Search

Intake of Western diet stimulates bile acid (BA) production and their conversion to secondary BAs by 7α-dehydroxylating (7αDH+) bacteria in the colon. Increased levels of 7αDH+ bacteria and the secondary BA deoxycholic acid (DCA) have been associated with sporadic colorectal cancer (CRC). However, causal proof of their tumour-promoting effects under detrimental dietary conditions is lacking. Therefore, we performed feeding studies in a transgenic pig model of CRC combined with multi-omics analyses and gnotobiotic mouse studies. Western diet worsened the disease phenotype inAPC1311/+pigs. This was accompanied by increased levels of DCA and colonic epithelial cell proliferation, which was counteracted by using the bile acid-scavenging drug colestyramine. Using a gnotobiotic mouse model of CRC, we demonstrate that colonization with different 7αDH+ bacterial isolates increased colonic tumour loads. These complementary approaches present clear evidence for the causal role of microbiome-derived DCA production in CRC, opening avenues for future preventive strategies.

Oxysterol-binding protein (OSBP) is a cholesterol/PI4P exchanger at contacts of the endoplasmic reticulum (ER) with trans-Golgi network (TGN) and endosomes. Several central endothelial cell (EC) functions depend on adequate cholesterol distribution in cellular membranes. Here we elucidated the effects of pharmacologic OSBP inhibition on the lipidome and transcriptome of human umbilical vein endothelial cells (HUVECs). OSBP was inhibited for 24 h with 25 nM Schweinfurthin G (SWG) or Orsaponin (OSW-1), followed by analyses of cellular cholesterol, 27-hydroxy-cholesterol, and triacylglycerol concentration, phosphatidylserine synthesis rate, the lipidome, as well as lipid droplet staining and western analysis of OSBP protein. Next-generation RNA sequencing of the SWG-treated and control HUVECs and angiogenesis assays were performed. Protein-normalized lipidomes of the inhibitor-treated cells revealed decreases in glycerophospholipids, the most pronounced effect being on phosphatidylserines and the rate of their synthesis, as well as increases in cholesteryl esters, triacylglycerols and lipid droplet number. Transcriptome analysis of SWG-treated cells suggested ER stress responses apparently caused by disturbed cholesterol exit from the ER, as indicated by suppression of cholesterol biosynthetic genes. OSBP was associated with the TGN in the absence of inhibitors and disappeared therefrom in inhibitor-treated cells in a time-dependent manner, coinciding with OSBP reduction on western blots. Prolonged treatment with SWG or OSW-1 inhibited angiogenesis in vitro. To conclude, inhibition of OSBP in primary endothelial cells induced multiple effects on the lipidome, transcriptome changes suggesting ER stress, and disruption of in vitro angiogenic capacity. Thus, OSBP is a crucial regulator of EC lipid homeostasis and angiogenic capacity.

Background and purpose The analysis of human faecal metabolites can provide an insight into metabolic interactions between gut microbiota and the host organism. The creation of metabolic profiles in faeces has received little attention until now, and reference values, especially in the context of dietary and therapeutic interventions, are missing. Exposure to xenobiotics significantly affects the physiology of the microbiome, and microbiota manipulation and short-chain fatty acid administration have been proposed as treatment targets for several diseases. The aim of the present study is to give concomitant concentration ranges of faecal sterol species, bile acids and short-chain fatty acids, based on a large cohort. Experimental approach Sterol species, bile acids and short-chain fatty acids in human faeces from 165 study participants were quantified by LC-MS/MS. For standardization, we refer all values to dry weight of faeces. Based on the individual intestinal sterol conversion, we classified participants into low and high converters according to their coprostanol/cholesterol ratio. Key results Low converters excrete more straight-chain fatty acids and bile acids than high converters; 5th and 95th percentile and median of bile acids and short-chain fatty acids were calculated for both groups. Conclusion and implications We give concentration ranges for 16 faecal metabolites that can serve as reference values. Patient stratification into high or low sterol converter groups is associated with significant differences in faecal metabolites with biological activities. Such stratification should then allow better assessment of faecal metabolites before therapeutic interventions. Linked articles This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Chronic inflammation is now a well-known precursor for cancer development. Infectious prostatitis are the most common causes of prostate inflammation, but emerging evidence points the role of metabolic disorders as a potential source of cancer-related inflammation. Although the widely used treatment for prostate cancer based on androgen deprivation therapy (ADT) effectively decreases tumor size, it also causes profound alterations in immune tumor microenvironment within the prostate. Here, we demonstrate that prostates of a mouse model invalidated for nuclear receptors liver X receptors (LXRs), crucial lipid metabolism and inflammation integrators, respond in an unexpected way to androgen deprivation. Indeed, we observed profound alterations in immune cells composition, which was associated with chronic inflammation of the prostate. This was explained by the recruitment of phagocytosis-deficient macrophages leading to aberrant hyporesponse to castration. This phenotypic alteration was sufficient to allow prostatic neoplasia. Altogether, these data suggest that ADT and inflammation resulting from metabolic alterations interact to promote aberrant proliferation of epithelial prostate cells and development of neoplasia. This raises the question of the benefit of ADT for patients with metabolic disorders., Mice lacking the liver X nuclear receptors (LXRs), crucial integrators of lipid metabolism, were used to study the response of the prostate to androgen deprivation. This reveals that lack of androgens leads to chronic inflammation due to impaired clearance of castration-induced apoptotic cells, allowing production of pro-inflammatory cytokines and promoting prostate neoplasia.

