Evaluation of a commercial liquid-chromatography high-resolution mass-spectrometry method for the determination of hepcidin-25

Introduction: Reliable determination of hepcidin-25, a key regulator of iron metabolism, is important. This study aimed at evaluating the performance of the Hepcidin-25 Liquid Chromatography-Tandem Mass-Spectrometry (LC-MS/MS) Kit (Immundiagnostik AG, Bensheim, Germany) for quantification of the hepcidin-25 protein. Materials and methods: Precision, accuracy, linearity, and preanalytical requirements of the liquid-chromatography high-resolution massspectrometry (LC-HR-MS) method were evaluated. The imprecision and bias acceptance criteria were defined ≤ 15%. We investigated sample stability at room temperature (RT) and after repeated freeze and thaw cycles. Additionally, we assessed serum hepcidin-25 concentrations of 165 healthy adults referred for a medical check-up. Results: The hepcidin-25 LC-MS/MS assay was linear over the concentration range of 3 – 200 ng/mL. Within- and between-run precision ranged between 1.9 – 8.6% and 5.1 – 12.4%, respectively. The mean bias of the low and high control material was - 2.7% and 2.1%, respectively. At RT, serum samples were stable for 3 h (mean bias + 0.3%). After two and three freeze and thaw cycles, hepcidin-25 concentrations showed a bias of +8.0 and + 20%, respectively. Of 165 healthy adults, 109 females had a significantly lower median of 8.42 (range: 1.00 – 60.10) ng/mL compared to 56 males with 15.76 (range: 1.50 – 60.50) ng/mL (P = 0.002). Conclusions: The hepcidin-25 LC-MS/MS kit shows a broad analytical range and meets the imprecision and bias acceptance criteria of ≤ 15%. Serum samples can be stored at RT for 3 h and resist up to two freeze and thaw cycles

Secondary Sclerosing Cholangitis in Critically Ill Patients Alters the Gut–Liver Axis: A Case Control Study

Secondary sclerosing cholangitis in critically ill patients (SC-CIP) occurs after long-term intensive care treatment. This study aimed to assess the gut–liver axis in SC-CIP. Stool microbiome composition, gut permeability, bacterial translocation and serum bile acid profiles of 18 SC-CIP patients compared to 11 patients after critical illness without liver disease (CIP controls), 21 patients with cirrhosis and 21 healthy controls were studied. 16S rDNA was isolated from stool and sequenced using the Illumina technique. Diamine oxidase, zonulin, soluble CD14 (sCD14) and lipopolysaccharide binding protein were measured in serum and calprotectin in stool. Serum bile acids were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Reduced microbiome alpha diversity and altered beta diversity were seen in SC-CIP, CIP controls and cirrhosis compared to healthy controls. SC-CIP patients showed a shift towards pathogenic taxa and an oralization. SC-CIP, CIP controls and cirrhotic patients presented with impaired gut permeability, and biomarkers of bacterial translocation were increased in SC-CIP and cirrhosis. Total serum bile acids were elevated in SC-CIP and cirrhosis and the bile acid profile was altered in SC-CIP, CIP controls and cirrhosis. In conclusions, observed alterations of the gut–liver axis in SC-CIP cannot solely be attributed to liver disease, but may also be secondary to long-term intensive care treatment

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5–73.3% (survey 1), 56.8–60.3% (survey 2); 36.2–35.8% (survey 1), 56.6–59.8, (survey 2); 61.1–197.7% (survey 1), 47.2–74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid- chromatography–Urgent need for harmonisation of analytical methods

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs

Efficacy of docosahexaenoic acid-choline-vitamin E (DHA-CHO-VE) in paediatric NASH: a randomized controlled clinical trial

