impaired paraoxonase-1 status in obese children. relationships with insulin resistance and metabolic...

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Clinical Biochemistry xxx (2013) xxx–xxx

CLB-08487; No. of pages: 7; 4C:

Contents lists available at ScienceDirect

Clinical Biochemistry

j ourna l homepage: www.e lsev ie r .com/ locate /c l inb iochem

Impaired paraoxonase-1 status in obese children. Relationships withinsulin resistance and metabolic syndrome

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OFNatàlia Ferré a,1, Albert Feliu a,b,1, Anabel García-Heredia c, Judit Marsillach d,e, Neus París a,b,

Marta Zaragoza-Jordana a, Bharti Mackness c, Michael Mackness c, Joaquín Escribano a,b,Ricardo Closa-Monasterolo a, Jorge Joven c, Jordi Camps c,⁎a Unitat de Pediatria, Institut d’Investigació Sanitària Pere Virgili, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spainb Servei de Pediatria, Hospital Universitari de Sant Joan, Reus, Spainc Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, Institut d’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Reus, Spaind Department of Medicine, University of Washington, Seattle, WA, USAe Department of Genome Sciences, University of Washington, Seattle, WA, USA

⁎ Corresponding author at: Unitat de Recerca BiomèdicJoan, C. Sant Joan s/n, 43201 Reus, Catalonia, Spain.

E-mail address: [email protected] (J. Camps).1 These authors contributed equally to the study.

0009-9120/$ – see front matter © 2013 The Canadian Sochttp://dx.doi.org/10.1016/j.clinbiochem.2013.08.020

Please cite this article as: Ferré N, et al, Impsyndrome, Clin Biochem (2013), http://dx.d

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TEDReceived 31 May 2013

Received in revised form 9 August 2013Accepted 30 August 2013Available online xxxx

Keywords:Insulin resistanceMetabolic syndromeObesityParaoxonase-1

Objectives: To investigate the relationships between serum paraoxonase-1 (PON1), insulin resistance, andmetabolic syndrome (MetS) in childhood obesity.

Design and methods: We studied 110 obese children and 36 non-obese children with a similar gender andage distribution. We measured serum PON1 activity against 5-thiobutyl butyrolactone (TBBLase) and againstparaoxon (paraoxonase). PON1 concentration was measured separately as were the levels of several standardmetabolic variables. The homeostasis model assessment (HOMA) index was calculated as an estimate of insulinresistance.

Results: TBBLase was significantly decreased in obese children (P = 0.008), while paraoxonase activity andPON1 concentrations showednon-significant trends towards decrease and increase, respectively (P = 0.054 andP = 0.060). TBBLase and paraoxonase specific activities were significantly decreased (P = 0.004 and P = 0.018,

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RRECrespectively). TBBLase specific activity was inversely associated with BMI, percentage body fat, insulin, HOMA,
triglycerides, and C-reactive protein, and directly associated with HDL-cholesterol. Paraoxonase specific activityshowed similar associations with BMI, percentage fat, HDL-cholesterol, and C-reactive protein. Obese childrenwith MetS had lower TBBLase activities than obese children without MetS (P = 0.018). Linear regression analy-ses showed that TBBLase was independently associated with HDL-cholesterol, BMI, percentage body fat andPON155 polymorphism, but paraoxonase activity was associated only with PON1192 polymorphism.

Conclusions:Our results suggest that PON1may play a role in the onset and development ofmetabolic alter-ations in childhood obesity leading to diabetes and cardiovascular disease later in life. However, being derivedfrom statistical association study, this finding cannot be seen as showing cause–effect.

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UNCIntroduction

Obesity in adults and children has become one of the most seriouspublic health problems worldwide, but particularly in developed coun-tries. Its prevalence has dramatically increased in the past few decades,and has reached epidemic proportions recently [1–3]. Spain has one ofthe highest childhood obesity rates in Europe, with about 15% of the ad-olescent population being affected [4,5]. The disease is often associatedwith the metabolic syndrome (MetS), and both have been demonstrat-ed to have a strong impact on cardiovascular mortality and morbidityrates [6]. Oxidative stress has been suggested as resulting from obesity

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aired paraoxonase-1 status inoi.org/10.1016/j.clinbiochem.2

and MetS, while cellular and tissue damage associated with peroxida-tion of lipids, proteins and nucleic acids contribute to the developmentof diabetesmellitus, hypertension, atherosclerosis, dyslipidemia, cancer,and coronary heart disease [7]. Therefore, investigation into theputativeprotective role played by endogenous antioxidants against the develop-ment of obesity and MetS in children is of considerable clinical interest.

