Atherosclerosis 177 (2004) 203–210

Impaired high density lipoprotein antioxidant activity in healthypostmenopausal women

Valeria Zago1, Silvia Sanguinetti1, Fernando Brites, Gabriela Berg, Julian Verona,Francisco Basilio, Regina Wikinski, Laura Schreier∗

Laboratory of Lipids and Lipoproteins, Departament of Clinical Biochemistry, School of Pharmacy and Biochemistry,University of Buenos Aires, Jun´ın 956 (C1113AAD), Buenos Aires, Argentina

Received 21 August 2003; received in revised form 1 March 2004; accepted 8 July 2004Available online 15 September 2004

Abstract

The high incidence of atherosclerosis in women after menopause is associated with a risk pattern including an increase in low densityl estrogensa ected afterm there ares onjugatedd d diabeteso e effect ofH presenceo lc n in PMWe ups.Ha ing HDLo ofH W (<p d only inP pairmenti nrichmento©

K

scu-soci-eva-L).

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ipoprotein (LDL), even though high density lipoprotein (HDL) cholesterol levels tend to be maintained or slightly decreased. Sincere considered potent antioxidants, an increase in lipid peroxidation and formation of reactive oxygen species would be expenopause. If HDL becomes oxidized, the ability to protect LDL against oxidation may be impaired. In postmenopausal women

carce reports concerning HDL oxidability and no data about its antioxidant activity. We studied copper-induced oxidation and cienes formation in HDL isolated from 58 women, 30 postmenopausal (PMW) and 28 premenopausal (PreMW). None presenter cardiovascular disease and none was receiving hormonal, hypolipidemic or antioxidant therapy either. In order to evaluate thDL on LDL oxidation we isolated LDL and HDL from the same subject and assessed copper-induced LDL oxidation in thef HDL, followed by thiobarbituric acid-reactive substances determination. Relationships with HDL chemical composition,�-tocopheroontent, cholesteryl ester transfer protein (CETP) and paraoxonase activity (PON) were investigated. HDL chemical compositioxhibited triglyceride enrichment when compared to PreMW (p< 0.05).�-Tocopherol content and CETP activity were similar in both groowever, CETP activity correlated positively with HDL triglyceride and negatively with HDL cholesterol percentage (r = 0.44,p < 0.01ndr = −0.32,p < 0.05, respectively). Paraoxonase activity did not show differences between PMW and PreMW. When evaluatxidability, PMW revealed a shorterlag time in comparison to PreMW, even after adjustment for age,p < 0.05. Moreover, when the effectDL on LDL oxidation was evaluated, HDL from PMW showed a reduction in its ability to inhibit LDL oxidation, compared to PreMp0.05). In addition, the extent of inhibition of LDL oxidation by HDL was positively correlated with HDL resistance to oxidation (r = 0.27,< 0.05). After women classification by paraoxonase phenotype, HDL ability to protect LDL against oxidation remained reduceMW belonging to the PON QR phenotype, in comparison to PreMW QR. These results suggest that HDL from PMW exhibits im

n its antioxidant ability, which is associated to a decreased HDL resistance to oxidation. In turn, this was related to triglyceride ef HDL particles. All these alterations were independent from HDL cholesterol plasma levels.2004 Elsevier Ireland Ltd. All rights reserved.

eywords:High density lipoprotein; Oxidation; Antioxidant activity; Paraoxonase; Postmenopause

∗ Corresponding author.E-mail address:lipids@ffyb.uba.ar (L. Schreier).

1 Contributed equally to the work.

1. Introduction

The high incidence of atherosclerosis and cardiovalar disease in women after menopause is mainly asated with a reduction in circulating estrogen and an eltion in the plasma levels of low density lipoprotein (LDAmong postmenopausal women an adverse lipoprotein

021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2004.07.011

204 V. Zago et al. / Atherosclerosis 177 (2004) 203–210

tern has been described, even though high density lipoprotein(HDL) cholesterol levels tend to be maintained or slightlydecreased[1,2]. We have previously reported no changes inHDL cholesterol plasma levels after menopause[3]. How-ever, compositional and structural alterations in HDL couldbe present, probably due to high very low density lipopro-tein (VLDL) triglycerides and changes in remodeling factorswhich include the cholesteryl ester transfer protein (CETP).These alterations could impact on HDL antiatherogenic prop-erties such as its capacity to inhibit LDL oxidation. Thehypothesis that LDL oxidation plays a significant role inatherogenesis is supported by epidemiological data as wellas studies in animal models receiving antioxidants[4]. How-ever, large-scale double-blind, placebo-controlled clinical tri-als with antioxidants have not shown benefits so far[5].

