Accuracy and biological variation of human serum paraoxonase 1 activity and polymorphism (Q192R) by kinetic enzyme assay

BACKGROUND: Paraoxonase 1 (PON1) phenotype is a better predictor of atherosclerosis risk than are PON1 genetic polymorphisms alone. Larger studies are required to determine the role of PON1 and there is a need for standardized PON1 assays between laboratories. METHODS: We have adapted 5 enzyme kinetic assays for high-throughput automated analysis of PON1 activity. Using different substrates and reaction conditions, we measured PON1 activity and used activity ratios to identify the PON1 Q192R genetic polymorphisms and assessed the accuracy of the genotype assignments in 79 adult study participants by comparing them with genotypes determined by AlwI restriction enzyme digestion of a 176-bp PCR amplification product from genomic DNA. Imprecision was determined using pooled serum and purified enzyme preparations. Biological variability was estimated by analysis of serial samples from 17 individuals. Variability parameters were compared with total cholesterol as a point of reference to a recognized biomarker of coronary heart disease risk. RESULTS: Salt stimulation and inhibition ratios were 97.4% and 94.7% correct in assigning Q192R genotype, respectively. Analytical imprecision (CV) was 1.0%-3.0% for phenylacetate and paraoxon substrate assays and 3.0%-8.0% for the para-nitrophenylacetate substrate assays. Combination of the 2 ratios into a double ratio resulted in 100% correct genotype classification. CONCLUSION: The described methods for measurement of PON1 activity and accurate genotype assignment are rapid and have potential to facilitate the efficient investigation of PON1 status in clinical and epidemiological studies.     

Paraoxonase 1 gene Q192R polymorphism affects stroke and myocardial infarction risk

OBJECTIVES: Paraoxonase (PON1), an enzyme associated with high-density lipoprotein (HDL) particles, inhibits oxidation and atherogenesis. We sought to investigate the association of the PON1 Q192R polymorphism with stroke and heart disease. DESIGN AND METHODS: In a case control study, we genotyped 242 ischemic stroke, 231 myocardial infarction (MI), and 310 healthy control subjects, all Chinese. RESULTS: R-containing genotypes (R+) were associated with vascular disease, OR = 1.5, P = 0.03. RR was increased in MI patients who were either smokers (OR = 3.1, P = 0.01), male, or younger than 60. R+ but not RR genotypes were increased in stroke patients, particularly large artery type (OR = 2.6 and P = 0.02 for R+, OR = 1.0 for RR) or among smokers. The relative dearth of RR in stroke might be due to earlier MI or death in at-risk people, such as smokers. R+ genotypes were increased with stroke in hypertensive (OR = 2.1, P = 0.02) but not normotensive (OR = 1.0) subjects. CONCLUSIONS: PON1 192R+ genotypes were associated with stroke and MI, particularly in subsets of patients, in patterns suggesting a possible survivor effect.     

The relationship between paraoxonase1-192 polymorphism and activity with coronary artery disease

ObjectiveWe tested the association between PON1 polymorphism, PON1 activity, oxidative susceptibility of LDL and coronary artery disease in Egyptians.MethodsPON1 polymorphism, serum PON1 activity, lipoprotein oxidation susceptibility and lipid profile were measured.ResultsLevels of HDL and paraoxonase activity were significantly decreased in CAD patients compared to control group, and in patients with three vessels compared to those of single or two vessels disease. High-activity allele (R) has a more atherogenic lipid profile than for the low activity allele (Q). PON1 RR genotype has nine fold risks to develop CAD in Egyptians while those with PON1 QR genotype have four fold risks.ConclusionThe PON1 activity is lower in subject with CAD and there is a significant relationship between activity of PON1 and the severity of coronary atherosclerosis. Also, we provide evidence of a significant association between R allele of the PON1 polymorphism and the development of coronary artery disease.     

Relationships between paraoxon and 2-coumaranone hydrolytic activities in sera genotyped for PON1 Q192R polymorphism

Objectives

To set-up a method for a direct evaluation in human serum of paraoxonase enzymatic activities, establishing a possible correlation with Q192R genotype polymorphism.

Design and methods

101 different human serum samples were genotyped for paraoxonase Q192R polymorphism by PCR restriction analysis, and evaluated spectrophotometrically with regard to paraoxon and 2-coumaranone hydrolytic activities. Both activities of paraoxonase were assayed, quantified through normalization by arylesterase activity, and compared with the data concerning Q/R genetic polymorphism.

