Paraoxonase-1 (PON-1) activity is a new inflammatory and oxidative marker. Technical effects and biological factors could affect the accuracy of PON-1 activity measurement. We investigated the effects of storage at different temperatures, repeated freeze–thaw cycles, interferences from hemolytic, lipemic, and icteric samples, and seasonal effects on PON-1 activity in horses. We evaluated 2 substrates with an automated spectrophotometer. Ten equine serum samples were stored under different conditions. Although storage at room (21°C) or refrigeration (4°C) temperature induced a statistically significant decrease ( p < 0.05) in PON-1 activity, this is not diagnostically relevant. PON-1 activity in frozen samples (−20°C) was stable for short-term storage; diagnostically significant ( p < 0.01) fluctuations were observed after 1 mo. Four repeated freeze–thaw cycles were assessed, and all cycles affected PON-1 activity ( p < 0.01); however, this was diagnostically significant only after the 4th cycle. Hemolysis induced an overestimation of PON-1 activity; lipemia and hyperbilirubinemia did not change PON-1 activity. Thirty-four horses were sampled monthly for 1 y, and PON-1 activity was higher in autumn ( p < 0.05) and winter ( p < 0.05) than in spring and summer.

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