EPR spectroscopic detection of free radical outflow from an isolated muscle bed in exercising humans.

There is no direct evidence to support the contention that contracting skeletal muscle and/or associated vasculature generates free radicals in exercising humans. The unique combination of isolated quadriceps exercise and the measurement of femoral arterial and venous free radical concentrations with the use of electron paramagnetic resonance (EPR) spectroscopy enabled this assumption to be tested in seven healthy men. Application of ex vivo spin trapping using alpha-phenyl-tert-butylnitrone (PBN) resulted in the detection of oxygen- or carbon-centered free radicals (a(N) = 1.38 +/- 0.01 mT and a(beta)(H) = 0.17 +/-0.01 mT, where a(N) and a(beta)(H) are the nitrogen and beta-hydrogen coupling constants, respectively) with consistently higher EPR signal intensities of the PBN spin adduct observed in the venous compared with the arterial circulation (P < 0.05). Incremental exercise further increased the venoarterial intensity difference [85 +/- 58 arbitrary units (AU) at 24 +/- 6% maximal work rate (WR(max)) vs. 387 +/- 214 AU at 69 +/- 7% WR(max); P < 0.05]. When combined with measured changes in femoral venous blood flow (Q), this resulted in a net adduct outflow of 130 +/- 118 and 1,146 +/- 582 AU/min (P < 0.05), which was positively associated with leg oxygen uptake (r(2) = 0.47, P < 0.05) and Q (r(2) = 0.47, P < 0.05). These results provide the first evidence for oxygen- or carbon-centered free radical outflow from an active muscle bed in humans.     

Electron paramagnetic spectroscopic evidence of exercise-induced free radical accumulation in human skeletal muscle

The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WR(MAX)) and 3 min at 100% WR(MAX). EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone (UQ*-) [0.167 +/- 0.055 vs. rest: 0.106 +/- 0.047 arbitrary units (AU)/g total protein (TP), P < 0.05] and alpha-phenyl-tert-butylnitrone-adducts (112 +/- 41 vs. rest: 29 +/- 9 AU/mg tissue mass, P < 0.05). Intramuscular lipid hydroperoxides also increased (0.320 +/- 0.263 vs. rest: 0.148 +/- 0.071 nmol/mg TP, P < 0.05) despite an uptake of alpha-tocopherol, alpha-carotene and beta-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct evidence for intramuscular free radical accumulation and lipid peroxidation following acute exercise in humans.     

Reduction of hexavalent chromium by human cytochrome b5: generation of hydroxyl radical and superoxide

The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b(5) and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure (53)Cr(VI). When 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO()) adduct was observed as well as a smaller signal for the superoxide (O(2)(-)) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 muM). NADPH, Cr(VI), O(2), and the PLs were all required for significant HO() generation. Superoxide dismutase eliminated the O(2)(-) adduct and resulted in a 30% increase in the HO() adduct. Catalase largely diminished the HO() adduct signal, indicating its dependence on H(2)O(2). Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H(2)O(2) was not needed, indicating that it was generated by the PLs. Adding exogenous H(2)O(2), however, did increase the amount of DEPMPO/HO() adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethyl sulfoxide (DMSO) plus the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO(). Quantification of the various species over time was consistent with a stoichiometric excess of HO() relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b(5) in the generation of HO(). Overall, the simultaneous generation of Cr(V) and H(2)O(2) by the PLs and the resulting generation of HO() at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.     

Evidence of O2 supply-dependent VO2 max in the exercise-trained human quadriceps.

