BIOLOGICAL TRACE ELEMENT RESEARCH 4, 95-104 (1982)
Magnesium, Calcium, Copper, and Zinc in Meals Correlations Related to the Epidemiology of Ischemic Heart Disease LESLIE M . K L E V A Y
United States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota 58202 Received November 22, 1981; Accepted December 8, 1981
Abstract Meals of 12 diets were prepared from conventional foods with precautions against contamination by metallic elements because of epidemiologic associations between ischemic heart disease and the metabolism of magnesium, calcium, copper, and zinc. Magnesium, calcium, copper, and zinc were measured by atomic absorption spectrometry with satisfactory accuracy and precision. The mean daily amount of copper in the diets was less than the apparent adult requirement. Mean amounts of magnesium and zinc were close to apparent requirements; however, adults consuming amounts less than one standard deviation below these means may be depleting body stores and be at risk of pathology. Magnesium and copper were highly correlated (r = 0.849, P = 0.0001) in meals. This and other significant correlations probably will prevent the relationships of these elements to ischemic heart disease from being elucidated by epidemiology. Metabolic experiments will be necessary to differentiate among several hypotheses. Index Entries: Calcium, and heart disease epidemiology; copper, and heart disease epidemiology; epidemiology, of Mg, Ca, Cu, and Zn in heart disease; ischemic heart disease, and Mg, Ca, Cu, and Zn; magnesium, and heart disease epidemiology; trace elements, in heart disease epidemiology; zinc, in heart disease epidemiology; heart disease epidemiology, of Mg, Ca, Cu, and Zn.
9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900-0095502.00
95
96
L . M . KLEVAY
Ischemic heart disease is the leading cause of death in the United States (1) and in much of the industrialized part of the world. The origin of this disease is mysterious; many factors have been implicated in its etiology and pathogenesis. Among the many dietary factors that have been associated with ischemic heart disease are the essential nutrients magnesium, calcium, copper, and zinc. Knox (2) found a negative correlation between dietary calcium and heart disease risk in England and Wales. Varo (3) found a high positive correlation between death rates for ischemic heart disease and the dietary ratio of calcium to magnesium in the countries comprising the Organization for Economic Cooperation and Development. Finnish children with the highest concentrations of cholesterol in serum, and presumably the highest risk of ischemic heart disease, ate less calcium than did those with the lowest concentrations of cholesterol (4). There are many similarities between animals deficient in copper and people with ischemic heart disease. Both are likely to die suddenly, to be hypercholesterolemic and hyperuricemic, to have arteries with abnormal connective tissue, and to have fibrotic, hypertrophied hearts low in copper with abnormal electrocardiograms (5). Copper and zinc can interact with each other in metabolism. High dietary zinc can increase copper requirements and lessen copper toxicity; high dietary copper can increase zinc requirements and lessen zinc toxicity (6). There is a positive correlation between the ratio of zinc to copper of milk available in 47 cities of the US and the mortality attributable to coronary heart disease in the cities (7). Among the epidemiologic characteristics associated with a high ratio of zinc to copper and either high risk of mortality or hypercholesterolemia are diets high in fat or sucrose, pregnancy, nephrotic syndrome, chronic hemodialysis, and hypertension. Associated with a low ratio of zinc to copper and either low risk of mortality or hypocholesterolemia are diets high in fiber, hepatic cirrhosis, infectious hepatitis, strenuous exercise, and the availability of hard water (8-12). Magnesium metabolism and magnesium deficiency also have been hypothesized to be important in the origin of ischemic heart disease (13, 14). Calcium, copper, and zinc can interact in metabolism, too. As calcium in the diet of rats was increased, the concentration of cholesterol in serum decreased and the concentrations of copper and zinc in liver increased and decreased, respectively (15). In adult rats there is an inverse correlation between the concentration of cholesterol in plasma and of copper in liver microsomes (16). Magnesium, calcium, copper, and zinc were measured in meals of 12 diets. Correlations were calculated between pairs of elements, between some important ratios of elements, and between ratios of elements and some elements. The null hypothesis that correlations were not significantly different from zero was tested. Significant correlations would identify nutritional associations of epidemiologic importance.
Methods Four (17) of these diets (AGK) were prepared in a university cafeteria according to menus designed to change the concentration of cholesterol in plasma by manipulating the intake of eggs, fat, fruits, meat, leguminous seeds, and vegeta-
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
97
bles (18). Five (19) of the diets (MCRH) were prepared in a local hospital and were typical of those served to patients. Three diets were prepared at home using usual culinary practices: the Kempner (20) rice-fruit diet for the treatment of hypertension and two diets (LMK) typical of those consumed by me. All diets were made from conventional foods with precautions against contamination. Details have been described (17, 19). A clean polyethylene container was provided for each meal. After individual foods were accurately weighed, each meal was freeze-dried. The dry meal was converted to a homogeneous powder by pulverization, grinding, being passed through a sieve, and mixing. Magnesium, calcium, copper, and zinc were measured in powdered meals by atomic absorption spectrometry in triplicate after digestion with nitric and sulfuric acids and hydrogen peroxide. Data on copper and zinc in the first nine diets were obtained similarly and have been reported (17, 19). Statistical calculations were done according to Ostle and Mensing (21).
