Metabolomics - PDF document

1 Metabolomics approach to assessing
Metabolomics approach to assessing plasma 13 - and 9 - hydroxy - octadecadienoic acid and linoleic acid metabolite responses to 75 - km cycling David C. Nieman, 1 R. Andrew Shanely, 1 Beibei Luo, 2 Mary Pat Meaney, 1 Dustin A. Dew, 1 and Kirk L. Pappan 3 1 Appalachian State University, Human Performance Lab, North Carolina Research Campus, Kannapolis, North Carolina; 2 Key Laboratory of Exercise and Health Sciences of Ministry of Education, Shanghai University of Sport, Shanghai, China; and 3 Metabolon Inc., Durham, North Carolina Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13 - and 9 - hydroxy - octadecadienoic acid (13 - HODE + 9 - HODE) and have been linked to oxidative stress, inflammation, and numerous pathological and physiological states. The purpose of this study was to measure changes in plasma 13 - HODE + 9 - HODE following a 75 - km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F 2 - isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists ( N = 19, age 38.0 ± 1.6 yr, watts max 304 ± 10.5) engaged in a 75 - km cycling time trial on their own bicycles using electromagnetically braked cycling ergometers (2.71 ± 0.07 h). Blood samples were collected preexer - cise, immediately post - , 1.5 h post - , and 21 h postexercise, and analyzed for plasma cytokines (IL - 6, IL - 8, IL - 10, tumor necrosis factor - ex , monocyte chemoattractant protein - 1, granulocyte colony - stimulating factor), F 2 - isoprostanes, and shifts in metabolites using global metabolomics procedures with gas chromatography mass spec - trometry (GC - MS) and liquid chromatography mass spectrometry (LC - MS). 13 - HODE + 9 - HODE increased 3.1 - fold and 1.7 - fold immediately post - and 1.5 h postexercise (both P 0.001) and returned to preexercise levels by 21 - h postexercise. Post - 75 - km cy - cling plasma levels of 13 - HODE + 9 - HODE were not significantly correlated with increases in plasma cytokines but were positively correlated with postexercise F 2 - isoprostanes ( r = 0.75, P 0.001), linoleate ( r = 0.54, P = 0.016), arachidate ( r = 0.77, P 0.001), 12,13 - dihydroxy - 9Z - octadecenoate (12,13 - DiHOME) ( r = 0.60, P = 0.006), dihomo - linolenate ( r = 0.57, P = 0.011), and adrenate ( r = 0.56, P = 0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1 - fold increase in the stable linoleic acid oxidation product 13 - HODE + 9 - HODE and was related to increases in F 2 - isoprostanes, linoleate, and fatty acids in the linoleate conver - sion pathway. These data support the use of 13 - HODE + 9 - HODE as an oxidative stress biomarker in acute exercise investigations. exercise; oxidative stress; inflammation; linoleate; metabolites THE RATE OF WHOLE BODY and muscle oxygen consumption increases during acute, high - intensity, and continuous exercise bouts and is accompanied by an increase in production of reactive oxygen species (ROS) (8, 26, 27). The causes of increased ROS during exercise include electron leakage in the mitochondrial electron transport chain, activation of neutro - Address for reprint requests and other correspondence: D. C. Nieman, DrPH, North Carolina Research Campus, 600 Laureate Way, Kannapolis, NC 28081 (e - mail: niemandc@appstate.edu). phils and other phagocytic cells, auto - oxidation of cat - echolamines, and activation of several enzymes including xan - thine oxidase, phospholipase A2, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (8). Direct measure - ment of free radical and ROS production is difficult, and assessment of oxidative stress during exercise is typically made using indirect methods (22). Reliable oxidative stress biomark - ers should be chemically unique and detectable, have relativ ely long half - lives, and be responsive to increases or decreases in ROS (26). The level of F 2 - isoprostanes (F 2 - IsoP) in blood or urine is widely regarded as an excellent indicator of exercise - induced oxidative stress and is formed via free radical - initiated peroxidation of arachidonic acid (22, 30). Linoleic acid (18:2n - 6) is the most common polyunsaturated fatty acid (PUFA) in human diets and mammalian tissue and is considered essential because humans lack delta - 12 and del - ta - 15 desaturase enzymes (9, 15, 29, 33). Ingested linoleic acid is converted to longer and more unsaturated fatty acids through enzyma tic desaturation and elongation in the endoplasmic reticulum of cells. Linoleic acid is the direct precursor to oxidized linoleic acid metabolites (OXLAMs) including 13 - and 9 - hydroxy - octadecadienoic acid (13 - HODE + 9 - HODE) (9, 15, 29, 33) (see http://www.genome.jp/kegg - bin/show_ pathway?map00591 for more detail). 13 - HODE + 9 - HODE are stable oxidation products and have been linked to patho - logical conditions including atherosclerosis, diabetes, Alzhei - ogt’u disease, non - alcoholic steatohepatitis, psoriasis, chronic inflammation, obesity, and cancer (14, 23, 29, 37). Plasma levels of 13 - HODE + 9 - HODE are responsive to lifestyle interventions, with decreases reported when subjects adopt healthy diets and lose excess body weight (2, 3, 7, 25). 13 - HODE + 9 - HODE are generated through the 15 - lipoxy - genase - 1 (15 - LOX) pathway in a variety of cell types (17, 25), are ligands of peroxisome proliferator - activated receptors (PPARs) (28), and can act through G protein - coupled receptor 132 (GPR132) to exert pro - inflammatory effects (37). Little information has been published on the influence of exercise on plasma 13 - HODE + 9 - HODE, and whether postexercise increases relate to changes in established bio - markers of oxidative stress and inflammation (19, 20). Using a metabolomics approach, we recently reported that plasma 13 - HODE + 9 - HODE increased 5.5 - fold in 15 runners following a 3 - day period of intensified training (2.5 h/day running at 70% V ˙ O 2max ), with levels returning to preexercise levels after 14 - h recovery (20). Metabolomics allows the simultaneous mea - surement of hundreds of metabolites and is especially valuable when focusing on complex interactions within the body during exercise or nutrition interventions. The purpose of this study was to measure the effect of prolonged and intensive exercise on plasma 13 - HODE + 9 - HODE and linoleic acid metabolism and correlations with postexercise increases in plasma F 2 - IsoP and inflammatory cytokines. MATERIALS AND METHODS Subjects. Subjects included 19 male cyclists (ages 27 – 49 yr) who regularly competed in road races (racing categories 1 to 5, with 5 assigned to cyclists with the lowest performance ability and experi - ence) and were capable of cycling 75 km at race pace. Subjects trained normally, maintained weight, and avoided the use of large - dose vitamin and mineral supplements (above 100% Daily Value), and all herbal supplements and medications for the 2 - wk period before the 75 - km cycling time trial. All subjects signed informed consent and all study procedures were approved by the Institutional Review Board at Appalachian State University. Research design. Two weeks before the 75 - km time trial, athletes completed orientation and baseline testing in the North Carolina Research Campus Human Performance Laboratory operated by Ap - palachian State University in Kannapolis, NC. Demographic and training histories were acquired with questionnaires. Maximal power, oxygen consumption, ventilation, and heart rate were measured during a graded exercise test (25 Watts increase every 2 min, starting at 150 Watts) with the Cosmed Quark CPET metabolic cart (Rome, Italy) and the Lode cycle ergometer (Lode Excaliber Sport, Lode, Gro - ningen, The Netherlands). Body composition was measured with the Bod Pod body composition analyzer (Life Measurement, Con - cord, CA). Two weeks after baseline testing, subjects returned to the Human Performance Laboratory at 6:45 AM in an overnight fasted state (no food or beverages other than water for at least 9 hours) and provided a preexercise blood sample. After warming up, subjects cycled on their own bicycles on CompuTrainer Pro model 8001 trainers (Rac - erMate,

