Clinical Biochemistry 43 (2010) 1230–1235

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Clinical Biochemistry

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HDL 2 Particles are associated with hyperglycaemia, lower PON1 activity andoxidative stress in type 2 diabetes mellitus patients

Aleksandra Stefanović ⁎, Jelena Kotur-Stevuljević, Slavica Spasić, Jelena Vekić, Aleksandra Zeljković,Vesna Spasojević-Kalimanovska, Zorana Jelić-IvanovićInstitute for Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

⁎ Corresponding author. Institute for Medical BiochVojvode Stepe 450, P. Box 146, 11000 Belgrade, Serbia.

E-mail address: alex@pharmacy.bg.ac.rs (A. Stefanov

0009-9120/$ – see front matter © 2010 The Canadiandoi:10.1016/j.clinbiochem.2010.08.005

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 26 February 2010Received in revised form 21 April 2010Accepted 5 August 2010Available online 13 August 2010

Keywords:Diabetes mellitus type 2Oxidative stressParaoxonaseHDL 2 particles

Objectives: In this study we examined the relationship of oxidative stress and hyperglycaemia toantioxidative capacity of high-density cholesterol (HDL-C) particles in type 2 diabetes mellitus (DM).

Design and methods: Oxidative stress status parameters (superoxide anion (O2−), superoxide dismutase

(SOD)activity andparaoxonase (PON1) statuswereassessed in114patientswith type2DMand91healthy subjects.HDL particle diameters were determined by non-denaturing polyacrylamide gradient (3–31%) gel electrophoresis.

Results: Patients had significantly higher concentrations of oxidative stress parameter O2−(pb0.001) and

antioxidative defence, SOD activity (pb0.001). Paraoxonase activity was significantly lower in diabetics (pb0.001).The PON1192 phenotype distribution among study groups was not significantly different. HDL 3 phenotype wassignificantly prevalent among patients (pb0.001). Paraoxonase activity was significantly lower in patients withpredominantly HDL 2 particles than in controls.

Conclusions: The results of our current study indicate that the diabetic HDL 2 phenotype is associated withhyperglycaemia, lower PON1 activity and elevated oxidative stress.

© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Type 2 diabetics often have increased risk of cardiovascular disease(CVD) development [1]. This risk in part is due to dislipidemia, whichis generally characterised by high plasma triglyceride (TG) concen-trations, reduced high-density lipoprotein (HDL)-cholesterol(HDL-C), and high or relatively normal low-density lipoprotein(LDL)-cholesterol (LDL-C) concentrations [2]. In addition, oxidativestress plays a significant role in the pathogenesis of atherosclerosis-related conditions including CVD, stroke and diabetes mellitus (DM)[3]. Oxidatively modified lipoproteins, especially LDLs, are formed viareactive oxygen species (ROS) activity and are implicated in processesof atherogenesis [4].

A number of studies suggest that HDL particles exert part of itsanti-atherogenic effect by counteracting LDL particles oxidation [5,6].This process is usually attributed to the high content of antioxidants inthis lipoprotein, but also to antioxidative properties of apolipoproteinA-I (Apo A-I) and especially to the presence of several antioxidativeenyzmes such as paraoxonase-1 (PON1), platelet activating factoracetylhydrolase (PAF-AH) and glutathione peroxidase (GPX) [6].Plasma HDL particles are highly heterogeneous in their chemicalcomposition, intravascular metabolism and biological function [7].

emistry, Faculty of Pharmacy,Fax: +381 11 39 72 840.ić).

Society of Clinical Chemists. Publish

Small, dense HDL 3 particles display a higher capacity to acceptcholesterol and to protect LDL particles from oxidative modificationsin comparison with large, light, lipid-rich HDL 2 particles in healthy,normolipidemic subjects [8]. In contrast, some reports suggest thatHDL 2 particles are significantly more efficient than HDL 3 ininhibiting LDL oxidation [9]. HDL particles from diabetics are knownto be compositionally abnormal, most likely due to the fact that theyare susceptible to structural modifications mediated by mechanismswhich are characteristic of DM including dyslipidemia, glucosehomeostasis and redox balance [10]. Structural alterations withinHDL particles may affect their functional and atheroprotectiveproperties. The ability of compositionally abnormal HDL particlespresent in individuals with type 2 DM to counteract oxidativemodifications within LDL particles remains incompletely understood.

