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Background. Human serum paraoxonase [(PON1) ; aryldialkylphosphatase (EC 3.1.8.1)] is associated with high density lipoprotein particles (HDL) responsible in part for the ability of HDL to prevent lipid peroxidation.The decreased serum paraoxonase (PON1) activity in patients with atherosclerosis disease may cause decreased HDL antioxidant capacity and therefore significantly influence the risk of the development of atherosclerosis. The enormous between-individual biological variability in serum PON1 activity seems to be regulated mainly by genetic determinants. PON1 is an esterase coded by pon1 gene on chromosome 7 and linked with two additional PON-like genes (pon2 and pon3). Polymorphisms in pon1 and pon2 genes (L55M and Q192R in pon1, and S311C in pon2) have been reported to be associated with the risk for the development of atherosclerosis as well as polymorphism in pon1 promoter region (-107C>T). Another lipoprotein-associated enzyme, the platelet-activating factor acetylhydrolase (PAF- AH) is an enzyme (EC 3.1.1.47) that may be involved in the degradation of oxidized phospholipids on low density lipoprotein particles (LDL) that are structurally similar to PAF and that are closely associated with atherosclerosis. We explored relations between serum PON1 and PAF- AH activities as well as the distribution of polymorphisms of pon1 and pon2 genes and atherosclerosis in well-characterized groups of patients with angiografically assessed severe stenosis of cerebral arteries and matched control no-stenosis group. Patients and methods. The study comprised 151 patients mean age of 66 years (range 31-83 years) with severe stenosis of extracranial cerebral arteries of more than 70%, established angiographically. The control no-stenosis group consisted of 186 apparently healthy individuals mean age of 64 years (range 44-82 years), with normal cerebral arteries on ultrasound examination. This study was approved by the Ethical Committee of Merkur University Hospital, Zagreb, Croatia. Serum total cholesterol and triacylglycerol were measured by enzymatic PAP- method. HDL-cholesterol was measured with direct method based on selective inhibition of the non- HDL fractions by means of polyanions. A homogeneous assay for the selective measurement of LDL-cholesterol in serum was used. Basal and NaCl- stimulated paraoxonase activities were determined by measuring the rate of paraoxon hydrolysis at 37 °C, based on the change of absorbance at 410 nm. The PAF-AH activity was measured in plain serum with the new automated spectrophotometric assay (AZWELL Auto PAF-AH, Osaka, Japan) at 37C. Enzyme activities are expressed in international units per liter of serum. All measurements were performed on the Olympus AU 600 automatic analyser (Olympus Mishima Co., Ltd., Shizuoka, Japan). Polymorphisms of pon1 and pon2 genes were determined by the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (PCR-RFLP). The PCR reaction was performed in a GeneAmp PCR System 2700 (Applied Biosystems) PCR machine. Amplified products were digested with BspPI (Q192R), Hin 1II (L55M), BsrBI (-108C>T) and DdeI (S311C) restriction enzymes and determinated by PCR-RFLP procedure. Results. Basal and stimulated PON1 activities differ significantly between patient’s group with stenosis and the control group (median values 96 U/L versus 149 U/L, p0.05 and 200 U/L versus 308 U/L, p0.05, respectively). There were no statistically significant relationships between PON1 activity and examined lipid and lipoprotein parameters (tryacylglicerol, total cholesterol, HDL cholesterol, LDL cholesterol). Genotype frequencies of pon1 and pon2 genes polymorphisms founded in the group of patients with angiografically assessed severe stenosis of cerebral arteries vs. control no-stenosis group were: QQ (47%), QR (45%), RR (8%) vs. QQ (47%), QR (48%), RR (5%) for Q192R ; LL (45%), LM (35%), MM (20%) vs. LL (41%), LM (51%), MM (9%) for L55M ; CC (25%), CT (45%) ; TT (30%) vs. CC (27%), CT (58%), TT (15%) for –108C>T and CC (3%), CS (34%), SS (63%) vs. CC (0%), CS (46%), SS (54%) for S311C respectively. Observed and expected genotype frequencies of all examined pon1 and pon2 genes polymorphisms were in Hardy-Weinberg equilibrium. There were no statistically significant differences between the most frequent alleles in patients group vs. control group (p>0, 05). The obtained results for the most frequent alleles were: Q (70%) for Q192R, L (63%) for L55M, T (52%) for –108C>T and S (79%) for S311C polymorphisms of the pon1 and pon2 genes in patients group vs. Q (71%) for Q192R, L (66%) for L55M, T (44%) for – 108C>T and S (77%) for S311C in the control group. The median serum PAF-AH activity did not differ significantly between patients with cerebrovascular stenosis and control group (median values 390 U/L versus 389 U/L, p>0, 05). The 95% range of the obtained results (198-671 U/L in the patient group vs. 247-575 U/L in the control group) indicated a great interindividual variation in both groups what is in agreement with the similar studies on patients with cardiovascular disease. Conclusions. Our study showed a relationship between decreased serum paraoxonase activity and the presence of severe stenosis of cerebral arteries. However, there were no significant differences in genotype or allele frequencies of pon1 and pon2 genes between patients with severe stenosis of cerebral arteries and controls indicating that changes in paraoxonase activity are determined by both genetic and environmental factors. The present data suggested that the level of serum PAF-AH activity was not significant determinant of susceptibility to cerebrovascular atherosclerosis. This study was supported by Ministry of Science Education and Sports of the Republic of Croatia (Grant No. 044-0061245-0551). |
