Lupus
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Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report F Bahrehmand, A Vaisi-Raygani, Z Rahimi, R Ahmadi, A Kiani, H Tavilani, H Vaisi-Raygani and T Pourmotabbed Lupus 2014 23: 263 originally published online 7 January 2014 DOI: 10.1177/0961203313518622 The online version of this article can be found at: http://lup.sagepub.com/content/23/3/263
Published by: http://www.sagepublications.com
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Lupus (2014) 23, 263–272 http://lup.sagepub.com
PAPER
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report F Bahrehmand1, A Vaisi-Raygani1,2,3, Z Rahimi4, R Ahmadi1, A Kiani1, H Tavilani5, H Vaisi-Raygani6 and T Pourmotabbed7 1
Molecular Diagnostic Research Center; 2Fertility and Infertility Research Center; 3Department of Clinical Biochemistry; 4Molecular Biology Research Center, Kermanshah University of Medical Sciences, Iran; 5Department of Clinical Biochemistry, Hamadan University of Medical Sciences, Kermanshah, Iran; 6Departments of Chemistry, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran; and 7Department of Microbiology, Immunology and Biochemistry, University of Tennessee, Health Science Center, Memefist, TN, USA
There is some evidence indicating lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and glomerulonephritis in systemic lupus erythematosus (SLE) patients. Human butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are two major bioscavenger enzymes that are associated with inflammation, oxidative stress and lipid metabolism. Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVDs in SLE. It has also been suggested that deficiency in the function of the antioxidant system and an increase in reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE. This study is the first investigation to examine the association of BuChE phenotypes, PON1 (L55M; PON-55-M) polymorphism, the levels of malondialdehyde (MDA), neopterin, lipidlipoprotein and activities of BuChE and arylesterase activity (ARE) of PON with severity of SLE. The present case-control study consisted of 109 SLE patients and 101 gender- and agematched, unrelated healthy control subjects from the population of west Iran. We found that the PON-55-M allele and BuChE non-UU act synergistically to increase the risk of SLE by 2.5 times (1.03–6.7, p ¼ 0.044). There was a significant negative correlation between severity of SLE with serum BuChE activity (R ¼ 0.31, p < 0.001) and positive correlation with serum neopterin level. The SLE patients with the PON-55-M (M/L þ M/M) allele or with BuChE non-UU phenotype had significantly lower serum ARE and BuChE activities than those with PON-55-L/L or BuChE-UU phenotypes, respectively. In addition, their serum levels of MDA, neopterin and LDL-C were significantly elevated, suggesting that these individuals are more susceptible to CVD. However, further studies are needed to shed more light on the contribution of the M allele of PON1 and non-UU phenotypes of BuChE in the development of SLE in different ethnicities. Lupus (2014) 23, 263–272. Key words: Butyrylcholinesterase (BuChE) activity and phenotype; paraoxonase (PON1) 55 polymorphism; arylesterase activity; systemic lupus erythematosus (SLE); oxidative stress
Introduction
Correspondence to: A Vaisi-Raygani, Department of Clinical Biochemistry, School of Medicine, Kermanshah University of Medical Sciences, Daneshgah Avenue, PO Box 6714869914, Kermanshah, Iran. Emails: [email protected]; [email protected] Received 28 January 2013; accepted 27 November 2013
Systemic lupus erythematosus (SLE) is an autoimmune disease associated with glomerulonephritis, pericarditis, hypertension, psychiatric disease and hematological disorder.1,2 The etiology of SLE is unknown, but evidence indicates that lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and
! The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav
Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
10.1177/0961203313518622
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
264