Although estrogens have not been shown to protect againstcardiovascular disease in prospective randomized placebo-controlled trials[6,7], there is a large piece of evidence onthe antioxidant properties of estrogens[8]. Thus, an increasein lipid peroxidation and formation of reactive oxygen specieswould be expected after menopause. The increase in oxidativestress may influence endothelial injury and the susceptibilityof lipoproteins to oxidation. If HDL becomes oxidized, itsability to protect LDL against oxidation in the subendothelialspace may be impaired.

d ber izedL )T e in-d wallb dL

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Aires, and their ages ranged from 43 to 60 years (mean age52.9 years), with at least one year of natural menopause andno more than 10 years of amenorrhea. In all the cases serumlevels of FSH > 40 IU/l confirmed the menopausal status.The control group comprised 28 women in reproductive age(23–44 years, mean age 33 years), with normal physical ex-amination and laboratory tests, recruited consecutively frompatients that attended the same Center for their routine healthcheck.

None of the women was included when presenting historyor underlying symptoms of diabetes or cardiovascular dis-ease, as well as if receiving hormonal, hypolipidemic or anyother drug known to modify lipid metabolism. Also womenwere excluded when receiving antioxidant supplementationor having history of hypothyroidism, neoplasia or renal dis-order. Women whose weight had varied more than 5% in thelast 6 months were also excluded. Four postmenopausal andfive premenopausal women consumed two to six cigarettesper day, the remaining subjects had been non-smokers forthe last 10 years. In no case did alcohol consumption surpass10 g per day. Post- and premenopausal women were not un-der regular training exercise. Both groups of women followedthe same diet with the following distribution: 20% protein,less than 30% fat and the rest in carbohydrate; calorie intakevaried according to individual body weight.

bjectb theE em-i

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Several enzyme systems associated with HDL coulesponsible for inactivating the reactive species in oxidDL and are mainly represented by paraoxonase (PON[9].his HDL-associated enzyme might protect against thuction of inflammatory responses in cells of the arterialy destroying biologically active lipids in mildly oxidizeDL.

In a previous work, we investigated the antioxidantion of HDL during copper-mediated LDL oxidation, as ws paraoxonase activity in fairly controlled type 2 diabatients[10]. In postmenopausal women there are scarcorts concerning HDL oxidability[11] and no data about intioxidant activity.

Our aim was to evaluate HDL characteristics and beor, beyond its cholesterol level. We initially investigatedusceptibility of isolated HDL to copper-induced oxidatnd then the effect of HDL on LDL oxidability in post- aremenopausal healthy women. Secondly, we assesseduence of different HDL biological active components ss CETP, paraoxonase and�-tocopherol on the oxidative presses previously studied.

. Methods

.1. Subjects

A total of 58 healthy women were studied. Thirty wostmenopausal women, clinically evaluated and cons

ively recruited at the Climateric Section of the Centertudies in Gynecology and Reproduction (CEGYR), Bue

-

Written informed consent was obtained from each suefore admission to the study, which was approved bythics Committee at the School of Pharmacy and Bioch

stry, University of Buenos Aires.

.2. Samples

After a 12-h overnight fast, blood samples were collento EDTA containing tubes (1 mg/ml). Samples were crifuged at 1500× g for 5 min at 4◦C and plasma was stort 4◦C for lipoprotein isolation and used within 24 h. For lipnd lipoprotein determination, blood was collected into

ubes and serum was separated at 4◦C. For paraoxonase aETP determination, a serum aliquot was stored at−70◦C.

.3. Lipoprotein isolation

Lipoproteins were isolated by sequential preparativeracentrifugation[12]. LDL [density (d): 1.019–1.063 g/mnd HDL (d: 1.063–1.210 g/ml) isolation was simultaneouerformed from two separate plasma aliquots in a XLeckman ultracentrifuge, with a type 90 Ti rotor. Eachas performed at 105,000× g, for 18 h, at 15◦C. Purity of

ipoprotein fractions was tested by agarose gel electropis [13] and albumin content tested by immunoturbidimeRoche Diagnostics, Mannheim, Germany). Albumin conn lipoprotein fractions was in no case higher than 2% ofrotein. Aliquots of isolated HDL were stored at−70◦C for-tocopherol analyses.

