Short-Term Supplementation of Sodium Nitrate vs. Sodium Chloride Increases Homoarginine Synthesis in Young Men Independent of Exercise.

The aim of the study was to investigate the effects of short-term oral administration of inorganic nitrate (NaNO3; n = 8) or placebo (NaCl; n = 9) (each 0.1 mmol/kg body weight/d for 9 days) on plasma amino acids, creatinine, and oxidative stress in healthy young men. At baseline, the plasma concentrations of amino acids did not differ between the groups. At the end of the study, the plasma concentrations of homoarginine (hArg; by 24%, p = 0.0001), citrulline and ornithine (Cit/Orn; by 16%, p = 0.015), and glutamine/glutamate (Gln/Glu; by 6%, p = 0.0003) were higher in the NaNO3 group compared to the NaCl group. The plasma concentrations of sarcosine (Sarc; by 28%, p < 0.0001), tyrosine (by 14%, p = 0.0051), phenylalanine (by 8%, p = 0.0026), and tryptophan (by 8%, p = 0.0047) were lower in the NaNO3 group compared to the NaCl group. These results suggest that nitrate administration affects amino-acid metabolism. The arginine/glycine amidinotransferase (AGAT) catalyzes two reactions: (1) the formation of l-homoarginine (hArg) and l-ornithine (Orn) from l-arginine (Arg) and l-lysine (Lys): Arg + Lys <−> hArg + Orn, with equilibrium constant Kharg; (2) the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly <−> GAA + Orn, with equilibrium constant Kgaa. The plasma Kgaa/KhArg ratio was lower in the NaNO3 group compared to the NaCl group (1.57 vs. 2.02, p = 0.0034). Our study suggests that supplementation of inorganic nitrate increases the AGAT-catalyzed synthesis of hArg and decreases the N-methyltransferase-catalyzed synthesis of GAA, the precursor of creatine. To our knowledge, this is the first study to demonstrate elevation of hArg synthesis by inorganic nitrate supplementation. Remarkably, an increase of 24% corresponds to the synthesis capacity of one kidney in healthy humans. Differences in the association between plasma concentrations of amino acids in the NaNO3 and NaCl groups suggest changes in amino-acid homeostasis. Plasma concentrations of the oxidative stress marker malondialdehyde (MDA) did not change after supplementation of NaNO3 or NaCl over the whole exercise time range. Plasma nitrite concentration turned out to be a more discriminant marker of NaNO3 ingestion than plasma nitrate (area under the receiver operating characteristic curve: 0.951 vs. 0.866, p < 0.0001 each).

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).

Simultaneous stable-isotope dilution GC-MS measurement of homoarginine, guanidinoacetate and their common precursor arginine in plasma and their interrelationships in healthy and diseased humans.

