Adenosine 5'-triphosphate (ATP), phosphocreatine (PCr), creatine (Cr), inorganic phosphate (Pi), lactate (LAC), pyruvate (PYR) and glycogen as glucose (GLU) were determined and free adenosine 5'-diphosphate (ADP) was calculated from ATP:creatine phosphokinase (CPK) reaction in the gracilis muscle of cold-acclimated rats in vivo, and in completely isolated muscles under medium perfusion and superfusion in vitro, using the freeze-clamping method. The mean in vivo levels (mumol/g w.w.) were: ATP 4.8, PCr 12.0, Cr 7.8, Pi 1.6, LAC 1.6, PYR 0.09, GLU 22.9, ADP 0.62 X 10(-3). Isolation of the muscle (about 11 min of anoxia followed by perfusion in the air with a high PO2 medium) decreased macroergic phosphate levels (ATP 3.0 , PCr 8.3). In isolated muscles perfused with a high pO2 Medium (99 kPa O2, perfusion rate 70 mul/min) and simultaneously superfused with a low pO2 medium (6.2 kPa O2, 2.3 ml/min) at 28-degrees-C in vitro the levels of metabolites were (mumol/g w.w.): ATP 3.1, PCr 8.5, Cr 5.6, Pi 0.9, LAC 2.1, PYR 0.19, GLU 6.6, ADP 0.44 x 10-3. The mean steady oxygen uptake of the isolated muscle was 97 nmol O2 x min-1 x g-1 w.w. Thus, the levels of macroergic phosphates and their derivatives are lower after isolation and perfusion of the muscle, but the creatine charge [PCr]/([PCr]+[Cr]] remains stable (0.61 in vivo versus 0.60 in the isolated muscle). This indicates that the steady-state and high energy status of the isolated perfused-superfused gracilis muscle is maintained.