2 Seattle, WA) with heart rate
Seattle, WA) with heart rate and rating of perceived exertion (RPE) recorded every 30 min and workload continuously monitored using the CompuTrainer MultiRider software system (version 3.0, RacerMate, Seattle, WA). A mountainous 75 - km course with moder - ate difficulty was chosen and programmed into the software system. Oxygen consumption and ventilation were measured using the Cosmed Quark CPET metabolic cart after 16 and 55 km cycling. Subjects were allowed to ingest water ad libitum during the 75 - km cycling time trial, but not any other beverage or food containing energy or nutrients. Blood samples were taken via venipuncture immediately and 1.5 h after completing the 75 - km time trial. Subjects returned in an overnight fasted state the next morning to provide a 21 - h postexercise blood sample. No dietary restrictions were imposed from 1.5 - h postexercise to the beginning of the overnight fast. F 2 - isoprostanes. Plasma F 2 - IsoP were determined using gas chro - matography mass spectrometry (GC - MS) (18). Plasma was collected from heparinized blood, flash - frozen in liquid nitrogen, and stored at - 80°C. Plasma samples were thawed, and free F 2 - IsoP was extracted with deuterated [ 2 H 4 ]prostaglandin F 2 ex added as an internal standard. Waters Sep - Pak C18 cartridges followed by Waters Sep - Pak Silica cartridges were used for solid phase extraction. F 2 - IsoP were con - verted to pentafluorobenzyl esters, subjected to thin layer chromatog - raphy, and converted to trimethylsilyl ether derivatives. Samples were analyzed by a negative ion chemical ionization GC - MS using an Agilent 6890N gas chromatography interfaced to an Agilent 5975B inert MSD mass spectrometer (Agilent Technologies, Santa Clara, CA). Cytokines. Total plasma concentrations of six inflammatory cyto - kines [monocyte chemoattractant protein - 1 (MCP - 1), tumor necrosis factor - ex (TNF - ex ), granulocyte colony - stimulating factor (GCSF), IL - 6, IL - 8, and IL - 10] were determined using an electrochemilumi - nescence based solid - phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD). All samples and provided standards were analyzed in duplicate, and the intra - assay coefficient of variation (CV) ranged from 1.7% to 7.5% and the interassay CV 2.4 to 9.6% for all cytokines measured. Pre - and postexercise samples for the cyto - kines were analyzed on the same assay plate to decrease interkit assay variab ility. Metabolomics. The nontargeted metabolic profiling instrumenta - tion employed for this analysis combined three independent plat - forms: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) optimized for basic species, UHPLC/ MS/MS optimized for acidic species, and GC/MS (1, 6). Blood samples were collected in EDTA tubes and centrifuged at 3,000 rpm for 10 min at 4°C, with the plasma aliquoted, snap frozen in liquid nitrogen, and stored at - 80°C until analysis. For each plasma sample, 100 µ l was used for analyses. With the use of an automated liquid handler (Hamilton LabStar, Salt Lake City, UT), protein was precip - itated from the plasma with methanol that contained four standards to report on extraction efficiency. The resulting supernatant was split into equal aliquots for analysis on the three platforms. Aliquots, dried under nitrogen and vacuum desiccated, were subsequently either reconstituted in 50 µ l 0.1% formic acid in water (acidic conditions) or in 50 µ l 6.5 mM ammonium bicarbonate in water, pH 8 (basic conditions) for the two UHPLC/MS/MS analyses or derivatized to a final volume of 50 µ l for GC/MS analysis using equal parts bistrim - ethyl - silyl - trifluoroacetamide and solvent mixture acetonitrile - dichlo - romethane - cyclohexane (5:4:1) with 5% triethylamine at 60°C for 1 h. In addition, three types of controls were analyzed in concert with the experimental samples: aliquots of a well - characterized human plasma pool served as technical replicates throughout the data set, extracted water samples served as process blanks, and a cocktail of standards spiked into every analyzed sample allowed instrument perfor - mance monitoring. Standards to monitor extraction were d6 - cho - lesterol, fl and tridecanoic acid. A standard to monitor GC/MS derivatization was 2 - tert - butyl - 6 - methylphenol (BHT). GC/MS standards to monitor GC and MS performance were C5 - C18 alkylbenzenes. Experimental samples and controls were ran - domized across platform run days. Instrument variability was deter - mined by calculating the median relative standard deviation (RSD) for the internal standards that were added to each sample before injection into the mass spectrometers. Overall process variability was deter - mined by calculating the median RSD for all endogenous metabolites (i.e., noninstrument standards) present in technical replicates of MTRX3. Values for instrument (5%) and process variability (12%) met Ogvcdqnqp’u acceptance criteria. For UHLC/MS/MS analysis, aliquots were separated using a Wa - ters Acquity UPLC (Waters, Millford, MA) instrument with separate a cid/base - dedicated 2.1 mm X 100 mm Waters BEH C18 1.7 - µ m particle columns heated to 40°C, and analyzed using an LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA), which con - sisted of an electrospray ionization (ESI) source and linear ion - trap (LIT) mass analyzer (6). Extracts reconstituted in formic acid were gradient eluted at 350 µ l/min using 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) (0% B to 70% B in 4 min, 70 – 98% B in 0.5 min, 98% B for 0.9 min), whereas extracts reconstituted in ammonium bicarbonate used 6.5 mM ammonium bicarbonate in Table 1. Subject characteristics Variable Means ± SE Age, yr 38.0 ± 1.6 Height, m 1.81 ± 0.02 Weight, kg 76.8 ± 2.3 Body fat, % 14.0 ± 1.0 Watts max 304 ± 10.5 V ˙ O 2max , ml·kg - 1 ·min - 1 51.7 ± 1.4 HR max , beats/min 179 ± 2.5 Training, km/wk 192 ± 18.0 n = 19 subjects; V ˙ O 2max , maximal oxygen consumed; HR, heart rate. Table 2. Average 75 - km metabolic and performance data Variable Means ± SE Time, h 2.71 ± 0.07 V O 2 , ml·kg - 1 ·min - 1 38.3 ± 1.2 V O 2 ,%V O 2max 69.3 ± 2.1 Watts 207 ± 7.6 % Watts max 68.3 ± 1.7 HR, beats/min 150 ± 2.5 %HR max 84.0 ± 1.1 Ventilation, l/min 74.9 ± 3.5 RPE 12.9 ± 0.3 V O 2 , volume of oxygen consumed; RPE, rating of perceived exertion. water, pH 8 (A) and 6.5 mM ammonium bicarbonate in 95/5 metha - nol - water (B) (same gradient profile as above) at 350 µ l/min. The MS instrument scanned 99 – 1,000 molecular weight ( m/z ) and alternated between MS and MS2 scans using dynamic exclusion with approxi - mately 6 scans per second. Derivatized samples for GC/MS were separated on a 5% diphenyl - 95% dimethyl polysiloxane - fused silica column with helium as the carrier gas and a temperature ramp from 60°C to 340°C and then analyzed on a Thermo - Finnigan Trace DSQ MS (Thermo Fisher Scientific) operated at unit mass resolving power with electron impact ionization and a 50 - to 750 - atomic mass unit scan range. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight ( m/z ), preferred adducts, and in - source fragments as well as associated MS spectra, and were curated by visual inspection for quality control using software developed at Metabolon (Durham, NC) (4). Common and biologically abundant isomers of unsaturated fatty acids containing the n3, n6, and n9 configuration are contained in Ogvcdqnqp’u chemical standard library. Liquid chromatography mass spectrometry (LC - MS) standards to monitor LC and MS performance were d 3 - leucine, chloro - and bromo - phenylalanine, d 2 - maleic acid, amitriptyline, and d10 - benzophenone (1). Biochemical identifications were based on three criteria: retention index within a narrow window of the proposed identification, accurate mass match to the library + / - 0.005 amu, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores were based on a comparison of