Maximal O2 delivery and O2 uptake (VO2) per 100 g of active muscle mass are far greater during knee extensor (KE) than during cycle exercise: 73 and 60 ml. min-1. 100 g-1 (2.4 kg of muscle) (R. S. Richardson, D. R. Knight, D. C. Poole, S. S. Kurdak, M. C. Hogan, B. Grassi, and P. D. Wagner. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1453-H1461, 1995) and 28 and 25 ml. min-1. 100 g-1 (7.5 kg of muscle) (D. R. Knight, W. Schaffartzik, H. J. Guy, R. Predilleto, M. C. Hogan, and P. D. Wagner. J. Appl. Physiol. 75: 2586-2593, 1993), respectively. Although this is evidence of muscle O2 supply dependence in itself, it raises the following question: With such high O2 delivery in KE, are the quadriceps still O2 supply dependent at maximal exercise? To answer this question, seven trained subjects performed maximum KE exercise in hypoxia [0.12 inspired O2 fraction (FIO2)], normoxia (0.21 FIO2), and hyperoxia (1.0 FIO2) in a balanced order. The protocol (after warm-up) was a square wave to a previously determined maximum work rate followed by incremental stages to ensure that a true maximum was achieved under each condition. Direct measures of arterial and venous blood O2 concentration in combination with a thermodilution blood flow technique allowed the determination of O2 delivery and muscle VO2. Maximal O2 delivery increased with inspired O2: 1.3 +/- 0.1, 1.6 +/- 0.2, and 1.9 +/- 0.2 l/min at 0.12, 0.21, and 1.0 FIO2, respectively (P < 0.05). Maximal work rate was affected by variations in inspired O2 (-25 and +14% at 0.12 and 1.0 FIO2, respectively, compared with normoxia, P < 0.05) as was maximal VO2 (VO2 max): 1.04 +/- 0.13, 1. 24 +/- 0.16, and 1.45 +/- 0.19 l/min at 0.12, 0.21, and 1.0 FIO2, respectively (P < 0.05). Calculated mean capillary PO2 also varied with FIO2 (28.3 +/- 1.0, 34.8 +/- 2.0, and 40.7 +/- 1.9 Torr at 0.12, 0.21, and 1.0 FIO2, respectively, P < 0.05) and was proportionally related to changes in VO2 max, supporting our previous finding that a decrease in O2 supply will proportionately decrease muscle VO2 max. As even in the isolated quadriceps (where normoxic O2 delivery is the highest recorded in humans) an increase in O2 supply by hyperoxia allows the achievement of a greater VO2 max, we conclude that, in normoxic conditions of isolated KE exercise, KE VO2 max in trained subjects is not limited by mitochondrial metabolic rate but, rather, by O2 supply.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Prior exercise speeds pulmonary O2 uptake kinetics by increases in both local muscle O2 availability and O2 utilization.

The effect of prior exercise on pulmonary O(2) uptake (Vo(2)(p)), leg blood flow (LBF), and muscle deoxygenation at the onset of heavy-intensity alternate-leg knee-extension (KE) exercise was examined. Seven subjects [27 (5) yr; mean (SD)] performed step transitions (n = 3; 8 min) from passive KE following no warm-up (HVY 1) and heavy-intensity (Delta50%, 8 min; HVY 2) KE exercise. Vo(2)(p) was measured breath-by-breath; LBF was measured by Doppler ultrasound at the femoral artery; and oxy (O(2)Hb)-, deoxy (HHb)-, and total (Hb(tot)) hemoglobin/myoglobin of the vastus lateralis muscle were measured continuously by near-infrared spectroscopy (NIRS; Hamamatsu NIRO-300). Phase 2 Vo(2)(p), LBF, and HHb data were fit with a monoexponential model. The time delay (TD) from exercise onset to an increase in HHb was also determined and an HHb effective time constant (HHb - MRT = TD + tau) was calculated. Prior heavy-intensity exercise resulted in a speeding (P < 0.05) of phase 2 Vo(2)(p) kinetics [HVY 1: 42 s (6); HVY 2: 37 s (8)], with no change in the phase 2 amplitude [HVY 1: 1.43 l/min (0.21); HVY 2: 1.48 l/min (0.21)] or amplitude of the Vo(2)(p) slow component [HVY 1: 0.18 l/min (0.08); HVY 2: 0.18 l/min (0.09)]. O(2)Hb and Hb(tot) were elevated throughout the on-transient following prior heavy-intensity exercise. The tauLBF [HVY 1: 39 s (7); HVY 2: 47 s (21); P = 0.48] and HHb-MRT [HVY 1: 23 s (4); HVY 2: 21 s (7); P = 0.63] were unaffected by prior exercise. However, the increase in HHb [HVY 1: 21 microM (10); HVY 2: 25 microM (10); P < 0.001] and the HHb-to-Vo(2)(p) ratio [(HHb/Vo(2)(p)) HVY 1: 14 microM x l(-1) x min(-1) (6); HVY 2: 17 microM x l(-1) x min(-1) (5); P < 0.05] were greater following prior heavy-intensity exercise. These results suggest that the speeding of phase 2 tauVo(2)(p) was the result of both elevated local O(2) availability and greater O(2) extraction evidenced by the greater HHb amplitude and HHb/Vo(2)(p) ratio following prior heavy-intensity exercise.     

