Journal of Clinical Neuroscience xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease Aynur Kirbas a,⇑, Serkan Kirbas b, Medine Cumhur Cure a, Ahmet Tufekci b a b
_ Department of Biochemistry, Recep Tayyip Erdog˘an University Faculty of Medicine, Islampas ßa mah., Merkez/Rize 53100, Turkey Department of Neurology, Recep Tayyip Erdog˘an University Faculty of Medicine, Rize, Turkey
a r t i c l e
i n f o
Article history: Received 11 February 2012 Accepted 13 April 2013 Available online xxxx Keywords: Arylesterase Oxidative stress index Paraoxonase Parkinson’s disease Total anti-oxidant status Total oxidant status
a b s t r a c t This study investigated serum paraoxonase (PON1) and arylesterase activity along with determination of oxidative status via measurement of total oxidant status (TOS), total anti-oxidant status (TAS) and oxidative stress index (OSI) in patients with Parkinson’s disease (PD) and compared results with data from healthy controls. A total of 82 subjects, including 42 patients with idiopathic PD, newly diagnosed and untreated (24 men, 18 women, aged 47–66 years) and 40 healthy controls were enrolled in this study. We aimed to evaluate the oxidative status of PD patients via measurement of serum TOS and TAS and estimation of OSI using new automated methods. PON1 and arylesterase activities were measured spectrophotometrically. Serum total cholesterol, high density lipoprotein cholesterol, low density lipoprotein (LDL) cholesterol and triglyceride levels were measured using routine methods. TAS levels of PD patients were significantly lower than that of controls (p < 0.05). TOS levels of PD patients were higher than those of controls (p < 0.05). PON1 and arylesterase activities of PD were lower than those of controls (p < 0.05). Serum levels of total and LDL cholesterol were significantly reduced in PD patients. In conclusion, the presence of high TOS and OSI levels together with low levels of TAS in PD patients supports the important role of oxidative stress in the pathophysiology of PD. Since oxidative stress is involved in neurodegeneration, selecting anti-oxidants, metal chelators or other compounds boosting endogenous enzymatic and non-enzymatic defense mechanisms seems to be an obvious choice as treatment for PD. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, tremor, progressive rigidity and postural instability, resulting from progressive degeneration of dopaminergic neurons in the substantia nigra of the midbrain. Both incidence and prevalence increase with age, with a prevalence of 0.3% and an estimated incidence of 8–18 per 100 000 person years [1]. Pathologically PD is characterized by loss of melanin-pigmented nigral neurons accompanied by depletion of dopamine in the striatum and the presence of Lewy bodies, which are deposits of specific cytoplasmic proteins such as ubiquitin, a-synuclein, and oxidized neurofilaments [2]. Similar to other neurodegenerative disorders, oxidative stress, inflammation, mitochondrial dysfunction, environmental factors and genetic predisposition may all be involved in PD [1,2]. Although oxidative stress plays a role in the pathogenesis of neuronal death, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or if it is a secondary manifestation of the neurodegenerative process [3]. ⇑ Corresponding author. Tel.: +90 46 4212 3009; fax: +90 46 4217 0367. E-mail address: [email protected] (A. Kirbas).
Many cellular reactions utilize molecular oxygen for catalysis and energy production [4]. These reactions in turn produce reactive oxygen species (ROS) including superoxide anions, hydrogen peroxide, hydroxyl radicals, peroxy radicals and, in the presence of nitric oxide, reactive nitrogen species (RNS) such as peroxynitrite and nitro-tyrosyl radicals. While these reactive species are important for the execution of physiological functions, excessive production is detrimental to cell membranes and can cause cell death [5]. The rate of production and destruction of ROS is in a state of balance, known as oxidative balance. In cases where this oxidative balance is maintained, ROS may have no impact on the organism but in cases where this balance is tipped in favor of free radicals, oxidative stress develops [6]. The defense system protecting against free radical damage involves enzymatic and non-enzymatic anti-oxidant systems. The enzymatic system includes superoxide dismutase, glutathione peroxidase, catalase, aldehyde dehydrogenases, and sulfiredoxin. The non-enzymatic system includes naturally occurring anti-oxidants such as vitamin A (retinol), vitamin C (ascorbic acid), vitamin E (tocopherol), beta-carotene and glutathione, as well as polyphenol anti-oxidants like flavonoids [7,8]. Under certain conditions, the oxidative or anti-oxidative balance shifts towards oxidative stress as a result of increase in ROS and/or impairment in the anti-oxidant
0967-5868/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2013.04.025
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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A. Kirbas et al. / Journal of Clinical Neuroscience xxx (2013) xxx–xxx