glomerulonephritis in SLE patients.3,4 Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVD in SLE.5–7 It has also been suggested that deficiency in the function of the antioxidant system and increased reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE.4,8 Human butyrylcholinesterase (BuChE, EC.3.1.1.8), also known as pseudo or non-neuronal cholinesterase, and paraoxonase-1 (PON1, aryldialkylphosphatase, EC 3.1.8.1), the two serum major bioscavengers,9–13 have recently received a lot of attention as antioxidants that attenuate oxidation of low-density lipoprotein (LDL), a key regulator in the pathogenesis of atherosclerosis leading to several CVDs. 10,14,15 BuChE cleaves cholinic esters including the muscle relaxants succinylcholine, mivacurium, and their derivatives, which are strong inhibitors of red cell acetylcholinesterase.15–17 More than 30 variants of serum cholinesterase have been described. Some of these (the atypical, the silent and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine.15–17 The association between BuChE activity with lipid and lipoprotein levels, type 2 diabetes mellitus (T2DM), stroke and CVD is well studied.9,11–20 We recently observed that the frequency of BuChE non-UU phenotypes (low activity) was high in stroke patients, who had high levels of cholesterol that might increase susceptibility to stroke.15 PON1, also referred to as arylestrase (ARE), hydrolyses organophosphate substrate paraoxon and aromatic esters, such as phenylacetate.10 PON activity is under genetic control and the variants in the PON1 gene have strong influences on PON activity.21 One of the major polymorphisms of PON1 is L55M, where Met (M) has replaced the amino acid Leu at position 55.22,23 The L55M have been reported to have lower paraoxonase and ARE activities than the 55 L allele isoforms. In humans, serum PON1 activity and serum LDL susceptibility to oxidation are inversely related.24 PON1 is synthesized mainly in the liver and secreted to blood and bound to high-density lipoprotein (HDL), and has been shown to inhibit LDL oxidation, and low PON1 activity has been associated with increased risk of coronary artery disease (CAD).23–25 PON1 activity is also reduced in individuals with SLE, hypercholesterolemia, noninsulin-dependent diabetes and in patients with vascular diseases, survivors of myocardial
infarction and rheumatoid arthritis (RA).3,5,6,26,27 However, the level of activities of BuChE and ARE of PON1 in SLE patients is unknown. To our knowledge, there are no published studies about possible influence of BuChE phenotypes and PON-1 L55M polymorphism on activities of these enzymes and lipid lipoproteins levels in SLE patients. In this study we demonstrate that BuChE non-UU phenotypes synergistically increase the risk of SLE in individuals carrying the PON-55 M allele.
Materials and methods Subjects The human subject study protocol was approved by the ethics committee of the Kermanshah University of Medical Sciences and was in accordance with the principles of the Declaration of Helsinki II, and all subjects provided written informed consent. One hundred and nine SLE patients (mean age, 37.3 11.3 years; range, 15–75 years; 87 females and 22 males) were recruited from the Rheumatology Division of the Imam Reza Hospital and a Rheumatology clinic of the Kermanshah University of Medical Sciences. All patients revealed at least four of the revised diagnostic criteria of the American College of Rheumatology (ACR) for SLE.28 Clinical and laboratory findings were defined using ACR criteria and collected from well-documented medical records during the follow-up. The Systemic Lupus International Collaborating Clinics/ACR (SLICC/ ACR) Damage Index (SLE activity) was calculated at the time of blood sampling.29 Clinical findings including age, gender, involved organs, duration and severity of the disease were all recorded. Cardiovascular and renal involvement in SLE were detected by chest X-ray, electrocardiogram, ultrasonic cardiogram, urinalysis, brain computed tomography scan or magnetic resonance imaging. Hypertension was defined as resting systolic blood pressure 160 mmHg and diastolic blood pressure 95 mmHg. SLE patients with a history of proteinuria greater than 500 mg/day or with diffuse or focal proliferative renal lesion were diagnosed with having lupus nephritis (LN). The 104 control subjects (mean age, 37.1 11.5 years; range, 15–72 years; 82 females and 22 males) were selected from healthy individuals during their annual check-ups at the Hospitals of the Kermanshah University of Medical Sciences. The patient and control groups were matched according
Lupus Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
265
to gender, age and race. All healthy donors were interviewed and examined for coronary heart disease, RA and SLE, and no specific diseases at the medical check-ups were noted. Individuals with a history of or having a family with hypertension, autoimmune disorders, rheumatic or CVDs were excluded from the study.