Low concentrations of L-homoarginine (hArg) in plasma or serum and urine have recently emerged as a novel cardiovascular risk factor. Previously, we reported gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS (GC-MS/MS) methods for the quantitative determination of hArg and Arg in plasma, serum, urine and other biological samples. In these methods, plasma and serum are ultrafiltered by means of commercially available cartridges (10 kDa), and 10-µL ultrafiltrate aliquots are subjected to a two-step derivatization procedure, yielding the methyl ester tri(N-pentafluoropropionyl) derivatives. De novo prepared trideuteromethyl ester hArg (d3Me-hArg) was used as an internal standard. To make the hArg analysis in plasma more convenient, straightforward and cheaper we performed two key modifications: (1) precipitation of plasma proteins by methanol and (2) use of newly prepared and d3Me-hArg as the internal standard. The method was validated and used for the quantitative determination of hArg in human plasma by GC-MS after electron-capture negative-ion chemical ionization (ECNICI) using methane as the reactant gas. Intra-assay accuracy (recovery) and imprecision (relative standard deviation) were within generally accepted ranges (93-109 and 2.3-10 %, respectively). Furthermore, we extended the applicability of this method to guanidinoacetate (GAA). This is of particular importance because hArg and GAA are produced from Arg by the catalytic action of arginine:glycine amidinotransferase (AGAT) also known as glycine:arginine transamidinase (GATM). Using this method, we quantitated simultaneously hArg, Arg and GAA in the selected-ion monitoring mode in 10-µL aliquots of plasma. In plasma samples of 17 non-medicated healthy young men, the concentration of hArg, GAA and Arg was determined to be (mean ± SD) 1.7 ± 0.6, 2.6 ± 0.8, 91 ± 29 µM, respectively. The correlation between hArg and Arg was borderline (r = 0.47, P = 0.06). GAA strongly correlated with Arg (r = 0.82, P < 0.0001) but did not correlate with hArg (r = 0.17, P = 0.52). The plasma concentrations of hArg, GAA and Arg measured in 9 patients suffering from stroke or transitory ischemic attack were 1.8 ± 0.6, 2.7 ± 0.4 and 82 ± 17 µM. The ratio values of the hArg, GAA and Arg concentrations measured after removal of plasma proteins by methanol precipitation or ultrafiltration were 0.94 ± 0.1, 0.94 ± 0.08, and 0.88 ± 0.07, respectively. Simultaneous measurement of hArg and GAA in human plasma may allow assessment of AGAT activity in vivo with respect both to GAA and to hArg and their relationship in health, disease, nutrition and pharmacotherapy.

Skeletal Muscle Function in Young Patients With Cystic Fibrosis.

PURPOSE: Defects in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) cause CF. Absence of the CFTR may result in skeletal muscle dysfunction. Here, we tested skeletal muscle function in male adolescent patients with CF. METHODS: Ten CF and 10 control participants (age: 16.8 ± 0.6 years) performed 7 repetitive sets of maximum voluntary contractions (MVCs) and underwent an isometric fatigue test of the knee extensors. Electromyography (EMG) activity was recorded from the m. vastus lateralis (VL) and m. vastus medialis (VM). RESULTS: In CF, the MVC torque was lower and correlated with the predicted forced expiratory volume in one second (r = .73, p = .012, n = 10). The M-wave in the VL was shorter in CF than in controls (18.6 ± 0.5 vs. 20.3 ± 0.5 ms, p < .028). In the VM, both the M-wave (4.96 ± 0.61 vs. 7.97 ± 0.60 mV, p = .001) and the EMG (0.29 ± 0.04 vs. 0.47 ± 0.04 mV, p = .004) amplitudes were smaller in CF. CONCLUSION: The differences in the VL and VM EMG signals between the groups indicate that the lower MVC torque in CF did not result from the direct impact of a CFTR defect on the sarcolemmal excitability; the differences more likely resulted from the less developed musculature in the patients with CF.

Two nights of sleep deprivation with or without energy restriction does not impair the thermal response to cold.

PURPOSE: In persons completing exhaustive daily exercise, sleep and energy restriction have been highlighted as risk factors for hypothermia in cold environments. The present study therefore sought to determine the effect of sleep deprivation (SDEP), with and without energy restriction, on the thermal response to cold. METHODS: In a random order, ten recreationally active men (mean ± SD: age 25 ± 6 years, body fat 17 ± 5 %) completed three 53 h trials: a control (CON: 436 min/night sleep), SDEP (0 min sleep), and sleep deprivation and energy restriction (SDEP + ER: 0 min sleep and 10% daily energy requirements). Exhaustive exercise was completed after 5 and 29 h. After 53 h participants completed a semi-nude seated cold air test (CAT, 0 °C), for 4 h or until rectal core temperature (T re) reached 36 °C. RESULTS: Two nights of sleep and energy restriction did not impair the thermal response to cold (T re, CON 36.15 ± 0.20 °C, SDEP 36.30 ± 0.15 °C, SDEP + ER 36.25 ± 0.20 °C, P = 0.25). Rewarming was also similar as indicated by 1 h post-CAT T re (P = 0.78). In contrast, perceived thermal discomfort during the initial hour of the CAT tended to be greater after SDEP and SDEP + ER (P ≤ 0.1). CONCLUSION: Sleep and energy restriction, at least as evaluated within this experiment, should be considered minimal risk factors for hypothermia. The greater perception of cold discomfort at the same body temperature suggests that sleep and energy restriction may actually reduce cold injury risk, as people are likely to engage earlier in normal behavioral cold adaptation.