Background & aims: Gut microbiota composition is linked to obesity and metabolic syndrome. The nutrients and doses required to modulate the gut microbiota towards beneficially influence components of the metabolic syndrome are unclear. This study aimed to investigate diet-induced effects on the gut microbiota and metabolic markers in overweight individuals with indices of the metabolic syndrome. Methods: A twelve-week randomized cross-over trial was conducted with two intervention periods separated by a washout period. The dietary intakes of interest were wheat bran extract, rich in arabinoxylan oligosaccharides (AXOS) (10.4 g/d AXOS) and polyunsaturated fatty acids (PUFA) (3.6 g/d n-3 PUFA). Dietary records, fecal and blood samples, as well as anthropometric data, were collected before and after intervention. Anthropometry and gastrointestinal symptoms were evaluated weekly. Gut microbiota composition was analyzed by massive sequencing of 16S ribosomal RNA gene V3V4 amplicons. Results: Twenty-seven participants completed the study (90%). Intake of AXOS induced an expected bifidogenic effect on gut microbiota (p < 0.01) and increased butyrate-producing bacterial species as well (p < 0.05). Beta-diversity analysis indicated that the structure of the gut microbiota only changed as a result of the AXOS intervention (Permanova = 1.90, p < 0.02) and no changes in metabolic markers were observed after any of the interventions. Conclusions: AXOS intake has a bifidogenic effect and also increases butyrate producers in the gut microbiota; even though this type of dietary fiber did not modulate lipid or glucose metabolic parameters related to metabolic syndrome. Four-week PUFA intake did not induce any notable effect on the gut microbiota composition or metabolic risk markers., This work was supported by the European Union's Seventh Framework Program [grant agreement no 613,979 (MyNewGut)]. Additional, funding to LK includes PhD grant from the Department of Nutrition, Exercise and Sports, SCIENCE, University of Copenhagen and support from the Innovative Medicines Initiative Joint Undertaking under EMIF [grant agreement n° 115,372], resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007–2013) and EFPIA companies' in kind contribution.

There has been an increasing interest during recent years in the role of the gut microbiome on health and disease. Therefore, metabolites in human feces related to microbial activity are attractive surrogate marker to track changes of microbiota induced by diet or disease. Such markers include 5α/β-stanols as microbiome-derived metabolites of sterols. Currently, reliable, robust, and fast methods to quantify fecal sterols and their related metabolites are missing. We developed a liquid chromatography-high-resolution mass spectrometry (LC-MS/HRMS) method for the quantification of sterols and their 5α/β-stanols in human fecal samples. Fecal sterols were extracted and derivatized to N, N-dimethylglycine esters. The method includes cholesterol, coprostanol, cholestanol and sitosterol, 5α/β-sitostanol, campesterol and 5α/β-campestanol. Application of a biphenyl column permits separation of isomeric 5α- and 5β-stanols. Sterols are detected in parallel reaction monitoring (PRM) mode and stanols in full scan mode. HRMS allows differentiation of isobaric β-stanols and the [M + 2] isotope peak of the coeluting sterol. Performance characteristics meet the criteria recommended by Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. Analysis of fecal samples from healthy volunteers revealed high interindividual variability of sterol and stanol fractions. Interestingly, cholesterol and sitosterol showed similar fractions of mainly 5β-stanols. In contrast, campesterol is substantially converted to 5α-campestanol and might be a poorer substrate for bacterial metabolism. Robust and fast quantification of fecal sterols and their related stanols by LC-MS/HRMS offers great potential to find novel microbiome-related biomarker in large-scale studies.

Abstract Bile acids (BA) are potent metabolic regulators influenced by diet. We studied effects of isoenergetic increases in the dietary protein and cereal-fiber contents on circulating BA and insulin resistance (IR) in overweight and obese adults. Randomized controlled nutritional intervention (18 weeks) in 72 non-diabetic participants (overweight/obese: 29/43) with at least one further metabolic risk factor. Participants were group-matched and allocated to four isoenergetic supplemented diets: control; high cereal fiber (HCF); high-protein (HP); or moderately increased cereal fiber and protein (MIX). Whole-body IR and insulin-mediated suppression of hepatic endogenous glucose production were measured using euglycaemic–hyperinsulinemic clamps with [6-62H2] glucose infusion. Circulating BA, metabolic biomarkers, and IR were measured at 0, 6, and 18 weeks. Under isoenergetic conditions, HP-intake worsened IR in obese participants after 6 weeks (M-value: 3.77 ± 0.58 vs. 3.07 ± 0.44 mg/kg/min, p = 0.038), with partial improvement back to baseline levels after 18 weeks (3.25 ± 0.45 mg/kg/min, p = 0.089). No deleterious effects of HP-intake on IR were observed in overweight participants. HCF-diet improved IR in overweight participants after 6 weeks (M-value 4.25 ± 0.35 vs. 4.81 ± 0.31 mg/kg/min, p = 0.016), but did not influence IR in obese participants. Control and MIX diets did not influence IR. HP-induced, but not HCF-induced changes in IR strongly correlated with changes of BA profiles. MIX-diet significantly increased most BA at 18 weeks in obese, but not in overweight participants. BA remained unchanged in controls. Pooled BA concentrations correlated with fasting fibroblast growth factor-19 (FGF-19) plasma levels (r = 0.37; p = 0.003). Higher milk protein intake was the only significant dietary predictor for raised total and primary BA in regression analyses (total BA, p = 0.017; primary BA, p = 0.011). Combined increased intake of dietary protein and cereal fibers markedly increased serum BA concentrations in obese, but not in overweight participants. Possible mechanisms explaining this effect may include compensatory increases of the BA pool in the insulin resistant, obese state; or defective BA transport.

A limited specificity is inherent to immunoassays for steroid hormone analysis. To improve selectivity mass spectrometric analysis of steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been introduced in the clinical laboratory over the past years usually with low mass resolution triple-quadrupole instruments or more recently by high resolution mass spectrometry (HR-MS). Here we introduce liquid chromatography-high resolution tandem mass spectrometry (LC-MS/HR-MS) to further increase selectivity of steroid hormone quantification. Application of HR-MS demonstrates an enhanced selectivity compared to low mass resolution. Separation of isobaric interferences reduces background noise and avoids overestimation. Samples were prepared by automated liquid-liquid extraction with MTBE. The LC-MS/HR-MS method using a quadrupole-Orbitrap analyzer includes eight steroid hormones i.e. androstenedione, corticosterone, cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, progesterone, and testosterone. It has a run-time of 5.3min and was validated according to the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines. For most of the analytes coefficient of variation were 10% or lower and LOQs were determined significantly below 1ng/ml. Full product ion spectra including accurate masses substantiate compound identification by matching their masses and ratios with authentic standards. In summary, quantification of steroid hormones by LC-MS/HR-MS is applicable for clinical diagnostics and holds also promise for highly selective quantification of other small molecules.

