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Differences in metal content of the heart muscle in death from ischemic heart disease
Differences in metal content bf the heart muscle in death from ischemic heart disease Barbara Chipperfield, M.A., Ph.D. J.R. Chipperfleld, M.A., Ph.D. Hull, England
The increased death rate from ischemic heart disease in soft water areas suggested that the mineral salt concentration in the drinking water affected the death rate from ischemic heart disease,and that there was a specific effect on the heart musc1e.l Since the increased death rate in soft water areas seems to be due to an increase in sudden deaths,’ the metal content of the heart muscle in those dying suddenly from ischemic heart disease is of particular interest. We have shown that there is a significant decreasein magnesium concentration in the heart muscle of those dying suddenly from ischemic heart disease.3This was confirmed by Behr and Burton,4 who found that although there was a decrease in magnesium in the heart muscle in those dying suddenly from heart disease, there was no decreasein chronic heart disease, or in the skeletal muscle of those dying suddenly. Anderson and co-worker9 have confirmed the decrease in magnesium in death from heart disease. They also found a significant increase in the calcium concentration and a decrease in the copper concentration in the heart muscle of those dying from ischemic heart disease, but no significant differences in the concentrations of zinc, chromium, cadmium, and lead. We have reported separately some work on the concentrations of silicon and aluminum in the heart muscle of those dying suddenly from ischemic heart disease.6This report compares the concentrations of several metals of interest in the From the Departments gity, Hull, England.
of Biochemistry
This work was supported Research Council.
and Chemistry,
by a Project
Received for publication
Sept. 13, 1977.
Accepted for publication
Oct. 17, 1977.
Grant
Reprint requests: Dr. B. Chipperfield, Department University of Hull, Hull, HU6 7RX England.
732
June, 1978, Vol. 95, No. 6
from
The Univerthe Medical
of Biochemistry,
heart muscle from three different groups: one control group of patients with apparently normal heart muscle, a group of “late” heart deaths, and a “sudden” heart death group. The causes of death in the normal control group included accidental and suicide deaths, pneumonia, and some deaths from cancer. We have shown that there was no significant difference in magnesium concentration of accident and suicide deaths as compared to those from sudden and chronic illness unrelated to heart disease.?The late death group represented chronic heart disease, and these were defined as patients who had suffered a coronary thrombosis and died more than three months later. The sudden death group were defined as in our previous report.3 Materials
and methods
Samples of left ventricle were taken at necropsy, and in the deaths from ischemic heart disease, were from uninfarcted areas of the ventricle. All the sudden death samples and some of the normal control group came from the Hull City Mortuary; they were stored at 4” C. for 1 to 3 days, then stored at -20” C. until they were prepared for analysis. All the late death samples and the other normal control samples came from the Hull Royal Infirmary, and were stored at -20” C. immediately after necropsy. There was no significant difference between the normal samples from these two different sources.’ Samples of heart muscle were wet-ashed as described previously.” The sulphuric acid which was present in solutions prepared by the wet-ash method interfered in analyses for calcium. For calcium analyses samples of ventricular muscle (cu. 5 Gm.) in platinum crucibles were dried to constant weight at 100”C. and were then ashed in a muffle furnace at 450’ C. The ash was dissolved
0002~8703/78/0695-0732$00.60/O
0 1978 The C. V. Mosby Co.
Metal content of heart muscle in IHD Table
I. Metal concentrations
in heart muscle Sudden heart death
Magnesium
Calcium
Sodium
Potassium
Iron
Copper
Manganese
Magnesium/ calcium
Age
Cont. (yg/Gm. wet weight) SD. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number
Late heart deuth
Normal controls
All
Ma&
Female
All
Male
Female
All
Male
Female
154
152
159
173
170
175
186
181
190
27 59
29 43
22 16
17 22
20 9
15 13
25 158
28 72
22 86
57.0
56.9
57.4
43.1
39.8
45.3
39.0
42.6
36.1
26.0 59
26.2 43
26.2 16
15.8 22
6.5 9
19.9 13
22.9
25.2 57
20.7 72
129
906
855
937
947
953
942
929
903
951
326 59
346 43
229 16
223 22
227 9
229 13
222 158
255 72
190 86
1580
1490
1830
2130
2160
2110
2080
1870
2250
670 36
690
530 9
370 22
470 9
300 13
700 158
680 72
680 86
27
38.5
37.6
41.6
48.6
51.4
46.6
11.0 36
10.9 28
10.8 9
8.1 22
9.3 9
6.8 13
.
