Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans.

The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results.

Fasting and Exercise Induce Changes in Serum Vitamin D Metabolites in Healthy Men.

BACKGROUND: Vitamin D plays pleiotropic roles in the body and hence, changes in its metabolism and distribution during starvation could play an important role in the adaptive response to famine. We aimed to identify the responses of some vitamin D metabolites to 8 d of fasting and exercise. METHODS: A repeated-measures design was implemented, in which 14 male volunteers fasted for 8 d and performed an exercise test before and after fasting. Serum samples were collected on day 1 after night fasting and after 8 d of complete food restriction, before and 1 h and 3 h after exercise. RESULTS: After 8 d of fasting, compared with baseline values, serum 24,25(OH)2D3 and 3-epi-25(OH)D3 levels significantly increased; those of 25(OH)D3 and 1,25(OH)2D3 were unaffected; and those of 25(OH)D2 decreased. Exercise on the first day of fasting induced an increase in serum 3-epi-25(OH)D3 levels, while exercise performed after 8 d of fasting induced an increase in 25(OH)D3, 24,25(OH)2D3, 25(OH)D2, and 3-epi-25(OH)D3 levels. CONCLUSION: Increases in 24,25(OH)2D3 and 3-epi-25(OH)D3 levels imply that fasting stimulates vitamin D metabolism. The effects of exercise on serum vitamin D metabolites, which are most pronounced after fasting and in subjects with serum 25(OH)D3 above 25 ng/mL, support the notion that fasting and exercise augment vitamin D metabolism.

Changes in Serum Iron and Leukocyte mRNA Levels of Genes Involved in Iron Metabolism in Amateur Marathon Runners-Effect of the Running Pace.

Iron is essential for physical activity due to its role in energy production pathways and oxygen transportation via hemoglobin and myoglobin. Changes in iron-related biochemical parameters after physical exercise in athletes are of substantial research interest, but molecular mechanisms such as gene expression are still rarely tested in sports. In this paper, we evaluated the mRNA levels of genes related to iron metabolism (PCBP1, PCBP2, FTL, FTH, and TFRC) in leukocytes of 24 amateur runners at four time points: before, immediately after, 3 h after, and 24 h after a marathon. We measured blood morphology as well as serum concentrations of iron, ferritin, and C-reactive protein (CRP). Our results showed significant changes in gene expression (except for TFRC), serum iron, CRP, and morphology after the marathon. However, the alterations in mRNA and protein levels occurred at different time points (immediately and 3 h post-run, respectively). The levels of circulating ferritin remained stable, whereas the number of transcripts in leukocytes differed significantly. We also showed that running pace might influence mRNA expression. Our results indicated that changes in the mRNA of genes involved in iron metabolism occurred independently of serum iron and ferritin concentrations.

Ferritin Genes Overexpression in PBMC and a Rise in Exercise Performance as an Adaptive Response to Ischaemic Preconditioning in Young Men.

OBJECTIVES: The proposal of this study was to evaluate the effect of acute and ten-day ischaemic preconditioning (IPC) training procedure on the Wingate Anaerobic Test (WAnT), the ferritin H (FTH), ferritin L (FTL), and transferrin receptor 1 (TFRC) mRNA expression in peripheral blood mononuclear cells (PBMC), and anaerobic performance. METHOD: 34 healthy men volunteers (aged 20.7 ± 1.22 years) participated in the study. The effects of bilateral upper limb IPC and sham controlled condition were assessed in two experimental protocols: (a) the influence of acute (one time) IPC based on an experimental crossover study design and (b) the influence of ten-day IPC training treatment based on a random group assignment. At the beginning and at the end of each experiment upper body WAnT was performed and blood samples were collected to assess gene expression via quantitative PCR (qPCR). RESULTS: No significant effect of one-time ischaemic preconditioning procedure was observed on upper body WAnT performance. Ten-day IPC training significantly increased upper limbs relative mean power (from 5.29 ± 0.50 to 5.79 ± 0.70 (W/kg), p < 0.05). One-time IPC caused significant decrease in FTH, FTL, and TFRC mRNA levels while 10 days of IPC resulted in significant increase of FTH and FTL mRNA (from 2 ∧254.2 to 2 ∧1678.6 (p = 0.01) for FTH and 2 ∧81.5 to 2 ∧923 (p = 0.01) for FTL) and decrease in TFRC mRNA. CONCLUSIONS: Our findings suggest that ten-day IPC training intervention significantly affects upper limb relative peak power. The observed overexpression of FTH and FTL genes could be associated with adaptation response induced by prolonged IPC.