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.     

Limitations to vasodilatory capacity and .VO2 max in trained human skeletal muscle

To further explore the limitations to maximal O(2) consumption (.VO(2 max)) in exercise-trained skeletal muscle, six cyclists performed graded knee-extensor exercise to maximum work rate (WR(max)) in hypoxia (12% O(2)), hyperoxia (100% O(2)), and hyperoxia + femoral arterial infusion of adenosine (ADO) at 80% WR(max). Arterial and venous blood sampling and thermodilution blood flow measurements allowed the determination of muscle O(2) delivery and O(2) consumption. At WR(max), O(2) delivery rose progressively from hypoxia (1.0 +/- 0.04 l/min) to hyperoxia (1.20 +/- 0.09 l/min) and hyperoxia + ADO (1.33 +/- 0.05 l/min). Leg .VO(2 max) varied with O(2) availability (0.81 +/- 0.05 and 0.97 +/- 0.07 l/min in hypoxia and hyperoxia, respectively) but did not improve with ADO-mediated vasodilation (0.80 +/- 0.09 l/min in hyperoxia + ADO). Although a vasodilatory reserve in the maximally working quadriceps muscle group may have been evidenced by increased leg vascular conductance after ADO infusion beyond that observed in hyperoxia (increased blood flow but no change in blood pressure), we recognize the possibility that the ADO infusion may have provoked vasodilation in nonexercising tissue of this limb. Together, these findings imply that maximally exercising skeletal muscle may maintain some vasodilatory capacity, but the lack of improvement in leg .VO(2 max) with significantly increased O(2) delivery (hyperoxia + ADO), with a degree of uncertainty as to the site of this dilation, suggests an ADO-induced mismatch between O(2) consumption and blood flow in the exercising limb.     

Comparison of oxygen uptake kinetics during knee extension and cycle exercise

The knee extension exercise (KE) model engenders different muscle and fiber recruitment patterns, blood flow, and energetic responses compared with conventional cycle ergometry (CE). This investigation had two aims: 1) to test the hypothesis that upright two-leg KE and CE in the same subjects would yield fundamentally different pulmonary O(2) uptake (pVo(2)) kinetics and 2) to characterize the muscle blood flow, muscle Vo(2) (mVo(2)), and pVo(2) kinetics during KE to investigate the rate-limiting factor(s) of pVo(2) on kinetics and muscle energetics and their mechanistic bases after the onset of heavy exercise. Six subjects performed KE and CE transitions from unloaded to moderate [< ventilatory threshold (VT)] and heavy (>VT) exercise. In addition to pVo(2) during CE and KE, simultaneous pulsed and echo Doppler methods, combined with blood sampling from the femoral vein, were used to quantify the precise temporal profiles of femoral artery blood flow (LBF) and mVo(2) at the onset of KE. First, the gain (amplitude/work rate) of the primary component of pVo(2) for both moderate and heavy exercise was higher during KE ( approximately 12 ml.W(-1).min(-1)) compared with CE ( approximately 10), but the time constants for the primary component did not differ. Furthermore, the mean response time (MRT) and the contribution of the slow component to the overall response for heavy KE were significantly greater than for CE. Second, the time constant for the primary component of mVo(2) during heavy KE [25.8 +/- 9.0 s (SD)] was not significantly different from that of the phase II pVo(2). Moreover, the slow component of pVo(2) evident for the heavy KE reflected the gradual increase in mVo(2). The initial LBF kinetics after onset of KE were significantly faster than the phase II pVo(2) kinetics (moderate: time constant LBF = 8.0 +/- 3.5 s, pVo(2) = 32.7 +/- 5.6 s, P < 0.05; heavy: LBF = 9.7 +/- 2.0 s, pVo(2) = 29.9 +/- 7.9 s, P < 0.05). The MRT of LBF was also significantly faster than that of pVo(2). These data demonstrate that the energetics (as gain) for KE are greater than for CE, but the kinetics of adjustment (as time constant for the primary component) are similar. Furthermore, the kinetics of muscle blood flow during KE are faster than those of pVo(2), consistent with an intramuscular limitation to Vo(2) kinetics, i.e., a microvascular O(2) delivery-to-O(2) requirement mismatch or oxidative enzyme inertia.     