V. Zago et al. / Atherosclerosis 177 (2004) 203–210 205

2.4. Analytical procedures

Cholesterol and triglycerides were measured in serumusing commercial enzymatic kits (Roche Diagnostics,Mannheim, Germany) in a HITACHI 727 autoanalyser. HDLand LDL cholesterol were determined by selective precipi-tation methods[14,15]. Serum lipid measurements were un-der good quality control [coefficient of variation (CV) rou-tinely <3%]. Serum apo A-I and apo B were determined byimmunoturbidimetry (Roche Diagnostics, Mannheim, Ger-many). Within-run and between-day precision (CV) were1.9% and 2.4% for apo A-I and 1.2% and 2.1% for apo B,respectively.

Cholesterol, triglycerides (by the methods previouslymentioned), phospholipids[16] and total protein[17] wereassessed in isolated HDL. As cholesteryl esters were not mea-sured, the chemical composition underestimates the percent-age of cholesterol in the particle.�-Tocopherol in HDL wasdetermined by HPLC following extraction with methanol andhexane[18].

2.5. CETP activity

Cholesteryl ester transfer protein activity was determinedin serum samples according to the general procedure pre-v -m acera( ruma werei d1 sw eres ,a thi n,a Re-s st erh ere4

2

seda e-s sala,S ighta er-ff no thea

tiono y thek RS

measurement, HDL samples were incubated with copper for120 min followed by addition of EDTA 2 mmol/l (final con-centration) to stop oxidation. TBARS were determined as pre-viously described[20]. The kinetics for the formation of con-jugated dienes was continuously monitored by the increasein absorbance at 234 nm[21] in a HITACHI U-1100 spec-trophotometer (Tokyo, Japan),lag time was calculated as theintercept of the linear least square slope of the curve withthex axis and expressed in min.Lag time is an indicator ofthe resistance to oxidation. Since fresh samples must be usedto perform this assay, only within-run precision was calcu-lated (CV). These were 6.5% and 6.9% for HDL oxidabilitymeasuring TBARS andlag time, respectively.

2.7. Effect of HDL during copper-mediated LDLoxidation

The inhibitory effect of HDL on LDL oxidability was as-sessed by measuring TBARS as before[10]. Briefly, isolatedLDL (10 mg protein/dl) was oxidized either in the presence orin the absence of HDL (20 mg protein/dl) obtained from thesame patient, in 0.16 mol/l NaCl (pH 7.4), 10�mol/l Cu2+,120 min, at 37◦C.

At the end of the incubation period oxidation was stoppedwith 2 mmol/l EDTA for TBARS determination.

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n ofT ea-s fol-lT Si

2

twod andp erema ea mol/lp aC n-t ute salt-s rateo inaa asn bya igmaC .8)c lg

iously described[19]. Briefly, the ability of serum to proote the transfer of tritiated cholesteryl esters from a trmount of biosynthetically labeled HDL3 (3H-CE-HDL3)NEN Life Science Products, Boston, USA) towards sepo B-containing lipoproteins was evaluated. Samples

ncubated with3H-CE-HDL3 (50�mol/l cholesterol) an.5 mmol/l iodoacetate, for 3 h, at 37◦C. After incubationere stopped by placing the tubes in ice, lipoproteins weparated by ultracentrifugation atd = 1.070 g/ml, for 18 ht 4◦C and 250,000× g. Radioactivity was measured bo

n the supernatant, containing the VLDL-IDL-LDL fractios well as in the subnatant, containing the HDL fraction.ults were expressed as percentage of3H-cholesteryl esterransferred from HDL3 to apo B-containing lipoproteins, p, per ml. Within-run and between-day precision (CV) w.9% and 6.0%, respectively.

.6. In vitro oxidability of HDL

Susceptibility of isolated HDL to oxidation was assesfter gel filtration of the lipoprotein fraction on PD-10 dalting columns (Amersham Pharmacia Biotech, Uppweden) to remove EDTA and plasma low molecular wentioxidants. Oxidation of HDL (20 mg protein/dl) was p

ormed in 0.16 mol/l NaCl (pH 7.4), at 37◦C, by addition of areshly prepared CuCl2 solution giving a final concentratiof 10�mol/l. Oxidation blanks were incubated withoutddition of copper.

