Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS
2007; Elsevier BV; Volume: 48; Issue: 7 Linguagem: Inglês
10.1194/jlr.m700097-jlr200
ISSN1539-7262
AutoresWeiying Yan, Gary D. Byrd, Michael W. Ogden,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoA simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF2α-III, 15-epi-iPF2α-III, iPF2α-VI, and 8,12-iso-iPF2α-VI along with the prostaglandin PGF2α and 2,3-dinor-iPF2α-III, a metabolite of iPF2α-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF2α-III obtained by our method were significantly correlated with results by an ELISA, although an ∼2-fold high bias was observed for the ELISA data. For iPF2α-III and its metabolite 2,3-dinor-iPF2α-III, smokers had significantly higher concentrations than nonsmokers (513 ± 275 vs. 294 ± 104 pg/mg creatinine; 3,030 ± 1,546 vs. 2,046 ± 836 pg/mg creatinine, respectively). The concentration of iPF2α-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents. A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF2α-III, 15-epi-iPF2α-III, iPF2α-VI, and 8,12-iso-iPF2α-VI along with the prostaglandin PGF2α and 2,3-dinor-iPF2α-III, a metabolite of iPF2α-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF2α-III obtained by our method were significantly correlated with results by an ELISA, although an ∼2-fold high bias was observed for the ELISA data. For iPF2α-III and its metabolite 2,3-dinor-iPF2α-III, smokers had significantly higher concentrations than nonsmokers (513 ± 275 vs. 294 ± 104 pg/mg creatinine; 3,030 ± 1,546 vs. 2,046 ± 836 pg/mg creatinine, respectively). The concentration of iPF2α-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents. collision-induced dissociation electrospray ionization Food and Drug Administration Center for Drug Evaluation and Research hydrochloric acid liquid chromatography-mass spectrometry liquid chromatography-tandem mass spectrometry limit of quantitation multiple reaction monitoring ammonium hydroxide quality control relative standard deviation Isoprostanes are prostaglandin-like compounds produced in vivo by nonenzymatic free radical-induced peroxidation of arachidonic acid (1.Morrow J.D. Hill K.E. Burk R.F. Nammour T.M. Badr K.F. Roberts L.J. A series of prostaglandin-F2-like compounds are produced in vivo in humans by a noncyclooxygenase, free radical-catalyzed mechanism.Proc. Natl. Acad. Sci. USA. 1990; 87: 9383-9387Crossref PubMed Scopus (1719) Google Scholar). 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A recent study investigating biomarkers of harm in smokers and nonsmokers found that nonsmokers had significantly reduced urinary iPF2α-III compared with smokers (29.Zedler B.K. Kinser R. Oey J. Nelson B. Roethig H.J. Walk R.A. Kuhl P. Rustemeier K. Schepers G. Holt K.Von et al.Biomarkers of exposure and potential harm in adult smokers of 3–7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers.Biomarkers. 2006; 11: 201-220Crossref PubMed Scopus (40) Google Scholar). On the other hand, some studies with small, targeted populations have revealed no significant differences in urinary iPF2α-III levels in smokers and nonsmokers (30.Tsikas D. Schwedhelm E. Suchy M.T. Niemann J. Gutzki F.M. Erpenbeck V.J. Hohfeld J.M. Surdacki A. Frolich J.C. 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Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods.J. Chromatogr. B. 2005; 827: 157-164Crossref PubMed Scopus (52) Google Scholar). Although isoprostanes occur in various biological fluids, a urine assay is attractive because it is noninvasive, minimizes subject discomfort, requires little expertise to collect the specimen, and thus may be more suitable for large clinical trials. Plus, it circumvents the problem of artifactual generation of isoprostanes compared with plasma or other lipid-containing tissue or fluid (59.Roberts L.J. Morrow J.D. Measurement of F2-isoprostanes as an index of oxidative stress in vivo.Free Radic. Biol. Med. 2000; 28: 505-513Crossref PubMed Scopus (855) Google Scholar, 60.Morrow J.D. Roberts L.J. Mass spectrometric quantitation of F2-isoprostanes in biological fluids and tissues as measurement of oxidant stress.Methods Enzymol. 