The aim of this current study was to evaluate the relationships ofoxidative stress and hyperglycaemia with LDL and HDL particle sizesand subclass distributions in type 2 DM patients. In addition, theeffects of different HDL phenotypes on PON1 activity in patients werealso explored.

Materials and methods

Subjects

We recruited 205 individuals who regularly attended annualmedical check-ups at two health centres in Belgrade. Control and

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1231A. Stefanović et al. / Clinical Biochemistry 43 (2010) 1230–1235

diabetic groups were matched for sex and age. The control groupconsisted of 91 healthy subjects. Exclusion criteria for controls wereinflammation, infection, and presence of neurological dysfunction,history of CVD, renal, hepatic, gastrointestinal, pulmonary, endocrineor oncological disease. 114were diagnosedwith type 2 DMaccording tothe National Diabetes Data Group criteria [11]. Inclusion criteria werewell-controlled diabetes, less than 5 years illness duration, undertreatment with diet and insulin or oral hypoglycaemic drugs. 26diabetics (23%) followed dietary control (1400–1600 kcal/day) in theabsence of prescribed medication. The others (77%) received oralhypoglycaemic agents (37%: sulfonylurea agents, 11%: bigvanidinagents and 19%: a combination of sulfonylurea and bigvanidin) inaddition to dietary control. Only 11 patients (9.5%) received insulin incombination with oral hypoglycaemic therapy. Patients were specifi-cally advised to consume a variety of fruit, vegetables, grains, low fat ornon-fat dairy products, fish, poultry and lean meats. Also, they weresuggested to limit foods high in saturated fat, trans fatty acids andcholesterol, to limit salt to 6 g/day by choosing foods low in salt andlimiting the amount of salt added to food and to limit alcohol intaketo≤2drinks formanandnomore thanonedrink/day forwomen. Thoseproposes are in agreement with the Dietary guidelines from theAmerican Diabetes Association and the American Heart Associationadapted from Franz et al. [12]. No participants were treated withantioxidant supplementation or lipid-lowering drugs. We chose type 2DM patients who did not receive lipid-lowering drugs in order topreserve their unique lipid profile. In addition, none of the patients hadany other systemic disorder (CVD, renal diseases, hepatic diseases,oncological diseases) or micro vascular complications of diabetes(diabetic nephropathy or retinopathy). The criterion for a diabeticnephropathy was if the microalbuminuria was above 300 mg/d, whilewe used fundus oculi examinations to rule out diabetic retinopathy [13].

At the point of study entry all individuals underwent a completeclinical and biochemical investigation revealing age, gender, bloodpressure and the assessment of risk factors (including familial historyof acute coronary syndrome, arterial hypertension, smoking, dyslip-idemia, current medication and other socioeconomic variables). Thestudy protocol also included height and weight measurement forbody-mass index calculation [BMI=weight (kg)/squared height(m2)]. Replies to a standard questionnaire were collected in-personby trained interviewers. Study participants were deemed hyperten-sive if they had a systolic pressure≥130 mm Hg and/or diastolicpressure≥85 mm Hg or were already taking any form of anti-hypertensivemedication [11]. The criterion for a “dyslipidemic status”was if the LDL-C concentration was above 3.36 mmol/L and/or the TGconcentration was above 1.69 mmol/L [14].

The whole study was planned according to the ethical standardsdetailed in the Declaration of Helsinki (as revised in 1983) andaccording to local institutional guidelines. The local institutionalreview committee approved the research proposal and informedconsent was obtained from all individuals involved in the study.

