(Hypertension. 1997;30:624.)
© 1997 American Heart Association, Inc.
Articles |
In Vivo Plasma Lipid Oxidation in Sugar-Induced Rat Hypertriglyceridemia and Hypertension
From the Department of Biochemistry, Instituto Nacional de Cardiología "Ignacio Chávez," Juan Badiano 1, Mexico.
Correspondence to Mohammed El Hafidi, Department of Biochemistry, Instituto Nacional de Cardiologia "Ignacio Chávez," Juan Badiano 1, México, DF 14080. E-mail florence{at}mail.internet.com.mx
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Abstract |
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Abstract Fe(II) and Fe(III) are required for the catalysis of lipid peroxidation through generation of reactive oxygen species that damage cell membranes. This study investigated the effect of free radicals and lipid peroxidation, induced by intraperitoneal injection of iron-dextran in vivo, in the plasma of the sugar-induced hypertriglyceridemic and hypertensive male and female rats. Lipid peroxidation was measured by the malondialdehyde (MDA) equivalent, using a fluorescence method of 2-thiobarbituric acid reactive substances (TBARS). Iron increased TBARS generation by fourfold (P<.0001) in male control rats and by twofold (P<.01) in female control rats, and the difference between TBARS concentration in female as compared with male animals was statistically significant (P<.05). In the case of the sugar-fed group, iron-dextran produced an increase of TBARS concentration by twofold in both male (P<.001) and female rats (P<.01), and no significant difference in TBARS concentration was observed between sugar-fed female and male rats. The analysis of fatty acid composition by gas chromatography showed a significant diminution of 50% in the proportion of arachidonic acid (C20:4n-6) in the male control group in comparison with the female group (P<.0001). In female control rats, a small diminution in the proportion of C20:4n-6 and in the other polyunsaturated fatty acids was observed (P<.05). A significant difference in the C20:4n-6 proportion was found between the male and female group of control rats. In the sugar-fed group, iron induced a significant diminution of arachidonic acid (P<.001) in both female and male rats in comparison with the sugar-fed group without iron.
Key Words: hypertriglyceridemia • iron-dextran • lipid peroxidation • arachidonic acid
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Introduction |
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Reactive oxygen species are important mediators of cellular injury via damage to membranes or alteration of enzyme activities. The polyunsaturated fatty acids of the membrane and of the lipoprotein particles are particularly susceptible to free radical attack, ultimately forming lipid hydroperoxides, lipid hydroxides, hydrocarbons, and aldehydes as their stable degradation products; these are implicated in many pathologies such as atherosclerosis, aging, cancer, etc.1 Recent data have suggested the possibility that the small, dense oxidized LDL are the more atherogenic particles.2 However, the increase in the small, dense LDL is due to the increase in the level of triglycerides in human plasma.3 Smaller, denser LDL are more susceptible to lipid peroxidation.4 5 Oxidation of LDL occurs in atherosclerotic lesions in experimental animals; antibodies to oxidized LDL have been found to correlate with the progression of atherosclerotic lesions in humans.6 7 In men and women, it has been suggested that antioxidant supplementation decreases the progression of atherosclerosis and that the high intake of vitamin E (an antioxidant ) lowers the risk of coronary heart disease in both women and men.8 9 10 It has been reported that the sex difference in cardiovascular diseases may be partially attributable to modulation of lipoprotein metabolism by estrogens11 12 13 and their antioxidant effect.14 15 16 It has been shown that estrogens and catecholestrogens inhibit microsomal lipid peroxidation stimulated by iron and NADPH.17 18 Iron catalyzes lipid peroxidation because of its ability to react with oxygen to form species capable of initiating peroxidative events. Both Fe(II) and Fe(III) are required for the catalysis of lipid peroxidation.19 The experiments described in this article were designed to investigate the effect of free radicals and lipid peroxidation induced by intraperitoneal injection of iron-dextran in male and female hypertriglyceridemic (HTG) Wistar rats. The hypertriglyceridemia was induced in rats by consumption of commercial sugar at 30% in their drinking water for a period of 18 to 20 weeks. Also we examine the sex difference in the lipid peroxidation in the HTG rat and the possible role of estrogens as radical trappers.
