True Blood – Understanding the impact of reduced oxygen carrying capacity using a physiological, haematological and metabolomics perspective.

Nathan Lawler

Oxygen carrying capacity has a positive association with endurance performance. As such, techniques such as altitude training can increase red cell mass (Hbmass) and have been shown to improve sea level performance. In addition to haematological changes (i.e. Hbmass), hypoxia can have a metabolic influence (e.g. increased oxidative and anaerobic enzyme concentration) resulting in changes to metabolites, which reflect systems level adaption to low oxygen carrying capacity environments. The use of metabolomics; therefore, represents an ideal analytical method to examine the physiological adaptations to hypoxia as this technique provides the ability to simultaneously analyse large number of metabolites present in human biological samples. The purpose of this thesis was to examine the acute (14 d) and chronic (42 d) influence of low oxygen carrying capacity on performance (Chapter Four), haematology (Chapters Three, Four and Five) and the metabolic profile (Chapter Three and Five). Chapter Three was conducted as an observational study to examine metabolic changes associated with moderate altitude exposure (~3000 m; 196.4 ± 25.6 h) during a 14 d Live High Train Low altitude camp in 10 endurance runners (six males, four females; 29 ± 7 y). Resting whole blood samples were collected before altitude (baseline), 3 d and 14 d of altitude camp and analysed using mass spectrometry. From this data, acute metabolic profiles to altitude exposure were developed, which demonstrated significant separation between measured time points. Specifically, through mass spectrometry 36 metabolites were identified from metabolite classes relating to amino acids, glycolysis, and purine metabolism and indicate a shift in substrate utilisation (i.e. greater carbohydrate use) during acute hypoxic exposure. From canonical variate analysis, trajectories of the 36 identified metabolites were identified, with two different trajectories observed during the acute moderate hypoxic exposure. Importantly, principal components analysis highlighted greater variance in measured metabolites between-person when compared to within-person. This finding is consistent with previous literature and indicates individual variability during the adaptive response to altitude exposure. During Chapter Four and Five, Australian Red Cross blood donation (~470 mL) was used to reduce the oxygen carrying capacity of 13 male participants, who were then monitored over a 42 d period. Measures of haemoglobin concentration and haematocrit % were obtained before blood removal (Baseline) and at seven time points (24 h, 7 d, 14 d, 21 d, 28 d, 35 d and 42 d) following blood removal. Additionally, exercise tests were conducted to assess fitness (V̇ O2max) and performance (4-minute self-paced cycling time trial [4MMP]) (Chapter Four and Five) and 4 mL blood samples were obtained for metabolomic analysis (Chapter Five). The purpose of Chapter Four was to examine the impact of reduced oxygen carrying capacity on V̇ O2max and both the performance and pacing during a 4MMP. During this study, only participants that were trained cyclists (n = 7) were used for the analyses. Blood removal resulted in a maximal decrease of 5.4 % in V̇ O2max (24 h), while the average power output during the 4MMP decreased significantly at 24 h (7 ± 6%), 7 d (6 ± 8%) and 21 d (4 ± 6%) when compared with baseline values. Furthermore, the aerobic contribution during the 4MMP was significantly reduced, when compared with baseline, by 5 ± 4%, 4 ± 5% and 4 ± 10% at 24 h, 7 d and 21 d, respectively. The rate of decline in power output upon commencement of the 4MMP was significantly attenuated and was 76 ± 20%, 72 ± 24% and 75 ± 35% lower than baseline at 24 h, 21 d and 42 d, respectively. These changes were unrelated to differences in haemoglobin concentration. Findings from this study indicated that reducing oxygen carrying capacity can influence pacing and the performance of middle-distance endurance events. Specifically, it appears that changes in pacing and performance during middle-distance endurance events are related to the ability to contribute power from aerobic metabolism, as an increase in anaerobic contribution was not observed. The focus of Chapter Five was to investigate, in addition to measures obtained in Chapter Four, the acute and chronic changes to the metabolic profile of individuals after blood donation. Untargeted metabolomic analyses was used on 4 mL whole blood samples obtained from 13 participants at baseline and at multiple time points over 42 d following the removal of some 470 mL of whole blood. Similar to findings in Chapter Four, whole blood removal resulted in a maximal decrease in V̇ O2max (-6%) and 4MMP (-7%), with both occurring at 7 d. However, with the inclusion of the additional six “untrained” participants, significant reductions in oxygen delivery (V̇ O2) only persisted for 24 h compared to 21 d in the subset of trained males (Chapter Four). The accelerated return is likely associated with participant fitness (i.e. V̇ O2max: 53.0 ± 10.9 mL・kg-1・min-1; (Chapter Five) vs. V̇ O2max: 60.7 ± 5.5 mL・kg-1・min-1 (Chapter Four)) with the addition of lesser trained males seemingly diminishing the influence of oxygen delivery in the later time points. The metabolomics analysis revealed multi-factorial changes with 40 metabolites deemed significant post blood removal. Through hierarchical cluster analysis, consistent with acute findings during Chapter Three, purine metabolites were significantly elevated immediately following blood removal (acute) and remained elevated throughout the 42 d monitoring period. This finding indicates a chronic adaptive response, which may occur to enhance oxygen delivery to the periphery. Overall, the series of studies have shown separation in plasma metabolites during acute moderate altitude exposure, which is likely linked to substrate utilisation and purine metabolism. Furthermore, decreasing oxygen carrying capacity does not appear to influence the anaerobic contribution to high-intensity middle-distance endurance exercise even with observed reductions in aerobic contribution (Chapter Four). The use of metabolomics throughout this thesis (Chapters Three and Five) has identified purine metabolism as an important marker of adaptation to hypoxia. Further research should focus on purine metabolism as a possible robust marker of hypoxic exposure and work to identify the unknown metabolites which were shown to be significant in the multivariate models.

True Blood – Understanding the impact of reduced oxygen carrying capacity using a physiological, haematological and metabolomics perspective.

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