Journal of the American College of Nutrition, Vol. 12, No. 2, 133-137 (1993)
*Present address: S.D.R.M., 64 rue de Longchamp 92200 Neuilly, France.
Address reprint requests to Y. Rayssiguier, DSc, INRA, Laboratoire des Maladies Métaboliques, Theix 63122 St-Genè-Champanelle, France.
Magnesium (Mg)-deficient and control diets were pair-fed to weanling Wistar rats for 8 days. Plasma lipoproteins were separated into various density classes by sequential preparative ultracentrifugation. The extent of lipid peroxidation was measured in terms of thiobarbituric acid reactive substances in lipoproteins and tissue homogenates before or after iron-induced lipid peroxidation. Hyperlipemia in Mg-deficient rats was accompanied by increased oxidation of very-low-density lipoproteins and low-density lipoproteins. Moreover, very-low-density lipoproteins and high-density lipoproteins from Mg-deficient rats were more susceptible to oxidative damage following iron incubation. Mg deficiency increased lipid peroxidation in liver, heart and skeletal muscles. Their homogenates were more susceptible to in vitro peroxidation. Mg deficiency has been discussed as a possible contributory factor in the development of cardiovascular disease and was associated with tissue damage and membrane alteration. These results demonstrate for the first time that Mg affects the susceptibility of lipoproteins to peroxidation and suggest that the mechanism responsible for the pathological consequences of Mg deficiency may be mediated by lipid peroxidation products.
Abbreviations: AAS = atomic absorption spectrophotometry, BHT = butylated hydroxytoluene, EDTA = ethylenediaminetetraacetic acid, HDL = high-density lipoprotein, LDL = low-density lipoprotein, MDA = malondialdehyde, Mg = magnesium, TBARS = thiobarbituric acid reactive substances, VLDL= very-low-density lipoprotein
Magnesium (Mg) deficit has been discussed as a possible contributory factor in the development of cardiovascular disease1-3. Several studies have provided evidences that Mg deficiency affects lipid metabolism3,4 and is associated with tissue injury, affecting the physical state of membrane bilayer lipids5. Defective membrane function could be the primary lesion underlying cellular disturbances in Mg-deficient animals6. While Mg deficiency has been shown to induce both hyperlipemia and vascular lesions in experimental animals, the extent of atherogenic lesions is poorly correlated with the level of serum cholesterol1,7. Lipid peroxidation has been proposed to contribute to various pathophysiological membranes and tissue abnormalities8, and the role of oxidative modification of lipoproteins in atherogenesis is well recognized9. The protection by vitamin E of myocardial necrosis induced by Mg deficiency as shown recently, suggests that free radicals participate in Mg deprivation-induced injury10. Our investigation represents an initial study in rats to determine the effect of Mg deficiency on susceptibility of lipoproteins and tissues to peroxidation.
Forty-eight weanling male Wistar rats (IFFA-CREDO, L'Arbresle, France) (~60 g, 3 weeks old) were divided at random into Mg-deficient and control groups (24 animals per group). The institution's guide for the care and use of laboratory animals was used. They were pair-fed with the appropriate diets for 8 days using an automatic feeding apparatus. Distilled water was provided ad libitum. Diets contained (g/kg) 200 casein, 3 DL-methionine, 702 sucrose, 50 corn oil, 35 mineral mixture, and 10 vitamin mixture as described previously11. Mg contents determined by flame atomic absorption spectrophotometric (AAS) analysis (model 400, Perkin Elmer, Norwalk, CT) were 35 (deficient) and 980 mg/kg (control). Experiments were performed on nonfasting rats. Plasma from 24 animals per group were obtained from rats anesthetized with sodium pentobarbital (40 mg/kg of body weight). Equal volumes of plasma samples from three animals were pooled for blood analysis and lipoprotein separations. The heart and liver (entire organs) and skeletal muscle (gastrocnemius) from eight animals per group were rapidly removed, washed in ice-cold saline, placed in liquid nitrogen, and stored at -80° C.