3 the ions present in the experi
the ions present in the experimental spectrum to the ions present in the library spectrum. Statistical analysis. Data are expressed as means ± SE. For the metabolomics statistical analyses and data display purposes, any missing values were assumed to be below the limits of detection and these values were imputed with the compound minimum (minimum value imputation). Statistical analysis of log - transformed data was performed using “T” (R Foundation, from http://cran.r - project.org/), which is a freely available, open - source software package. One - way ANOVA with post hoc contrasts ( t - tests) was performed to compare data across time points. An estimate of the false discovery rate ( q value) was calculated to take into account the multiple comparisons that normally occur in metabolomic - based studies, with q 0.05 used as an indication of high confidence in a result (34). Fold changes across time points were calculated using group averages of the median scaled intensity values. Plasma cytokines and F 2 - IsoP were analyzed using one - way ANOVA with post hoc contrasts ( t - tests) performed to compare data across time points. Correlations between 13 - HODE + 9 - HODE and other variables were made using Rgctuqp’u product - moment correlation coefficients. RESULTS Subjects included 19 competitive male cyclists (ages 27 to 49 yr) who successfully adhered to all aspects of the study design (see Table 1). Metabolic and performance data from the 75 - km mountainous cycling time trial are summarized in Table 2. The cyclists were able to maintain a high power output (69% Watts max ) during the 75 - km trial, with an average duration o f 2.7 1 h. The metabolomics analysis revealed 423 detectable com - pounds of known identity. After log transformation and impu - tation with minimum observed values for each compound, repeated measures ANOVA contrasts identified significant Pre - 21 h - Post - 75 km Pre - 1.5 h - Post - 75 km Pre - Post Exerc Fig. 1. Fold changes in metabolites grouped ac - cording to subpathways. Numbers in parentheses represent the number of metabolites in each sub - pathway that increased twofold or greater after the 75 - km cycling time trial. Nucleotide, purine (2) Carb metabolism (3) Kreb cycle (2) Hemo globin (3) Carnitines (8) Amino acids (8) Sterol/steroid (1) Dihydroxy FA (2) Bile acid (2) Branched FA (2) Essential FA (7) Lipid Metabolism (2) Monohydroxy FA (5) Medium chain FA (4) Long chain FA (20) Dicarboxylate FA (7) Ketones (2) Fold Change Table 3. Exercise effects on linoleic acid and long chain polyunsaturated fatty acids in the conversion pathway (all time main effects, q 0.001) Variable (Median Scaled Intensity) Preexercise Post - 75 km 1.5 - h Post - 75 km 21 - h Post - 75 km Linoleate (18:2n6) 0.76 ± 0.06 2.86 ± 0.15* (3.7 - fold) 1.96 ± 0.11* (2.6 - fold) 0.83 ± 0.05 Linolenate [ ex or 'Y ; (18:3n3 or 6)] 0.72 ± 0.07 4.23 ± 0.29* (5.8 - fold) 2.43 ± 0.20* (3.4 - fold) 0.76 ± 0.06 Dihomo - linolenate (20:3n3 or n6) 0.77 ± 0.04 2.66 ± 0.16* (3.4 - fold) 2.18 ± 0.16* (2.8 - fold) 0.84 ± 0.04 Arachidonate (20:4n6) 0.80 ± 0.06 1.83 ± 0.12* (2.3 - fold) 1.55 ± 0.08* (1.9 - fold) 0.84 ± 0.06 Adrenate (22:4n6) 0.62 ± 0.08 2.13 ± 0.12* (3.4 - fold) 1.68 ± 0.17* (2.7 - fold) 0.78 ± 0.11 Docosapentaenoate (22:5n6) 0.81 ± 0.06 1.59 ± 0.22* (2.0 - fold) 1.87 ± 0.18* (2.3 - fold) 0.94 ± 0.06 Values are means ± SE. * P 0.01 vs. preexercise. post - 75 km cycling time effects for 221 metabolites. Fold changes were calculated from the median scaled intensity values for pre - to - post - 75 - km cycling time points and then rank ordered. A total of 80 metabolites of known identity had a twofold or higher increase following 75 km cycling (all q values 0.01). These were grouped by metabolic subpath - ways, and three mean fold changes were calculated (preexer - cise to immediately - , 1.5 h post - , and 21 h postexercise) (see Fig. 1 and Supplemental Table S1). All but 26 of these metabolites were related to lipid and carnitine metabolism, with the largest fold changes seen for ketones, dicarboxylate fatty acids, and long chain fatty acids. Table 3 summarizes exercise - induced changes in median scaled intensity (MSI) values for linoleic fatty acid (18:2n6) and five other long chain polyunsaturated fatty acids in the conversion pathway. Fold changes for all of these fatty acids ranged from 2.0 to 5.8 immediately post - 75 km cycling and 1.9 to 3.4 after 1.5 h recovery and were not different from preex - ercise after 21 h recovery. Plasma 13 - HODE + 9 - HODE increased 3.1 - and 1.7 - fold immediately and 1.5 h following the 75 - km cycling time trial, respectively (time main effect, q 0.001) (Fig. 2). Similar patterns of postexercise increases were seen for ( Z ) - 9,10 - dihydroxyoctadec - 12 - enoic acid (9,10 - DiHOME) and ( Z ) - 12,13 - dihydroxyoctadec - 9 - enoic acid 13 - HODE + 9 - HODE 3.0 (12,13 - DiHOME) (both time main effects, q 0.001)(Fig. 3) and F 2 - IsoP (time main effect, P 0.001) (Fig. 4). Plasma cytokine data are summarized in Table 4. Significant postexercise increases were measured for IL - 6 (8.6 - fold), IL - 8 (2.3 - fold), IL - 10 (9.1 - fold), TNF - ex (1.10 - fold), MCP - 1 (1.44 - fold), and GCSF (1.38 - fold), with values near preexercise levels after 21 h recovery (all time main effects, P 0.001). Immediate postexercise MSI values for 13 - HODE + 9 - HODE were significantly correlated with postexercise ara - chidonate ( r = 0.77, P 0.001), 12,13 - DiHOME ( r = 0.60, P = 0.006), and F 2 - IsoP ( r = 0.75, P 0.001) (Fig. 5, A – C ). Postexercise 13 - HODE + 9 - HODE was also significantly correlated with postexercise linoleate ( r = 0.54, P = 0.016), dihomo - linolenate ( r = 0.57, P = 0.011), and adrenate (r = 0.56, P = 0.013), and marginally to linolenate ( r = 0.44, P = 0.058) and 9,10 - DiHOME ( r = 0.41, P = 0.081). Postex - ercise levels of 13 - HODE + 9 - HODE were not correlated with any of the postexercise cytokine concentrations listed in Table 4. Immediate postexercise MSI values for linoleate were sig - nificantly correlated with 9,10 - DiHOME ( r = 0.64, P = 0.003), 12,13 - DiHOME ( r = 0.58, P = 0.10), and each fatty 9,10 DiHOME 12,13 DiHOME 2.0 1.8 1.6 2.5 1.4 1.2 2.0 1.5 1.0 1.0 0.8 0.6 0.4 0.5 0.0 0.2 0.0 Pre - Exercise Post - 75 km 1.5 - h Post - 75 km 21 - h Post - 75 km Fig. 2. Changes in 13 - and 9 - hydroxy - octadecadienoic acid (13 - HODE + 9 - HODE) across four time points: preexercise, immediately post - 75 - km cy - cling time trial, and 1.5 - h and 21 - h postexercise. * P 0.01 compared with preexercise. Time main effect, P 0.001. Fig. 3. Changes in ( Z ) - 9,10 - dihydroxyoctadec - 12 - enoic acid (9,10 - DiHOME) and ( Z ) - 12,13 - dihydroxyoctadec - 9 - enoic acid (12,13 - DiHOME) and across four time points: preexercise, immediately post - 75 - km cycling time trial, and 1.5 - h and 21 - h postexercise. * P 0.01 compared with preexercise. Time main effect, P 0.001. * * * * * * Median Scaled Intensity Median Scaled Intensity * * Post - Exercise F 2 - isoprostanes (pg/ml) 80 Oleic (18:1), palmitic (16:0), and linoleic (18:2) are the three major free fatty acids (FFA) in adipose tissue (9, 15). Upper 70 body subcutaneous adipose tissue is the primary source of FFA during prolonged and intensive exercise (11), and relative 60 availability is the predominant determinant of individual FFA 50 use by contracting muscles (13). Ketone bodies increased strongly in overnight fasted cyclists following the 75 - km cy - 40 cling time trial, indicating a high degree of FFA 13 - oxidation. 30 20 10 0 Pre - Exercise Post - 75 km 1.5 - h Post - 75 km 21 - h Post - 75 km A 3.5 3 2.5 2 Fig. 4. Changes in F 2 - isoprostanes across four time points: preexercise, immediately post - 75 - km cycling time trial, and 1.5 - h and 21 - h postexercise. * P 0.01 compared with preexercise. Time main effect, P 0.001. acid listed in the conversion pathway except for docosapen - taenote

