Facilitated micromethod for measurement of metabolically generated 14CO2, with application to measurement of ornithine decarboxylase.
A modified microassay for the determination of metabolically generated 14CO2 is described and is applied to the measurement of ornithine decarboxylase in animal tissue preparations. In this technique, the reaction takes place in a microcentrifuge tube inside a 20-ml scintillation vial that also contains a center well with a CO2-trapping agent. The vial is sealed with a silicone septum-lined plastic screw cap. After the initial incubation period during which the enzymatic reaction occurs, acid is injected through the septum into the reaction tube, and 14CO2 is released during a second incubation period. The reaction vial is then removed, counting solution is added to the scintillation vial, and radioactivity is measured. Linearity is present with respect to both increasing amounts of tissue and incubation time. Recovery of evolved 14CO2 was greater (97.5 vs 91.5%) and variation between replicate samples was less (coefficient of variation 2.7 vs 8.5%) when the modified microassay was compared with an assay system that requires removal of the screw cap from the vial before acid injection. The modification allows greater safety and facilitated assays, which could result in savings of both time and laboratory personnel. Special precautions for the use of NaH14CO3 as the recovery marker are noted.
Gaines
D W
DW
Food and Drug Administration, Division of Toxicology, Beltsville, Maryland 20708.
Friedman
L
L
Braunberg
R C
RC
D000818:Animals; D002245:Carbon Dioxide / Q000032:analysis*; D002250:Carbon Radioisotopes; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009955:Ornithine Decarboxylase / Q000032:analysis*; D051381:Rats; D013997:Time Factors
D002250:Carbon Radioisotopes; D002245:Carbon Dioxide; D009955:Ornithine Decarboxylase