Plasma lipoproteins were separated into density classes by sequential preparative ultracentrifugation12 at 15° C. in a Beckman L-5-50 model ultracentrifuge (Beckman Instruments Inc, Palo Alto, CA) with a 50 titanium rotor. Ethylenediaminetetraacetic acid (EDTA) (1 mg/ml) and butylated hydroxytoluene (BHT) (4.4 µg/ml) were added immediately before lipoprotein separation. Samples were overlayered with 0. 15 M NaCl (d = 1.006 g/ml) and chylomicrons were discarded following two centrifugations for 30 minutes at 12,000 x g. Very-low-density lipoproteins (VLDL) were isolated, under the same density conditions by centrifugation for 18 hours at 100,000 x g. Centrifugation was done at 100,000 x g for 20 and 48 hours to isolate low-density lipoprotein (LDL, d = 1.0061-1.050 g/ml) and high-density lipoprotein (HDL, d = 1.050-1.21 g/ml), respectively. Densities were adjusted with solid KBr. Fractions were again centrifuged at the same density. The purified lipoprotein fractions were dialyzed against 0.01 M phosphate buffer (pH 7.4) containing 0.16 M NaCl which was made oxygen free by vacuum degassing followed by purging with nitrogen. These EDTA and BHT-free lipoproteins were used for all oxidation studies. VLDL and LDL were diluted to a final concentration of 0.1 mg protein/ml and HDL to 0.5 mg protein/ml. The same concentration was used for both experimental groups.
Thiobarbituric acid reactive substances (TBARS) were determined in lipoproteins supplemented with BHT (10 mM), as reported by Dousset et al13, with slight modifications. A mixture (750 µL of thiobarbituric acid (Merck) at 8 g/L and 7% perchloric acid (Merck) (2:1 v/v) was added to 100 µL of lipoprotein. After agitation, the mixture was placed in a 95° C water bath for 60 minutes and then cooled in an ice bath. The fluorescent compound was extracted by mixing with n-butanol for 2 minutes. The n-butanol layer containing the TBARS was read in a spectrofluorometer (Perkin Elmer LS 5, excitation wavelength 532 nm, emission 553 nm) and expressed as nmol TBARS per mg of protein. Tissue homogenates were prepared on ice in a ratio of 1 g wet tissue to 9 ml 15% KC] using a Polytron homogenizer. TBARS were measured in tissue homogenates supplemented with BHT (10 mM) as previously described14. TBARS were also determined in BHT-free lipoprotein fractions and in BHT-free tissue homogenates after lipid peroxidation induced with FeSO4 (10 µM)/ascorbate (250 µM) for 30 minutes in a 37° C water bath in an oxygen-free medium, using a standard of 1,1,3,3 tetraethoxy propane.
Plasma Mg was determined with a series 400 AAS after dilution in lanthanum chloride solution containing 1 g/l La. Triglycerides (Biotrol, Paris, France), cholesterol and phospholipids (Biomérieux, Charbonnières-les-Bains, France) were determined in plasma by enzymatic procedures15-17. Protein concentration of isolated lipoproteins was determined by a modified Lowry method using bovine serum albumin, fraction V (Sigma, L'Isle d'Abeau, France) as a standard18.
Results were expressed as means ± SEM. Statistical significance of differences between means was assessed by Student's t test.