mechanisms [7]. The body’s oxidant/anti-oxidant status can be ascertained by measuring the activity of individual molecules or enzymes; likewise the overall oxidant/anti-oxidant status can be assessed more easily by measuring total anti-oxidant status (TAS) [9] and total oxidant status (TOS) [10]. Environmental factors may influence PD risk [11]. One oftendiscussed risk factor is pesticide exposure [12]. Paraoxonase 1 (PON1), a 43–45 kDa glycoprotein, is synthesized mainly in the liver, which hydrolyzes organophosphates such as pesticides, neurotoxins, and arylesters [11,12]. The paraoxonase (PON) gene family consists of three members – PON1, PON2, and PON3 – located adjacent to each other on the long arm of chromosome 7 in humans (q21.3–q22.1) [13]. These three human PON genes share approximately 60% identity at the amino acid level and approximately 70% identity at the nucleotide level [14]. PON is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated in the anti-oxidant and anti-inflammatory properties exerted by HDL. Many investigations have provided considerable evidence for PON1 anti-atherogenicity [13,14]. Studies have shown that PON1 inhibits oxidation of HDL and low-density lipoproteins (LDL) that preserve HDL function, increases cellular cholesterol efflux from macrophages, ameliorates effects of oxidized LDL, and decreases lipid peroxides in atherosclerotic lesions [13,14]. There are two polymorphisms in the PON1 coding region: leucine/methionine at position 55 (M55L) and glutamine/arginine at position 192 (Q192R). These polymorphisms are associated with a number of pathophysiological conditions, including coronary artery disease, PD, stroke, familial hypercholesterolemia, type 2 diabetes mellitus, late-onset Alzheimer’s disease and reduced bone mass in post-menopausal women [12–14]. The activity of PON1 in patients with PD has been very different to results obtained in studies on populations without PD [11,15,16]. The aim of this study was to investigate the relationship between serum paraoxonase and arylesterase activity, TOS, TAS and oxidative stress index (OSI) in patients with PD. 2. Methods 2.1. Subjects This study was conducted at the Neurology Clinic of Recep Tayyip Erdog˘an University School of Medicine, Turkey. Forty-two newly diagnosed and untreated idiopathic PD patients (24 men, 18 women, mean age 59.3 ± 4.9 years [standard deviation], range 47–66) were included in this study. The PD patients were diagnosed according to the Parkinson’s Disease Society Brain Bank clinical diagnostic criteria for idiopathic PD [17]. Severity ratings for PD between 0 and 5 used the Hoehn and Yahr scale, and all patients were between Hoehn and Yahr stage 1 and 2. The procedures were in accordance with the revised form of the Declaration of Helsinki 2008 and all participants signed an informed consent form. The study protocol was approved by the local Ethical Committee. The control group consisted of 40 healthy individuals (24 men, 16 women, mean age 57.0 ± 4.9 years [standard deviation], range 45–67). All subjects were informed about the study. Body mass index (weight/height2) was obtained through height and weight measurements using a wall-mounted ruler and a digital scale.
fugation at 3000 g for 10 minutes, and then stored at –80 °C until further analysis of paraoxonase and arylesterase activities along with determination of oxidative status via measurement of TOS, TAS and OSI. Serum total cholesterol, HDL cholesterol and triglyceride levels were measured with routine methods (Architect C1600; Abbott Laboratories, Abbott Park, IL, USA). LDL was calculated using the Friedewald formula. 2.3. Measurement of paraoxonase and arylesterase activity Paraoxonase activity was measured in absence (basal activity) and presence of NaCl (salt-stimulated activity) [18]. Briefly, the rate of paraoxon hydrolysis was measured by the increase of absorbance at 412 nm at 25 °C. The amount of generated p-nitrophenol was calculated from the molar absorptivity coefficient at pH 8, which was 17.100 M 1 cm 1. Paraoxonase activity was expressed as U/L serum. Phenylacetate was used as a substrate to measure arylesterase activity. The reaction was initiated by addition of serum and the increase in absorbance was read at 270 nm. Blanks were included to correct spontaneous hydrolysis of phenylacetate. Enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol, 1310 M 1 cm 1. One unit of arylesterase activity was defined as 1 mmol phenol generated/ minute under the above conditions and expressed as U/L serum. Phenotype distribution of paraoxonase was determined in presence of 1 mol/L NaCl (salt-stimulated paraoxonase). The ratio of salt-stimulated paraoxonase activity to arylesterase activity was used to assign individuals to one of the three possible phenotypes [19]. 2.4. Measurement of the TOS Serum TOS was determined using a novel automated measurement method previously described [9]. Oxidants present in the sample oxidize ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. Ferric ion reacts with xylenol orange in an acidic medium to produce a colored complex. The intensity of color, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules in the sample. The assay was calibrated with hydrogen peroxide and results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (lmol H2O2 equiv./L). The assay has excellent precision with error values lower than 2%. 2.5. Measurement of the TAS Serum TAS was determined using an automated measurement method previously described [10]. Briefly, potent free radical reactions were initiated with the production of a hydroxyl radical via the Fenton reaction and the rate of reactions was monitored by following the absorbance of colored dianisidyl radicals. Using this method, the anti-oxidative effect of the sample against potent free radical reactions, which were initiated by synthesized hydroxyl radical, was measured. This method was applied to an automated analyzer (Architect C1600; Abbott Laboratories). Both intra- and inter-assay coefficients of variations were lower than 3%. Data were expressed as lmol equiv./L Trolox (Hoffman-LaRoche, Basel, Switzerland).
2.2. Blood sample collection 2.6. Calculation of the OSI After overnight fasting, peripheral venous blood samples were taken from patients and controls into empty tubes. After coagulation, samples were immediately separated from the cells by centri-
The OSI was calculated by dividing the TOS by the TAS, that is, OSI = (TOS, lmol H2O2 equiv./L)/(TAS, lmol Trolox equiv./L).