Paraoxonase-1 (PON1) is an enzyme found in the circulation associ-ated with high-density lipoproteins (HDL) [8,9]. The native functionattributed to PON1 is that of a lactonase, and lipophilic lactones consti-tute its primary substrates [10]. In addition, PON1 has an esteraseactivity and degrades organophosphate xenobiotics such as paraoxon(paraoxonase activity), phenylacetate (arylesterase activity) and nerveagents [9]. PON1 also hydrolyzes oxidized phospholipids and, as such,plays a role in an organism's antioxidant system [8]. Several studieshave demonstrated that PON1 possesses anti-atherogenic and anti-inflammatory properties [9]. PON1 levels are genetically determined,

y Elsevier Inc. All rights reserved.

obese children. Relationships with insulin resistance and metabolic013.08.020

http://dx.doi.org/10.1016/j.clinbiochem.2013.08.020

mailto:[email protected]


http://www.sciencedirect.com/science/journal/02637296


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2 N. Ferré et al. / Clinical Biochemistry xxx (2013) xxx–xxx

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and several polymorphisms in the coding and promoter regions ofthe PON1 gene (including PON1192, PON155, PON1−162, PON1−832,PON1−909, PON1−1076, and PON1−1741) have been associated withchanges in the enzyme's activity and/or concentration [9].

Despite these potentially important pointers, there is a dearth of in-formation on the possible alterations of PON1 activity and their meta-bolic consequences in obese children. Our study sought to investigatethe relationships between serum PON1, insulin resistance, MetS, andbiochemical markers of inflammation in a group of obese children andadolescents. As a distinguishing feature, we measured PON1 proteinconcentrations and serum PON1 activity against a synthetic lactonewhich mimics native enzyme activity.

Participants and methods

Participants

The study was performed with 110 obese children and adolescents(48male, 62 female)with amean age of 12 years (range: 9–15) attend-ing the Child Endocrinology Unit of Hospital Universitari de Sant Joan. Allparticipants had a body mass index (BMI) above the 97% percentileof the corresponding age and gender of the Spanish population [11]. Ex-clusion criteria were: an active infection, having been vaccinatedwithinthe two weeks prior to blood extraction for the current study, havingbeen diagnosed with any chronic inflammatory disease, diabetesmellitus, immunodepression, congenital heart disease, or an alteredthyroid function. As a control group, we recruited 36 lean, sports practi-tioners (boys: n = 15; girls: n = 21) with a mean age of 12 years(range: 9–15) attending the Sports Medicine Unit of our Hospital.There were no statistically significant differences between groupswith respect to pubertal status, estimated by the Tanner scale [12](Supplementary Table 1).

Fasting blood samples were obtained from all the participants and,following centrifugation, the serum, EDTA-plasma, and leukocyteswere stored in aliquots at −80 °C. All participants and their parentsprovided fully-informed consent to participation in the study on the un-derstanding that anonymity of all data was guaranteed. The study wasapproved by the Ethics Committee (Institutional Review Board) of theHospital Universitari de Sant Joan.

Anthropometric and biochemical measurements

Weight, height and blood pressures (systolic and diastolic) weremeasured in all participants using standard methods. BMI was calculat-ed as weight (kg)/height (m)2. Percentage body fat was estimated bythemethod of Siri [13]. MetSwas diagnosed in obese children accordingto the criteria and definitions of the National Heart, Lung, and Blood In-stitute and the American Heart Association [14,15] according to which,the waist circumference needed to be ≥90% percentile for the corre-sponding age and gender, and at least two among the following criterianeeded to be fulfilled: (1) systolic pressure ≥130 mm Hg or diastolicpressure ≥85 mm Hg; (2) serum triglycerides ≥1.7 mmol/L; (3) HDL-cholesterol ≤1.03 mmol/L; (4) serum glucose ≥5.6 mmol/L.