Speeding of VO2 kinetics during moderate-intensity exercise subsequent to heavy-intensity exercise is associated with improved local O2 distribution

The relationship between the adjustment of muscle deoxygenation (Δ[HHb]) and phase II V(O(2p)) during moderate-intensity exercise was examined before (Mod 1) and after (Mod 2) a bout of heavy-intensity "priming" exercise. Moderate intensity V(O(2p)) and Δ[HHb] kinetics were determined in 18 young males (26 ± 3 yr). V(O(2p)) was measured breath-by-breath. Changes in Δ[HHb] of the vastus lateralis muscle were measured by near-infrared spectroscopy. V(O(2p)) and Δ[HHb] response profiles were fit using a monoexponential model, and scaled to a relative % of the response (0-100%). The Δ[HHb]/Vo(2) ratio for each individual (reflecting the local matching of O(2) delivery to O(2) utilization) was calculated as the average Δ[HHb]/Vo(2) response from 20 s to 120 s during the exercise on-transient. Phase II τV(O(2p)) was reduced in Mod 2 compared with Mod 1 (P < 0.05). The effective τ'Δ[HHb] remained the same in Mod 1 and Mod 2 (P > 0.05). During Mod 1, there was an "overshoot" in the Δ[HHb]/Vo(2) ratio (1.08; P < 0.05) that was not present during Mod 2 (1.01; P > 0.05). There was a positive correlation between the reduction in the Δ[HHb]/Vo(2) ratio and the smaller τV(O(2p)) from Mod 1 to Mod 2 (r = 0.78; P < 0.05). This study showed that a smaller τV(O(2p)) during a moderate bout of exercise subsequent to a heavy-intensity priming exercise was associated with improved microvascular O(2) delivery during the on-transient of exercise, as suggested by a smaller Δ[HHb]/Vo(2) ratio.     

Hyperbaric hyperoxia reduces exercising forearm blood flow in humans

Hypoxia during exercise augments blood flow in active muscles to maintain the delivery of O(2) at normoxic levels. However, the impact of hyperoxia on skeletal muscle blood flow during exercise is not completely understood. Therefore, we tested the hypothesis that the hyperemic response to forearm exercise during hyperbaric hyperoxia would be blunted compared with exercise during normoxia. Seven subjects (6 men/1 woman; 25 ± 1 yr) performed forearm exercise (20% of maximum) under normoxic and hyperoxic conditions. Forearm blood flow (FBF; in ml/min) was measured using Doppler ultrasound. Forearm vascular conductance (FVC; in ml·min(-1)·100 mmHg(-1)) was calculated from FBF and blood pressure (in mmHg; brachial arterial catheter). Studies were performed in a hyperbaric chamber with the subjects supine at 1 atmospheres absolute (ATA) (sea level) while breathing normoxic gas [21% O(2), 1 ATA; inspired Po(2) (Pi(O(2))) ≈ 150 mmHg] and at 2.82 ATA while breathing hyperbaric normoxic (7.4% O(2), 2.82 ATA, Pi(O(2)) ≈ 150 mmHg) and hyperoxic (100% O(2), 2.82 ATA, Pi(O(2)) ≈ 2,100 mmHg) gas. Resting FBF and FVC were less during hyperbaric hyperoxia compared with hyperbaric normoxia (P < 0.05). The change in FBF and FVC (Δ from rest) during exercise under normoxia (204 ± 29 ml/min and 229 ± 37 ml·min(-1)·100 mmHg(-1), respectively) and hyperbaric normoxia (203 ± 28 ml/min and 217 ± 35 ml·min(-1)·100 mmHg(-1), respectively) did not differ (P = 0.66-0.99). However, the ΔFBF (166 ± 21 ml/min) and ΔFVC (163 ± 23 ml·min(-1)·100 mmHg(-1)) during hyperbaric hyperoxia were substantially attenuated compared with other conditions (P < 0.01). Our data suggest that exercise hyperemia in skeletal muscle is highly dependent on oxygen availability during hyperoxia.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Effect of arterial oxygenation on quadriceps fatigability during isolated muscle exercise