4 ( r = 0.32, P = 0.18; all
( r = 0.32, P = 0.18; all other relationships, P 0.02). DISCUSSION 1.5 1 0.5 0 0 1 2 3 4 5 6 Cyclists experienced a transient 3.1 - fold increase in the oxidized linoleic acid derivative 13 - HODE + 9 - HODE follow - ing the 75 - km cycling time trial. The postexercise increase in plasma concentration of HODEs was related to increases in the well - established oxidative stress biomarker, F 2 - IsoP, but not to increases in inflammatory cytokines. Postexercise HODEs were also positively correlated with exerci se - induced mobili - zation of most components of the linoleic acid conversion pathway including linoleate, dihomo - linolenate, arachidonate, and adrenate, and the oxidized isoleukotoxin 12,13 - DiHOME. Metabolomics data revealed a twofold or greater postexer - cise increase in 80 metabolites of known identity, and two - thirds were related to lipid and carnitine metabolism. These results are very similar to those of our previous metabolomics - based investigation of 15 runners who ran 2.5 h/day at 70% V ˙ O 2max 3 days in a row (20). Together, these two studies indicate that following prolonged and intensive exercise, en - durance athletes experience profound systemic shifts in blood metabolites, especially those from the lipid pathway. In agree - ment with Lehman et al. (17), the data support substantial fatty acid transport across the mitochondrial membrane and oxida - tion during prolonged exercise. B 3.5 3 2.5 2 1.5 1 0.5 0 C 160 140 120 100 Post - Exercise 13 - HODE + 9 - HODE (MSI) 0 1 2 3 4 5 6 Post - Exercise 13 - HODE + 9 - HODE (MSI) 80 Table 4. Exercise effects on plasma cytokines (all time main effects, P 0.001) 60 Variable, pg/ml Preexercise Post - 75 km 1.5 - h Post - 75 km 21 - h Post - 75 40 km 20 IL - 6 1.02 ± 0.28 8.77 ± 0.69* 5.97 ± 0.97* 0.63 ± 0.05 IL - 8 5.22 ± 0.42 11.83 ± 1.1* 7.91 ± 0.74* 4.70 ± 0.33 IL - 10 2.05 ± 0.21 18.6 ± 2.6* 10.6 ± 1.8* 2.14 ± 0.27 TNF - ex 3.90 ± 0.16 4.30 ± 0.20* 4.10 ± 0.20 3.61 ± 0.16 MCP - 1 267 ± 9.1 385 ± 18.9* 340 ± 22.5* 230 ± 7.1 GCSF 12.8 ± 1.0 17.7 ± 1.8* 18.7 ± 2.2* 15.3 ± 1.5 0 0 1 2 3 4 5 6 Post - Exercise 13 - HODE + 9 - HODE (MSI) Fig. 5. Scatterplot relationships between at the postexercise time point for Values are means ± SE. IL, interleukin; TNF - ex , tumor necrosis factor - ex ; MCP - 1, monocyte chemoattractant protein - 1; GCSF, granulocyte colony - stimulating factor. * P 0.01 vs. preexercise. 13 - HODE + 9 - HODE and arachidonate ( r = 0.77, P 0.001) ( A ), 12,13 - DiHOME ( r = 0.60, P = 0.006) ( B ), and F 2 - isoprostanes ( r = 0.75, P 1.1) ( C ). MSI, median scaled intensity. Plasma F2 - soprostanes (pg/ml) Post - Exercise 12,13 DiHOME (MSI) Post - Exercise Arachidate (MS) Plasma linoleate increased 3.7 - fold postexercise in concert with other fatty acids in the conversion pathway, indicating a high degree of mobilization and availability. Linoleic acid is the predominant PUFA in the diet and adipose tissue stores and can be metabolized by cyclooxygen - ase, lipoxygenase, and P450 enzymes (32). 13 - HODE + 9 - HODE are monohydroxy lipoxygenation products and the most widely distributed of linoleic acid metabolites (17, 32). The HODEs are secreted by a variety of cells including macrophages, endothelial cells, platelets, and smooth muscle cells, and exert biological and signaling activities as PPAR and GPR132 ligands (12, 21, 28, 37). Cell injury activates lipoxy - genases and may be one pathway through which intensive exercise increases production of HODES (33). Several cell culture studies indicate that HODES induce pro - inflammatory responses including generation of inflammatory cytokines such as IL - 1 13 and IL - 8 (16, 35), chemotactic activity of neutrophils (10), and stimulation of NF - K B activity (24). The data from this study, however, do not support that HODES are related to inflammatory cytokines within an exercise context despite similar patterns of change over time and peaking immediately postexercise. Isoprostanes are prostaglandin - like compounds produced by the free radical catalyzed peroxidation of arachidonic acid independent of cyclooxygenase (22, 30). A specific class of isoprostanes, the F 2 - IsoP, are an accurate indicator of in vivo oxidant stress and have been related to a variety of human disorders (30). The tight linkage between postexercise levels of 13 - HODE + 9 - HODE and F 2 - IsoP is a novel finding from this study. Plasma concentrations of F 2 - IsoP are much lower than 13 - HODE + 9 - HODE (39). Linoleate is the most abundant fatty acid in atherosclerotic plaques, and high levels of HODEs accumulate in the low - density lipoprotein (LDL) and plaque of patients with atherosclerosis compared with healthy controls (14). Similar to F 2 - IsoP, the HODEs are stable lipid peroxida - tion products that are elevated in diseased individuals and can be lowered through positive lifestyle changes (2, 3, 7, 25, 29, 37). The data from this study support the use of 13 - HODE + 9 - HODE as biomarkers of oxidative stress during exercise trials. Postexercise 13 - HODE + 9 - HODE was significantly corre - lated with 12,13 - DiHOME, another derivative of linoleic acid diol. 12,13 - DiHOME is produced via the oxidation of the isoleukotoxin 12,13 - epoxyoctadecenoic acid (12,13 - EpOME). Both 12,13 - DiHOME and 12,13 - EpOME are PPAR - 'Y ligands with potentially wide - ranging effects. In addition to its role as a PPAR ligand, 12,13 - DiHOME exerts toxic and oxidative effects, inhibits mitochondrial function, stimulates neutrophil chemotactic activity, and suppresses neutrophil respiratory burst activity (5, 31, 36, 38). Perspectives and Significance We recently reported that a 3 - day period of intensified training elicited large changes in the human serum metabolome of runners (20). Athletes ran for 2.5 h/day on treadmills at ~ 70% V ˙ O 2max for 3 days in a row, with blood samples collected preexercise and immediately and 14 h postexercise. Immediately after the 3 - day exercise period, significant two - fold or higher increases in 75 metabolites were measured, with all but 22 of these metabolites related to lipid/carnitine metab - olism. We reported a fivefold postexercise increase in plasma 13 - HODE + 9 - HODE, with levels returning to preexercise levels within 14 - h recovery (20). Although this was the first published information on the acute increase in plasma 13 - HODE + 9 - HODE following intensive exercise, other inves - tigators using disease models had reported that higher HODE concentrations were excellent indicators of oxidative stress and should be considered in future studies because they were much more abundant than F 2 - isoprostanes (37, 39). A major objec - tive in the current study was to determine whether 13 - HODE + 9 - HODE could be related to F 2 - isoprostanes within an exercise context, and this was established. In this study, cyclists com - pleting a 75 - km time trial experienced twofold or higher increases in 80 metabolites, including linoleate and other fatty acids in the conversion pathway and the oxidized derivatives 13 - HODE + 9 - HODE. Although 13 - HODE + 9 - HODE play proinflammatory roles under certain conditions, no relationship to postexercise increases in six cytokines was established in this study. The 3.1 - fold postexercise increase in HODEs was signi ficantly correlated with F 2 - IsoP, supporting the inclusion of HODEs as a stable oxidative stress biomarker in future exercise trials. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: D.C.N., R.A.S., and M.P.M. conception and design of research; D.C.N., R.A.S., B.L., M.P.M., D.A.D., and K.L.P. performed exper - iments; D.C.N. and K.L.P. analyzed data; D.C.N., R.A.S., B.L., M.P.M., D.A.D., and K.L.P. interpreted results of experiments; D.C.N. and K.L.P. prepared figures; D.C.N. drafted manuscript; D.C.N., B.L., M.P.M., D.A.D., and K.L.P. edited and revised manuscript; D.C.N., R.A.S., B.L., M.P.M., D.A.D., and K.L.P. approved final version of manuscript. REFERENC

