Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

Liver phenotypes in PCOS: Analysis of exogenous and inherited risk factors for liver injury in two European cohorts.

BACKGROUND & AIMS: Fatty liver disease (FLD) is common in women with polycystic ovary syndrome (PCOS). Here, we use non-invasive tests to quantify liver injury in women with PCOS and analyse whether FLD-associated genetic variants contribute to liver phenotypes in PCOS. METHODS: Prospectively, we recruited women with PCOS and controls at two university centres in Germany and Poland. Alcohol abuse was regarded as an exclusion criterion. Genotyping of variants associated with FLD was performed using TaqMan assays. Liver stiffness measurements (LSM), controlled attenuation parameters (CAP) and non-invasive HSI, FLI, FIB-4 scores were determined to assess hepatic steatosis and fibrosis. RESULTS: A total of 42 German (age range 18-53 years) and 143 Polish (age range 18-40 years) women with PCOS, as well as 245 German and 289 Polish controls were recruited. In contrast to Polish patients, Germans were older, presented with more severe metabolic profiles and had significantly higher LSM (median 5.9 kPa vs. 3.8 kPa). In the German cohort, carriers of the PNPLA3 p.I148M risk variant had an increased LSM (p = .01). In the Polish cohort, the minor MTARC1 allele was linked with significantly lower serum aminotransferases activities, whereas the HSD17B13 polymorphism was associated with lower concentrations of 17-OH progesterone, total testosterone, and androstenedione (all p < .05). CONCLUSIONS: FLD is common in women with PCOS. Its extent is modulated by both genetic and metabolic risk factors. Genotyping of variants associated with FLD might help to stratify the risk of liver disease progression in women suffering from PCOS.

Antidepressant effects of direct-acting antivirals against hepatitis C virus-Results from a pilot study.

BACKGROUND AND AIMS: The new direct-acting antiviral agents (DAA) have revolutionized the treatment of patients with chronic hepatitis C virus (HCV) infection. This study investigates to which extent DAA affect fatigue and mood and, if so, whether this results from changes to tryptophan (TRP) metabolism, as reflected by two critical biosynthetic pathways, serotonin (SRT) generation from TRP and TRP degradation through kynurenines (KYN) via indoleamine 2,3-dioxygenase (IDO). METHODS: This study assessed 24 patients with chronic HCV infection, before (T1), during (T2: at 4 weeks) and 12 weeks post-treatment with DAA (T3) with respect to viral load, fatigue and depressive symptoms (BDI-II questionnaire), physical activity (actigraph) and plasma serotonin-tryptophan metabolites (LC/MS). The KYN:TRP ratio reflected IDO activity. RESULTS: All participants achieved sustained virological response (SVR12) with DAA treatment (79% sofosbuvir-based). Fatigue (scores at T1:0.83 ± 0.70, T2:0.48 ± 0.70, T3:0.30 ± 0.50; P = 0.023) and depressive symptoms (scores at T1:9.8 ± 10.2, T2:6.0 ± 7.3, T3:5.0 ± 7.6; P = 0.005) improved significantly on therapy, whereas no changes were noted in five untreated controls. TRP plasma concentrations markedly decreased (T1:306 ± 179 mg/L, T2:283 ± 84 mg/L), whereas 5-HTP levels increased (T1:0.08 ± 0.01 mg/L, T2:0.10 ± 0.06 mg/L). KYN concentrations (T1:2.4 ± 2.0 mg/L, T2:3.7 ± 1.4 mg/L, P = 0.003) increased significantly during treatment, as did IDO activity (T1:0.008 ± 0.006 mg/L, T2:0.014 ± 0.004 mg/L; P < 0.001). CONCLUSIONS: In this study, DAA exert positive and persistent effects on both fatigue and mood in patients with chronic HCV infection. These extrahepatic benefits are, at least in part, related to the modulation of TRP metabolism. The robust elevation of KYN concentrations challenges the current paradigm of low KYN levels as prerequisite for mental health.

Effects of Gene Variants Controlling Vitamin D Metabolism and Serum Levels on Hepatic Steatosis.

