Identification Of Metabolites In Plasma Research Articles

Identification of Plasma Metabolites and Dipeptides as Diagnostic Biomarkers for Psoriasis Vulgaris through Liquid Chromatography-High Resolution Mass Spectrometry-Based Metabolomics.

Psoriasis, an immune-mediated chronic inflammatory skin disease, is primarily diagnosed through clinical assessment. Currently, specific markers for the accurate diagnosis and prediction of psoriatic disease are lacking. Here, we employed a three-step designed study to perform untargeted metabolomics, with the aim of identifying candidate biomarkers for psoriasis. Through comprehensive multivariate and univariate statistical analyses, we screened eight potential biomarkers specific to psoriasis, with five structurally identified. Two dipeptide biomarkers, γ-GluSer and ThrGly, along with a lysine glycation metabolite, Nα-fructosyl-lysine (Fruc-Lys), were found to be psoriasis biomarkers for the first time. Receiver operating characteristic curve analysis revealed that the area under the curve (AUC) values of these eight metabolites/features ranged from 0.68 to 0.94. A biomarker panel comprising ThrGly and feature m/z 120.0656 (C4H9NO3) demonstrated high diagnostic accuracy (AUC = 0.97) in distinguishing psoriasis patients from healthy controls. Overall, our study identified and validated a panel of plasma metabolites/features that could serve as potential biomarkers for the diagnosis of psoriasis, providing new insights into the diagnosis and pathogenesis of this disease.

Data-Independent Acquisition for the Quantification and Identification of Metabolites in Plasma.

A popular fragmentation technique for non-targeted analysis is called data-independent acquisition (DIA), because it provides fragmentation data for all analytes in a specific mass range. In this work, we demonstrated the strengths and weaknesses of DIA. Two types of chromatography (fractionation/3 min and hydrophilic interaction liquid chromatography (HILIC)/18 min) and three DIA protocols (variable sequential window acquisition of all theoretical mass spectra (SWATH), fixed SWATH and MSALL) were used to evaluate the performance of DIA. Our results show that fast chromatography and MSALL often results in product ion overlap and complex MS/MS spectra, which reduces the quantitative and qualitative power of these DIA protocols. The combination of SWATH and HILIC allowed for the correct identification of 20 metabolites using the NIST library. After SWATH window customization (i.e., variable SWATH), we were able to quantify ten structural isomers with a mean accuracy of 103% (91–113%). The robustness of the variable SWATH and HILIC method was demonstrated by the accurate quantification of these structural isomers in 10 highly diverse blood samples. Since the combination of variable SWATH and HILIC results in good quantitative and qualitative fragmentation data, it is promising for both targeted and untargeted platforms. This should decrease the number of platforms needed in metabolomics and increase the value of a single analysis.

Identification of metabolites in plasma for predicting survival in glioblastoma.

Circulating metabolomics profiling holds prognostic potential. However, such efforts have not been extensively carried out in glioblastoma. In this study, two-step (training and testing) metabolomics profiling was conducted from the plasma samples of 159 glioblastoma patients. Metabolomics profiling was tested for correlation with 2-year overall and disease-free survivals. Arginine, methionine, and kynurenate levels were significantly associated with 2-year overall survival in both the training and testing sets. In the combined sets, elevated levels of arginine and methionine were associated with a 34% and 37% increased probability whereas kynurenate was associated with a 55% decreased probability of 2-year overall survival. These three metabolites were also significantly associated with 2-year disease-free survival. Risk scores were generated using the linear combination of levels of these significant metabolites. Glioblastoma patients with a high-risk score exhibited a 2.41-fold decreased probability of 2-year overall survival (hazard ratio (HR) = 2.41; 95% Confidence Interval (CI) = 1.20-4.93) and a 3.17-fold decreased probability of 2-year disease free survival (HR = 3.17, 95%CI = 1.42-7.54) relative to those with a low-risk score. In conclusion, we identified a unique plasma metabolite profile that is predictive of glioblastoma prognosis.

PM165 Identification of Plasma Metabolites for Patients on Warfarin With Unstable and Stable International Normalized Ratio

PM165 Identification of Plasma Metabolites for Patients on Warfarin With Unstable and Stable International Normalized Ratio

Identification of domperidone metabolites in plasma and urine of gastroparesis patients with LC–ESI-MS/MS