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Methods |
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Study Design
Weanling male and female Wistar rats, aged 25 days and weighing 50±5 g, were used and randomly distributed into two groups: one was given 30% commercially refined sugar in their drinking tap water for 18 to 20 weeks; the other group received only tap water. Both groups were fed standard laboratory rat chow ad libitum. At the end of the treatment period, measurements of the rat’s blood pressure were taken by the tail-cuff method: the cuff was connected to a pneumatic pulse transducer (Narco Bio-Systems from Healthdyne Co) and a Programmed Electro-Sphygmomanometer from the same company. The recordings were obtained in duplicate by means of a Narco Bio-Systems polygraph.
Each group was then divided into eight subgroups of five rats each and treated as follows: male and female groups without iron or sugar (groups 1M and 1F), male and female groups with sugar but without iron (groups 2M and 2F), male and female experimental groups with iron overload and without sugar (groups 3M and 3F), and male and female groups with sugar and with iron overload (groups 4M and 4F). Iron overload was induced by intraperitoneal injection of iron-dextran at 10, 25, and 50 mg/kg body weight on day 1, day 3, and day 5, after 18 weeks of sugar treatment. Two days after the last injection, the rats were killed by decapitation, taking care to avoid hemolysis. Rats not treated with iron-dextran received the corresponding volume of saline with dextran-500 in order to obtain the same final injected liquid volume.
TBARS Activity
After decapitation, blood was collected from the animals
in a tube containing 2% EDTA plus 0.05% BHT. Then it was
centrifuged at 3000 rpm at 4°C during 20 minutes. The plasma
obtained was stored at -70°C until the lipid analysis was
carried out. We used 0.1 mL plasma for the determination of lipid
peroxidation, measuring TBARS by a fluorescence
method.20 Briefly, we added 0.05 mL of 4% [wt/vol] BHT
and 1 mL phosphate buffer to 0.1 mL of plasma. After incubation at
37°C for 30 minutes, 1.5 mL of 20% acetic acid and 1.5 mL 0.8%
2-thiobarbituric acid were added. The mixture was heated for 45 minutes
in boiling water and TBARS were extracted into 5x10–3 L
of n-butanol. After a brief centrifugation the
fluorescence of the butanol layer was measured at 515 nm
excitation and 553 nm emission in a spectrofluorometer (Aminco
Bowman Series 2 Luminescence Spectrometer). The value is expressed
as mmol TBARS (MDA equivalents) per liter of plasma. An MDA
standard was prepared from 1,1,3,3-tetraethoxypropane.
Lipid Extraction and FA Composition Determination
Plasma lipid extraction was performed as described by Folch et
al.21 The fat was hydrolyzed in a KOH/MetOH (1
mol/L) solution containing 0.02% BHT at 90°C for 30 minutes.
The free FA, in the presence of heptadecanoic acid (C17:0) as internal
standard, was extracted with hexane-diethylether (1/1, vol/vol)
and dried over anhydrous sulfate sodium. After evaporation to dryness
of the solvent under a gentle stream of nitrogen, FA were esterified at
laboratory temperature overnight to their corresponding methyl esters
in methanol containing 2% of concentrated sulfuric acid and 0.005% of
BHT. FA methyl esters were separated and identified by gas liquid
chromatography on a model Carlo Erba Fratovap 2300
fitted with a 25 mx0.25 mm interior diameter fused-silica
capillary column coated with CP-Sil 88 (film thickness,
0.25x10-3 mm) at an isotherm temperature
of 195°C and helium gas flow rate 1 mL/min.
Data Analysis
FA are expressed as percentage of each individual FA of the
total FA identifiable by gas liquid chromatography.
Peak areas and retention time of FA were measured by means of a
computer program (Gold, Beckman). Identification of individual methyl
ester components was made by comparison of the retention time with a
standard mixture.