BACKGROUND/AIMS: Common genetic variations in vitamin D metabolism are associated with liver stiffness. Whether these genes are implicated in hepatic steatosis remains unclear. Here we aimed to analyse the association of common vitamin D pathway gene variants with liver steatosis. METHODS: Liver steatosis was assessed non-invasively in 241 patients with chronic liver conditions by controlled attenuation parameter (CAP). The following polymorphisms were genotyped using TaqMan assays: group-specific component (GC) rs7041, 7-dehydrocholesterol reductase (DHCR7) rs12785878, cytochrome P450 2R1 (CYP2R1) rs10741657, -vitamin D receptor (VDR) rs7974353. Chemiluminescence immunoassay determined serum 25-hydroxyvitamin D (25(OH) D) concentrations. RESULTS: Vitamin D deficiency (defined by 25(OH)D concentrations <20 ng/mL) occurred in 66% of patients. Median CAP was 296 (100-400) dB/m. Patients with advanced steatosis (CAP ≥280 dB/m) had significantly (p = 0.033) lower 25(OH)D levels as compared to patients with CAP <280 dB/m. Moreover, the rare allele [T] in GC rs7041 was significantly (p = 0.018) associated with higher 25(OH)D levels in patients with CAP <280 dB/m. However, GC, DHCR7, CYP2R1, and VDR polymorphisms were not related to liver steatosis and obesity traits. CONCLUSIONS: Higher CAP values are associated with low serum 25(OH)D concentrations but not with common vitamin D pathway gene variants.

L-ornithine L-aspartate for prevention and treatment of hepatic encephalopathy in people with cirrhosis.

BACKGROUND: Hepatic encephalopathy is a common complication of cirrhosis and has high associated morbidity and mortality. The condition is classified as overt if it is clinically apparent or minimal if only evident though psychometric testing. The exact pathogenesis of this syndrome is unknown although ammonia is thought to play a key role. L-ornithine L-aspartate has ammonia-lowering properties and may, therefore, benefit people with cirrhosis and hepatic encephalopathy. OBJECTIVES: To evaluate the beneficial and harmful effects of L-ornithine L-aspartate versus placebo, no intervention, or other active interventions in people with cirrhosis and hepatic encephalopathy. SEARCH METHODS: We undertook electronic searches of The Cochrane Hepato-Biliary Group Controlled Trials Register, CENTRAL, MEDLINE, Embase, LILACS and Science Citation Index Expanded to December 2017 and manual searches of meetings and conference proceedings; checks of bibliographies; and corresponded with investigators and pharmaceutical companies. SELECTION CRITERIA: We included randomised clinical trials, irrespective of publication status, language, or blinding. We included participants with cirrhosis who had minimal or overt hepatic encephalopathy or who were at risk for developing hepatic encephalopathy. We compared: L-ornithine L-aspartate versus placebo or no intervention; and L-ornithine L-aspartate versus other active agents such as non-absorbable disaccharides, antibiotics, probiotics, or branched-chain amino acids. DATA COLLECTION AND ANALYSIS: Two review authors, working independently, retrieved data from published reports and correspondence with investigators and pharmaceutical companies. The primary outcomes were mortality, hepatic encephalopathy, and serious adverse events. We undertook meta-analyses and presented the results as risk ratios (RR) and mean differences (MD) with 95% confidence intervals (CI). We assessed bias control using the Cochrane Hepato-Biliary Group domains; we evaluated the risk of publication bias and other small trial effects in regression analyses; conducted subgroup and sensitivity analyses; and performed Trial Sequential Analyses. We determined the quality of the evidence using GRADE. MAIN RESULTS: We identified 36 randomised clinical trials, involving at least 2377 registered participants, which fulfilled our inclusion criteria including 10 unpublished randomised clinical trials. However, we were only able to access outcome data from 29 trials involving 1891 participants. Five of the included trials assessed prevention, while 31 trials assessed treatment. Five trials were at low risk of bias in the overall assessment of mortality; one trial was at low risk of bias in the assessment of the remaining outcomes.L-ornithine L-aspartate had a beneficial effect on mortality compared with placebo or no intervention when including all trials (RR 0.42, 95% CI 0.24 to 0.72; I2 = 0%; 19 trials; 1489 participants; very low quality evidence), but not when the analysis was restricted to the trials at low risk of bias (RR 0.47, 95% CI 0.06 to 3.58; 4 trials; 244 participants). It had a beneficial effect on hepatic encephalopathy compared with placebo or no intervention when including all trials (RR 0.70, 95% CI 0.59 to 0.83; 22 trials; 1375 participants; I2 = 62%; very low quality evidence), but not in the one trial at low risk of bias (RR 0.96, 95% CI 0.85 to 1.07; 63 participants). The analysis of serious adverse events showed a potential benefit of L-ornithine L-aspartate when including all randomised clinical trials (RR 0.63, 95% CI 0.45 to 0.90; 1 trial; 1489 participants; I2 = 0%; very low quality evidence), but not in the one trial at low risk of bias for this outcome (RR 0.83, 95% CI 0.15 to 4.65; 63 participants). The Trial Sequential Analyses of mortality, hepatic encephalopathy, and serious adverse events found insufficient evidence to support or refute beneficial effects. Subgroup analyses showed no difference in outcomes in the trials evaluating evaluating the prevention or treatment of either overt or minimal hepatic encephalopathy or trials evaluating oral versus intravenous administration We were unable to undertake a meta-analysis of the three trials involving 288 participants evaluating health-related quality of life. Overall, we found no difference between L-ornithine L-aspartate and placebo or no intervention in non-serious adverse events (RR 1.15, 95% CI 0.75 to 1.77; 14 trials; 1076 participants; I2 = 40%). In comparison with lactulose, L-ornithine L-aspartate had no effect on mortality (RR 0.68, 95% CI 0.11 to 4.17; 4 trials; 175 participants; I2 = 0%); hepatic encephalopathy (RR 1.13, 95% CI 0.81 to 1.57); serious adverse events (RR 0.69, 95% CI 0.22 to 2.11); or non-serious adverse events (RR 0.05, 95% CI 0.01 to 0.18). In comparison with probiotics, L-ornithine L-aspartate had no effect on mortality (RR 1.01, 95% CI 0.11 to 9.51); serious adverse events (RR 1.07, 95% CI 0.23 to 4.88); or changes in blood ammonia concentrations from baseline (RR -2.30 95% CI -6.08 to 1.48), but it had a possible beneficial effect on hepatic encephalopathy (RR 0.71, 95% CI 0.56 to 0.90). Finally, in comparison with rifaximin, L-ornithine L-aspartate had no effect on mortality (RR 0.33, 95% CI 0.04 to 3.03; 2 trials; 105 participants); hepatic encephalopathy (RR 1.06, 95% CI 0.57 to 1.96); serious adverse events (RR 0.32, 95% CI 0.01 to 7.42), or non-serious adverse events (RR 0.32, 95% CI 0.01 to 7.42). AUTHORS' CONCLUSIONS: The results of this review suggest a possible beneficial effect of L-ornithine L-aspartate on mortality, hepatic encephalopathy, and serious adverse events in comparisons with placebo or no-intervention, but, because the quality of the evidence is very low, we are very uncertain about these findings. There was very low quality evidence of a possible beneficial effect of L-ornithine L-aspartate on hepatic encephalopathy, when compared with probiotics, but no other benefits were demonstrated in comparison with other active agents. Additional access to data from completed, but unpublished trials, and new randomised placebo-controlled, double-blind clinical trials are needed.