Sample collection

Two samples (each of 20 mL) of venous blood were drawn fromthe antecubital vein after night-time fasting (N10 h). The blood wascollected into one EDTA sample tube (for plasma) and one serumsample tube (for serum) before immediate centrifugation at 1500×gfor 10 min at 4 °C. Plasma and serum samples were stored at −80 °Cin aliquots for up to one month until the analysis. Only for themeasurement of O2

−plasma from heparinised blood samples was usedimmediately.

Oxidative stress, antioxidative defence and PON1 status determination

The rate of nitroblue tetrazolium (NBT) reduction in plasma wasused to measure the rate of superoxide anion (O2

−) generation, as a

parameter of oxidative stress [15]. The intra-assay and inter-assaycoefficients of variance were 5.6% and 9.5%, respectively. Plasmasuperoxide dismutase (SOD) activity of a plasma marker of antiox-idative defence was measured according to the previously publishedmethod [16]. We monitored SOD-mediated inhibition of adrenalinauto-oxidation to adrenochrome. One unit of SOD activity is definedas the activity that inhibits the auto-oxidation of adrenalin by 50%. Theintra-assay and inter-assay coefficients of variance were 6.3% and9.2%, respectively. The concentration of sulphydryl (SH) groups inplasma was determined using 0.2 mmol/L 5.5′-dithiobis (2-nitroben-zoic acid) (DTNB) [17]. DTNB reacts with aliphatic thiols (at pH 9.0)producing 1 mol of p-nitrophenol per mole of thiol. p-Nitrophenolwas measured by spectrophotometry at 412 nm.

PON1192 status was assessed using a two-substrate activity(paraoxon/diazoxon) method [18]. PON1 paraoxon (POase activity)and diazoxon (DZOase activity) hydrolyses rates were measuredspectrophotometrically in serum using a continuous spectrophotom-eter (Pharmacia LKB, Cambridge, UK) according to Richter andFurlong [18]. The concentrations of paraoxon and diazoxon were1.2 mmol/L and 1 mmol/L, respectively. Both paraoxon and diazoxonwere purchased from Chem Service (West Chester, PA, USA). ThePON1192 phenotype was determinate by calculating the DZOase/POase activity ratio [19]. PON1Q192R isoforms show substrate-dependent differences. The Q variant has a higher DZOase activitybut the R allele variant has predominantly POase activity [20]. QQphenotypes have a DZOase/POase activity ratio above 60 while QRphenotype subjects have a DZOase/POase activity ratio between 21and 59. Subjects with a DZOase/POase activity below 20 exhibited theRR phenotype.

Biochemical analyses

Fasting glucose levels, glycated haemoglobin (HbA1c) and lipidstatus parameters [total cholesterol (t-C), LDL-C, HDL-C, TG, Apo A-Iand apolipoprotein B (Apo B)] were measured in serum using aHitachi 912 autoanalyser employing commercial kits (Roche Diag-nostics, Mannheim, Germany).

LDL and HDL particle size and subclass analysis

LDL and HDL subclasses were separated in EDTA plasma using amodified version of one described previously [21]. A detaileddescription of the modified procedure has been published elsewhere[22]. In brief, electrophoresis was performed at 8 °C in a Hoefer SE 600Ruby electrophoresis unit (GE Healthcare, Vienna, Austria) using Tris(90 mM)-boric acid (80 mM)-Na2EDTA (2.7 mM) buffer, pH 8.35 for20 h. Gels were calibrated using the Pharmacia High MolecularWeight protein standards, carboxylated polystyrene microspherebeads and human plasma with two LDL subclasses, standardised inDr David Rainwater's laboratory (South-West Foundation for Bio-medical Research, San Antonio, TX, USA). After electrophoresisseparation, the gels were stained for proteins with Coomassie brilliantblue G-250 and for lipids with Sudan black. Gels were analysed usingImage Scanner (GE Healthcare, Vienna, Austria) with Image Quantsoftware (version 5.2; 1999; Molecular Dynamics). The migrationdistance for each absorbance peak was determined and the particlediameter corresponding to each peak was calculated from thecalibration curve. The estimated diameter of the major peak in theLDL and HDL size region of each scan was referred to as the dominantparticle diameter. We classified LDL subclass phenotypes as pheno-type A (≥25.5 nm) or phenotype B (b25.5 nm). The relative contentof each HDL subclasses was estimated by determining the areas underthe peaks of densitometric scans of the sample. To achieve this, weused previously defined regions with the following cut-off values:HDL 2b (9.70–12.00 nm), HDL 2a (8.80–9.69 nm), HDL 3a (8.20–8.79 nm), HDL 3b (7.80–8.19 nm) and HDL 3c (7.20–7.79 nm) [23].