HDL oxidation products were assessed by determinaf thiobarbituric acid reactive substances (TBARS) and binetics for the formation of conjugated dienes. For TBA

TBARS obtained in separately oxidized HDL was stracted from the value obtained in the mixtures HDL/Lhich corresponds to LDL oxidation in the presence of HResults were expressed as percentage of inhibitio

BARS-LDL, in relation to its respective baseline value mured in separately oxidized LDL. It was calculated asows: % inhibition = (TBARS in separately oxidized LDL−BARS in LDL oxidized in the presence of HDL)/(TBAR

n separately oxidized LDL)× 100.

.8. Paraoxonase/arylesterase activity

The enzyme paraoxonase was evaluated employingifferent substrates: paraoxon (paraoxonase activity)henylacetate (arylesterase activity). Both activities weasured following the original method of Furlong et al.[22],nd previously validated in our laboratory[10]. Paraoxonasctivity was assessed by adding serum samples to 2.6 maraoxon (O,O-diethyl-O-p-nitrophenylphosphate, Sigmhemical Co.) in Tris–HCl buffer (100 mmol/l, pH 8.0) co

aining 2 mmol/l CaCl2, at 25◦C. The assay was carried oither in the presence or in the absence of 1 mol/l NaCl (timulated and non-stimulated PON, respectively). Thef generation ofp-nitrophenol was determined at 405 nmHITACHI U-1100 spectrophotometer. Theε270 for the re-ction is 17,000 M−1 cm−1 and results were expressedmol/ml min. Arylesterase (ARE) activity was measureddding serum samples to 4.4 mmol/l phenylacetate (Shemical Co.) in Tris/Acetate buffer (50 mmol/l, pH 7ontaining 20 mmol/l CaCl2, at 25◦C. The rate of phenoeneration was determined spectrophotometrically (ε270 =

206 V. Zago et al. / Atherosclerosis 177 (2004) 203–210

1310 M−1 cm−1). Blanks were included to correct for thespontaneous hydrolysis of phenylacetate. Results were ex-pressed as�mol/ml min. Since measurements were all car-ried out within the same assay, only within-run precision (CV)was calculated. These were 2.9%, 5.5% and 4.8% for PON,PON 1M and ARE, respectively.

Paraoxonase, unlike arylesterase, exhibits substrate-dependent activity polymorphism in humans, thus pheno-typic distribution of paraoxonase in the subjects was graph-ically estimated by the dual substrate method[23], whichallows to assign one of the following phenotypes to each sub-ject: QQ (homozygous-low activity), QR (heterozygous ac-tivity) and RR (homozygous-high activity). Only six womenbelonging to the RR phenotype were disregarded becausedistinction between QR and RR phenotypes was not com-pletely reliable[23]. The phenotypic distribution was similarbetween post- and premenopausal women [QQ: 50% (n= 15)and QR: 40% (n= 12) versus QQ: 43% (n= 12) and QR: 46%(n = 13), respectively,p = 0.12].

2.9. Statistical analysis

Results are expressed as mean± S.E.M. for normallydistributed data and as median and range for skewed data.Differences between groups were tested using the un-p heM be-t Spear-m com-p nal-y vari-a5

3

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We tion,w Pa ity

TH eryl es W) andp

in % (w l)

P 10.9P 10.6

Table 1Physical and biochemical features of post- and premenopausal women (mean± S.E.M.)

Postmenopausalwomen

Premenopausalwomen

n 30 28Age 52.9 ± 1.3b 32.7 ± 1.3Body mass index (kg/m2) 26.0 ± 0.7a 21.4 ± 0.4Waist (cm) 86.6 ± 1.9b 74.0 ± 1.2Triglycerides (mg/dl) 137.0 ± 11.7b 69.0 ± 6.8Total cholesterol (mg/dl) 244.3 ± 8.8b 187.1 ± 5.9HDL cholesterol (mg/dl) 52.0 ± 2.3c 60.1 ± 2.5LDL cholesterol (mg/dl) 167.1 ± 8.7b 108.5 ± 5.9Apo A-I (mg/dl) 126.9 ± 2.8 137.8 ± 5.7Apo B (mg/dl) 120.8 ± 6.3b 79.4 ± 4.2

a vs. premenopausal women,p < 0.001.b vs. premenopausal women,p < 0.0001.c vs. premenopausal women,p < 0.05.

correlated positively with HDL triglyceride content and neg-atively with HDL cholesterol percentage (r = 0.44,p < 0.01andr = −0.32,p < 0.05, respectively).