Stored platelet concentrates (PLCs) for therapeutic purpose, develop a platelet storage lesion (PSL), characterized by impaired platelet (PLT) viability and function, platelet extracellular vesicle (PL-EV) release and profound lipidomic changes. Whereas oxidized low-density lipoprotein (oxLDL) activates PLTs and promotes atherosclerosis, effects linked to oxidized high-density lipoprotein (oxHDL) are poorly characterized. PLCs from blood donors were treated with native (nHDL) or mildly oxidized HDL (moxHDL) for 5days under blood banking conditions. Flow cytometry, nanoparticle tracking analysis (NTA), aggregometry, immunoblot analysis and mass spectrometry were carried out to analyze PL-EV and platelet exosomes (PL-EX) release, PLT aggregation, protein expression, and PLT and plasma lipid composition. In comparison to total nHDL, moxHDL significantly decreased PL-EV release by -36% after 5days of PLT storage and partially reversed agonist-induced PLT aggregation. PL-EV release positively correlated with PLT aggregation. MoxHDL improved PLT membrane lipid homeostasis through enhanced uptake of lysophospholipids and their remodeling to corresponding phospholipid species. This also appeared for sphingomyelin (SM) and d18:0/d18:1 sphingosine-1-phosphate (S1P) at the expense of ceramide (Cer) and hexosylceramide (HexCer) leading to reduced Cer/S1P ratio as PLT-viability indicator. This membrane remodeling was associated with increased content of CD36 and maturation of scavenger receptor-B1 (SR-B1) protein in secreted PL-EVs. MoxHDL, more potently than nHDL, improves PLT-membrane lipid homeostasis, partially antagonizes PL-EV release and agonist-induced PLT aggregation. Altogether, this may be the result of more efficient phospho- and sphingolipid remodeling mediated by CD36 and SR-B1 in the absence of ABCA1 on PLTs. As in vitro supplement in PLCs, moxHDL has the potential to improve PLC quality and to prolong storage.

The human gut microbiome plays a crucial role in both health and disease. Metabolites in human faeces related to microbial activity might therefore be attractive surrogate markers to track changes of microbiota induced by diet or disease. The hyphenation of gas chromatography with triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad MS instruments. The versatility of gas chromatography-tandem mass spectrometry (GC-MS/MS) can be advantageously exploited in clinical laboratory medicine, e.g. for quantification of sterols in biological material. In this paper, we present the application of GC-MS/MS for determination of sterol components in human faeces. A serious problem of analysis of faeces is preanalytics. Uncontrolled degradation of metabolites during transport and storage of faeces before entering the clinical laboratory might occur. In our experiments we did not observe any increasing or decreasing concentration after storage of native faeces material even at room temperature. Furthermore, we answer the question of how personal metabolic responses with respect to sterols are and address the importance of sampling strategies. From a pilot study it is concluded that differentiation between high and low metabolizers is independent of the type of sampling and constant over several days.

International audience; Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5alpha-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 (5alpha-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.

Long-term consumption of dietary fiber is generally considered beneficial for weight management and metabolic health, but the results of interventions vary greatly depending on the type of dietary fibers involved. This study provides a comprehensive evaluation of the effects of a specific dietary fiber consisting of a wheat-bran extract enriched in arabinoxylan-oligosaccharides (AXOS) in a human intervention trial. An integrated multi-omics analysis has been carried out to evaluate the effects of an intervention trial with an AXOS-enriched diet in overweight individuals with indices of metabolic syndrome. Microbiome analyses were performed by shotgun DNA sequencing in feces; in-depth metabolomics using nuclear magnetic resonance in fecal, urine, and plasma samples; and massive lipid profiling using mass spectrometry in fecal and serum/plasma samples. In addition to their bifidogenic effect, we observed that AXOS boost the proportion of Prevotella species. Metagenome analysis showed increases in the presence of bacterial genes involved in vitamin/cofactor production, glycan metabolism, and neurotransmitter biosynthesis as a result of AXOS intake. Furthermore, lipidomics analysis revealed reductions in plasma ceramide levels. Finally, we observed associations between Prevotella abundance and short-chain fatty acids (SCFAs) and succinate concentration in feces and identified a potential protective role of Eubacterium rectale against metabolic disease given that its abundance was positively associated with plasma phosphatidylcholine levels, thus hypothetically reducing bioavailability of choline for methylamine biosynthesis. The metagenomics, lipidomics, and metabolomics data integration indicates that sustained consumption of AXOS orchestrates a wide variety of changes in the gut microbiome and the host metabolism that collectively would impact on glucose homeostasis. (This study has been registered at ClinicalTrials.gov under identifier NCT02215343.) IMPORTANCE The use of dietary fiber food supplementation as a strategy to reduce the burden of diet-related diseases is a matter of study given its cost-effectiveness and the positive results demonstrated in clinical trials. This multi-omics assessment, on different biological samples of overweight subjects with signs of metabolic syndrome, sheds light on the early and less evident effects of short-term AXOS intake on intestinal microbiota and host metabolism. We observed a deep influence of AXOS on gut microbiota beyond their recognized bifidogenic effect by boosting concomitantly a wide diversity of butyrate producers and Prevotella copri, a microbial species abundant in non-Westernized populations with traditional lifestyle and diets enriched in fresh unprocessed foods. A comprehensive evaluation of hundreds of metabolites unveiled new benefits of the AXOS intake, such as reducing the plasma ceramide levels. Globally, we observed that multiple effects of AXOS consumption seem to converge in reversing the glucose homeostasis impairment., This study was supported by the EU Project MyNewGut (no. 613979) from the European Commission 7th Framework Program.