Mean final body weights of Mg-deficient and pair-fed control rats were 84 ± 2 and 93 ± 3 g (mean ± SEM; n = 24; p < 0.01), respectively, at the end of the experimental period. Hypomagnesemia was usually observed in Mg-deficient rats (Table 1). Turbidity, visible in plasma of deficient animals, was due to hyperlipemia. Triglyceride and phospholipid plasma levels were significantly higher in Mg-deficient rats than in controls, whereas plasma cholesterol was only modestly elevated. As shown in Table 2, VLDL and LDL from Mg-deficient rats exhibited increased oxidation in vivo as monitored by TBARS compared to controls (p < 0.01 and p < 0.05). After exposure to oxidative stress, in vitro TBARS of the VLDL fraction from Mg-deficient rats were significantly higher than that observed in the VLDL from control rats (p < 0.001). There were insignificantly higher levels of TBARS in LDL from Mg-deficient rats as compared to control animals following the oxidation experiment. In contrast to our findings for VLDL and LDL fractions, TBARS of the HDL fraction were not altered in vivo but were significantly higher (p < 0.001) in vitro than those of control rats following oxidative stress. Concentrations of TBARS were significantly increased in liver (p < 0.05), heart (p < 0.00 1), and skeletal muscle (p < 0.05) from Mg-deficient rats as compared to control rats. After exposure of tissue homogenates to iron-induced lipid peroxidation, TBARS were significantly higher in liver (p < 0.001), heart (p < 0.01) and skeletal muscle (p < 0.05) from Mg-deficient rats as compared to control rats (Table 3).
These results demonstrate for the first time that Mg deficiency affects susceptibility of lipoproteins to peroxidation. Previous studies from our laboratory have provided evidence that experimental Mg deficiency in rats produced a dyslipoproteinemia characterized by increased plasma levels of VLDL, LDL and decreased plasma HDL3,11. The hypertriglyceridemia observed in Mg-deficient animals was largely associated with the d < 1.006 g/ml lipoproteins made up of VLDL and VLDL-like particles; cholesterol was redistributed toward the lower density lipoproteins19. Marked alterations in lipoprotein composition20, and the mechanisms underlying hyperlipemia of Mg-deficient rats have been addressed elsewhere21,22. Hyperlipemia can result from excessive production and release of lipids into the circulation, from their impaired removal from the circulation, or both. Decreased clearance of circulating triglycerides is a major mechanism contributing to hyperlipemia in Mg-deficient rats19. In contrast to the slight modification in plasma cholesterol from short-term Mg deficiency, plasma cholesterol significantly increased during long-term Mg deficiency21.
Early studies indicate that Mg deficiency enhances vascular lipid infiltration in rats, rabbits and monkeys on atherogenic diets1,22. Recent studies confirm that Mg deficiency can intensify cardiovascular lipid deposition and lesions in animals on atherogenic diets and that dietary Mg supplementation prevents atherosclerosis7. We have demonstrated that marked changes occur in the concentration and composition of lipoproteins in Mg-deficient animals and that in turn may affect vascular lipid infiltration. However, there has not been agreement that cardiovascular lesions are necessarily correlated with lipemia in Mg-deficient animals1,7. Oxidative modifications of lipoproteins can be of importance in the development of atheromatous lesions9. Modified LDL, not recognized by apolipoprotein B/E receptors, bind to macrophages which act as scavenger cells. The uptake of oxidized LDL by macrophages is not down regulated by internalized LDL cholesterol. This leads to accumulated levels of esterified cholesterol in foam cells9.
Our results clearly indicate that Mg deficiency increases lipid peroxidation of lipoproteins in vivo. Lipid peroxidation in plasma from Mg-deficient rats (measured by thiobarbituric assay) has been reported to increase plasma lipid peroxides in one study4, whereas another report23 does not indicate higher plasma malondialdehyde (MDA) levels in Mg-deficient rats than in control animals. In neither study was lipoprotein fractionation performed. Since plasma contains many substances that react in the thiobarbituric assay, the value of MDA in plasma as an indicator for lipid peroxidation is limited24; measurements of TBARS in isolated lipoproteins has significant advantages in oxidation studies. In the present experiment, triglyceride-rich lipoproteins from Mg-deficient rats exhibited increased oxidation as monitored by TBARS compared to that from control rats, whereas the TBARS of HDL were unmodified. These findings indicate that the enhanced plasma lipid peroxides found in Mg-deficient rats4 are primarily in the lower density lipoproteins since the hyperlipemia observed in Mg-deficient rats is largely associated with increased levels of lower density lipoproteins. Plasma lipid peroxides have been reported to be elevated in diabetic rats when plasma triglycerides are increased, TBARS levels being elevated in a lipoprotein fraction containing VLDL and LDL25. Hypertriglyceridemia in Mg-deficient rats was similarly accompanied by increased oxidation of lipoprotein-rich lipoproteins.
