Enzymatic determination of total CO 2 in freeze-clamped animal tissues and plasma

Abstract

An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO 2] was defined as the sum of the dissolved CO 2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 m m 2-amino-2-methyl-1-propanol, pH 9.5 at 25°C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO 2 was determined as bicarbonate/carbonate by monitoring the oxidation of NADH at 340 nm using the coupled assay of phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). In the coupled assay system, 1 μmol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 μmol NADH at 340 nm. The assay medium comprised 50 m m 2-amino-2-methyl-1-propanol, pH 9.0 at 25°C, 5 m m phosphoenolpyruvate (PEP), 0.25 m m NADH, 5 m m MgCl 2, 5 m m mercaptoethanol, 0.02% bovine serum albumin, 10 m m oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO 2 content measured in freeze-clamped rat heart, liver, brain, and skeletal muscle was 20.53 ± 0.64, 17.34 ± 0.67, 17.00 ± 0.48, 16.06 ± 0.53 μmol/g wet wt tissue, respectively ( n = 5). The total CO 2 in the crusher muscle of the lobster was found to be 5.0 ± 0.33 μmol/g wet wt. Total CO 2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 ± 1.81 μmol/ml plasma. It was concluded that the enzymatic method of total CO 2 determination can be used to measure bicarbonate in amounts of tissue ranging in weight from 50 to 100 mg or 50 to 100 μl of plasma. By substituting the bicarbonate concentration into the Henderson-Hasselbalch equation, and knowing the venous plasma pCO 2, the method can be used to calculate the intracellular pH in a variety of tissues.