46.8
45.7
47.8
9.6
11.0 72
8.1 86
158
3.11
3.08
3.19
2.84
2.77
2.88
3.32
3.30
3.35
0.44 31
0.48 23
0.29 8
0.36 22
0.32 9
0.38 ’ 13
0.66 56
0.69 31
0.63 25
0.96
0.97
0.91
0.76
0.72
0.80
1.07
1.15
0.96
0.18 31
0.19 23
0.15 8
0.23 -22
0.21 9
0.25 13
0.31 56
0.36 31
0.20 25
3.60
3.60
3.58
4.40
4.42
4.37
6.13
5.72
6.46
SD. Number
2.35 59
2.46 43
2.11 16
1.32 22
1.12 9
1.49 13
2.64 129
2.71 57
2.56 72
Mean S.D. Number
66.5 9.9 59
64.3 9.6 43
72.6 8.1 16
70.7 11.8 22
75.2 9.5 13
58.1 20.7 158
55.4 20.5 72
60.4 20.7 86
Cont. (pg/Gm. wet weight) SD. Number Cont. (pg/Gm. wet weight) S.D. Number Ratio
in the minimum quantity of hydrochloric acid. Appropriate quantitiesof the wet ash (Mg”, Na+, K+, Fe3+, Cu2+, MnZ+) or dry ash (Mg*+, Ca*+, Na+, K+, Fe3+, CuZ+, Mn2+) solutions were analyzed by atomic absorption spectrophotometry using a Perkin Elmer Model 305B spectrophotometer with an air/acetylene flame. Analyses for magnesium, sodium, potassium, manganese and copper of solutions prepared by wet ashing and dry ashing the same sample gave excellent agreement. Analyses for iron on wet ashed samples were corrected for the blank reading found for this element.
American Heart Journal
64.3 12.3 9
Results
The concentrations of lithium, lead, chromium, cobalt, strontium, cadmium, and mercury in our solutions were so low that they were not measurable by flame atomic absorption techniques. Table I gives the concentrations (in pg/Gm. wet weight) of magnesium, calcium, sodium, potassium, iron, manganese and copper in our three groups, and Fig. 1 indicates the percentage difference from the normal control group in the two heart death groups for all these elements and for the magnesium /calcium ratio. There was a highly significant (t = 8.14)
733
Chipperfield
and Chippefield
61 Controls
225
Cl Sudden heart deaths @ Late heart deaths
Fig. 1. Comparative metal concentrations of heart muscle (controls = 100 per cent). Bar lines show standard deviations; *probably significant 0.05 > P > 0.01;. **significant 0.01 > P > 0.001; ***highly significant 0.001
> P.
Late
Cekitm Fig.
daaths
ratio
1 ,B. For legend, see above.
decrease in the magnesium concentration in the sudden death group as expected from earlier work. This difference was still highly significant (t = 5.33) when normal males were compared with males from the sudden heart death group, and when normal females were compared with females of the sudden heart death group (t = 5.14). There was a much.smaller decrease in magnesium in the late death group which was only significant at the 5 per cent level (t = 2.30) and was only probably significant in females (t = 2.34) when the sexes were considered separately.
734
heart
There was no significant difference between the calcium concentrations of the normal and late death groups, but the sudden death group showed a highly significant (t = 4.81) increase in calcium. The difference in females was also highly significant (t = 3.55), but in males the difference was only significant at the 1 per cent level (t = 2.77).
For both potassium and iron concentrations there was no significant difference between the normal and late death groups, but a highly significant decrease in the sudden death group (t = 4.10 for potassium, 4.58 for iron). The potas-
June, 1978, Vol. 95, No. 6
Metal
sium in normal males was significantly (t = 3.55) lower than in normal females. When males and females were considered separately, the decrease in potassium in the sudden death group was only probably significant for the males (t = 2.42) and not significant for the females (t = 1.81). The decreasein iron concentration was still significant when males and females were considered separately, though t was higher for the males (3.32) than for the females (2.10). The results for copper and manganese concentrations were very different from those for the other elements. The slight decrease in both elements in the sudden death group was not statistically significant, but the late death group showed a highly significant decrease in manganese (t = 4.24) and a significant difference in the copper concentration (t = 3.21). When males and females were considered separately, the decreased manganese in the males was significant at the 1 per cent level (t = 3.39) and the other comparisons were all probably significant. There was no significant difference in the magnesium/potassium ratio between the groups, but a highly significant decrease in the magnesium/calcium ratio of the sudden death group compared with the normals (t = 6.30). The decreased magnesium/calcium ratio in the sudden death group was highly significant for both males (t = 4.02) and females (t = 4.18) compared separately. This ratio was also significantly decreased in the late death group compared with the normal group (t= 3.00), but was only significant in females (t = 2.84) when the sexes were compared separately. The differences between the sudden death and late death groups were highly significant for the potassium concentration, significant for the magnesium, iron, manganese and copper concentrations, and probably significant for the calcium concentration. Measurements of the watercontent of some of the heart muscle samples indicated that there was no significant difference in the water content of the heart muscle from the three groups, so that these wet weight comparisons are also valid as indications of the relative concentrations per gram dry weight. Statistical analysis indicated that there was no significant variation with age for the concentrations of any of the elements. Histograms of the
American
Heart Journal
conted
of heart muscle in IHfi
results indicated that the distributions were reasonably normal and Wilcoxon-Mann-Whitney significance tests gave similar results to the t tests. Discussion