[Updated S3-Guideline for Prophylaxis, Diagnosis and Treatment of Gallstones. German Society for Digestive and Metabolic Diseases (DGVS) and German Society for Surgery of the Alimentary Tract (DGAV) - AWMF Registry 021/008]. / Aktualisierte S3-Leitlinie der Deutschen Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten (DGVS) und der Deutschen Gesellschaft für Allgemein- und Viszeralchirurgie (DGAV) zur Prävention, Diagnostik und Behandlung von Gallensteinen.

This guideline provides evidence-based key recommendations for the prevention, diagnosis and therapy of gallstones and upgrades the 2007 version. The guideline was developed by an interdisciplinary team of gastroenterologists and surgeons, and patient support groups under the auspice of the German Society for Gastroenterology and Metabolic Diseases and the German Society for General Surgery and Surgery of the Alimentary Tract. The guideline used structural S3 consensus-based methodology and includes statements on clinical practice, medical education, prevention, quality assurance, outcome analysis, and integration of outpatient and inpatient care for patients with gallstone diseases.

Design and validation of a German version of the GSRS-IBS - an analysis of its psychometric quality and factorial structure.

BACKGROUND: Currently, a suitable questionnaire in German language is not available to monitor the progression and evaluate the severity of irritable bowel syndrome (IBS). Therefore, this study aimed to translate the Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS) into German and to evaluate its psychometric qualities and factorial structure. METHODS: This study is based on a total sample of 372 participants [62.6% female, mean age = 41 years (SD = 17 years)]. 17.5% of the participants had a diagnosis of IBS, 19.9% were receiving treatment for chronic inflammatory bowel disease, 12.1% of the participants were recruited from a psychosomatic clinic, and 50.5% belonged to a control group. All participants completed the German version of GSRS-IBS (called Reizdarm-Fragebogen, RDF), as well as the Gießen Subjective Complaints List (GBB-24) and the Hospital Anxiety and Depression Scale - German version (HADS-D). RESULTS: The internal consistency of the RDF total scale was at least satisfactory in all subsamples (Cronbach's Alpha between .77 and .92), and for all subscales (Cronbach's Alpha between .79 and .91). The item difficulties (between .25 and .73) and the item-total correlations (between .48 and .83) were equally satisfactory. Principal axis analysis revealed a four-factorial structure of the RDF items, which mainly resembled the structure of the English original. Convergent validity was established based on substantial and significant correlations with the stomach-complaint scale of the GBB-24 (r = .71; p < .01) and the anxiety (r = .42; p < .01) and depression scales (r = .43; p < .01) of the HADS-D. CONCLUSION: The German version of the GSRS-IBS RDF proves to be an effective, reliable, and valid questionnaire for the assessment of symptom severity in IBS, which can be used in clinical practice as well as in clinical studies.

