Atherosclerosis 154 (2001) 475–483

The relationships between post-prandial lipaemia, endothelialfunction and oxidative stress in healthy individuals and patients

with type 2 diabetes

Richard A. Anderson a,*, Marc L. Evans b, Gethin R. Ellis a, J. Graham f, K. Morris e,Simon K. Jackson c, Malcolm J. Lewis d, Alan Rees b, Michael P. Frenneaux a

a Department of Cardiology, Wales Heart Research Institute, Uni6ersity of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UKb Department of Medicine, Wales Heart Research Institute, Uni6ersity of Wales College of Medicine, Cardiff, UK

c Department of Microbiology, Wales Heart Research Institute, Uni6ersity of Wales College of Medicine, Cardiff, UKd Department of Pharmacology, Wales Heart Research Institute, Uni6ersity of Wales College of Medicine, Cardiff, UK

e School of Biomedical Studies, Uni6ersity of Wales Institute, Cardiff, UKf Department of Biochemistry, Li6erpool John Moores Uni6ersity, Li6erpool, UK

Received 21 September 1999; received in revised form 13 January 2000; accepted 24 March 2000

Abstract

Post-prandial lipaemia (PPL) is a factor in atherogenesis and results in reversible endothelial dysfunction in healthy individuals.Oxidative stress and triglyceride (TG)-rich lipoproteins have been implicated. Type 2 diabetes (NIDDM) results in exaggeratedPPL. We attempted to delineate the mechanisms of PPL induced, endothelial dysfunction (EF) and oxidative stress in 12 NIDDMand 12 matched healthy subjects. Subjects underwent a fat tolerance test, with endothelial function assessed by flow-mediatedvasodilatation and oxidative stress measured by venous lipid-derived free radicals ex vivo and lipid peroxidation products over thepostprandial phase. Fasting TG, post-prandial hypertriglyceridaemia and the TG enrichment of all lipoproteins was significantlygreater in NIDDM. Post-prandial endothelial function inversely correlated with fasting HDL-C (r= −0.84, P=0.001) in boththe control and NIDDM groups. The deterioration in EF in the NIDDM group also correlated with TG enrichment of VLDLand LDL. PPL in both groups also resulted in increased oxidative stress. The increment in free radicals correlated with TGenrichment of VLDL in both groups and was, therefore, greater in NIDDM. Thus, PPL — with the production ofTG-enrichment of VLDL — results in endothelial dysfunction by an oxidative stress mechanism in both groups. The magnitudeis greater in NIDDM. Fasting HDL-C appears to contribute to the protection of the endothelium against this phenomenon.Hence, exaggerated PPL associated with reduced HDL-C may be important in the pathogenesis of vascular disease, particularlyin NIDDM. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Oxidative stress; Endothelial dysfunction; Post-prandial lipoproteins; Triglyceride-rich lipoproteins

www.elsevier.com/locate/atherosclerosis

1. Introduction

Post-prandial lipaemia (PPL) represents the state ofabsorption during which the TG metabolic capacity isunder challenge. The magnitude of PPL may be definedas the extent and duration of the rise in plasma TGafter a standard fatty meal, peaking at 4 h and inNIDDM lasting up to 12 h. After a fatty meal, healthysubjects demonstrate endothelial dysfunction for up to4 h, which can be attenuated by the antioxidant vitaminC [1], suggesting a possible free radical mediated mech-anism. Endothelial dysfunction was not observed how-

Abbre6iations: AUC, area under the curve; CAD, coronary arterydisease; EF, endothelial function; EPR, electron paramagnetic reso-nance; FFA, free fatty acids; FMD; flow mediated dilatation; FR,free radicals; HDL, high density lipoprotein; LDL, low densitylipoprotein; NIDDM, type 2 diabetes; PPL, post-prandial lipaemia;TBARS, thiobarbituric acid reacting substances; TG, triglycerides;UKPDS, UK Prospective Diabetes Study; VLDL, very low densitylipoprotein; WHO, World Health Organisation.

* Corresponding author. Tel.: +44-1222-744430; fax: +1-1222-743500.

E-mail address: raanderson@ukgateway.net (R.A. Anderson).

