Energy Metabolism in Continuous, High-Intensity, and Sprint Interval Training Protocols With Matched Mean Intensity.

ABSTRACT: Eigendorf, J, Maassen, M, Apitius, D, and Maassen, N. Energy metabolism in continuous, high-intensity, and sprint interval training protocols with matched mean intensity. J Strength Cond Res 35(11): 3104-3110, 2021-To evaluate acute physiological reactions and energy metabolism with 3 different training regimes, 7 subjects performed a high-intensity interval training (HIT), a sprint interval training (SIT), and a continuous training (CT) in a cross-over design. All training sessions were matched for relative mean intensity (50% Pmax). Stress-to-pause-ratios were chosen as 6-24 seconds (SIT) and 30-30 seconds (HIT) for interval protocols. No significant differences (significance level p ≤ 0.05) were found for oxygen uptake (V̇o2), respiratory exchange ratio (RER), slope of RER (RERslope), and heart rate between the different training regimes. Lactate concentrations ([Lac]) in CT were significantly lower (p < 0.01) compared with HIT and SIT. No significant differences were found for free fatty acids ([FFA], p = 0.41) and glycerol ([GLY], p = 0.26) levels during all 3 training protocols (CT 0.27 mmol·L-1, SIT 0.22 mmol·L-1, and HIT 0.22 mmol·L-1). Ammonia (NH3, p > 0.05) levels did not show significant differences between the 3 training protocols during exercise phase. The comparable physiological reactions of [FFA], [GLY], and RER show that the activation of fat metabolism is not different between training regimes with different stress-to-pause-ratios. Moreover, mean intensity and time of exercise influence activation of fat metabolism. Increases in [NH3] suggest similar sources between the 3 training protocols and the need for further research concerning amino acid deamination. The better understanding of the acute reactions and changing of the energy metabolism during training sessions will help athletes in planning and executing their training sessions more efficiently and more precisely in the context of periodization.

GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV.

The most widely recognized activity of the large family of the metalloenzyme carbonic anhydrases (CAs) is the diffusion-controlled hydration of CO2 to HCO3- and one proton, and the less rapid dehydration of HCO3- to CO2: CO2 + H2O ⇆ HCO3- + H+. CAs also catalyze the reaction of water with other electrophiles such as aromatic esters, sulfates and phosphates, thus contributing to lending to CAs esterase, sulfatase and phosphatase activity, respectively. Renal CAII and CAIV are involved in the reabsorption of nitrite, the autoxidation product of the signalling molecule nitric oxide (NO): 4 NO + O2 + 2 H2O → 4 ONO- + 4 H+. Bovine and human CAII and CAIV have been reported to exert nitrite reductase and nitrous anhydride activity: 2 NO2- + 2 H+ ⇆ [2 HONO] ⇆ N2O3 + H2O. In the presence of L-cysteine, NO may be formed. In the literature, these issues are controversial, mainly due to analytical shortcomings, i.e., the inability to detect authentic HONO and N2O3. Here, we present a gas chromatography-mass spectrometry (GC-MS) assay to unambiguously detect and quantify the nitrous anhydrase activity of CAs. The assay is based on the hydrolysis of N2O3 in H218O to form ON18O- and 18ON18O-. After pentafluorobenzyl bromide derivatization and electron capture negative-ion chemical ionization of the pentafluorobenzyl nitro derivatives, quantification is performed by selected-ion monitoring of the anions with mass-to-charge (m/z) ratios of 46 (ONO-), m/z 48 (ON18O- and 18ONO-), m/z 50 (18ON18O-) and m/z 47 (O15NO-, internal standard).

Even and carbon dioxide independent distribution of nitrite between plasma and erythrocytes of healthy humans at rest.

In the literature, the distribution of nitrite and nitrate, the major metabolites of nitric oxide (NO), between plasma and erythrocytes and its dependency on partial CO2 pressure (pCO2) in mammalian blood are uncertain. By means of a previously reported fully validated stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) method, we measured nitrite and nitrate concentrations in heparinized plasma from venous, arterial and arterialized blood donated by five healthy non-exercising volunteers at three different time points (0, 15, 30 min). pCO2, pH and oxygen saturation were measured by standard techniques. The nitrite and nitrate concentrations and the nitrite-to-nitrate ratio in plasma did not correlate with pCO2 (r=-0.272, P=0.07). Nitrite was found to be almost evenly distributed between plasma and erythrocytes of another eleven healthy non-exercising subjects. In a rabbit model of ARDS, no differences were found in the plasma nitrite and nitrate concentrations comparing normoventilation with hypercapnia. Our studies suggest that the distribution of nitrite between plasma and erythrocytes at rest is largely even and independent of pCO2 in blood of healthy humans and rabbits with ARDS.

Long-term effects of NO3- on the relationship between oxygen uptake and power after three weeks of supplemented HIHVT.

The aim of this study was to investigate the later effects of daily NO3- supplementation over 3 wk of training on the relationship between O2 uptake and power at different intensities with an incremental test (IT), a double-wingate test (WT), and an endurance capacity test at 80% Wmax (ECT) before and after the supplementation period. Seventeen male recreational athletes participated in this double-blind placebo (PL)-controlled study. Subjects participated in a 3-wk intermittent high-intensity, high-volume training period with 45 intervals of Wmax - 10 W and an active recovery period of 10 W in between with dietary NO3- (NaNO3) or placebo supplementation (NaCl) (both 8.5 mg·kg-1·day-1) on a cycle ergometer. During a training session, plasma [ NO3- ] ( P < 0.001) and plasma [ NO2- ] ( P < 0.01) were higher in nitrate (N), whereas in pre- and posttests mean plasma [ NO3- ] and [ NO2- ] were not different between groups. In the WT [48 h after cessation of supplementation (C)], the ratio between V̇o2 and power decreased in N ( P < 0.01) with no changes in PL. Endurance capacity (4-5 days after C) similarly increased in both groups ( P < 0.01). However, the total oxygen consumption decreased by 5% ( P < 0.01) in N, with no change in PL. The slope of V̇o2·W-1 in IT (5-7 days after C) decreased in N ( P < 0.01), whereas no changes were found in PL. During low- and moderate-intensity workloads, no changes and differences in V̇o2 could be detected. We conclude that nitrate supplementation causes a sustaining reduction of the oxygen cost per watt during exercise with a large recruitment of type II muscle fibers without affecting endurance capacity. NEW & NOTEWORTHY Because most studies focused on the acute effects of NO3- supplementation on exercise performance during a supplementation period, the sustainability of the effects of the NO3- supplementation remain unknown. We followed the development of V̇o2/W at different intensities during the first week after cessation of daily NO3- supplementation over 3 wk. The results indicate that NO3- supplementation has a long-term effect for at least 7 days after cessation during heavy all-out workloads without affecting endurance capacity.

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO-) and nitrate (ONO2-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO-, O15NO-, ONO2-, O15NO2-, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO-), m/z 47 (O15NO-), m/z 62 (ONO2-), m/z 63 (O15NO2-), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97min for PFB-ONO2/PFB-O15NO2, 3.1min for PFB-NO2/PFB-15NO2, and 6.7min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO3 (n=9) supplementation increased UNOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.