Master's Thesis, 2011, 33 Pages
Metabolomics is the comprehensive analysis of the metabolite profiles within a biological system. r-HuEPO stimulates red blood cell production, thereby enhancing maximal oxygen delivery to the tissues. The ergogenic potential of r-HuEPO, demonstrated through improved aerobic capacity and performance has reported r-HuEPO to be the most widely abused erythropoietic stimulant till date. This study was aimed at determining the effects of r-HuEPO on the human metabolome by analyzing metabolites from blood plasma and urine. Analysis of these metabolites was then used to determine how such metabolic interactions affected physiological status and exercise performance. Three well trained individuals participated in the study. Blood and urine were collected from the subjects. Plasma and urine samples were analyzed for metabolites by LC-MS Orbitrap and data was analyzed in the form of heatmaps and PCA plots. Further data interpretations for metabolites of interest were performed by a software, mzMatch/PeakML. Analysis of the human metabolome revealed 1000 metabolites which were manually categorized into 190 human metabolites. Significant metabolite patterns in response to r-HuEPO suggested the physiological effects of r-HuEPO on certain metabolites. A better understanding of the metabolic changes mediated by r-HuEPO might provide an insight into the metabolic signals in response to exercise performance.
r-HuEPO- Recombinant Human Erythropoietin
MS- Mass Spectrometry
LC-MS- Liquid Chromatography-Mass Spectrometry
NMR- Nuclear Magnetic Resonance
GC-TOF/MS- Gas Chromatography-Time of Flight/Mass Spectrometry
EPS- Erythropoietic Stimulant
ETT- Exercise Treadmill Test
WADA- World Anti-Doping Agency
PCA- Perchloric Acid
HMDB- Human Metabolite Database
KEGG- Kyoto Encyclopaedia of Genes and Genomes
NEG- Negative Ionization Mode
RSD- Relative Standard Deviation
PCA- Principal Component Analysis
Metabolomics, a part of ‘OMICS’ sciences, is the identification and quantification of all metabolites (lipids, vitamins, small peptides and protein co-factors) in a biological system [33, 38]. Metabolomics is progressing as an important tool in systems biology in combination with genomics, transcriptomics and proteomics [2, 4] (figure 1). In relation to exercise physiology, metabolomics focuses on metabolite-rich body fluids such as blood plasma and urine [18, 33]. Plasma plays a crucial role in metabolite transportation throughout the body while urine samples are easy to collect for metabolomics analysis [3, 10]. Since metabolites are difficult to analyze in a single analysis they could be grouped into specific classes, analyzed and data restored electronically to provide necessary qualitative and quantitative information . MS and NMR are the main technologies used in metabolomic studies [10, 33]. Based on the nature of metabolites, LC-MS Orbitrap was used for metabolite analysis due to high chromatographic resolution, sensitivity and superior quantitative analysis [22, 23]. EPO is a hormone produced primarily by the kidneys [11, 13]. EPO is the key regulator of RBC formation by stimulating differentiation of erythroid progenitor cells, a process known as erythropoiesis [11, 13]. r-HuEPO, a genetically modified form of EPO, has been found to significantly improve aerobic performance in athletes by accelerating RBC production and enhancing maximal oxygen delivery to the tissues [1, 13, 37].
Figure 1.Outline of the ‘OMICS’ cascade: The diagram represents the stages from genes to metabolites which are eventually analyzed by LC-MS. The relay of genetic information to subsequent higher levels is demonstrated. Differences in the metabolite levels may reflect their corresponding enzymatic activities. Metabolite profiles may also be altered by changes in drug concentrations or kinetics (pharmacokinetics) and/or by disease onset or progression. In association with the other ‘OMICS’ technologies, metabolomics aims to combine data and provide useful information on the physiological status of an individual (i.e. the phenotype) [33, 45].
In conjunction with exercise, metabolic interactions can be assessed on the basis of complex nutrients in combination with advanced multivariate statistics and interpretation tools [7, 15, 47]. In a study performed on professional athletes subjected to strength-endurance training (i.e. rowing), metabolomic differences between rowers and control subjects with prolonged training was determined by monitoring the level of endogenous metabolites by GC-TOF/MS during the training program . The study demonstrated how metabolites such as alanine, lactate, cysteine, glutamic acid, free fatty acids, pyroglutamic acid, tyrosine and glutamine affected glucose and energy metabolism, oxidative stress, lipolysis and amino acid metabolism, thus providing an understanding of the rowers’ physiological status during intensive exercise .