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.

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

BACKGROUND: 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. METHODS: Hepatic steatosis was quantified by transient elastography, using the controlled attenuation parameter (CAP) in 174 patients with chronic liver diseases (50% women, age 18-77 years). In addition, a cohort of 174 gender-matched healthy controls (50% women, age 32-77 years) was recruited. The PNPLA3 mutation as well as the novel NAFLD-predisposing genetic variant (TM6SF2 p.E167K) were genotyped with allele-specific probes. RESULTS: The PNPLA3 genotype correlated significantly (P = 0.001) with hepatic CAP measurements. The p.148M risk allele increased the odds of developing liver steatosis (OR = 2.39, P = 0.023). In multivariate models, BMI and PNPLA3 mutation were both independently associated with CAP values (P < 0.001 and P = 0.007, respectively). Carriers of the TM6SF2 risk allele presented with increased aminotransferase activities (ALT: P = 0.007, AST: P = 0.004), but the presence of this variant did not affect CAP values. CONCLUSIONS: The PNPLA3 p.I148M variant represents the most important prosteatotic genetic risk factor. NAFLD carriers of this variant should be followed up carefully, with elastography and CAP being ideally suited for this purpose.

How to prepare a manuscript fit-for-purpose for submission and avoid getting a 'desk-reject'.

First impressions are very important and, when these are negative, they can adversely affect a manuscript's journey through the scientific publication system. This short guide highlights some crucial factors to take into consideration before submitting a manuscript for review in a scientific journal. The aim is to advise authors on the best way to present their research, to comply with formal requirements of a journal and to optimize the first impression made. Copyright © 2016 John Wiley & Sons, Ltd.

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.

BACKGROUND: 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. METHODS: Sixty-five patients with liver cirrhosis (median age, 58 years; range, 19-76 years; 66% male; Child-Pugh stage C, 46%) were included in our prospective single-centre survival study. Serum 25-hydroxyvitamin D concentrations were measured by chemiluminescence immunoassay. The optimal cut-off was determined using receiver operating characteristic (ROC) and Kaplan-Meier analysis. Chi-square statistics and multivariate binary logistic regression analysis were also conducted. RESULTS: Median serum vitamin D levels were 8·2 ng/mL (range <4·0-95·8 ng/mL). Overall, 48% of patients (31/65) died during a 24-month follow-up period. ROC analysis determined a vitamin D level of 6·0 ng/mL as optimal cut-off for discriminating survivors from nonsurvivors. Kaplan-Meier analysis of survival confirmed low vitamin D levels as significant predictor of death (P = 0·012). Finally, multivariate analysis identified low vitamin D levels (OR = 6·3; 95% CI, 1·2-31·2; P = 0·012) and MELD scores (OR = 1·4; 95% CI, 1·2-1·7; P < 0·001) as independent predictors of survival. CONCLUSION: Low vitamin D levels are associated with increased mortality in patients with advanced liver disease. Thus, serum levels of vitamin D might represent a critical marker of survival in advanced liver cirrhosis.

An Overview of Different Vitamin D Compounds in the Setting of Adiposity.

A large body of research shows an association between higher body weight and low vitamin D status, as assessed using serum 25-hydroxyvitamin D concentrations. Vitamin D can be metabolised in adipose tissue and has been reported to influence gene expression and modulate inflammation and adipose tissue metabolism in vitro. However, the exact metabolism of vitamin D in adipose tissue is currently unknown. White adipose tissue expresses the vitamin D receptor and hydroxylase enzymes, substantially involved in vitamin D metabolism and efficacy. The distribution and concentrations of the generated vitamin D compounds in adipose tissue, however, are largely unknown. Closing this knowledge gap could help to understand whether the different vitamin D compounds have specific health effects in the setting of adiposity. This review summarises the current evidence for a role of vitamin D in adipose tissue and discusses options to accurately measure vitamin D compounds in adipose tissue using liquid chromatography tandem mass spectrometry (LC/MS-MS).