Non-alcoholic steatohepatitis (NASH), a progressive form of non-alcoholic fatty liver disease (NAFLD), is one of the most common hepatic diseases in children. We conducted a randomized controlled clinical trial on children with biopsy-proven NASH based on a combinatorial nutritional approach compared to placebo. Participants were assigned to lifestyle modification plus placebo, or lifestyle modification plus a mix containing docosahexaenoic acid, choline and vitamin E (DHA-CHO-VE). Forty children and adolescents concluded the trial. The primary outcome was the improvement of liver hyperechogenicity. Secondary outcomes included alterations of ALT and other metabolic parameters. Furthermore, changes of serum bile acids (BA) and plasma fibroblast growth factor 19 (FGF19) levels were evaluated as inverse biomarkers of disease severity. At the end of the study, we observed a significant decrease in severe steatosis in the treatment group (50% to 5%, p=0.001). Furthermore, although the anthropometric and biochemical measurements in the placebo and DHA-CHO-VE groups were comparable at baseline, at the end of the study ALT and fasting glucose levels improved only in the treatment group. Finally, we found that BA levels were not influenced whereas FGF19 levels were significantly increased by DHA-CHO-VE. The results suggest that a combination of DHA, vitamin E and choline could improve steatosis and reduce ALT and glucose levels in children with NASH. However, further studies are needed to assess the impact of a DHA and vitamin E combination on repair of liver damage in paediatric NASH.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Serum Bile Acids in Repaired Tetralogy of Fallot: A Marker for Liver and Heart?

Patients with repaired tetralogy of Fallot may develop chronic right ventricular dysfunction and hepatic congestion over time. We hypothesized that bile acid metabolism is altered in repaired tetralogy of Fallot patients and therefore sought to correlate right ventricular indices with serum bile acid levels.Indexed right ventricular end diastolic volume, as assessed by cardiac magnetic-resonance imaging, was classified as 150ml/m2 (Group 3, n = 6) in 29 patients with repaired tetralogy of Fallot. Pulmonary regurgitation fraction and right ventricular ejection fraction were calculated. The serum bile acid profile, including 15 species, in these patients was determined by liquid chromatography coupled with mass spectrometry.Serum bile acid levels increased from Group 1 to Group 3 (2.5 ± 0.7; 4.1 ± 2.5; 6.0 ± 2.8 μmol/l, respectively) with significantly increased bile acid values in Group 3 compared to Group 1 (p≤0.05). In Group 3, but not in Group 1 and 2, a significant increase in glycine-conjugated bile acids was observed. Pulmonary regurgitation fraction increased (12 ± 1; 28 ± 16; 43 ± 3%, Groups 1-3, respectively) and right ventricular ejection fraction decreased (48.4 ± 6.4; 48.5 ± 6.5; 42.1 ± 5.3%, Groups 1-3, respectively) with rising indexed right ventricular end diastolic volume.These preliminary results suggest that serum bile acid levels are positively correlated with indexed right ventricular end-diastolic volume in patients with repaired tetralogy of Fallot; however, this needs to be confirmed in a larger patient cohort

Serum Bile Acid Levels in Children with Nonalcoholic Fatty Liver Disease

Objective: Because the prevalence of obesity in children is increasing, the frequency of pediatric nonalcoholic fatty liver disease (NAFLD) is growing. A reliable noninvasive biomarker for monitoring progression of liver fibrosis would be useful. In cirrhotic persons serum bile acid (BA) levels are significantly elevated. We hypothesized that BA levels and composition in pediatric NAFLD vary depending on the stage of fibrosis. Methods: Children with NAFLD were compared with controls and classified by stages of fibrosis (NAFLD-F0, n = 27; NAFLD-F≥1, n = 65) based on liver-biopsy findings. Fasted metabolic and cholestasis status was assessed by several blood tests. BA profiles were measured by tandem mass spectrometry and compared with healthy controls (n = 105). Results: Compared with controls, all of the NAFLD patients were overweight and showed significantly elevated glucose, insulin, aspartate transaminase, and alanine transaminase levels. Total serum BAs were lower in nonfibrotic NAFLD children than in a control cohort (1.73 vs 3.6 μmol/L) because low glycine-conjugated BA levels were incompletely compensated by increases in taurine-conjugated or unconjugated BA. In patients with fibrotic NAFLD, BA levels were lower than in controls (2.45 vs 3.6 μmol/L) but higher than in nonfibrotic patients (2.45 vs 1.73 μmol/L), and the BA pattern resembled that of healthy controls. Fibroblast growth factor 19 levels were significantly lower in both NAFLD groups than in controls (P ≤ 0.001) and were positively correlated with ursodeoxycholic acid levels. Conclusions: Our data indicate that serum BA levels decrease in early NAFLD and increase during progression to fibrosis. Given that BA levels are increased in cirrhotic adults, we postulate a continuous rise as NAFLD advances. BA may have a value as a noninvasive biomarker in pediatric NAFLD progression. © 2015 by ESPGHAN and NASPGHAN