/-) mice a high vitamin D diet enriched serum vitamin D levels, lowered liver enzyme activities, altered expression levels of profibrogenic genes and ameliorated, in part, liver injury. CONCLUSIONS: This is the first report to demonstrate that fibrogenesis in the established Abcb4 (-/-) model is influenced by vitamin D supplementation. Since vitamin D modulates sclerosing cholangitis in vivo, we speculate that sufficient vitamin D intake might improve liver damage and induce antifibrotic effects in chronic cholestasis in humans.

Vitamin D in chronic liver disease.

UNLABELLED: Chronic liver disease (CLD) and several related extrahepatic manifestations such as hepatic osteodystrophy are associated with deficiency of vitamin D, which has therefore been suggested as therapeutic target. Vitamin D undergoes hepatic 25-hydroxylation, rendering the liver critical to the metabolic activation of this vitamin. Vitamin D deficiency is highly prevalent in CLD patients, and vitamin D levels are inversely related to the severity of CLD. Declining levels of carrier proteins such as albumin and vitamin D-binding protein might also be critical in CLD. Intervention studies report improvements of CLD following supplementation, and benefits to health outcomes in particular with respect to hepatitis C virus infection have recently been documented. CONTENT: We discuss vitamin D sources, functions and metabolism with a focus on the inherent complications of analytical measurements, such as the interference of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D C-3 epimers. Global discrepancies in the definition of optimal serum 25-hydroxyvitamin D levels are covered, and the prevalence of vitamin D deficiency in CLD is reviewed. We also address the functional mechanisms underlying this deficiency, and refer to associations between genetic variation in vitamin D metabolism and CLD. Lastly, we consider the health implications of a vitamin D deficiency in CLD and consider therapeutic options. SUMMARY: Herein, we focus on the epidemiological and functional relationships between vitamin D deficiency and CLD, followed by a discussion of the potential implications for therapeutic interventions.