Background\ud \ud Metabolic profiling is becoming increasingly popular to identify subtle metabolic variations induced by diet alterations and to characterize the metabolic impact of variations of the gut microbiota. In this context, fecal samples, that contain unabsorbed metabolites, offer a direct access to the outcome of diet - gut microbiota metabolic interactions. Hence, they are a useful addition to measure the ensemble of endogenous and microbial metabolites, also referred to as the hyperbolome.\ud \ud Scope and Approach\ud \ud Many reviews have focused on the metabolomics analysis of urine, plasma and tissue biopsies; yet the analysis of fecal samples presents some challenges that have received little attention. We propose here a short review of current practices and some practical considerations when analyzing fecal material using metabolic profiling of small polar molecules and lipidomics.\ud \ud Key Findings and Conclusions: To allow for a complete coverage of the fecal metabolome, it is recommended to use a combination of analytical techniques that will measure both hydrophilic and hydrophobic metabolites. A clear set of guidelines to collect, prepare and analyse fecal material is urgently needed.

Oxysterols are important intermediates in numerous metabolic and catabolic pathways and their biological significance is also proven. The present paper describes a reliable and short liquid chromatography-high-resolution mass spectrometry method (LC-MS/HR-MS) for the quantification of 8 different oxysterols (24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol and cholestan-3β,5α,6β-triol) in human plasma and red blood cells. Oxysterols were extracted with iso-octane after saponification of esterified sterols. Due to the poor ionization efficiency of the target compounds in electrospray ionization (ESI) derivatization of the molecules has been performed with N,N-dimethylglycine (DMG). Within less than 8 min we were able to achieve baseline separation of the isobaric 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol and 7β-hydroxycholesterol. Moreover, high mass resolution was advantageously applied to resolve quasi-isobaric interferences. The method was validated based on the recommendations of US Food and Drug Administration and the European Medicines Agency guidelines. Oxysterol concentrations were determined in human plasma and red blood cells from healthy volunteers. Furthermore, the applicability for clinical use has been proven by the analysis of oxysterols as biomarkers in Niemann-Pick type C or cerebrotendinous xanthomatosis patients.

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.

International audience; 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 7alpha-, 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 7alpha-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.

A fast and sensitive GC–MS/MS method is proposed to determine pregnenolone, dehydroepiandrosterone (DHEA), testosterone and dihydrotestosterone from human plasma. Steroids were extracted by liquid/liquid extraction, and derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Electron ionization at 30 eV was applied to generate high abundant precursor ions. Specific precursor/product transitions were generated a priori for each compound of interest and these assays were then used to selectively detect and quantify these steroids. Using the selected reaction monitoring mode, detection limits in the pg/ml (sub nmol/l) range could be achieved for all four steroid compounds. The method was validated for imprecision, and recovery and applied in a pilot study in patients with hypercholesterolemia. Associations of increased cholesterol biosynthesis and low testosterone and dihydrotestosterone concentrations were found.

In the present paper we have performed comparative lipidomic analysis of two prototypic atherogenic LDL modifications, oxidized LDL and enzymatically modified LDL. Oxidization of LDL was carried out with different chemical modifications starting from the same native LDL preparations: (i) by copper oxidation leading to terminally oxidized LDL (oxLDL), (ii) by moderate oxidization with HOCl (HOCl LDL), (iii) by long term storage of LDL at 4 °C to produce minimally modified LDL (mmLDL), or (iv) by 15-lipoxygenase, produced by a transfected fibroblast cell line (LipoxLDL). The enzymatic modification of LDL was performed by treatment of native LDL with trypsin and cholesteryl esterase (eLDL). Free cholesterol (FC) and cholesteryl esters (CE) represent the predominant lipid classes in all LDL preparations. In contrast to native LDL, which contains about two-thirds of total cholesterol as CE, enzymatic modification of LDL decreased the proportion of CE to about one-third. Free cholesterol and CE in oxLDL are reduced by their conversion to oxysterols. Oxidization of LDL preferentially influences the content of polyunsaturated phosphatidylcholine (PC) and polyunsaturated plasmalogen species, by reducing the total PC fraction in oxLDL. Concomitantly, a strong rise of the lysophosphatidylcholine (LPC) fraction can be found in oxLDL as compared to native LDL. This effect is less pronounced in eLDL. The mild oxidation of LDL with hypochlorite and/or lipoxygenase does not alter the content of the analyzed lipid classes and species in a significant manner. The lipidomic characterization of modified LDLs contributes to the better understanding their diverse cellular effects.

Background: Fibroblast growth factor 19 (FGF19) is an enteric hormone regulating bile acid de novo synthesis by sensing ileal bile acid flux. However, the role of FGF19 in cholelithiasis has not yet been elucidated and therefore is investigated in the present study. Methods: Total mRNA and protein were isolated from ileal biopsies and used for tissue expression analysis. FGF19, 7α-hydroxycholesterol (7α-OH-Chol), 27-hydroxycholesterol (27-OH-Chol), and different bile acids were determined in the blood samples. Results: FGF19 serum levels did not differ between gallstone carriers and controls but were significantly decreased in the overweight individuals (−32%, p = 0.0002), irrespective of gallstone status (normalweight to overweight controls −29%, p = 0.0017; normalweight to overweight gallstone carriers −44%, p = 0.0338), and correlated inversely with bodyweight (p

The application of Gas Chromatography (GC)-Atmospheric Pressure Chemical Ionization (APCI)-Time-of-Flight Mass Spectrometry (TOF-MS) is presented for sterol analysis in human plasma. A commercial APCI interface was modified to ensure a well-defined humidity which is essential for controlled ionization. In the first step, optimization regarding flow rates of auxiliary gases was performed by using a mixture of model analytes. Secondly, the qualitative and quantitative analysis of sterols including oxysterols, cholesterol precursors, and plant sterols as trimethylsilyl-derivatives was successfully carried out. The characteristics of APCI together with the very good mass accuracy of TOF-MS data enable the reliable identification of relevant sterols in complex matrices. Linear calibration lines and plausible results for healthy volunteers and patients could be obtained whereas all mass signals were extracted with an extraction width of 20 ppm from the full mass data set. One advantage of high mass accuracy can be seen in the fact that from one recorded run any search for m/z can be performed.