1232 A. Stefanović et al. / Clinical Biochemistry 43 (2010) 1230–1235

HDL phenotypes were classified as HDL 2 phenotype, which includedHDL 2b and HDL 2a particles and HDL 3 phenotype, which includedHDL 3a, HDL 3b and HDL 3c particles [22].

Statistical analysis

Data are shown as mean±standard deviation for normallydistributed variables and as relative or absolute frequencies forcategorical variables. Comparisons of continuous variables wereperformed using the Student's t-test. Analyses of categorical variablesused the Chi-square test for contingency tables. A logarithmictransformation of TG levels was performed because of the skeweddistribution in analysis using Student's t-test analysis [24]. Todeterminate possible correlation between parameters of oxidativestress and lipoprotein particle sizes and subclasses Spearman'snonparametric correlation analysis was employed. We used multipleregression analysis to estimate the independent contribution ofpredictors to the variance in PON1 activity. All statistical analyseswere performed using STATGRAPHIC Plus (version 4.2), CBstat(version 4.3.2) and Medcalc software's. All statistical tests wereconsidered significant at the 0.05 probability level.

Results

Clinical and laboratory parameters, LDL and HDL sizes andphenotypes, oxidative stress/antioxidative defence parameters,PON1 activities and PON1 phenotypes distribution within the studygroups are all shown in Table 1. As expected, the diabetics hadsignificantly higher BMI and glucose concentration (pb0.001) andwere dyslipidemic, as indicated by significantly higher plasma TG(pb0.001), lower HDL-C (Pb0.05) and lower Apo A-I (pb0.001).

Table 1Clinical and laboratory parameters, LDL and HDL sizes and phenotypes, oxidativestress/antioxidative defence parameters, PON1 activities and PON1 phenotypesdistribution in the two study groups.

Parameter Diabetics Controls P1

(n=114) (n=91)

Gender, m/f 58/56 45/46 0.319Age, years 52.3±6.01 50.5±6.03 0.683Smoking, % 26 20 0.313Hypertension, % 55 16 b0.001BMI, kg/m2 28.7±4.81 26.2±3.55 b0.001Glucose, mmol/L 9.2±2.76 5.6±0.64 b0.001Hb A1c, mmol/L,% 58.3±4.7 33.5±1.3 b0.05

7.7±1.9 5.4±0.3TC, mmol/L 5.9±1.57 5.6±0.94 0.059LDL-C, mmol/L 3.6±1.18 3.7±0.82 0.534HDL-C, mmol/L 1.1±0.29 1.2±0.33 b0.05TG, mmol/L 2.2±1.71 1.5±0.73 b0.001Apo A-I, g/L 1.3±0.23 1.9±0.45 b0.001Apo B, g/L 1.3±0.32 1.1±0.27 b0.01Dyslipidemia, % 71 54 b0.001LDL size, nm 26.3±1.39 26.6±1.16 0.097LDL B phenotype, % 28.9 12.1 b0 .001HDL size, nm 9.9±1.21 10.5±0.92 b0 .001HDL 3 phenotype, % 28.1 4.4 b0.001O2−, μmol/min/L 666±241.4 103±48.1 b0.001

SOD, kU/L 215.8±73.55 153.5±35.61 b0.001SH-groups, g/L 0.38±0.06 0.44±0.11 b0.001POase, U/L 199.4±154.73 299.5±285.61 b0.01DZOase, U/L 9389±3925.1 13248±5808.6 b0.001PON1 phenotype, %QQ 59% 57% 0.936QR 26% 28%RR 15% 15%

Data are expressed as mean±SD.1 Continuous variables were compared using Student's t-test and categorical

variables by Chi-square test.

