Recombinant human erythropoietin (rHuEpo) increases haemoglobin mass (Hb_mass) and maximal oxygen uptake ( O_{2 max}). This study defined the time course of changes in Hb_mass, O_{2 max} as well as running time trial performance following 4 weeks of rHuEpo administration to determine whether the laboratory observations would translate into actual improvements in running performance in the field. 19 trained men received rHuEpo injections of 50 IU•kg⁻¹ body mass every two days for 4 weeks. Hb_mass was determined weekly using the optimized carbon monoxide rebreathing method until 4 weeks after administration. O_{2 max} and 3,000 m time trial performance were measured pre, post administration and at the end of the study. Relative to baseline, running performance significantly improved by ∼6% after administration (10∶30±1∶07 min:sec vs. 11∶08±1∶15 min:sec, p<0.001) and remained significantly enhanced by ∼3% 4 weeks after administration (10∶46±1∶13 min:sec, p<0.001), while O_{2 max} was also significantly increased post administration (60.7±5.8 mL•min⁻¹•kg⁻¹ vs. 56.0±6.2 mL•min⁻¹•kg⁻¹, p<0.001) and remained significantly increased 4 weeks after rHuEpo (58.0±5.6 mL•min⁻¹•kg⁻¹, p = 0.021). Hb_mass was significantly increased at the end of administration compared to baseline (15.2±1.5 g•kg⁻¹ vs. 12.7±1.2 g•kg⁻¹, p<0.001). The rate of decrease in Hb_mass toward baseline values post rHuEpo was similar to that of the increase during administration (−0.53 g•kg⁻¹•wk⁻¹, 95% confidence interval (CI) (−0.68, −0.38) vs. 0.54 g•kg^−1•wk⁻¹, CI (0.46, 0.63)) but Hb_mass was still significantly elevated 4 weeks after administration compared to baseline (13.7±1.1 g•kg⁻¹, p<0.001). Running performance was improved following 4 weeks of rHuEpo and remained elevated 4 weeks after administration compared to baseline. These field performance effects coincided with rHuEpo-induced elevated O_{2 max} and Hb_mass.

Aerobic exercise, in spite of its multi-organ benefit and potent effect on the metabolome, has yet to be investigated comprehensively via an untargeted metabolomics technology. We conducted an exploratory untargeted liquid chromatography mass spectrometry study to investigate the effects of a one-h aerobic exercise session in the urine of three physically active males. Individual urine samples were collected over a 37-h protocol (two pre-exercise and eight post-exercise). Raw data were subjected to a variety of normalization techniques, with the most effective measure dividing each metabolite by the sum response of that metabolite for each individual across the 37-h protocol expressed as a percentage. This allowed the metabolite responses to be plotted on a normalised scale. Our results highlight significant metabolites located in the following systems: purine pathway, tryptophan metabolism, carnitine metabolism, cortisol metabolism, androgen metabolism, amino acid oxidation, as well as metabolites from the gastrointestinal microbiome. Many of the significant changes observed in our pilot investigation mirror previous research studies, of various methodological designs, published within the last 15 years, although they have never been reported at the same time in a single study.

Glucocorticoids (GCs) are metabolic hormones with immunosuppressive effects that have proven effective drugs against childhood acute lymphoblastic leukemia (ALL). Yet, the role of metabolic reprogramming in GC-induced ALL cell death is poorly understood. GCs efficiently block glucose uptake and metabolism in ALL cells, but this does not fully explain the observed induction of autophagy and cell death. Here, we have performed parallel time-course proteomics, metabolomics, and isotope-tracing studies to examine in detail the metabolic effects of GCs on ALL cells. We observed metabolic events associated with growth arrest, autophagy, and catabolism prior to onset of apoptosis: nucleotide de novo synthesis was reduced, while certain nucleobases accumulated; polyamine synthesis was inhibited; and phosphatidylcholine synthesis was induced. GCs suppressed not only glycolysis but also entry of both glucose and glutamine into the TCA cycle. In contrast, expression of glutamine-ammonia ligase (GLUL) and cellular glutamine content was robustly increased by GC treatment, suggesting induction of glutamine synthesis, similar to nutrient-starved muscle. Modulating medium glutamine and dimethyl-α-ketoglutarate (dm-αkg) to favor glutamine synthesis reduced autophagosome content of ALL cells, and dm-αkg also rescued cell viability. These data suggest that glutamine synthesis affects autophagy and possibly onset of cell death in response to GCs, which should be further explored to understand mechanism of action and possible sources of resistance.

Recombinant human erythropoietin (rHuEPO) is frequently abused by athletes as a performance-enhancing drug, despite being prohibited by the World Anti-Doping Agency. Although the methods to detect blood doping, including rHuEPO injections, have improved in recent years, they remain imperfect. In a proof-of-principle study, we identified, replicated, and validated the whole blood transcriptional signature of rHuEPO in endurance-trained Caucasian males at sea level (n = 18) and Kenyan endurance runners at moderate altitude (n = 20), all of whom received rHuEPO injections for 4 wk. Transcriptional profiling shows that hundreds of transcripts were altered by rHuEPO in both cohorts. The main regulated expression pattern, observed in all participants, was characterized by a “rebound” effect with a profound upregulation during rHuEPO and a subsequent downregulation up to 4 wk postadministration. The functions of the identified genes were mainly related to the functional and structural properties of the red blood cell. Of the genes identified to be differentially expressed during and post-rHuEPO, we further confirmed a whole blood 34-transcript signature that can distinguish between samples collected pre-, during, and post-rHuEPO administration. By providing biomarkers that can reveal rHuEPO use, our findings represent an advance in the development of new methods for the detection of blood doping.

Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capacity (VO_2max) was determined and the second was a 45 min submaximal exercise test. Urine samples were collected separately the day before (day 1) , the day of (day 2), and the day after (day 3) the submaximal exercise test (12 samples per subject). Metabolomic profiling of the samples was carried out using hydrophilic interaction chromatography (HILIC) coupled to an Orbitrap Exactive mass spectrometer. Data were extracted, database searched and then subjected to principle components (PCA) and orthogonal partial least squares (OPLSDA) modelling. The best results were obtained from pre-treating the data by normalising the metabolites to their mean output on days 1 and 2 of the trial. This allowed PCA to separate the day 2 first void samples (D2S1) from the day 2 post-exercise samples (D2S3) PCA also separated the equivalent samples obtained on day 1 (D1S1 and D1S3). OPLSDA modelling separated both the D2S1 and D2S3 samples and D1S1 and D1S3 samples. The metabolites affected by the exercise samples included a range of purine metabolites and several acyl carnitines. Some metabolites were subject to diurnal variation these included bile acids and several amino acids, the variation of these metabolites was similar on day 1 and day 2 despite the exercise intervention on day 2. Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO_2max when the levels in the D2S3 samples were considered.