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SERUM-CHOLESTEROL LEVELS AFTER CONSUMING EGGS WITH INCREASED CONTENT OF UNSATURATED LIPIDS
244 saturated fatty acids in high concentration may be consumed without raising cholesterol levels above the " basal value " for the individual.* Our experience indicates that the basal values are about 30% below the levels ordinarily found in Western society. Perhaps modification of foodstuffsmay enable us to achieve the blood-cholesterol levels now seen in populations which have low morbidity and mortality from atherosclerosis. We are indebted to Dr. A. T. James, of the National Institute for Medical Research, Mill Hill, for the analytical data. One of us (L. H.) receives a grant from the National Research Council, Canada.
Department of Medicine Department of Poultry Husbandry, University of Saskatchewan,
Saskatoon,
Canada
L. HORLICK M.D. McGill, F.R.C.P.(C.) J. B. O’NEIL M.S.A.
Brit. Columbia
SERUM-CHOLESTEROL LEVELS AFTER CONSUMING EGGS WITH INCREASED CONTENT OF UNSATURATED LIPIDS THE rise in serum-cholesterol level which follows consumption of hens’ eggs is something of an enigma.2 It is not due to their cholesterol or protein content,’ and it is generally attributed to a fatty-acid factor. Eggyolk lipids, however, are only moderately saturated (iodine value 70-80) 3; and their cholesterogenic effect is greater than would be expected on the " degree of saturation" hypothesis of Ahrens et al.4 Furthermore, egg-yolk lipids contain about 20% of linoleic acid5 (in addition to 32% of saturated fatty acids, 47% of oleic acid, and 2% of linolenic acid) and thus their cholesterogenic effect cannot readily be explained in terms of the essential-fatty-acid deficiency postulated by Sinclair6 and by Kinsell et al.7 While considering this we recalled an old report by Cruickshank,5 who found that when hens were fed on a diet rich in hempseed, the linoleic-acid content of their
yolk lipids rapidly rose from 19 to 44%, with a corresponding fall in saturated and oleic acids; at the same time, the iodine value of the yolk lipids rose from 80 to 120. Accordingly, in the hope of shedding some light on their cholesterogenic mechanism, we decided to compare the effects of ordinary and " unsaturated " hens’ eggs on the human serum-cholesterol level. A flock of Black
Australorp hens was chosen for our experiDuring an initial 2-week control period they had a stock diet containing 3% of fat with an iodine value of 93. Eggs were collected on four occasions, and the iodine value of their yolk lipids averaged 72 (S.D. 1). Half the hens were then placed in a separate henhouse and were given a special feed which included sunflower seeds (80% dehulled). The other half continued with their ordinary diet. The composition of the control and the experimental feeds is shown in the table. From now on four eggs were gathered from each hen house ments.
* Since this communication was first submitted, two more subjects have been tested. The ingestion of ten eggs daily (high in unsaturated fatty acids) resulted in a gradual return of the serum-cholesterol levels from lqw-fat basal levels to control levels in a 2-week test period. With stock eggs, the serum-cholesterol rose more abruptly and reached hohef levels. 1. Messinger, W. J., Prosowska, Y., Steele, J. M. Arch. intern. Med. 1950, 86, 189. 2. Bronte-Stewart, B., Antonis, A., Eales, L., Brock, J. F. Lancet, 1956,
i, 521. 3. Deuel, H. J., Jr. The Lipids: Their Chemistry and Biochemistry; vol. 2. London, 1951. 4. Ahrens, E. H., Jr., Hirsch, J., Insult, W., Tsaltas, T. T., Blomstrand, R., Peterson, M. L. Lancet, 1957, i, 943. 5. Cruickshank, E. M. Biochem. J. 1934, 28, 965. 6. Sinclair, H. M. Lancet, 1956, i, 381. 7. Kinsell, L. W., Michaels, G. D., Friskey, R. W., Splitter, S. ibid. 1958, i, 334.