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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2.7. Statistical analysis
Table 2 Studied parameters in patients with Parkinson’s disease and healthy controls
All statistical analyses were performed using the Statistical Package for the Social Sciences, version 15.0 (SPSS, Chicago, IL, USA). Data are expressed as mean ± standard deviation. The normality of the distribution for all variables was assessed by the Kolmogorov–Smirnov test. Student’s t-test was used for normally distributed variables and the Mann–Whitney U test was used for non-parametric variables. Relationships between variables were analyzed by Pearson or Spearman correlation analysis according to the distribution type of the variables. p < 0.05 was considered to be statistically significant. 3. Results The demographic and clinical data of the subjects are summarized in Table 1. There were no significant differences in age, sex or body mass index between patients with PD and healthy controls. As compared to controls, serum levels of total cholesterol and LDL cholesterol activity were significantly reduced in PD patients. Serum levels of HDL cholesterol and triglyceride showed no significant differences between PD patients and controls. Paraoxonase and arylesterase activity, OSI, TAS and TOS levels are shown in Table 2. Paraoxonase and arylesterase activity were lower in the PD patients compared to healthy controls and the differences were statistically significant (p = 0.001 and p = 0.001, respectively). Plasma TOS and OSI values were higher in patients with PD than healthy controls and again the differences were statistically significant (p = 0.001 and p = 0.001, respectively). TAS was significantly lower in PD patients (p = 0.001). There was no correlation between serum PON1 activity and OSI in patients with PD (Fig. 1, p = 0.09). There was no significant difference between serum PON1 activity and OSI in male and female PD patients (Supplementary Fig. 1, p = 0.854). 4. Discussion We recruited patients who were in the relatively early stages of disease, were of a young age and had not yet begun treatment. These factors are important for two reasons: first, the oxidant and anti-oxidant balance changes over time and in favor of oxidative damage during disease progression, and it is known that PON1 activity decreases with age [2,19]. Second, we removed the impact of drugs on oxidant and anti-oxidant mechanisms by sampling sera before starting treatment on L-dopa and/or dopamine agonist drugs. One of the most metabolically active organs of the body is the brain. When the spinal cord is included to make up the central
Table 1 Demographic characteristics and results of biochemical analyses in patients with Parkinson’s diseases and healthy controls
Age (years) Male, n Female, n BMI (kg/m2) Total cholesterol (mg/dL) LDL cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mg/dL)
PD (n = 52)
Controls (n = 40)
p value*
59.3 ± 4.9 32 20 24.8 ± 4.3 181.9 ± 31.7 97.7 ± 32.3 58.3 ± 12.2 134.5 ± 52.1
57.0 ± 4.9 24 16 25.3 ± 4.9 202.0 ± 17.5 116.4 ± 17.2 56.5 ± 13.4 128.9 ± 46.6
NS NS NS NS 0.001 0.001 NS NS
Data are presented as mean ± standard deviation, unless otherwise stated. BMI = body mass index, HDL = high-density lipoprotein, LDL = low-density lipoprotein, NS = not significant, PD = Parkinson’s disease. * Student’s t-test.
Paraoxonase (U/L) Arylesterase (U/L) TAS (mmol Trolox equiv./L) TOS (lmolH2O2 equiv./L) OSI (arbitrary unit)
PD (n = 52)
Controls (n = 40)
p value
112.3 ± 48.7 141.5 ± 26.4 1.50 ± 0.13 9.39 ± 2.34 6.28 ± 1.73
200.9 ± 56.5 198.8 ± 24.7 1.66 ± 0.10 8.04 ± 1.46 4.84 ± 0.95
0.001 0.001 0.001 0.001 0.001
Data are presented as mean ± standard deviation. equiv. = equivalent, OSI = oxidative stress index, TAS = total antioxidant status, TOS = total oxidant status.
nervous system it, even at rest, utilizes an estimated 20% of total oxygen uptake [2]. During the active state this percentage substantially increases and it requires an uninterrupted oxygen-rich blood supply to carry out normal physiological actions. Any blockage or deprivation of this oxygen supply even for a few seconds can have severe and irreversible detrimental effect to the cells of brain (both neurons and glia). Consumption of oxygen leads to the production of free radicals, and the brain’s requirement for a higher amount of oxygen leads to even higher numbers of reactive oxygen/nitrogen species [3]. ROS and RNS can attract and damage a variety of critical biological molecules, including lipids, essential cellular proteins and DNA, and may be involved in the pathogenesis of many neurodegenerative disorders including PD [2,3]. Products of lipid peroxidation can be easily and reliably detected in biological fluids and tissues, yielding sensitive and specific signals of lipid peroxidation occurring in vivo. These products are isoprostanes, dimalonealdehyde, 4-hydroxynonenal, and 8-hydroxy-2-deoxy-guanosine (a marker of oxidized DNA) [20]. Several protein kinases including protein kinase C and mitogen activated protein kinases have been implicated in activation of NADPH oxidase and therefore contribute to oxidative stress [2]. These oxidant enzymes and molecules have been studied in vitro and in vivo in patients with PD with reports that oxidative stress generally increased following an increase in the levels of these enzymes and molecules. [1–4]. Reported deficiencies in the major anti-oxidant enzyme systems in the brain (catalase, superoxide dismutase, and glutathione peroxidase), along with a reduction in the levels of glutathione, suggest that oxidative stress in PD is real and contributing to pathogenesis [2,3]. Levels of many oxidant and anti-oxidant parameters present in serum may be measured individually. Since oxidant and anti-oxidant parameters show an additive effect, individual values may not correctly reflect TOS or TAS [9,10]. Therefore, TOS and TAS are more accurate indicators of the oxidative and anti-oxidative status of individuals. The OSI is calculated as the ratio of TOS to TAS. We found the TOS levels, a general indicator of oxidant molecules, were significantly higher in the PD group compared with the control group, and the TAS levels were significantly lower in the PD group than in the control group. The reason for this finding is unknown, but it could be suggested that the endogenous anti-oxidants are reduced through consumption by the increasing oxidants seen in PD patients. Paraoxonase enzymes have attracted growing interest in the last few years due to their wide applications in environmental and genetic toxicology. PON1 is a particularly important anti-oxidant enzyme in the human body [20]. Lipid peroxidation leads to toxic aldehydes that are highly reactive and can be hydrolyzed by PON1 [21]. PON1 therefore constitutes a possible link between environmental toxins and genetic susceptibility to PD [11,22]. This enzyme has a significant role in the defense of LDL and HDL from oxidation by hydrolyzing lipid peroxide products. In our study, we found that total cholesterol and LDL cholesterol activity was significantly lower in PD patients than the control group. However,
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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A. Kirbas et al. / Journal of Clinical Neuroscience xxx (2013) xxx–xxx
Fig. 1. Graph showing the non-significant correlation between serum PON1 activity and oxidative stress index in patients with Parkinson’s disease (p = 0.09). AU = arbitrary unit.
