Analytical errors caused by suboptimal performance of the chosen platform for a number of metabolites and instrumental drift are a major issue in large-scale metabolomics studies. Especially for ...MS-based methods, which are gaining common ground within metabolomics, it is difficult to control the analytical data quality without the availability of suitable labeled internal standards and calibration standards even within one laboratory. In this paper, we suggest a workflow for significant reduction of the analytical error using pooled calibration samples and multiple internal standard strategy. Between and within batch calibration techniques are applied and the analytical error is reduced significantly (increase of 25% of peaks with RSD lower than 20%) and does not hamper or interfere with statistical analysis of the final data.
A large metabolomics study was performed on 600 plasma samples taken at four time points before and after a single intake of a high fat test meal by obese and lean subjects. All samples were analyzed ...by a liquid chromatography−mass spectrometry (LC−MS) lipidomic method for metabolic profiling. A pragmatic approach combining several well-established statistical methods was developed for processing this large data set in order to detect small differences in metabolic profiles in combination with a large biological variation. Such metabolomics studies require a careful analytical and statistical protocol. The strategy included data preprocessing, data analysis, and validation of statistical models. After several data preprocessing steps, partial least-squares discriminant analysis (PLS-DA) was used for finding biomarkers. To validate the found biomarkers statistically, the PLS-DA models were validated by means of a permutation test, biomarker models, and noninformative models. Univariate plots of potential biomarkers were used to obtain insight in up- or downregulation. The strategy proposed proved to be applicable for dealing with large-scale human metabolomics studies.
The prevalence of overweight is increasing globally and has become a serious health problem. Low-grade chronic inflammation in overweight subjects is thought to play an important role in disease ...development. Novel tools to understand these processes are needed. Metabolic profiling is one such tool that can provide novel insights into the impact of treatments on metabolism.
To study the metabolic changes induced by a mild anti-inflammatory drug intervention, plasma metabolic profiling was applied in overweight human volunteers with elevated levels of the inflammatory plasma marker C-reactive protein. Liquid and gas chromatography mass spectrometric methods were used to detect high and low abundant plasma metabolites both in fasted conditions and during an oral glucose tolerance test. This is based on the concept that the resilience of the system can be assessed after perturbing a homeostatic situation.
Metabolic changes were subtle and were only detected using metabolic profiling in combination with an oral glucose tolerance test. The repeated measurements during the oral glucose tolerance test increased statistical power, but the metabolic perturbation also revealed metabolites that respond differentially to the oral glucose tolerance test. Specifically, multiple metabolic intermediates of the glutathione synthesis pathway showed time-dependent suppression in response to the glucose challenge test. The fact that this is an insulin sensitive pathway suggests that inflammatory modulation may alter insulin signaling in overweight men.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dietary medium chain fatty acids (MCFA) and linoleic acid follow different metabolic routes, and linoleic acid activates PPAR receptors. Both these mechanisms may modify lipoprotein and fatty acid ...metabolism after dietary intervention. Our objective was to investigate how dietary MCFA and linoleic acid supplementation and body fat distribution affect the fasting lipoprotein subclass profile, lipoprotein kinetics, and postprandial fatty acid kinetics. In a randomized double blind cross-over trial, 12 male subjects (age 51±7 years; BMI 28.5±0.8 kg/m2), were divided into 2 groups according to waist-hip ratio. They were supplemented with 60 grams/day MCFA (mainly C8:0, C10:0) or linoleic acid for three weeks, with a wash-out period of six weeks in between. Lipoprotein subclasses were measured using HPLC. Lipoprotein and fatty acid metabolism were studied using a combination of several stable isotope tracers. Lipoprotein and tracer data were analyzed using computational modeling. Lipoprotein subclass concentrations in the VLDL and LDL range were significantly higher after MCFA than after linoleic acid intervention. In addition, LDL subclass concentrations were higher in lower body obese individuals. Differences in VLDL metabolism were found to occur in lipoprotein lipolysis and uptake, not production; MCFAs were elongated intensively, in contrast to linoleic acid. Dietary MCFA supplementation led to a less favorable lipoprotein profile than linoleic acid supplementation. These differences were not due to elevated VLDL production, but rather to lower lipolysis and uptake rates.