While the LDL-C levels were similar in both study groups the Apo Bconcentration was significantly elevated in the diabetics (pb0.01).

The diabetics had a smaller mean LDL particle size than controls,although the difference was not statistically significant (p=0.097).However, small, dense LDL particles were more frequent in thediabetics, as revealed by a significantly greater prevalence of the LDL Bphenotype (pb0.001). Furthermore, the diabetics had a significantlysmaller mean HDL size (pb0.001). We also found that the diabeticshad a significantly higher percentage of subjects with the HDL 3phenotype (pb0.001).

The diabetics exhibited an elevated state of oxidative stress. Theyhad significantly higher O2

−(pb0.001) and significantly higher SODactivity (pb0.001) compared with the controls. In addition, thediabetics had a significantly lower concentration of total SH-groupscompared with controls. Higher PON1 activity towards both sub-strates (paraoxon and diazoxon) was found in controls. The PON1192phenotype frequencies in both groups were similar (χ2=3.19,p=0.936).

Thereafter, we calculated correlations between oxidative stressand PON1 status parameters and the measured characteristics of LDLand HDL particles in the diabetics (Table 2). We found that O2

−wasinversely associated with HDL particle size (pb0.01) but positivelyassociated with the proportion of the HDL 3b subclasses (pb0.01).Significant positive correlations were found between DZOase activityand both HDL-C (pb0.05) and HDL particle size (pb0.05). A significantnegative correlation between DZOase activity and the abundance ofthe HDL 3c subclasses was noted.

Next we performed multiple linear regression analysis to identifythe determinants of DZOase activity among the variables that gavesignificant association in univariate analysis. The significant predic-tors of DZOase activity were still HDL-C concentrations (β=0.228,pb0.05) and HDL particle size (β=0.207, pb0.05).

The effect of hyperglycaemia on LDL and HDL particle sizes,oxidative stress and PON1 status was examined by comparing theseparameters in diabetics with a blood glucose level less than 10 mmol/L with those in diabetics with blood glucose ≥10 mmol/L (Table 3).Although we found no significant differences in serum lipidsparameters, diabetics with a glucose level ≥10 mmol/L had signifi-cantly smaller LDL and HDL particle sizes. Plasma O2

−level and SODactivity positively correlated with glucose concentration, but thesetendencies were not statistically significant. Interestingly, POaseactivity was significantly lower in diabetics whose blood glucosewas ≥10 mmol/L.

Biochemical parameters, oxidative stress status parameters andPON1 activities in diabetics and controls were compared according toHDL phenotypes distribution (Table 4). By analysing individuals withthe HDL 3 phenotype, we failed to find differences in TG and HDL-Cconcentrations between diabetics and controls, although the diabeticshad significantly reduced Apo A-I concentrations. No significantdifference in POase and DZOase activities between diabetics andcontrols with the HDL 3 phenotype were noted (p=0.071 andp=0.614, respectively). The reverse was true when we compareddiabetics and controls with the HDL 2 phenotype. The diabetics hadsignificantly higher TG concentrations (pb0.001) and lower levels ofboth HDL-C (Pb0.05) and Apo A-I (pb0.001) compared with controls.Moreover, we found, significantly lower POase and DZOase activities(pb0.05 and pb0.001, respectively) in diabetics with the HDL 2phenotype compared with controls. Serum glucose concentrations,plasma O2

−levels and SOD activities were significantly higher in thediabetics irrespective of HDL phenotype.

Discussion

Type 2 DM is unmistakably a multifactorial disease [2]. Affectedindividuals are characterised by a cluster of CVD factors, such asabdominal obesity, insulin resistance, impaired glucose tolerance,

Table 2Spearman's non-parametric correlations between LDL and HDL cholesterol contents, particle sizes and subclasses and oxidative stress and PON1 status parameters in diabetics.