Results

The mean normalized paraoxonase activity was found to be significantly higher in RR than in QQ human sera (3.99 ± 0.6 versus 1.32 ± 0.44; P  < 0.0001); instead, the 2-coumaranone hydrolysis showed an opposite trend (0.10 ± 0.02 versus 0.23 ± 0.04, in RR and QQ sera respectively; P  <  0.0001).

Conclusions

These methods were successfully applied to the whole serum, suggesting a possible use of this approach for a clinically relevant phenotypic characterization.     

脑梗死与对氧磷酶1基因192G/A位点多态性的关系

目的:评估脑梗死患者对氧磷酶1(paraoxonase 1,PON1)的基因192G/A多态性与脑梗死间的关系。方法:采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法分析295例脑梗死患者(脑梗死组)及117名正常对照者(对照组)的PON1基因192G/A多态性。结果:PON1基因192G/A多态性在对照组和脑梗死组中的GG型、GA型和AA型基因型分布分别为36.75%、50.43%、12.82%和28.14%、50.85%、21.02%。G、A等位基因分布在对照组和脑梗死组中分别为61.97%、38.03%和53.56%、46.44%。经检验,PON1基因192G/A位点基因型频率在2组人群中的分布差异无统计学意义(P>0.05),而等位基因频率在2组人群中的分布差异有统计学意义(P     

郑州地区2型糖尿病肾功能衰竭患者芳香酯酶与基因Q192R多态性的关系

目的 探讨郑州地区汉族人群2型糖尿病慢性肾功能衰竭(DN-CRF)与血清芳香酯酶(ArE／PON1)活性及其192位基因多态性的关系。方法 通过检测2型糖尿病组(DM，121例)、DN-CRF组(123例)、健康对照组(127例)等观察对象的血清ArE／PONl活性及其192位基因多态性、血脂和脂蛋白等，进行分析研究。结果 郑州地区汉族人群中存在有ArE／PON1 192位等位基因Q与R，DN-CRF组Q、R基因频率为0．45和0．55，与DM、对照组比较，差异无统计学意义；两病例组患者血清酶活性均低于对照组，DN-CFR组降低幅度最大；DN-CFR组内RR基因型患者高密度脂蛋白胆固醇(HDL-C)、高密度脂蛋白2胆固醇(HDL2-C)水平低于QQ基因型，总胆固醇(TC)、甘油三酯(TG)和氧化型低密度脂蛋白(oxLDL)高于QQ基因型。结论 DN-CRF组ArE／PONl192位基因多态性与DM、健康对照组间虽差异无统计学意义，但不能排除DM合并DN-CRF与ArE／PON1的192位基因多态性有关；DN-CRF患者血清ArE／PON1活性降低，可能是DM合并DN的危险因素。     

Relationship between the paraoxonase 1 gene glutamine 192 to arginine polymorphism and gestational diabetes mellitus in Saudi women

Objectives

Gestational diabetes mellitus (GDM) is recognized as an imbalance between insulin resistance and insulin secretion, leading to maternal hyperglycemia. Previous studies in a Saudi population indicated a high frequency of Paraoxonase 1 glutamine 192 to arginine (PON1 Q192R) polymorphism, suggesting this polymorphism as an additional risk factor. The present study was designed to explore the possible association between the PON1 Q192R polymorphism and GDM in a Saudi population.

Methods

This case–control study was carried out in 500 pregnant women, including 200 GDM cases and 300 non-GDM women. Genotyping for PON1 Q192R (rs662) variants was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).

Results

The results of the present study indicates that Q192R polymorphism was significantly associated with GDM in a Saudi population with the minor allele frequency (MAF) (p = 0.0007). Q192R genotypes and alleles showed a strong association with GDM (p = 0.009 and p = 0.0007, respectively).

Conclusion

In conclusion, these findings suggest that the PON1 Q192R polymorphism has high MAF in GDM in the studied Saudi population.     