0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.PII: S 0 0 2 1 -9150 (00 )00499 -8

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483476

ever, with low fat meals [2], suggesting a role for TGand potentially TG-rich lipoproteins in thisphenomenon.

In the fasting state, subjects with elevated triglyce-rides but normal LDL-cholesterol levels exhibit en-dothelial dysfunction [3] and elevated fasting remnantlipoprotein levels are associated with impaired coronaryvasomotor function [4]. A significant relationship be-tween PPL and coronary artery disease (CAD) has beenreported in association with both normal and abnormalcarbohydrate metabolism [5]. TG-rich lipoprotein rem-nant particles have been independently associated withthe presence, severity and progression of atherosclerosis[6], with higher levels of remnant lipoprotein particlesin the fasting state being shown to predict future coro-nary events in subjects with CAD [7].

NIDDM is associated with a markedly increased riskof macrovascular disease, particularly affecting coro-nary artery disease (CAD). The UKPDS 33 demon-strated that intensive glycaemic control has nosignificant impact on macrovascular disease incidence[8]. The Paris Prospective Study demonstrated thatplasma triglyceride (TG) level was the most importantpredictor of coronary death in NIDDM [9]. This infersthat abnormal lipid handing in NIDDM may be morelikely to impart the large macrovascular risk associatedwith NIDDM. In NIDDM, there is a consistent, pro-longed and exaggerated post-prandial (PP) excursion inplasma TG with subsequent TG enrichment of lipo-protein particles [10], resulting in an ‘atherogenic’ lipidprofile (including abnormal PPL) and fasting endothe-lial dysfunction.

The object of this study was, therefore, to test thehypothesis that TG-rich lipoproteins, produced duringPPL, induce transient endothelial dysfunction and oxi-dative stress in healthy individuals and to compare thiseffect to NIDDM subjects, where there are decreasedantioxidant defences and an excess of these TG-richlipoproteins both in the fasting and post-prandial state.

2. Materials and methods

2.1. Subjects

Twelve consecutive patients with NIDDM (mean age47.3 years; range 35–53; seven men) (diagnosed onWHO criteria) with moderate glycaemic control (HbA1c

6.5–10%, mean 8.5%) were enrolled in this study andcompared with 12 age-matched healthy volunteers(mean age 43 years; range 30–63; five men). All sub-jects were non-smokers, normotensive and had no his-tory or clinical evidence of overt vascular disease; hadfasting cholesterol levels B6.5 mmol/l, and were nottaking aspirin, lipid lowering or hormone replacementtherapy, or supplemental vitamins. All women in both

groups were studied at the same time in their menstrualcycles (follicular phase). The diabetic group had asignificantly higher body mass index (32.294.1 vs.27.593.2, PB0.05). All NIDDM subjects were eitherdiet controlled or were on oral hypoglycaemic agents.Those subjects taking oral agents omitted their medica-tion on the morning of the test. Written informedconsent was obtained from all subjects and the localethics committee approved the study.

2.2. Protocol

Studies began at 09:00 after a 12-h overnight fast.Blood was drawn for serum total, LDL and HDLcholesterol, triglycerides, lipoprotein subfractionation,glycosylated haemoglobin, glucose, and venous FRmeasurements and brachial artery EF was measured.Each subject was then given a standard fat tolerancetest meal [6], in the form of homogenised milk-shakecontaining 80 g of saturated fat, with a calorific valueof 1480 Kcal. Lipid profiles and plasma glucose wererepeated at 2-h intervals over the following 8 h. Lipo-protein subfractions were assessed at baseline and 4 and8 h post-prandially, with lipid-derived FRs and EF atbaseline and during the peak lipaemic phase, 4 h fol-lowing meal ingestion.

2.3. Ultrasound of the brachial artery

Changes in brachial artery diameter in response toreactive hyperaemia, (flow mediated dilatation; FMD)were measured non-invasively using a high-resolutionultrasonic wall-tracking system, (Valdirec Wall-trackSystem™, resolution 93 mm) as previously describedand validated [11]. Studies were performed at a temper-ature of 21–23°C, with subjects supine with their armheld outstretched on a pneumatic cushion. Baselinemeasurements of internal brachial artery diameter andblood flow were taken after \10 min of supine rest.Reactive hyperaemia was produced by releasing a pae-diatric sphygmomanometer wrist cuff inflated to sys-tolic pressure plus 50 mmHg for 5 min. Blood flow wasrecorded from 15 s until before 90 s after cuff release.Internal brachial artery diameter was measured 60 safter cuff release. Further measurements were madeafter 15 min to confirm vessel recovery. Repeat mea-surements of brachial artery diameter and blood flowwere taken 3 min after sublingual glyceryl trinitate(GTN) (400 mg). Flow and GTN-mediated vasodilata-tion data are presented as the percentage diameterchange from baseline in the brachial artery both pre-prandially and 4 h post-prandially.

2.4. Lipoprotein separation

Chylomicron-free plasma was analysed to assess

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483 477

compositional variation in the three major lipo-protein classes (VLDL, LDL and HDL) during PPLin both groups. Lipoproteins were fractionated inself-generated gradients of iodixanol, as pre-viously described [12]. Briefly, chylomicron-freeplasma was mixed with Liposep™ to produce aniodixanol concentration of 12% (w/v) and 2.8 ml(overlayered with 0.3 ml of Hepes-buffered saline)centrifuged at 350 000×g for 3 h in a BeckmanTLN100 rotor. The gradients were collected by tubepuncture in 20 fractions whose volumes weremeasured. Location of HDL, LDL and VLDL withinthe gradient was confirmed by agarose gel elec-trophoresis.

2.5. Lipid assay and biochemical measurements

Cholesterol and TG concentrations (mmol/ml) ineach gradient fraction were measured spectrophoto-metrically using standard diagnostic reagent kits[13,14]. Amounts of cholesterol and TG (mmoles) ineach gradient fraction were calculated and the choles-terol and TG content of HDL, LDL and VLDLcomputed by summation of the appropriate fractions(as determined by agarose gel electrophoresis) [12].Plasma glucose was measured by a hexokinase-basedtechnique and HbA1c via enzyme immunoassay [15].In appropriate samples, HDL-C concentrations werealso measured after precipitation of apolipoprotein Bwith phosphotungstate/magnesium [16], as an internalcontrol.

2.6. Measurement of 6enous free radicals

The formation of lipid radicals follows oxidativedamage in vivo [17]. In this study, we used the tech-nique of EPR spectroscopy by ex vivo spin trappingto investigate the formation of free radicals in venousblood [18,19]. Reactive free radicals decay too quicklyto be observed under normal laboratory conditions.However, they can be ‘trapped’ by some chemicals(‘spin traps’) to form long-lived stable radical adductswhich can be observed and identified by EPR spec-troscopy. While such spin traps are too toxic to beinjected into humans, they can be conveniently usedto trap free radicals in blood samples ex-vivo. Thistechnique has recently been used and validated toidentify lipid-derived free radicals generated post-exer-cise in humans [20] and in the coronary sinus of pa-tients undergoing elective cardioplegia [21] andcoronary angioplasty [22]. Reactive oxygen species in-duce lipid peroxidation producing secondary lipidradicals, which are spin-trapped and detectable inblood samples ex vivo [20]. Lipid derived free radicalswere measured ex vivo in venous blood. Venousblood was taken directly into a sealed glass tube,

containing 1 ml of the spin trap, a-phenyl N-tert-bu-tyl nitrone (PBN) in saline (0.125 mol/l). Followingcentrifugation, the PBN adduct was extracted withtoluene, dried under nitrogen gas and reconstituted indegassed chloroform. EPR spectra were recorded on aVarian E104 spectrometer operating at 9.1 GHz at 10mW power, 1 Gauss modulation, 0.25 s time constantand 100 G scan range. EPR spectral parameters wereobtained from data acquisition and processing usingin-house EPR computational software. EPR spectralpeak heights were taken as a good correlation ofspin-adduct concentration after confirmation of peakto peak line width conformity and double integrationon samples. The separation between sets of peaks inthe EPR spectrum (coupling constant) was used toidentify the lipid radical species trapped. These wereidentified as carbonyl (L−) and alkoxyl (LO−) freeradicals.

2.7. Thiobarbituric acid-reacti6e substances (TBARS)

The plasma level of TBARS, an indicator of lipidperoxides in plasma was determined as previously de-scribed [23], using a spectrophotometric assay (Oxis).The results are expressed as mol/l and give values formalonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE) combined. The reproducibility of this assay us-ing the above protocol over the period analysing oursamples is S.E.M. B5%.

2.8. Statistics

Statistical analysis was performed on Minitab (ver-sion 12). Conventional methods were used for calcu-lating mean, S.D. and checks for normal dis-tribution. Group differences in continuous variableswere determined by two-tailed t-test. Statistical sig-nificance for differences in continuous variables be-tween groups was tested by one way analysis ofvariance. As a measure of plasma glucose and thetotal amounts of lipid and lipoprotein present inplasma during the 8-h post-prandial period, areas un-der the curve (AUC) were calculated for plasma con-centrations without subtraction of baseline values.AUC was calculated using Simpson’s integration rule[24]. This measure of AUC, without subtraction ofbaseline values was used since the principal aim ofthis study was to investigate the effect of post-prandial lipaemia on measures of endothelial functionand oxidative stress. A two sample Mann–WhitneyU-test was used for non-parametric data where iden-tified in the results. Multiple-regression analysis wasused to study the independent relationship betweenvariables with logarithmic transformation of skeweddata. Statistical significance was inferred when PB0.05.

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483478

Table 1Glucose and lipid parameters in NIDDM and control subjects,fasting, 4 and 8 h after consumption of a fatty meala

Baseline 4 h 8 h

2.290.7*Plasma TG controls 1.3590.31.3390.59.198.2*,† 4.893.6*,†Plasma TG NIDDM 3.592.1†

5.790.75.790.75 5.691Cholesterol–controls5.991.1Cholesterol–NIDDM 6.191.1 691

3.290.4LDL-C–controls 3.190.53.290.253.790.73.790.8 3.690.6LDL-C–NIDDM

1.3990.3HDL-C–controls 1.3890.3 1.3990.3HDL-C–NIDDM 1.0490.22† 0.9990.25† 1.0290.21†

4.890.24.790.4 591Glucose–controls11.993.3†Glucose–NIDDM 11.293.4† 9.492.7†

a All measurements in mmol/l.* Denotes PB0.05 versus baseline; † Denotes PB0.05 versus con-

trols at the same time points.

Table 3Distribution of cholesterol and TG amongst major lipoprotein sub-classes in both groups in the fasting statea

HDLVLDL LDL

0.790.14 0.1590.110.3190.11Controls–TG content0.5790.48*2.6891.12*NIDDM–TG content 0.9790.44*

4.591.71.0691.25Controls–cholesterol 1.8590.9**2.0691.21*NIDDM–cholesterol 1.1490.94.3191.16

a Data expressed as mean9S.D. Lipoprotein cholesterol and TGcontent represented in mol/ml.

* Denotes PB0.05 NIDDM versus controls at baseline.** Denotes PB0.05 controls versus NIDDM at baseline.

TG rich lipoproteins, with AUC for TG content of HDL,LDL and VLDL being significantly greater in theNIDDM group than controls (Table 3). As expected, TGenrichment of HDL-C and VLDL-C correlated withAUC TG, r=0.55, 0.52; P=0.002 and P=0.03, respec-tively.

Cholesterol distribution amongst the lipoproteins re-mained unchanged at all post-prandial time points inboth groups. The cholesterol content of VLDL in theNIDDM subjects remained significantly elevated com-pared to controls, whereas, the cholesterol content ofHDL remained significantly higher in controls than theNIDDM subjects (Table 4).

3.2. Endothelial function

Fasting FMD in healthy controls demonstrated nosignificant correlation with any measured metabolicparameter. There was a significant decrement in FMDpost-prandially in controls (Table 5). In the diabeticsubjects, there was lower fasting FMD, which signifi-cantly deteriorated post-prandially. Baseline brachialartery diameters were 3.4990.23 and 3.3890.42 mm inthe control and NIDDM groups, respectively. In theNIDDM group, FMD in the fasting state demonstrateda significant correlation with a variety of parametersincluding total, HDL and LDL-cholesterol, plasma TG,chronic glycaemia and TBARS (Table 6). No correla-

3. Results

3.1. Post-prandial lipaemia

There were no significant quantitative changes in total,LDL or HDL cholesterol or plasma glucose during thePP period in either group, although, as expected, plasmaglucose remained consistently higher in the NIDDMgroup compared to controls (Table 1). Table 1 alsoillustrates the changes in plasma TG over 8 h; plasma TGincreased significantly in response to the meal, with peaklevels attained after 4 h and in the NIDDM groupremaining elevated after 8 h. The total AUC-TG (mmol/lper 8 h) was significantly higher in the NIDDM groupthan in controls (191.89108 (NIDDM) vs. 64.25910.75 (controls), PB0.001), fasting plasma TG levelscorrelating with total AUC in both groups (r=0.883,PB0.001 (NIDDM), r=0.71, P=0.003 (controls)).

Table 2 illustrates the compositional changes in HDL,LDL and VLDL as a result of PPL. There was significantTG enrichment of all lipoproteins in the NIDDMsubjects at both 4 and 8 h following the fatty meal. Inthe healthy subjects, there was TG-enrichment of VLDL.PPL in the NIDDM group resulted in the production of

Table 2Cholesterol and TG distribution in the major subclasses during PPL in both groupsa

HDLLDLVLDL

8 h8 h 4 h 8 h4 h 4 h

0.1290.050.1490.070.3190.170.3490.14Controls–TG 0.7290.590.8690.564.7392.26* 3.3291.65* 1.0490.78* 0.9290.66* 0.6890.65* 0.6690.61*NIDDM–TG

4.3291.634.191.680.9991.031.1191.02Controls–cholesterol 1.7190.76**1.7290.81**2.5491.11*NIDDM–cholesterol 1.2190.521.1590.514.5191.764.7891.812.3991.08*

a Data is expressed as mean9S.D. Lipoprotein cholesterol and TG content is represented as mol/ml.* Denotes PB0.05 NIDDM versus controls.** Denotes PB0.05 controls versus NIDDM.

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483 479

Table 4Post-prandial AUC for TG content (mol/ml per 8 h) of lipoproteinsin both groups, following a fatty meala

AUC AUC LDL-TG AUC HDL-TGVLDL-TG

NIDDM 1.2290.35*1.7990.22* 1.0190.43*0.5690.271.1390.41 0.8190.28Controls

a Data is expressed as mean9S.D.* Denotes PB0.05 NIDDM\controls, when analysed using two

sample Mann–Whitney U-test.

Table 6Correlation of fasting FMD with various parameters in the NIDDMgroup

Correlation coefficient P value

0.912Age (years) −0.026−0.375HbA1c (%) 0.094

Total cholesterol (mmol/l) −0.53 0.014*0.005*−0.55TG (mmol/l)

−0.52Glucose (mmol/l) 0.016*Insulin (Iu/l) −0.23 0.118

0.006*LDL-C (mmol/l) −0.58HDL-C (mmol/l) 0.034*0. 47

−0.32 0.16EPR peak heights (U)0.008*TBARS (mol/l) −0.72

* PB0.05

tion was noted with compositional analyses of lipo-protein content.

3.3. Post-prandial endothelial function

In healthy controls, the reduction in FMD associatedwith PPL correlated inversely with fasting HDL-C lev-els (r= −0.84, P=0.001). There was no correlationwith any other measured variable including the magni-tude of post-prandial hypertriglyceridaemia. In theNIDDM group, the reduction in FMD associated withPPL was related to several parameters (Table 7). Themagnitude of post-prandial hypertriglyceridaemia(AUC TG), fasting plasma TG levels and the TGenrichment of both VLDL and LDL demonstrated thestrongest correlation with reduction in FMD in theNIDDM group. Total plasma HDL-C however, corre-lated inversely with the reduction in FMD during PPLin both groups. Post-prandially, there were no signifi-cant changes in endothelium independent vasodilata-tion in either group (Table 5).

3.4. Oxidati6e stress

Lipid-derived free radicals increased post-prandiallyin both controls (2.490.1 to 3.390.2, PB0.05) andNIDDM (2.2590.4 to 4.591.3 U, PB0.05), the in-crease being greater in NIDDM (PB0.05) (Table 5).Analysis of the EPR spectra from spin-trapped radicalsderived from venous blood samples suggested that theradicals trapped were alkoxyl radicals (coupling con-

stants aN=13.9 Gauss, abH=2.2 Gauss) and car-bonyl radicals (aN=14.1 Gauss; abH=4.0 Gauss).These assignments, which agree with previous studies[20], suggest that these radicals were derived from de-composition of lipid hydroperoxides in the extracellularcompartment. Detection of secondarily formed lipidfree radicals strongly supports the presence of peroxida-tive damage. Fasting markers of lipid peroxidationwere significantly greater in NIDDM subjects com-pared to controls, whilst fasting lipid derived free radi-cals were similar in both groups. (Table 5) In NIDDM,fasting TBARS positively correlated with HbA1c,VLDL-C (r=0.535, PB0.05) and LDL-C (r=0.529,P=0.05). There was also a negative correlation withfasting FMD (r= −0.72, PB0.05). There was no rela-tionship between fasting TBARS and any measuredvariable in control subjects.

3.5. Correlation between PPL and oxidati6e stress

PPL in both groups resulted in augmented oxidativestress, the rise in free radicals correlated significantlywith the extent of VLDL TG enrichment, AUC forVLDL-TG content (r=0.59, P=0.02, NIDDM), (r=0.55, P=0.03, controls). Both groups also exhibitedsignificant increases in TBARS post-prandially, indicat-

Table 5Oxidative stress and vascular data

EPR height (arbitrary units) TBARS (mol/l) FMD (%) GTN-induced vasodilatation (%)

Controls11.591.12.490.1 3.592.1 6.391.3Baseline10.890.83.390.2* 4.7791.4*4 h 6.293.6*

NIDDM2.6590.96†2.390.4 11.190.7Baseline 7.593.6†

4.5391.3*,†4 h 10.5919.693.5*,† 1.4591*,†

* Denotes PB0.05 versus baseline.† Denotes PB0.05 versus controls.

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483480

Table 7Correlation of PPL and metabolic parameters with the reduction inFMD in the NIDDM group

Correlation P valuecoefficient

0.160.15Age (years)0.52Fasting TG (mmol/l) 0.015*0.51AUC plasma TG 0.03*

0.790.060Fasting insulin (Iu/l)0.33HbA1c (%) 0.14

0.033*−0.49AUC HDL-C (mmol/l/8 h)0.099AUC LDL-C (mmol/l/8 h) 0.370.330.22AUC total cholesterol (mmol/l/8 h)

0.26 0.22Increase in oxidative stress(arbitrary units)

−0.33AUC HDL-TG content (ml/ml/8 0.11h)

AUC LDL-TG content (ml/ml/8 h) 0.03*0.490.58 0.02*AUC VLDL-TG content (ml/ml/8

h)

* PB0.05

rich VLDL may induce oxidative stress and isassociated with endothelial dysfunction after a fattymeal.

The present study shows fasting endothelial dysfunc-tion in NIDDM subjects, a finding that correlates withglucose, plasma TG, total and LDL-cholesterol andinversely with HDL-C. This is consistent with previousdata indicating impaired agonist-induced vasodilatationcorrelating with plasma TG levels and HDL/totalcholesterol levels [25]. Secondly, the deterioration inFMD in the NIDDM group most strongly correlateswith TG enrichment of both VLDL and LDL (thepotential atherogenic molecules). Thirdly, PP oxidativestress correlated strongly with TG-enrichment ofVLDL. These observations suggest that after a fattymeal, the atherogenic insult may be TG-rich VLDL andLDL, involving endothelial dysfunction and oxidativestress in NIDDM. The elevations of alkoxyl and car-bonyl free radicals post-prandially can cause endothe-lial dysfunction by a number of mechanisms. They caninhibit cellular processes and react rapidly with nitricoxide (NO) to terminate chain propagation in vivo [26]and can also subsequently form reactive nitrogen spe-cies [27], resulting in blunted flow-mediated vasodilata-tion. This transient increase in oxidative stress issuperimposed on existing elevations in lipid peroxida-tion, which relates to fasting endothelial function.

