The determination of Q192R polymorphism of paraoxonase 1 by using non-toxic substrate p-nitrophenylacetate

Abstract

CONTEXT:The human serum paraoxonase 1 (PON1) is calcium-dependent esterase and associates with the high density serum lipoproteins. PON1 plays a major role in oxidation of high density lipoprotein and low density lipoprotein and prevention of atherogenesis in coronary heart disease. PON1Q and R allele hydrolyses number of substrates like paraoxon (PO) (diethyl p-nitrophenyl phosphate) and phenylacetate.AIMS:The aim of the study is to the determination of Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate and compares it with the phenotype determined by using PO as substrate.MATERIALS AND METHODS:The study group consists of 60 healthy normal patients. Paraoxonase activity was measured using the procedure described by Eckerson (Reference method) and for phenotyping; the ratio of hydrolysis of PO in the presence of 1 M NaCl (salt-stimulated PON1, SALT) to the hydrolysis of phenylacetate (PA) is calculated. In new method (Haagen et al.) arylesterase activity measured using p-nitrophenylacetate and for phenotyping arylesterase, the ratio of inhibition of enzymatic hydrolysis of p-nitrophenylacetate (substrate) by phenyl acetate to non-inhibited hydrolysis of p-nitrophenylacetate (inhibited arylesterase activity (IA-IA0)/non-inhibited arylesterase activity (NIA).RESULTS:It was found that paraoxonase activity is trimodally distributed in both the methods. There is no significant difference in the distribution of PON1 phenotypes of both reference method and new method being frequencies 0.946 and 0.376 respectively and there was no significant difference for phenotypic polymorphism for an individual by both methods (χ2= 0.15 and P = 0.9262).CONCLUSION:The Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate showed trimodal distribution of QQ (homozygous), QR (heterozygous), and RR (homozygous) phenotype and it is comparable with reference method. This method can be used for PON1 phenotype in different pathological and complex disease conditions.

Serum paraoxonase 1 (PON1) measurement: an update

“…– Paraoxon . It is very unstable and extremely toxic [ 6 ] and can cause a rapid and severe organophosphate poisoning [ 22 ]. In addition to the European hazard codes described in Table 2 , paraoxon possesses the European risk statements number 26 (very toxic by inhalation), 27 (very toxic in contact with skin) and 28–50 (in which there is included that may cause cancer and hereditable genetic damage).…”

“…Paraoxon stock solutions should be handled in an air-extraction fumehood and the operator should take appropriate safety precautions such as wearing a face mask and double nitrile gloves to protect against accidental contact or inhalation of the toxic fumes. In addition it can produce pollution to the laboratory equipment, therefore dedicated sample and reagent needles should be used, and waste water receptacle as well waste disposal and pipettes should be treated with NaOH [ 6 , 22 , 23 ]. In particular paraoxon presents problems for automation in biochemical analysers, since it can contaminate them producing difficulties to perform assays for other analytes on the same instrument, and the removal of its traces requires the use of high concentration of NaOH which could damage instruments [ 24 ].…”

Serum biomarkers of oxidative stress in cats with feline infectious peritonitis

“…Lactones are considered as the natural substrates of PON1 (Billecke et al, 2000), and other artificial substrates can also be used for measurement of this enzyme as paraoxon, phenyl acetate (PA) or p-nitrophenyl acetate (pNA; Ceron et al, 2014). However, use of paraoxon as substrate would not be optimal due to its toxicity (Camps et al, 2009) and the possibility of analyzer contamination (Mogarekar and Chawhan, 2013). Divergences in the diagnostic performance between different substrates have been described in certain diseases (Dantoine et al, 1998;Keskin et al, 2009).…”

Paraoxonase 1 Activity and its Polymorphism in Type 2 Diabetic Nephropathy

“…The rate of formation of p-nitrophenol was determined at 405 nm at a temperature of 25 ºC over 225 s, following 100 slag time. The activity was expressed in kU/L, based on the molar absorptivity (14000 M -1 cm -1 min -1 ) of p-nitrophenol at 405 nm and a pH 7.4 (21).…”