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6 r 22: 502 – 510, 2003. 39. Yo
r 22: 502 – 510, 2003. 39. Yoshida Y, Kodai S, Takemura S, Minamiyama Y, Niki E. Simulta - neous measurement of F2 - isoprostane, hydroxyoctadecadienoic acid, hy - droxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples. Anal Biochem 379: 105 – 115, 2008. Archived ersiofrom CKS Institutional Repository ttp://libres.uncg.edu/ir/asu/ Gomes EC, Silva Oliveira MR. Oxidants, antioxidants, and the beneficial roles exercise-induced production reactive species. Oxid Med Cell Longev 2012: 756132. doi:10.1155/2012/756132, 2012.Halliwell KJ, Fielding BA, Samra JS, Humphreys Frayn KN. Release individual fatty acids from human adipose tissue vivo after overnight fast. J Lipid Res 37: 18421848, 1996.10.Henricks PA, Engels van der Vliet Nijkamp 9- and - hydroxy-linoleic acid possess chemotactic activity for bovine and human polymorphonuclear leukocytes. Prostaglandins 27, 1991.11.Horowitz JF. Fatty acid mobilization from adipose tissue during exercise.Trends Endocrinol Metab 14: 386 392, 2003.12.Itoh Fairall Amin Inaba Szanto Balint BL, Nagy Yamamoto Schwabe JW. Structural basis for the activation of PPARgamma oxidized fatty acids. Nat Struct Mol Biol 15: 924 931, 2008.13.Jacobs KA, Krauss RM, Fattor JA, Horning MA, Friedlander AL, Bauer TA, Hagobian TA, Wolfel EE, Brooks GA. Endurance training has little effect active muscle free fatty acid, lipoprotein cholesterol, ortriglyceride net balances. J Physiol Endocrinol Metab 291: E656 E665, 2006.14.Jira Spiteller Carson Schramm Strong increase hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients. Chem Phys Lipids 111, 1998.15.Kokatnur MG, Oalmann MC, Johnson WD, Malcom GT, Strong JP.Fatty acid composition human adipose tissue from two anatomical sites a biracial community. J Clin Nutr 32: 2198 2205, 1979.16. Thomas CE, Akeson AL, Jackson RL. Induction interleukin beta expression from human peripheral blood monocyte-derived macro- phages 9-hydroxyoctadecadienoic acid. J Biol Chem 267: 1418314188, 1992.17.Lehmann WD, Stephan Fürstenberger Profiling assay for lipoxy- genase products linoleic and arachidonic acid gas chromatography- mass spectrometry. Anal Biochem 204: 158 170, 1992.18. Morrow JD, Yin Quantification F-isoprostanes a reliable index oxidative stress vivo using gas chromatography-massspectrometry (GC-MS) method. Free Radic Biol Med 11011107, 2009.19.Markworth JF, Vella Lingard BS, Tull DL, Rupasinghe TW, Sinclair AJ, Maddipati KR, Cameron-Smith Human inflammatoryand resolving lipid mediator responses resistance exercise and ibuprofen treatment. J Physiol Regul Integr Comp Physiol 305: R1281R1296, 2013.20.Nieman Shanely Gillitt Pappan KL, Lila MA. Serum metabolic signatures induced a three-day intensified exercise periodpersist after h recovery runners. J Proteome Res 12: 45774584, 2013.21.Niki Biomarkers lipid peroxidation clinical material. Biochim Biophys Acta 1840: 809 817, 2014.22.Nikolaidis MG, Kyparos Vrabas IS. -isoprostane formation, mea- surement and interpretation: the role of exercise. Prog Lipid Res 50: 103, 2011.23.O’Hncjgtvy JT, Wooten RE, Samuel MP, Thomas MJ, Levine EA,Case LD, Akman SA, Edwards IJ. Fatty acid metabolites rapidly proliferating breast cancer. PLos One e63076, 2013. 24.Ogawa Owada Ikawa Adachi Egawa Nemoto Suzuki Hishinuma Kawashima Kondo Aiba OkuyamaEpidermal FABP (FABP5) regulates keratinocyte differentiation by 13(S)--mediated activation the B signaling pathway. Invest Dermatol 131: 604 612, 2011.25.Pasman WJ, van Erk MJ, Klöpping WA, Pellis Wopereis S, Bijlsma Hendriks HF, Kardinaal AF. Nutrigenomics approach elu- cidates health-promoting effects high vegetable intake lean and obese men. Genes Nutr 507521, 2013.26.Powers SK, Jackson MJ. Exercise-induced oxidative stress: cellular mechanisms and impact muscle force production. Physiol Rev 88: 12431276, 2008.27.Powers SK, Nelson WB, Hudson MB. Exercise-induced oxidative stress humans: cause and consequences. Free Radic Biol Med 51: 942950, 2011.28.Rago Development a high-throughput ultra performance liquid chromatography-mass spectrometry assay profile eicosanoids exploratory biomarkers for atherosclerotic diseases. J Chromatogr Analyt Technol Biomed Life Sci 936: 32, 2013.29.Ramsden CE, Ringel Feldstein AE, Taha MacIntosh BA, Hibbeln JR, Majchrzak-Hong SF, Faurot KR, Rapoport SI, Cheon Y, Chung YM, Mann JD. Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites humans. Prostaglandins Leukot Essent Fatty Acids 87: 135141, 2012.30.Roberts LJ, Milne GL. Isoprostanes. J Lipid Res 50, Suppl: S219 S223, 2009.31.Sisemore MF, Zheng Yang JC, Thompson Plopper CG, Cor- topassi GA, Hammock BD. Cellular characterization leukotoxin diol- induced mitochondrial dysfunction. Arch Biochem Biophys 392: 37, 2001.32.Spindler SA, Clark KS, Callewaert DM, Reddy RG. Significance and immunoassay 9- and -hydroxyoctadecadienoic acids. Biochem Bio- phys Res Commun 218: 187191, 1996.33.Spiteller Linoleic acid peroxidationthe dominant lipid peroxidation process in low density lipoproteinand its relationship to chronic diseases. Chem Phys Lipids 162, 1998.34.Storey JD, Tibshirani Statistical significance for genomewide studies.Proc Natl Acad Sci USA 100: 9440 9445, 2003.35.Terkeltaub Banka CL, Solan Santoro Brand Curtiss LK. Oxidized LDL induces monocytic cell expression interleukin-chemokine with T-lymphocyte chemotactic activity. Arterioscler Thromb 53, 1994.36.Thompson Hammock BD. Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst. J Biosci 32: 279 291, 2007.37.Vangaveti Baune BT, Kennedy RL. Hydroxyoctadecadienoic acids: novel regulators macrophage differentiation and atherogenesis. Ther Adv Endocrinol Metab 60, 2010.38.Viswanathan Hammock BD, Newman JW, Meerarani Toborek Hennig Involvement CYP 2C9 mediating the proinflammatoryeffects linoleic acid vascular endothelial cells. J Coll Nutr 22: 502510, 2003.39.Yoshida Kodai Takemura Minamiyama Niki Simulta- neous measurement -isoprostane, hydroxyoctadecadienoic acid, - droxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples. Anal Biochem 379: 105115, 2008. * * Post - Exercise F 2 - isoprostanes (pg/ml) Oleic (18:1), palmitic (16:0), and linoleic (18:2) are the three major free fatty acids (FFA) adipose tissue (9, 15). Upper subcutaneous adipose tissue the primary source during prolonged and intensive exercise (11), and relativeavailability the predominant determinant individual use by contracting muscles (13). Ketone bodies increased strongly overnight fasted cyclists following the cling time trial, indicating a high degree -oxidation. 