The paraoxonase L55M and Q192R gene polymorphisms and myocardial infarction in a Tunisian population

ObjectivesIn the present study, we examined a possible association between the PON1 Q192R and L55M polymorphisms and myocardial infarction (MI) in a sample of the Tunisian population.Design and methodsThree hundred and ten patients with MI and 375 controls were recruited. Paraoxonase gene polymorphisms at codon 192 and 55 were analyzed by PCR-RFLP.ResultsGenotype distributions and allele frequencies of L55M were similar among the control and MI groups. For the Q192R polymorphism patients with MI had significantly higher frequency of the RR genotype compared to controls [17.1% vs. 10.9%; OR (95% CI), 1.93 (1.24–3.02); p = 0.004]. The MI patient group showed a significantly higher frequency of the R allele compared to the controls [38% vs. 30%; χ² = 10.74, p = 0.001]. The association between the PON1 Q192R polymorphism and MI remained significant after adjustment for other well-established cardiovascular risk factors.ConclusionsThe present study showed a significant and independent association between the PON1 Q192R polymorphism (presence of R allele) and MI in the Tunisian population.     

动脉粥样硬化候选基因屏氧酶1基因192位Gln-Arg多态性对血脂的影响

目的:探讨屏氧酶1基因192位Gln-Arg多态性与血脂水平的关系。方法:从社区正常人群、健康体检者和患者中抽取1019例受试者,其中680例脑卒中患者及339例正常对照组人群,男606例,女413例。采用聚合酶链式反应-限制性片段长度多态性方法测定屏氧酶1基因多态性。结果:非心脑血管病、脑出血、脑梗死患者年龄、性别、体质量指数、总胆固醇和低密度脂蛋白胆固醇比较,差异无显著性意义(P>0.05),三者血清空腹血糖、三酰甘油、低密度脂蛋白胆固醇比较,差异有显著性意义(P<0.05)。屏氧酶13种基因型(QQ,QR,RR基因型)各血脂水平差异无显著性意义(P>0.05),其中QQ基因型的三酰甘油、胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇水平分别为(1.58±0.89),(4.82±1.01),(1.39±0.38)和(2.84±0.75)mmol/L;QR基因型分别为(1.59±0.99),(4.78±1.00),(1.36±0.38)和(2.82±0.80)mmol/L;RR基因型分别为(1.57±0.87),(4.84±1.05),(1.34±0.39)和(2.87±0.87)mmol/L。同一性别不同基因型之间各血脂水平差异无显著性意义(P>0.05)。男女两性屏氧酶1各基因型的比例相近(P>0.05)。男女不同性别之间屏氧酶1基因各基因型的血脂水平存在差异,尤其以高密度脂蛋白胆固醇明显(P<0.05)。结论:屏氧酶1基因192位Gln-Arg各基因型间血脂水     

糖尿病合并冠心病与芳香酯酶基因Q192R多态性分析

目的 :探讨糖尿病 (DM)芳香酯酶 (ArE/PON1)基因Q192R多态性与糖尿病合并冠心病 (DM -CAD)的关系 ,为防治DM -CAD提供理论依据。方法 :调查对象为郑州地区汉族健康人群 6 4例 (对照组 )、2型糖尿病 6 0例 (DM组 )与DM -CAD6 7例 (DM -CAD组 )。测定其血清ArE/PON1活性 ,并用聚合酶链式反应 -单链构型多态性 (PCR -SSCP)分析技术检测ArE/PON1基因Q192R的多态性 ,进行分析研究。结果 :发现对照组、DM组和DM -CAD组均存在ArE/PON1基因 192位点 (Q/R)多态性。对照组基因频率Q为 0 .5 5、R为 0 .4 5 ;DM组为 0 .5 3与 0 .4 7;DM -CAD组为 0 .38与 0 .6 2。DM -CAD组R基因频率明显高于DM组和对照组 (P <0 .0 5 ) ,DM组与对照无显著差异 (P >0 .0 5 ) ,DM与DM -CAD组血清ArE/PON1活性 (分别为 0 .2 2 5± 0 .0 18μ/ml与 0 .2 0 0± 0 .0 19μ/ml)均低于对照组 (0 .2 5 9± 0 .0 18μ/ml) ,DM -CAD组低于DM组 (P <0 .0 5 )。 3组中每组内每种基因型组间酶活性无显著差异。结论 :郑州汉族人群存在ArE/PON1Q192R(GIn/Arg)多态性 ,基因频率分布不同于白种人 ,提示R基因可能是DM合并CAD的危险因素。     