Serum PON1 lactonase activity was measured as the hydrolysis of5-thiobutyl butyrolactone (TBBL) as previously described [16,17].TBBLase activity was measured in an assay reagent containing 1mMCaCl2, 0.25 mM TBBL and 0.5 mM 5,5′-dithio-bis-2-nitrobenzoic acid(DTNB) in 0.05 mMTris–HCl buffer, pH = 8.0. The change in absorbancewas monitored at 412 nm. Activities were expressed as U/L (1 U =1 mmol of TBBL hydrolyzed per minute). Serum PON1 paraoxonase ac-tivity was determined as the rate of hydrolysis of paraoxon at 410 nmand 37 °C in a 0.05 mM glycine buffer, pH 10.5 with 1 mM CaCl2 [18].Activities were expressed as U/L (1 U = 1 μmol of paraoxon hydrolyzedper minute). Serum PON1 concentrations were determined by in-houseELISA with rabbit polyclonal antibodies generated against the syntheticpeptide CRNHQSSYQTRLNALREVQ which is a sequence specific for

Please cite this article as: Ferré N, et al, Impaired paraoxonase-1 status insyndrome, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2

mature PON1 [19,20]. PON1 specific activities were calculated as the ra-tios between the activity and the corresponding concentration, andwereexpressed as U/mg.

Plasma concentrations of adiponectin were determined withFlowCytomix™ reagents (eBioscience®, Affymetrix, San Diego, CA,USA) in a Coulter® EpicsXL-MLC™flowcytometer (Beckman-Coulter®,Fullerton, CA, USA). Serum concentrations of insulin, thyrotropin, high-sensitivity C-reactive protein, glucose, cholesterol, triglycerides, HDL-cholesterol, aminotransferases, lactate dehydrogenase, bilirubin, andcreatinine concentrations were measured in an automated analyzer(UniCel™ DxI 800, Beckman Coulter®). LDL-cholesterol concentrationswere estimated by the Friedewald formula [21]. The homeostasis modelassessment (HOMA) index was calculated as an estimate of insulin re-sistance, as previously reported [22].

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PON1 genotyping

Genomic DNA was obtained from leukocytes (Puregene DNA Isola-tion reagent set, Gentra Systems Inc., Minneapolis, MN). PON1192,PON155, PON1−162, PON1−832, PON1−909, PON1−1076, and PON1−1741,single nucleotide polymorphisms were analyzed by the Iplex GoldMassArray™ method (Sequenom Inc., San Diego, CA) at the SpanishNational Genotyping Center (Centro Nacional de Genotipado, of theUniversitat Pompeu Fabra, Barcelona, Spain).

ED PStatistical analyses

All calculations were performed with the SPSS 18.0 statistical pack-age (SPSS Inc., Chicago, IL, USA). Normality of distributions was deter-mined with the Kolmogorov–Smirnov test. Homogeneity of varianceswas examined by the Levene test. Differences between two groupswere assessed with the Student's t-test (parametric) or the Mann–Whitney U test (non-parametric). Results are shown as medians and95%CI. Spearman correlation coefficientwas used to evaluate the degreeof association between variables. A linear regression analysis was fittedto evaluate the variables thatwere independently associatedwith PON1activities (dependent variables were log10-transformed since they werenon-normally distributed). Differences in genotype frequencies be-tween obese and non-obese children were assessed by the χ2 test. Toevaluate haplotype blocks in genetic analyses, linkage disequilibrium(LD) between loci expressing genetic variation and pairwise measure-ments (D′ and r2) were calculated using the Haploview 4.0 softwarepackage [23]. A value of P ≤ 0.05was considered statistically significant.

Results

PON1-related variables and anthropometric and biochemical indices inobese and non-obese children

Results on the anthropometric and biochemical variables in obeseand non-obese children are summarized in Table 1. As expected,obese children had a significant increase in BMI and percentagebody fat. They also had higher systolic and diastolic arterial pressures,as well as lower HDL-cholesterol and higher serum triglyceride concen-trations. Serum insulin, the HOMA index, and C-reactive protein weresignificantly increased in obese children. Plasma adiponectin concentra-tions were significantly decreased. Serum PON1 TBBLase activity wassignificantly decreased, and paraoxonase activity and PON1 concentra-tions showed non-significant trends to decrease and increase, respec-tively. When we calculated the PON1 specific activities (i.e. theenzyme activities per milligram of PON1 protein), we observed signifi-cant decreases in both TBBLase and paraoxonase activities. There wereno statistically significant differences in PON1-related variables be-tween boys and girls (Supplementary Table 2).



