Caroline S. Stokes

Vitamin D in chronic liver disease.

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.

Analysis of vitamin D metabolic markers by mass spectrometry: Current techniques, limitations of the “gold standard” method, and anticipated future directions

Vitamin D compounds belong to a group of secosteroids, which occur naturally as vitamin D3 in mammals and D2 in plants. Vitamin D is vital for bone health but recent studies have shown a much wider role in the pathologies of diseases such as diabetes, cancer, autoimmune, neurodegenerative, mental and cardiovascular diseases. Photosynthesis of vitamin D in the human skin and subsequent hepatic and renal metabolism generate a wide range of transformation products occurring over a large dynamic range spanning from picomolar to nanomolar levels. This necessitates selective and sensitive analytical methods to quantitatively capture these low concentration levels in relevant tissues such as blood. Ideally, vitamin D assessment would be performed using a universal and standardized analytical method available to clinical laboratories that provides reliable and accurate quantitative results for all relevant vitamin D metabolites with sufficiently high throughput. At present, LC-MS/MS assays are the most promising techniques for vitamin D analysis. The present review focuses on developments in mass spectrometry methodologies of the past 12 years. It will highlight detrimental influences of the biological matrix, epimer contributions, pitfalls of specific mass spectrometry data acquisition routines (in particular multiple reaction monitoring, MRM), influence of ionization source, derivatization reactions, inter-laboratory comparisons on precision, accuracy, and application range of vitamin D metabolites.

Gallstones: Environment, Lifestyle and Genes

Gallstone disease represents one of the most common and costly gastroenterological disorders. In Germany, 0.25% of the population undergo cholecystectomy per year, and cholelithiasis incurs annual medical expenses of more than USD 6.5 billion in the United States. The paradigm of environmental risk factors for gallstones has lately been challenged by genetic studies in experimental models and humans. The analysis of more than 40,000 Swedish twin pairs with gallstones demonstrated that genetic factors account for 25% of the phenotypic variance. Since then, studies employing genome-wide association analysis, case-control cohorts and analysis of sib-pairs in families with gallstones have expanded our knowledge of ‘gallstone genes’. Indeed, gallstone disease phenotypes are likely to result from the complex interaction of genetic factors, chronic overnutrition with carbohydrates, depletion of dietary fibre and other not fully defined environmental factors including physical inactivity and infections. This hypothesis is supported by the profound increases of cholesterol gallstone prevalence rates in Native Americans, post-war European countries and current urban centres in East Asia, all of which were associated with ‘westernized’ nutrition. Herein, we summarise the spectrum of environmental and genetic risk factors which should pave the way to ‘personalised’ strategies for the prevention and therapy of gallstones.

Ursodeoxycholic Acid and Diets Higher in Fat Prevent Gallbladder Stones During Weight Loss: A Meta-analysis of Randomized Controlled Trials

BACKGROUND & AIMS The prevalence of gallstones is increasing in association with the obesity epidemic, but rapid weight loss also increases the risk of stone formation. We conducted a systematic review of the efficacy of strategies to prevent gallbladder stones in adults as they lose weight. METHODS Randomized controlled trials of nonsurgical strategies to prevent gallstones were identified by electronic and manual searches. Our final analysis included 13 trials, comprising 1836 participants undergoing weight loss through dieting (8 trials) or bariatric surgery (5 trials). The trials compared ursodeoxycholic acid (UDCA) or high-fat weight loss diets with control interventions. We performed random-effects meta-analyses and evaluated heterogeneity and bias with subgroup, sensitivity, regression, and sequential analysis. RESULTS UDCA reduced the risk of ultrasound-verified gallstones compared with control interventions (risk ratio, 0.33; 95% confidence interval [CI], 0.18-0.60; number needed to treat, 9). This effect was significantly larger in trials of diets alone (risk ratio, 0.17; 95% CI, 0.11-0.25) than in trials of patients who underwent bariatric surgery (risk ratio, 0.42; 95% CI, 0.21-0.83) (test for subgroup differences, P =.03). UDCA reduced the risk of cholecystectomy for symptomatic stones (risk ratio, 0.20; 95% CI, 0.07-0.53). Diets high in fat content also reduced gallstones, compared with those with low fat content (risk ratio, 0.09; 95% CI, 0.01-0.61). The meta-analyses were confirmed in trials with a low risk of bias but not in sequential analysis. No additional beneficial or harmful outcomes were identified. CONCLUSIONS On the basis of a meta-analysis of randomized controlled trials, during weight loss, UDCA and/or higher dietary fat content appear to prevent formation of gallstones.