The effect of various levels of oxygenation on quadriceps muscle fatigability during isolated muscle exercise was assessed in six male subjects. Twitch force (Q(tw)) was assessed using supramaximal magnetic femoral nerve stimulation. In experiment 1, maximal voluntary contraction (MVC) and Q(tw) of resting quadriceps muscle were measured in normoxia [inspired O(2) fraction (Fi(O(2))) = 0.21, percent arterial O(2) saturation (Sp(O(2))) = 98.4%, estimated arterial O(2) content (Ca(O(2))) = 20.8 ml/dl], acute hypoxia (Fi(O(2)) = 0.11, Sp(O(2)) = 74.6%, Ca(O(2)) = 15.7 ml/dl), and acute hyperoxia (Fi(O(2)) = 1.0, Sp(O(2)) = 100%, Ca(O(2)) = 22.6 ml/dl). No significant differences were found for MVC and Q(tw) among the three Fi(O(2)) levels. In experiment 2, the subjects performed three sets of nine, intermittent, isometric, unilateral, submaximal quadriceps contractions (62% MVC followed by 1 MVC in each set) while breathing each Fi(O(2)). Q(tw) was assessed before and after exercise, and myoelectrical activity of the vastus lateralis was obtained during exercise. The percent reduction of twitch force (potentiated Q(tw)) in hypoxia (-27.0%) was significantly (P < 0.05) greater than in normoxia (-21.4%) and hyperoxia (-19.9%), as were the changes in intratwitch measures of contractile properties. The increase in integrated electromyogram over the course of the nine contractions in hypoxia (15.4%) was higher (P < 0.05) than in normoxia (7.2%) or hyperoxia (6.7%). These results demonstrate that quadriceps muscle fatigability during isolated muscle exercise is exacerbated in acute hypoxia, and these effects are independent of the relative exercise intensity.     

Glucose kinetics and substrate oxidation during exercise in the follicular and luteal phases.

The purpose of this investigation was to determine whether plasma glucose kinetics and substrate oxidation during exercise are dependent on the phase of the menstrual cycle. Once during the follicular (F) and luteal (L) phases, moderately trained subjects [peak O(2) uptake (V(O(2))) = 48.2 +/- 1.1 ml. min(-1). kg(-1); n = 6] cycled for 25 min at approximately 70% of the V(O(2)) at their respective lactate threshold (70%LT), followed immediately by 25 min at 90%LT. Rates of plasma glucose appearance (R(a)) and disappearance (R(d)) were determined with a primed constant infusion of [6,6-(2)H]glucose, and total carbohydrate (CHO) and fat oxidation were determined with indirect calorimetry. At rest and during exercise at 70%LT, there were no differences in glucose R(a) or R(d) between phases. CHO and fat oxidation were not different between phases at 70%LT. At 90%LT, glucose R(a) (28.8 +/- 4.8 vs. 33.7 +/- 4.5 micromol. min(-1). kg(-1); P < 0.05) and R(d) (28.4 +/- 4.8 vs. 34.0 +/- 4.1 micromol. min(-1). kg(-1); P < 0.05) were lower during the L phase. In addition, at 90%LT, CHO oxidation was lower during the L compared with the F phase (82.0 +/- 12.3 vs. 93.8 +/- 9.7 micromol. min(-1) .kg(-1); P < 0.05). Conversely, total fat oxidation was greater during the L phase at 90%LT (7.46 +/- 1.01 vs. 6.05 +/- 0.89 micromol. min(-1). kg(-1); P < 0.05). Plasma lactate concentration was also lower during the L phase at 90%LT concentrations (2.48 +/- 0.41 vs. 3.08 +/- 0.39 mmol/l; P < 0.05). The lower CHO utilization during the L phase was associated with an elevated resting estradiol (P < 0.05). These results indicate that plasma glucose kinetics and CHO oxidation during moderate-intensity exercise are lower during the L compared with the F phase in women. These differences may have been due to differences in circulating estradiol.     

Frequency domain analysis of ventilation and gas exchange kinetics in hypoxic exercise.

The kinetics of O2 up-take (VO2), CO2 output (VCO2), ventilation (VE), and heart rate (HR) were studied during exercise in normoxia and hypoxia [inspired O2 fraction (FIO2) 0.14]. Eight male subjects each completed 6 on- and off-step transitions in work rate (WR) from low (25 W) to moderate (100-125 W) levels and a pseudorandom binary sequence (PRBS) exercise test in which WR was varied between the same WRs. Breath-by-breath data were linearly interpolated to yield 1-s values. After the first PRBS cycle had been omitted as a warm-up, five cycles were ensemble-averaged before frequency domain analysis by standard Fourier methods. The step data were fit by a two-component (three for HR) exponential model to estimate kinetic parameters. In the steady state of low and moderate WRs, each value of VO2, VCO2, VE, and HR was significantly greater during hypoxic than normoxic exercise (P less than 0.05) with the exception of VCO2 (low WR). Hypoxia slowed the kinetics of VO2 and HR in on- and off-step transitions and speeded up the kinetics of VCO2 and VE in the on-transition and of VE in the off-transition. Frequency domain analysis confined to the range of 0.003-0.019 Hz for the PRBS tests indicated reductions in amplitude and greater phase shifts in the hypoxic tests for VO2 and HR at specific frequencies, whereas amplitude tended to be greater with little change in phase shift for VCO2 and VE during hypoxic tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Effects of arterial oxygen content on peripheral locomotor muscle fatigue.