30 20 10 Pre-Exercise Post- 1.5-h Post- km -h Post- 3.5 2.5 Fig. 4. Changes in F-isoprostanes across four time points: preexercise, immediately post- cycling time trial, and 1.5-h and -h postexercise.P 0.01 compared with preexercise. Time main effect, P 0.001. acid listed the conversion pathway except for docosapen-taenote (r = 0.32, P = 0.18; all other relationships, P 0.02). DISCUSSION 1.5 1 0.5 0 1 2 3 4 5 Cyclists experienced transient 3.1-fold increase in the oxidized linoleic acid derivative 13-+ 9-HODE follow- ing the cycling time trial. The postexercise increase in plasma concentration HODEs was related increases the well-established oxidative stress biomarker, F-IsoP, but not to increases in inflammatory cytokines. Postexercise HODEs were also positively correlated with exerci-induced mobili- zation of most components the linoleic acid conversion pathway including linoleate, dihomo-linolenate, arachidonate, and adrenate, and the oxidized isoleukotoxin 12,13-DiHOME. Metabolomics data revealed a twofold greater postexer- cise increase metabolites known identity, and two- thirds were related lipid and carnitine metabolism. These results are very similar those our previous metabolomics- based investigation runners who ran 2.5 h/day 2max days in row (20). Together, these two studiesindicate that following prolonged and intensive exercise, - durance athletes experience profound systemic shifts blood metabolites, especially those from the lipid pathway. agree- ment with Lehman et al. (17), the data support substantial fatty acid transport across the mitochondrial membrane and oxida- tion during prolonged exercise. 3.5 2.5 2 1.5 1 0.5 0 C 160140 120 Post-Exercise -HODE + 9-HODE (MSI) 0 1 2 3 4 5 Post-Exercise -HODE + 9-HODE (MSI) Table 4. Exercise effects plasma cytokines (all time maineffects, P 0.001)Variable,pg/ml Preexercise Post-75 1.5-h Post-75 21-h Post-75-6 1.02 ± 0.28 8.77 ± 0.69* 5.97 ± 0.97* 0.63 ± 0.05-8 5.22 ± 0.42 11.83 ± 1.1* 7.91 ± 0.74* 4.70 ± 0.332.05 ± 0.21 18.6 ± 2.6* 10.6 ± 1.8* 2.14 ± 0.27TNF-3.90 ± 0.16 4.30 ± 0.20* 4.10 ± 0.20 3.61 ± 0.16MCP-1 267 ± 9.1 385 ± 18.9* 340 ± 22.5* 230 ± 7.1GCSF 12.8 ± 1.0 17.7 ± 1.8* 18.7 ± 2.2* 15.3 ± 1.5 0 1 2 3 4 5 Post-Exercise -HODE + 9-HODE (MSI) Fig. Scatterplot relationships between the postexercise time point for Values are means ± SE. IL, interleukin; TNF-, tumor necrosis factor-; MCP- monocyte chemoattractant protein- GCSF, granulocyte colony- stimulating factor. *P 0.01 vs. preexercise.-HODE + and arachidonate (r = 0.77, 0.001) ( 12,13-DiHOME (= 0.60, = 0.006) ( and F-isoprostanes (= 0.75, 1.1) MSI, median scaled intensity. Plasma F2 - soprostanes (pg/ml) Post - Exercise 12,13 DiHOME (MSI) Post - Exercise Arachidate (MS) Table Exercise effects linoleic acid and long chain polyunsaturated fatty acids the conversion pathway (all time main effects, q 0.001) Variable (Median Scaled Intensity) Preexercise Post - 75 km 1.5 - h Post - 75 km 21 - h Post - 75 km Linoleate (18:2n6) 0.76 ± 0.06 2.86 ± 0.15* (3.7 - fold) 1.96 ± 0.11* (2.6 - fold) 0.83 ± 0.05 Linolenate [ ex or 'Y ; (18:3n3 or 6)] 0.72 ± 0.07 4.23 ± 0.29* (5.8 - fold) 2.43 ± 0.20* (3.4 - fold) 0.76 ± 0.06 Dihomo - linolenate (20:3n3 or n6) 0.77 ± 0.04 2.66 ± 0.16* (3.4 - fold) 2.18 ± 0.16* (2.8 - fold) 0.84 ± 0.04 Arachidonate (20:4n6

7 ) 0.80 ± 0.06 1.83 ± 0.12*
) 0.80 ± 0.06 1.83 ± 0.12* (2.3 - fold) 1.55 ± 0.08* (1.9 - fold) 0.84 ± 0.06 Adrenate (22:4n6) 0.62 ± 0.08 2.13 ± 0.12* (3.4 - fold) 1.68 ± 0.17* (2.7 - fold) 0.78 ± 0.11 Docosapentaenoate (22:5n6) 0.81 ± 0.06 1.59 ± 0.22* (2.0 - fold) 1.87 ± 0.18* (2.3 - fold) 0.94 ± 0.06 Values are means ± * vs. preexercise. post- cycling time effects for metabolites. Fold changes were calculated from the median scaled intensity values for pre- -post- cycling time points and then rank ordered. A total metabolites known identity had twofold higher increase following cycling (all values 0.01). These were grouped metabolic subpath- ways, and three mean fold changes were calculated (preexer- cise immediately-, 1.5 h post-, and h postexercise) (see Fig. 1 and Supplemental Table S1). All but these metabolites were related lipid and carnitine metabolism, with the largest fold changes seen for ketones, dicarboxylate fatty acids, and long chain fatty acids.Table 3 summarizes exercise-induced changes median scaled intensity (MSI) values for linoleic fatty acid (18:2n6) and five other long chain polyunsaturated fatty acids the conversion pathway. Fold changes for all these fatty acids ranged from 2.0 5.8 immediately post- cycling and 1.9 3.4 after 1.5 h recovery and were not different from preex- ercise after h recovery. Plasma + 9-HODE increased 3.1- and 1.7-fold immediately and 1.5 h following the cycling time trial, respectively (time main effect, q 0.001) (Fig. 2). Similar patterns postexercise increases were seen for ()-9,10-dihydroxyoctadec--enoic acid (9,10- DiHOME) and ()-12,13-dihydroxyoctadec-9-enoic acid + 9-3.0(12,13-DiHOME) (both time main effects, q 0.001)(Fig. and F-IsoP (time main effect, P 0.001) (Fig. 4).Plasma cytokine data are summarized Table Significantpostexercise increases were measured for -6 (8.6-fold), (2.3-fold), (9.1-fold), TNF-(1.10-fold), MCP-1 (1.44-fold), and GCSF (1.38-fold), with values near preexerciselevels after h recovery (all time main effects, P 0.001).Immediate postexercise MSI values for -HODE 9- were significantly correlated with postexercise ara-chidonate (r = 0.77, P 0.001), 12,13-DiHOME (r = 0.60,P = 0.006), and F-IsoP (r = 0.75, P 0.001) (Fig. 5, Postexercise + 9- was also significantlycorrelated with postexercise linoleate (r = 0.54, P = 0.016),dihomo-linolenate (r = 0.57, P = 0.011), and adrenate0.56, P = 0.013), and marginally linolenate (r = 0.44,P = 0.058) and 9,10-DiHOME (r = 0.41, P = 0.081). Postex-ercise levels of -HODE + 9- were not correlated with any the postexercise cytokine concentrations listed TableImmediate postexercise MSI values for linoleate were sig-nificantly correlated with 9,10-DiHOME (r = 0.64, P 0.003), 12,13-DiHOME (r = 0.58, P = 0.10), and fatty9,10 DiHOME 12,13 DiHOME2.0 1.8 1.6 2.5 1.4 1.2 2.0 1.5 1.0 1.0 0.8 0.6 0.4 0.5 0.0 0.2 0.0 Pre-Exercise Post- 1.5-h Post--h Post- Fig. Changes in 13- and 9-hydroxy-octadecadienoic acid (13-9-HODE) across four time points: preexercise, immediately post-- cling time trial, and 1.5-h and -h postexercise. *P 0.01 compared with preexercise. Time main effect, P 0.001.Fig. Changes ()-9,10-dihydroxyoctadec--enoic acid (9,10-DiHOME) and ()-12,13-dihydroxyoctadec-9-enoic acid (12,13-DiHOME) and across four time points: preexercise, immediately post- cycling time trial, and 1.5-h and -h postexercise. *P 0.01 compared with preexercise. Time main effect, P 0.001. * * * * * * Median Scaled Intensity Median Scaled Intensity Table 2. Average metabolic performance data Variable Means ± Time, h 2.71 ± 0.07, ml·kg·min38.3 ± 1.2,%V2max69.3 ± 2.1Watts 207 ± 7.6% Wattsmax68.3 ± HR, beats/min 150 ± max84.0 ± 1.1Ventilation, l/min 74.9 ± RPE 12.9 ± 0.3 , volume oxygen consumed; RPE, rating perceived exertion. water, 8 (A) and 6.5 ammonium bicarbonate 95/5 metha- -water (B) (same gradient profile as above) at 350 l/min. The MS instrument scanned 1,000 molecular weight () and alternated between and MS2 scans using dynamic exclusion with approxi- mately 6 scans per second. Derivatized samples for GC/MS were separated a diphenyl-95% dimethyl polysiloxane-fused silica column with helium the carrier gas and a temperature ramp from 60°C 340°C and then analyzed a Thermo-Finnigan Trace DSQ (Thermo Fisher Scientific) operated unit mass resolving power with electron impact ionization and a 750-atomic mass unit scan range. Metabolites were identified automated comparison of the ion features the experimental samples a reference library of chemical standard entries that included retention time, molecular weight ( preferred adducts, and -source fragments well associated spectra, and were curated visual inspection for quality control using software