a limitation of the present study is that it was not a prospective study and included no diet information for PD patients and controls. The causative mechanism for lowering serum total cholesterol and LDL cholesterol levels in PD patients remains unclear [1]. Previous studies have investigated the complex relationship between serum cholesterol levels and risk of PD. A Finnish cohort study suggested that higher serum levels of total cholesterol increased the risk of developing PD in men and women aged 25– 54 years at baseline [23]. In contrast, the Rotterdam Study reported that higher serum levels of total cholesterol decreased the risk of PD in women [24]. Ikeda et al. reported that serum levels of total cholesterol and LDL cholesterol were inversely correlated with disease progression in female PD patients [1]. It has been reported that the primary effect of PON1 is to protect lipoproteins from oxidation, but it is sensitive to oxidative stress and enzyme activity is inactivated by oxidants [11,13,14]. In our study, we found that PON1 enzyme activity was significantly lower in PD patients than in the control group. On the other hand we concluded that due to the higher TOS levels in PD, the factors that increase oxidative stress (including lipid peroxidation products) decrease PON1 enzyme activity. However, in the literature, association studies of PON1 have given different results in different populations [16,25–28]. This could have several explanations. First, although the incidence of PD is geographically rather uniform, the relative importance of different genetic risk factors may vary between different populations. Second, the lack of a given risk factor in a certain cohort may be balanced by a more severe exposure to environmental toxins. In conclusion, our data provides evidence for the presence of oxidative stress in PD patients. Decreased anti-oxidant enzymes, paraoxonase and arylesterase activities with lowered TAS levels against raised oxidative stress may be useful for the clinical diagnosis and/or monitoring of PD. On the other hand, metabolism of lipid, oxidant and anti-oxidant related substances may contribute to the pathogenesis and progression of PD. For an optimal therapeutic strategy for PD, all these causes and their interplay need to be taken into consideration.
Conflict of interest/disclosure The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2013.04.025. References [1] Ikeda K, Nakamura Y, Kiyozuka T, et al. Serological profiles of urata, paraoxonase-1, ferritin and lipid in parkinson’s diseases changes linked to diseases progression. Neurodegener Dis 2011;8:252–8. [2] Miller RL, James-Kracke M, Sun GY, et al. Oxidative and inflammatory pathways in parkinson’s disease. Neurochem Res 2009;34:55–65. [3] Shukla V, Mishra SK, Pant HC. Oxidative Stress in Neurodegeneration. Advances in Pharmacological Sciences 2011;2011:572634. [4] Betarbet R, Sherer TB, Greenamyre JT. Ubiquitin-proteasome system and Parkinson’s diseases. Exp Neurol 2005;191:S17–27. [5] Loh KP, Huang SH, De Silva R, et al. Oxidative stress: apoptosis in neuronal injury. Curr Alzheimer Res 2006;3:327–37. [6] Serafini M, Del Rio D. Understanding the association between dietary antioxidants redox status and disease: is the total antioxidant capacity the right tool? Redox Rep 2004;9:145–52. [7] Barbieri E, Sestili P. Reactive oxygen species in skeletal muscle signaling. J Signal Transduct 2012;2012:982794. [8] Mattson MP. Neuronal life-and-death signaling, apoptosis, and neurodegenerative disorders. Antioxid Redox Signal 2006;8:1997–2006. [9] Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38:1103–11. [10] Erel O. A novel automated method to measure total anti-oxidant response against potent free radical reactions. Clin Biochem 2004;37:112–9. [11] Carmine A, Buervenich S, Sydow O, et al. Further evidence for an association of the Paraoxonase 1 (PON1) Met-54 allele with Parkinson’s disease. Mov Disord 2002;17:764–6. [12] Androutsopoulos VP, Kanavouras K, Tsatsakis AM. Role of paraoxonase 1(PON 1) in organophosphate metabolism: implications in neurodegenerative diseases. Toxicol Appl Pharmacol 2011;256:418–24. [13] She ZG, Chen HZ, Yan Y, et al. The humanparaoxonase gene cluster as a target in the treatment of atherosclerosis. Antioxid Redox Signal 2012;16:597–632. [14] Précourt LP, Amre D, Denis MC, et al. The three-gene paraoxonase family: physiologic roles, actions and regulatıon. Atherosclerosis 2011;214:20–36.
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
A. Kirbas et al. / Journal of Clinical Neuroscience xxx (2013) xxx–xxx [15] Akhmedova SN, Yakimovsky AK, Schwartz EI. Paraoxonase 1 Met-Leu54 polymorphism is associated with Parkinson’s disease. J Neurol Sci 2001;184: 179–82. [16] Wang J, Liu Z. No association between paraoxonase 1 (PON1) gene polymorphisms and susceptibility to Parkinson’s disease in a Chinese population. Mov Disord 2000;15:1265–7. [17] Daniel SE, Lees AJ. Parkinson’s Disease Society Brain Bank, London: overview and research. J Neural Transm. 1993;39(suppl):165–72. [18] Gan KN, Smolen A, Eckerson HV, et al. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab Dispos 1991;19:100–6. [19] Seres I, Paragh G, Deschene E, et al. Study of factors influencing the decreased HDL associated PON1 activity with aging. Exp Gerontol 2004;39:59–66. [20] Taylor MC, Le Couteur DG, Mellick GD, et al. Paraoxonase polymorphisms, pesticide exposure and Parkinson’s disease in a Caucasian population. J Neural Transm 2000;107:979–83. [21] Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 1991;286:152–4.