The augmented oxidative stress seen in diabetics maybe the result of greater free radical production thancontrols or decreased antioxidant defenses. In subjectswith impaired glucose tolerance (IGT) and NIDDM,there is a deficit in antioxidant capacity, particularlyvitamin C and urate [28]. Depressed vitamin E levelshave also been found in diabetic patients with increasedlipid peroxides [29] and when associated with hyper-triglyceridaemia [30]. Our TBAR data concurs withthese findings. In vitro data would seem to indicate thatPP lipid changes (via TG enriched VLDL) increase theoxidative milieu present in the arterial wall. Monocytesand polymorphonuclear cells release more superoxideanions when exposed to plasma from hypertriglyceri-daemic patients [31,32], a phenomenon also positivelycorrelated with plasma TG and negatively with HDL.Furthermore, leucocyte activation secondary to hyper-triglyceridaemia may contribute to the increased oxida-tive stress seen in NIDDM [33].

In our study, the source of the increase in oxidativestress seen is unclear at present. We used two measuresof oxidative stress, EPR spectroscopy measurementsand TBARS. In the fasting state, direct measurementsof carbonyl and alkoxyl radicals were similar to normalcontrols in the fasting state, indicating that ‘active’ lipidperoxidation was similar at baseline. However, TBARSgive evidence of previous lipid peroxidation with stableend product accumulation and were significantlygreater in fasting NIDDM subjects than those of con-

ing increased oxidative stress post-prandially. Therewere no correlations between the post-prandial rise inoxidative stress and the magnitude of PPL as defined byAUC for plasma TG in both groups. Due to thecomplex nature of the interaction between PP lipidmetabolism and its effect on FMD in NIDDM, multi-ple regression analysis was applied to assess the associa-tions between FMD and measured variables. In astepwise model, the change in FMD during PPL wasassessed as the dependent variable against all measuredfactors. The change in oxidative stress, HbA1c, theAUC for plasma TG and for plasma glucose werefound to be significant determinants (P=0.002) of theobserved reduction in FMD, accounting for 64.2% ofobserved variation in the change in FMD. Using fast-ing FMD as the dependent variable, age, fasting TG,HbA1c and fasting LDL-C levels (PB0.0001) were seento be the most significant determinants, accounting for75.7% of variation.

4. Discussion

Previous reports of endothelial dysfunction in associ-ation with PPL in healthy individuals [1] have suggestedthat TG-rich lipoproteins and oxidative stress may beinvolved. This study demonstrates that in healthy con-trols post-prandial lipaemia induces endothelial dys-function, which is inversely correlated with fastingHDL-C, but not with the magnitude of post-prandialhypertriglyceridaemia. Therefore, HDL-C may have aprotective role against PP endothelial dysfunction. Sec-ondly, venous lipid-derived free radicals increase inassociation with endothelial dysfunction, which corre-lates with VLDL TG enrichment, implying that TG-

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483 481

trols, consistent with previous data [29,30]. The PP risein carbonyl and alkoxyl radicals indicates a transientrise in reactive species at the 4-h time point, accompany-ing ED. This suggests an acute oxidative milieu post-prandially, a finding substantiated by the rise inTBARS. This data supports in vitro data indicating thatoxidative damage caused by lipid components in rem-nant lipoproteins, especially peroxidized phospholipids,deteriorates cell surface membranes and may be partlyresponsible for remnant induced impairment of en-dothelial-dependent relaxation [33]. In the presentstudy, the free radicals were found to increase post-prandially and this was found to be more significant inthe NIDDM group. Our study showed that this rise infree radicals was associated with increased triglyceridelevels in the lipoprotein classes and not with plasmatriglycerides or other lipids per se. We do not believe,therefore, that the increase in free radicals detected wassimply a reflection of increased lipid load. Furthermore,our recent findings have shown that plasma lipid-derived free radicals do not show a simple correlationwith lipid content (unpublished data).