1999; 300: 3-12Crossref PubMed Scopus (339) Google Scholar). The goal of our work was to develop a simple, accurate, and fast method for the determination of isoprostanes in human urine by LC-MS/MS to permit high-throughput sample analysis. The isomers selected for study were based on commercially available standards and include iPF2α-III, 8,12-iso-iPF2α-VI, 15-epi-iPF2α-III, PGF2α, iPF2α-VI, and 2,3-dinor-iPF2α-III, a major metabolite of iPF2α-III (structures shown in Fig. 1). These compounds have been identified in human urine, and some have been characterized based on their biological activity (11.Cracowski J.L. Isoprostanes: an emerging role in vascular physiology and disease?.Chem. Phys. Lipids. 2004; 128: 75-83Crossref PubMed Scopus (53) Google Scholar, 61.Marliere S. Cracowski J.L. Durand T. Chavanon O. Bessard J. Guy A. Stanke-Labesque F. Rossi J.C. Bessard G. The 5-series F2-isoprostanes possess no vasomotor effects in the rat thoracic aorta, the human internal mammary artery and the human saphenous vein.Br. J. Pharmacol. 2002; 135: 1276-1280Crossref PubMed Scopus (24) Google Scholar). The prostaglandin PGF2α is an isomer of iPF2α-III and was included in this assay even though it is not an isoprostane. The method described here was validated according to Food and Drug Administration Center for Drug Evaluation and Research (FDA/CDER) guidelines (62.FDA/CDER.Guidance for Industry on Bioanalytical Methods Validation. FDA/CDER, Rockville, MD2001Google Scholar). The method was applied in a study of 24 h urine samples from smokers and nonsmokers on controlled diets. Results from smokers and nonsmokers were compared along with the degree of smoking as determined by total nicotine uptake. iPF2α-III, 15-epi-iPF2α-III, iPF2α-VI, 8,12-iso-iPF2α-VI, PGF2α, 2,3-dinor-iPF2α-III, and iPF2α-III-d4 were obtained from Cayman Chemicals (Ann Arbor, MI). Urea, potassium chloride, hippuric acid, ammonium chloride, anhydrous magnesium sulfate, anhydrous calcium chloride, oxalic acid, anhydrous sodium silicate, creatinine, anhydrous sodium sulfate, and pepsin were from Sigma-Aldrich (St. Louis, MO). Citric acid, anhydrous sodium dihydrogen phosphate, and glucose were from Aldrich (Milwaukee, WI). Lactic acid was obtained from Fluka (Lausanne, Switzerland). High-purity water from a Milli-Q A10 Synthesis purification system (Millipore, Bedford, MA) was used for all solutions. High-purity acetonitrile, methanol, and hexane were obtained from Burdick and Jackson (Muskegon, MI). Ammonium hydroxide (NH4OH) and sodium chloride were obtained from Acros Organics (Fair Lawn, NJ), and hydrochloric acid (HCl) was obtained from Fisher Scientific (Fair Lawn, NJ). Urine samples from 24 h collections were portioned into 50 ml plastic (modified polystyrene) centrifuge tubes (Corning, Corning, NY) and stored at −80°C until analysis. After thawing, 2 ml was divided into aliquots on deep 96-well plates (Phenomenex, Torrance, CA). Centrifugation of samples was done on an IEC Centra-GP8R centrifuge (Needham Heights, MA). Oasis® HLB and MAX 96-well extraction plates (10 and 30 mg) were from Waters (Milford, MA). Extraction was performed on a Phenomenex 96-well plate manifold with a customized insert to adapt shallow 0.5 ml 96-well plates (Agilent Technologies, Palo Alto, CA) for collection. For each sample, 2 ml of urine was fortified with 50 μl of iPF2α-III-d4 internal standard solution (20 ng/ml) to a concentration of 500 pg/ml. It was then acidified to approximately pH 3 by the addition of 40 μl o
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