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E-mail: kcbtan{at}hkucc.hku.hkSir,
We thank Dr Philip W. Connelly for his interest in our paper, and for raising valuable comments on our methodology of measuring paraoxonase activity in HDL-containing supernatant from serum after the precipitation of apolipoprotein B-containing lipoproteins using the dextran-sulfate method. Paraoxonase 1 is an important HDL-associated antioxidative enzyme and there are data showing that paraoxonase 1 is active in the HDL-containing supernatant after dextran-sulfate precipitation. Navab et al. have reported that adding purified human paraoxonase 1 to HDL-containing supernatant after dextran-sulfate precipitation of plasma from patients with coronary heart disease can restore the antioxidative function of HDL [1].
Paraoxonase has multiple activities including organophosphatase, phosphotriesterase, arylesterase and thiolactonase [2]. The EnzChek® Paraoxonase Assay from InvitrogenTM used in our study is a homogeneous fluorometric assay for the organophosphatase activity of paraoxonase. The substrate is a fluorogenic organophosphate analogue as indicated in the protocol provided by the manufacturer. As pointed out by Dr Connelly, the EnzChek® Paraoxonase Assay has also been used by Rector et al. for the measurement of paraoxonase-1 activity in serum and in HDL [3], and the results are corroborated by the measurement of enzyme mass but not by serum arylesterase activity. We welcome Dr Connelly's suggestion that arylesterase activity should also be measured. We have shown that the organophosphatase activity in HDL was significantly reduced in diabetic subjects with microalbuminuria or proteinuria. Since we compared diabetic subjects with healthy controls in our study, and serum, plasma and HDL samples from diabetic subjects and healthy controls were all handled in the same manner, any methodological problem would apply to both groups and therefore unlikely to bias our results.
Conflict of interest statement. None declared.
Department of Medicine, University of Hong Kong, Honk Kong
References
- Navab M, Hama SY, Hough GP, et al. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids. J Lipid Res (2001) 42:1308–1317.
[Abstract/Free Full Text] - James RW. A long and winding road: defining the biological role and clinical importance of paraoxonases. Clin Chem Lab Med (2006) 44:1052–1059.[CrossRef][Web of Science][Medline]
- Rector RS, Warner SO, Liu Y, et al. Exercise and diet induced weight loss improves measures of oxidative stress and insulin sensitivity in adults with characteristics of the metabolic syndrome. Am J Physiol Endocrinol Metab (2007) 293:E500–E506.
[Abstract/Free Full Text]
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