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Table 1t1:1

t1:2 Anthropometric and biochemical variables in non-obese and obese children.

t1:3 Variable Non-obese (n = 36) Obese (n = 110) P

t1:4 Age; years 11.6 (8.8–14.7) 12.2 (9.1–14.8) 0.288t1:5 Gender; male/female 15/21 48/62 0.831t1:6 BMI; kg/m2 18.4 (14.2–23.3) 29.2 (24.9–37.6) b0.001t1:7 Body fat; % 21.5 (13.4–31.3) 37.5 (33.2–40.5) b0.001t1:8 Systolic arterial pressure;

mm Hg95 (80–120) 113 (95–138) b0.001

t1:9 Diastolic arterial pressure;mm Hg

55 (40–70) 70 (50–87) b0.001

t1:10 Cholesterol; mmol/L 4.10 (3.30–5.84) 4.00 (3.00–5.16) 0.530t1:11 HDL-cholesterol; mmol/L 1.28 (0.87–2.00) 1.02 (0.73–1.47) b0.001t1:12 LDL-cholesterol; mmol/L 2.60 (1.98–3.92) 2.67 (1.80–3.52) 0.494t1:13 Triglycerides; mmol/L 0.50 (0.30–0.73) 0.70 (0.30–1.80) b0.001t1:14 Glucose; mmol/L 5.2 (4.6–5.7) 5.1 (4.6–5.8) 0.831t1:15 Insulin; pmol/L 43.2 (18.3–90.2) 91.4 (38.8–238.1) b0.001t1:16 HOMA index 1.41 (0.53–3.11) 2.86 (1.15–7.37) b0.001t1:17 C-reactive protein; mg/L 0.20 (0.20–2.47) 2.93 (0.26–14.06) b0.001t1:18 Alanine aminotransferase;

μkat/L0.31 (0.21–1.35) 0.34 (0.23–0.63) 0.085

t1:19 Aspartate aminotransferase;μkat/L

0.39 (0.31–0.65) 0.37 (0.25–0.51) 0.120

t1:20 Lactate dehydrogenase; μkat/L 2.95 (2.20–4.07) 2.90 (2.04–3.86) 0.825t1:21 Bilirubin; μmol/L 11.0 (6.2–18.0) 12.1 (7.2–26.0) 0.022t1:22 Creatinine; μmol/L 55.0 (44.6–72.3) 53.0 (41.5–71.5) 0.269t1:23 Thyrotropin; mIU/L 2.27 (0.91–5.89) 2.00 (0.95–4.29) 0.496t1:24 Adiponectin; mg/L 2.51 (0.36–9.14) 1.78 (0.40–4.32) 0.014t1:25 TBBLase activity; U/L 7.3 (3.4–12.2) 6.4 (3.6–10.8) 0.008t1:26 Paraoxonase activity; U/L 357.2 (190.0–551.3) 294.0 (185.9–525.5) 0.054t1:27 PON1 concentration; mg/L 32.2 (17.8–69.8) 38.7 (22.9–66.6) 0.060t1:28 TBBLase specific activity; U/mg 0.22 (0.09–0.44) 0.17 (0.07–0.33) 0.004t1:29 Paraoxonase specific activity;

U/mg10.68 (3.75–24.55) 7.89 (3.78–16.71) 0.018

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Relationships between PON1-related variables, metabolic alterations andMetS

We observed significant associations between PON1-related vari-ables and the anthropometric and biochemical measurements; themost significant among them referring to the TBBLase specific activity(Fig. 1). This parameter was inversely associated with BMI, the percent-age of fat, insulin, HOMA index, triglycerides, and C-reactive protein,and directlywithHDL-cholesterol. Paraoxonase specific activity showedsimilar associations with BMI, percentage fat, and HDL-cholesterol, butnot with any of the other parameters analyzed (Fig. 2).

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Fig. 1. Relationships between serum TBBLase specific activity and anthropometric and


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In a further analysis, the obese children were segregated with re-spect presence or absence of MetS. We found that obese children withMetS had lower TBBLase activities and TBBLase specific activities thanobese children without MetS. However, we did not observe any signifi-cant differences in paraoxonase activities or concentrations betweenthe two groups (Fig. 3).

PON1 genotyping

We did not observe any significant differences between obese andnon-obese children with respect to genotype frequencies of the ana-lyzed PON1 gene polymorphisms (Table 2). We also evaluated PON1gene haplotypes with the aim of including them in the multivariateanalysis of factors influencing serum PON1 activities. Results showedthat polymorphisms PON1−1741 and PON1192 segregated independently,while the other genotypes were transmitted as a single block (Supple-mentary Table 3). Again, we did not observe any statistically significantdifferences between obese and non-obese children.