Inhalation is one of the entry ports for different chemicals into the human body. In order to investigate this application route and its negative health effect to humans, the presence of volatile organic compounds (VOCs) in indoor air is monitored since many years. To assess global trends and changes of the distribution and disposition of VOCs and the corresponding personal exposure, this study analyzed annual indoor air concentrations collected over a period of 9 (2006–2014) years in the context of a birth control cohort study of 72 VOCs. Additionally, Short Time-series Expression Model (STEM) was used to identify certain correlation for VOCs from different compound classes. For ~42 % of the compounds, a tendency to lower annual median indoor air concentrations was found, and for ~10 % of the VOCs, a trend to higher annual median indoor air concentrations. No such tendencies were observed for ~22 % of the investigated compounds. For ~26 % of the VOCs, the applied linear regression model was not suitable to predict global trends as annual median values were not linearly distributed. Mann-Kendall test was used to (i) confirm the results from the linear regression model and to (ii) calculate trends for those compounds, where linear regression was found to be unsuitable. Thus, for only approximately four of the investigated VOCs, no prediction was possible using both statistical approaches. STEM analysis revealed the connection of benzene, ethylbenzene, m+p xylene, α-pinene, 3-carene, pentadecane, and decamethlycyclopentasiloxane, in addition to the correlation of 1-butanol, chlorobenzene, heptanal, and 2-ethyl-1-hexanol concentrations.

The hyphenation of gas chromatography (GC) and triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad mass spectrometry. We present in this paper the application of GC-triple quadrupole mass spectrometry for determination of sterol components in biological samples. Due to the fact that sterols are quite small molecules an appropriate ionization mode has to be found for advantageous exploitation of the triple quad function. Electron ionization (EI), positive and negative chemical ionization (PCI, NCI) have been tested regarding sensitivity improvement in oxysterol and bile acid analysis in plasma samples. Target analytes were 24-, 25- and 27-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 3β,5α,6β-cholestanetriol, cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. In contrast to bile acids, oxysterols could be analyzed with the highest degree of sensitivity by application of PCI in multiple reaction monitoring mode whereas 7β-hydroxycholesterol and 7-ketocholesterol showed even better results with NCI.

Monitoring bile acids as signal molecules in combination with a bile acid synthesis marker and the FXR regulator fibroblast growth factor 19 (FGF19), this study addresses significant postprandial changes. The efficacy of the different pathways to regulate bile acid synthesis through short heterodimer partner (SHP) dependent FXR modulation in liver, and SHP independent activation via FGF19 is demonstrated. Characteristic changes of the bile acid profile during an oral glucose tolerance test (oGTT) were investigated in 73 individuals. 15 bile acid species including conjugated and unconjugated forms were quantitatively determined with LC-MS/MS in serum samples collected at three time points during the oGTT. All conjugated bile acid species showed the same time course, a significant increase at 60 min after the glucose intake and an incline at 120 min. In contrast, a consistent decline of all unconjugated bile acids was monitored. 7α-Hydroxy-4-cholesten-3-one, an early bile acid synthesis marker, showed an inverse response with a significant decrease at 60 min which proves the efficient and rapid downregulation of CYP7A1 via FXR activation through bile acid signaling. Significantly higher levels of FGF19 were observed 120 min after glucose intake and 60 min after bile acid excursion.

Volatile organic compounds (VOC) play an important role indoors since they have been linked to health symptoms and disorders. Particularly, after renovation activities, high indoor VOC concentrations have been observed. The study will give an indication, for the first time under real conditions, of the to-be-expected time frame for renovation-derived indoor pollution decreases when the exposure to it will reach a reference level. The decrease in the concentrations of investigated 26 VOC after renovations was assessed under real-life situations. Both the daily VOC concentration was measured by active sampling for 30 days in selected homes which had undergone various renovations and, as part of an epidemiologic study, the same VOC were collected monthly using passive samplers in 243 homes. An exponential function was used to interpret the concentration decay. The average time range which has to elapse following renovation activities before a guideline value or reference load is reached showed a time range between 2 and 8 weeks. This waiting time had at least be applicable to public buildings and institutions (especially relevant in case of nurseries, playschools etc.) with increasingly being implemented in private homes as well. Practical Implications After renovation an optimal waiting period had to be up to 60 days before the rooms will be used again. Fourteen days are possible, but increased ventilation is recommended. These had to be applicable at least for public buildings used by risk groups like young children. Renovations had to be carried out in summer season to ensure optimal ventilation to reduce the waiting time.

Monthly measurements of volatile organic compounds (VOCs) were carried out in a field study in Greater Cairo in the period of 2005–2007. Ten apartments were chosen as sites for passive sampling of VOCs in different parts of Greater Cairo, Egypt taking into consideration the traffic volume profile across the city and the suburbs. The concentrations of 29 VOCs, belonging to the groups of alkanes, cycloalkanes, aromatic hydrocarbons, halogenated hydrocarbons and terpenes were measured indoor and outdoor at the same time. A strong correlation was found between sites with high traffic volume and the concentration of aromatic compounds. The measured VOC burden in the outdoor environment of the whole city is dominated by compounds related to vehicle emissions, e.g. the BTEX compounds (benzene, toluene, ethyl benzene and xylene). The spatial variation of the indoor BTEX exposure is similar to the outdoor exposure. This implies a strong infiltration of outdoor ambient air pollution into the indoor environment. The highest indoor BTEX concentration was measured in the city centre (172 μg/m3). Significant seasonal cycles with high values in winter and lower values in summer months could be modelled for indoor concentrations of BTEX and terpenes. This is in accordance with findings in Middle Europe (Germany) with completely different climate and building characteristics.