Chemical analysis Determination of serum BuChE activity and phenotypes BuChE activity and phenotypes were determined spectrophotometrically using benzoylcholine chloride (50 mmol/l) as substrate in the presence or absence of the inhibitors, dibucaine hydrochloride (10 mmol/l) and sodium fluoride (50 mmol/l) (for determination of dibucaine (DN) and fluoride (FN) numbers), as previously described.15,30 Measurement of serum ARE activity of PON1 Serum ARE activity of PON1 was measured spectrophotometrically using phenylacetate as substrate according to protocol previously described.31,32 Malondialdehyde (MDA) and neopterin Plasma MDA and serum neopterin was measured by an Agilent Technologies 1200 Series HPLC system (Agilent Corp. Germany) using EC 250/ 4.6 Nucleodur 100-5 C18ec column MachereyNagel, Duren, Germany) as we previously described.32–34 Plasma lipids Total serum cholesterol (TC), triacylglycerol (TG), LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) levels were measured by the standard enzymatic method (Pars Azmon kit, Iran), using an automated Erba XL-600 (Mannheim, Germany).
and the reverse primer, 5’-TTT,AAT,CCA,GAG, CTA,ATG,AAA,GCC-3’.32 The 25 ml amplification reaction contained 50–100 ng genomic DNA, 10 pmol of each primer, a 200 mmol L–1 solution of each deoxyribonucleotide triphosphate, 2.5 ml of 10 reaction buffer and 0.5 U Taq DNA polymerase. After the DNA was denatured at 95 C for five minutes, the reaction mixture was subjected to 35 cycles of 94 C for one minute, 58.5 C for 1.5 minutes and 72 C for 1.5 minutes, with a final extension time of 10 minutes. The PCR products (179 base pairs (bps)) were digested with Hsp92II restriction endonuclease at 37 C overnight and the product(s) were separated on a 2.5% agarose gel and visualized by staining with ethidium bromide. The PCR products were assigned to the PON1 55 Leu (L) or Met (M) allele based on the presence of a 170 bp (undigested) fragment or 126 and 44 bp digested fragments, respectively. Statistical analysis The allelic frequencies were calculated by the gene counting method. The 2 test was used to verify the agreement of the observed genotype frequencies with those expected according to the HardyWeinberg equilibrium. The BuChE variants, genotypes and allele frequencies of PON1 55 in SLE patients were compared to the control group using the 2 test. Odds ratios (OR) were calculated as estimates of relative risk for disease and 95% confidence intervals obtained by SPSS logistic regression. The correlation of serum BuChE and ARE activities, neopterin, MDA, HDL-C, LDLC, TC, TG and SLE with the PON1 55 polymorphism between SLE patients and control groups were calculated using linear regression and an unpaired t test. A two-tailed Student’s t test, analysis of variance (ANOVA) and nonparametric independent-sample Mann-Whitney analysis were used to compare quantitative data. Statistical significance was assumed at p < 0.05.
Results
DNA analysis Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform extraction method.26 Genotyping of all individuals was performed without knowledge of their groups or disease. The PON1 55 Met>Leu (M > L) polymorphism was detected by polymerase chain reaction (PCR) using the forward primer, 5’-GAA,GAG,TGA,TGT,ATA,AGC,CCC,A-3’
Characteristics of patients and control groups are demonstrated in Table 1. The means of age and gender distribution were the same for SLE patients and the control group. Except for HDL-C, TC and TG, all the other lipid variables were significantly different in SLE patients compared to the control group. The patients with SLE had significantly lower serum BuChE activity (930 226 vs. Lupus
Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
266
1011 231 U/l, p < 0.001) and ARE activity (132480 31496 vs. 142550 26708 U/L, p < 0.001) and higher plasma MDA (18.4 12.7 vs. 14.1 6.4 m mol/l, p ¼ 0.003), neopterin and LDL-C concentration than those of the control group, respectively.
Table 1 The demographic and distribution of risk factors in SLE patients and control subjects Parameter
SLE patients (n ¼ 109)
Control subjects (n ¼ 101)
p values
Age (years) Sex (M/F) BuChE activity (U/l)a ARE activity (U/l)b Neopterin (nmol/l) MDA (mmol/l) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Total cholesterol (mg/dl) TG (mg/dl) SLE activity
35.6 16.3 19/90 930 226 132480 31496 25.8 38.1 18.4 12.7 119.7 29.2 42.1 21.1 196 37 179 141 21 12.2
37.1 11.5 22/82 1011 231 142550 26708 6.5 2.9 14.1 6.4 86.4 37 42.7 11 188 61 173 117 –
0.74 0.38 ¼0.011
http://lup.sagepub.com/
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report F Bahrehmand, A Vaisi-Raygani, Z Rahimi, R Ahmadi, A Kiani, H Tavilani, H Vaisi-Raygani and T Pourmotabbed Lupus 2014 23: 263 originally published online 7 January 2014 DOI: 10.1177/0961203313518622 The online version of this article can be found at: http://lup.sagepub.com/content/23/3/263
Published by: http://www.sagepublications.com
Additional services and information for Lupus can be found at: Email Alerts: http://lup.sagepub.com/cgi/alerts Subscriptions: http://lup.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Feb 14, 2014 OnlineFirst Version of Record - Jan 7, 2014 What is This?
Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
Lupus (2014) 23, 263–272 http://lup.sagepub.com
PAPER
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report F Bahrehmand1, A Vaisi-Raygani1,2,3, Z Rahimi4, R Ahmadi1, A Kiani1, H Tavilani5, H Vaisi-Raygani6 and T Pourmotabbed7 1
Molecular Diagnostic Research Center; 2Fertility and Infertility Research Center; 3Department of Clinical Biochemistry; 4Molecular Biology Research Center, Kermanshah University of Medical Sciences, Iran; 5Department of Clinical Biochemistry, Hamadan University of Medical Sciences, Kermanshah, Iran; 6Departments of Chemistry, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran; and 7Department of Microbiology, Immunology and Biochemistry, University of Tennessee, Health Science Center, Memefist, TN, USA
There is some evidence indicating lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and glomerulonephritis in systemic lupus erythematosus (SLE) patients. Human butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are two major bioscavenger enzymes that are associated with inflammation, oxidative stress and lipid metabolism. Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVDs in SLE. It has also been suggested that deficiency in the function of the antioxidant system and an increase in reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE. This study is the first investigation to examine the association of BuChE phenotypes, PON1 (L55M; PON-55-M) polymorphism, the levels of malondialdehyde (MDA), neopterin, lipidlipoprotein and activities of BuChE and arylesterase activity (ARE) of PON with severity of SLE. The present case-control study consisted of 109 SLE patients and 101 gender- and agematched, unrelated healthy control subjects from the population of west Iran. We found that the PON-55-M allele and BuChE non-UU act synergistically to increase the risk of SLE by 2.5 times (1.03–6.7, p ¼ 0.044). There was a significant negative correlation between severity of SLE with serum BuChE activity (R ¼ 0.31, p < 0.001) and positive correlation with serum neopterin level. The SLE patients with the PON-55-M (M/L þ M/M) allele or with BuChE non-UU phenotype had significantly lower serum ARE and BuChE activities than those with PON-55-L/L or BuChE-UU phenotypes, respectively. In addition, their serum levels of MDA, neopterin and LDL-C were significantly elevated, suggesting that these individuals are more susceptible to CVD. However, further studies are needed to shed more light on the contribution of the M allele of PON1 and non-UU phenotypes of BuChE in the development of SLE in different ethnicities. Lupus (2014) 23, 263–272. Key words: Butyrylcholinesterase (BuChE) activity and phenotype; paraoxonase (PON1) 55 polymorphism; arylesterase activity; systemic lupus erythematosus (SLE); oxidative stress
Introduction
Correspondence to: A Vaisi-Raygani, Department of Clinical Biochemistry, School of Medicine, Kermanshah University of Medical Sciences, Daneshgah Avenue, PO Box 6714869914, Kermanshah, Iran. Emails: [email protected]; [email protected] Received 28 January 2013; accepted 27 November 2013
Systemic lupus erythematosus (SLE) is an autoimmune disease associated with glomerulonephritis, pericarditis, hypertension, psychiatric disease and hematological disorder.1,2 The etiology of SLE is unknown, but evidence indicates that lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and
! The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav
Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
10.1177/0961203313518622
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
264
glomerulonephritis in SLE patients.3,4 Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVD in SLE.5–7 It has also been suggested that deficiency in the function of the antioxidant system and increased reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE.4,8 Human butyrylcholinesterase (BuChE, EC.3.1.1.8), also known as pseudo or non-neuronal cholinesterase, and paraoxonase-1 (PON1, aryldialkylphosphatase, EC 3.1.8.1), the two serum major bioscavengers,9–13 have recently received a lot of attention as antioxidants that attenuate oxidation of low-density lipoprotein (LDL), a key regulator in the pathogenesis of atherosclerosis leading to several CVDs. 10,14,15 BuChE cleaves cholinic