Most biological studies of modified lipoproteins were performed with lipoproteins oxidized by iron or copper ions8. For oxidative modification lipoproteins were incubated with iron/ascorbate, the pro-oxidant activity of ascorbate resulting from its ability to reduce Fe3+ complexes to forms that react with O2 to form initiators of lipid peroxidation8. Results indicate that Mg deficiency increases lipoprotein oxidizability. Differences in the VLDL susceptibility to lipid peroxidation in vitro indicate that the particles from Mg-deficient rats are less protected against oxidative modification than are those from control rats. Rats have relatively small amounts of LDL26. Thus, results concerning VLDL oxidation are of greater significance than are those concerning LDL in contrast to studies with human lipoproteins.
Our results also clearly show that the amount of lipid peroxide increased on incubation of HDL with iron/ascorbate, indicating oxidative modification of HDL; Mg deficiency increased HDL susceptibility to oxidative damage in rats. Data have accumulated concerning oxidized lipoproteins, but whether oxidative modification could also occur in HDL has been questioned27. A recent report ascertained that oxidative modification occurred in vitro in HDL and that HDL loses its effect to stimulate efflux of cholesterol from foam cells after oxidative modification27.
The enhanced production of TBARS may be attributable either to a depleted antioxidant system of the lipoproteins or to a modification in the lipid composition28. In lipoproteins from Mg-deficient animals, the percent composition of triglycerides was elevated and that of protein reduced. Fatty acid analysis showed increased percent composition of linoleic acid20. These changes may affect the susceptibility of lipoproteins to oxidative modifications. Since no information is available concerning the influence of Mg on antioxidant concentrations, further studies are required to clarify the mechanism by which lipoprotein oxidation increases in Mg-deficient animals.
Several data indicate that oxidized VLDL and LDL are toxic to cells in culture25. Cytotoxicity of oxidized lipoproteins in vitro suggests the potential for lipoprotein-mediated tissue damage in vivo. A cytotoxic activity of HDL after oxidative modification has also been recently suggested27. Thus oxidized lipoproteins from Mg-deficient animals might play an important role in development of atherosclerosis and might be responsible for some of the tissue damage characteristic of Mg deficiency1. Little information is available regarding the susceptibility of various tissues to lipid peroxidation in Mg-deficient animals. Mg-deficient rats as compared to those control-fed ad libitum showed increased lipid peroxidation in the liver but not in other tissues23. Lipid peroxidation was also increased in isolated liver mitochondria incubated in vitro, whereas in microsomes and mitochondria from other Mg-deficient tissues, lipid peroxidation was unmodified29. Our experiment clearly indicates that Mg deficiency increases lipid peroxidation in liver, heart and skeletal muscle and that these tissues are more susceptible to in vitro peroxidation. Of particular significance is the observation that Mg deficiency has a very harmful effect on lipid peroxidation in the cardiovascular system.
TBARS, although imperfect, appears valuable for comparative data24. An important proof of the increased formation of oxygen free radicals are the results showing the influence of antioxidant treatment of Mg-deficient animals. Various antioxidant drugs and nutrients, vitamin E, sulphydryl-containing angiotensin converting enzyme inhibitors, probucol and β-blockers provide protection against the cardiomyopathy of Mg deficiency10,30,31. Together, these studies suggest that Mg deficiency enhances the susceptibility to free radical-mediated injury. Mg deficiency has been discussed as a possible contributory factor in the development of cardiovascular disease and it is well established that Mg deficiency induces tissue injury and damage to the integrity of cellular membranes. The results suggest that the mechanisms responsible for the pathological consequences of Mg deficiency may be mediated by lipid peroxidation products.
We thank C. Lab and A. Bellanger for their helpful assistance.
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