A miniaturized sample preparation method is presented that is based on microextraction by packed sorbent (MEPS) to determine metabolites of monoterpenes in small volumes of human urine. A protocol including syringe modifications has been developed to exploit the concept of MEPS for application in gas chromatography—mass spectrometry with splitless injection. Only 210 μL of enzymatically hydrolysed urine is needed to carry out a complete extraction protocol. Elution volumes between 1 and 10 μL can be transferred completely or partly to the GC-MS system. All steps of filling and injection of the MEPS syringes are performed automatically. The utility of this approach was demonstrated in terms of determinations of the monoterpene metabolites cis-verbenol, trans-verbenol, α-terpineol, myrtenol and perillyl alcohol in a urine sample of a volunteer who lived in a dwelling with high indoor air exposure of monoterpenes.

Microbial volatile organic compounds (MVOCs) emitted from the mould species Penicillium expansum, P. chrysogenum, Aspergillus versicolor, A. fumigatus, A. niger and Cladosporium cladosporoides were analyzed by means of solid phase microextraction (SPME) and GCMS. The mould species were cultivated on the synthetic agar dichloran chloramphenicol (DG 18) and on wet wall paper. The production of MVOCs was monitored over several weeks to detect changes in the emission rates between the initial stage and later periods of growth. The cultivation on the synthetic agar resulted in MVOC patterns with a wide variety of signals. In contrast, the growth on wet wall paper led to changed MVOC patterns with less signals. The emission rates were drastically reduced. Components emitted by all six fungi species on wall paper were 2-pentanol and 2-pentanone. 1-Octen-3-ol was emitted by five fungi species. 2-Pentanol was only detected in considerable amounts during the first days of growth whereas 1-octen-3-ol had a more constant emission rate over the whole period of growth. On the basis of our studies some MVOCs could be proposed as specific for single fungi on wall paper, e.g. 1,3-dimethoxybenzene for A. versicolor and 2,4-pentandione for A. fumigatus.

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Temporal analysis of products (TAP), XPS, and DRIFTS were used to study the activity and deactivation of acid sites on a sulfated zirconia catalyst for the n-butane isomerization. The activity of the catalyst was altered by selective poisoning of active centres by sulfation with sodium sulfate under the condition of a constant surface sulfate content. Depending on the surface sodium concentration the Bronsted acid sites were selectively poisoned, which was reflected by a stepwise decrease in the isomerization activity and an altered Bronsted-to-Lewis acid ratio. It could be shown, that only a very small amount of Bronsted acid sites were responsible for the initiation of the catalytic reaction. TAP measurements revealed that with a decreasing number of available strong Bronsted sites, the adsorption of the reactant on the surface dramatically decreased. The altered adsorption surface places prevented the formation of reactive intermediates which seemed to be necessary to start the isomerization reaction cycle. The samples were additionally characterized by N2-physisorption, X-ray diffraction, and temperature programmed desorption of ammonia. Moreover, the number of Bronsted sites was estimated by a solid-phase exchange of sulfated zirconia with NH4Y zeolite which could be directly correlated to the DRIFTS results.

Two sulfated zirconia catalysts were prepared via sulfation and calcination of zirconium hydroxide at 873 K; the zirconium hydroxide had been aged at room temperature for 1 h (SZ-1) or aged at 373 K for 24 h (SZ-2). SZ-1 was active for n-butane isomerization at 373 K; SZ-2 reached a similar performance level only at 473 K. Both materials contained about 9 wt% sulfate and were tetragonal. Because of a lower BET surface area (105 vs. 148 m2/g) SZ-1 featured a higher sulfate density, and XRD and EXAFS analysis showed larger (ca. 10 nm) and more well ordered crystals than for SZ-2. n-Butane TPD on SZ-1 showed a butene desorption peak at low temperature, whereas no obvious butene desorption was observed with SZ-2, suggesting that SZ-1 has a higher oxidizing power at low temperature than SZ-2. The number of sites capable of dehydrogenation are less than 5 μmol/g, because the differential heats of n-butane adsorption as measured by microcalorimetry were 45–60 kJ/mol for higher coverages, indicating weak and reversible interaction. TAP experiments describe the adsorption and desorption behavior of n-butane at different activity states and are the basis for a simple adsorption model. Reactant pulses and purge experiments show that the active species, presumably formed in an oxidative dehydrogenation step, are stable at the surface under reaction conditions.

Electrochemical impedance spectroscopy (EIS) was applied to study charge transfer and ion diffusion processes of all-solid-state ion-sensitive electrodes based on zeolite–polydimethylsiloxane (PDMS) membranes. In order to investigate the effects of zeolites embedded into a polymer matrix membranes with different types and contents of zeolites were characterised. On the basis of EIS studies, zeolites of the faujasite type were found most suitable for potentiometric determinations. This result can be attributed to the large pore size of 7.4 A and a low Si/Al ratio. The differences in selectivity of potentiometric responses towards alkali metal cations and tetrabutylammonium cations were found to be reflected by differences in the bulk resistance of the membrane. The impedance behaviour of the zeolite–polymer electrodes was modelled by an appropriate equivalent electrical circuit.

A planar all-solid-state electrode for the titrimetric analysis of ionic surfactants has been developed and characterised. The electrode is fabricated by lamination of suitable electrode structures that are sandwiched between flexible plastic foils. The zeolite–polymer membrane containing 50% (w/w) zeolite of the faujasite type is cast onto an exposed gold film surface to form the surfactant-sensitive electrode. The very fast potentiometric response of the sensor can be exploited for potentiometric titrations of surfactants. Without any further pre-treatment, reproducible and well-defined titration curves are obtained for the titrimetric analysis of anionic surfactants using a cationic surfactant and vice versa. According to the fabrication strategy applied, the developed devices have the character of disposable electrodes. The electrode assembly exhibits shelf-lives longer than 3 months under room temperature and dry conditions. The practical utility of the disposable all-solid-state electrodes is demonstrated in terms of titrimetric determinations of anionic surfactants in commercial detergents.

Background: The indoor air quality is among other things determined by air pollutants such as volatile organic compounds (VOCs) which are associated with health effects. The severity of these health effects depends on the kind and part of the specific chemicals within the VOC group. Since the 90th the expected health risk has been evaluated by means of the VOC sum concentration under the assumption of a fixed composition with a clear defined part of every single VOC. The question is whether the VOC sum and whether the share of individual VOCs have been changed over the recent years. Methods: Indoor VOCs were measured within the frame of epidemiological studies from 1994 until 2008. Additionally, a large number of apartments were investigated depend on inquiries by physicians, health offices, or inhabitants in a period from 2009 to 2014. The human health risk was assessed both based on currently valid criteria and based on adapted criteria considering potential composition of the VOC pattern. Results: A substantial reduction in the sum of VOCs (about 60%) could be demonstrated caused by a clear decreasing trend of aromatics and alkanes. No such changes could be observed for cycloalkanes, chlorinated hydrocarbons and terpenes. The resulting pattern of VOC is quite different compared with the start situation in 1994. The part of terpenes at the VOC sum increased by two from approximately 25% (1994) to 55% (2014). Due to the decrease of the VOC sum, the human health risk might be underestimated if the VOC sum is the only criterion. Conclusions: Based on our findings special attention should be paid to groups of VOCs and/or single VOCs.

Using temperature-programmed desorption (TPD), we have investigated the desorption behavior after subsequent co-adsorption of methanol and water and after adsorption of their mixtures on a NaZSM-5 zeolite. The course of desorption indicates that a strong mutual displacement of both components occurs. However, on the strongest adsorption sites methanol is preferentially adsorbed, and already the addition of small amounts of methanol leads to a displacement of water. Our results support the idea of a subdivision of the pore space for adsorption of water/methanol mixtures. Above all, the experiments show that in the part of the pore space where both components are adsorbed, different sites are of importance which vary significantly in their interaction strength.

Potentiometric electrodes based on the incorporation of zeolite particles into polydimethylsiloxane (PDMS) membranes are described. The electrode characteristics are evaluated regarding the response towards alkali metal cations. Zeolite/PDMS-coated iridium disk electrodes offer lower limits of detection (3 × 10−5 M for Cs+) and a more extended concentration range with Nernstian slope than zeolite-PDMS electrodes with inner reference solution. Both types of electrodes respond rapidly to concentration changes (within a few seconds or less). Zeolite-PDMS electrodes exhibit almost the same sensitivity towards all alkali metal cations independent of the use of either zeolite X or zeolite A. Furthermore, zeolite-PDMS electrodes are applied to the determination of ionic surfactants. In contrast to the results obtained for alkali metal cations zeolites of the faujasite type (X and Y) lead to superior electrode function when compared to A zeolites. The determination of surfactants is accomplished by potentiometric titration using a zeolite-PDMS indicator electrode. Practical applications of surfactant determination in commercial detergents are presented and verified by performing the two-phase titration as independent reference method.

We have investigated the interaction of water with Na+-ion exchanged zeolites of different structures (LTA, FAU, ERI, MOR and MFI) by means of temperature-programmed desorption (TPD). The non-isothermal desorption of water shows, depending on the zeolite type, differently structured desorption profiles. In every case the profiles have, however, two main ranges. Using a regularization method, desorption energy distribution functions have been calculated. The desorption energy distributions between 42–60 kJ mol−1, which can be attributed to a non-specific interaction of water, show two clearly distinguished energy ranges. The water desorption behaviour of this range correlates with the electronegativity of the zeolites and the average charge of the lattice oxygen atoms calculated by means of the electronegativity equalization method (EEM). The part of the desorption energy distributions in the range of 60–90 kJ mol−1, reflecting interactions of water with Na+ cations, shows two more or less pronounced maxima. In agreement with vibrational spectroscopic studies in the far infrared region, it may be concluded that all samples under study possess at least two different cation sites.

The high mass transport caused by ultrasonic radiation in the preconcentration of trace metal species in anodic stripping voltammetry (ASV), leads to an order of magnitude enhancement in sensitivity. A Nafion-coated mercury thin-film working electrode is placed in a specially designed small-volume sonovoltammetric cell. The preparation and curing procedure for the Nafion film on a glassy carbon electrode substrate as well as ultrasound-enhanced mercury deposition through the Nafion film are optimized. The resulting electrode assembly is stable under ultrasonic irradiation. Optical microscopy observations show the formation of closely spaced mercury droplets of micrometer dimensions under the Nafion film. The performance characteristics of ultrasound-enhanced ASV at the Nafion-coated mercury thin-film electrode are studied in detail. The detection limit obtained for lead and cadmium test species is 3 × 10-11 M (30 s preconcentration in the presence of ultrasound). A relative standard deviation of 1.2% is obtained for repetitive determinations of 10 nM Cd2+ (n = 9).

Indoor volatile organic compounds (VOCs) may be associated with adverse health effects. The aim of this study was to investigate whether the indoor VOCs exposure burden has undergone changes during a past 15-year period, exemplarily in a German city. The study was limited to homes with families. To avoid possible regional-specific confounder effects, this assessment was conducted for this urban centre with a sufficiently long and continuous time series (1994–2008). VOCs were determined using passive sampling over 4 weeks followed by liquid extraction and analysis with gas chromatography-mass spectrometry. All measurement periods were equally distributed over the seasons and years. A substantial reduction in the total sum of VOCs and aromatics could be demonstrated as well as a trend of decrease concentrations for alkanes. No such changes could be observed for cycloalkanes, chlorinated hydrocarbons and terpenes group. Possible reasons for the reduction of indoor VOCs exposure are most likely due to the applied regulations/guidelines for the VOCs reduction in indoor-relevant products. Problems are, however, regarding the slight increase in terpene exposure. These chemicals have been found to be associated with quite specific health effects. Therefore, future emphasis should be directed not only towards the general reduction of anthropogenically generated VOC exposures in the outdoor environment but also on the reduction of health-relevant VOCs, especially indoors.

