NUTRITION RESEARCH, Vol. 2, pp. 699-714, 1982 0271-5317/82/060699-16503.00/0 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved. RELATIONSHIP OF SUFFICIENT AND ABUNDANTLEVEL OF MINERALS TO LIPID AND LIPOPROTEIN METABOLISM IN MALE RATS Lalitha Murthy, Ph.D., V.N. Finelli, Ph.D., P.S. Gartside, Ph.D., and H,G. Petering, Ph.D. Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267. ABSTRACT Recent studies have shown the effects of inidividual essential metals, or simple combinations of them, on lipid metabolism. Perturbations in blood or liver lipids due to 1% dietary cholesterol were seen in rats fed purified diets containing National Research Council (NRC) recommended levels of minerals, while rats fed chow were protected against these effects. To determine the effects of dietary minerals on lipids and lipoproteins, a study was conducted using male rats fed purified diets with two mineral mixtures, one equivalent to NRC recom- mendations and the other with levels similar to those present in chow. The effects of 1% dietary cholesterol in these groups were compared with those observed in chow-fed rats. The added mineral mixture affected the cholesterol distribution in the lipoprotein fractions by increasing the ratio of high den- sity lipoprotein (HDL) to very low density + low density lipoprotein (VLDL + LDL) from 0.2 to 0.9. Moreover, it significantly reduced the total liver lipid, thus minimizing the fatty liver formation. In view of the above findings, dietary levels of essential mineral nutrients must be considered in studying the relationship of cholesterol to lipid and lipoprotein metabolism. KEY WORDS: Rat, minerals, lipid, lipoprotein, cholesterol. INTRODUCTION During the last few decades hypercholesterolemia has been considered to be one of the primary risk factors in the development of atherosclerosis or ische- mic heart disease. Recently, however, the distribution of cholesterol in plasma lipoproteins is considered to be a more valid parameter in the assessment of the risk than is the total plasma cholesterol. There is sufficient evidence to indicate that increased levels of ~lasma high-density lipoproteins (HDL) are protective in the pathogenesis of atherosclerosis (1-4), whereas elevated plasma levels of low-density lipoporteins (LDL) are associated with a high degree of risk for this disease (4-7). Reports on the effects of a variety of dietary components on plasma cho- lesterol and lipoproteins are too numerous to cite. It is, however, clear that the most prominent role in hypercholesterolemia has been ascribed to the amount and type of dietary fat, in general, and to cholesterol, in particular. There are also indications that, at least in laboratory animals, other constituents of the diet may moderate or eliminate the effects of dietary fat and/or cho- lesterol. During nutritional studies conducted over the past few years we have repeatedly observed that animals fed purified diets showed higher serum and liver lipids than the ones fed commercial chow. Upon examination of the mineral 699
Transcript
NUTRITION RESEARCH, Vol. 2, pp. 699-714, 1982 0271-5317/82/060699-16503.00/0 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
RELATIONSHIP OF SUFFICIENT AND ABUNDANT LEVEL OF MINERALS TO LIPID AND LIPOPROTEIN METABOLISM IN MALE RATS
Lalitha Murthy, Ph.D., V.N. Finel l i , Ph.D., P.S. Gartside, Ph.D., and H,G. Petering, Ph.D.
Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267.
ABSTRACT
Recent studies have shown the effects of inidividual essential metals, or simple combinations of them, on l ip id metabolism. Perturbations in blood or l iver l ipids due to 1% dietary cholesterol were seen in rats fed purified diets containing National Research Council (NRC) recommended levels of minerals, while rats fed chow were protected against these effects. To determine the effects of dietary minerals on l ipids and lipoproteins, a study was conducted using male rats fed purified diets with two mineral mixtures, one equivalent to NRC recom- mendations and the other with levels similar to those present in chow. The effects of 1% dietary cholesterol in these groups were compared with those observed in chow-fed rats. The added mineral mixture affected the cholesterol distribution in the lipoprotein fractions by increasing the ratio of high den- sity lipoprotein (HDL) to very low density + low density lipoprotein (VLDL + LDL) from 0.2 to 0.9. Moreover, i t significantly reduced the total l iver l ip id, thus minimizing the fatty l iver formation. In view of the above findings, dietary levels of essential mineral nutrients must be considered in studying the relationship of cholesterol to l ip id and lipoprotein metabolism.