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.

Relationship between Blood Volume, Blood Lactate Quantity, and Lactate Concentrations during Exercise.

We wanted to determine the influence of total blood volume (BV) and blood lactate quantity on lactate concentrations during incremental exercise. Twenty-six healthy, nonsmoking, heterogeneously trained females (27.5 ± 5.9 ys) performed an incremental cardiopulmonary exercise test on a cycle ergometer during which maximum oxygen uptake (V·O2max), lactate concentrations ([La-]) and hemoglobin concentrations ([Hb]) were determined. Hemoglobin mass and blood volume (BV) were determined using an optimised carbon monoxide-rebreathing method. V·O2max and maximum power (Pmax) ranged between 32 and 62 mL·min-1·kg-1 and 2.3 and 5.5 W·kg-1, respectively. BV ranged between 81 and 121 mL·kg-1 of lean body mass and decreased by 280 ± 115 mL (5.7%, p = 0.001) until Pmax. At Pmax, the [La-] was significantly correlated to the systemic lactate quantity (La-, r = 0.84, p < 0.0001) but also significantly negatively correlated to the BV (r = -0.44, p < 0.05). We calculated that the exercise-induced BV shifts significantly reduced the lactate transport capacity by 10.8% (p < 0.0001). Our results demonstrate that both the total BV and La- have a major influence on the resulting [La-] during dynamic exercise. Moreover, the blood La- transport capacity might be significantly reduced by the shift in plasma volume. We conclude, that the total BV might be another relevant factor in the interpretation of [La-] during a cardio-pulmonary exercise test.

Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men.

Several studies suggest that exercise is associated with elevated oxidative stress which diminishes NO bioavailability. The aim of the present study was to investigate a potential link between NO synthesis and bioavailability and oxidative stress in the circulation of subjects performing high-intensive endurance exercise. Twenty-two male healthy subjects cycled at 80% of their maximal workload. Cubital venous blood was taken before, during and after exercise, and heparinized plasma was generated. Plasma concentrations of nitrite and nitrate were quantified by GC-MS and of the oxidative stress biomarker 15(S)-8-iso-PGF(2alpha) by GC-MS/MS. pH and pCO(2) fell and HbO(2) increased upon exercise. The duration of the 80% phase (d80) was 740+/-210s. Subjects cycled at 89.2+/-3.3% of their peak oxygen uptake. Plasma concentration of nitrite (P<0.01) and 15(S)-8-iso-PGF(2alpha) (P<0.05) decreased significantly during exercise. At the end of exercise, plasma nitrite concentration correlated positively with d80 and performed work (w80) (each P<0.05). Changes in nitrate concentration also correlated positively with d80 (P<0.05) and w80/kg (P<0.01). These findings provide evidence of a favorable effect of nitrite on high-intensive endurance exercise. The lack of association between 15(S)-8-iso-PGF(2alpha) and NO bioavailability (nitrite concentration) and NO biosynthesis (nitrate concentration) suggest that oxidative stress, notably lipid peroxidation, is not linked to the l-arginine/NO pathway in healthy male subjects being on endurance exercise.

The effects of two nights of sleep deprivation with or without energy restriction on immune indices at rest and in response to cold exposure.