Results The 134 foods used in these diets were classified according to the basic groups (22) and are tabulated in Table 1. Accuracy and precision of analysis can be assessed from the data in Table 2. Recovery (accuracy) of standard amounts of the elements to samples of meals was approximately 98%. Values were generally within 3% of true values (accuracy) from the National Bureau of Standards for liver samples. Coefficients of variation (precision) for replicate analyses of meals were approximately 4%. Table 3 contains the amounts of the elements in the meals and the mean daily amounts of the elements for each diet. Table 4 contains the correlations between pairs of data: elements, element vs ratio, and ratio vs ratio.
Discussion The diets generally contained carbohydrate, fat, and protein in amounts similar to the average percentages (of energy) in diets in the United States of 46, 42, and 12%, respectively (23). The amount of energy generally exceeded the mean values for young adult females (2100 kcal); several were similar to the corresponding value for males (2900 kcal). Some diets were quite different from average. The Kempner diet contained approximately 2% of the energy as fat and 5% as protein. It also contained only 150 mg sodium, an amount lower than usual in the United States (23) and lower than the low sodium diet (MCRH), which contained 2 g. Two diets (Kempner, AGK: L-2) were low in cholesterol (less than 181 mg) in comparison to the usual amounts found in a large dietary survey (24). The Kempner diet contained no foods of animal origin. The mean daily amounts of magnesium, calcium, copper, and zinc in these diets were 281, 1201, 1.12, and 11.2 mg, respectively. This amount of calcium is suffi-
98
L, M. KLEVAY
TABLE 1 Foods in the Diets ~ Milk and dairy products Butter Buttermilk Cheese (Colby, Parmesan) Half-and-half Ice cream Ice cream bar Milk (dried skim, skim, whole) Meat, poultry, fish, eggs, legumes, and nuts Beef (ground) Bologna (beef) Chicken Coconut Egg Ham Link sausage Pea Peanut butter Roast beef Seeds: caraway, poppy Steak (Swiss) Turkey Veal Walnut Cereals and their products Bran cereal (Bran Buds) Bread (Italian, white) Corn flakes Cracker (saltine) Cream of wheat Flour (All purpose; Dakota Maid, Rye) Noodle: egg Oat cereal (Cheerios) Rice Spaghetti Wheat wafer (Triscuit)
Fruits and vegetables Apple Apricotb Banana Blueberry Blueberry~ Broccoli Cabbage Carrot Carrot ~ Celery Corn~ Grape Grapefruit Juices: Apple~ Grapefruit Lemon Orange" Tomato ~ (salt-free and regular) Lemon Lettuce Melon: honey dew Onion Orange Parsley (dried) Peach b Peaff Pineapple Pineapple ~ Potato Prune ~ Radish Raisin Tomato Tomato ~
Mixed foods and other Baking powder Basil Bay leaf Beer (pilsner) Boullion (chicken) Brandy Candy (creme de menthe) Catsup Cinnamon Coffee: Espresso Instant, regular Dressing b (French, mayonnaise) Fruit cocktaiP Garlic Garlic spread Gravy (drippings: beef, ham Gravy' (turkey) Honey Jam: strawberry Jelly: apple, grape Margarine: Corn oil, Vegetable, Other Mushrooms b Mustard (dry) Oil: Corn Cottonseed Salad Vegetable Orange ice Oregano Paprika Pepper Pickles (dill, sweet) Pie (lemon meringue) Pudding: lemon (continued)
99
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
TABLE 1 (continued)
Mixed foods and other Salt Salt (iodized) SoupS: Minestrone Vegetable Sugar (brown, white) Sweetener, artificial Syrupb Tomato paste Vanilla Vanilla bean Vinegar Vodka Whipped topping Wine (sweet vermouth) Yeast (dry) ~ bCanned. ~Frozen.
indicate type of processing. A colon precedes a member of a class of foods.
cient to satisfy the Recommended Dietary Allowance (23) for adults. These daily intakes of magnesium and zinc are less than the adult Recommended Dietary Allowances of 350 and 15 mg, respectively (23). A magnesium intake of 281 mg is close to some estimates of the adult magnesium requirement (23). The daily adult requirement for zinc seems to be approximately 11-13 mg (25; for other references, see 19). However this requirement can be higher or lower than this range because of effects of other dietary materials on TABLE 2 Accuracy and Precision of Analysisa
Ca Mg Cu Zn Mean
Recovery, %
True value, %
Coefficient of variation, %
98.3 98.4 96.6 97.1 97.6
99.2 98.5 96.9 99.8 98.6
5.47 2.90 4.77 2.73 3.97
aAccuracy was assessed by measurement of the recovery of standard amounts of elements to samples of meals and by comparison of results with the true values for bovine liver (NBS reference material #1577). Precision was assessed by calculation of the coefficient of variation for triplicate analyses.
100
L . M . KLEVAY
TABLE 3 Amounts of the Elements in Meals and Daily Diets (mg) a
AGK: R
AGK: S
AGK: L-I
AGK: L-2
MCRH: General
MCRH: Soft
MCRH: Low Na
MCRH: Low Cal.
MCRH: Puree
Kempner
Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total
Magnesium
Calcium
Copper
Zinc
43 106 116 265 40 125 159 324 68 125 168 361 79 121 177 377 75 94 118 287 38 74 62 174 22 55 52 129 46 63 69 178 56 70 77 203 88 73 60 221
424 368 274 1066 263 725 675 1663 595 787 855 2237 773 326 547 1646 638 412 576 1626 377 275 303 955 208 275 167 650 440 310 332 1083 557 492 498 1547 82 56 25 163
0.27 0.50 0.32 1.09 0.13 0.44 0.73 1.30 0.16 0.43 0.94 1.53 0.18 0.50 0.65 1.33 0.17 0.29 0.40 0.86 0.22 0.32 0.28 0.83 0.14 0.26 0.42 0.82 0.11 0.25 0.31 0.67 0.16 0.22 0.23 0.60 0.49 0.50 0.44 1.43
2.2 1.9 6.3 10.4 1.2 4.8 10.7 16.7 1.3 5.3 11.4 18.0 1.7 2.8 5.3 9.8 2.6 2.7 5.8 11.1 2.2 2.7 4.3 9.2 1.4 4.0 5.1 10.5 1.6 2.3 4.5 8.4 1.5 2.1 4.0 7.6 2.0 1.8 1.0 4.9
(continued)
101
METALLIC ELEMENTS A N D HEART DISEASE EPIDEMIOLOGY
TABLE 3
LMK: Fr 78
(continued)
Magnesium
Calcium
Copper
Zinc
152 44 229 425 125 128 180 433 281 102
222 44 216 482 327 471 501 1299 1201 586
0.47 0.14 0.95 1.56 0.32 0.46 0.61 1.39 1.12 0.35
4.5 1.8 7.3 13.5 2.8 4.5 7.0 14.2 11.2 3.8
Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total
LMK: Sa 78
Mean daily amounts Standard deviation
aTotal daily amounts do not alwaysequal the sum of the values for each meal because of rounding error. bioavailability (25) and retention (26). If these diets are representative of diets in the United States, adults who regularly consume diets containing amounts between one and two standard deviations below mean daily amounts may be depleting body stores. They may be at risk for pathology. There is no Recommended Dietary Allowance for copper (23); the suggested safe and adequate daily intake for adults is 2-3 mg (23). The adult daily requirement for copper seems to be about 2 mg (27); the most recent measurement of the requirement of men is 1.30 mg/day (28). If the copper requirement actually exceeds 1.3 mg/day, average adults who regularly consume a diet represented by the average diet here minus one standard deviation probably will deplete their body stores and be at risk for pathology. The Kempner diet contained the lowest amount of zinc. Perhaps this amount of zinc (4.9 mg) contributes to its efficacy in lowering blood pressure. Converting
TABLE 4 Correlation Coefficients for 36 Mealsa Mg Mg Ca Cu Zn Ca/Mg
--
Ca
Cu
Zn
Ca/Mg
Zn/Cu
0.322 --
0.849 ~ 0.128 --
0.731~ 0.4168 0.762 c --
-0.478 D 0.540 e -0.566 v -0.272 --
-0.021 0.333 A -0.202 0.438 c 0.265
aProbabilities have been rounded to one figure: 0.05A, 0.01B, 0.008c, 0.003~ 0.0007 E, 0.0003 F, 0.0001a.
102
L . M . KLEVAY
enzyme, which produces the potent pressor agent angiotension II from an inactive precursor (29), contains zinc at its active site (30). The long-term consumption of this diet may result in zinc depletion and impaired enzyme activity. This hypothetical mechanism may supplement the beneficial effect of the low salt intake. Some of the meals contained cheese, eggs, or milk, but no meat. Neither fish nor nutritional supplements were included. Only three meals contained small amounts of alcoholic beverages. Some of the meals were considerably higher in fat and protein than others. It seems likely that correlations calculated for 36 meals would be representative of a greater diversity of diets than correlations calculated for 12 diets. That is, meals of these 12 diets may provide information on characteristics of diets not prepared and analyzed in this work. Correlations were calculated for the 12 diets, however. Table 4 shows signficant correlations between pairs of nutrients and between nutrients and ratios of nutrients for meals. The significant correlations between calcium and the ratio of calcium to magnesium or zinc and the ratio of zinc to copper may be biologically trivial. Alternatively, the biological importance remains undiscovered. These correlations became insignificant when calculated for diets (n = 12) instead of meals (n = 36): Mg vs Ca, P = 0.3; Mg vs Ca/Mg, P = 0.2; Ca vs Zn/Cu, P = 0.08; Cu vs Zn, P = 0.09; Zn vs Zn/Cu, P = 0.02. No correlations that were significant for meals had a different sign when calculated for diets. The correlation between CaJMg vs Zn/Cu was the only one significant for diets (r = 0.804, P = 0.002) that was insignificant for meals. The significant correlations between magnesium and copper, magnesium and the ratio of calcium to magnesium, copper and the ratio of calcium to magnesium, and the ratio of zinc to copper and the ratio of calcium to magnesium are consistent with epidemiology. Low intakes of magnesium and copper and high dietary ratios of calcium to magnesium and zinc to copper are hypothesized to be harmful. Comparison of the sizes of correlation coefficients between magnesium and copper, copper and zinc, copper and the ratio of calcium to magnesium, and so on will not be epidemiologically informative. For example, even though magnesium and copper are highly correlated, a diet could contain adequate magnesium and insufficient copper or vice versa in comparison to requirements. Nutritional adequacy is measured by comparison to standards independent (ideally) of the amounts in diets. The large correlations between some of these elements and between elements and ratios of elements probably will prevent the relationship(s) of these nutrients to ischemic heart disease from being elucidated by epidemiology. Associations between elements, etc., and disease have been useful in the formulation of hypotheses. Causality and pathophysiology can be determined by experimentation. Experiments, for example on the effects of copper deficiency, can decrease the number of variables so that results can be interpreted. Then series of experiments can be used to build an hypothesis supported by more than statistics.
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
103
Acknowledgment I wish to thank my wife, Martha N. Klevay, for diet preparation.
References 1. Anon., Vital Statistics of the United States1970. Mortality, US Department of Health, Education and Welfare, Washington, DC, 1974, Vol. 2, Part A, Table 1-7. 2. E. G. Knox, Lancet 1, 1465 (1973). 3. P. Varo, Int. J. Vit. Nutr. Res. 44, 267 (1974). 4. L. R~is~inen, M. Wilska, R.-L. Kantero, V. N~intr, A. Ahlstrrm, and N. Hallman, Am. J. Clin. Nutr. 31, 1050 (1978). 5. L. M. Klevay, Ann. NYAcad. Sci. 355, 140 (1980). 6. E.J. Underwood, Trace Elements in Human and AnimaI Nutrition, 4th ed., Academic Press, New York, 1977, pp. 61,226, 231. 7. L. M. Klevay, in Trace Substances in Environmental Health, vol. 8, D. D. Hemphill, ed., University of Missouri Press, Columbia, Missouri, 1974, pp. 9-14. 8. L. M. Klevay, Am. J. Clin. Nutr. 26, 1060 (1973). 9. L. M. Klevay, Am. J. Clin. Nutr. 28, 764 (1975). 10. L. M. Klevay, Perspect. Biol. Med. 20, 186 (1977). 11. L. M. Klevay, in Advances in Nutritional Research, vol. 1, H. H. Draper, ed., Plenum, New York, 1977, pp. 227-252. 12. L. M. Klevay, Proceedings of the 19th Annual Meeting of the American College of Nutrition, S.P. Medical and Scientific Books, Jamaica, N.Y., 1981. 13. M. S. Seelig, and H. A. Heggtveit, Am. J. Clin. Nutr. 27, 59 (1974). 14. M. S. Seelig, Magnesium Deficiency in the Pathogenesis of Disease: Early Roots of Cardiovascular, Skeletal, and Renal Abnormalities, Plenum, New York, 1980. 15. R. S. Romasz, E. A. Lemmo, and J. L. Evans, in Trace Substances in Environmental Health, vol. 11, D. D. Hemphill, ed., University of Missouri Press, Columbia, Missouri, 1977, pp. 289-296. 16. R. A. Jacob, L. M. Klevay, and G. M. Logan, Jr.,Am. J. Clin. Nutr. 31,477 (1978). 17. L. M. Klevay, K. P. Vo-Khactu, and R. A. Jacob, in Trace Substances in Environmental Health, vol. 9, D. D. Hemphill, ed., University of Missouri Press, Columbia, Missouri, 1975, pp. 131-138. 18. J. T. Anderson, F. Grande, and A. Keys, J. Am. Dietet. Assoc. 62, 133 (1973). 19. L. M. Klevay, S. J. Reck, and D. F. Barcome, J. Am. Med. Assoc. 241, 1916 (1979). 20. E. Peschel, and R. L. Peschel, J. Clin. Invest. 29, 455 (1950). 21. B. Ostle, and R. W. Mensing, Statistics in Research, 3rd ed., The Iowa State University Press, Ames, Iowa, 1975, pp. 165-180. 22. F. J. Stare and M. McWilliams, Living Nutrition, 2nd ed., Wiley, 1977, pp. 7-8. 23. Anon., Recommended Dietary Allowances, 9th ed., Food and Nutrition Board, National Research Council, National Academy of Sciences, Washington, DC, 1980, pp. 6, 23, 31, 129, 135, 146, 153, 170. 24. A. B. Nichols, C. Ravenscroft, D. E. Lamphiear, and L. D. Ostrander, Jr., Am. J. Clin. Nutr. 29, 1384 (1976).
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25. W. J. Darby et al., Trace Elements in Human Nutrition, World Health Organization
Technical Report Series, No. 532, Geneva, 1973, pp. 9-15. 26. H. Sandstead, L. Klevay, J. Mahalko, L. Johnson, and D. Milne, Am. J. Clin. Nutr.
34, 617 (1981). 27. Anon., Recommended Dietary Allowances, 8th ed., Food and Nutrition Board, Na-
tional Research Council, National Academy of Sciences, Washington, DC, 1974, pp. 95-96. 28. L. M. Klevay, S. J. Reck, R. A. Jacob, G. M. Logan, Jr., J. M. Mufioz, and H. H. Sandstead, Am. J. Clin. Nutr. 33, 45 (1980). 29. Y. S. Bakhle, in Angiotension, I. H. Page and F. M. Bumpus, eds., Springer Verlag, New York, 1974, pp. 41-80. 30. M. Das and R. L. Softer, J. Biol. Chem. 250, 6762 (1975).
Magnesium, Calcium, Copper, and Zinc in Meals Correlations Related to the Epidemiology of Ischemic Heart Disease LESLIE M . K L E V A Y
United States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota 58202 Received November 22, 1981; Accepted December 8, 1981
Abstract Meals of 12 diets were prepared from conventional foods with precautions against contamination by metallic elements because of epidemiologic associations between ischemic heart disease and the metabolism of magnesium, calcium, copper, and zinc. Magnesium, calcium, copper, and zinc were measured by atomic absorption spectrometry with satisfactory accuracy and precision. The mean daily amount of copper in the diets was less than the apparent adult requirement. Mean amounts of magnesium and zinc were close to apparent requirements; however, adults consuming amounts less than one standard deviation below these means may be depleting body stores and be at risk of pathology. Magnesium and copper were highly correlated (r = 0.849, P = 0.0001) in meals. This and other significant correlations probably will prevent the relationships of these elements to ischemic heart disease from being elucidated by epidemiology. Metabolic experiments will be necessary to differentiate among several hypotheses. Index Entries: Calcium, and heart disease epidemiology; copper, and heart disease epidemiology; epidemiology, of Mg, Ca, Cu, and Zn in heart disease; ischemic heart disease, and Mg, Ca, Cu, and Zn; magnesium, and heart disease epidemiology; trace elements, in heart disease epidemiology; zinc, in heart disease epidemiology; heart disease epidemiology, of Mg, Ca, Cu, and Zn.
9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900-0095502.00
95
96
L . M . KLEVAY
Ischemic heart disease is the leading cause of death in the United States (1) and in much of the industrialized part of the world. The origin of this disease is mysterious; many factors have been implicated in its etiology and pathogenesis. Among the many dietary factors that have been associated with ischemic heart disease are the essential nutrients magnesium, calcium, copper, and zinc. Knox (2) found a negative correlation between dietary calcium and heart disease risk in England and Wales. Varo (3) found a high positive correlation between death rates for ischemic heart disease and the dietary ratio of calcium to magnesium in the countries comprising the Organization for Economic Cooperation and Development. Finnish children with the highest concentrations of cholesterol in serum, and presumably the highest risk of ischemic heart disease, ate less calcium than did those with the lowest concentrations of cholesterol (4). There are many similarities between animals deficient in copper and people with ischemic heart disease. Both are likely to die suddenly, to be hypercholesterolemic and hyperuricemic, to have arteries with abnormal connective tissue, and to have fibrotic, hypertrophied hearts low in copper with abnormal electrocardiograms (5). Copper and zinc can interact with each other in metabolism. High dietary zinc can increase copper requirements and lessen copper toxicity; high dietary copper can increase zinc requirements and lessen zinc toxicity (6). There is a positive correlation between the ratio of zinc to copper of milk available in 47 cities of the US and the mortality attributable to coronary heart disease in the cities (7). Among the epidemiologic characteristics associated with a high ratio of zinc to copper and either high risk of mortality or hypercholesterolemia are diets high in fat or sucrose, pregnancy, nephrotic syndrome, chronic hemodialysis, and hypertension. Associated with a low ratio of zinc to copper and either low risk of mortality or hypocholesterolemia are diets high in fiber, hepatic cirrhosis, infectious hepatitis, strenuous exercise, and the availability of hard water (8-12). Magnesium metabolism and magnesium deficiency also have been hypothesized to be important in the origin of ischemic heart disease (13, 14). Calcium, copper, and zinc can interact in metabolism, too. As calcium in the diet of rats was increased, the concentration of cholesterol in serum decreased and the concentrations of copper and zinc in liver increased and decreased, respectively (15). In adult rats there is an inverse correlation between the concentration of cholesterol in plasma and of copper in liver microsomes (16). Magnesium, calcium, copper, and zinc were measured in meals of 12 diets. Correlations were calculated between pairs of elements, between some important ratios of elements, and between ratios of elements and some elements. The null hypothesis that correlations were not significantly different from zero was tested. Significant correlations would identify nutritional associations of epidemiologic importance.
Methods Four (17) of these diets (AGK) were prepared in a university cafeteria according to menus designed to change the concentration of cholesterol in plasma by manipulating the intake of eggs, fat, fruits, meat, leguminous seeds, and vegeta-
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
97
bles (18). Five (19) of the diets (MCRH) were prepared in a local hospital and were typical of those served to patients. Three diets were prepared at home using usual culinary practices: the Kempner (20) rice-fruit diet for the treatment of hypertension and two diets (LMK) typical of those consumed by me. All diets were made from conventional foods with precautions against contamination. Details have been described (17, 19). A clean polyethylene container was provided for each meal. After individual foods were accurately weighed, each meal was freeze-dried. The dry meal was converted to a homogeneous powder by pulverization, grinding, being passed through a sieve, and mixing. Magnesium, calcium, copper, and zinc were measured in powdered meals by atomic absorption spectrometry in triplicate after digestion with nitric and sulfuric acids and hydrogen peroxide. Data on copper and zinc in the first nine diets were obtained similarly and have been reported (17, 19). Statistical calculations were done according to Ostle and Mensing (21).
Results The 134 foods used in these diets were classified according to the basic groups (22) and are tabulated in Table 1. Accuracy and precision of analysis can be assessed from the data in Table 2. Recovery (accuracy) of standard amounts of the elements to samples of meals was approximately 98%. Values were generally within 3% of true values (accuracy) from the National Bureau of Standards for liver samples. Coefficients of variation (precision) for replicate analyses of meals were approximately 4%. Table 3 contains the amounts of the elements in the meals and the mean daily amounts of the elements for each diet. Table 4 contains the correlations between pairs of data: elements, element vs ratio, and ratio vs ratio.
Discussion The diets generally contained carbohydrate, fat, and protein in amounts similar to the average percentages (of energy) in diets in the United States of 46, 42, and 12%, respectively (23). The amount of energy generally exceeded the mean values for young adult females (2100 kcal); several were similar to the corresponding value for males (2900 kcal). Some diets were quite different from average. The Kempner diet contained approximately 2% of the energy as fat and 5% as protein. It also contained only 150 mg sodium, an amount lower than usual in the United States (23) and lower than the low sodium diet (MCRH), which contained 2 g. Two diets (Kempner, AGK: L-2) were low in cholesterol (less than 181 mg) in comparison to the usual amounts found in a large dietary survey (24). The Kempner diet contained no foods of animal origin. The mean daily amounts of magnesium, calcium, copper, and zinc in these diets were 281, 1201, 1.12, and 11.2 mg, respectively. This amount of calcium is suffi-
98
L, M. KLEVAY
TABLE 1 Foods in the Diets ~ Milk and dairy products Butter Buttermilk Cheese (Colby, Parmesan) Half-and-half Ice cream Ice cream bar Milk (dried skim, skim, whole) Meat, poultry, fish, eggs, legumes, and nuts Beef (ground) Bologna (beef) Chicken Coconut Egg Ham Link sausage Pea Peanut butter Roast beef Seeds: caraway, poppy Steak (Swiss) Turkey Veal Walnut Cereals and their products Bran cereal (Bran Buds) Bread (Italian, white) Corn flakes Cracker (saltine) Cream of wheat Flour (All purpose; Dakota Maid, Rye) Noodle: egg Oat cereal (Cheerios) Rice Spaghetti Wheat wafer (Triscuit)
Fruits and vegetables Apple Apricotb Banana Blueberry Blueberry~ Broccoli Cabbage Carrot Carrot ~ Celery Corn~ Grape Grapefruit Juices: Apple~ Grapefruit Lemon Orange" Tomato ~ (salt-free and regular) Lemon Lettuce Melon: honey dew Onion Orange Parsley (dried) Peach b Peaff Pineapple Pineapple ~ Potato Prune ~ Radish Raisin Tomato Tomato ~
Mixed foods and other Baking powder Basil Bay leaf Beer (pilsner) Boullion (chicken) Brandy Candy (creme de menthe) Catsup Cinnamon Coffee: Espresso Instant, regular Dressing b (French, mayonnaise) Fruit cocktaiP Garlic Garlic spread Gravy (drippings: beef, ham Gravy' (turkey) Honey Jam: strawberry Jelly: apple, grape Margarine: Corn oil, Vegetable, Other Mushrooms b Mustard (dry) Oil: Corn Cottonseed Salad Vegetable Orange ice Oregano Paprika Pepper Pickles (dill, sweet) Pie (lemon meringue) Pudding: lemon (continued)
99
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
TABLE 1 (continued)
Mixed foods and other Salt Salt (iodized) SoupS: Minestrone Vegetable Sugar (brown, white) Sweetener, artificial Syrupb Tomato paste Vanilla Vanilla bean Vinegar Vodka Whipped topping Wine (sweet vermouth) Yeast (dry) ~ bCanned. ~Frozen.
indicate type of processing. A colon precedes a member of a class of foods.
cient to satisfy the Recommended Dietary Allowance (23) for adults. These daily intakes of magnesium and zinc are less than the adult Recommended Dietary Allowances of 350 and 15 mg, respectively (23). A magnesium intake of 281 mg is close to some estimates of the adult magnesium requirement (23). The daily adult requirement for zinc seems to be approximately 11-13 mg (25; for other references, see 19). However this requirement can be higher or lower than this range because of effects of other dietary materials on TABLE 2 Accuracy and Precision of Analysisa
Ca Mg Cu Zn Mean
Recovery, %
True value, %
Coefficient of variation, %
98.3 98.4 96.6 97.1 97.6
99.2 98.5 96.9 99.8 98.6
5.47 2.90 4.77 2.73 3.97
aAccuracy was assessed by measurement of the recovery of standard amounts of elements to samples of meals and by comparison of results with the true values for bovine liver (NBS reference material #1577). Precision was assessed by calculation of the coefficient of variation for triplicate analyses.
100
L . M . KLEVAY
TABLE 3 Amounts of the Elements in Meals and Daily Diets (mg) a
AGK: R
AGK: S
AGK: L-I
AGK: L-2
MCRH: General
MCRH: Soft
MCRH: Low Na
MCRH: Low Cal.
MCRH: Puree
Kempner
Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total
Magnesium
Calcium
Copper
Zinc
43 106 116 265 40 125 159 324 68 125 168 361 79 121 177 377 75 94 118 287 38 74 62 174 22 55 52 129 46 63 69 178 56 70 77 203 88 73 60 221
424 368 274 1066 263 725 675 1663 595 787 855 2237 773 326 547 1646 638 412 576 1626 377 275 303 955 208 275 167 650 440 310 332 1083 557 492 498 1547 82 56 25 163
0.27 0.50 0.32 1.09 0.13 0.44 0.73 1.30 0.16 0.43 0.94 1.53 0.18 0.50 0.65 1.33 0.17 0.29 0.40 0.86 0.22 0.32 0.28 0.83 0.14 0.26 0.42 0.82 0.11 0.25 0.31 0.67 0.16 0.22 0.23 0.60 0.49 0.50 0.44 1.43
2.2 1.9 6.3 10.4 1.2 4.8 10.7 16.7 1.3 5.3 11.4 18.0 1.7 2.8 5.3 9.8 2.6 2.7 5.8 11.1 2.2 2.7 4.3 9.2 1.4 4.0 5.1 10.5 1.6 2.3 4.5 8.4 1.5 2.1 4.0 7.6 2.0 1.8 1.0 4.9
(continued)
101
METALLIC ELEMENTS A N D HEART DISEASE EPIDEMIOLOGY
TABLE 3
LMK: Fr 78
(continued)
Magnesium
Calcium
Copper
Zinc
152 44 229 425 125 128 180 433 281 102
222 44 216 482 327 471 501 1299 1201 586
0.47 0.14 0.95 1.56 0.32 0.46 0.61 1.39 1.12 0.35
4.5 1.8 7.3 13.5 2.8 4.5 7.0 14.2 11.2 3.8
Breakfast Lunch Dinner Total Breakfast Lunch Dinner Total
LMK: Sa 78
Mean daily amounts Standard deviation
aTotal daily amounts do not alwaysequal the sum of the values for each meal because of rounding error. bioavailability (25) and retention (26). If these diets are representative of diets in the United States, adults who regularly consume diets containing amounts between one and two standard deviations below mean daily amounts may be depleting body stores. They may be at risk for pathology. There is no Recommended Dietary Allowance for copper (23); the suggested safe and adequate daily intake for adults is 2-3 mg (23). The adult daily requirement for copper seems to be about 2 mg (27); the most recent measurement of the requirement of men is 1.30 mg/day (28). If the copper requirement actually exceeds 1.3 mg/day, average adults who regularly consume a diet represented by the average diet here minus one standard deviation probably will deplete their body stores and be at risk for pathology. The Kempner diet contained the lowest amount of zinc. Perhaps this amount of zinc (4.9 mg) contributes to its efficacy in lowering blood pressure. Converting
TABLE 4 Correlation Coefficients for 36 Mealsa Mg Mg Ca Cu Zn Ca/Mg
--
Ca
Cu
Zn
Ca/Mg
Zn/Cu
0.322 --
0.849 ~ 0.128 --
0.731~ 0.4168 0.762 c --
-0.478 D 0.540 e -0.566 v -0.272 --
-0.021 0.333 A -0.202 0.438 c 0.265
aProbabilities have been rounded to one figure: 0.05A, 0.01B, 0.008c, 0.003~ 0.0007 E, 0.0003 F, 0.0001a.
102
L . M . KLEVAY
enzyme, which produces the potent pressor agent angiotension II from an inactive precursor (29), contains zinc at its active site (30). The long-term consumption of this diet may result in zinc depletion and impaired enzyme activity. This hypothetical mechanism may supplement the beneficial effect of the low salt intake. Some of the meals contained cheese, eggs, or milk, but no meat. Neither fish nor nutritional supplements were included. Only three meals contained small amounts of alcoholic beverages. Some of the meals were considerably higher in fat and protein than others. It seems likely that correlations calculated for 36 meals would be representative of a greater diversity of diets than correlations calculated for 12 diets. That is, meals of these 12 diets may provide information on characteristics of diets not prepared and analyzed in this work. Correlations were calculated for the 12 diets, however. Table 4 shows signficant correlations between pairs of nutrients and between nutrients and ratios of nutrients for meals. The significant correlations between calcium and the ratio of calcium to magnesium or zinc and the ratio of zinc to copper may be biologically trivial. Alternatively, the biological importance remains undiscovered. These correlations became insignificant when calculated for diets (n = 12) instead of meals (n = 36): Mg vs Ca, P = 0.3; Mg vs Ca/Mg, P = 0.2; Ca vs Zn/Cu, P = 0.08; Cu vs Zn, P = 0.09; Zn vs Zn/Cu, P = 0.02. No correlations that were significant for meals had a different sign when calculated for diets. The correlation between CaJMg vs Zn/Cu was the only one significant for diets (r = 0.804, P = 0.002) that was insignificant for meals. The significant correlations between magnesium and copper, magnesium and the ratio of calcium to magnesium, copper and the ratio of calcium to magnesium, and the ratio of zinc to copper and the ratio of calcium to magnesium are consistent with epidemiology. Low intakes of magnesium and copper and high dietary ratios of calcium to magnesium and zinc to copper are hypothesized to be harmful. Comparison of the sizes of correlation coefficients between magnesium and copper, copper and zinc, copper and the ratio of calcium to magnesium, and so on will not be epidemiologically informative. For example, even though magnesium and copper are highly correlated, a diet could contain adequate magnesium and insufficient copper or vice versa in comparison to requirements. Nutritional adequacy is measured by comparison to standards independent (ideally) of the amounts in diets. The large correlations between some of these elements and between elements and ratios of elements probably will prevent the relationship(s) of these nutrients to ischemic heart disease from being elucidated by epidemiology. Associations between elements, etc., and disease have been useful in the formulation of hypotheses. Causality and pathophysiology can be determined by experimentation. Experiments, for example on the effects of copper deficiency, can decrease the number of variables so that results can be interpreted. Then series of experiments can be used to build an hypothesis supported by more than statistics.
METALLIC ELEMENTS AND HEART DISEASE EPIDEMIOLOGY
103
Acknowledgment I wish to thank my wife, Martha N. Klevay, for diet preparation.
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25. W. J. Darby et al., Trace Elements in Human Nutrition, World Health Organization
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