Thermochemical analysis of intermediary metabolism in resting 2C11-12 mouse macrophage hybridoma cells

Abstract

The calorimetric-respirometric (CR) ratio is a thermochemical device to establish whether given living organisms and cells possess a balanced aerobic energy (enthalpy) budget. For those with a CR ratio more exothermic than −500 kJ per mol O 2, the only tenable explanation must lie in simultaneous aerobic and anaerobic catabolism. The subject of the current investigation, resting 2C11-12 mouse macrophage hybridoma cells, went through nearly four generations when cultured for 48 h and in the final 12 h increased in average protein biomass from 0.45 to 0.68 ng per cell and in mean diameter from 12.6 to 15.4 μm. Singular to this period only, cellular mass density decreased by 15% and luminol-enhanced chemiluminescence, which had peaked at 24 h (7.2cpm per cell), declined by over 50% to 2.8 cpm per cell. Heat flux was 68 μW per g protein at 12 h but reduced from 60 to 38 μW per g protein between 36 and 48 h. Oxygen flux was above 7nmol min −1 per g protein but declined to 5.8 at 36 h and 2.8nmol min −1 per g protein at 48 h. These data resulted in CR ratios becoming more negative with time, being −514 kJ per mol O 2 at 24 h and −843 kJ per mol 0; at 48h. Lactate flux increased from 9.0 nmol min −1 per g protein at 12 h to 13.3 nmol min −1 per g protein at 48h. Succinate flux was the most constant parameter at 0.6nmol min −1 per g protein but did show a peak at 36 h of 1.2 nmol min −1 per g protein. The molar amount of lactate and succinate produced per unit amount of oxygen consumed (Lac/O 2 and Succ/O 2 ratios) is used to calculate the catabolic heat changes per mol O 2 and gives results very close to the observed CR ratios. This indicates that catabolism in resting 2C11-12 cells is by integrated aerobic and anaerobic processes which become more intensively anaerobic as the cells increase in mass late in the growth curve.