Development and validation of an HPLC based kinetic enzyme assay of human serum paraoxonase 3 (PON3)
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Paraoxonase 3 (PON3) is thought to play a role in the prevention of atherosclerosis through inhibition of oxidation of low-density lipoproteins (LDL). Hence it is important from a clinical point of view to develop an assay to measure the activity of PON3 in humans. Research has shown that PON3 alone has simvastatinase activity (when compared to PON1 or 2), thus a simple and relatively easy HPLC method has been proposed to analyze PON3 activity in human blood serum by measuring the conversion of simvastatin (SV) to β,δ-dihydroxyacid (SVA). However, the complete method performance characteristics and specificity of the assay have not been established. We have developed and validated an HPLC based assay for the measurement of serum simvastatinase activity. The method was optimized for time of reaction (30 min), substrate concentration (96μM), serum pre-dilution (1:4), pH (8.5) and calcium concentration (10μM). The within-run precision was < 6%CV, however the between-run imprecision was >50%CV. On further investigations, based on chromatographic purification and isolation, we found that the majority of simvastatinase activity could be attributed to a 68kDa serum protein consistent with human serum albumin (HSA). We demonstrate that not only does purified HSA have significant simvastatinase activity but also that the simvastatinase activity of human serum could be largely inhibited by treatment with an anti-human serum albumin antibody. We conclude that the simvastatinase activity of human serum is mostly due to the non-specific lactonase activity of HSA and is not a useful marker of PON3 activity. This is the first description of this particular HSA activity to our knowledge and may have important implications for drug metabolism and disposition studies of the statins and other lactone drugs.