LDL size,nm

HDL size,nm

HDL 2b,%

HDL 2a,%

HDL 3a,%

HDL 3b,%

HDL 3c,%

O2−,

μmol/min/LPOase,IU/L

DZOase,IU/L

HDL-C, mmol/L 0.238⁎ −0.049 0.032 −0.041 0.104 −0.219⁎⁎ 0.036 −0.048 −0.029 0.190⁎LDL-C, mmol/L 0.067 −0.105 −0.053 0.119 0.058 −0.324⁎⁎ 0.08 −0.032 0.077 0.147LDL size, nm 0.279⁎⁎ 0.07 −0.055 −0.044 −0.037 −0.112 −0.158 −0.040 0.133HDL size, nm 0.506⁎⁎ −0.065 −0.484⁎⁎ −0.181 −0.198⁎ −0.223⁎⁎ 0.004 0.174⁎

HDL 2b, % −0.391⁎⁎ −0.392⁎⁎ 0.033 −0.137 −0.013 −0.191⁎ 0.184⁎

HDL 2a, % −0.439⁎⁎ −0.196⁎ −0.083 0.03 0.218⁎ −0.036HDL 3a, % −0.193⁎ 0.114 −0.099 −0.022 0.022HDL 3b, % −0.107 0.238⁎ −0.105 −0.117HDL 3c, % −0.093 0.125 −0.179⁎

O2−, μmol/min/L −0.040 −0.233⁎

POase, IU/L −0.062

⁎ Pb0.05.⁎⁎ Pb0.01.

1233A. Stefanović et al. / Clinical Biochemistry 43 (2010) 1230–1235

hypertension, dyslipidemia and inflammation. Furthermore, theextent of CVD risk in diabetics is strongly related to the degree ofhypertiglyceridemia and hyperglycaemia [25]. In the present study,we also noted a significantly higher percentage of subjects withhypertension and dyslipidemia in the diabetics group compared withcontrols (Table 1). In addition, BMI values were significantly higher indiabetics. We failed to find differences in TC and LDL-C concentrationsbetween the two study groups. Despite this, glucose and TGconcentrations were statistically higher, while HDL-C and Apo A-Iconcentrations were significantly lower in the diabetics (Table 1).Overall, our results are in agreement with previous data for diabeticdyslipidemia [26].

Type 2 diabetics are characterised by qualitative changes inlipoprotein composition and structure,which is particularly noticeablein the case of LDL andHDL particles [27]. Small, dense LDL particles areusually associated with increased TG and Apo B and decreased HDL-Cand Apo A-I concentrations [28]. Traditionally, accumulation of small,dense LDL particles has been considered to reflect atherogenicdyslipidemia of the metabolic syndrome, closely associated withtype 2 DM. We confirmed that DM type 2 patients had prevalence ofsmall, dense LDL particles which are known to be more susceptible tooxidation. In our current study, mean LDL particle size was smaller indiabetics, but the difference was not statistically significant, despiteobvious dyslipidemia. Nevertheless, the percentage of subjects withthe atherogenic LDL B phenotype was significantly higher in diabetics,indicating the accumulation of small, dense LDL particles. Increasedsmall HDL particles have been observed in subjects with very-highcardiovascular risk, such as those with type 2 DM [29]. In contrast toLDL, mean HDL size was significantly lower in diabetics. Furthermore,

Table 3Lipid parameters, oxidative stress status parameters and PON1 activities in diabeticswith different blood glucose levels.

