Quantification of human serum paraoxonase by enzyme-linked immunoassay: population differences in protein concentrations

Abstract

Paraoxonase is a serum protein bound to high-density lipoproteins (HDLs). The physiological function of the enzyme is unknown, but a role in lipid metabolism has been postulated. To date, studies of the protein have had to rely on measurements of enzyme activity with various substrates. We have developed a highly specific, competitive e.l.i.s.a. using a previously characterized monoclonal antibody. The assay can detect 20 ng of paraoxonase with a working range of 75-600 ng. Intra- and interassay coefficients of variation were 6.5 and 7.9% respectively. Serum concentrations of paraoxonase in healthy subjects from Geneva and Manchester ranged from 25 to 118 micrograms/ml. There were significant differences in mean concentrations between the two groups (Geneva, 79.3 +/- 18.7 micrograms/ml; Manchester, 59.9 +/- 24.1 micrograms/ml: P < 0.001), differences also apparent when subjects were compared according to paraoxonase phenotype. These appeared to be largely a consequence of differences in apolipoprotein A-I concentrations between the two populations, suggesting that HDL particle number may be important in determining serum levels of paraoxonase. Paraoxonase specific activities were also significantly different between the two groups of subjects (Geneva, 2.08 +/- 0.96 units/mg; Manchester, 3.08 +/- 1.73 units/mg: P < 0.001), which may reflect differences in HDL particle composition. The e.l.i.s.a. should furnish the necessary complement to studies of paraoxonase enzymic activity and has already provided evidence for differences with respect to serum levels of the protein both between populations and between phenotypes within populations.

Enzymatically Active Paraoxonase-1 Is Located at the External Membrane of Producing Cells and Released by a High Affinity, Saturable, Desorption Mechanism

“…In each case transfected and control cells were photographed and printed under identical conditions. PON1 Activity and Peptide Mass-PON1 activity (arylesterase (ARE)) was measured in conditioned medium using phenylacetate as substrate (28) (CV 1-2%). Unless otherwise stated, activity is expressed as ⌬OD 270 /min.…”

“…Stain was removed by thorough washing with HBSS, and cells were fixed in formaldehyde (3%, 30 min), washed in HBSS, and mounted in glycerol (90%) for photographic analysis. PON1 mass was quantified by competitive immunoassay (CV 7-8%) (28).…”

The paraoxonase PON1 promoter polymorphism C(-107)T is associated with increased serum glucose concentrations in non-diabetic patients

“…Plasma lipid, lipoprotein and apolipoprotein concentrations, as well as glucose concentrations were analysed [29]. Paraoxonase serum activities and concentrations were assayed [31] and gene polymorphisms affecting coding and promoter regions were determined by restriction isotyping and allele specific hybridisation [32,33].…”

Promoter Polymorphisms of Human Paraoxonase PON1 Gene and Serum Paraoxonase Activities and Concentrations

“…PON enzymatic activities were assayed in human serum samples as described previously 11 by using both phenylacetate and paraoxon as substrates. A control pool of human sera, independently calibrated by Dr M. Mackness (University of Manchester, School of Medicine, Manchester, UK), was used to standardize activity assays.…”