Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase

Rationale: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. Results: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2–3 months old) 3-MST−/− mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST−/− mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST−/− mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST−/− mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST−/− mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. Conclusions: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy

Metabolite ratios as potential biomarkers for type 2 diabetes:a DIRECT study

Aims/hypothesis Circulating metabolites have been shown to reflect metabolic changes during the development of type 2 diabetes. In this study we examined the association of metabolite levels and pairwise metabolite ratios with insulin responses after glucose, glucagon-like peptide-1 (GLP-1) and arginine stimulation. We then investigated if the identified metabolite ratios were associated with measures of OGTT-derived beta cell function and with prevalent and incident type 2 diabetes. Methods We measured the levels of 188 metabolites in plasma samples from 130 healthy members of twin families (from the Netherlands Twin Register) at five time points during a modified 3 h hyperglycaemic clamp with glucose, GLP-1 and arginine stimulation. We validated our results in cohorts with OGTT data (n = 340) and epidemiological case–control studies of prevalent (n = 4925) and incident (n = 4277) diabetes. The data were analysed using regression models with adjustment for potential confounders. Results There were dynamic changes in metabolite levels in response to the different secretagogues. Furthermore, several fasting pairwise metabolite ratios were associated with one or multiple clamp-derived measures of insulin secretion (all p Conclusion/interpretation In this study we have shown that the Val_PC ae C32:2 metabolite ratio is associated with an increased risk of type 2 diabetes and measures of insulin secretion and resistance. The observed effects were stronger than that of the individual metabolites and independent of known risk factors.</p

Design, baseline characteristics, and retention of African American light smokers into a randomized trial involving biological data

<p>Abstract</p> <p>Background</p> <p>African Americans experience significant tobacco-related health disparities despite the fact that over half of African American smokers are light smokers (use ≤10 cigarettes per day). African Americans have been under-represented in smoking cessation research, and few studies have evaluated treatment for light smokers. This paper describes the study design, measures, and baseline characteristics from <it>Kick It at Swope III </it>(KIS-III), the first treatment study of bupropion for African American light smokers.</p> <p>Methods</p> <p>Five hundred forty African American light smokers were randomly assigned to receive bupropion (150mg bid) (n = 270) or placebo (n = 270) for 7 weeks. All participants received written materials and health education counseling. Participants responded to survey items and provided blood samples for evaluation of phenotype and genotype of CYP2A6 and CYP2B6 enzymes involved in nicotine and bupropion metabolism. Primary outcome was cotinine-verified 7-day point prevalence smoking abstinence at Week 26 follow-up.</p> <p>Results</p> <p>Of 2,628 individuals screened, 540 were eligible, consented, and randomized to treatment. Participants had a mean age of 46.5 years and 66.1% were women. Participants smoked an average of 8.0 cigarettes per day, had a mean exhaled carbon monoxide of 16.4ppm (range 1-55) and a mean serum cotinine of 275.8ng/ml. The mean Fagerström Test for Nicotine Dependence was 3.2, and 72.2% of participants smoked within 30 minutes of waking. The average number of quit attempts in the past year was 3.7 and 24.2% reported using pharmacotherapy in their most recent quit attempt. Motivation and confidence to quit were high.</p> <p>Conclusion</p> <p>KIS-III is the first study designed to examine both nicotine and bupropion metabolism, evaluating CYP2A6 and CYP2B6 phenotype and genotype in conjunction with psychosocial factors, in the context of treatment of African American light smokers. Of 1629 smokers screened for study participation, only 18 (1.1%) were ineligible to participate in the study because they refused blood draws, demonstrating the feasibility of recruiting and enrolling African American light smokers into a clinical treatment trial involving biological data collection and genetic analyses. Future evaluation of individual factors associated with treatment outcome will contribute to advancing tailored tobacco use treatment with the goal of enhancing treatment and reducing health disparities for African American light smokers.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="URL">NCT00666978</a></p

Causes of changing earnings inequality

'This paper surveys major empirical reqularities concerning changes in earnings inequality in Europe and the U.S. over the past 25 years. Next, it indicates which of these regularities can be explained within the competitive demand-supply framework of analysis and what is left unexplained. Finally, it considers the implications of organizational change as a possible rationale for recent inequality developments.' (author's abstract)Der Autor stellt die Entwicklung von Einkommensunterschieden in den USA und Westeuropa dar, analysiert sie modelltheoretisch und untersucht die Bedeutung des Wandels der Arbeitsorganisation. (IAB)German title: Ursachen der sich aendernden EinkommensungleichheitSIGLEAvailable from IAB-90-1US0-111300 BH 323 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDiskussionsprotokollDEGerman

Targeted metabolomics of dried blood spot extracts.