缺血性脑卒中患者半胱氨酸与PON-1 Q192R基因多态性的研究

目的探讨缺血性脑卒中患者同型半胱氨酸(Hcy)与PON-1 Q192R基因多态性的相关性。方法采用循环酶联免疫法检测血浆Hcy浓度以及使用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测140例缺血性脑卒中患者(病例组)和117名健康者(对照组)PON-1 Q192R基因多态性。结果病例组和对照组平均空腹血浆Hcy浓度分别为(14.02±4.94)μmol/L和(11.64±4.18)μmol/L,两组差异有统计学意义(t=4.09,P<0.001);将病例组和对照组作为一个研究整体发现:PON-1QQ型、QR型、RR型的平均空腹血浆Hcy浓度分别是(13.34±3.91)μmol/L、(13.14±4.98)μmol/L、(13.30±4.31)μmol/L,经方差分析,PON-1各基因型间血浆Hcy浓度差异无统计学意义(F=0.033,P=0.967)。结论血浆Hcy浓度升高与缺血性卒中有关,并且缺血性脑卒中患者Hcy与PON-1 Q192R基因多态性无关。     

Asthma and multiple sclerosis: an inverse association in a case-control general practice population

BACKGROUND: Th1/Th2 imbalance is hypothesized to up-regulate some diseases and down-regulate others. Compared to controls, multiple sclerosis (MS) (Th1-mediated) has been linked to a reduced risk of allergy and asthma (Th2-mediated), based on patient questionnaire studies and a review of asthma medication. AIM: To investigate whether MS is associated with a reduced risk of Th2-associated diseases and an increased risk of Th1-associated diseases. DESIGN: Retrospective matched case-control study. METHODS: Three hundred and twenty MS patients and controls matched for age, gender, location and smoking were selected from the Welsh General Practice Morbidity Database from 1995-99. Case and control records were assessed for Th1-mediated and Th2-mediated diseases. RESULTS: Overall, 346 MS patients were identified, giving a prevalence of 127 per 100 000. There was an inverse relationship between multiple sclerosis (MS) and asthma (OR 0.33; 95%CI 0.15-0.77). No statistically significant relationships emerged between other Th2-associated (eczema, dermatitis) or any Th1-associated (rheumatoid arthritis, thyroid disorders, inflammatory bowel disease [IBD], type 1 diabetes) diseases and MS, although no patient in either group had treated type 1 diabetes. A trend existed for IBD, with 5/320 of cases affected and no controls; OR infinity; 95%CI 1.30-infinity; p=0.063. DISCUSSION: This inverse association between MS and asthma is compatible with a Th1/Th2 imbalance. Although the Th1/Th2 theory is probably an over-simplification in MS, a shift from Th1 cytokine dominance towards Th2 may provide drug-targeting routes for MS.     

Matrix metalloproteinase‐9 and paraoxonase 1 Q/R192 gene polymorphisms and the risk of coronary artery stenosis in Iranian subjects

Purpose: To investigate the association of matrix metalloproteinase‐9 (MMP‐9) and paraoxonase 1 (PON1) 192 polymorphisms with susceptibility to coronary artery stenosis (CAS) and the number of diseased vessels in patients with CAS. Methods: The study population comprised 302 unrelated Iranian individuals, including 145 patients with CAS and 157 control subjects. Genotypes for MMP‐9 and PON1 192 polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: In our study, distributions of the TT genotype of MMP‐9 and the RR genotype of PON1 192 were significantly higher in patients compared with healthy control subjects (P<0.05). Subsequent analysis demonstrated that a significant difference existed in the male (TT+TC vs. CC and RR+QR vs. QQ, P<0.01) but not in the female. The associations of these polymorphisms with the severity of stenosis were also evaluated, which according to results distribution of MMP‐9 and PON1 192 genotypes were not significantly different compared with the severity of stenosis (P>0.05). Conclusions: The observation indicates that the polymorphisms in the MMP‐9 and PON1 192 genes potentially play a role in the manifestation of coronary atherosclerosis but does not have any effect on the number of diseased vessels in Iran. J. Clin. Lab. Anal. 24:305–310, 2010. © 2010 Wiley‐Liss, Inc.     