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This open-label, single-period study describes the human absorption, distribution, metabolism, excretion, and pharmacokinetics of velsecorat (AZD7594). Healthy subjects received inhaled velsecorat ...(non-radiolabeled; 720
g) followed by intravenous infusion of carbon 14 (
C)-velsecorat (30
g). Plasma, urine, and feces were collected up to 168 hours post-dose. Objectives included identification and quantification of velsecorat and its metabolites (i.e., drug-related material) in plasma and excreta, and determining the elimination pathways of velsecorat by measuring the rate and route of excretion, plasma half-life (t
), clearance, volume of distribution and mean recovery of radioactivity. On average, 76.0% of administered
C dose was recovered by the end of the sampling period (urine = 24.4%; feces = 51.6%), with no unchanged compound recovered in excreta, suggesting that biliary excretion is the main elimination route. Compared with intravenous
C-velsecorat, inhaled velsecorat had a longer t
(27 versus 2 hours), confirming that plasma elimination is absorption-rate-limited from the lungs. Following intravenous administration, t
of
C-drug-related material was longer than for unchanged velsecorat, and 20% of the
C plasma content was related to unchanged velsecorat. The geometric mean plasma clearance of velsecorat was high (70.7 l/h) and the geometric mean volume of distribution at steady state was 113 l. Velsecorat was substantially metabolized via O-dealkylation of the indazole ether followed by sulfate conjugation, forming the M1 metabolite, the major metabolite in plasma. There were 15 minor metabolites. Velsecorat was well tolerated, and these results support the progression of velsecorat to phase 3 studies. SIGNIFICANCE STATEMENT: This study describes the human pharmacokinetics and metabolism of velsecorat, a selective glucocorticoid receptor modulator, evaluated via co-administration of a radiolabeled intravenous microtracer dose and a non-radiolabeled inhaled dose. This study provides a comprehensive assessment of the disposition of velsecorat in humans. It also highlights a number of complexities associated with determining human absorption, distribution, metabolism, and excretion for velsecorat, related to the inhaled route, the high metabolic clearance, sequential metabolite formation and the low intravenous dose.
Therapeutic drug monitoring of mesalazine (5-ASA) in patients with ulcerative colitis is unavailable. Mucosal 5-ASA concentrations are assumed to be higher during remission, but biopsy is not ...practical. Therefore, we investigated the feasibility of measuring mesalazine levels in feces. To explore the potential role of fecal mesalazine measurements in therapeutic drug monitoring, we compared the dry fecal concentration and daily fecal excretion of 5-ASA and its metabolite N-acetyl-5-ASA in patients with ulcerative colitis with active and quiescent disease.
Adults with ulcerative colitis on oral mesalazine and scheduled for colonoscopy were eligible for inclusion in this cross-sectional study. Stool and urine samples were collected for 48 and 24 hours, respectively, and rectal biopsies were performed. (N-acetyl-)5-ASA was measured using mass spectrometry. Biochemically active disease was defined as a fecal calprotectin level above 100 mcg/g and endoscopically active disease as any activity following the endoscopic Mayo score (≥1).
Approximately 28 patients were included in the study. Daily fecal excretion of (N-acetyl-)5-ASA did not differ between patients with (n = 13) and without (n = 15) endoscopically active disease median 572 mg/d versus 597 mg/d (P = 0.86) for 5-ASA and 572 mg/d versus 554 mg/d (P = 0.86) for N-acetyl-5-ASA. The same applied to the fecal concentration median 9.7 mcg/mg dry weight versus 10.3 (P = 0.53) and 12.0 versus 9.9 (P = 0.89). The results were comparable when the biochemical disease activity definition was used. The mucosal concentrations and urinary excretion of (N-acetyl-)5-ASA did not differentiate between quiescent and active activity.
Fecal (N-acetyl-)5-ASA measurements do not correlate with disease activity, which renders it an unsuitable tool for therapeutic drug monitoring of mesalazine.