Multiple linear regression analysis

We then performed multiple linear regression analyses to investi-gate which of the metabolic and genetic variables (which had shownsignificant associations on univariate analysis) were significantly andindependently associated with TBBLase or paraoxonase specific activi-ties. TBBLase activity was significantly and independently related toHDL-cholesterol concentrations, BMI, percentage body fat and PON155polymorphism, while paraoxonase specific activity was significantly as-sociatedwith PON1192 polymorphism; almost statistically significant as-sociations were observed between HDL-cholesterol and PON1−1741

polymorphism (Table 3).

Discussion

Obesity-related diseases are a socio-economic strain on society, andare a significant cause of death [24]. As well as an increase in adult obe-sity, evidence shows that a high, progressively increasing, percentage ofchildren are becoming obese, especially in developed countries [1–3].However, although the influence of obesity on diabetes, cardiovasculardisease, and cancer is well established in adults, its effects in childrenare less well understood. Nevertheless, a growing body of evidence sug-gests a similar association [25]. Childhood obesity has been linked to an

biochemical variables in obese (gray dots) and non-obese children (white dots).



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Fig. 2. Relationships between serumparaoxonase specific activity and anthropometric andbiochemical variables in obese (gray dots) and non-obese children (white dots).


increase in fasting serum insulin concentration [26]. The present studyconcurs with these data i.e. 47 of the 110 obese children (42.7%) had aserum fasting insulin concentration above the upper limit of the non-


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obese children (N94 pmol/L), and 42 children (38.2%) had a HOMAindex above the upper limit of the non-obese children (N3). One ofour main findings is that a decreased serum PON1 activity is related tohyperinsulinemia and insulin resistance in obese children and adoles-cents, and suggests that this endogenous antioxidant may be involvedin themetabolic alterations leading to the future development of diabe-tes mellitus.

We also observed significant associations between impaired serumPON1 activity and higher triglyceride and lower HDL-cholesterol con-centrations, indicating a possible underlying mechanism associatingthis enzyme with an increased risk of cardiovascular disease. Indeed,raised fasting insulin concentration has been linked to a two-fold in-crease in the future incidence of type 2 diabetes mellitus [27], whileraised serum triglycerides increase the incidence of cardiovascular dis-ease around four-fold [28].

The present study showed an association between decreased serumPON1 activity andMetS in childhood obesity. Our results confirm previ-ous reports in children [29], and in adults with MetS [30,31]. MetS ischaracterized by several metabolic abnormalities that lead to an in-creased risk of cardiovascular disease. The hypothesis is that MetSpresents when an excess of body fat accumulates in subjects with aspecific metabolic susceptibility, most likely insulin resistance [32].MetS is known to be associated with a pro-oxidant and pro-inflammatory status, as well. Further, oxidative stress is considered toplay a pivotal role in MetS pathophysiology. The magnitude and direc-tion of the associations between low PON1 activity and metabolic ab-normalities associated with MetS are consistent with a progressiveworsening of the antioxidant/oxidant balance. Free radicals occur dis-proportionately in metabolic abnormalities such as chronic hyperglyce-mia and dyslipidemia. In addition, pancreatic β-cells exposed tohyperglycemia produce free radicals that can suppress glucose-induced insulin secretion [33].

A caveat of the present study is the low coefficient of determinationof the multiple regression analyses for the factors influencing serumPON1 activities. This suggests that factors other than those measuredalso play important roles. A possible explanation for serum PON1 activ-ity being decreased in obese children is that PON1 is inactivated byoxidized lipids, as has been shown by Aviram et al. [34] who demon-strated that the incubation of PON1 in vitro with oxidized palmitoylarachidonoyl phosphatidylcholine, lysophosphatidylcholine, oxidizedcholesteryl arachidonate and oxidized LDL, resulted in inactivation ofPON1 arylesterase activity. Indeed, obesity is associated with increasedoxidative stress in adults and children [35,36]. These data support thehypothesis of a direct inhibition of the PON1 enzyme active site bylipid peroxides. However, an alternative possibility (and one whichdoes not exclude an inhibition by lipid peroxidation products) is thatchanges in HDL structure and composition influence PON1 activity.This would be feasible since it is well documented that PON1 activityis closely dependent on the lipid and protein compositional environ-ment of the HDL particles [37]. Our finding of a decreased HDL-cholesterol concentration together with an unmodified PON1 proteinconcentration in obese children supports this hypothesis.