Vitamin D deficiency is associated with mortality in patients with advanced liver cirrhosis

Chronic liver disease is the fifth most common cause of mortality in Europe. Recently, vitamin D deficiency has been associated with an increased risk of mortality in the general population. As patients with advanced liver disease frequently exhibit vitamin D deficiency, we assessed for a possible association of vitamin D deficiency with survival in a cohort of patients with advanced liver disease.

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.

The common PNPLA3 variant p.I148M is associated with liver fat contents as quantified by controlled attenuation parameter (CAP).

Non‐alcoholic fatty liver disease (NAFLD) is becoming the most prevalent liver disorder. The PNPLA3 (adiponutrin) variant p.I148M has been identified as common genetic modifier of NAFLD. Our aim was to assess the relationships between genetic risk and non‐invasively measured liver fat content.

HCC and liver disease risks in homozygous PNPLA3 p.I148M carriers approach monogenic inheritance.

[1] Kessler Sonja M, Barghash Ahmad, Laggai Stephan, Helms Volkhard, Kiemer Alexandra K. Hepatic hepcidin expression is decreased in cirrhosis and HCC. J Hepatol 2015;62:977–979. [2] Harrison-Findik DD, Schafer D, Klein E, Timchenko NA, Kulaksiz H, Clemens D, et al. Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression. J Biol Chem 2006;281:22974–22982. [3] Mueller K, Sunami Y, Stuetzle M, Guldiken N, Kucukoglu O, Mueller S, et al. CHOP-mediated hepcidin suppression modulates hepatic iron load. J Pathol 2013;231:532–542. [4] Meynard D, Babitt JL, Lin HY. The liver: conductor of systemic iron balance. Blood 2014;123:168–176. [5] Ramm GA, Ruddell RG. Iron homeostasis, hepatocellular injury, and fibrogenesis in hemochromatosis: the role of inflammation in a noninflammatory liver disease. Semin Liver Dis 2010;30:271–287. [6] Lunova M, Goehring C, Kuscuoglu D, Mueller K, Chen Y, Walther P, et al. Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload. J Hepatol 2014;61:633–641. [7] Valenti L, Fracanzani AL, Bugianesi E, Dongiovanni P, Galmozzi E, Vanni E, et al. HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology 2010;138: 905–912. [8] Ganz T. Iron homeostasis: fitting the puzzle pieces together. Cell Metab 2008;7:288–290. [9] Nahon P, Sutton A, Rufat P, Charnaux N, Mansouri A, Moreau R, et al. A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis. J Hepatol 2012;56:426–432. [10] Boyault S, Rickman DS, de Reyniès A, Balabaud C, Rebouissou S, Jeannot E, et al. Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology 2007;45:42–52.

A simple micro‐extraction plate assay for automated LC‐MS/MS analysis of human serum 25‐hydroxyvitamin D levels

This short application note describes a simple and automated assay for determination of 25-hydroxyvitamin D (25(OH)D) levels in very small volumes of human serum. It utilizes commercial 96-well micro-extraction plates with commercial 25(OH)D isotope calibration and quality control kits. Separation was achieved using a pentafluorophenyl liquid chromatography column followed by multiple reaction monitoring-based quantification on an electrospray triple quadrupole mass spectrometer. Emphasis was placed on providing a simple assay that can be rapidly established in non-specialized laboratories within days, without the need for laborious and time consuming sample preparation steps, advanced calibration or data acquisition routines. The analytical figures of merit obtained from this assay compared well to established assays. To demonstrate the applicability, the assay was applied to analysis of serum samples from patients with chronic liver diseases and compared to results from a routine clinical immunoassay.

Genetics of biliary lithiasis from an ethnic perspective.

Gallstone disease represents one of the most common gastroenterological disorders worldwide. Gallstones affect over 15% of adults in Europe and 25-30% of Hispanic populations in Central and South America. The heritability of gallstones varies considerably according to ethnicity, with Native Americans and Hispanics with Amerindian admixture being the most susceptible populations. Genetic factors have been shown to account for 25-30% of total gallstone risk in Europe, however, in Hispanic populations, this risk percentage may increase to 45-65%. Recent genome-wide association and candidate gene studies have identified common polymorphisms in enterohepatic transporters (ABCG5/8, SLC10A2) and the Gilbert syndrome UGT1A1 variant as genetic determinants of gallstone formation. Together, these polymorphisms cover a significant proportion of the previously predicted genetic background of gallstones in European populations. New lithogenic genes need to be discovered in future studies in high-risk populations. In this review, we address the latest developments in the genetic analysis of gallstones and discuss the ethnic background of this condition in European, Central and South American and Asian populations.