NADH and NADPH in human skeletal muscle at rest and during ischaemia

A method for determining the content of NADH and NADPH in biopsy specimens from human skeletal muscle is described. It is based on the bioluminescent technique, utilizing oxidoreductases specific for NADH and NADPH, respectively. Muscle samples were taken from the lateral portion of the quadriceps muscle in the basal state and following local circulatory occlusion. In resting human skeletal muscle, the content of NADH was 91 +/- 6 mumol/kg dry muscle (mean +/- SE, n = 11) and the NAD/NADH ratio was 18.8 +/- 1.3. The content of NADPH determined in four subjects was 108 +/- 2 mumol/kg dry muscle. After 5 min of circulatory occlusion, NADH had risen about 100% and a further increase was found after 10 and 20 min, to a plateau about 150% above the basal value. The muscle content of NADPH also increased but to a minor extent (about 30% above the basal value). The changes in muscle lactate after 5 and 10 min occlusion were not uniform between subjects but an increase was obtained in all subjects after 20 min occlusion. No relation was found between the ratios pyruvate/lactate and NAD/NADH and the latter ratio was smaller than the estimated value, calculated from the LDH equilibrium in the cytoplasm. The results indicate that the major part of NADH in muscle tissue is confined to the mitochondrial compartment. It is concluded that measurement of NADH provides information primarily about the mitochondrial redox state rather than the cytosolic and that changes in NADH precede lactate formation and thus are a more sensitive index of tissue hypoxia than increases in lactate.