Ⅰ型对氧磷酯酶基因Gln/Arg192遗传多态性与冠心病相关性的初步研究

目的 探讨中国北方地区I型对氧磷酯酶(paraoxonase l，PON1)基因Gln／Arg192遗传多态性与冠心病发病的关系。方法 应用聚合酶铁反应(polymerase chain reaction，PCR)及限制片段长度多态性(restriction fragment length polymorphisms，RFLP)技术，检测49例老年冠心病患者和38例健康老年对照者的PONl—Gln／Arg192基因多态性，等位基因以A／B表示。结果 冠心病组与健康组比较各基因型分布差异具有显著性意义(χ^2＝6．35，P＝0.042)。B等位基因在冠心病组明显增高(0．56vs0．37)。B等位基因是中国北方地区冠心病发病的危险因素(OR＝2.19，95％CI：1．19～4.05)。结论 PONl基因Gln／Arg192遗传多态性与中国北方地区冠心病发病明显相关。该酶切位点多态性具有明显的种族差异。     

Abnormal subcortical deep-gray matter susceptibility-weighted imaging filtered phase measurements in patients with multiple sclerosis: a case-control study

Objective

To investigate abnormal phase on susceptibility-weighted imaging (SWI)-filtered phase images indicative of iron content, in subcortical deep-gray matter (SDGM) of multiple sclerosis (MS) patients and healthy controls (HC), and to explore its relationship with MRI outcomes.

Methods

169 relapsing-remitting (RR) and 64 secondary-progressive (SP) MS patients, and 126 age- and sex-matched HC were imaged on a 3 T scanner. Mean phase of the abnormal phase tissue (MP-APT), normal phase tissue volume (NPTV) and normalized volume were determined for total SDGM, caudate, putamen, globus pallidus, thalamus, pulvinar nucleus of thalamus (PVN), hippocampus, amygdala, nucleus accumbens, red nucleus and substantia nigra. 63 HC were used for establishment of normal reference phase values, while additional 63 HC were used for blinded comparisons with MS patients.

Results

Increased MP-APT, decreased normalized volume and decreased NPTV were detected in total SDGM, caudate, putamen, globus pallidus, thalamus and PVN in MS patients compared to HC (p < .0004). MS patients also showed decreased volume in hippocampus (< .0001) and decreased NPTV in the hippocampus, amygdala and accumbens (< .0004). SPMS patients had increased MP-APT, decreased volume and decreased NPTV in total SDGM, caudate and amygdala compared to RRMS (p < .005), while individual measure differences were also detected in putamen, thalamus, hippocampus and accumbens (p < .006). RRMS patients showed a significant relationship between increased MP-APT and increased lesion burden and more advanced brain atrophy (p < .004).

Conclusions

Abnormal phase, indicative of higher iron content was significantly increased in MS patients compared to HC, and was related to more severe lesion burden and brain atrophy.     

Headache and multiple sclerosis: a population-based case-control study in Catania, Sicily

We carried out a population-based case-control study to evaluate the association between multiple sclerosis (MS) and headache. We had previously determined the incidence of MS during 1990-1999 in Catania, Sicily, identifying 155 incident MS patients; these subjects underwent a telephone interview using a standardized questionnaire for headache. Diagnosis and classification of headaches were made according to International Headache Society criteria (1988). A control group was selected from the general population through random digit dialling. One hundred and one (65.2%) MS patients, of the 155 identified, and 101 controls were screened for headaches. Fifty-eight (57.4%) MS patients and 38 (37.2%) controls fulfilled the diagnostic criteria of headache. A significant association between MS and headache was found with an adjusted odds ratio, estimated by logistic regression, of 2.18 (95% confidence interval 1.27, 3.93). Frequency of headaches in our MS population is higher than in the general population, supporting the hypothesis of a possible association between these two conditions.     

心肌梗死患者对氧磷酶-1 Q/R192基因多态性及活性检测的临床意义

目的探讨心肌梗死（AMI）患者对氧磷酶-1（PON1）Q/R192基因多态性及其活性检测的临床意义。方法分别采用紫外线分光光度法和聚合酶链式反应-限制性片段长度多态性（PCR-RELP）法检测65例AMI患者和70名健康体检者PON1活性及PON1Q/R192基因多态性。结果 AMI组血清PON1活性[（78.56±16.69）U/mL]明显低于健康对照组[（118.65±30.25）U/mL]（P〈0.01）。AMI组与健康对照组3种基因型及2种等位基因频率分布差异无统计学意义（P〉0.05）;AMI组与健康对照组间不同PON1 Q/R192基因型间血清PON1活性相比差异有统计学意义（P〈0.01）;AMI组内及健康对照组内PON1 Q/R192不同基因型间血清PON1活性相比差异无统计学意义（P〉0.05）。结论血清PON1活性降低是AMI的危险因素之一;PON1Q/R192基因多态性与AMI的发生无相关性。