Effects of muscle contraction on cytochrome a,a3 redox state.

The relationships among mitochondrial O2 availability, O2 delivery, and lactate formation in exercising skeletal muscle remain unclear. Some data suggest that muscle O2 provision is sufficient at maximal O2 consumption (VO2max) to challenge the concept of a mitochondrial O2 limitation at VO2max. The relationships among VO2, mitochondrial O2 availability, and net lactate production were studied over a wide range of exercise intensities. Using near-infrared spectroscopy, the oxidation-reduction state of cytochrome a,a3 was monitored in the canine gracilis in vivo. Twenty adult dogs were anesthetized with alpha-chloralose, intubated, and mechanically ventilated on room air. Five-minute stimulation periods at rates of 2, 3, 4, 5, 7, 8, 10, or 12 stimuli/s were performed. VO2max generally was achieved at a stimulation rate of 8 stimuli/s; mean VO2max was 0.12 +/- 0.09 (SE) ml.min-1 x g-1. The concentration of oxidized mitochondrial cytochrome a,a3 decreased at all work loads relative to resting state and demonstrated a near-linear relationship with muscle VO2 (r2 = 0.99). Muscle lactate efflux and the lactate-pyruvate ratio also were correlated positively with cytochrome a,a3 reduction, suggesting a common regulatory mechanism coupling the processes of aerobic glycolysis and oxidative phosphorylation. At VO2max, the corresponding cytochrome oxidation was not significantly different from that observed at death. Thus, in the gracilis maximal exercise leads to near-complete reduction of cytochrome a,a3 secondary to deficient O2 provision. We conclude that VO2max is limited primarily by O2 delivery to this muscle and not by other factors limiting mitochondrial ATP production or substrate oxidation.

Cerebrocortical oxygenation and ventilatory response during sustained hypoxia.

Cerebrocortical oxygenation was monitored in 8 healthy adults during exposure to sustained isocapnic hypoxia. Subjects were maintained at an arterial oxygen saturation (SaO2) of 80% for 12 min with a rebreathing circuit while cerebrocortical oxygenation was assessed non-invasively using near-infrared (NIR) spectroscopy to measure changes in the oxidation state of cytochrome a,a3 (Cyt a,a3) and changes in cortical blood volume (tBV). During sustained hypoxia, subjects demonstrated a biphasic ventilatory response. The mean minute ventilation (VE) peak response was 255% of baseline at an average of 3.4 +/- 0.5 min (mean +/- SE) after the initiation of hypoxia. A subsequent significant attenuation of VE to 163% (P less than 0.05) of baseline occurred after an additional 8.6 min. NIR monitoring revealed a significant (P less than 0.05) decrease in oxidized Cyt a,a3 as well as a significant (P less than 0.05) increase in tissue blood volume (tBV) at the time of peak VE. Both Cyt a,a3 and tBV remained stable during the remainder of the hypoxic period, despite attenuation of VE during sustained hypoxia. The data suggest that cerebrocortical oxygenation and blood flow remain constant when the ventilatory attenuation is observed during sustained hypoxia.

Reduction of quinones with zinc metal in the presence of zinc ions: application of post-column reactor for the fluorometric detection of vitamin K compounds.

A simple and effective chemical method has been developed for quantitatively reducing quinones, which is based on their reaction with zinc metal and zinc ions. Comparison of this method with conventional electrochemical reduction revealed the chemical method to be considerably superior. The reduction of quinones to their corresponding hydroquinones was verified by ultraviolet spectrophotometry. The reduction methodology has been applied to derivatize phylloquinone and its metabolite, namely phylloquinone 2,3-epoxide, 'on-line', with subsequent fluorometric detection of the generated hydroquinones.

Chemical reduction system for the detection of phylloquinone (vitamin K1) and menaquinones (vitamin K2).

Both isocratic and gradient elution systems for fluorometric detection of K vitamins after post-column reduction with zinc metal to their hydroquinones are described. The reaction detection system for K vitamins (phylloquinone and menaquinones) in liquid chromatography is based on reduction of K vitamins to their corresponding hydroquinones with zinc metal in the presence of zinc ions. It was found that 95% of the injected quinones (K vitamins) could be reduced to their corresponding hydroquinones with zinc metal compared to 60% reduction for electrochemical detectors. Menaquinones could be detected down to 100 pg with relative ease during gradient elution.

Liquid-chromatographic determination of vitamin K1 in plasma, with fluorometric detection.

This assay for phylloquinone (vitamin K1) in plasma requires a single liquid-chromatographic step. Much smaller volumes of plasma (0.5-1.0 mL) are required than in previous assays. Before liquid chromatography, we purified crude lipid extracts by conventional chromatography on silica, then extracted the lipid fraction by dissolving it in an acidic mixture of hexane/acetonitrile (1/4 by vol) containing 70 mmol of zinc chloride per liter. The vitamin K1 was selectively extracted into acetonitrile after being converted to vitamin K1 hydroquinone by addition of zinc metal. This procedure removes greater than 99% of contaminating lipids. We injected the lipid extract directly onto a reversed-phase column after re-converting the vitamin K1 hydroquinone to vitamin K1. Vitamin K1 was quantified by comparison with the internal standard (dihydro-vitamin K1) and detected fluorometrically after post-column "on-line" reduction to the hydroquinone with zinc metal. The lower limit of detection for vitamin K1 in the final reversed-phase system was about 0.05 microgram/L plasma; CVs for replicates were less than 10%. The mean concentration of vitamin K1 in plasma from 22 healthy fasting adults was 0.55 (range 0.09-2.12) micrograms/L.