Batch-injection analysis exhibits the advantages of rapid and simple electroanalysis of microlitre samples. Nafion-coated mercury thin film electrodes have been evaluated for use in batch-injection analysis with anodic stripping voltammetry (BIA-ASV). The advantages of Nafion-coated electrodes in reducing electrode contamination by components of complex matrices are combined with the analysis of small microlitre sample volumes. The measurement of traces of lead and cadmium is used to illustrate the approach. An optimised procedure for formation of Nafion-coated mercury thin film electrodes is evolved. The relative sensitivity for BIA-ASV at electrodes with and without Nafion coatings is 0.9 and 0.8 for cadmium and lead respectively; detection limits are 2 x 10(-9) M and 4 x 10(-9) M. Studies were done concerning the influence of surfactants and their effect was found to be much less with the Nafion film coating. Applications to real environmental samples are demonstrated.

BACKGROUND Cholesterol precursors and plant sterols have considerable potential as plasma biomarkers in several disorders of sterol metabolism and intestinal sterol absorption. Oxysterols are associated with atherogenesis, neurodegeneration, and inflammation. We developed a GC-MS method for the simultaneous analysis of these species in human plasma, including 24-, 25-, 27-hydroxycholesterol; 7-ketocholesterol; lanosterol; lathosterol; 7-dehydrocholesterol; desmosterol; stigmasterol; sitosterol; and campesterol. METHODS Sterols were hydrolyzed with ethanolic potassium hydroxide solution, extracted by liquid/liquid extraction with n-hexane, and derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Positive chemical ionization with ammonia, as reagent gas, was applied to generate high abundant precursor ions. RESULTS The definition of highly sensitive precursor/product ion transitions, especially for coeluting substances, allowed fast gas chromatography run times of under 8.5 min. Using the multiple reaction monitoring mode, detection limits in the picogram per milliliter range could be achieved for most compounds. The method was validated for precision and recovery. Intraassay and interassay CVs were mostly CONCLUSIONS This method allows high-throughput sterol profiling in various diseases.

Farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, plays an important role in the regulation of cholesterol and more specifically high-density lipoprotein (HDL) homeostasis. Activation of FXR is reported to lead to both pro- and anti-atherosclerotic effects. In the present study we analyzed the impact of different FXR agonists on cholesterol homeostasis, plasma lipoprotein profiles, and transhepatic cholesterol efflux in C57BL/6J mice and cynomolgus monkeys and atherosclerosis development in cholesteryl ester transfer protein transgenic (CETPtg) low-density lipoprotein receptor (LDLR) (-/-) mice. In C57BL/6J mice on a high-fat diet the synthetic FXR agonists isopropyl 3-(3,4-difluorobenzoyl)-1,1-dimethyl-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (FXR-450) and 4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl]benzoic acid (PX20606) demonstrated potent plasma cholesterol-lowering activity that affected all lipoprotein species, whereas 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) and 6-ethyl chenodeoxycholic acid (6-ECDCA) showed only limited effects. In FXR wild-type mice, but not FXR(-/-) mice, the more efficacious FXR agonists increased fecal cholesterol excretion and reduced intestinal cholesterol (re)uptake. In CETPtg-LDLR(-/-) mice PX20606 potently lowered total cholesterol and, despite the observed HDL cholesterol (HDLc) reduction, caused a highly significant decrease in atherosclerotic plaque size. In normolipidemic cynomolgus monkeys PX20606 and 6-ECDCA both reduced total cholesterol, and PX20606 specifically lowered HDL(2c) but not HDL(3c) or apolipoprotein A1. That pharmacological FXR activation specifically affects this cholesterol-rich HDL(2) subclass is a new and highly interesting finding and sheds new light on FXR-dependent HDLc lowering, which has been perceived as a major limitation for the clinical development of FXR agonists.

Cerebrotendinous xanthomatosis (CTX) is a rare, inherited autosomal-recessive lipid-storage disorder caused by 27-hydroxylase deficiency. In this study, we report of a 30-year old man with this disorder who was treated using chenodeoxycholic acid, simvastatin, and low-density lipoprotein (LDL) apheresis. The LDL apheresis was performed weekly for nine months. The first subjective improvement was reported by the patient after his fourth LDL-apheresis. Spasticity, gait disturbances, and his entire psychomotoric test results had improved tremendously. His fine motoric skills have been regained. The efficacy of LDL-apheresis was monitored using quantitative determination of 7α-OH-4-cholesten-3-one in plasma based on a LC–MS/MS method. The clearance efficacy of 7α-hydroxy-4-cholesten-3-one from the patient's plasma per LDL-apheresis varied between 8% and 53% but returned to the initial high levels after seven days (mean value 241 ng/mL). A slight negative trend in the plasma concentration could be derived over the period of nine months.

Passive sampling combined with GC-MS was applied for the detection of microbial volatile organic compounds (MVOCs) emitted by mould cultures. The fungi species Penicillium expansum, Penicillium chrysogenum, Penicillium brevicompactum, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus niger, and Cladosporium cladosporoides were cultivated on dichloran glycerol agar. The emitted volatiles were collected by charcoal-based passive diffusion monitors during the first four weeks of growth. Several alcohols, mainly 2-methyl-1-propanol and 3-methyl-1-butanol, alkenes, ketones, ethers and esters could be identified and quantified after external calibration. Species-specific compounds like 1,3-dimethoxybenzene for A. versicolor could be proposed. Associations between the concentrations of some of the identified MVOCs (e.g. methylfurans and dimethyldisulfide) and indoor mould exposure in dwellings were found in a field study comprising more than 500 dwellings in Leipzig/Germany. It could be concluded that passive sampling onto charcoal adsorbents for the determination of MVOCs is particularly suitable in epidemiological studies with a high number of participants.

Ultrasonically-enhanced mass transport was exploited to increase preconcentration efficiency in anodic stripping voltammetry. We developed a Nafion-coated mercury thin-film working electrode which is stable under ultrasonic irradiation, making it possible to achieve very low limits of detection for relatively short preconcentration times. This allows the investigation of a variety of biological and environmental samples.