Dried blood spot (DBS) samples are already successfully used in newborn screening and pharmacological analyses. The application of DBS matrix to further metabolomic methods will considerably extend the analytical options for the diagnostics of metabolic diseases. We present an MS/MS based method for the simultaneous extraction and quantification of 188 metabolites from dried blood spots. We provide a sensitive and reproducible method that adapts the Absolute IDQ&trade; p180 kit of Biocrates to the DBS matrix for the quantification of metabolites of different substance classes including amino acids, biogenic amines, free carnitine, acylcarnitines, hexoses, glycerophospholipids, lysophosphatidylcholines, phosphatidylcholines, and sphingolipids

Important roles of the AKR1C2 and SRD5A1 enzymes in progesterone metabolism in endometrial cancer model cell lines.

Endometrial cancer is the most frequently diagnosed gynecological malignancy. It is associated with prolonged exposure to estrogens that is unopposed by progesterone, whereby enhanced metabolism of progesterone may decrease its protective effects, as it can deprive progesterone receptors of their active ligand. Furthermore, the 5&alpha;-pregnane metabolites formed can stimulate proliferation and may thus contribute to carcinogenesis. The aims of our study were to: 1) identify and quantify progesterone metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and 2) pinpoint the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism studies combined with liquid chromatography-tandem mass spectrometry enabled identification and quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-interfering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20&alpha;-hydroxy-pregn-4-ene-3-one, 20&alpha;-hydroxy-5&alpha;-pregnane-3-one, and 5&alpha;-pregnane-3&alpha;/&beta;,20&alpha;-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6,800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5&alpha;-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased progesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5&alpha;-reduction is catalyzed by 5&alpha;-reductase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progesterone metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment

Unemployment traps Do financial dis-incentives matter?

'This paper analyses the importance of financial dis-incentives for workers in Denmark. Based on a panel survey which is merged to a number of administrative registers it is possible to calculate precise measures of the economic incentives for labour force participants between employment in a full time job and being on unemployment insurance benefits and considering also the fixed costs of work. The results indicate large dis-incentives effects for some groups, especially low paid women. In 1996, 6 per cent of Danish men and 13 per cent of the women had a lower disposable net income if working in a full-time job compared to being on unemployment benefits. The effect of these financial dis-incentives is analysed in simple reduced form models of on-the-job search, unemployed search behaviour, unemployment risk, and transitions out of the labour force. We find that the net compensation rate in unemployment has a significant impact on women's propensity to leave the labour force, on measures of search intensity, on the risk of being hit by unemployment and on one of our flexibility measures, i.e. the maximum acceptable commuting time to a job. The net compensation rate has no impact on the willingness to move to another place to get a job. However, here we find a significant impact from job attitude related measures. We end the paper reporting the results from including attitude variables along with economic variables. We find a number of significant effects from attitude variables. However, the main conclusion is that economic incentives dominate the present analysis of unemployment traps.' (author's abstract)Das Papier befasst sich mit der Bedeutung der Arbeitslosigkeitsfalle in Daenemark. Dabei werden die moeglichen negativen Arbeitsanreize analysiert, die vom Sozial- und Steuersystem ausgehen. Die Datengrundlage bilden zwei Arbeitskraeftebefragungen (Erwerbstaetige und Arbeitslose) von 1993 und 1996. (IAB)German title: Arbeitslosigkeitsfalle: spielen fehlende finanzielle Anreize eine Rolle?SIGLEAvailable from ftp://ftp.iza.org/dps/dp274.pdf / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Combined liquid chromatography - tandem mass spectrometry analysis of progesterone metabolites.

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5&alpha;-pregnane-3-one and (20S)-5&alpha;-pregnane-3&alpha;,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing

High-throughput extraction and quantification method for targeted metabolomics in murine tissues.

Introduction Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction. Objectives We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ (TM) p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput. Methods We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. Results We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma. Conclusion We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays