Semiautomated method for determination of serum paraoxonase activity using paraoxon as substrate.

BACKGROUND Serum paraoxonase (PON1) is an enzyme associated with HDL, and its ability to protect LDL from oxidation is one mechanism by which HDL protects against atherosclerosis. Low concentrations of PON1 are found in patients with type 2 diabetes or coronary heart disease. Serum PON1 activity may also be important in avoidance of organophosphate toxicity in industry. METHODS The generally accepted method for determining PON1 activity requires use of a recording spectrophotometer and is not suited to large numbers of samples; in addition, automation presents particular problems because of the extreme toxicity of substrates such as paraoxon. We established a relatively safe microtiter plate method that facilitates the determination of PON1 activity at a rate of 120 samples per hour. RESULTS PON1 activity was determined by the generally accepted method (x) and the new method (y); results correlated with a slope close to unity (y = 0.93x + 8; r = 0.97; P < 0.0001; n = 101). Examination of differences by Bland-Altman plots showed a weak concentration-dependent difference (r = 0.33; P < 0.0001; n = 101). The intra- and interassay sample CVs, obtained with samples with PON1 activities ranging from 41 to 348 nmol x min(-1) x mL(-1), were 3.5% and 2.7%, respectively (n = 16). CONCLUSION The proposed method for determination of PON1 activity is simple, relatively safe, and inexpensive and is suitable for analysis of large numbers of samples.