KEY WORDS: Rat, minerals, l ip id, lipoprotein, cholesterol.
INTRODUCTION
During the last few decades hypercholesterolemia has been considered to be one of the primary risk factors in the development of atherosclerosis or ische- mic heart disease. Recently, however, the distribution of cholesterol in plasma lipoproteins is considered to be a more valid parameter in the assessment of the risk than is the total plasma cholesterol. There is sufficient evidence to indicate that increased levels of ~lasma high-density lipoproteins (HDL) are protective in the pathogenesis of atherosclerosis (1-4), whereas elevated plasma levels of low-density lipoporteins (LDL) are associated with a high degree of risk for this disease (4-7).
Reports on the effects of a variety of dietary components on plasma cho- lesterol and lipoproteins are too numerous to cite. I t is, however, clear that the most prominent role in hypercholesterolemia has been ascribed to the amount and type of dietary fat, in general, and to cholesterol, in particular. There are also indications that, at least in laboratory animals, other constituents of the diet may moderate or eliminate the effects of dietary fat and/or cho- lesterol. During nutritional studies conducted over the past few years we have repeatedly observed that animals fed purified diets showed higher serum and l iver l ipids than the ones fed commercial chow. Upon examination of the mineral
699
700 L. Murthy et al.
content of the various diets, i t was evident that chow diets contained much higher levels of certain micro and macro essential minerals than the purif ied diets meeting the National Research Council (NRC) recommendations (8). Thus, i t was hypothesized that one or more of the essential minerals, at the levels con- tained in commercial chow, were responsible for the protective effects against cholesterol-induced hyperli~idemia and fatty l iver . This prompted us to investigate the effects of dietary cholesterol, as influenced by the total mineral content of the diet, on l i p id and lipoprotein metabolism in the rat.
MATERIALS AND METHODS
Male weanling Sprague-Dawley rats with a mean body weight of 52g were ran- domly assigned to eight groups of seven animals. Rats were individual ly housed in an environmentally controlled room (9), provided with d is t i l l ed demineralized water and fed ad l ibitum. Groups 1,2,3 and 4 were fed a purif ied basal diet (Table 1) containing al l essential nutrients based on the recommendation of the National Academy of Science Committee on Animal Nutrit ion (8). Groups 1 and 2 received a dietary mineral supplement equal to that recommended by NRC (suf f ic ient) , and groups 3 and 4 received minerals approximately equivalent to those present in Purina Laboratory chow (abundant). In addition groups 5 and 6 were fed powdered Purina Laboratory chow (lab chow) to which 5% corn oi l had been added (f inal concentration of fat 10%). Final ly, groups 2,4 and 6 had 1% cholesterol added to their diet. The mineral composition of al l diets is shown in Table 2. The supplemental mineral mixture was prepared by adding 312.5g potassium ci t rate (K3C6H507.H20); 160.Og potassium phosphate (K2HP04); 187.5g calcium carbonate (CaC03) ; 62.5g, magnesium oxide (MgO); 24.125g ferr ic c i t rate (FeC6HsO7"5H20); 1.9g sodium fluoride (NaF) ; 3 1 . 9 g zinc ci t rate (Zn3(C6HsO7)2"2H20); 0.815g cupric c i t rate (Cu2C6H407.2 1/2H20) ; O.04g cobalt chloride (COC12.6H20); 0.019 potassium iodide (KI); with starch as the carrier to make 1 kg. This mix was used at 4% of the diet at the expense of starch in addition to the specially prepared Bernhart and Tomarelli (10) mineral mix which also supplied 5 ppm chromium (Cr(C2H3Op)3.H20) , 0.04 ppm selenium (Na2SeO 3) and 1.0 ppm molybdenum ((NH4)6Mo7024.4H20 F. The final diets were analyzed for all minerals, except f luorine, iodine, cobalt and molybdenum, by atomic absorption spectrophotometry after digesting the samples with 4:1 n i t r ic :perchlor ic acids (11). The values obtained in this laboratory on several batches of Purina Laboratory chow were found to be approximately equivalent to those reported by the supplier (Ralston Purina Company, St. Louis, MO). Similarly, metal analyses of the purif ied diets yielded results in agreement with the values calculated from the mineral content of the salt mix and other dietary ingredients.
Rats were weighed weekly. After 169 days, they were fasted overnight and k i l led by exsanguination (heart puncture, after sodium pentobarbital anesthesia with 65 mg/kg body wt.). Blood was drawn from the heart for hematology, serum metals and l i p id analyses. Hematocrits were obtained by centrifugation. Livers were quickly excised, blotted, weighed and stored frozen in food grade polyethy- lene bags unti l they were analyzed for l ip ids and metals. Tissue (approximately lg), dried in a convection current oven at I05O-I08 oC to constant weight and wet ashed with 4:1 n i t r ic :perchlor ic acids (12), and serum, diluted with d is t i l l ed deionized water (13), were analyzed for zinc, copper and iron by atomic absorp- tion spectrophotometry.
Effects of Minerals on Lipids 701
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Serum high density lipoprotein (HDL) was obtained by centrifugation following precipitation with sodium phosphotungstate-magnesium chloride (17). This precipation method gave HDL associated with the supernatant. Total serum cholesterol and HDL cholesterol were determined enzymatically using a commer- c ial ly available k i t (18). Values for cholesterol associated with very low den- sity and low density lipoproteins (VLDL + LDL) were obtained by subtracting HDL cholesterol from total cholesterol.
STATISTICAL ANALYSIS
The response variables were checked for outliers and non-normality. Outliers were deleted and logarithmic transformations were used for serum copper, cholesterol and high density lipoprotein cholesterol, l iver total l ip ids, and l iver cholesterol. These values are reported in the tables as geometric means while the values of remaining parameters are given as arithmetic means. The data were then analyzed by two-way analysis of variance (ANOVA) and Scheffe comparisons (19). The stat ist ical significance by ANOVA for the overall model, cholesterol and diet effects were determined. Their interactions are reported in the tables. A p value of 0.05 or less was considered as s igni f i - cant.
RESULTS
Periodic examination of the rats throughout the experiment did not reveal any symptoms of deficiency or toxici ty in any of the groups. Growth rate as well as hematopoiesis were normal and similar in all groups. No significant changes were observed in the hematocrits and final body weights at sacrifice. However, there was an overall significant increase (p < O.O001) in l iver wet weight as well as specific l iver weight in the cholesterol-fed groups (Table 3). Furthermore, the l iver weight of chow-fed rats was greater than those of rats fed purif ied diets independently from the cholesterol and the mineral contents of these diets. The least increment in l iver weight was evident in group 4. The l iver total l ipids and cholesterol, as shown in Table 4, were also elevated in the cholesterol-fed group. These increases were minimized in groups 4 and 6 indicating the protective effect of dietary minerals. Liver phospholipids, when expressed in terms of fat-free wet weight (range 35-39 mg/g), were not altered by dietary manipulations.
The data on serum cholesterol, HDL cholesterol, and the calculated values for HDL cholesterol as percent of total cholesterol, (VLDL + LDL) cholesterol, and the ratio of HDL/(VLDL + LDL) cholesterol, along with the stat ist ical signi- ficance of effect by ANOVA, are given in Table 5. Analysis of variance of the serum cholesterol values did not show a significant effect due to dietary cho- lesterol. However, there was a significant effect on serum cholesterol due to the diet, to the interaction of cholesterol and diet, as well as an overall effect which indicates a disturbance in cholesterol metabolism by dietary mani- pulations. Among the cholesterol-fed groups the only one that showed a s ign i f i -
704 L. Murthy et al.
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Effects of Minerals on Lipids 711
cant increase (p < O.OOl) in serum cholesterol was group 2. Furthermore, dietary cholesterol as well as the mineral composition of the diet affected signif icantly the cholesterol bound to HDL. Similarly, (VLDL + LDL) cholesterol as well as the percentage of HDL cholesterol were also affected by the dietary manipulations. There was a signif icant effect of dietary cholesterol in the lipoprotein fractions and this was mainly contributed by the group 2. As expected, dietary cholesterol lowered the ratio of HDL/(VLD + LDL) cholesterol. This effect was more pronounced in the rats fed the diet with suff icient minerals (group 2), while i t was minimized in the groups receiving the diet with abundant minerals or chow (groups 4 and 6). I t is evident that the presence of abundant levels of minerals in the diet caused a favorable alteration in l ip id metabolism in cholesterol-fed rats. Thus, our hypothesis has been confirmed.
Zinc, copper and iron content of serum and l iver are presented in Tables 6 and 7, respectively. Analysis of variance of the data indicated no major changes in serum metals due to the dietary manipulation. The only exception is a significant (p < 0.022) decrease in serum zinc due to dietary cholesterol. Again, i t can be seen here that this significance of effect was mainly contri- buted by the group fed suff icient levels of minerals (group 2).