Non-stimulated paraoxonase, salt-stimulated paraoxonase(PON 1M), and arylesterase activities are shown inTable 3.No differences between PMW and PreMW were detected inany of the three enzymatic activities, even if women wereclassified by paraoxonase phenotype.

When evaluating copper-mediated HDL oxidation, PMWrevealed an increased HDL oxidability, evidenced by ashorterlag time, in comparison to PreMW (Fig. 1), even af-ter adjustment for age (p < 0.05). On the other hand, HDLoxidability assessed by measuring TBARS 120 min post-oxidation showed no difference between groups, PMW: 35.3± 2.1 versus PreMW: 34.1± 1.8 nmol MDA/mg HDL pro-tein (p = 0.677).

In searching for factors potentially linked to the decreasein HDL resistance to oxidation, correlations with HDL com-ponents were calculated. A significant and negative associ-ation between HDLlag time and HDL triglyceride contentwas found,r = −0.30,p < 0.05. However, no correlationswere found with HDL�-tocopherol content (p = 0.19) orparaoxonase activity (p = 0.56).

To test HDL antioxidant ability during LDL oxidation,co-incubation assays with both lipoproteins of the same sub-j x-i d inP ofw izedL

aired Student’st-test for normally distributed data and tann–WhitneyU-test for skewed data. Correlations

ween variables were assessed using the Pearson oran correlation tests. The chi-square test was used toare proportions of PON phenotypic frequencies. Asis of covariance was used, including age as cote. Differences were considered significant atp less than%.

. Results

In the present work, postmenopausal women (PMhowed an abnormal lipid-lipoprotein profile, as welligher obesity degree and abdominal fat distribution, in carison to premenopausal women (PreMW) (Table 1). Al-

hough HDL cholesterol concentration was lower in PMpo A-I remained unchanged.

Table 2shows that HDL chemical composition in PMxhibits triglyceride enrichment and cholesterol deplehen compared to PreMW.�-Tocopherol content and CETctivity were similar in both groups. However, CETP activ

able 2DL percent chemical composition,�-tocopherol content and cholestremenopausal women (PreMW) (mean± S.E.M.)

Cholesterol triglycerides phospholipids total prote

MW (n = 30) 16.4± 0.4a 4.4± 0.4b 21.8± 4.1 57.4±reMW (n = 28) 19.3± 0.7 3.5± 0.2 21.8± 4.2 55.2±

a vs. PreMW,p < 0.005.b vs. PreMW,p < 0.05.

ter transfer protein (CETP) activity in postmenopausal women (PM

/w) �-Tocopherol (nmol/mg protein) CETP (% CE/h m

5.8± 0.3 125.4± 8.16.1± 0.6 105.0± 7.8

ect were performed. In all cases HDL inhibited LDL odation, but the extent of the inhibition was decreaseMW (p< 0.05) (Fig. 2). It is noteworthy that both groupsomen presented similar oxidability of separately oxidDL (Table 4).

V. Zago et al. / Atherosclerosis 177 (2004) 203–210 207

Table 3Serum paraoxonase activities in postmenopausal women (PMW) and premenopausal women (PreMW), median (range)

PON PON 1M ARE

PMW (n = 30) 119 (77–510) 246 (111–1042) 153 (96–204)PMW QQ (n = 15) 78 (55–171) 151 (110–246) 132 (96–207)PMW QR (n = 12) 224 (32–447) 552 (37–763) 172 (13–209)

PreMW (n = 28) 183 (60–343) 374 (127–629) 129 (106–189)PreMW QQ (n = 12) 74 (37–148) 142 (73–230) 122 (64–141)PreMW QR (n = 13) 268 (98–343) 538 (341–629) 136 (106–194)

PON: non-stimulated paraoxonase activity; PON 1M: salt-stimulated paraoxonase activity, assessed in 1 mol/l NaCl; ARE: arylesterase activity, assessed withphenylacetate. Results are expressed as nmol/ml min, for PON and PON 1M and expressed as�mol/ml min, for ARE. No statistical differences between groupswere found. Patients were classified according to homozygous-low activity (QQ) and heterozygous paraoxonase activity (QR) phenotypes, which limited thesample size.