We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of ...several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R 2 > 0.995), sensitivity (limit of detection 0.1−1 ng), repeatability, and reproducibility (relative standard deviation <10%). The IP-LC-ESI-MS method was shown to be a useful tool for microbial metabolomics, i.e., the comprehensive quantitative analysis of metabolites in extracts of microorganisms, and for the determination of the energy charge, i.e., the cellular energy status, as an overall quality measure for the sample workup and analytical protocols.
Increased dietary cholesterol intake is associated with atherosclerosis. Atherosclerosis development requires a lipid and an inflammatory component. It is unclear where and how the inflammatory ...component develops. To assess the role of the liver in the evolution of inflammation, we treated ApoE*3Leiden mice with cholesterol-free (Con), low (LC; 0.25%) and high (HC; 1%) cholesterol diets, scored early atherosclerosis and profiled the (patho)physiological state of the liver using novel whole-genome and metabolome technologies.
Whereas the Con diet did not induce early atherosclerosis, the LC diet did so but only mildly, and the HC diet induced it very strongly. With increasing dietary cholesterol intake, the liver switches from a resilient, adaptive state to an inflammatory, pro-atherosclerotic state. The liver absorbs moderate cholesterol stress (LC) mainly by adjusting metabolic and transport processes. This hepatic resilience is predominantly controlled by SREBP-1/-2, SP-1, RXR and PPARalpha. A further increase of dietary cholesterol stress (HC) additionally induces pro-inflammatory gene expression, including pro-atherosclerotic candidate genes. These HC-evoked changes occur via specific pro-inflammatory pathways involving specific transcriptional master regulators, some of which are established, others newly identified. Notably, several of these regulators control both lipid metabolism and inflammation, and thereby link the two processes.
With increasing dietary cholesterol intake the liver switches from a mainly resilient (LC) to a predominantly inflammatory (HC) state, which is associated with early lesion formation. Newly developed, functional systems biology tools allowed the identification of novel regulatory pathways and transcriptional regulators controlling both lipid metabolism and inflammatory responses, thereby providing a rationale for an interrelationship between the two processes.
Scope
The drug fenofibrate and dietary fish oils can effectively lower circulating triglyceride (TG) concentrations. However, a detailed comparative analysis of the effects on the plasma metabolome ...is missing.
Methods and Results
Twenty overweight and obese subjects participate in a double‐blind, cross‐over intervention trial and receive in a random order 3.7 g day‐1 n‐3 fatty acids, 200 mg fenofibrate, or placebo treatment for 6 weeks. Four hundred twenty plasma metabolites are measured via gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography–mass spectrometry (LC‐MS). Among the treatments, 237 metabolites are significantly different, of which 22 metabolites change in the same direction by fish oil and fenofibrate, including a decrease in several saturated TG‐species. Fenofibrate additionally changes 33 metabolites, including a decrease in total cholesterol, and total lysophosphatidylcholine (LPC), whereas 54 metabolites are changed by fish oil, including an increase in unsaturated TG‐, LPC‐, phosphatidylcholine‐, and cholesterol ester‐species. All q < 0.05.
Conclusion
Fenofibrate and fish oil reduce several saturated TG‐species markedly. These reductions have been associated with a decreased risk for developing cardiovascular disease (CVD). Interestingly, fish oil consumption increases several unsaturated lipid species, which have also been associated with a reduced CVD risk. Altogether, this points towards the power of fish oil to change the plasma lipid metabolome in a potentially beneficial way.
In a double‐blind, cross‐over trial, 20 overweight and obese subjects receive in a random order 3.7 g day‐1 n‐3 fatty acids, 200 mg fenofibrate, or placebo treatment for 6 weeks. Four hundred twenty plasma metabolites are measured via GC‐MS and LC‐MS. Among the treatments, 237 metabolites are significantly different. Fenofibrate and fish oil reduce saturated TG‐species (containing <5 double bonds) markedly. Fish oil additionally increases several unsaturated lipid species. Altogether, this points towards the power of fish oil to change the plasma lipid metabolome in a potentially beneficial way.
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