Another study demonstrated the comprehensive metabolic profiling of the human plasma suggesting significant changes in twenty-three metabolites in the plasma at peak exercise . Up-regulation in the glycolytic and lipolytic pathways, amino acids and purine catabolism was observed and these results were in conjunction with rises in lactate, pyruvate, glycerol, alanine and glutamine and a reduction in acetoacetate respectively [20, 26]. Increased levels of 3-phosphoglyceric acid and glucose-6 phosphate in plasma were also observed after an ETT . Plasma metabolic profiles of fumarate, malate and succinate were individually affected in the tri-carboxylic acid cycle by exercise while α-ketoglutarate remained unchanged [8, 16, 32]. In concordance with such interesting findings from the human metabolome, the aim of this study was to investigate the complex interactions between multiple metabolites obtained from plasma and urine in the human metabolome and determine how these metabolites get affected by r-HuEPO administration. A careful analysis of these metabolites would further help interpret the physiological status of athletes, prevent misuse of r-HuEPO and determine alternative strategies to doping detection .
Design and Methods
The study involved three well trained individuals (mean ± SD, age: 25.7 ± 5.7 years, weight: 69.7 ± 2.3 kg, height: 177.3 ± 1.7 cm). All subjects were university students and were allowed to continue their respective training sessions and maintain a healthy lifestyle throughout the study period. However, none of the subjects participated in a competition during the course of the study . The subjects underwent an initial medical examination a week before the study commenced. The study was conducted according to the World Medical Association (Declaration of Helsinki) and was reviewed and approved by WADA and the Glasgow University Ethics Committee. All subjects were informed of the duration, experimental procedures and risks involved in the study and were included after having given their written consent [19, 35, 43].
The trial was divided into three phases: pre-treatment, treatment with r-HuEPO, and posttreatment/wash-out phase. The experimental protocol lasted 10 weeks with two weeks for baseline and four weeks each for r-HuEPO treatment and wash-out phases (figure 2). Following initial testing, blood and urine samples were collected from the subjects twice during baseline for metabolomics (figure 2). During the r-HuEPO administration phase, the subjects received subcutaneous injections of r-HuEPO (Epoietin beta, NeoRecormon®, Roche, Welwyn Garden City, UK) every two days for a period of 4 weeks (i.e. a total of 15 injections in 4 weeks) at a dose of 50 IU/kg body mass . Iron was administered orally in the form of a 200 mg ferrous sulphate tablet (Almus Pharmaceuticals, Actavis, Barnstaple, UK) for haem synthesis [19, 43]. Blood and urine was collected thrice during this phase
(figure 2). The post-treatment phase was focused on demonstrating the effects of r-HuEPO on the metabolic status of the subject, based on which the physiological conditions of the subjects were also determined. There were three sampling points for blood and urine (figure 2). This phase alleviated r-HuEPO concentrations gradually from the circulatory system of the subjects allowing them to participate in future competitions without the risk of being caught.
Figure 2.10 weeks trial protocol of the r-HuEPO study: The diagram not only shows the sample collection points (total of 20, red arrows) for all subjects throughout the study, but also the sampling points for blood and urine collection for metabolomics during the three phases (i.e. Baseline/pre-treatment:(2), treatment with r-HuEPO:(3) and post-treatment:(3)) represented by purple arrows. The time points at which r-HuEPO was administered to the subjects during the treatment phase is also indicated by thick blue arrows.
Phlebotomy was performed either by canulation or venipuncture depending upon the experimental tests the subjects underwent on that particular visit. Each blood sample was collected into a 4 ml K 2 EDTA tube (Vacuette®, Greiner bio-one, Austria), a 3.5 ml serum tube (Vacutainer, BD, Belliver Industrial Estate, Plymouth, UK) and a 5 ml clear tube (Sterilin, UK) from an anticubital vein before the trial protocol-twice during baseline, thrice during the acceleration and wash-out phases. The subject was asked to lie down facing up
(supine position) for venipuncture . Following blood collection, 1 ml of plasma from the EDTA-vacutainer-system was extracted after centrifugation at 4000 g for 15 minutes on a bench top centrifuge (Universal 320R, Zentrifugen, Germany) into a 1.8 ml cryotube (Alpha Laboratories, Eastleigh, Hampshire, UK), placed in a cryobox and stored at -80°C (figure 3). Urine samples collected from the subjects in a 20 ml clear tube was divided into three aliquots of 1 ml each in1.8 ml cryotubes, placed in the cryobox and stored at -80°C (figure 3). Urine aliquots were prepared immediately after their collection and stored at -80°C to inhibit metabolic reactions completely before proceeding to collect plasma samples. The protocol for blood collection also involved addition of 2.5% (0.3 N) PCA to blood in the EDTAvacutainer-system (figure 3).
Figure 3.Protocol for sample collection for metabolomics: Blood plasma and urine were extracted from the subjects for metabolite analysis. Perchloric Acid, abbreviated as PCA was used as a deproteinization agent to inhibit the metabolic reactions immediately after blood collection in the EDTA tube. A modified protocol (see discussion) involved replacing PCA with HPLC-grade Acetonitrile (ACN) in the ratio of 3:1 (3 ml of ACN:1 ml of whole blood). The plasma and urine samples were stored at -80°C for further analysis by LC-MS.
Sample analysis by LC-MS and Statistical interpretation
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