1Institute of Lipid and Atherosclerosis Research and 2Department of Medicine 'B', Sheba Medical Center,. Tel-Hashomer, and Sackler Faculty of Medicine,.
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Atherogenesis Inhibition Induced by Magnesium-Chloride Fortification of Drinking Water HOFIT COHEN,1 YANIV SHERER,1,2 AVIV SHAISH,1 YEHUDA SHOENFELD,2 HANA LEVKOVITZ,1 RAFAEL BITZUR,1 AND DROR HARATS*,1 1
Institute of Lipid and Atherosclerosis Research and 2Department of Medicine ‘B’, Sheba Medical Center, Tel-Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv Israel Received March 21, 2002; Accepted April 15, 2002
ABSTRACT Magnesium (Mg) modulates blood lipid levels, atherogenesis, and atherosclerosis in rabbits, when supplemented to diet. We have recently reported that a high concentration (50 g/L) of Mg sulfate fortification of drinking water attenuates atherogenesis in male and female LDL-receptordeficient mice fed a high-cholesterol diet. The aims of the current study were to examine whether lower concentrations and another Mg salt could also have such an antiatherogenic effect. Thirty male LDL-receptor-deficient mice were divided into three groups (n=10 in each group). The mice received either distilled water or water fortified with 0.83 g or with 8.3 g Mg-chloride per liter. In the first (27 wk) and second (5 wk) stages of the experiment, the mice received normal chow and Western-type diet, respectively. Blood was drawn for determination of plasma Mg, calcium, and lipid levels. The extent of atherosclerotic lesions was determined at the aortic sinus. Magnesium-chloride fortification of drinking water did not result in higher plasma Mg concentrations, whereas a trend toward lower plasma calcium concentrations did not reach statistical significance. Even though plasma lipid levels were similar at the beginning and the end of the study, there were decreased plasma cholesterol and triglyceride levels in the Mg groups after stage I. The atherosclerosis extent at the aortic sinus was significantly decreased in the 8.3-g Mg-chloride/L group (23,437±10,083 µm2) compared with the control group (65,937±31,761 µm2). There was also a trend toward lower atherosclerosis extent at the aortic sinus in the 0.83-g *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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Cohen et al. Mg-chloride/L group. An additional Mg salt (Mg-chloride) fortification of drinking water is capable of inhibiting atherogenesis in male LDL-receptor-deficient mice. That is done in a lower concentration of Mg than previously reported. Index Entries: Atherogenesis; cholesterol; drinking water; magnesium.
INTRODUCTION Magnesium (Mg) plays an important role as a cofactor of more than 300 enzymatic reactions (1). Mg deficiency is not uncommon among the general population of Western society because the average Western diet does not provide the recommended daily intake of Mg (2,3); also its intake has deceased over the years. The antiatherogenic effect of Mg was reported previously in rabbits (4–6). These studies (4,5) emphasized that both Mg aspartate and Mg sulfate were equipotent in atherogenesis inhibition. Our group has investigated whether Mg fortification of drinking water (without change in the diet content of Mg) can also inhibit atherogenesis. We have found that fortification of drinking water with Mg-sulfate resulted in atherogenesis inhibition to a level of about one-third of the controls in male low density lipoprotein (LDL)-receptor-deficient mice (7). In addition, it has recently been demonstrated that Mg fortification of drinking water was also capable of inhibiting atherogenesis in female LDL-receptor-deficient mice fed a high-cholesterol diet (8). These LDL-receptor-deficient mice develop atherosclerotic lesions throughout the arterial tree that have many similarities with human atherosclerosis regarding the distribution pattern and morphological features; thus, they provide an adequate model for human atherosclerosis research (9). Upon feeding with a low-cholesterol diet (chow diet) the mice do not develop significant atherosclerotic lesions, whereas upon feeding a high-cholesterol diet the mice develop marked hypercholesterolemia and lesions throughout the aorta within 3–4 mo (10). The aim of this study was to examine whether another Mg salt, namely Mg-chloride, has antiatherogenic effects as well. More importantly, we intend to find out whether fortification of drinking water with lower levels of Mg could also inhibit atherogenesis. Therefore, we test two concentrations of Mg-chloride in this study, both lower than the concentrations of Mg-sulfate used by us earlier (7,8).