weekly. The yolk lipids from the hens on the experimental feed showed a progressive increase in iodine value, and by the end of 3 weeks this had reached 100, at which level it remained for the duration of the experiment. The iodine value of the yolk lipids from the control hens did not change and at 3 weeks averaged 73 (standard deviation 1-5). At this stage all the eggs from the control and the experimental hens were collected and used for human feeding trials. The experimental egg yolks were a deeper yellow than the controls, but when they were cooked they could not be distinguished by taste. The experimental hens throve on their diet, and their eggproduction was in no way inferior to that of the controls. It interested us to find that, after the 6 weeks, the mean serumcholesterol level of the ten experimental hens was 117 mg. per 100 ml. (S.D. 23), compared with 166 mg. (s.D. 34): these mean values were significantly different (p less than 002). By this time the body fats of the experimental hens had become relatively unsaturated: the values for the control and experimental hens respectively were: abdominal fat, 76 and 94; subcutaneous fat, 76 and 85; muscle fat, 77 and 91; liver fat, 77 and 90. Too few analyses were performed, however, to permit of any statistical comparison. The effect of feeding these eggs was studied in 2 healthy Bantu male volunteers, each 34 years old. During the
experiments they lived in the metabolism ward and received the basal diet, very low in fat (11 g. daily), which 8 we have previously described, Except as indicated their diets were constant with respect to below, kept calories (2500 daily) and protein (88 g. daily). Experiment 1 (fig. 1).-On the basal diet, the mean serumcholesterol level was 75 mg. per 100 ml. Introduction of ten boiled " control " egg-whites into the daily diet for 10 days did not alter it. When they were replaced by ten boiled control egg yolks daily for 10 days, it rose to 100 mg. These were then replaced by ten boiled " unsaturated " yolks, and during the next 10 days the serum-cholesterol level fell to 95 mg. per 100 ml.-a trivial change. In the next period, ten whole " unsaturated " eggs fried in 100 g. of sunflower-seed
245
oil were eaten daily (3100 calories, 112 9th day the serum-cholesterol level practically to basal level. On returning diet there was a temporary rise in the
protein), and by the fell to 77 mg.-i.e., the subject to his basal serum-cholesterol, but
g.
returned to the basal level. In this first experiment there was no difference in the effect on the serum-cholesterol level of " control " and " It remained possible, however, unsaturated " eggs. that if the latter had been eaten for more than 10 days the cholesterogenic effect of the control eggs might have been reversed. In the next experiment, therefore, the unsaturated eggs were fed without a preliminary period on control eggs. Experiment 2 (fig. 2).-During an initial 6-day basal period, the serum-cholesterol level averaged 188 mg. per 100 ml. Then ten boiled unsaturated egg yolks daily were introduced into the diet and fed for 7 days; the serum-cholesterol level promptly rose to 218 mg. The subject was then returned to his basal diet and 3 g. of cholesterol daily (i.e., the amount in ten yolks) was mixed with his food for the remaining 8 days of the experiment. The serum-cholesterol quickly returned to the basal level, and this showed again that the serum-cholesterol-increasing action of eggs is not due to their cholesterol
it
quickly
content.
It appears from these two experiments that increase of the iodine value of egg yolks from 72 to 100 does not materially affect their cholesterogenic action. Although egg-yolk lipids with iodine value of 100 markedly raise the serum-cholesterol level, we have frequently observed that certain other more saturated fats, such as hydrogenated sunflower seed oil (iodine value 74), do not raise the serum-cholesterol, and that some, such as olive oil (iodine value 88) and peanut oil (iodine value 89), sometimes lower it. Thus we conclude that the effect of all dietary fats on the serum-cholesterol level is not simply a function of their degree of saturation. The suggestion of Keys et al.8-that, to offset the cholesterogenic effect of saturated fatty acids, at least twice as much polyethenoid fatty acid is required-might appear to be a satisfactory explanation of our findings; thus, the additional linoleic acid introduced into the unsaturated eggs may not have sufficed to neutralise their saturated-fatty-acid In Cruickshank’s experiments,5 the polyethenoidcontent. fatty-acid content of her unsaturated eggs was more than double their saturated-fatty-acid content; had we been able " to unsaturate " our eggs beyond iodine value 100, it is conceivable that we might have produced a " cholesterol-decreas8. Keys,
A., Anderson, J. T., Grande, F. ibid. p. 742.