Parameter Glucoseb10 mmol/L(n=80)

Glucose≥10 mmol/L(n=34)

P1

TC, mmol/L 5.9±1.57 6.2±1.60 0.362LDL-C, mmol/L 3.6±1.19 3.7±1.18 0.655HDL-C, mmol/L 1.1±0.29 1.0±0.28 0.714TG, mmol/L 2.1±1.47 2.7±2.26 0.231Apo A-I, g/L 1.3±0.20 1.3±0.27 0.564Apo B, g/L 1.1±0.32 1.1±0.32 0.354LDL size, nm 26.5±1.37 25.9±1.36 b0.05HDL size, nm 10.0±1.22 9.5±1.11 b0.05O2−, μmol/min/L 643±250.2 723±211.4 0.089

SOD, kU/L 209.9±74.82 229.6±69.60 0.183SH-groups, g/L 0.38±0.05 0.38±0.06 0.759POase, U/L 215.9±164.35 160.8±123.01 b0.05DZOase, U/L 9148±3955.4 9956±3850.8 0.314

Data are expressed as mean±SD.1 Variables were compared using Student's t-test.

the percentage of subjects with smaller HDL particles (HDL 3phenotype) was significantly greater in diabetics.

Oxidative stress plays a key role in the pathogenesis andprogression of atherosclerosis in type 2 DM [3,4]. In our currentstudy, we measured the level of O2

−, as a marker of oxidative stress,together with plasma SOD activity and the total SH-groups content(the major portion of the latter being reduced glutathione), as indicesof antioxidative defence potential. We found a significant increase inthe activity of SOD and O2

−, in combination with a decrease in the totalSH group's content in the diabetics when compared with the controls(Table 1). The increase in SOD activity could be explained by the factthat diabetics suffered chronic oxidative stress and as a consequence acompensatory increase in their antioxidative potential ensues. Otherstudies have also found increased SOD activity in some pathologicalsituations, caused by high levels of oxidative stress [30,31].

PON1 activity and PON1192 phenotype distribution in diabetics andcontrols were also considered. PON1 is a calcium dependent esterasethat is exclusively bound to the Apo A-I containing HDL fraction inserum [6]. It has been well documented that PON1 has the ability todecrease the susceptibility of LDL to lipid peroxidation [32]. Ourdiabetics showed a reduction in both, POase and DZOase activities,which is in agreement with previous findings of reduced serum PON1activity is in diabetics [33]. Opinions regarding association betweenthe PON1Q192R phenotypes and DM are controversial in that someresearch groups found a positive association implicating that the RRphenotype correlates with DM and CVD development [34]. In ourcurrent study, the PON1Q192R phenotype distribution didn't differbetween the two study groups (Table 1). This agreed with otherstudies proposing that the variation in PON1 specific activity indiabeticswas not caused by differences in phenotype distribution [35].

We also confirmed some earlier findings [35] that serum PON1activity does not correlate with the serum concentrations of mostlipids and lipoproteins in diabetics (data not shown). Indeed, weonly found statistically significant positive correlations betweenDZOase activity and HDL size and HDL-C concentration (Table 2). Inaddition, we found significant negative correlation between DZOaseactivity and HDL 3c subclasses abundance. In order to definecharacteristics of HDL particles most associated with DZOase activity,we performed multiple regression analysis. DZOase activity in thediabetics was strongly influenced by HDL particle size and itscholesterol content.

The influence of blood glucose levels on serum lipids, oxidativestress parameters and PON1 activity in diabetics was investigated. Nosignificant differences in serum lipid parameters or in oxidative stressstatus between diabetics with blood glucose below 10 mmol/L andthose with blood glucose were ≥10 mmol/L were found (Table 3).However, diabetics with glucose levels exceeding 10 mmol/L hadsignificantly reduced LDL and HDL particles sizes. We could speculatethat elevated glucose levels (above10 mmol/L) could be associated

Table 4Biochemical parameters, oxidative stress status parameters and PON1 activities in controls and diabetics according to HDL phenotypes.

