Ornithine decarboxylase assay in human colorectal mucosa. Methodologic issues of importance to quality control.

Abstract

Ornithine decarboxylase may be a useful biomarker for risk of neoplasia in colorectal tissues. Investigators have reported enzyme activities varying by as much as 10- to 20-fold using variations of the usual 14CO2 release assay. We have examined the effect of different methodologic factors on calculated ornithine decarboxylase activity. Major effects on the assay result (greater than 20% change) were produced by: (1) use of Tris vs. phosphate buffer, the former yielding 1.5- to 4-fold greater activity; (2) protein content of the reaction mixture with significant error if less than 50 micrograms; (3) use of alpha-difluoro-methylornithine-inhibited blank versus buffer-only blank. Other changes in assay conditions, including addition of sucrose, detergent, protease inhibitors, specific activity of 14C-ornithine, the nature of the trapping agent used, and incorporation of a sonication step, did not have a significant effect on ODC quantification (less than or equal to 20%). Thus, seemingly minor variations in assay conditions can greatly affect the results, which may provide a partial explanation for the variability of ODC activities reported in the literature. Strict quality control measures are mandatory in the interpretation of clinical observations utilizing this marker as an endpoint.