esters including the muscle relaxants succinylcholine, mivacurium, and their derivatives, which are strong inhibitors of red cell acetylcholinesterase.15–17 More than 30 variants of serum cholinesterase have been described. Some of these (the atypical, the silent and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine.15–17 The association between BuChE activity with lipid and lipoprotein levels, type 2 diabetes mellitus (T2DM), stroke and CVD is well studied.9,11–20 We recently observed that the frequency of BuChE non-UU phenotypes (low activity) was high in stroke patients, who had high levels of cholesterol that might increase susceptibility to stroke.15 PON1, also referred to as arylestrase (ARE), hydrolyses organophosphate substrate paraoxon and aromatic esters, such as phenylacetate.10 PON activity is under genetic control and the variants in the PON1 gene have strong influences on PON activity.21 One of the major polymorphisms of PON1 is L55M, where Met (M) has replaced the amino acid Leu at position 55.22,23 The L55M have been reported to have lower paraoxonase and ARE activities than the 55 L allele isoforms. In humans, serum PON1 activity and serum LDL susceptibility to oxidation are inversely related.24 PON1 is synthesized mainly in the liver and secreted to blood and bound to high-density lipoprotein (HDL), and has been shown to inhibit LDL oxidation, and low PON1 activity has been associated with increased risk of coronary artery disease (CAD).23–25 PON1 activity is also reduced in individuals with SLE, hypercholesterolemia, noninsulin-dependent diabetes and in patients with vascular diseases, survivors of myocardial
infarction and rheumatoid arthritis (RA).3,5,6,26,27 However, the level of activities of BuChE and ARE of PON1 in SLE patients is unknown. To our knowledge, there are no published studies about possible influence of BuChE phenotypes and PON-1 L55M polymorphism on activities of these enzymes and lipid lipoproteins levels in SLE patients. In this study we demonstrate that BuChE non-UU phenotypes synergistically increase the risk of SLE in individuals carrying the PON-55 M allele.
Materials and methods Subjects The human subject study protocol was approved by the ethics committee of the Kermanshah University of Medical Sciences and was in accordance with the principles of the Declaration of Helsinki II, and all subjects provided written informed consent. One hundred and nine SLE patients (mean age, 37.3 11.3 years; range, 15–75 years; 87 females and 22 males) were recruited from the Rheumatology Division of the Imam Reza Hospital and a Rheumatology clinic of the Kermanshah University of Medical Sciences. All patients revealed at least four of the revised diagnostic criteria of the American College of Rheumatology (ACR) for SLE.28 Clinical and laboratory findings were defined using ACR criteria and collected from well-documented medical records during the follow-up. The Systemic Lupus International Collaborating Clinics/ACR (SLICC/ ACR) Damage Index (SLE activity) was calculated at the time of blood sampling.29 Clinical findings including age, gender, involved organs, duration and severity of the disease were all recorded. Cardiovascular and renal involvement in SLE were detected by chest X-ray, electrocardiogram, ultrasonic cardiogram, urinalysis, brain computed tomography scan or magnetic resonance imaging. Hypertension was defined as resting systolic blood pressure 160 mmHg and diastolic blood pressure 95 mmHg. SLE patients with a history of proteinuria greater than 500 mg/day or with diffuse or focal proliferative renal lesion were diagnosed with having lupus nephritis (LN). The 104 control subjects (mean age, 37.1 11.5 years; range, 15–72 years; 82 females and 22 males) were selected from healthy individuals during their annual check-ups at the Hospitals of the Kermanshah University of Medical Sciences. The patient and control groups were matched according
Lupus Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
265
to gender, age and race. All healthy donors were interviewed and examined for coronary heart disease, RA and SLE, and no specific diseases at the medical check-ups were noted. Individuals with a history of or having a family with hypertension, autoimmune disorders, rheumatic or CVDs were excluded from the study.