Parametera HDL 2 phenotype HDL 3 phenotype

Diabetics(n=82)

Controls(n=87)

P Diabetics(n=32)

Controls(n=4)

P

Glucose, mmol/L 9.1±2.79 5.6±0.64 b0.001 9.5±2.70 5.2±0.22 b0.01HDL-C, mmol/L 1.1±0.31 1.2±0.33 b0.01 1.1±0.21 0.97±0.26 0.469TG, mmol/L 2.3±1.85 1.4±0.71 b0.001 2.2±1.23 2.5±0.46 0.561Apo A-I, g/L 1.3±0.24 1.9±0.46 b0.001 1.31±0.17 1.8±0.43 b0.001O2−, μmol/min/L 648±235.2 104±49.1 b0.001 710±257.2 86±14.8 b0.001

SOD, kU/L 213.9±67.65 152.9±35.28 b0.001 218.5±88.47 163.2±44.17 b0.05SH-groups, g/L 0.38±0.06 0.44±0.10 b0.01 0.38±0.06 0.46±0.25 0.167POase, IU/L 204.7±159.46 295.1±290.64 b0.05 142.5±25.60 375.9±180.86 0.071DZOase, IU/L 9645±4266 13,384±5625 b0.001 8726±2868 10,916±8919 0.614

Data are expressed as mean±SD.a Variables were compared using Student's t-test.

1234 A. Stefanović et al. / Clinical Biochemistry 43 (2010) 1230–1235

with compositional changes in LDL and HDL particles. It is known thatglycated lipoproteins in diabetics plasma, significantly contribute toatherogenesis [36]. Glycated LDL particles are more susceptible tooxidation, whereas HDL glycation results in attenuated anti-athero-genic potential [36]. Ferretti et al. [37] noted that HDL glycation isaccompanied by increased lipid peroxidation, suggesting that glyca-tion clearly impairs the antioxidative potential of HDL. Indeed, wefound a significantly lower PON1 activity in diabetics having glucoselevels greater than 10 mmol/L. Therefore, elevated glucose could beassociated with defective antioxidative capacity of HDL particles indiabetics.

The interconnectivity of antioxidativemechanisms and lipoproteindisorders was evaluated by examining the association of altered HDLparticle size and distribution with changes of PON1 activity. PON1activities in diabetics and controls were evaluated taking into accountHDL phenotypes. Diabetics with the HDL 2 phenotype had signifi-cantly higher TG concentrations and reduced both HDL-C and Apo A-Icompared with controls. In addition, serum glucose concentrations,plasma O2

− levels and SOD activities were significantly higher amongdiabetics bearing the HDL 2 phenotype. In contrast, the same diabeticshad significantly lower POase and DZOase activities compared withthe corresponding controls (Table 4).We can assume that lower PON1activity in HDL 2 phenotype diabetics could be a consequence ofaltered HDL composition. Abbot et al . [35] suggested that theconformation of PON1 within the hydrophobic environment of HDLcould be critical for its enzymatic activity. HDL particles in diabeticsare enriched in TG but deficient in both cholesterol and Apo A-I [5].Therefore, HDL composition could affect PON1 binding or change itssubstrate accessibility resulting in reduced PON1 activity despitelarger HDL size. Our results could complement previously publisheddata by Gowri et al. [10], in that isolated HDL 2 particles, rather thanHDL 3 particles, exhibited decreased protection against macrophage-mediated LDL oxidation in poorly controlled diabetics.

In conclusion, the results of our current study indicate that type 2DM is characterised by intense oxidative stress, reduced PON1 activityand impaired lipoprotein metabolism of LDL and HDL particles. Wehave confirmed the association of elevated oxidative stress andchanged antioxidative capacity of HDL particles as reflected by lowerPON1 activity in type 2 DM patients. In addition, we have shown thatthe diabetic HDL 2 phenotype is associated with hyperglycaemia,lower PON1 activity and elevated oxidative stress. Future studies willshed light on whether large HDL 2 particles in diabetics could berelated to increased risk of CVD development.

Acknowledgments

We appreciate financial support from the Ministry of Science andEnvironmental Protection, Republic of Serbia (project number

145036B). The authors would like to thank Dr. David R. Jones forthe help in editing the manuscript. We are also grateful to VericaMilanović and Marina Baranin for their excellent support with thelaboratory analyses.

References

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