Abstract
Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins. We optimized buffer conditions to obtain quantitative recovery of PON1 (arylesterase) activity and analyzed the distribution of PON1 in mice using a combination of size-exclusion chromatography and ultracentrifugation. Size-exclusion chromatography of mouse serum separated the esterase activity into two peaks, one overlapping the high density lipoproteins and a second peak of lower molecular weight, consistent with serum carboxylesterase, which accounted for approximately 20% of the total esterase activity of normal mouse serum. Using conditions for the quantitative recovery of arylesterase activity, we fractionated serum by ultracentrifugation into d < 1.21 g/ml, d < 1.25 g/ml, d > 1.21 g/ml, and d > 1.25 g/ml fractions. We observed that PON1 arylesterase activity and mass were isolated in the d < 1.21 g/ml fraction and that serum carboxylesterase was recovered in the d > 1.25 g/ml fraction. The significance of the confounding of PON1 arylesterase activity by serum carboxylesterase was demonstrated by studying mice challenged with a high-fat, high-cholate diet for 14 days. It was shown that all of the decrease in arylesterase activity in response to this diet is attributable to the HDL-associated arylesterase activity (PON1). We conclude that mouse PON1 is quantitatively associated with high density lipoproteins. The contribution of serum carboxylesterase to the total esterase activity significantly confounds the interpretation of total arylesterase activity in mouse serum.
Highlights
Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins
We report that the lipoprotein-associated serum arylesterase is inactivated during dialysis when time and buffer conditions typical for lipoprotein isolation are used
We focus on mouse serum arylesterase activity and show that it could be quantitatively preserved by decreasing the dialysis time and using buffers containing 2 mM calcium
Summary
Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins. We optimized buffer conditions to obtain quantitative recovery of PON1 (arylesterase) activity and analyzed the distribution of PON1 in mice using a combination of size-exclusion chromatography and ultracentrifugation. As part of our studies of various transgenic and knockout mouse strains [7], we observed a quantitative association of PON1 arylesterase activity with HDL from C57Bl/6 mice when serum was separated by size-exclusion chromatography using Superose-6 but a poor recovery of PON1 arylesterase activity from serum after ultracentrifugation to isolate HDL. The peak of PON1 arylesterase activity precedes the peak of HDL cholesterol by size-exclusion chromatography
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