The molecular mechanisms underlying the protective effects ofPON1 in insulin resistance and MetS have not been investigated inhumans. However, recent studies in mice and in cultured cells showedthat PON1 attenuates diabetes development and stimulates β-cell re-lease [38], and that oxidative stress decreases [39], and PON1 increases[40] glucose transport 4 (GLUT4) expression in plasma membranes.This, in turn, increases glucose uptake following insulin stimulation.Also, PON1 decreases the activity of p38 mitogen-activated kinases(p38MAPK), a component of the stress response that contributes todesensitizing insulin signaling [40]. These results suggest that lowPON1 levels in MetS and insulin resistance can be a causal factor in dia-betes development and, as well, its measurement can be a usefuldiagnostic tool for diabetes predisposition in subjects with insulin resis-tance and MetS.



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Fig. 3. TBBLase and paraoxonase activities in relation to metabolic syndrome (MetS) in obese children.

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Table 3 t3:1

t3:2Linear regression analyses of the variables independently associated with PON1 TBBLaset3:3and paraoxonase specific activities in obese and non-obese children.

t3:4Log10 TBBLase specific activity; U/mga B 95% CI of B P

t3:5Constant −0.761 −1.124 to−0.399 b0.001t3:6HDL-cholesterol; mmol/L 0.210 0.054–0.365 0.009


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There is a paucity of data on PON1 status alterations in obesity. Fur-ther, inter-laboratory comparisons are inconclusive due, probably, totwo factors. Firstly, most studies havemeasured PON1 esterase activity,which is not commonly accepted as the native activity of PON1; otherinvestigators have measured PON1 as its hydrolytic activity towardsparaoxon (paraoxonase activity), while yet others have measured theactivity against phenylacetate (arylesterase activity). Secondly, moststudies have not taken into account the possible influence of geneticpolymorphisms on the enzyme's activity. This is a major confoundingfactor since, in studieswith a low number of participants, it is very likelythat different distributions of genotype frequencies between cases andcontrols may result from chance alone [41]. Data on PON1 levels in obe-sity were first provided by a report from Ferreti et al. [42] in which de-creased PON1 paraoxonase activities and increased lipid peroxidationwere observed in isolated HDL from adult obese women. Since then,several studies have consistently described decreased serum PON1arylesterase activity in obese adults [43,44] and children [45–47].However, reports measuring PON1 paraoxonase activity are not soconsistent. A decrease in paraoxonase activity was described by someauthors [48–50], while others did not observe any significant changes[7,51–53]. The present study is novel in that we measured thethiolactonase PON1 activity by the hydrolysis of TBBL, an enzymatic

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Table 2Genotype frequencies (%) of the analyzed PON1 gene polymorphisms in obese and non-obese children. Differences were assessed by the χ2 test.

Non-obese Obese Non-obese Obese Non-obese Obese P

PON1−1741 CC CT TT43.9 44.4 42.8 37.1 13.3 18.5 0.733

PON1−1076 GG AG AA4.2 3.8 34.4 29.6 61.4 66.6 0.232

PON1−909 CC CG GG16.5 18.5 40.2 37.0 43.2 48.1 0.976

PON1−832 CC CT TT54.1 57.1 41.8 35.7 4.0 7.4 0.524

PON1−162 GG GA AA62.2 67.9 33.7 28.6 4.1 3.7 0.220

PON155 LL LM MM33.7 33.3 48.9 48.1 17.3 22.2 0.462

PON1192 QQ QR RR47.9 42.9 45.9 46.4 6.1 10.7 0.393


ED Pactivity that has been reported to resemble the native physiological ac-

tivity of PON1 [16]. When using this substrate, we observed decreasedserum PON1 activity (expressed both as total and as specific activity)in obese children and adolescents, and both univariate and multipleregression analysis showed significant associations with metabolicalterations related to MetS. However, a slightly different picture isobserved when paraoxon was used as the enzyme's substrate. Totalparaoxonase activities were only marginally lower than those of non-obese children, and only the specific activities showed a clear, statisti-cally significant, decrease. Although univariate analysis showed thatparaoxonase specific activity was related to BMI, percentage fat, andHDL-cholesterol concentrations, multiple regression analysis showed aclear association only with PON1192 polymorphism. These results, andthose of other investigators, suggest that the measurements of serum