A general feature of our results is the similarity of the late heart death group to the normal control group, apart from the significant decreases in manganese and copper concentrations and the magnesium/calcium ratio in the late heart death group. This suggests that, other factors being equal, the concentrations of some metals in the heart muscle may be important in deciding whether a patient survives a coronary thrombosis. In the sudden heart death group there were significant decreases in the concentration of magnesium, potassium, and iron and a significant increase in the concentration of calcium. The decrease in magnesium confirms the results from earlier studi;es.3-5Seelig” has suggested that the Western diet is deficient in magnesium, and that lack of magnesium may be a cause of the increased death rate from ischemic heart disease in soft water areas,9slo and Bradshaw and De&l’ have suggested that a prospective study of those regularly taking magnesium salts should be carried out to show whether magnesium has a specific protective effect in ischemic heart disease. Anderson and colleagues”found that the magnesium concentration of normal heart muscle was slightly lower in soft-water areas of Canada compared with hard-water areas, whereas our comparison of English hard- and soft-water areas showed the reverse.‘* We suggested that possibly the magnesium/potassium ratio, which was lower in the soft-water area, m ight be important in susceptibility to death from ischemic heart disease. In the present survey, however, there is no significant difference in magnesium/potassium ratio between the sudden death group and the normal group. Previous studies of both animals and men9 have usually found a decrease in potassium accompanying the decreased magnesium in hearts affected by ischemic heart disease and related conditions. Our earlier investigation” showed a slight, but not statistically significant, decrease in potassium concentration in the hearts of those dying suddenly from ischemic heart disease, and
735
Chipper!leld
and Chipperfield
in this larger study the potassium concentration in the sudden heart deaths was significantly reduced. There was a negative correlation between the cardiovascular mortality rate in South Wales and the potassium content of the drinking water, but Elwood, Abernethy, and Morton13 considered that this was negligible as compared with the correlation with the calcium content of the drinking water. In comparing normal hearts from Hull and Burnley, we found that there was a significant increase in the potassium concentration in the heart muscle samples from the soft-water area (Burnley),‘* which makes it unlikely that any increased death rate in soft-water areas is directly related to potassium metabolism. Calcium also seems to be ruled out as the “water factor” by these results. Our sudden death group, like the Canadian heart deaths, had a significantly increased calcium concentration, which makes it unlikely that the decreased calcium content of soft water is the factor increasing heart deaths. It is not likely that this higher calcium is due to deposition of calcium salts in atheromatous plaques, since there was no significant increase in the calcium in our late death group, where such deposits m ight also be expected. Since there are established interrelations between calcium and magnesium,14 it is possible that, as suggested by Anderson and associates,5the increased calcium in the sudden heart deaths may be secondary to the decrease in magnesium concentration. Calculations on Anderson and colleagues’data5 show that there is a highly significant decrease in the magnesium/ calcium ratio in the heart deaths in the Canadian survey, like the decrease in this ratio we report here. In the soft-water areas the magnesium/ calcium ratio in the heart deaths was 2.47, while in the accident cases it was 4.10 (t = 7.45); in the hard-water areas the ratio in heart deaths was 2.47 and the accident death ratio was 4.23 (t = 5.04). The slight decrease in the accident deaths in the soft-water area was not statistically significant, but it is in the direction one would expect if this ratio change contributed to the increased death rate in soft-water areas. This ratio may vary in different populations as the means in our investigation are nearly 50 per cent greater than the values for the corresponding Canadian groups. The decreasediron concentration in the sudden
736
heart death group is unexpected. The iron will be almost entirely bound in the heme groups of myoglobin and the cytochromes.15These proteins are most unlikely to leak out of cells after death, as m ight occur with the small cations, so the iron decreases must have occurred before death. Decreases in the concentration of these heme proteins will decrease the cell’s capacity to react with oxygen and thus the rate of production of adenosine triphosphate will be lower. A lower capacity to produce adenosine triphosphate may be a great disadvantage in conditions of oxygen shortage after a myocardial infarction. We found a decrease in copper concentration for the late heart death group but not for the sudden heart death group. Anderson and coworkers5 found a decrease in copper concentration in the heart muscle of those dying from ischemic heart disease. About 85 per cent of their heart deaths occurred less than 24 hours after the first symptoms, so their patients could be considered as intermediate between our sudden and late death groups. Severe copper deficiency has been associated with effects on the heart in cows and pigs.