The purpose of the study was to determine the effects of two nights of sleep deprivation with or without energy restriction on immune indices at rest and in response to cold exposure. On three randomised occasions ten males slept normally [mean (SD): 436 (21) min night(-1); CON], were totally sleep-deprived (SDEP), or were totally sleep-deprived and 90% energy-restricted (SDEP + ER) for 53 h. After 53 h (1200 h) participants performed a seated cold air test (CAT) at 0.0 degrees C until T (re) decreased to 36.0 degrees C. Circulating leucocyte counts, neutrophil degranulation, stress hormones and saliva secretory IgA (S-IgA) were determined at 0 h, 24 h, 48 h, pre-CAT, post-CAT, 1-h and 2-h post-CAT. One night on SDEP increased bacterially stimulated neutrophil degranulation (21%, P < 0.05), and two nights on SDEP and SDEP + ER increased S-IgA concentration (40 and 44%; P < 0.01). No other significant effects were observed for immuno-endocrine measures prior to CAT. CAT duration was not different between trials [mean (SD): 133 (53) min] and T (re) decreased to 35.9 (0.3) degrees C. Modest whole-body cooling decreased circulating lymphocyte counts (25%; P < 0.01), S-IgA concentration (36%; P < 0.01) and secretion rate (24%; P < 0.05). A neutrophilia occurred post-CAT on CON and SDEP and 2-h post-CAT on SDEP + ER (P < 0.01). Modest whole-body cooling also decreased neutrophil degranulation on CON (22%) and SDEP (18%; P < 0.05). Plasma cortisol and norepinephrine increased post-CAT (31 and 346%, P < 0.05), but modest whole-body cooling did not alter plasma epinephrine. In conclusion, two nights of SDEP or SDEP + ER did not compromise resting immune indices. However, modest whole-body cooling (T(re) 35.9 degrees C) decreased circulating lymphocytes, neutrophil degranulation and S-IgA, but responses were not amplified by prior SDEP or SDEP + ER.

Human blood neutrophil responses to prolonged exercise with and without a thermal clamp.

The purpose of this study was to investigate the effects of prolonged exercise with and without a thermal clamp on neutrophil trafficking, bacterial-stimulated neutrophil degranulation, stress hormones, and cytokine responses. Thirteen healthy male volunteers (means +/- SE: age 21 +/- 1 yr; mass 74.9 +/- 2.1 kg; maximal oxygen uptake 58 +/- 1 ml x kg(-1) x min(-1)) completed four randomly assigned, 2-h water-immersion trials separated by 7 days. Trials were exercise-induced heating (EX-H: water temperature 36 degrees C), exercise with a thermal clamp (EX-C: 24 degrees C), passive heating (PA-H: 38.5 degrees C), and control (CON: 35 degrees C). EX-H and EX-C was comprised of 2 h of deep water running at 58% maximal oxygen uptake. Blood samples were collected at pre-, post-, and 1 h postimmersion. Core body temperature was unaltered on CON, clamped on EX-C (-0.02 degrees C), and rose by 2.23 degrees C and 2.31 degrees C on EX-H and PA-H, respectively. Exercising with a thermal clamp did not blunt the neutrophilia postexercise (EX-C postexercise: 9.6 +/- 1.1 and EX-H postexercise: 9.8 +/- 1.0 x 10(9)/liter). Neutrophil degranulation decreased (P < 0.01) similarly immediately after PA-H (-21%), EX-C, and EX-H (-28%). EX-C blunted the circulating norepinephrine, cortisol, granulocyte-colony stimulating factor, and IL-6 response (P < 0.01) but not the plasma epinephrine and serum growth hormone response. These results show a similar neutrophilia and decrease in neutrophil degranulation after prolonged exercise with and without a thermal clamp. As such, the rise in core body temperature does not appear to mediate neutrophil trafficking and degranulation responses to prolonged exercise. In addition, these results suggest a limited role for cortisol, granulocyte-colony stimulating factor, and IL-6 in the observed neutrophil responses to prolonged exercise.

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.