The results on l iver metals, expressed in terms of ug/g wet weight as well in terms of ug/g fat-free dry weight, are presented in Table 7. Effects of dietary cholesterol on l iver zinc and copper were evident only when the concen- trations of these metals were expressed on tissue wet weight basis, but not when they were based on fat-free dry weight. Thus, this effect is only secondary to the increased l iver weight due to fat in f i l t ra t ion . The effect of cholesterol on l iver iron, however, remained significant independently of the fat content of l iver . Cholesterol lowered l iver iron in all three groups with the lowest value occurring in rats fed diet containing suff icient level of minerals. Liver manganese values, either expressed on wet weight basis (range, 2.1 to 2.4 ug/g) or on fat-free dry weight basis (range 9.7-I0.9 ug/g), were not affected by any of the dietary manipulations, since the level of dietary manganese was the same in all of the diets tested (Table 2).
DISCUSSION
The most significant findings of this study were the protective effects of the mineral content of diet on serum cholesterol and i ts lipoprotein distribu- tion. I t is evident from this study that dietary cholesterol decreased HDL and increased VLDL + LDL cholesterol in the rat. I t is widely accepted that the partit ion of serum cholesterol into i ts lipoprotein fractions improves the pre- d ic tab i l i ty of the risk for cardiovascular diseases in humans (20). Thus, i f HDL cholesterol is negatively correlated to the risk of coronary heart disease while the cholesterol bound to LDL and VLDL is positively associated with this risk, then the ratio HDL/(LDL + VLDL) may be a better indicator of the risk than either of these values alone. In any case, upon examination of total serum cho- lesterol or i ts lipoprotein fraction or HDL/(LDL + VLDL), i t is evident that the increased mineral content of the diet offers protections against the adverse effect of dietary cholesterol. In addition to the alteration of cholesterol distribution in the lipoprotein fractions, the dietary minerals also affected fat in f i l t ra t ion in the l iver of cholesterol-fed rats. Whether this effect is due to the altered intestinal absorption of cholesterol or to the mobilization
712 L. Murthy et al.
of cholesterol from peripheral tissues remains to be studied. In regard to the trace metal content of tissues, the data did not suggest drastic changes in the distribution of metals in the cholesterol-fed groups, possibly with the excep- tion of iron. I t is unclear at this point how the added minerals affect l ipids and lipoproteins without any major changes of the trace metal content of l iver and plasma. Whether the essential macro minerals are the ones responsible for moderating the effects on lipids due to dietary cholesterol, rather than the micro minerals, remains to be investigated.
We have repeatedly found that chow, in relation to purified diets, is pro- tective against the effects of dietary cholesterol on l ip id metabolism in rats (21). Others have also demonstrated the protective effects of chow diets on l ip id metabolism (22) and on experimental atherosclerosis (23) in rabbits. Although there may be other dietary factors in chow influencing l ip id metabo- lism, in this study we have definitely shown that the total mineral mixture in the diet plays a major role in the modification of the effects of dietary cho- lesterol on l ip id levels and lipoprotein distribution.
I t is fe l t that reduced levels of HDL cholesterol may constitute a major risk factor in the development of atherosclerosis, since HDL seems to play an important role in the mobilization of cholesterol from peripheral tissue (1). Thus, in view of the above findings, i t is necessary to consider the essential mineral content of the diet in studying the relationship of dietary cholesterol and/or fat to l ip id and lipoprotein metabolism. Moreover, i t is important to evaluate further the role of dietary minerals, as well as that of dietary cho- lesterol and fat intake, in the prevention and control of cardiovascular diseases. Finally, the elevated l iver weight of chow-fed rats, over that observed in the groups fed purified diets, may be attributable to the presence of hepatotoxic contaminants in the natural products which are constituents of the closed formula chow diet.
ACKNOWLEDGMENTS
The authors wish to thank Mr. Charles Broge, Mr. James Boyle and Mrs. Deborah Keaton for their excellent technical assistance. The authors also appreciate the help of Mrs. Rebecca Davis, Mrs. Jane Onslow, and Mrs. Nancy Knapp in typing the manuscript. This research was partly supported by NIH ES-00159.
Effects of Minerals on Lipids 713
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