Statistical analysis was performed on a personal computer using a statistical and graphic system (SigmaPlot, SigmaStat 1.0, Jandel Co, 1992-1994). Data are presented as the mean±SD. Significance of differences was determined by Student’s t test.
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Results |
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Body Weight and Triglyceride Level
Table 1 shows that weight and weight gain were significantly greater in males than in females; within the same sex group there was no difference in weight due to the type of treatment. Total food consumption was the same in both male and female groups; therefore, the only difference was the intake of sucrose. The triglyceride level was significantly higher in both female and male sugar-fed rats in comparison with the female and male control groups (P<.05).
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Blood Pressure
Mean blood pressure for all four groups of rats for 18 weeks is
shown in Table 1. Blood pressure in sugar-fed males (group 2M) was
significantly greater than in control males (group 1M)
(P<.001) at the end of the 18-week period. No significant
difference in blood pressure between sugar-fed females (group 2F) and
control females (group 1F) was observed. However, blood pressure in
sugar-fed males (group 2M) was significantly higher than in sugar-fed
females (group 2F) (P<.05).
Plasma Thiobarbituric Acid Reactivity
Iron-dextran produced a significant increase in the concentration
of TBARS, an index of high lipid peroxidation, in plasma of control
groups (Figure panel A) as described in
the literature22 and in the plasma of the sugar-fed rats
(Figure panel B). During the preparation of the plasma, the addition of
BHT did not increase the concentration of TBARS, which suggests that
the increased concentration of TBARS in the plasma with iron overload,
in comparison to the plasma without iron, reflects in vivo formation of
MDA. It is important to point out that iron increased TBARS generation
by fourfold (P<.0001) in control males and by twofold
(P<.01) in control females and the difference between
TBARS concentration in females as compared with males was statistically
significant (P<.05). In the case of the sugar-fed group,
iron-dextran produced an increase of TBARS concentration by twofold in
both male (P<.001) and female (group 4F and 4M) rats
(P<.01), and no significant difference in the index of
lipid peroxidation was observed between sugar-fed female and male
rats.
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Plasma FA Composition
Tables 2 and 3 show the
analyses of FA composition of plasma from control and sugar-fed
groups, respectively, and from rats exposed to iron compared with the
same groups without iron overload. A decrease in polyunsaturated FA was
observed in both groups of rats exposed to oxidative stress. A
significant diminution of 50% in the proportion of
arachidonic acid (C20:4n-6) was noted in the male
control group (group 1M) in comparison to that of the female group
(group 1F) (P<.0001). Also, a proportion of
dihomo-
-linolenic (C20:3n-6), eicosapentaenoic (C20:5n-3),
and docosahexaenoic acid (C22:6n-3) decreased to 27.85%, 56.87%, and
35.65%, respectively, in the male control group (group 1M)
(P<.01). In the female control group (group 1F), a small
lowering in the proportion of C20:4n-6 and in the other polyunsaturated
FA was observed (P<.05). A significant difference in the
C20:4n-6 proportion was found between the male and female groups of
control rats (Table 2). In the sugar-fed group (Table 3), the same
phenomenon was observed. Indeed iron induced a significant diminution
of arachidonic acid to 35.76% (P<.001) in
both sugar-fed female and male rats in comparison with the other
sugar-fed group without iron. A proportion of C20:3n-6, C20:5n-3, and
C22:6n-3 decreased to 17.64%, 26.15%, and 25.53%, respectively, in
male controls ( group 1M) (P<.01).
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Discussion |
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It has been well established that a diet rich in sucrose induces hypertriglyceridemia and hypertension in rats, but the mechanism by which hypertriglyceridemia induces hypertension is not clear.23 24 25 Our study showed that there is no difference in triglyceride concentration and in blood pressure between male and female sugar-fed rats. The higher blood pressure cannot be explained by larger total body weight in the sugar-fed animals, because there was no statistically significant difference in weight between control and sugar-fed rats. Body composition was not examined; it is not known whether sucrose feeding resulted in an increased percentage of adipose tissue at the expense of other tissues. The analysis of the plasma FA composition in both sugar-fed male and female rats in comparison with the control group showed a significant increase in palmitoleic and oleic acid and a significant decrease of linoleic and arachidonic acid. It has been reported that oleic acid exerts a pressor effect in rats when administered by venous infusion26 and that the linoleic acid has a hypotensive effect when administered in the diet.27 28 However, alteration in plasma FA composition in sugar-fed rats may be associated with high blood pressure.