High Intensity High Volume Interval Training Improves Endurance Performance and Induces a Nearly Complete Slow-to-Fast Fiber Transformation on the mRNA Level.

We present here a longitudinal study determining the effects of two 3 week-periods of high intensity high volume interval training (HIHVT) (90 intervals of 6 s cycling at 250% maximum power, Pmax/24 s) on a cycle ergometer. HIHVT was evaluated by comparing performance tests before and after the entire training (baseline, BSL, and endpoint, END) and between the two training sets (intermediate, INT). The mRNA expression levels of myosin heavy chain (MHC) isoforms and markers of energy metabolism were analyzed in M. vastus lateralis biopsies by quantitative real-time PCR. In incremental tests peak power (Ppeak) was increased, whereas V˙ O2peak was unaltered. Prolonged time-to-exhaustion was found in endurance tests with 65 and 80% Pmax at INT and END. No changes in blood levels of lipid metabolites were detected. Training-induced decreases of hematocrit indicate hypervolemia. A shift from slow MHCI/ß to fast MHCIIa mRNA expression occurred after the first and second training set. The mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a master regulator of oxidative energy metabolism, decreased after the second training set. In agreement, a significant decrease was also found for citrate synthase mRNA after the second training set, indicating reduced oxidative capacity. However, mRNA expression levels of glycolytic marker enzyme glyceraldehyde-3-phosphate dehydrogenase did not change after the first and second training set. HIHVT induced a nearly complete slow-to-fast fiber type transformation on the mRNA level, which, however, cannot account for the improvements of performance parameters. The latter might be explained by the well-known effects of hypervolemia on exercise performance.

Effect of exercise with and without a thermal clamp on the plasma heat shock protein 72 response.

The contribution of heat and exercise related stress to the release of heat shock protein 72 (HSP72) is currently unknown. The purpose of the present study was to determine the combined and independent effects of heat and exercise on the extracellular (e)HSP72 response. Eleven moderately trained male volunteers [means +/- SD: age 21 +/- 4 yr; body mass 75.7 +/- 7.7 kg; maximal oxygen uptake ((.)Vo(2 max)) 57.8 +/- 3.3 ml.kg(-1).min(-1)] completed four 2-h, heat-manipulated, water-immersion trials. Trials were exercise-induced heat (EIH; rectal temperature change +2.2 degrees C), clamped exercise (CEx; 0 degrees C), passive heating (PHT; +2.3 degrees C), and control (Con; 0 degrees C). Exercise trials (EIH and CEx) comprised deep-water running at 58.5 +/- 2.4 and 59.1 +/- 1.7% (.)vo(2)max. eHSP72 and catecholamine concentrations were determined by ELISA and HPLC, respectively, pre- and postimmersion. All trials induced an eHSP72 response (P < 0.05) with postimmersion values significantly greater on EIH compared with other trials (6.0 +/- 3.4; CEx 3.8 +/- 2.6; PHT 2.7 +/- 2.1; Con 2.2 +/- 1.9 ng/ml). Exercising with a thermal clamp blunted the eHSP72 response, but postimmersion values were also greater than Con. PHT induced a large catecholamine response, but postimmersion eHSP72 values did not reach significance vs. Con. Given that exercising with a thermal clamp evoked a significant increase in plasma eHSP72 concentration, exercise-related stressors other than heat appeared influential in stimulating HSP72 release. Moreover, the catecholamine data from PHT suggest neither epinephrine nor norepinephrine was solely responsible for eHSP72 release.

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.

Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

Malondialdehyde (MDA, CH2(CHO)2) is one of the best investigated and most frequently measured biomarkers of lipid peroxidation in biological fluids, a constituent of the so called thiobarbituric acid reactive substances (TBARS). The reaction of thiobarbituric acid with MDA and other carbonyl compounds is the basis for the batch TBARS assay, one of the most commonly and widely used assays of oxidative stress. Yet, the TBARS assay lacks specificity even if combined with HPLC separation prior to visible absorbance or fluorescence detection. In this article, we report highly specific and sensitive stable-isotope dilution GC-MS and GC-MS/MS methods for the quantitative determination of MDA in human plasma (0.1 mL). These methods utilize the acidity (pKa, 4.46) of the two methylene H protons of MDA in aqueous solution, which are as acidic as acetic acid. Endogenous MDA in native plasma and the externally added internal standard [1,3-(2)H2]-MDA (d2-MDA, CH2(CDO)2) are derivatized in aqueous acetone (400 µL) with pentafluorobenzyl (PFB) bromide (10 µL). The reaction products were identified as C(PFB)2(CHO)2 (molecular weight, 432) and C(PFB)2(CDO)2) (molecular weight, 434), respectively. After solvent extraction with toluene (1 mL) quantification is performed by selected-ion monitoring (SIM) in GC-MS and by selected-reaction monitoring (SRM) in GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode. In the SIM mode, the anions [M-PFB](-) at m/z 251 for MDA and m/z 253 for d2-MDA are detected. In the SRM mode, the mass transitions m/z 251 to m/z 175 for MDA and m/z 253 to m/z 177 for d2-MDA are monitored. The method was thoroughly validated in human plasma. Potential interfering substances including anticoagulants and commercially available monovettes commonly used for blood sampling were tested. The lowest MDA concentrations were measured in serum followed by heparinized and EDTA plasma. The GC-MS and GC-MS/MS methods were found to be specific, precise, accurate and sensitive. Thus, the LOD of the GC-MS/MS method was determined to be 2 amol (2 × 10(-18)mol) MDA. The GC-MS/MS method is exceedingly useful in clinical settings. We report several biomedical applications and discuss the utility of circulating MDA as a biomarker of lipid peroxidation, especially in long-term clinical studies, and its relation to the F2-isoprostane 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).

Human plasma concentrations of malondialdehyde (MDA) and the F2-isoprostane 15(S)-8-iso-PGF(2alpha) may be markedly compromised by hemolysis: evidence by GC-MS/MS and potential analytical and biological ramifications.

OBJECTIVES: Malondialdehyde (MDA) and the F(2)-isoprostane 15(S)-8-iso-prostaglandin F(2alpha) (15(S)-8-iso-PGF(2alpha)) belong to the most frequently analyzed biomarkers of oxidative stress in basic and clinical research. The objective of the present study was to examine the effect of hemolysis on free MDA and total (free+esterified) 15(S)-8-iso-PGF(2alpha) concentrations in human plasma. DESIGN AND METHODS: MDA and 15(S)-8-iso-PGF(2alpha) were determined by GC-MS/MS in plasma samples from venous heparinized blood drawn under resting conditions (n=22) as well as under physical exercise (n=158) in 22 healthy young subjects. In vitro, we prepared plasma samples with hemolysis degrees up to 0.8% using artificially hemolyzed, freshly obtained heparinized blood. RESULTS: In some plasma samples of the exercise study both under resting and exercise conditions, clinically significant hemolysis was macroscopically visible. Both in vivo (r=0.74) and in vitro (r=0.87), we found a significant positive correlation between hemolysis degree (0-0.2%) and MDA plasma concentrations (50-250 nmol/L). Unlike in vitro (r=0.84), in vivo, 15(S)-8-iso-PGF(2alpha) and MDA plasma concentrations correlated weakly (r=0.50). CONCLUSIONS: We hypothesize that free hemoglobin catalyzes the formation of MDA and 15(S)-8-iso-PGF(2alpha) from free and esterified arachidonic acid. Plasma concentrations of MDA and total 15(S)-8-iso-PGF(2alpha) may be markedly compromised by hemolysis. Measurements of MDA and 15(S)-8-iso-PGF(2alpha) should be treated with caution regarding involvement of oxidative stress in disease as well as in health both under resting conditions and under exercise.