MATERIALS AND METHODS Mice and Diets LDL-receptor deficient mice were bred in the local animal house of the Institute of Lipid and Atherosclerosis Research (Sheba Medical Center, TelBiological Trace Element Research
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Hashomer, Israel). The mice were created by homologous recombination as described by Ishibashi et al. (9). Three groups of 10 male mice were studied. The mice were 5 wk old when entered into the study. They were on 12-h dark/light cycles and were allowed access to food and water ad libitum. The study included two stages: stage I lasted 27 wk, in which the mice were fed a normal chow diet containing 4.5% fat by weight and 0.02% cholesterol. One group of male mice was allowed access to distilled water (Mg concentration < 10 mg/L), whereas the other groups were given water containing two different concentrations of Mg: 8.3 g Mg-chloride/L (40 mM) and 0.83 g Mg-chloride/L (4 mM). Stage II lasted 5 wk, and, in this period, the mice were fed an atherogenic Western-type diet containing 0.15% cholesterol, 21% anhydrous milk fat, and 19.5% casein, with the same Mg treatment for each group. At the end of stage II, the mice were sacrificed. The mineral content of both diets was measured in an ICP machine (Spectro, Germany): the chow diet contained 2470 mg/kg diet of Mg and 15,400 mg/kg diet of calcium, whereas the Western-type diet contained 510 mg/kg diet of Mg and 6400 mg/kg diet of calcium.
Measurement of Plasma Mg and Calcium At the end of stage II, blood was obtained from the retroorbital plexus, and the plasma Mg and calcium were determined; 1000 U of heparin/mL blood was added to each sample for plasma Mg determination. Briefly, 10 µL of plasma is deposited on either Mg or calcium slides (Vitros Chemistry Products) that have a correlation coefficient of 0.999. Mg, both free and protein bound, from the sample reacts with the formazan dye derivative in the reagent layer, the high Mg affinity of the dye dissociates Mg from its binding proteins, and the Mg–dye complex formed is read at a wavelength of 630 nm. Calcium forms a complex with Arsenazo III dye, and this complex is read at a wavelength of 680 nm.
Determination of Lipid Profile At the beginning and the end of both stages I and II, blood was collected from the retroorbital plexus, after 12 h of fasting. One milligram of ethylenediaminetetraacetic acid (EDTA) per milliliter of blood was added to each sample. The total plasma cholesterol and triglycerides levels were determined by using an automated enzymatic technique (BoehringerMannheim, Germany) (11).
Assessment of the Extent of Atherosclerosis at the Aortic Sinus Quantification of atherosclerotic fatty streak lesions was carried out by calculating the lesion size in the aortic sinus, as previously described Biological Trace Element Research
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(12) but with a few modifications. Briefly, the heart and upper section of the aorta were removed from the animals and the peripheral fat was cleaned carefully. The upper section was embedded in optimal cutting tempertature (OCT) medium and frozen. Every other section (5 µm thick) throughout the aortic sinus (400 µm) was taken for analysis. The distal portion of the aortic sinus is recognized by the three valve cusps, which are the junctions of the aorta to the heart. Sections were evaluated for fatty streak lesions after staining with oil red O. Lesion areas per sections were counted using a grid by an observer unfamiliar with the tested specimen. The extent of atherosclerosis was evaluated at the level of the aortic sinus. Processing and staining of the tissue was carried out according to Paigen et al. (12). Lesion area was quantified by the modified method of Rubin et al. (13).
Statistical Analysis Statistical analysis was carried out using the analysis of variance (ANOVA) test. p < 0.05 was accepted as statistically significant.
RESULTS The body weights of both mouse groups were compared at the end of the study, and no significant difference was found (see Fig. 1).
Plasma Mg and Calcium Levels At the end of the study, plasma Mg levels were 2.4±0.1 mg/dL in the mice that received distilled water and was similar in the two Mg-chloride groups (see Fig. 2). Plasma calcium levels were 9.1±0.26 mg/dL in the control group, but they were lower for the 0.83-g Mg-chloride/L group and the 8.3-g Mg-chloride/L group (8.1±0.2 and 8.4±0.26 mg/dL, respectively); however, no significant difference was found among the groups (see Fig. 2).
Lipid Profile Lipid levels were determined at the end of stages I and II (see Table 1). No differences were found among mouse groups at the beginning of the study, and similarly triglycerides and cholesterol levels did not differ significantly among the groups when the study ended. Of note is that all mouse groups developed severe hypercholesterolemia after the second stage of feeding with a high-cholesterol diet. However, after the first stage (feeding with chow diet) ended, significantly lower cholesterol levels were found in the 8.3-g Mg-chloride/L and 0.83-g Mg-chloride/L group, in comparison to the control group (p