In this report we have shown that a baseline of plasma lipid peroxidation measured by TBARS activity was not significantly different between sugar-fed and control groups. But a stimulatory effect of iron-dextran on plasma TBARS concentration in both groups was observed. The lipid peroxidation induced by iron is well documented in the literature.19 Iron may initiate the lipoperoxidation by the Fenton reaction with the oxygen-forming reactive oxygen species such as the superoxide anion, which reacts with polyunsaturated FA to form FA hydroperoxides, hydroxides, and their degradation products such as hydrocarbons and aldehydes. However, no difference was noted between lipid peroxidation in sugar-fed and control groups. In the control group (group 1), the stimulation of lipid peroxidation was greater in the male than in the female group, whereas it was not statistically different between male and female in the sugar-fed group. The increase of plasma TBARS concentration was well correlated with plasma polyunsaturated FA composition in the groups both with and without iron. Iron overload induces changes in the plasma polyunsaturated FA such as arachidonic acid, one of the major polyunsaturated FA susceptible to lipid peroxidation. The analysis of FA composition permits us to validate the determination of lipid peroxidation measured by TBARS activity. Indeed, arachidonic acid, the major precursor of MDA formation, was present at a lower proportion (-50%) in males than in females in both sugar-fed and control groups treated with iron. If we compare the proportions of plasma arachidonic acid in both sugar-fed and control groups without treatment, we find a lower proportion of this acid in the sugar-fed group than in the control group and a relatively lower proportion in male than in female rats. Therefore, we can postulate that the lack of difference in lipid peroxidation between sugar-fed and control animals in the male group may be due to a lower proportion of polyunsaturated FA, such as linoleic and arachidonic acids, in the sugar-fed groups. Between male and female animals of the control group, the difference in the TBARS concentration may be due to a protective effect of the many antioxidant substances in the plasma. The gender difference points to estrogens to explain the difference in lipid peroxidation between female sugar-fed and female control rats. Indeed estrogens and catecholestrogens (hydroxylation products of estrogens, exhibit a protective effect against oxidative membrane damage in vitro,17 18 and their antioxidant effects are greater when peroxidation is initiated by complexes containing iron and less when peroxidation involves peroxyl radicals.18
Other studies have shown an antioxidant effect of estrogens against peroxidation induced by ultraviolet irradiation and other pro-oxidant systems.15 16 17 18
In many reports, it has been stated that the sex difference in cardiovascular diseases may be partially attributable to modulation of lipid metabolism by estrogens13 ; the evidence that lipid peroxidation is implicated in cardiovascular diseases6 7 suggests that the combination of the hypertriglyceridemia and hypertension induced by a sugar-rich diet and an oxidative stress induced by iron as a pro-oxidant system may increase the risk of cardiovascular complications in sugar-fed female rats.
In brief, our results show no significant difference in the degree of iron-induced lipoperoxidation between sugar-fed male and female rats, and it was comparable to the level found in male controls, the female controls having a lower degree of lipoperoxidation. This suggests that in female controls there might be a protective factor or factors that reduces peroxidation. On the other hand, since sugar-fed female animals now exhibit the same degree of peroxidation as the males, the female protecting factor or factors are not operating. The mechanisms involved and the full identification of such factor or factors require further study.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Carlos Posadas, Head of the Endocrinology Department, kindly gave us facilities for the measurement of blood triglycerides. Part of this work was carried out with equipment obtained through grant No. F554 from the National Council for Science and Technology (Conacyt), whom we should also like to thank.
Received March 18, 1997; first decision April 28, 1997; accepted May 19, 1997.
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