Chemical analysis Determination of serum BuChE activity and phenotypes BuChE activity and phenotypes were determined spectrophotometrically using benzoylcholine chloride (50 mmol/l) as substrate in the presence or absence of the inhibitors, dibucaine hydrochloride (10 mmol/l) and sodium fluoride (50 mmol/l) (for determination of dibucaine (DN) and fluoride (FN) numbers), as previously described.15,30 Measurement of serum ARE activity of PON1 Serum ARE activity of PON1 was measured spectrophotometrically using phenylacetate as substrate according to protocol previously described.31,32 Malondialdehyde (MDA) and neopterin Plasma MDA and serum neopterin was measured by an Agilent Technologies 1200 Series HPLC system (Agilent Corp. Germany) using EC 250/ 4.6 Nucleodur 100-5 C18ec column MachereyNagel, Duren, Germany) as we previously described.32–34 Plasma lipids Total serum cholesterol (TC), triacylglycerol (TG), LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) levels were measured by the standard enzymatic method (Pars Azmon kit, Iran), using an automated Erba XL-600 (Mannheim, Germany).
and the reverse primer, 5’-TTT,AAT,CCA,GAG, CTA,ATG,AAA,GCC-3’.32 The 25 ml amplification reaction contained 50–100 ng genomic DNA, 10 pmol of each primer, a 200 mmol L–1 solution of each deoxyribonucleotide triphosphate, 2.5 ml of 10 reaction buffer and 0.5 U Taq DNA polymerase. After the DNA was denatured at 95 C for five minutes, the reaction mixture was subjected to 35 cycles of 94 C for one minute, 58.5 C for 1.5 minutes and 72 C for 1.5 minutes, with a final extension time of 10 minutes. The PCR products (179 base pairs (bps)) were digested with Hsp92II restriction endonuclease at 37 C overnight and the product(s) were separated on a 2.5% agarose gel and visualized by staining with ethidium bromide. The PCR products were assigned to the PON1 55 Leu (L) or Met (M) allele based on the presence of a 170 bp (undigested) fragment or 126 and 44 bp digested fragments, respectively. Statistical analysis The allelic frequencies were calculated by the gene counting method. The 2 test was used to verify the agreement of the observed genotype frequencies with those expected according to the HardyWeinberg equilibrium. The BuChE variants, genotypes and allele frequencies of PON1 55 in SLE patients were compared to the control group using the 2 test. Odds ratios (OR) were calculated as estimates of relative risk for disease and 95% confidence intervals obtained by SPSS logistic regression. The correlation of serum BuChE and ARE activities, neopterin, MDA, HDL-C, LDLC, TC, TG and SLE with the PON1 55 polymorphism between SLE patients and control groups were calculated using linear regression and an unpaired t test. A two-tailed Student’s t test, analysis of variance (ANOVA) and nonparametric independent-sample Mann-Whitney analysis were used to compare quantitative data. Statistical significance was assumed at p < 0.05.
Results
DNA analysis Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform extraction method.26 Genotyping of all individuals was performed without knowledge of their groups or disease. The PON1 55 Met>Leu (M > L) polymorphism was detected by polymerase chain reaction (PCR) using the forward primer, 5’-GAA,GAG,TGA,TGT,ATA,AGC,CCC,A-3’
Characteristics of patients and control groups are demonstrated in Table 1. The means of age and gender distribution were the same for SLE patients and the control group. Except for HDL-C, TC and TG, all the other lipid variables were significantly different in SLE patients compared to the control group. The patients with SLE had significantly lower serum BuChE activity (930 226 vs. Lupus
Downloaded from lup.sagepub.com at UNIVERSITY OF WINDSOR on October 23, 2014
Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of SLE F Bahrehmand et al.
266
1011 231 U/l, p < 0.001) and ARE activity (132480 31496 vs. 142550 26708 U/L, p < 0.001) and higher plasma MDA (18.4 12.7 vs. 14.1 6.4 m mol/l, p ¼ 0.003), neopterin and LDL-C concentration than those of the control group, respectively.
Table 1 The demographic and distribution of risk factors in SLE patients and control subjects Parameter
SLE patients (n ¼ 109)
Control subjects (n ¼ 101)
p values
Age (years) Sex (M/F) BuChE activity (U/l)a ARE activity (U/l)b Neopterin (nmol/l) MDA (mmol/l) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Total cholesterol (mg/dl) TG (mg/dl) SLE activity
35.6 16.3 19/90 930 226 132480 31496 25.8 38.1 18.4 12.7 119.7 29.2 42.1 21.1 196 37 179 141 21 12.2
37.1 11.5 22/82 1011 231 142550 26708 6.5 2.9 14.1 6.4 86.4 37 42.7 11 188 61 173 117 –
0.74 0.38 ¼0.011