‘” Our results suggest that copper deficiency is not important in those dying suddenly from ischemic heart disease, but may be important to those who survive the initial myocardial infarction. Although we found a decrease in manganese in the late deaths, the total concentration of this element is very small. Manganese can normally replace magnesium in vitro as an activator of enzymes acting on adenosine triphosphate, so the decrease in manganese may add to the harmful effects of the slight decrease in magnesium in the late deaths. Cardiovascular mortality has been shown to have a positive association with manganese in water,13 however, which suggests that manganese may have a harmful effect on the heart. These results suggest that the factor most likely to increase the death rate in soft-water areas may be a decrease in the magnesium/ calcium ratio, as these two cations are vitally important in the control of muscular contraction.‘? The iron concentration in the heart was not measured in the Canadian study, and Elwood, Abernethy, and Morton13 did not report on the iron concentrations in their water samples. It is possible that a decrease in the iron in the heart muscle in soft-water areas m ight also
June, 1978, Vol. 95, No. 6
Metal content of heart muscle in IHD
contribute to the increased death rate. Preliminary results6 suggest that possible toxic effects from aluminum are also’worth investigation. The sudden heart death group showed significant decreases in the concentration of magnesium, iron, and potassium. Decreases in magnesium, iron, and potassium have recently been established in Kwashiorkor.‘* Magnesium deficiency has been particularly associated with the electrocardiographic changes in Kwashiorkorls and in alcoholic heart disease.20 Sudden death from ischemic heart disease may be another result of “empty calorie malnutrition.” Diets which are high in fats and refined carbohydrates will probably be low in mineral salts.** It is therefore possible that the harmful effect of Western diets on ischemic heart disease rates is not due to the increase in saturated fats, cholesterol, or sucrose, but to a deficiency of mineral salts. This deficiency could lead to the decreased concentration of several vital metals we have observed in those dying suddenly from ischemic heart disease. Summary
In a group of patients dying suddenly from ischemic heart disease, the uninfarcted heart muscle contained significantly lower concentrations of magnesium, iron, and potassium and a significantly higher concentration of calcium than the heart muscle from a group of normal controls and a group of patients dying more than three months after a coronary thrombosis. The late death group had significantly lower concentrations of manganese and copper than the normal group, and a slight decrease in magnesium concentration which was probably significant. There was no significant difference in the sodium concentration between the three groups. The results are discussed in relation to the increased death rate from ischemic heart disease in areas with soft drinking water, and possible dietary deficiencies in mineral salts. We thank Mrs. Y. P. Thompson for her skillful technical help, the Coroner, pathologists, and mortuary staff at the Hull City Mortuary and Hull Royal Infirmary for help in collection of samples, and Mr. A. G. Memon for statistical help.
American Heart Journal
REFERENCES
1. Crawford, M. D., Gardner, M. J., and Morris, J. N.: Cardiovascular disease and the mineral content of drinking water, Br. Med. Bul. 27:21, 1971. 2. Anderson, T. W., Le Riche, W. H., and MaeKay, J. S.: Sudden death and ischemic heart disease. Correlation with hardness of local water supply, N. Engl. J. Med. 280:&X5,1969. 3. Chipperfleld, B., and Chipperfield, J. R.: Heart-muscle magnesium, potassium, and zinc concentrations after sudden death from heart disease, Lancet 2293, 1973. 4. Behr, G., and Burton, P.: Heart-muscle magnesium, Lancet 2:456, 1973. 5. Anderson, T. W., Neri, L. C., Schreiber, G. B., Talbot, F. D. F., and Zdrojewski, A.: Ischemic heart disease, water hardness and myocardial magnesium, Can. Med. Assoc. J. 113:199, 1975. 6. ChipperEeld, B., Chipperiield, J. R., and Bower, N. R.: Silicon and aluminium in heart deaths, Lancet 1:755, 1977. 7. Chipperfield, B., Chipperfield, J. R., Behr, G., and Burton P.: Magnesium in heart muscle, Lancet 1:1X54, 1976. 8. Seeling, M. S.: The requirement of magnesium by the normal adult, Am. J. Clin. Nutr. 14542, 1964. 9. Seelig, M. S.: Myocardial loss of functional magnesium. II. In cardiomyopathies of diverse etiology, Recent Adv. Stud. Cardiac Struct. Metab. 1:626, 1972. 10. Seelig, M. S., and Heggtveit, H. A.: Magnesium interrelationships in ischemic heart disease, Am. J. Clin. Nutr. 27:59, 1974. 11. Bradshaw, J. S., and Dean, G.: Epsom salts and coronary heart disease, Practitioner 218:673, 1976. 12. Chipperfield, B., Chipperfield, J. R., Behr, G., and Burton, P.: Magnesium and potassium content of normal heart muscle in areas of hard and soft water, Lancet 1:121, 1976. 13. Elwood, P. C., Abernethy, M., and Morton, M.: Mortality in adults and trace elements in water, Lancet 2:1470, 1974. 14. Irving, J. T.: Calcium and phosphorus metabolism, New York, 1973, Academic Press. 15. Jacobs, A., and Worwood, M., editors: Iron in biochemistry and medicine, London, 1974, Academic Press. 16. Adelstein, S. J., and Vallee, B. L., in Mineral metabolism, Vol. 2B, edited by C. L. Comar, and F. Bronner, New York, 1962, Academic Press, p. 371. 17. Nayler, W. G., editor: Contraction and relaxation in the myocardium, London, 1975, Academic Press. 18. Davidson, S., Passmore, R., and Brock, J. F., editors: Human nutrition and dietetics, fifth ed., Edinburgh and London, 1972, Churchill-Living&one. 19. Caddell, J. L.: Magnesium deficiency in protein-calorie malnutrition, Ann. N. Y. Acad. Sci. 182:874, 1969. 20. Seelig, M. S.: Electrographic patterns of magnesium depletion appearing in alcoholic heart disease, Ann. N. Y. Acad. Sci. 182:906,1969. 21. McCance, R. A., and Widdowson, E. M.: The composition of foods, M. R. C. Special Report No. 297, London, 1969, Her Majesty’s Stationery Office.
737
The increased death rate from ischemic heart disease in soft water areas suggested that the mineral salt concentration in the drinking water affected the death rate from ischemic heart disease,and that there was a specific effect on the heart musc1e.l Since the increased death rate in soft water areas seems to be due to an increase in sudden deaths,’ the metal content of the heart muscle in those dying suddenly from ischemic heart disease is of particular interest. We have shown that there is a significant decreasein magnesium concentration in the heart muscle of those dying suddenly from ischemic heart disease.3This was confirmed by Behr and Burton,4 who found that although there was a decrease in magnesium in the heart muscle in those dying suddenly from heart disease, there was no decreasein chronic heart disease, or in the skeletal muscle of those dying suddenly. Anderson and co-worker9 have confirmed the decrease in magnesium in death from heart disease. They also found a significant increase in the calcium concentration and a decrease in the copper concentration in the heart muscle of those dying from ischemic heart disease, but no significant differences in the concentrations of zinc, chromium, cadmium, and lead. We have reported separately some work on the concentrations of silicon and aluminum in the heart muscle of those dying suddenly from ischemic heart disease.6This report compares the concentrations of several metals of interest in the From the Departments gity, Hull, England.
of Biochemistry
This work was supported Research Council.
and Chemistry,
by a Project
Received for publication
Sept. 13, 1977.
Accepted for publication
Oct. 17, 1977.
Grant
Reprint requests: Dr. B. Chipperfield, Department University of Hull, Hull, HU6 7RX England.
732
June, 1978, Vol. 95, No. 6
from
The Univerthe Medical
of Biochemistry,
heart muscle from three different groups: one control group of patients with apparently normal heart muscle, a group of “late” heart deaths, and a “sudden” heart death group. The causes of death in the normal control group included accidental and suicide deaths, pneumonia, and some deaths from cancer. We have shown that there was no significant difference in magnesium concentration of accident and suicide deaths as compared to those from sudden and chronic illness unrelated to heart disease.?The late death group represented chronic heart disease, and these were defined as patients who had suffered a coronary thrombosis and died more than three months later. The sudden death group were defined as in our previous report.3 Materials
and methods
Samples of left ventricle were taken at necropsy, and in the deaths from ischemic heart disease, were from uninfarcted areas of the ventricle. All the sudden death samples and some of the normal control group came from the Hull City Mortuary; they were stored at 4” C. for 1 to 3 days, then stored at -20” C. until they were prepared for analysis. All the late death samples and the other normal control samples came from the Hull Royal Infirmary, and were stored at -20” C. immediately after necropsy. There was no significant difference between the normal samples from these two different sources.’ Samples of heart muscle were wet-ashed as described previously.” The sulphuric acid which was present in solutions prepared by the wet-ash method interfered in analyses for calcium. For calcium analyses samples of ventricular muscle (cu. 5 Gm.) in platinum crucibles were dried to constant weight at 100”C. and were then ashed in a muffle furnace at 450’ C. The ash was dissolved
0002~8703/78/0695-0732$00.60/O
0 1978 The C. V. Mosby Co.
Metal content of heart muscle in IHD Table
I. Metal concentrations
in heart muscle Sudden heart death
Magnesium
Calcium
Sodium
Potassium
Iron
Copper
Manganese
Magnesium/ calcium
Age
Cont. (yg/Gm. wet weight) SD. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number Cont. (pg/Gm. wet weight) S.D. Number
Late heart deuth
Normal controls
All
Ma&
Female
All
Male
Female
All
Male
Female
154
152
159
173
170
175
186
181
190
27 59
29 43
22 16
17 22
20 9
15 13
25 158
28 72
22 86
57.0
56.9
57.4
43.1
39.8
45.3
39.0
42.6
36.1
26.0 59
26.2 43
26.2 16
15.8 22
6.5 9
19.9 13
22.9
25.2 57
20.7 72
129
906
855
937
947
953
942
929
903
951
326 59
346 43
229 16
223 22
227 9
229 13
222 158
255 72
190 86
1580
1490
1830
2130
2160
2110
2080
1870
2250
670 36
690
530 9
370 22
470 9
300 13
700 158
680 72
680 86
27
38.5
37.6
41.6
48.6
51.4
46.6
11.0 36
10.9 28
10.8 9
8.1 22
9.3 9
6.8 13
.
46.8
45.7
47.8
9.6
11.0 72
8.1 86
158
3.11
3.08
3.19
2.84
2.77
2.88
3.32
3.30
3.35
0.44 31
0.48 23
0.29 8
0.36 22
0.32 9
0.38 ’ 13
0.66 56
0.69 31
0.63 25
0.96
0.97
0.91
0.76
0.72
0.80
1.07
1.15
0.96
0.18 31
0.19 23
0.15 8
0.23 -22
0.21 9
0.25 13
0.31 56
0.36 31
0.20 25
3.60
3.60
3.58
4.40
4.42
4.37
6.13
5.72
6.46
SD. Number
2.35 59
2.46 43
2.11 16
1.32 22
1.12 9
1.49 13
2.64 129
2.71 57
2.56 72
Mean S.D. Number
66.5 9.9 59
64.3 9.6 43
72.6 8.1 16
70.7 11.8 22
75.2 9.5 13
58.1 20.7 158
55.4 20.5 72
60.4 20.7 86
Cont. (pg/Gm. wet weight) SD. Number Cont. (pg/Gm. wet weight) S.D. Number Ratio
in the minimum quantity of hydrochloric acid. Appropriate quantitiesof the wet ash (Mg”, Na+, K+, Fe3+, Cu2+, MnZ+) or dry ash (Mg*+, Ca*+, Na+, K+, Fe3+, CuZ+, Mn2+) solutions were analyzed by atomic absorption spectrophotometry using a Perkin Elmer Model 305B spectrophotometer with an air/acetylene flame. Analyses for magnesium, sodium, potassium, manganese and copper of solutions prepared by wet ashing and dry ashing the same sample gave excellent agreement. Analyses for iron on wet ashed samples were corrected for the blank reading found for this element.
American Heart Journal
64.3 12.3 9
Results
The concentrations of lithium, lead, chromium, cobalt, strontium, cadmium, and mercury in our solutions were so low that they were not measurable by flame atomic absorption techniques. Table I gives the concentrations (in pg/Gm. wet weight) of magnesium, calcium, sodium, potassium, iron, manganese and copper in our three groups, and Fig. 1 indicates the percentage difference from the normal control group in the two heart death groups for all these elements and for the magnesium /calcium ratio. There was a highly significant (t = 8.14)
733
Chipperfield
and Chippefield
61 Controls
225
Cl Sudden heart deaths @ Late heart deaths
Fig. 1. Comparative metal concentrations of heart muscle (controls = 100 per cent). Bar lines show standard deviations; *probably significant 0.05 > P > 0.01;. **significant 0.01 > P > 0.001; ***highly significant 0.001
> P.
Late
Cekitm Fig.
daaths
ratio
1 ,B. For legend, see above.
decrease in the magnesium concentration in the sudden death group as expected from earlier work. This difference was still highly significant (t = 5.33) when normal males were compared with males from the sudden heart death group, and when normal females were compared with females of the sudden heart death group (t = 5.14). There was a much.smaller decrease in magnesium in the late death group which was only significant at the 5 per cent level (t = 2.30) and was only probably significant in females (t = 2.34) when the sexes were considered separately.
734
heart
There was no significant difference between the calcium concentrations of the normal and late death groups, but the sudden death group showed a highly significant (t = 4.81) increase in calcium. The difference in females was also highly significant (t = 3.55), but in males the difference was only significant at the 1 per cent level (t = 2.77).
For both potassium and iron concentrations there was no significant difference between the normal and late death groups, but a highly significant decrease in the sudden death group (t = 4.10 for potassium, 4.58 for iron). The potas-
June, 1978, Vol. 95, No. 6
Metal
sium in normal males was significantly (t = 3.55) lower than in normal females. When males and females were considered separately, the decrease in potassium in the sudden death group was only probably significant for the males (t = 2.42) and not significant for the females (t = 1.81). The decreasein iron concentration was still significant when males and females were considered separately, though t was higher for the males (3.32) than for the females (2.10). The results for copper and manganese concentrations were very different from those for the other elements. The slight decrease in both elements in the sudden death group was not statistically significant, but the late death group showed a highly significant decrease in manganese (t = 4.24) and a significant difference in the copper concentration (t = 3.21). When males and females were considered separately, the decreased manganese in the males was significant at the 1 per cent level (t = 3.39) and the other comparisons were all probably significant. There was no significant difference in the magnesium/potassium ratio between the groups, but a highly significant decrease in the magnesium/calcium ratio of the sudden death group compared with the normals (t = 6.30). The decreased magnesium/calcium ratio in the sudden death group was highly significant for both males (t = 4.02) and females (t = 4.18) compared separately. This ratio was also significantly decreased in the late death group compared with the normal group (t= 3.00), but was only significant in females (t = 2.84) when the sexes were compared separately. The differences between the sudden death and late death groups were highly significant for the potassium concentration, significant for the magnesium, iron, manganese and copper concentrations, and probably significant for the calcium concentration. Measurements of the watercontent of some of the heart muscle samples indicated that there was no significant difference in the water content of the heart muscle from the three groups, so that these wet weight comparisons are also valid as indications of the relative concentrations per gram dry weight. Statistical analysis indicated that there was no significant variation with age for the concentrations of any of the elements. Histograms of the
American
Heart Journal
conted
of heart muscle in IHfi
results indicated that the distributions were reasonably normal and Wilcoxon-Mann-Whitney significance tests gave similar results to the t tests. Discussion
A general feature of our results is the similarity of the late heart death group to the normal control group, apart from the significant decreases in manganese and copper concentrations and the magnesium/calcium ratio in the late heart death group. This suggests that, other factors being equal, the concentrations of some metals in the heart muscle may be important in deciding whether a patient survives a coronary thrombosis. In the sudden heart death group there were significant decreases in the concentration of magnesium, potassium, and iron and a significant increase in the concentration of calcium. The decrease in magnesium confirms the results from earlier studi;es.3-5Seelig” has suggested that the Western diet is deficient in magnesium, and that lack of magnesium may be a cause of the increased death rate from ischemic heart disease in soft water areas,9slo and Bradshaw and De&l’ have suggested that a prospective study of those regularly taking magnesium salts should be carried out to show whether magnesium has a specific protective effect in ischemic heart disease. Anderson and colleagues”found that the magnesium concentration of normal heart muscle was slightly lower in soft-water areas of Canada compared with hard-water areas, whereas our comparison of English hard- and soft-water areas showed the reverse.‘* We suggested that possibly the magnesium/potassium ratio, which was lower in the soft-water area, m ight be important in susceptibility to death from ischemic heart disease. In the present survey, however, there is no significant difference in magnesium/potassium ratio between the sudden death group and the normal group. Previous studies of both animals and men9 have usually found a decrease in potassium accompanying the decreased magnesium in hearts affected by ischemic heart disease and related conditions. Our earlier investigation” showed a slight, but not statistically significant, decrease in potassium concentration in the hearts of those dying suddenly from ischemic heart disease, and
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Chipper!leld
and Chipperfield
in this larger study the potassium concentration in the sudden heart deaths was significantly reduced. There was a negative correlation between the cardiovascular mortality rate in South Wales and the potassium content of the drinking water, but Elwood, Abernethy, and Morton13 considered that this was negligible as compared with the correlation with the calcium content of the drinking water. In comparing normal hearts from Hull and Burnley, we found that there was a significant increase in the potassium concentration in the heart muscle samples from the soft-water area (Burnley),‘* which makes it unlikely that any increased death rate in soft-water areas is directly related to potassium metabolism. Calcium also seems to be ruled out as the “water factor” by these results. Our sudden death group, like the Canadian heart deaths, had a significantly increased calcium concentration, which makes it unlikely that the decreased calcium content of soft water is the factor increasing heart deaths. It is not likely that this higher calcium is due to deposition of calcium salts in atheromatous plaques, since there was no significant increase in the calcium in our late death group, where such deposits m ight also be expected. Since there are established interrelations between calcium and magnesium,14 it is possible that, as suggested by Anderson and associates,5the increased calcium in the sudden heart deaths may be secondary to the decrease in magnesium concentration. Calculations on Anderson and colleagues’data5 show that there is a highly significant decrease in the magnesium/ calcium ratio in the heart deaths in the Canadian survey, like the decrease in this ratio we report here. In the soft-water areas the magnesium/ calcium ratio in the heart deaths was 2.47, while in the accident cases it was 4.10 (t = 7.45); in the hard-water areas the ratio in heart deaths was 2.47 and the accident death ratio was 4.23 (t = 5.04). The slight decrease in the accident deaths in the soft-water area was not statistically significant, but it is in the direction one would expect if this ratio change contributed to the increased death rate in soft-water areas. This ratio may vary in different populations as the means in our investigation are nearly 50 per cent greater than the values for the corresponding Canadian groups. The decreasediron concentration in the sudden
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heart death group is unexpected. The iron will be almost entirely bound in the heme groups of myoglobin and the cytochromes.15These proteins are most unlikely to leak out of cells after death, as m ight occur with the small cations, so the iron decreases must have occurred before death. Decreases in the concentration of these heme proteins will decrease the cell’s capacity to react with oxygen and thus the rate of production of adenosine triphosphate will be lower. A lower capacity to produce adenosine triphosphate may be a great disadvantage in conditions of oxygen shortage after a myocardial infarction. We found a decrease in copper concentration for the late heart death group but not for the sudden heart death group. Anderson and coworkers5 found a decrease in copper concentration in the heart muscle of those dying from ischemic heart disease. About 85 per cent of their heart deaths occurred less than 24 hours after the first symptoms, so their patients could be considered as intermediate between our sudden and late death groups. Severe copper deficiency has been associated with effects on the heart in cows and pigs.‘” Our results suggest that copper deficiency is not important in those dying suddenly from ischemic heart disease, but may be important to those who survive the initial myocardial infarction. Although we found a decrease in manganese in the late deaths, the total concentration of this element is very small. Manganese can normally replace magnesium in vitro as an activator of enzymes acting on adenosine triphosphate, so the decrease in manganese may add to the harmful effects of the slight decrease in magnesium in the late deaths. Cardiovascular mortality has been shown to have a positive association with manganese in water,13 however, which suggests that manganese may have a harmful effect on the heart. These results suggest that the factor most likely to increase the death rate in soft-water areas may be a decrease in the magnesium/ calcium ratio, as these two cations are vitally important in the control of muscular contraction.‘? The iron concentration in the heart was not measured in the Canadian study, and Elwood, Abernethy, and Morton13 did not report on the iron concentrations in their water samples. It is possible that a decrease in the iron in the heart muscle in soft-water areas m ight also
June, 1978, Vol. 95, No. 6
Metal content of heart muscle in IHD
contribute to the increased death rate. Preliminary results6 suggest that possible toxic effects from aluminum are also’worth investigation. The sudden heart death group showed significant decreases in the concentration of magnesium, iron, and potassium. Decreases in magnesium, iron, and potassium have recently been established in Kwashiorkor.‘* Magnesium deficiency has been particularly associated with the electrocardiographic changes in Kwashiorkorls and in alcoholic heart disease.20 Sudden death from ischemic heart disease may be another result of “empty calorie malnutrition.” Diets which are high in fats and refined carbohydrates will probably be low in mineral salts.** It is therefore possible that the harmful effect of Western diets on ischemic heart disease rates is not due to the increase in saturated fats, cholesterol, or sucrose, but to a deficiency of mineral salts. This deficiency could lead to the decreased concentration of several vital metals we have observed in those dying suddenly from ischemic heart disease. Summary
In a group of patients dying suddenly from ischemic heart disease, the uninfarcted heart muscle contained significantly lower concentrations of magnesium, iron, and potassium and a significantly higher concentration of calcium than the heart muscle from a group of normal controls and a group of patients dying more than three months after a coronary thrombosis. The late death group had significantly lower concentrations of manganese and copper than the normal group, and a slight decrease in magnesium concentration which was probably significant. There was no significant difference in the sodium concentration between the three groups. The results are discussed in relation to the increased death rate from ischemic heart disease in areas with soft drinking water, and possible dietary deficiencies in mineral salts. We thank Mrs. Y. P. Thompson for her skillful technical help, the Coroner, pathologists, and mortuary staff at the Hull City Mortuary and Hull Royal Infirmary for help in collection of samples, and Mr. A. G. Memon for statistical help.
American Heart Journal
REFERENCES
1. Crawford, M. D., Gardner, M. J., and Morris, J. N.: Cardiovascular disease and the mineral content of drinking water, Br. Med. Bul. 27:21, 1971. 2. Anderson, T. W., Le Riche, W. H., and MaeKay, J. S.: Sudden death and ischemic heart disease. Correlation with hardness of local water supply, N. Engl. J. Med. 280:&X5,1969. 3. Chipperfleld, B., and Chipperfield, J. R.: Heart-muscle magnesium, potassium, and zinc concentrations after sudden death from heart disease, Lancet 2293, 1973. 4. Behr, G., and Burton, P.: Heart-muscle magnesium, Lancet 2:456, 1973. 5. Anderson, T. W., Neri, L. C., Schreiber, G. B., Talbot, F. D. F., and Zdrojewski, A.: Ischemic heart disease, water hardness and myocardial magnesium, Can. Med. Assoc. J. 113:199, 1975. 6. ChipperEeld, B., Chipperiield, J. R., and Bower, N. R.: Silicon and aluminium in heart deaths, Lancet 1:755, 1977. 7. Chipperfield, B., Chipperfield, J. R., Behr, G., and Burton P.: Magnesium in heart muscle, Lancet 1:1X54, 1976. 8. Seeling, M. S.: The requirement of magnesium by the normal adult, Am. J. Clin. Nutr. 14542, 1964. 9. Seelig, M. S.: Myocardial loss of functional magnesium. II. In cardiomyopathies of diverse etiology, Recent Adv. Stud. Cardiac Struct. Metab. 1:626, 1972. 10. Seelig, M. S., and Heggtveit, H. A.: Magnesium interrelationships in ischemic heart disease, Am. J. Clin. Nutr. 27:59, 1974. 11. Bradshaw, J. S., and Dean, G.: Epsom salts and coronary heart disease, Practitioner 218:673, 1976. 12. Chipperfield, B., Chipperfield, J. R., Behr, G., and Burton, P.: Magnesium and potassium content of normal heart muscle in areas of hard and soft water, Lancet 1:121, 1976. 13. Elwood, P. C., Abernethy, M., and Morton, M.: Mortality in adults and trace elements in water, Lancet 2:1470, 1974. 14. Irving, J. T.: Calcium and phosphorus metabolism, New York, 1973, Academic Press. 15. Jacobs, A., and Worwood, M., editors: Iron in biochemistry and medicine, London, 1974, Academic Press. 16. Adelstein, S. J., and Vallee, B. L., in Mineral metabolism, Vol. 2B, edited by C. L. Comar, and F. Bronner, New York, 1962, Academic Press, p. 371. 17. Nayler, W. G., editor: Contraction and relaxation in the myocardium, London, 1975, Academic Press. 18. Davidson, S., Passmore, R., and Brock, J. F., editors: Human nutrition and dietetics, fifth ed., Edinburgh and London, 1972, Churchill-Living&one. 19. Caddell, J. L.: Magnesium deficiency in protein-calorie malnutrition, Ann. N. Y. Acad. Sci. 182:874, 1969. 20. Seelig, M. S.: Electrographic patterns of magnesium depletion appearing in alcoholic heart disease, Ann. N. Y. Acad. Sci. 182:906,1969. 21. McCance, R. A., and Widdowson, E. M.: The composition of foods, M. R. C. Special Report No. 297, London, 1969, Her Majesty’s Stationery Office.
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