1. Domperidone is a prokinetic agent used to treat gastroparesis. Previous studies reported oxidative metabolites of domperidone, detected by radiometric high-performance liquid chromatography or single quadrupole mass spectrometric techniques. Our aim was to identify domperidone Phase I and Phase II metabolites using liquid chromatography combined with electrospray ionization-enabled tandem mass spectrometry.2. Domperidone metabolites were identified in the plasma and urine of 11 gastroparesis patients currently being treated with domperidone. In addition, oxidative and conjugative metabolites of domperidone were characterized in human liver subcellular fractions.3. Seven metabolites were detected in vivo. Domperidone was metabolized to two mono-hydroxylated metabolites (M1 and M2), a de-alkylated metabolite (M5) and a di-hydroxylated metabolite (M7). The mono-hydroxylated metabolites were further glucuronidated to M8, M9 and sulfated to M11. To the best of our knowledge, M7, M8, M9 and M11 have not been reported previously. Five additional metabolites were identified in vitro in human subcellular fractions which comprise two additional mono-hydroxylated metabolites (M3 and M4), an alcohol metabolite (M6) possibly formed from an aldehyde intermediate, and other conjugative metabolites (M10 and M12). M6, M10 and M12 have not been characterized previously.4. In total, 12 domperidone metabolites including 7 new metabolites were identified in the present study. These results allow a better understanding of domperidone disposition in humans.

Abstract 3501: Identification of plasma metabolites as biomarker candidates for the diagnosis of pancreatic ductal adenocarcinoma (PDAC).

Abstract Background and objective: Pancreatic cancer (PDAC) is characterized by very poor prognosis mostly caused by late diagnosis. Although modern imaging techniques have pushed the detection limit to lesions below 10mm. The combination of diagnostic imaging and biomarkers can distinguish between PDAC and chronic pancreatitis in only 67% (Carriere et al. J Eval Clin Pract. 2009). We have therefore conducted discovery and confirmation studies to identify metabolite plasma biomakers for the detection of pancreatic cancer and the differentiation from chronic pancreatitis. Material and method:The retrospective study was conducted in three phases: An initial pilot study on plasma samples was followed by analysis of a second plasma sample collection and a serum sample collection from pancreatic cancer, chronic pancreatitis and liver cirrhosis patients as well as blood donors. Metabolomic profiles of plasma and serum samples were generated applying a high throughput polar and lipid GC-MS and LC-MS/MS technology (MxP™ Broad Profiling). In addition, targeted platforms for steroids and lipids (MxP™ Steroid, MxP™ Lipids) were applied. Up to 477 metabolites were analyzed semi-quantitatively or quantitatively per study, 90% of them with an identified chemical structure. Statistical data analysis was done by linear models (ANOVA) on log10 transformed data considering age, gender, BMI and sample storage time as fixed effects. A panel of metabolites was selected for the creation of a diagnosis biomarker. The predictive ability of the biomarker was evaluated through the estimation of ROC characteristics and AUC values from Bootstrap-based Cross-Validation. Result within the three consecutive studies, sphingolipids (sphingomyelins and ceramides) were consistently and significantly increased in the pancreatic cancer group relative to the corresponding pancreatitis group whereas certain amino acids, amino acid related metabolites and coenzyme Q9 were consistently and significantly decreased. A multi-marker panel consisted of 10 metabolites and provided an AUC=0.85 when discriminating between pancreatic cancer and pancreatitis. When the CA19-9 data was included in the analysis, an AUC= 0.92 was reached. Conclusions: The results from the metabolomics study indicate that a plasma metabolite biomarker panel can be used to distinguish between pancreatic cancer and chronic pancreatitis with a high degree of accuracy. The most discriminating metabolites showed robustness with respect to transferability of their diagnostic potential from plasma to serum. A multicenter validation study has now been initiated to establish a diagnostic assay with a targeted negative predictive value (NPV) > 95%. Such a test would allow the exclusion of suspicious patients from further more invasive diagnostic procedures. Citation Format: Julia Mayerle, Holger Kalthoff, Regina Reszka, Beate Kamlage, Christian Pilarsky, Robert Grützmann, Bodo Schniewind, F. Ulrich Weiss, Markus M. Lerch. Identification of plasma metabolites as biomarker candidates for the diagnosis of pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3501. doi:10.1158/1538-7445.AM2013-3501

A Single Dose Mass Balance Study of the Hedgehog Pathway Inhibitor Vismodegib (GDC-0449) in Humans Using Accelerator Mass Spectrometry

Vismodegib (GDC-0449), a small-molecule Hedgehog pathway inhibitor, was well tolerated in patients with solid tumors and showed promising efficacy in advanced basal cell carcinoma in a Phase I trial. The purpose of the study presented here was to determine routes of elimination and the extent of vismodegib metabolism, including assessment and identification of metabolites in plasma, urine, and feces. Six healthy female subjects of nonchildbearing potential were enrolled; each received a single 30-ml oral suspension containing 150 mg of vismodegib with 6.5 μg of [(14)C]vismodegib to yield a radioactivity dose of approximately 37 kBq (1000 nCi). Plasma, urine, and feces samples were collected over 56 days to permit sample collection for up to 5 elimination half-lives. Nonradioactive vismodegib was measured in plasma using liquid chromatographic-tandem mass spectrometry, and total radioactivity in plasma, urine, and feces was measured using accelerator mass spectrometry. Vismodegib was slowly eliminated by a combination of metabolism and excretion of parent drug, most of which was recovered in feces. The estimated excretion of the administered dose was 86.6% on average, with 82.2 and 4.43% recovered in feces and urine, respectively. Vismodegib was predominant in plasma, with concentrations representing >98% of the total circulating drug-related components. Metabolic pathways of vismodegib in humans included oxidation, glucuronidation, and uncommon pyridine ring cleavage. We conclude that vismodegib and any associated metabolic products are mainly eliminated through feces after oral administration in healthy volunteers.