HDL ability to protect LDL against oxidation was relatedto its oxidability, since a significant correlation was observedbetween inhibition of LDL oxidation (% inhibition TBARS-LDL) and HDL resistance to oxidation (lag time-HDL) (r =0.27,p < 0.05). In contrast, no significant correlations wereobtained between HDL inhibitory action and HDL choles-terol (p= 0.71), apo A-I (p= 0.55) or PON/ARE activities (p= 0.76 for PON 1M), in PMW and PreMW.

Taking into account the QQ and QR phenotypes previouslyassigned to PMW and PreMW, we investigated the influenceof each phenotype in HDL inhibitory action.Fig. 3 revealsthat only PMW belonging to the QR phenotype, showed a de-crease in HDL protective effect in comparison to PreMW QR(p= 0.060). On the other hand, within each women group, nodifferences were observed in HDL protective effect betweenQQ and QR phenotype.

F atedf eMW)w atedw e-t dfP

Fig. 2. Box plots showing HDL inhibition of LDL oxidation in post- andpremenopausal women, HDL and LDL were isolated from postmenopausalwomen (PMW) and premenopausal women (PreMW). LDL was diluted to10 mg protein/dl in 0.16 mol/l NaCl (pH 7.4) and incubated with�mol/lCu2+, at 37◦C, for 120 min, either in the presence or in the absence of20 mg protein/dl HDL. At the end of the incubation period thiobarbituricacid reactive substances (TBARS) were determined. Percent inhibition ofLDL oxidation was calculated as explained inSection 2. The boxes representthe interquartile ranges of values and contain 50% of values. The line acrosseach box indicates the median. The error bars indicate the highest and lowestvalues when outliers and extremes are excluded. The square indicate outliers(values between 1.5 and 3 times the box length from the upper or lower edgeof the box). (∗) vs. PreMW,p < 0.05.

Table 4LDL oxidability in post- and premenopausal women (mean± S.E.M.)

Postmenopausalwomen

Premenopausalwomen

LDL TBARS (nmolMDA/mg protein)

53.5± 2.2 50.5± 2.5

LDA lag time (min) 61.2± 3.6 66.5± 1.9

LDL (10 mg protein/dl) was oxidized in 0.16 mol/l NaCl (pH 7.4) with10�mol/l CuCl2, at 37◦C. Thiobarbituric acid reactive substances (TBARS)were measured after 120 min oxidation.Lagtime was calculated from the ki-netics of conjugated diene formation, as explained in methods. No statisticaldifferences between groups were found.

ig. 1. HDL oxidability in post- and premenopausal women, HDL isolrom postmenopausal women (PMW) and premenopausal women (Pras diluted to 20 mg protein/dl in 0.16 mol/l NaCl (pH 7.4) and incubith 10�mol/l Cu2+, at 37◦C. Oxidation was kinetically monitored by d

ermination of conjugated diene formation at 234 nm.Lagtime was obtainerom the kinetic curves. Results are expressed as mean± S.E.M. (∗) vs.reMW,p < 0.0005.

208 V. Zago et al. / Atherosclerosis 177 (2004) 203–210

Fig. 3. Box plots showing HDL inhibition of LDL oxidation in post- andpremenopausal women classified by paraoxonase phenotype, HDL and LDLwere isolated from postmenopausal women (PMW) and premenopausalwomen (PreMW) and inhibition of LDL oxidation in the presence of HDLwas performed as explained inSection 2. Either QQ or QR paraoxonasephenotype were assigned to each women by the dual substrate method, asdescribed inSection 2. Subgroups were conformed as follows: QQ PMW(n = 15); QR PMW (n = 12); QQ PreMW (n = 12); QR PreMW (n = 13).The boxes represent the interquartile ranges of values and contain 50% ofvalues. The line across each box indicates the median. The error bars indicatethe highest and lowest values when outliers and extremes are excluded. Thesquare indicate outliers (values between 1.5 and 3 times the box length fromthe upper or lower edge of the box). (∗) vs. PreMW QR,p = 0.060.