t3:7Triglycerides; mmol/L 0.017 −0.075–0.110 0.713t3:8C-reactive protein; mg/L −0.001 −0.007–0.005 0.369t3:9Insulin; pmol/L 0.000 −0.001–0.001 0.736t3:10BMI; kg/m2 −0.013 −0.026 to−0.001 0.040t3:11Body fat; % 0.005 −0.005 to−0.014 0.014t3:12PON1−1741 0.023 −0.041–0.088 0.476t3:13PON155 −0.071 −0.137 to−0.004 0.037t3:14PON1192 0.028 −0.039–0.095 0.408t3:15PON1 haplotype −0.040 −0.120–0.039 0.317t3:16Log10 paraoxonase specific activity; U/mgb

t3:17Constant 0.826 0.531–1.121 b0.001t3:18HDL-cholesterol; mmol/L 0.121 −0.004–0.246 0.058t3:19BMI; kg/m2 −0.006 −0.017–0.004 0.210t3:20Body fat; % 0.001 −0.007–0.009 0.207t3:21PON1−1741 0.054 0.000–0.108 0.051t3:22PON155 −0.024 −0.080–0.031 0.386t3:23PON1192 0.190 0.133–0.247 b0.001t3:24PON1 haplotype −0.022 −0.089–0.045 0.512

t3:25TBBlase and paraoxonase specific activities were log10 transformed to normalize thet3:26distributions for statistical analyses.

a Model summary: r2 = 0.273; P b 0.001. t3:27b Model summary: r2 = 0.487; P b 0.001. t3:28



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TBBLase or arylesterase activities may bemore appropriate than the de-termination of the enzyme's paraoxonase activity, when investigatingassociations between PON1, obesity and MetS.

We did not observe any significant differences in PON1 polymor-phisms between obese and non-obese children. The number of casesanalyzed is too low for a reliable gene-association study. However, webelieve it is important to mention these data so as to address the possi-bility that the observed differences in enzyme activities could be due tothe disease per se, or whether the differences result from the distribu-tions of allelic frequencies between cases and controls. Our resultsshow that genotype differences do not explain the observed differencesin PON1 activities between obese and non-obese children. Further, thegenotypic frequencies were similar in both groups of children to thosewe had previously reported for the healthy adult Mediterranean popu-lation [17]. Nevertheless, it needs to be highlighted that recent studiesobserved significant associations between PON1 gene polymorphismsand obesity in children [54,55].

The relationship between serum PON1 activity and adipokines inobese children has been investigated recently. Koncsos et al. [45–47]found decreased PON1 paraoxonase and arylesterase activities, lowerleptin and higher adiponectin concentrations, and a positive correlationbetween adiponectin and arylesterase, but not with paraoxonase, inobese children. The authors interpreted their data with the suggestionthat the production of the PON1 protein (which is indirectly estimatedby arylesterase activity) might be affected by adiponectin. Our studycannot confirm this hypothesis since we did not observe any significantassociation between adiponectin levels and either PON1 concentrationor TBBLase activity (which is considered an estimate of the nativePON1 lactonase activity). Overall, the results suggest that the relation-ship between PON1 and adipokines is substrate-dependent. Previousstudies had noted that the associations between serum PON1 activityand other biochemical markers may be divergent, depending on thesubstrate employed for PON1 activity measurement [56]. The diver-gence highlights the need for more caution in interpreting the findingsuntil more specific methods using physiologically-akin substrates aredeveloped for PON1 measurement.

In summary, we observed a significant decrease in serum PON1TBBLase and paraoxonase specific activities in obese children and ado-lescents, and significant associations between these parameters andmetabolic markers of insulin resistance and MetS. These associationswere stronger with TBBLase activity, which mimics the native activityof this enzyme. Our results suggest that PON1 may play a role in theonset and development of metabolic alterations in childhood obesityleading to diabetes and cardiovascular disease later in life. However,being derived from a statistical association study, this finding cannotbe seen as showing cause–effect.
O
465466467468469470471472

NCAcknowledgements

This studywas supported by grants from the Instituto de Salud CarlosIII (PI 08/1381, 08/1032, 10/0082, 11/2187), Madrid, Spain. Editorialassistance was by Dr. Peter R. Turner of Tscimed.com.

473474475476477478479480

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Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clinbiochem.2013.08.020.

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