Genetics and treatment of bile duct stones: new approaches.

PURPOSE OF REVIEW: The global burden of gallstones is increasing. Although the gallbladder is the most common site for gallstone formation, 10-25% of patients display concurrent gallbladder and bile duct stones. Secondary stones are differentiated from primary stones that develop de novo in the biliary tree. Overall, the natural history of bile duct stones is less well defined and their diagnosis and treatment are more complex as compared to gallbladder stones. RECENT FINDINGS: Elevated liver function tests are not always reflective of bile duct stones, and noninvasive diagnosis by endoscopic ultrasound or MRI should be pursued in ambiguous cases. For treatment, recent studies report endoscopic dilation to result in similar clearance but lower complication and recurrence rates as with sphincterotomy. Pharmacological adjuvants such as ursodeoxycholic acid with sphincterotomy and stenting have been suggested for elderly patients. Indication and timing of cholecystectcomy after endoscopic treatment of bile duct stones is critical, and early cholecystectomy within 3-7 days prevents recurrent biliary events. SUMMARY: In this review we address the pathophysiology of bile duct stones and present the latest developments in the diagnosis and treatment of this challenging condition, with a consideration of stone recurrence.

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.

Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization.

In this study, we report a one-pot double derivatization scheme, which used acetylation after a Diels-Alder reaction using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to improve separation efficiency and provide baseline separations of the five vitamin D metabolites 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 3ß-25-hydroxyvitamin D3 (3ß-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3) and vitamin D3 on a C-18 stationary phase. Vitamin D metabolites are often very challenging to measure quantitatively using mass spectrometry, due to their low serum concentration levels and low ionization efficiencies. Moreover, some of these species are isomers with virtually identical mass spectral dissociation behavior. To overcome the low ionization efficiency and unspecific fragmentation behavior, derivatization using Diels-Alder reactions with Cookson-type reagents such as PTAD are common. These derivatization reactions generally result in more complicated liquid chromatography separations, because both 6R- and 6S-isomers are formed during Diels-Alder reactions. It has been shown that separations have been particularly challenging for the 3α-25(OH)D3 and 3ß-25(OH)D3 epimers. Here, we optimized the PTAD derivatization and the esterification using acetic anhydride. By utilizing the esterification catalyst 4-dimethylaminopyridine, we avoided quenching and evaporation between the two derivatization steps, but were also able to perform the esterification at room temperature without heating. The optimized one-pot double derivatization LC-MS/MS assay was validated with respect to inter/intra-day precision, accuracy, recovery and linear dynamic range and applied to metabolic fingerprinting of vitamin D3 metabolites in serum samples. The metabolites 3α-25(OH)D3, 3ß-25(OH)D3 and 24,25(OH)2D3, were readily quantified in all investigated samples. The method was, in principle, also fit for purpose for quantification of the native vitamin D3 species; the relatively high blank concentration of the commercial vitamin D-depleted serum used for calibration, however, limited the limits of quantification for this metabolite. The method provided insufficient limits of quantification for serum levels of 1,25(OH)2D3.

Transporters in cholelithiasis.

Gallstones are a common and costly disease with a projected increase in prevalence due to the increasing ageing population. Numerous endogenous and environmental factors are aetiologically related to this multifactorial disease, and genetic studies continue to unravel the pathobiological mechanisms related to gallstone formation. In particular, variants of genes encoding hepatobiliary transporters have been implicated in gallstone disease and, given their ability to influence biliary lipid composition, have undergone considerable investigation. Here we summarize the role of enterohepatic transporters in cholelithogenesis with a particular focus on pertinent ATP-binding cassette transporters (ABCB4, ABCB11, ABCC7, and ABCG5/G8).

Analytical considerations for accurately capturing the relevant species contributing to vitamin D status in liquid chromatography-tandem mass spectrometry assays.

This tutorial review focuses on analytical challenges encountered with the liquid chromatography-tandem mass spectrometry determination of 25-hydroxyvitamin D, which is currently still considered the metabolite that is most representative of vitamin D status. It describes how multiple binding states of circulating 25-hydroxyvitamin D (phase II metabolites, epimers, free/bioavailable/protein-bound species) can influence the accuracy of the analytical determination. It also summarizes important chemical species that can inadvertently contribute to vitamin D status and thus cause systematic errors. These interfering endogenous and exogenous compounds might be isomers of vitamin D, constitutional isomers or isobars and the article outlines techniques to eliminate or minimize these interferences, including chromatographic separations, ion mobility spectrometry, and high-resolution mass spectrometry.

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.