4. Discussion

We have evaluated HDL from healthy postmenopausalwomen, beyond its cholesterol content. The role of HDLpreventing LDL oxidation and the potential consequencesof HDL oxidation on its activity have not been studied inpostmenopause before. In the present study, HDL from post-menopausal women was less resistant to oxidation, whichwas evidenced by a shorterlag time, which in turn was as-sociated to an increase in HDL triglyceride content. More-over, HDL inhibition of LDL oxidation was diminished inpostmenopausal women, as compared to controls, and thisfinding was inversely related to HDL oxidability. In addition,the decrease in HDL inhibitory action was only observed inpostmenopausal women belonging to the QR paraoxonasephenotype.

It is known that women become more susceptible to coro-nary artery disease with age and estrogen reduction[24].This may be linked, in part, to an increase in the oxida-tive status[25], which in turn can enhance LDL and HDLoxidation. The higher HDL oxidability shown herein in post-menopausal women was evidenced by a shortening in thelag time when kinetically monitoring conjugated dienes pro-duction during copper-induced oxidation. However, whenTBARS HDL were measured after 120 min oxidation, dif-f bablyd netic

measurement – may provide an incomplete evaluation of thewhole oxidative process.

Even though oxidative stress increases with age[26], thedecrease in HDLlag time shown herein remained signifi-cant even after adjustment for age, indicating a relation withmenopausal status. Moreover, the decrease in HDL resis-tance to oxidation correlated with tryglicerides enrichmentin HDL. This is in accordance to the fact that triglyceride-enriched lipoproteins are prone to oxidative modification[27]. On the other hand, the increase in HDL triglycerideswas associated with CETP activity, which exchanges triglyc-erides and cholesteryl esters between HDL and triglyceride-rich lipoproteins. In postmenopausal women, the increasein plasma triglycerides and presumably in VLDL particleswould promote exchanges between lipoproteins, remodelingtheir composition.

Although�-tocopherol within HDL may contribute to itsself-protective effect, no association between HDL oxidabil-ity and�-tocopherol content was found herein. In addition,no differences in�-tocopherol concentration were found be-tween groups, according to their similar diet composition.The role of�-tocopherol in relationship with HDL oxidationremains controversial as some reports indicate this antiox-idant molecule is able to exert a pro-oxidant effect duringHDL oxidation induced by copper or other oxidants[10,28].M enta-t e toi tiono i-c E)h nT hesisb f nat-u oni

st-m otecta tionr es-t tiono -vl re-s con-c entedl se inH e inL ev-i , thea rtherm anta

ainsto DLl Lw t, it

erences between groups could not be appreciated, proue to the fact that a single end point – rather than a ki

oreover, other authors observed that dietary supplemion with vitamins C and E did not increase HDL resistancn vitro oxidation when determined by measuring formaf conjugated dienes (lag time). Above all, large-scale clinal trials with antioxidant supplementation (i.e., Vitaminave not shown benefits for heart disease risk reductio[5].hese results not necessarily refute the oxidation hypotut they can raise some questions about the capacity orally occurring antioxidants to inhibit lipoprotein oxidati

n vivo, at least in the conditions employed so far.An important finding of this study is that HDL from po

enopausal women revealed an impaired ability to prgainst LDL oxidation. The relative protein concentraatio of HDL to LDL assayed in our experiments wasablished as 2 to 1, close to the physiological proporf HDL/LDL found in the arterial wall, which varies inersely with respect to circulating lipoproteins[29]. Sinceipoprotein concentration was fixed in the assays, theults observed are independent from lipoprotein plasmaentration. Nevertheless, postmenopausal women presipoprotein quantitative alterations such as a decreaDL, expressed by its cholesterol content, an increasDL cholesterol and in LDL particle number, the latter

denced by higher apo B concentration. Consequentlyctual state in postmenopausal women would be fuore unfavorable, beyond the reduction in HDL antioxidctivity.

In the present investigation, decreased protection agxidation by HDL was related to the shortening of Hag time, indicating that oxidative modifications on HDould influence its protective action. In a recent repor

V. Zago et al. / Atherosclerosis 177 (2004) 203–210 209

was demonstrated that in vitro HDL oxidation was accom-panied by a loss of its antioxidant activity towards LDL ox-idation, which was attributed to an inactivation of paraox-onase during oxidation[30]. It has also been reported thatHDL oxidation impairs other roles, such as interaction withlecithin-cholesterol acyltransferase[31] and the HDL facultyto accept cholesterol from cell membranes[32]. These pro-cesses, in addition to our observation of an impaired antiox-idant action, could potentially weaken HDL antiatherogenicrole.