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[22] Berbecki J, Mitosek-Szewczyk K, Kurzepa J, et al. Processes of free radical lipid peroxidation with a particular regard to the role of paraoxonase -1 in the pathogenesis of multiple sclerosis. Wiad Lek 2011;64:31–6. [23] Hu G, Antikainen R, Jousilahti P, et al. Total cholesterol and the risk of Parkinson disease. Neurology 2008;70:1972–9. [24] De Lau LM, Koudstaal PJ, Hofman A, et al. Serum cholesterol levels and the risk of Parkinson’s disease. Am J Epidemiol 2006;164:998–1002. [25] Manthripragada AD, Costello S, Cockburn MG, et al. Paraoxonase 1, agricultural organophosphate exposure, and Parkinson’s diseases. Epidemiology 2010;21: 87–94. [26] Fong CS, Cheng CW, Wu RM. Pesticides exposure and genetic polymorphisms of paraoxonase in the susceptibility Parkinson’s disease. Acta Neurol Taiwan 2005;14:55–60. [27] Furlong CE, Suzuki SM, Stevens RC, et al. Human PON1, a biomarker of risk of diseases and exposure. Chem Biol Interact 2010;187:355–61. [28] Wingo TS, Rosen A, Cutler DJ, et al. Paraoxonase 1 polymorphisms in Alzheimer’s diseases, Parkinson’s disease, and AD-PD spectrum diseases. Neurobiol Aging 2012;33:204.e13-5.
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease Aynur Kirbas a,⇑, Serkan Kirbas b, Medine Cumhur Cure a, Ahmet Tufekci b a b
_ Department of Biochemistry, Recep Tayyip Erdog˘an University Faculty of Medicine, Islampas ßa mah., Merkez/Rize 53100, Turkey Department of Neurology, Recep Tayyip Erdog˘an University Faculty of Medicine, Rize, Turkey
a r t i c l e
i n f o
Article history: Received 11 February 2012 Accepted 13 April 2013 Available online xxxx Keywords: Arylesterase Oxidative stress index Paraoxonase Parkinson’s disease Total anti-oxidant status Total oxidant status
a b s t r a c t This study investigated serum paraoxonase (PON1) and arylesterase activity along with determination of oxidative status via measurement of total oxidant status (TOS), total anti-oxidant status (TAS) and oxidative stress index (OSI) in patients with Parkinson’s disease (PD) and compared results with data from healthy controls. A total of 82 subjects, including 42 patients with idiopathic PD, newly diagnosed and untreated (24 men, 18 women, aged 47–66 years) and 40 healthy controls were enrolled in this study. We aimed to evaluate the oxidative status of PD patients via measurement of serum TOS and TAS and estimation of OSI using new automated methods. PON1 and arylesterase activities were measured spectrophotometrically. Serum total cholesterol, high density lipoprotein cholesterol, low density lipoprotein (LDL) cholesterol and triglyceride levels were measured using routine methods. TAS levels of PD patients were significantly lower than that of controls (p < 0.05). TOS levels of PD patients were higher than those of controls (p < 0.05). PON1 and arylesterase activities of PD were lower than those of controls (p < 0.05). Serum levels of total and LDL cholesterol were significantly reduced in PD patients. In conclusion, the presence of high TOS and OSI levels together with low levels of TAS in PD patients supports the important role of oxidative stress in the pathophysiology of PD. Since oxidative stress is involved in neurodegeneration, selecting anti-oxidants, metal chelators or other compounds boosting endogenous enzymatic and non-enzymatic defense mechanisms seems to be an obvious choice as treatment for PD. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, tremor, progressive rigidity and postural instability, resulting from progressive degeneration of dopaminergic neurons in the substantia nigra of the midbrain. Both incidence and prevalence increase with age, with a prevalence of 0.3% and an estimated incidence of 8–18 per 100 000 person years [1]. Pathologically PD is characterized by loss of melanin-pigmented nigral neurons accompanied by depletion of dopamine in the striatum and the presence of Lewy bodies, which are deposits of specific cytoplasmic proteins such as ubiquitin, a-synuclein, and oxidized neurofilaments [2]. Similar to other neurodegenerative disorders, oxidative stress, inflammation, mitochondrial dysfunction, environmental factors and genetic predisposition may all be involved in PD [1,2]. Although oxidative stress plays a role in the pathogenesis of neuronal death, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or if it is a secondary manifestation of the neurodegenerative process [3]. ⇑ Corresponding author. Tel.: +90 46 4212 3009; fax: +90 46 4217 0367. E-mail address: [email protected] (A. Kirbas).
