Enzymatic determination of total CO2 in freeze-clamped animal tissues and plasma.
An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO2] was defined as the sum of the dissolved CO2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 mM 2-amino-2-methyl-1-propanol, pH 9.5 at 25 degrees C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO2 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 mumol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 mumol NADH at 340 nm. The assay medium comprised 50 mM 2-amino-2-methyl-1-propanol, pH 9.0 at 25 degrees C, 5 mM phosphoenolpyruvate (PEP), 0.25 mM NADH, 5 mM MgCl2, 5 mM mercaptoethanol, 0.02% bovine serum albumin, 10 mM oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO2 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 mumol/g wet wt tissue, respectively (n = 5). The total CO2 in the crusher muscle of the lobster was found to be 5.0 +/- 0.33 mumol/g wet wt. Total CO2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 +/- 1.81 mumol/ml plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
Dobson
G P
GP
Laboratory of Metabolism and Molecular Biology, NIAAA, Rockville, Maryland 20852.
Veech
R L
RL
Hoeger
U
U
Passonneau
J V
JV
D000818:Animals; D001639:Bicarbonates / Q000032:analysis; D002245:Carbon Dioxide / Q000032:analysis* / Q000097:blood; D008291:Malate Dehydrogenase / Q000032:analysis; D008297:Male; D008121:Nephropidae; D010730:Phosphoenolpyruvate Carboxylase / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D014020:Tissue Extracts
D001639:Bicarbonates; D014020:Tissue Extracts; D002245:Carbon Dioxide; D008291:Malate Dehydrogenase; D010730:Phosphoenolpyruvate Carboxylase