The effect of arterial O2 content (Ca(O2)) on quadriceps fatigue was assessed in healthy, trained male athletes. On separate days, eight participants completed three constant-workload trials on a bicycle ergometer at fixed workloads (314 +/- 13 W). The first trial was performed while the subjects breathed a hypoxic gas mixture [inspired O2 fraction (Fi(O2)) = 0.15, Hb saturation = 81.6%, Ca(O2) = 18.2 ml O2/dl blood; Hypo] until exhaustion (4.5 +/- 0.4 min). The remaining two trials were randomized and time matched with Hypo. The second and third trials were performed while the subjects breathed a normoxic (Fi(O2) = 0.21, Hb saturation = 95.0%, Ca(O2) = 21.3 ml O2/dl blood; Norm) and a hyperoxic (Fi(O2) = 1.0, Hb saturation = 100%, Ca(O2) = 23.8 ml O2/dl blood; Hyper) gas mixture, respectively. Quadriceps muscle fatigue was assessed via magnetic femoral nerve stimulation (1-100 Hz) before and 2.5 min after exercise. Myoelectrical activity of the vastus lateralis was obtained from surface electrodes throughout exercise. Immediately after exercise, the mean force response across 1-100 Hz decreased from preexercise values (P < 0.01) by -26 +/- 2, -17 +/- 2, and -13 +/- 2% for Hypo, Norm, and Hyper, respectively; each of the decrements differed significantly (P < 0.05). Integrated electromyogram increased significantly throughout exercise (P < 0.01) by 23 +/- 3, 10 +/- 1, and 6 +/- 1% for Hypo, Norm, and Hyper, respectively; each of the increments differed significantly (P < 0.05). Mean power frequency fell more (P < 0.05) during Hypo (-15 +/- 2%); the difference between Norm (-7 +/- 1%) and Hyper (-6 +/- 1%) was not significant (P = 0.32). We conclude that deltaCa(O2) during strenuous systemic exercise at equal workloads and durations affects the rate of locomotor muscle fatigue development.     

Respiratory muscle blood flows during physiological and chemical hyperpnea in the rat.

Whether the diaphragm retains a vasodilator reserve at maximal exercise is controversial. To address this issue, we measured respiratory and hindlimb muscle blood flows and vascular conductances using radiolabeled microspheres in rats running at their maximal attainable treadmill speed (96 +/- 5 m/min; range 71-116 m/min) and at rest while breathing either room air or 10% O(2)-8% CO(2) (balance N(2)). All hindlimb and respiratory muscle blood flows measured increased during exercise (P < 0.001), whereas increases in blood flow while breathing 10% O(2)-8% CO(2) were restricted to the diaphragm only. During exercise, muscle blood flow increased up to 18-fold above rest values, with the greatest mass specific flows (in ml. min(-1). 100 g(-1)) found in the vastus intermedius (680 +/- 44), red vastus lateralis (536 +/- 18), red gastrocnemius (565 +/- 47), and red tibialis anterior (602 +/- 44). During exercise, blood flow was higher (P < 0.05) in the costal diaphragm (395 +/- 31 ml. min(-1). 100 g(-1)) than in the crural diaphragm (286 +/- 17 ml. min(-1). 100 g(-1)). During hypoxia+hypercapnia, blood flows in both the costal and crural diaphragms (550 +/- 70 and 423 +/- 53 ml. min(-1). 100 g(-1), respectively) were elevated (P < 0.05) above those found during maximal exercise. These data demonstrate that there is a substantial functional vasodilator reserve in the rat diaphragm at maximal exercise and that hypoxia + hypercapnia-induced hyperpnea is necessary to elevate diaphragm blood flow to a level commensurate with its high oxidative capacity.