Many cellular reactions utilize molecular oxygen for catalysis and energy production [4]. These reactions in turn produce reactive oxygen species (ROS) including superoxide anions, hydrogen peroxide, hydroxyl radicals, peroxy radicals and, in the presence of nitric oxide, reactive nitrogen species (RNS) such as peroxynitrite and nitro-tyrosyl radicals. While these reactive species are important for the execution of physiological functions, excessive production is detrimental to cell membranes and can cause cell death [5]. The rate of production and destruction of ROS is in a state of balance, known as oxidative balance. In cases where this oxidative balance is maintained, ROS may have no impact on the organism but in cases where this balance is tipped in favor of free radicals, oxidative stress develops [6]. The defense system protecting against free radical damage involves enzymatic and non-enzymatic anti-oxidant systems. The enzymatic system includes superoxide dismutase, glutathione peroxidase, catalase, aldehyde dehydrogenases, and sulfiredoxin. The non-enzymatic system includes naturally occurring anti-oxidants such as vitamin A (retinol), vitamin C (ascorbic acid), vitamin E (tocopherol), beta-carotene and glutathione, as well as polyphenol anti-oxidants like flavonoids [7,8]. Under certain conditions, the oxidative or anti-oxidative balance shifts towards oxidative stress as a result of increase in ROS and/or impairment in the anti-oxidant
0967-5868/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2013.04.025
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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A. Kirbas et al. / Journal of Clinical Neuroscience xxx (2013) xxx–xxx
mechanisms [7]. The body’s oxidant/anti-oxidant status can be ascertained by measuring the activity of individual molecules or enzymes; likewise the overall oxidant/anti-oxidant status can be assessed more easily by measuring total anti-oxidant status (TAS) [9] and total oxidant status (TOS) [10]. Environmental factors may influence PD risk [11]. One oftendiscussed risk factor is pesticide exposure [12]. Paraoxonase 1 (PON1), a 43–45 kDa glycoprotein, is synthesized mainly in the liver, which hydrolyzes organophosphates such as pesticides, neurotoxins, and arylesters [11,12]. The paraoxonase (PON) gene family consists of three members – PON1, PON2, and PON3 – located adjacent to each other on the long arm of chromosome 7 in humans (q21.3–q22.1) [13]. These three human PON genes share approximately 60% identity at the amino acid level and approximately 70% identity at the nucleotide level [14]. PON is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated in the anti-oxidant and anti-inflammatory properties exerted by HDL. Many investigations have provided considerable evidence for PON1 anti-atherogenicity [13,14]. Studies have shown that PON1 inhibits oxidation of HDL and low-density lipoproteins (LDL) that preserve HDL function, increases cellular cholesterol efflux from macrophages, ameliorates effects of oxidized LDL, and decreases lipid peroxides in atherosclerotic lesions [13,14]. There are two polymorphisms in the PON1 coding region: leucine/methionine at position 55 (M55L) and glutamine/arginine at position 192 (Q192R). These polymorphisms are associated with a number of pathophysiological conditions, including coronary artery disease, PD, stroke, familial hypercholesterolemia, type 2 diabetes mellitus, late-onset Alzheimer’s disease and reduced bone mass in post-menopausal women [12–14]. The activity of PON1 in patients with PD has been very different to results obtained in studies on populations without PD [11,15,16]. The aim of this study was to investigate the relationship between serum paraoxonase and arylesterase activity, TOS, TAS and oxidative stress index (OSI) in patients with PD. 2. Methods 2.1. Subjects This study was conducted at the Neurology Clinic of Recep Tayyip Erdog˘an University School of Medicine, Turkey. Forty-two newly diagnosed and untreated idiopathic PD patients (24 men, 18 women, mean age 59.3 ± 4.9 years [standard deviation], range 47–66) were included in this study. The PD patients were diagnosed according to the Parkinson’s Disease Society Brain Bank clinical diagnostic criteria for idiopathic PD [17]. Severity ratings for PD between 0 and 5 used the Hoehn and Yahr scale, and all patients were between Hoehn and Yahr stage 1 and 2. The procedures were in accordance with the revised form of the Declaration of Helsinki 2008 and all participants signed an informed consent form. The study protocol was approved by the local Ethical Committee. The control group consisted of 40 healthy individuals (24 men, 16 women, mean age 57.0 ± 4.9 years [standard deviation], range 45–67). All subjects were informed about the study. Body mass index (weight/height2) was obtained through height and weight measurements using a wall-mounted ruler and a digital scale.
fugation at 3000 g for 10 minutes, and then stored at –80 °C until further analysis of paraoxonase and arylesterase activities along with determination of oxidative status via measurement of TOS, TAS and OSI. Serum total cholesterol, HDL cholesterol and triglyceride levels were measured with routine methods (Architect C1600; Abbott Laboratories, Abbott Park, IL, USA). LDL was calculated using the Friedewald formula. 2.3. Measurement of paraoxonase and arylesterase activity Paraoxonase activity was measured in absence (basal activity) and presence of NaCl (salt-stimulated activity) [18]. Briefly, the rate of paraoxon hydrolysis was measured by the increase of absorbance at 412 nm at 25 °C. The amount of generated p-nitrophenol was calculated from the molar absorptivity coefficient at pH 8, which was 17.100 M 1 cm 1. Paraoxonase activity was expressed as U/L serum. Phenylacetate was used as a substrate to measure arylesterase activity. The reaction was initiated by addition of serum and the increase in absorbance was read at 270 nm. Blanks were included to correct spontaneous hydrolysis of phenylacetate. Enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol, 1310 M 1 cm 1. One unit of arylesterase activity was defined as 1 mmol phenol generated/ minute under the above conditions and expressed as U/L serum. Phenotype distribution of paraoxonase was determined in presence of 1 mol/L NaCl (salt-stimulated paraoxonase). The ratio of salt-stimulated paraoxonase activity to arylesterase activity was used to assign individuals to one of the three possible phenotypes [19]. 2.4. Measurement of the TOS Serum TOS was determined using a novel automated measurement method previously described [9]. Oxidants present in the sample oxidize ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. Ferric ion reacts with xylenol orange in an acidic medium to produce a colored complex. The intensity of color, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules in the sample. The assay was calibrated with hydrogen peroxide and results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (lmol H2O2 equiv./L). The assay has excellent precision with error values lower than 2%. 2.5. Measurement of the TAS Serum TAS was determined using an automated measurement method previously described [10]. Briefly, potent free radical reactions were initiated with the production of a hydroxyl radical via the Fenton reaction and the rate of reactions was monitored by following the absorbance of colored dianisidyl radicals. Using this method, the anti-oxidative effect of the sample against potent free radical reactions, which were initiated by synthesized hydroxyl radical, was measured. This method was applied to an automated analyzer (Architect C1600; Abbott Laboratories). Both intra- and inter-assay coefficients of variations were lower than 3%. Data were expressed as lmol equiv./L Trolox (Hoffman-LaRoche, Basel, Switzerland).
2.2. Blood sample collection 2.6. Calculation of the OSI After overnight fasting, peripheral venous blood samples were taken from patients and controls into empty tubes. After coagulation, samples were immediately separated from the cells by centri-
The OSI was calculated by dividing the TOS by the TAS, that is, OSI = (TOS, lmol H2O2 equiv./L)/(TAS, lmol Trolox equiv./L).
