Characterization of Red Blood Cell Metabolism in Rainbow Trout

Abstract

Red blood cell metabolism was studied in vitro using whole blood obtained by catheter from resting rainbow trout (Oncorhynchus mykiss). Preparations were viable as shown by stable NTP, metabolite and catecholamine levels and acid-base status, all of which remained at in vivo levels over the 2 h incubation period. Enzymes diagnostic of glycolysis, the tricarboxylic acid (TCA) cycle and phosphagen metabolism were all present in significant amounts in red blood cells. In direct comparisons of 14C-labelled substrates at normal resting plasma concentrations, rates of CO2 production were in the order: glucose greater than lactate greater than alanine greater than oleate. Total CO2 production rates from these four oxidative substrates did not equal directly measured O2 consumption rates, indicating that other substrates may also be important in vivo. Oxidative pathway Km values for glucose (8.4 mmol l-1), lactate (3.3 mmol l-1) and alanine (0.8 mmol l-1) were well within the normal physiological ranges of plasma concentrations. Glucose concentration did not affect lactate oxidation rates, but there was some inhibition (27%) of glucose oxidation by high lactate concentrations (20 mmol l-1). The observed Km values and competitive interactions suggest that changes in plasma concentrations associated with environmental stresses can considerably alter the relative rates of oxidation of glucose and lactate in vivo. Considerable pentose-phosphate shunt activity was detected in red cells, as indicated by high activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and high CO2 production rates from (1-14C)-labelled glucose. Even in the presence of normal O2 levels, a significant percentage (28%) of glucose metabolism was directed to lactate production. Taken together, these results demonstrate that rainbow trout whole blood incubated in vitro constitutes a dynamic and viable system for metabolic studies at the pathway level.