When HDL-associated paraoxonase was evaluated, wecould not discern any difference in PON/ARE activities be-tween groups or paraoxonase phenotype subgroups. It is no-ticeable, however, that the findings on paraoxonase pheno-type subgroups are somewhat limited because of the smallsample size. As regards the lack of difference in paraoxonaseactivity, it must be highlighted that either pre- as well aspostmenopausal women included in the present investigationwere healthy, while decreases in paraoxonase activities previ-ously demonstrated by others were only reported in subjectssuffering from diseases associated with coronary heart dis-ease such as diabetes[33], end-stage renal disease[34] ordiabetic neuropathy[35]. Furthermore, a study of a clinicallyhealthy population failed to show an association betweenparaoxonase and carotid lesions[36]. Perhaps, as previouslys tiond roticp

ngedd omenc HDLc aysc enc ribedf de-c re-f raox-ow DLc thorsa o A-I usalw se inp

aox-o lu-a oundt edi type.T QRp ct inc pe, as phe-n auset ro-t ere

obtained with a small sample size, this finding needs to beconfirmed by larger studies.

It must be highlighted that the postmenopausal womenstudied here were healthy and did not exhibit clinical man-ifestations of cardiovascular disease. In addition, they pre-sented an altered lipoprotein profile in comparison to pre-menopausal women linked to their age and menopausal sta-tus. Moreover, the impairment in HDL antioxidant activityindicates that its antiatherogenic role may be deficient, eventhough HDL cholesterol was within desirable values (10thpercentile: 37 mg/dl; 90th percentile: 70 mg/dl).

Several experimental and epidemiological studies haveshown a decrease in LDL cholesterol in women underestrogen treatment[41]. However, these treatments havebeen highly discouraged as a therapy for primary or sec-ondary prevention of cardiovascular disease since controlled-randomized clinical trials showed that continuous combinedtreatment with estrogen plus progestin failed to decrease,but in some cases even increased, the incidence of cardio-vascular events[6,7]. Nevertheless, the treatment of healthypostmenopausal women without preexisting cardiovasculardisease, with unopposed micronized 17-� estradiol, showeda decrease in the rate of progression in the intima-mediathickness[42]. Therefore, investigation of the mechanismspromoting the higher atherogenic risk observed in post-m

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uggested[36], the effect of paraoxonase on plaque formaiffers between early and later stages of the atherosclerocess.

Our observation that paraoxonase remained unchaespite HDL cholesterol decrease in postmenopausal would be explained by the fact that correlations betweenholesterol, apo A-I, and HDL particle number is not alwlose[37]. Probably, HDL exhibits a disconnection betweomponents and particle number, as was clearly descor LDL [38]. In fact, postmenopausal women showed arease in HDL cholesterol but not in apo A-I levels. Theore, we should not necessarily expect a reduction in panase activity. This is in accordance to Durrington et al.[39]ho affirm that there is not a clear correlation between Hholesterol levels and paraoxonase activity. Other aulso showed that a decrease in HDL cholesterol and apobtained after treatment with tibolone in postmenopaomen was practically not accompanied by a decreaaraoxonase activity[40].

Even though no correlations were found between parnase activity and the HDL inhibitory effect, when evating paraoxonase by means of their phenotypes, we f

hat reduction in HDL inhibitory action was only observn postmenopausal women belonging to the QR phenoaking into account that premenopausal women fromhenotype did not show a reduced HDL protective effeomparison to premenopausal women from QQ phenotytill unsolved question is raised as regards which are theomena which take place during the transition to menop

hat determine the manifestation of a reduction in HDL pective effect in QR women. Anyway, since these results w

enopausal women is still relevant.In summary, our results show that postmenopausal wo

xhibited a reduction in the HDL inhibitory effect which wssociated to a decreased resistance to oxidation. In turas related to triglyceride-enrichment of HDL particles.ether, these results suggest that HDL from postmenopomen may not exert its antioxidant action efficiently, inendently from HDL cholesterol plasma levels.

cknowledgments

This study was supported by grants from the Univerf Buenos Aires (B089 and B069). The authors thanksenter for Studies in Gynecology and Reproduction.

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