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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2.7. Statistical analysis
Table 2 Studied parameters in patients with Parkinson’s disease and healthy controls
All statistical analyses were performed using the Statistical Package for the Social Sciences, version 15.0 (SPSS, Chicago, IL, USA). Data are expressed as mean ± standard deviation. The normality of the distribution for all variables was assessed by the Kolmogorov–Smirnov test. Student’s t-test was used for normally distributed variables and the Mann–Whitney U test was used for non-parametric variables. Relationships between variables were analyzed by Pearson or Spearman correlation analysis according to the distribution type of the variables. p < 0.05 was considered to be statistically significant. 3. Results The demographic and clinical data of the subjects are summarized in Table 1. There were no significant differences in age, sex or body mass index between patients with PD and healthy controls. As compared to controls, serum levels of total cholesterol and LDL cholesterol activity were significantly reduced in PD patients. Serum levels of HDL cholesterol and triglyceride showed no significant differences between PD patients and controls. Paraoxonase and arylesterase activity, OSI, TAS and TOS levels are shown in Table 2. Paraoxonase and arylesterase activity were lower in the PD patients compared to healthy controls and the differences were statistically significant (p = 0.001 and p = 0.001, respectively). Plasma TOS and OSI values were higher in patients with PD than healthy controls and again the differences were statistically significant (p = 0.001 and p = 0.001, respectively). TAS was significantly lower in PD patients (p = 0.001). There was no correlation between serum PON1 activity and OSI in patients with PD (Fig. 1, p = 0.09). There was no significant difference between serum PON1 activity and OSI in male and female PD patients (Supplementary Fig. 1, p = 0.854). 4. Discussion We recruited patients who were in the relatively early stages of disease, were of a young age and had not yet begun treatment. These factors are important for two reasons: first, the oxidant and anti-oxidant balance changes over time and in favor of oxidative damage during disease progression, and it is known that PON1 activity decreases with age [2,19]. Second, we removed the impact of drugs on oxidant and anti-oxidant mechanisms by sampling sera before starting treatment on L-dopa and/or dopamine agonist drugs. One of the most metabolically active organs of the body is the brain. When the spinal cord is included to make up the central
Table 1 Demographic characteristics and results of biochemical analyses in patients with Parkinson’s diseases and healthy controls
Age (years) Male, n Female, n BMI (kg/m2) Total cholesterol (mg/dL) LDL cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mg/dL)
PD (n = 52)
Controls (n = 40)
p value*
59.3 ± 4.9 32 20 24.8 ± 4.3 181.9 ± 31.7 97.7 ± 32.3 58.3 ± 12.2 134.5 ± 52.1
57.0 ± 4.9 24 16 25.3 ± 4.9 202.0 ± 17.5 116.4 ± 17.2 56.5 ± 13.4 128.9 ± 46.6
NS NS NS NS 0.001 0.001 NS NS
Data are presented as mean ± standard deviation, unless otherwise stated. BMI = body mass index, HDL = high-density lipoprotein, LDL = low-density lipoprotein, NS = not significant, PD = Parkinson’s disease. * Student’s t-test.
Paraoxonase (U/L) Arylesterase (U/L) TAS (mmol Trolox equiv./L) TOS (lmolH2O2 equiv./L) OSI (arbitrary unit)
PD (n = 52)
Controls (n = 40)
p value
112.3 ± 48.7 141.5 ± 26.4 1.50 ± 0.13 9.39 ± 2.34 6.28 ± 1.73
200.9 ± 56.5 198.8 ± 24.7 1.66 ± 0.10 8.04 ± 1.46 4.84 ± 0.95
0.001 0.001 0.001 0.001 0.001
Data are presented as mean ± standard deviation. equiv. = equivalent, OSI = oxidative stress index, TAS = total antioxidant status, TOS = total oxidant status.
nervous system it, even at rest, utilizes an estimated 20% of total oxygen uptake [2]. During the active state this percentage substantially increases and it requires an uninterrupted oxygen-rich blood supply to carry out normal physiological actions. Any blockage or deprivation of this oxygen supply even for a few seconds can have severe and irreversible detrimental effect to the cells of brain (both neurons and glia). Consumption of oxygen leads to the production of free radicals, and the brain’s requirement for a higher amount of oxygen leads to even higher numbers of reactive oxygen/nitrogen species [3]. ROS and RNS can attract and damage a variety of critical biological molecules, including lipids, essential cellular proteins and DNA, and may be involved in the pathogenesis of many neurodegenerative disorders including PD [2,3]. Products of lipid peroxidation can be easily and reliably detected in biological fluids and tissues, yielding sensitive and specific signals of lipid peroxidation occurring in vivo. These products are isoprostanes, dimalonealdehyde, 4-hydroxynonenal, and 8-hydroxy-2-deoxy-guanosine (a marker of oxidized DNA) [20]. Several protein kinases including protein kinase C and mitogen activated protein kinases have been implicated in activation of NADPH oxidase and therefore contribute to oxidative stress [2]. These oxidant enzymes and molecules have been studied in vitro and in vivo in patients with PD with reports that oxidative stress generally increased following an increase in the levels of these enzymes and molecules. [1–4]. Reported deficiencies in the major anti-oxidant enzyme systems in the brain (catalase, superoxide dismutase, and glutathione peroxidase), along with a reduction in the levels of glutathione, suggest that oxidative stress in PD is real and contributing to pathogenesis [2,3]. Levels of many oxidant and anti-oxidant parameters present in serum may be measured individually. Since oxidant and anti-oxidant parameters show an additive effect, individual values may not correctly reflect TOS or TAS [9,10]. Therefore, TOS and TAS are more accurate indicators of the oxidative and anti-oxidative status of individuals. The OSI is calculated as the ratio of TOS to TAS. We found the TOS levels, a general indicator of oxidant molecules, were significantly higher in the PD group compared with the control group, and the TAS levels were significantly lower in the PD group than in the control group. The reason for this finding is unknown, but it could be suggested that the endogenous anti-oxidants are reduced through consumption by the increasing oxidants seen in PD patients. Paraoxonase enzymes have attracted growing interest in the last few years due to their wide applications in environmental and genetic toxicology. PON1 is a particularly important anti-oxidant enzyme in the human body [20]. Lipid peroxidation leads to toxic aldehydes that are highly reactive and can be hydrolyzed by PON1 [21]. PON1 therefore constitutes a possible link between environmental toxins and genetic susceptibility to PD [11,22]. This enzyme has a significant role in the defense of LDL and HDL from oxidation by hydrolyzing lipid peroxide products. In our study, we found that total cholesterol and LDL cholesterol activity was significantly lower in PD patients than the control group. However,
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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Fig. 1. Graph showing the non-significant correlation between serum PON1 activity and oxidative stress index in patients with Parkinson’s disease (p = 0.09). AU = arbitrary unit.
