Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.
An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.
Costello
J F
JF
Division of Nephrology and Hypertension, Allegheny-Singer Research Institute, Allegheny General Hospital/Medical College of Pennsylvania, Pittsburgh.
Smith
M
M
D000818:Animals; D002245:Carbon Dioxide / Q000032:analysis*; D002250:Carbon Radioisotopes; D002262:Carboxy-Lyases / Q000737:chemistry*; D005243:Feces / Q000737:chemistry; D007263:Infusions, Parenteral; D008297:Male; D010070:Oxalates / Q000008:administration & dosage / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D002250:Carbon Radioisotopes; D010070:Oxalates; D002245:Carbon Dioxide; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase