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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.33 n.2 Santiago  2000

http://dx.doi.org/10.4067/S0716-97602000000200010 

Lipid peroxidation and antioxidants in hyperlipidemia
and hypertension

PATRICIA MORIEL1, FRIDA L PLAVNIK2, MARIA T ZANELLA2, MARCELO C
BERTOLAMI3, DULCINEIA SP ABDALLA1

1Department of Clinical and Toxicological Analyses, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil.
2Department of Nefrology, Hospital do Rim, Universidade Federal de São Paulo.
3Instituto Dante Pazzanese de Cardiologia, São Paulo, Brazil

ABSTRACT

Lipid peroxidation and lipid-derived oxidized products have been implicated in the pathogenesis of a variety of human diseases. To clarify the role of oxidative stress in essential hypertension and hypercholesterolemia the in vitro oxidative susceptibility of LDL, the antioxidant status and the lipid peroxide content of blood plasma were examined in hypercholesterolemic (HC), hypertensive (H), hypercholesterolemic/hypertensive (HH) and normolipidemic/normotensive subjects (N). Plasma ascorbate and lipid-soluble antioxidants were lower, while LDL oxidizability, CE-OOH and TL-OOH were higher in H, HC, and HH groups than in the N group. No difference was observed among groups for PL-OOH and isoprostanes. In summary, the results show that: 1) lipid- and water-soluble antioxidants are lower in hypercholesterolemic and hypertensive patients as compared to normal subjects, whereas the lipid peroxide content and the LDL susceptibility to oxidation were higher; 2) total cholesterol, LDL-cholesterol, apoB and CE-OOH were negatively correlated with the content of a-tocopherol; 3) there was a positive correlation between the content of lipid-soluble antioxidants and the resistance of LDL to oxidation; and 4) CE-OOH and TL-OOH were positively correlated with total cholesterol and LDL-cholesterol.

KEY TERMS: Lipoprotein oxidation, lipid peroxides, isoprostanes, antioxidants, atherosclerosis, oxidative stress

INTRODUCTION

Hypercholesterolemia is a significant risk factor for atherosclerosis (Sloop, 1999). However, there is great inter-individual variation in the development of atherosclerotic lesions at a given cholesterol level. The reasons for this phenomenon are not yet completely clear. It has been suggested that beyond the known risk markers for cardiovascular disease, oxidation of LDL could play an important role in the development and progression of atherosclerosis (Esterbauer et al, 1992; Witzum and Steinberg, 1991; Berliner et al, 1995 ; Lynch and Frei, 1994). It may be clinically relevant whether an elevated blood cholesterol concentration is associated with enhanced LDL oxidation, which would represent an additional risk.

The view that an excess of lipid peroxidation products is present and relevant in the pathogenesis of human hypertension or in hypertension-induced damage has not yet received definitive support. Evidence suggests that hypertension is associated with enhanced oxidative stress, although it is not yet clear whether this phenomenon occurs before or after the development of hypertension (Kumar and Das, 1993). Ascorbic acid and a-tocopherol represent the major water- and lipid-soluble antioxidants in the several tissues and organs and they protect against oxidative damage (Frei, 1991; Frei et al, 1988; 1989; Traber et al, 1992). Moreover, ascorbic acid is reported to exert an antihypertensive effect, and epidemiological studies have shown that a strong correlation exists between a-tocopherol ingestion and reduced risk of cardiovascular diseases (Marchioli, 1999; Akpaffiong and Taylor, 1998).

Conflicting results are reported concerning the LDL oxidation, lipid peroxidation and antioxidants in hypercholesterolemia and hypertension. This may be due primarily to the different nutritional habits of the groups studied, as well as the distinct analytical methodologies used to evaluate these variables. In the present study the ex vivo LDL oxidation, as well as the content of lipid peroxides and the concentrations of water- and lipid-soluble antioxidants were determined in blood plasma of hypercholesterolemic and hypertensive patients in comparison with normolipidemic subjects.

METHODS

Subjects

Hypercholesterolemic normotensive patients (HC; n=18; total cholesterol >240 mg/dL; systolic <140 mmHg and diastolic <90 mmHg blood pressure), normolipidemic hypertensive patients (H; n=11; total cholesterol <200 mg/dL; 140< systolic <179 mmHg and 90< diastolic <109 mmHg blood pressure), and hypercholesterolemic hypertensive (HH; n=9; total cholesterol >240 mg/dL; 140< systolic <179 mmHg and 90< diastolic <109 mmHg blood pressure) were screened at the Instituto Dante Pazzanese de Cardiologia, São Paulo, SP, Brazil and at the Hypertension Ambulatory of the Hospital do Rim, Universidade Federal de São Paulo (UNIFESP). Exclusion criteria included myocardial infarction, unstable angina, smoking habits, diabetes, significant valvular heart disease, use of antioxidant vitamins, cholesterol-lowering therapy and estrogen replacement therapy. Normolipidepic subjects were normal healthy volunteers with similar exclusion criteria (N; n = 12; total cholesterol <200 mg/dL; systolic <40 mmHg and diastolic <90 mmHg blood pressure). The study was approved by the Ethics Committees of the Instituto Dante Pazzanese de Cardiologia and Universidade Federal de São Paulo. All subjects provided written informed consent, and the study was conducted in conformance with the principles embodied in the Declaration of Helsinki.

Biochemical Analyses

Blood samples were collected in tubes containing EDTA, and the blood plasma was immediately separated. The concentrations of total cholesterol, triglyceride and high density lipoprotein (HDL)-cholesterol were determined by enzymatic analyses using commercial kits (Biosystem, Barcelona, Spain). LDL-cholesterol was calculated by the Friedwald equation (Friedwald et al, 1972). Isoprostane concentration was determined by ELISA using a commercial kit (8EPGF2 Immunoassay Kit, Oxford Biomedical Research, USA). Apolipoprotein B (apoB) was determined by nephelometric immunoassays (Dade Behring, São Paulo, Brazil).

The content of cholesteryl ester (CE-OOH)-, trygliceride (TL-OOH)- and phospholipid (PL-OOH)-derived hydroxy/hydroperoxides in plasma was determined by HPLC (LC10 Shimadzu Corp., Tokyo, Japan) using a C-8 Inertsil column (GL Sciences, Inc., Japan) and eluted with methanol containing 20 mM lithium acetate at a flow rate of 1.0 ml/min. through a LC-10AD pump (Shimadzu Corp., Tokyo, Japan) and a diode array detector (SPD-M10A, Shimadzu Corp., Tokyo, Japan) set at 234 nm. Peaks were identified using external standards prepared from their respective photosensitized oxidation products by the procedure previously described (Terao et al, 1985; Yamamoto et al, 1987) and quantified using the Class-LC10 software package (Shimadzu Corp., Tokyo, Japan). The samples were first extracted with methanol:hexane (1:3, v/v). The contents were vortexed for 2 minutes and centrifuged at 2500 rpm for 10 minutes for phase separation. The hexane phase was collected and evaporated with N2. To the methanol phase 3 ml of chloroform were added for PL-OOH extraction. The contents were vortexed by 2 minutes and centrifuged at 2500 rpm for 10 minutes for phase separation. The chloroform phase was collected and evaporated under N2. The residue was dissolved with mobile phase, filtered through a 22 mm Millex filter (Millipore, São Paulo, Brazil) and analyzed by HPLC.

The content of a-tocopherol, ß-carotene, lycopene (lipid-soluble antioxidants) and ascorbate in blood plasma were determined by HPLC using a C-18 ODS column (Shimadzu Corp., Tokyo, Japan). Lipid-soluble antioxidants were extracted from plasma by adding to each sample (250 mL), 4% sodium dodecyl sulfate (250 mL) and methanol:hexane (1:3, v/v). The contents were vortexed for 2.5 minutes and centrifuged at 2500 rpm for 10 minutes for phase separation. The hexane phase was collected and evaporated under N2. The residue was dissolved in mobile phase (Thurnham et al, 1988). For ascorbate determination 2.0 mg/dL DL-Dithiothreitol (Sigma Chem. Co., St. Louis, MO, USA) was added to the plasma to preserve the ascorbate in the reduced form (Margolis et al, 1990). Plasma proteins were precipitated with 0.75% perchroric acid by vortexing the mixture for 1 minute and centrifuging it at 2500 rpm for 10 minutes, at 4"-C for phase separation (Pachla and Kissinger, 1979). After extraction, the samples were filtered through a 22 mm Millex filter (Millipore, São Paulo, Brazil) and analyzed by HPLC. The lipid soluble antioxidants were eluted with methanol:acetonitrile:chroroform (35:35:30,v/v/v), containing 20 mM lithium perclorate at a flow rate of 1.0 ml/min. Ascorbate was eluted with sodium acetate trihydrate (0.04M), n-decylamine (1mL) and EDTA (0.200 mg/dL) in water (pH = 5.0), at a flow rate of 0.8 ml/min. Detection was made electrochemically (potential = + 600 mV, L-ECD-6A, Shimadzu Corp., Tokyo, Japan) and by UV absorption. Peaks were identified using external standards (Sigma Chem. Co., St. Louis, MO, USA) and quantified from their electrochemical signal.

The kinetics of LDL oxidation was monitored by incubating dense LDL (1.045<density <1.063; 0.5 mg of protein/mL) with 60 mM CuN2+ at 37ºC and following the absorbance in 234 nm for analyzing the lag time, lag rate, log rate and peak time (Esterbauer et al, 1992).

Statistical Analysis

Data are represented as mean ± SEM. The statistical analysis was done using a non-parametric test (Wilcoxon) and the statistical significance was defined as p<0.05. The correlations were made using Pearson Product Moment Correlation (Sigma Stat software) and the statistical significance was defined as p<0.05.

RESULTS

The concentrations of cholesterol and triglycerides of blood plasma and lipoproteins, apo B, as well as the age, sex and blood pressure of the groups are shown in Table I. The cholesterol of blood plasma and LDL were increased in hypercholesterolemic patients in comparison with hypertensive and normolipidemic groups. Lag phase and peak time were significantly shorter in hypercholesterolemic and hypertensive patients than in normolipidemic controls (Fig. 1). The cholesteryl ester (CE-OOH)- and trygliceride-derived (TL-OOH) hydroxy/hydroperoxydes were higher in the blood plasma of hypercholesterolemic and hypertensive patients than in normolipidemic/normotensive subjects, although no difference was found for the phospholipid-derived hydroxy/hydroperoxides among the studied groups (Fig. 2). The a-Tocopherol, ß-carotene, lycopene and ascorbate contents were significantly lower in both hypercholesterolemic and hypertensive patients than in normotensive/normocholesterolemic group (Figs. 3A, 3B, 3C). Isoprostane (Fig. 4) was not significantly different among the groups. The correlation analysis (Table II) showed that 1) total cholesterol, LDL-cholesterol, apoB and CE-OOH were negatively correlated with the content of a-tocopherol; 2) there was a positive correlation of a-tocopherol and lycopene concentrations with the resistance of LDL to oxidation; 4) CE-OOH and TL-OOH were positively correlated with total cholesterol and LDL-cholesterol.

TABLE I

Age, sex, blood pressure and lipid profile of hypercholesterolemic (HC), hypertensive (H), hypercholesterolemic/hypertensive (HH) and normolipidemic/ normotensive subjects (N).


HC 
HH 

age 
48.4 ±7.0 
55.8± 11.1 
57.8± 9.7 
55.0 ±1.41 
sex (M/F) 
3/8 
7/11 
4/7 
3/5 
blood pressure 
systolic 
119 ±.4 9.4 
121.3±15.8 
148.3±24.8 
146.6±35.1 
diastolic 
75.0± 8.0 
70.9±7.4 
90.8±10.2 
93.3±11.5 
Cholesterol (mg/dL) 
166.7± 23.7 
314.5±76.9* 
178.4± 11.4 
261.1±16.0* 
VLDL-cholesterol (mg/dL) 
24.2± 14.6 
24.5 ±11.5 
23.3±12.5 
31.2±16.9 
LDL-cholesterol (mg/dL) 
104.5± 35.3 
246.7±88.8* 
111.5±17.0 
186.4±43.1* 
HDL-cholesterol (mg/dL) 
38.8 ±12.4 
36.7±11.9 
40.3±15.7 
31.2±15.7 
Triglycerides (mg/dL) 
120.9±73.2 
122.8±57.7 
108.9±29.6 
135.4±68.3 
Apo B (mg/dL) 
84.8 ±15.1 
152.8±34.1* 
96.6±20.9 
125.2±38.6* 

The results are mean S.E.M.; * = statistically significant in relation to N

FIGURE 1. - In vitro oxidability of LDL monitored by lag time and peak time. Hypercholesterolemic normotensive (light gray), hypertensive norcholesterolemic (medium gray), hypercholesterolemic hypertensive (white) and normolipidemic normotensive subjects (black). * Significant in relation to normolipidemic normotensive group. FIGURE 2. Cholesteryl ester (CE-OOH), triglyceride (TL-OOH) and phospholipid (PL-OOH) peroxides in blood plasma. Hypercholesterolemic normotensive (light gray), hypertensive norcholesterolemic (medium gray), hypercholesterolemic hypertensive (white) and normolipidemic normotensive subjects (black). * Significant in relation to normolipidemic normotensive group.

DISCUSSION

In this study we found that LDL from hypercholesterolemic and hypertensive patients has a higher susceptibility to oxidation (lag phase and peak time, Fig. 1) and that the plasma lipid-dderived hydroxy/hydroperoxide content (Fig. 2) is increased in these patients as compared with normolipidemic and normotensive subjects. Lag phase is a measure of LDL resistance to oxidation in vitro. It depends on LDL antioxidant content and amounts of preformed lipid hydroxy/hydroperoxides. The lower content of lipid-soluble antioxidants (Fig. 3) and the higher content of lipid-derived hydroxy/hydroperoxides (Fig. 2) in hypercholesterolemic and hypertensive patients could account for the lower duration of the lag phase of LDL in these patients in comparison with normocholesterolemic and normotensive subjects. In fact, the positive correlation of the antioxidants a-tocopherol and lycopene with LDL lag time (Table II) indicates that the lower these antioxidants are, the faster the oxidation of LDL particles is.

FIGURE 3. a-Tocopherol, lycopene and ß-carotene in blood plasma expressed as mM (A) and after normalizing the concentration of lipid-soluble antioxidants by the content of blood plasma cholesterol, which indicates the antioxidant content of lipoprotein particles (B). Ascorbate of blood plasma (C). Hypercholesterolemic normotensive (light gray), hypertensive norcholesterolemic (medium gray), hypercholesterolemic hypertensive (white) and normolipidemic normotensive subjects (black). * Significant in relation to normolipidemic normotensive group.

Lipid hydroperoxides in LDL isolated from blood plasma is indicative of oxidation that has already occurred in vivo. Free radical mediated oxidative damage of LDL particles may occur in arterial wall (Quinn et al, 1987) or in the intravascular compartment by oxidant species generated by mononuclear and polymorphonuclear leukocytes (Araujo et al, 1995). The positive correlation between cholesterol concentration and the content of cholesteryl ester- and triglyceride-derived hydroperoxides (Table II) indicates that lipid peroxidation is facilitated in hyperlipidemic states due to a high availability of oxidizable lipid substrates.

Isoprostanes (iPs) are stable prostaglandin isomers formed by free radical-catalyzed oxidation of arachidonic acid and have been considered as biomarkers of in vivo lipid peroxidation (Esterbauer et al, 1990). They are initially formed in the phospholipid domain of cell membranes and lipoproteins, from which they are cleaved by phospholipases, circulate in esterified and unesterified forms and are excreted in urine (Roberts et al, 1999). In the case of isomers of PGF2a (F2-iPs), up to 64 compounds in four structural classes may be formed. It is unknown whether their relative predominance depends on their site of formation, their affinity for phospholipase cleavage, their metabolism, or their clearance from plasma into urine. In the present study, no difference was found for the plasma F2-iP levels among the studied groups (Fig. 4). As commercially available immunoassays detect only the iPF 2a-III isomers, which may not be the main F2-iPs formed in blood plasma, this finding does not exclude the possibility that other F2-iPs isomers are enhanced in hypercholesterolemia and hypertension.


FIGURE 4. Concentration of isoprotanes in bood plasma of hypercholesterolemic normotensive (light gray), hypertensive norcholesterolemic (medium gray), hypercholesterolemic hypertensive (white) and normolipidemic normotensive subjects (black).

The lipid-soluble antioxidants, a-tocopherol, ß-carotene and lycopene, can protect lipoproteins from oxidative damage by free radicals and excited oxygen species (McCall and Frei, 1999). Several epidemiological studies have shown that there is a strong correlation between a-tocopherol and ß-carotene ingestion and reduced risk of cardiovascular diseases (Marchioli, 1999). The negative correlation of total cholesterol, LDL-cholesterol and apo B with a-tocopherol observed in this study (Table II) suggests that the consumption of this antioxidant is enhanced in hypercholesterolemic states. a-Tocopherol is a chain-breaking antioxidant that scavenges lipid peroxyl radicals (Halliwell and Gutteridge, 1989). It is the major antioxidant in LDL, and its concentration in LDL particles is the result of exogenous intake, its transfer from LDL to other lipoproteins or cells, and its redox reactions in LDL. It can be suggested that the one-electron oxidation of a-tocopherol via formation of a-tocopheroxyl radical would reduce its content in LDL. The inverse correlation between the concentrations of cholesteryl ester-derived hydroperoxydes and a-tocopherol (Table II) reinforces the idea that a-tocopherol is consumed during peroxidation of LDL particles due to its chain-breaking antioxidant activity. Ascorbate is a water-soluble chain breaking antioxidant that reacts with oxygen free radicals and regenerates the a-tocopheroxyl radical (Halliwell and Gutteridge, 1990). Although LDL lipid peroxidation may occur via a-tocopherol-mediated peroxidation, the concomitant decrease of a-tocopherol and ascorbate in plasma of hypercholesterolemic and hypertensive patients (Fig. 3) suggests that ascorbate is acting as a co-antioxidant efficiently reducing the a-tocopheroxyl radical and exporting this radical from the lipoprotein particle into the aqueous phase.

TABLE II

Pearson Product Moment Correlations
 


   
CE-OOH
(mM) 
TL-OOH
(mM) 
Lag Time
(minutes) 
a-tocopherol
(nmol/mg
of cholesterol) 

Total Cholesterol
(mg/dL) 
r
0.5157
0.0006 
0.4955
0.0012 
 
-0.5950
>0.0001 
LDL Cholesterol
(mg/dL) 
r
p
0.5064
0.0007 
0.4625
0.0027 
 
-0.5833
>0.0001 
a-tocopherol
(nmol/mg of cholesterol) 
r
-0.43627
0.0043 
-0.5034
0.0009 
0.4208
0.0055 
 
Apo B (mg/dL) 
r
p
0.3768
0.0152 
0.4103
0.0086 
 
-0.5457
0.0002 
Lycopene
(nmol/mg of cholesterol)
r
   
0.4128
0.0066 
0.5376
0.0002 

Ascorbate is reported to exert and anti-hypertensive effect (Akpaffiong and Taylor, 1998). A number of epidemiological studies have shown a negative correlation between blood pressure and ascorbate (Sakai et al, 1998). Ascorbate might influence blood pressure by several mechanisms, including a free radical-scavenging action preventing prostacyclin syntethase inhibition (Simon, 1992). Moreover, as the superoxide radical reacts rapidly with nitric oxide generating the oxidant peroxynitrite (Radi et al, 1991), ascorbate could increase nitric oxide bioavailability by reacting with superoxide radicals and consequently preserving the vasodilatatory action of nitric oxide. Reactions of ascorbate with free radicals could stop several pathways that contribute to the maintenance of hypertension, resulting in an anti-hypertensive effect.

In summary, our findings support the hypothesis that hypercholesterolemia and hypertension are associated with greater than normal lipid peroxidation and antioxidant consumption, suggesting that oxidative stress is important in the pathogenesis of both conditions.

ACKNOWLEDGEMENTS

Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 97/05090-3 to D.S.P.A. and Doctoral fellowship to P.M.).

Correspondence to: Dulcineia S. P. Abdalla Faculdade de Ciências. Farmacêuticas,Universidade de São Paulo. Av. Prof. Lineu Prestes, 580. Cidade Universitária - Butantã. 05508-900 São Paulo - S.P. - Brasil. Phone: (5511) 818-3637. Fax: (5511) 813-2197. E-mail: dspa@usp.br

Received: December 8, 1999. Accepted May 24, 2000

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