Metabolism and transport pathways of the blood stable camptothecin AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin)

2553 Background: AR-67 is a 3rd generation camptothecin analog with superior stability of its active lactone form (>85%). This stability has been credited in part to the -R10 hydroxyl group (-OH); a group also present on the two clinically approved analogs, topotecan and irinotecan. As with SN-38, the active metabolite of irinotecan, the -OH of AR-67 may be a site for glucuronidation by a UDP glucuronosyltransferase. Moreover, many camptothecins are substrates for the efflux transporter BCRP (ABCG2) and the anionic carboxylate may be a substrate for the liver uptake transporter OATP1B1 (SLCO1B1). Pharmacogenetic studies have established that significant portions of the population have polymorphisms in these genes, which lead to altered pharmacokinetics and toxicity profiles of their substrates. This report describes in-vitro metabolism and transport studies with AR-67 as well as identification of metabolites in plasma and urine from patients enrolled in a phase I study. Methods: AR-67 and its metabolites were detected using chromatography methods with fluorescence or MS/MS detection. In vitro metabolism studies were done with UGT and CYP450 enzyme isoforms expressed in Sf9 cells. In vivo AR-67 metabolic products were identified in clinical plasma and urine specimens. Collection of clinical specimens has been described (control #09-AB-30336-ASCOAM). AR-67 uptake and efflux studies were performed in cells transfected with ABCG2 or SLCO1B1. Results: AR-67 glucuronidation was demonstrated to occur in vitro, primarily by UGT1A8. AR-67 was also found to be a substrate of CYP3A4 and CYP3A5. Only limited glucuronide and oxidation metabolite peaks were observed in patient samples at the highest concentrations. Diminished accumulation of AR-67 lactone was observed in BCRP expressing Saos-2 cells and significant uptake of AR-67 carboxylate was observed in OATP1B1 expressing cells. Conclusions: Unlike SN-38, AR-67 was not found to be a major substrate of UGT1A1, but was rapidly processed by UGT1A8, an isoform that is highly expressed in the GI tract. These data may explain the absence of diarrhea during this phase I study. The pharmacologic significance of ABCG2 and SLCO1B1 polymorphisms should be explored further in the context of population studies. [Table: see text]

Identification of metabolites in plasma and urine of Uruguayan propolis-treated rats.

Propolis is a resinous substance collected by honeybees from various plant sources. It is extensively used in food and beverages to improve health and prevent diseases such as heart disease, diabetes, and cancer. To investigate the absorption and metabolism of the components in propolis, in the present study, we administered ethanol extracts of Uruguayan propolis (poplar type propolis) orally to rats and analyzed their plasma and urine by high-performance liquid chromatography with photodiode array and mass spectrometric detection. After deconjugation of the components by beta-glucuronidase/sulfatase treatment of the specimen, pinobanksin 5-methyl ether, pinobanksin, kaempferol, chrysin, pinocembrin, and galangin were detected in plasma of rats orally administered propolis. These compounds were detected also in urine after beta-glucuronidase/sulfatase treatment. Furthermore, pinobanksin 5-methyl ether, pinobanksin, chrysin, pinocembrin, and galangin were present in the urine also in free form. These results suggest that flavonoids in propolis are metabolized and circulate in the body after oral administration of propolis.

Identification of Some Abnormal Metabolites in Plasma from Uremic Subjects

We describe a method for comparing plasma samples from healthy subjects and from chronic uremic patients before and after dialysis. It was used to determine the nature of those metabolites that appear to characterize the uremic state. Preliminary fractionation of the metabolites by gel chromatography was followed by removal of the aqueous effluent by lyophilization and preparation of volatile trimethylsilyl derivatives, which were then examined by gas-liquid chromatography. Gas-liquid chromatography/mass spectrometry was used to characterize and identify individual metabolites. Gas-liquid chromatographic patterns of plasma from healthy and uremic subjects differ markedly, more so than do individual plasma samples within the same class of subjects. Concentrations of many metabolites are increased in uremia, but after dialysis of the patient's blood, the concentrations become about the same as those in healthy plasma. We have observed some 150-200 metabolites in each category of plasma. We have tentatively identified about a tenth of the compounds that appear to be specific to or increased in uremia, including lactic acid, glycerol, erythritol, erythronic acid, 2-deoxy erythro pentonic acid, arabinitolarabinonic acid, inositol, and lactose. Some of these are present in concentrations greater than 20 mg/liter and have not been previously reported as occurring in the uremic state.