Kinetic characteristics of pyridine nucleotide reduction as an indicator of energy production

Abstract

Changes in NADH fluorescence in brain mitochondria of rats exposed to hypercapnic hypoxia were studied. The status of the succinate oxidation system determines the level and kinetic characteristics of pyridine nucleotide reduction. The kinetic characteristics of energy production are more susceptible to hypoxia than changes in NADH level. Key Words: brain mitochondria; NADH; hypoxia; transamination; succinate oxidationThe state of the energy production system is evaluated by the characteristics suggested by B. Chance [5]. We consider that such an approach provides insufficient intbrmation about the system in general and does not allow a comparison between the contribution of the fast and slow metabolic cluster [1,2] to the maintenance of energy homeostasis. We investigated the level of NADH fluorescence in brain mitochondria (MC) of rats exposed to hypercapnic hypoxia. Special attention was paid to the relationship between the rate of reduction of pyridine nucleotide (PN) oxidized with ADP and the oxidation substrate and functional state of the organism. MATERIALS AND METHODSMale Wistar rats aged 2 months (200-250 g) from Rassvet Breeding Center (Tomsk) were used. During 2-week acclimatization and the experiment the rats were kept in standard plastic cages (no more than 15 per cage) at 20-25~ humidity no more than 50%, outflow/inflow 8:10 air exchange, and day-night light regimen. Hypercapnic hypoxia was modeled by placing the animal tot 2 h into a sealed 5-liter vessel. The brain MC oxidation system was evaluated fluorimically by the level of PN reduction (PNR) on a Hitachi [7]. The oxidation substrates were succinic acid (0.5 raM), malate-glutamate mixture (3 mM each), their combination with SDH inhibitor malonate (2 raM) or aminotransferase inhibitor aminooxyacetic acid (0.5 raM). PNR was studied during oxidation of endogenous substrates (arbitrary metabolic state 2), denoted as the "silent" status 2 (2S), and the PNR was consequently denoted as F2s (Fig. 1). PNR changes were studied after addition of 50 ~tM ADP to the medium (transition from the 2S metabolic status to active phosphorylation 2AP and then to a resting status 2R, i.e., the F2s---~F2Ap-~F2R transitions were examined).Effects of oxidation substrate (transition from 2R to 4S, i.e. F2R--~Fgs) and of subsequent addition of ADP during utilization of exogenous substrates (metabolic states 4S, 3, 4R, i.e., Fes--~F3--~Fek transformations) were examined. The time of 2S to 2R transition (Tr2) and 4S to 4R transition (Tr3) and the rate of transition from 2AP to 2R metabolic state and from 3 to 4R (V 2 and V 3, respectively) were studied. The results were processed by paired Wilcoxon--Mann--Whitney's test. RESULTSThe results provide a new interpretation of changes in PNR in MC during transition from one metabolic stale to another. Due to a better preservation of the MC structure [4], we demonstrated stability of PNR and

Effects of Succinic and Glutamic Acid Combination on Energy Metabolism in the Liver of Mice under Conditions of Hypoxia

“…The course of these reactions conforms to the regularities of the common adaptation syndrome and are aimed at the maintenance of high level of energy supply and its prolongation. Urgent adaptation to slight hypoxia involves activation of energy metabolism via intensification of NAD-dependent substrates oxidation [7,8]. Under conditions of our experiment, augmenting hypoxia involves inhibtion of the MC respiratory chain electron transporting function [6], which leads to exhaustion of adaptation potentialities of organelles with progressive reduction of NAD-dependent energy production and suppression of oxidative phosphorylation.…”

“…Presumably, this was paralleled by modification of the physical and chemical characteristics of membrane lipids, membrane-bound proteins, and increase of proton conductivity of MC membranes [2][3][4], which suggests dissociation of oxidative phosphorylation [8].…”

“…In addition to the stationary levels of NADH fluorescence, the kinetic characteristics (V 3 velocity and Tr 3 time of NAD reduction in MC after addition of ADP into incubation medium to the concentration of 5×10 -5 M) were evaluated, reflecting the proportion between delivery of reducing equivalents to NADH dehydrogenase and their oxidation by the respiratory chain [8]. Succinate, malate-glutamate mixture, their combination with SDH inhibitor malonate, and a combination of malate, glutamate, and amino-oxyacetate (aminotransferase inhibitor) served as the oxidation substrates (Fig.…”

Kinetic characteristics of the energy production system in rat brain under conditions of posthypoxic encephalopathy and its correction withBergeniae crassifolia extract

“…The functional state of the energy production system was evaluated by the level of reduced pyridine nucleotides [9].…”