We suggest that oxidative stress results from TGenrichment of lipoproteins, as indicated by the correla-tion with TG enrichment of VLDL. Oxidative stress isprobably causally related to the post-prandial impair-ment of EF, since antioxidants attenuate this transientimpairment of EF in normal subjects in previous studies[1] and both our measures of oxidative stress are ele-vated post-prandially in both groups. In our study, TGenriched VLDL was a significant determinant of thedeterioration in PP endothelial dysfunction, which sup-ports fasting observations by Lewis et al. [3] but is atvariance with the observation of normal EF in patientswith profound hypertriglyceridaemia, (type V hyperlipi-daemia) [34]. These findings suggest that functioninglipoprotein lipase is required for the endothelial damageby these lipoproteins. AUC for plasma glucose andbasal metabolic control (HbA1c) were also significantdeterminants of PP endothelial dysfunction. These ob-servations are supported by data demonstrating en-dothelial dysfunction with acute hyperglycaemia [35].Poor overall metabolic control may play a role in PPendothelial dysfunction by several potential mecha-nisms. Increased supply of atherogenic free fatty acids(FFA’s) including production of reactive aldehydes,enhanced production of advanced glycation end prod-ucts and glycation of lipoproteins.

PPL affects lipoprotein composition by TG enrich-ment. LDL-cholesterol enriched with TG results insmaller dense LDL-C particles and large VLDL-C par-ticles. These have a prolonged endothelial residence timeand thus, increased propensity to both absorption bymacrophages and endothelial cell injury [36], a viewsupported by the present data. VLDL particles enrichedwith TG are toxic to endothelial cells in vitro. Recent

data show that VLDL particle exposure from hyper-triglyceridemic patients (equivalent to post-prandial lev-els) results in NF-kB activation in endothelial cells after6 h, implying that TG-rich lipoproteins and oxidativestress are intimately linked [37]. In addition, lipid en-riched HDL particles have an impaired endothelialprotective capacity in hypertriglyceridaemia, in particu-lar diminished antioxidant potential, particularly theability to shuttle reactive hydroperoxides from the en-dothelium to the liver for excretion [38]. HDL inhibitsendothelial cell injury mediated by TG-rich lipoproteinremnants [39], a factor which may explain our observa-tion of reduced FMD and fasting HDL-C levels in bothgroups.

TG-rich lipoproteins can also influence EF indirectlyby changing the distribution of LDL particles to asmaller, denser LDL population. Plasma TG is thedominant factor in reducing LDL size [40], a factor thatmay be important chronically. An acute increase inFFA fluxes in the PP phase can contribute to theendothelial dysfunction and oxidative stress seen. Thiscould be mediated by either facilitation of excess choles-terol-rich lipoproteins entering the intima [41,42] or bydecreasing the endothelial protective properties of albu-min [43], as VLDL causes endothelial cell toxicity whenFFA/albumin ratio is elevated [42]. FFAs have alsobeen shown to impair cultured endothelial cell respon-siveness to NO and prostacyclin [44]. Furthermore, theyhave been shown to increase markers of lipid peroxida-tion [42] and therefore, may underly our observations ofgreater deterioration in endothelial dysfunction andelevated TBARS seen in our diabetic subjects. Further-more, it may explain the non-significant fall in endothe-lial independent vasodilatation post-prandially in bothgroups.

This study shows that free radicals increase post-prandially in normal individuals. HDL may offer pro-tection against the transient impairment of endothelialfunction by TG-rich VLDL lipoproteins during PPL. InNIDDM subjects where fasting ED is present, PPLappears to result in further decrements in endothelialfunction, which may be related to TG-rich VLDL, LDLand a greater increase in oxidative stress. These phe-nomena may be related to the compounding effects ofhyperglycaemia and dyslipidaemia. Therapeutic inter-ventions aimed at decreasing TG-VLDL and oxidativestress post-prandially may, therefore, attenuate en-dothelial dysfunction and atherogenesis in both healthysubjects and appears particularly important in NIDDM.

Acknowledgements

We would like to thank the British Heart Foundationwho supported this study and sponsored RAA, GREand MPF.

R.A. Anderson et al. / Atherosclerosis 154 (2001) 475–483482

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