developed at Metabolon (Durham, NC) (4). Common and biologically abundant isomers unsaturated fatty acids containing the n6, and configuration are contained in Ogvcdqnqp’u chemical standard library. Liquid chromatography mass spectrometry (LC- standards monitor and performance were -leucine, chloro- and bromo-phenylalanine, -maleic acid, amitriptyline, and -benzophenone (1). Biochemical identificationswere based three criteria: retention index within a narrow window the proposed identification, accurate mass match the library 0.005 amu, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores were based a comparison the ions present the experimental spectrum the ions present the library spectrum.Statistical analysis. Data are expressed means ± SE. For the metabolomics statistical analyses and data display purposes, any missing values were assumed below the limits detection and these values were imputed with the compound minimum (minimum value imputation). Statistical analysis log-transformed data was performed using “T” Foundation, from http://cran.r-project.org/),which a freely available, open-source software package. One-way ANOVA with post contrasts (-tests) was performed compare data across time points. estimate the false discovery rate (q value) was calculated take into account the multiple comparisons that normally occur in metabolomic-based studies, with q 0.05 used indication high confidence a result (34). Fold changes across time points were calculated using group averages the median scaled intensity values. Plasma cytokines and F-IsoP were analyzed using -way ANOVA with post contrasts (-tests) performed compare data across time points. Correlations between 9- and other variables were made using Rgctuqp’u product- moment correlation coefficients.RESULTS Subjects included competitive male cyclists (ages to yr) who successfully adhered all aspects the study design (see Table 1). Metabolic and performance data from the mountainous cycling time trial are summarized Table The cyclists were able maintain a high power output (69% Wattsmax) during the trial, with average duration of 2.71 The metabolomics analysis revealed detectable com- pounds known identity. After log transformation and impu- tation with minimum observed values for compound, repeated measures ANOVA contrasts identified significant Pre- h-Post- km Pre-1.5 h-Post- Pre-Post Exerc Fig. Fold changes metabolites grouped - cording subpathways. Numbers parentheses represent the number metabolites each sub- pathway that increased twofold greater after the cycling time trial.Nucleotide, purine (2)Carb metabolism (3)Kreb cycle (2)Hemoglobin (3)Carnitines (8)Amino acids (8)Sterol/steroid (1)Dihydroxy FA (2)Bile acid (2)Branched FA (2)Essential FA (7)Lipid Metabolism (2)Monohydroxy FA (5) Medium chain FA (4)Long chain FA (20) Dicarboxylate FA (7)Ketones (2) Fold Change was measure the effect prolonged and intensive exercise plasma + 9-HODE and linoleic acid metabolism and correlations with postexercise increases plasma F-IsoP and inflammatory cytokines.MATERIALS METHODS Subjects. Subjects included male cyclists (ages yr) who regularly competed road races (racing categories 1 with assigned cyclists with the lowest performance ability and experi- ence) and were capable cycling race pace. Subjects trained normally, maintained weight, and avoided the use large-dose vitamin and mineral supplements (above Daily Value), and all herbal supplements and medications for the 2- period before the -km cycling time trial. All subjects signed informed consent and all study procedures were approved the Institutional Review Board at Appalachian State University.Research design. Two weeks before the time trial, athletes completed orientation and baseline testing the North Carolina Research Campus Human Performance Laboratory operated - palachian State University Kannapolis, NC. Demographic and training histories were acquired with questionnaires. Maximal power, oxygen consumption, ventilation, and heart rate were measured during a graded exercise test (25 Watts increase every 2 min, starting 150 Watts) with the Cosmed Quark CPET metabolic cart (Rome, Italy) and the Lode cycle ergometer (Lode Excaliber Sport, Lode, Gro- ningen, The Netherlands). Body composition was measured with the Bod Pod body composition analyzer (Life Measurement, Con- cord, CA).Two weeks after baseline testing, subjects returned the Human Performance Laboratory 6:45 overnight fasted state (no food beverages other than water for least 9 hours) and provided a preexercise blood sample. After warming subjects cycled their own bicycles CompuTrainer Pro model 8001 trainers (Rac- erMate, Seattle, WA) with heart rate and rating perceived exertion (RPE) recorded every min and workload continuously monitored using the CompuTrainer MultiRider software system (version 3.0, RacerMate, Seattle, WA). A mountainous course with moder- ate difficulty was chosen and programmed into the software system. Oxygen consumption and ventilation were measured using the Cosmed Quark CPET metabolic cart after and cycling. Subjects were allowed ingest water libitum during the -km cycling time trial, not any other beverage food containing energy nutrients. Blood samples were taken via venipuncture immediately and 1.5 h after completing the time trial. Subjects returned overnight fasted state the next morning provide -h postexercise

8 blood sample. dietary restrictions wer
blood sample. dietary restrictions were imposed from 1.5-h postexercise the beginning the overnight fast.-isoprostanes. Plasma F-IsoP were determined using gas chro- matography mass spectrometry (GC- (18). Plasma was collected from heparinized blood, flash-frozen liquid nitrogen, and stored 80°C. Plasma samples were thawed, and free F-IsoP was extracted with deuterated []prostaglandin Fadded internal standard. Waters Sep-Pak C18 cartridges followed Waters Sep-Pak Silica cartridges were used for solid phase extraction. F-IsoP were con- verted pentafluorobenzyl esters, subjected thin layer chromatog- raphy, and converted trimethylsilyl ether derivatives. Samples were analyzed by a negative ion chemical ionization using an Agilent gas chromatography interfaced Agilent inert MSD mass spectrometer (Agilent Technologies, Santa Clara, CA).Cytokines. Total plasma concentrations six inflammatory cyto- kines [monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-(TNF- granulocyte colony-stimulating factor (GCSF), and were determined using electrochemilumi- nescence based solid-phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD). All samples and provided standards were analyzed duplicate, and the intra-assay coefficient variation(CV) ranged from 1.7% 7.5% and the interassay 2.4 9.6% for all cytokines measured. Pre- and postexercise samples for the cyto- kines were analyzed the same assay plate decrease interkit assay variability.Metabolomics. The nontargeted metabolic profiling instrumenta- tion employed for this analysis combined three independent plat- forms: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) optimized for basic species, UHPLC/ MS/MS optimized for acidic species, and GC/MS (1, 6). Blood samples were collected EDTA tubes and centrifuged 3,000 rpm for min 4°C, with the plasma aliquoted, snap frozen liquid nitrogen, and stored 80°C until analysis. For plasma sample, l was used for analyses. With the use automated liquid handler (Hamilton LabStar, Salt Lake City, UT), protein was precip- itated from the plasma with methanol that contained four standards to report extraction efficiency. The resulting supernatant was split into equal aliquots for analysis the three platforms. Aliquots, dried under nitrogen and vacuum desiccated, were subsequently either reconstituted 50 l 0.1% formic acid in water (acidic