a limitation of the present study is that it was not a prospective study and included no diet information for PD patients and controls. The causative mechanism for lowering serum total cholesterol and LDL cholesterol levels in PD patients remains unclear [1]. Previous studies have investigated the complex relationship between serum cholesterol levels and risk of PD. A Finnish cohort study suggested that higher serum levels of total cholesterol increased the risk of developing PD in men and women aged 25– 54 years at baseline [23]. In contrast, the Rotterdam Study reported that higher serum levels of total cholesterol decreased the risk of PD in women [24]. Ikeda et al. reported that serum levels of total cholesterol and LDL cholesterol were inversely correlated with disease progression in female PD patients [1]. It has been reported that the primary effect of PON1 is to protect lipoproteins from oxidation, but it is sensitive to oxidative stress and enzyme activity is inactivated by oxidants [11,13,14]. In our study, we found that PON1 enzyme activity was significantly lower in PD patients than in the control group. On the other hand we concluded that due to the higher TOS levels in PD, the factors that increase oxidative stress (including lipid peroxidation products) decrease PON1 enzyme activity. However, in the literature, association studies of PON1 have given different results in different populations [16,25–28]. This could have several explanations. First, although the incidence of PD is geographically rather uniform, the relative importance of different genetic risk factors may vary between different populations. Second, the lack of a given risk factor in a certain cohort may be balanced by a more severe exposure to environmental toxins. In conclusion, our data provides evidence for the presence of oxidative stress in PD patients. Decreased anti-oxidant enzymes, paraoxonase and arylesterase activities with lowered TAS levels against raised oxidative stress may be useful for the clinical diagnosis and/or monitoring of PD. On the other hand, metabolism of lipid, oxidant and anti-oxidant related substances may contribute to the pathogenesis and progression of PD. For an optimal therapeutic strategy for PD, all these causes and their interplay need to be taken into consideration.
Conflict of interest/disclosure The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2013.04.025. References [1] Ikeda K, Nakamura Y, Kiyozuka T, et al. Serological profiles of urata, paraoxonase-1, ferritin and lipid in parkinson’s diseases changes linked to diseases progression. Neurodegener Dis 2011;8:252–8. [2] Miller RL, James-Kracke M, Sun GY, et al. Oxidative and inflammatory pathways in parkinson’s disease. Neurochem Res 2009;34:55–65. [3] Shukla V, Mishra SK, Pant HC. Oxidative Stress in Neurodegeneration. Advances in Pharmacological Sciences 2011;2011:572634. [4] Betarbet R, Sherer TB, Greenamyre JT. Ubiquitin-proteasome system and Parkinson’s diseases. Exp Neurol 2005;191:S17–27. [5] Loh KP, Huang SH, De Silva R, et al. Oxidative stress: apoptosis in neuronal injury. Curr Alzheimer Res 2006;3:327–37. [6] Serafini M, Del Rio D. Understanding the association between dietary antioxidants redox status and disease: is the total antioxidant capacity the right tool? Redox Rep 2004;9:145–52. [7] Barbieri E, Sestili P. Reactive oxygen species in skeletal muscle signaling. J Signal Transduct 2012;2012:982794. [8] Mattson MP. Neuronal life-and-death signaling, apoptosis, and neurodegenerative disorders. Antioxid Redox Signal 2006;8:1997–2006. [9] Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38:1103–11. [10] Erel O. A novel automated method to measure total anti-oxidant response against potent free radical reactions. Clin Biochem 2004;37:112–9. [11] Carmine A, Buervenich S, Sydow O, et al. Further evidence for an association of the Paraoxonase 1 (PON1) Met-54 allele with Parkinson’s disease. Mov Disord 2002;17:764–6. [12] Androutsopoulos VP, Kanavouras K, Tsatsakis AM. Role of paraoxonase 1(PON 1) in organophosphate metabolism: implications in neurodegenerative diseases. Toxicol Appl Pharmacol 2011;256:418–24. [13] She ZG, Chen HZ, Yan Y, et al. The humanparaoxonase gene cluster as a target in the treatment of atherosclerosis. Antioxid Redox Signal 2012;16:597–632. [14] Précourt LP, Amre D, Denis MC, et al. The three-gene paraoxonase family: physiologic roles, actions and regulatıon. Atherosclerosis 2011;214:20–36.
Please cite this article in press as: Kirbas A et al. Paraoxonase and arylesterase activity and total oxidative/anti-oxidative status in patients with idiopathic Parkinson’s disease. J Clin Neurosci (2013), http://dx.doi.org/10.1016/j.jocn.2013.04.025
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