conditions) or l 6.5 ammonium bicarbonate water, 8 (basic conditions) for the two UHPLC/MS/MS analyses derivatized final volume l for GC/MS analysis using equal parts bistrim- ethyl-silyl-trifluoroacetamide and solvent mixture acetonitrile-dichlo- romethane-cyclohexane (5:4:1) with triethylamine 60°C for 1 h. addition, three types controls were analyzed concert with the experimental samples: aliquots a well-characterized human plasma pool served technical replicates throughout the data set, extracted water samples served process blanks, and a cocktail standards spiked into every analyzed sample allowed instrument perfor- mance monitoring. Standards monitor extraction were -cho- lesterol, and tridecanoic acid. A standard monitor GC/MS derivatization was 2-tert-butyl-6-methylphenol (BHT). GC/MS standards monitor and performance were -C18 alkylbenzenes. Experimental samples and controls were ran- domized across platform run days. Instrument variability was deter- mined calculating the median relative standard deviation (RSD) for the internal standards that were added sample before injection into the mass spectrometers. Overall process variability was deter- mined calculating the median RSD for all endogenous metabolites (i.e., noninstrument standards) present technical replicates of MTRX3. Values for instrument (5%) and process variability (12%) Ogvcdqnqp’u acceptance criteria.For UHLC/MS/MS analysis, aliquots were separated using a - ters Acquity UPLC (Waters, Millford, MA) instrument with separate acid/base-dedicated 2.1 X Waters BEH C18 1.7-m particle columns heated 40°C, and analyzed using LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA), which con- sisted electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer (6). Extracts reconstituted formic acid were gradient eluted 350 l/min using 0.1% formic acid water (A) and 0.1% formic acid methanol (0% B B 4 min, 98% 0.5 min, 98% B for 0.9 min), whereas extracts reconstituted in ammonium bicarbonate used 6.5 mM ammonium bicarbonate Table 1. Subject characteristics Variable Means ± Age, 38.0 ± 1.6Height, m 1.81 ± Weight, 76.8 ± Body fat, % 14.0 ± 1.0Watts304 ± 10.52max, ml·kg·min1 51.7 ± 1.4, beats/min 179 ± Training, km/wk 192 ± 18.0 n = 19 subjects; V2max, maximal oxygen consumed; HR, heart rate. Metabolomics approach assessing plasma - and 9-hydroxy-octadecadienoic acid and linoleic acid metabolite responses cyclingDavid Nieman,1 Andrew Shanely,1 Beibei Luo,2 Mary Pat Meaney,1 Dustin Dew,and Kirk PappanAppalachian State University, Human Performance Lab, North Carolina Research Campus, Kannapolis, North Carolina; Key Laboratory Exercise Health Sciences Ministry Education, Shanghai University Sport, Shanghai, China; Metabolon Inc., Durham, North Carolina Bioactive oxidized linoleic acid metabolites (OXLAMs) include - and 9-hydroxy-octadecadienoic acid (13-+ 9-HODE) and have been linked oxidative stress, inflammation, and numerous pathological and physiological states. The purpose this study was measure changes plasma + 9-HODE following a cycling and identify potential linkages linoleate metabolism and established biomarkers of oxidative stress -isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N = 19, age38.0 ± 1.6 yr, wattsmax ± 10.5) engaged a cycling timetrial their own bicycles using electromagnetically braked cyclingergometers (2.71 ± 0.07 h). Blood samples were collected preexer-cise, immediately post-, 1.5 h post-, and h postexercise, andanalyzed plasma cytokines (IL--10, tumor necrosisfactor-, monocyte chemoattractant protein- granulocyte colony- stimulating factor), F-isoprostanes, and shifts metabolites usingglobal metabolomics procedures with gas chromatography mass spec- trometry (GC-MS) and liquid chromatography mass spectrometry(LC-MS). + 9- increased 3.1-fold and 1.7-foldimmediately post- and 1.5 h postexercise (both P 0.001) andreturned preexercise levels -h postexercise. Post-cling plasma levels + 9-HODE were not significantlycorrelated with increases plasma cytokines but were positivelycorrelated with postexercise F-isoprostanes (r = 0.75, P 0.001),linoleate (r = 0.54, P 0.016), arachidate (r 0.77, P 0.001),12,13-dihydroxy--octadecenoate (12,13-DiHOME) (r = 0.60, P 0.006), dihomo-linolenate (r = 0.57, P = 0.011), and adrenate (r 0.56, P = 0.013). These findings indicate that prolonged and intensiveexercise caused a transient, 3.1-fold increase the stable linoleic acidoxidation product -HODE + 9- and was related increases F-isoprostanes, linoleate, and fatty acids the linoleate conver-sion pathway. These data support the use + 9- oxidative stress biomarker acute exercise investigations.exercise; oxidative stress; inflammation; linoleate; metabolites THE RATE OF WHOLE BODY and muscle oxygen consumption increases during acute, high-intensity, and continuous exercise bouts and accompanied increase production of reactive oxygen species (ROS) (8, 27). The causes of increased ROS during exercise include electron leakage the mitochondrial electron transport chain, activation neutro- Address for reprint requests and other correspondence: Nieman, DrPH, North Carolina Research Campus, 600 Laureate Way, Kannapolis, NC 28081 -mail: niemandc@appstate.edu).phils and other phagocytic cells, auto-oxidation cat- echolamines, and activation several enzymes including xan- thine oxidase, phospholipase A2, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (8). Direct measure- ment free radical and ROS production difficult, and assessment oxidative stress during exercise typically made using indirect methods (22). Reliable oxidative stress biomark- ers should chemically unique and detectable, have relatively long half-lives, and responsive increases decreases in ROS (26). The level F-isoprostanes -IsoP) blood or urine widely regarded excellent indicator exercise- induced oxidative stress and formed via free radical-initiated peroxidation arachidonic acid (22, 30).Linoleic acid (18:2n- the most common polyunsaturated fatty acid (PUFA) in human diets and mammalian tissue and is considered essential because humans lack delta- and del- -15 desaturase enzymes (9, 15, 29, 33). Ingested linoleic acid converted longer and more unsaturated fatty acids through enzyma desaturation and elongation the endoplasmic reticulum cells. Linoleic acid the direct precursor to oxidized linoleic acid metabolites (OXLAMs) including - and 9-hydroxy-octadecadienoic acid (13-+ 9-HODE) (9, 33) (see http://www.genome.jp/kegg-bin/show_pathway?map00591 for more detail). + 9-HODE are stable oxidation products and have been linked patho- logical conditions including atherosclerosis, diabetes, Alzhei- ogt’u disease, non-alcoholic steatohepatitis, psoriasis, chronic inflammation, obesity, and cancer (14, 37). Plasma levels + 9-HODE are responsive lifestyle interventions, with decreases reported when subjects adopt healthy diets and lose excess weight (2, 25). 13-+ 9- are generated through the -lipoxy- genase-1 (15-LOX) pathway a variety cell types (17, 25), are ligands peroxisome proliferator-activated receptors (PPARs) (28), and through G protein-coupled receptor (GPR132) exert pro-inflammatory effects (37).Little information has been published the influence of exercise plasma -HODE + 9-HODE, and whether postexercise increases relate changes established bio- markers oxidative stress and inflammation (19, 20). Using metabolomics approach, recently reported that plasma - + 9- increased 5.5-fold runners followinga 3-day period intensified training (2.5 h/day running 70% 2max with levels returning preexercise levels after -hrecovery (20). Metabolomics allows the simultaneous mea- surement hundreds metabolites and especially valuable when focusing complex interactions within the during exercise nutrition interventions. The purpose this