ing " egg. It must be noted, however, that in several other dietary experiments in man, a fall in serum-cholesterol has been produced with far less than a 2 : 1 ratio of polyethenoid to saturated fatty acids. For example, fig. 8 of our paper last December,9 shows that the cholesterogenic effect of 100 g. of hydrogenated coconut oil (almost all saturated fatty acids) was abolished by 100 g. of sunflower-seed oil-i.e., by only 65 g. of linoleic acid. In a number of long-term studies of active men consuming their ordinary diets, which were often rich in saturated fats, we found that by simply administering 50 g. of sunflower-seed oil (about 33 g. of linoleic acid) daily, substantial decrease in the serum-cholesterol level
a
was
usually produced and maintained.10 It may be that the rule of Keys et al.8 is valid when applied to their type of group " experiment but we agree with Kinsell 11 that the individual response to a dietary fat is variable and cannot be predicted by a mathematical formula. "
We have no data on the individual fatty-acid components of our control and unsaturated egg yolks, but on the basis of Cruickshank’s findings 5 we have assumed that the unsaturation of the latter is achieved by an increase in the proportion of linoleic acid at the expense of the monoethenoid and saturated fatty acids. Failure of this presumed rise in the linoleic-acid content of our eggs to inhibit the cholesterogenic effect seems to be at variance with the " essential fatty acid deficiency " hypothesis. We must point out, however, that it is not known whether the " linoleic acid " of egg yolks is the biologically active It is known that the " linoleic cis-cis isomer or not. acid " of a number of animal fats is in the inactive form,12 isomerisation apparently having taken place in the process of digestion, absorption, or deposition of the active linoleic acid of the diet. Thus, Hilditch and Longenecker 13 found that, despite an abundance of natural linoleic acid in pasture grasses, the depot fats of ruminants contain very little of it; most of the octadecadienoic acids of these fats are in the trans-cis or other isomeric forms. It is possible that the failure of our unsaturated egg yolks to decrease the serum-cholesterol level is due to isomerisation of the linoleic acid of the dietary sunflower seeds in the course of its incorporation into the egg yolk. The above speculation implies acceptance of the " essential fatty acid deficiency " hypothesis. This hypothesis, however, is far from proven: hypercholesterolxmia does not occur in animals fed on a diet deficient in essential fatty acids,14 nor is there any direct evidence of such deficiency in hypercholesterolaemic human beings. It is true that Kinsell et al. have shown that 95% pure linoleic acid can lower the, serum-cholesterol level of man, but the effect of its " non-essential " isomers has not been studied. We must conclude that the factor or factors in dietary fats which regulate the serumcholesterol level are still unknown. These experiments were performed with the cooperation of Dr. M. Finlayson, the owner, and Mr. J. Mirvish, the manager, of the farm Kelvin Grove in the Constantia valley. We are grateful to
them for their kindness.
H. GORDON M.D.,
Department of Medicine, University of Cape Town, and the Clinical
Nutrition Research Unit of the Council for Scientific and Industrial Research, Union of South Africa 9. 10. 11. 12. 13. 14.
B.SC.
Cape
Town
J. WILKENS B.SC.
W’srand
J. F. BROCK D.M. Oxon., F.R.C.P.
Gordon, H., Lewis, B., Eales, L., Brock, J. F. ibid. 1957, ii, 1299. Gordon, H., Brock, J. F. S. Afr. med. J. 1958, 32, 397. Kinsell, L. W. Lancet, 1958, i, 857. Hilditch, T. P. Chemical Constitution of Natural Fats. London, 1956. Hilditch, T. P., Longenecker, H. E. Biochem. J. 1937, 31, 1805. Deuel, H. J., Jr., Alfin-Slater, R. B., Wells, A. F., Kryder, G. D., Aftergood, L. J. Nutr. 1955, 55, 337.
Department of Medicine Department of Poultry Husbandry, University of Saskatchewan,
Saskatoon,
Canada
L. HORLICK M.D. McGill, F.R.C.P.(C.) J. B. O’NEIL M.S.A.
Brit. Columbia
SERUM-CHOLESTEROL LEVELS AFTER CONSUMING EGGS WITH INCREASED CONTENT OF UNSATURATED LIPIDS THE rise in serum-cholesterol level which follows consumption of hens’ eggs is something of an enigma.2 It is not due to their cholesterol or protein content,’ and it is generally attributed to a fatty-acid factor. Eggyolk lipids, however, are only moderately saturated (iodine value 70-80) 3; and their cholesterogenic effect is greater than would be expected on the " degree of saturation" hypothesis of Ahrens et al.4 Furthermore, egg-yolk lipids contain about 20% of linoleic acid5 (in addition to 32% of saturated fatty acids, 47% of oleic acid, and 2% of linolenic acid) and thus their cholesterogenic effect cannot readily be explained in terms of the essential-fatty-acid deficiency postulated by Sinclair6 and by Kinsell et al.7 While considering this we recalled an old report by Cruickshank,5 who found that when hens were fed on a diet rich in hempseed, the linoleic-acid content of their
yolk lipids rapidly rose from 19 to 44%, with a corresponding fall in saturated and oleic acids; at the same time, the iodine value of the yolk lipids rose from 80 to 120. Accordingly, in the hope of shedding some light on their cholesterogenic mechanism, we decided to compare the effects of ordinary and " unsaturated " hens’ eggs on the human serum-cholesterol level. A flock of Black
Australorp hens was chosen for our experiDuring an initial 2-week control period they had a stock diet containing 3% of fat with an iodine value of 93. Eggs were collected on four occasions, and the iodine value of their yolk lipids averaged 72 (S.D. 1). Half the hens were then placed in a separate henhouse and were given a special feed which included sunflower seeds (80% dehulled). The other half continued with their ordinary diet. The composition of the control and the experimental feeds is shown in the table. From now on four eggs were gathered from each hen house ments.
* Since this communication was first submitted, two more subjects have been tested. The ingestion of ten eggs daily (high in unsaturated fatty acids) resulted in a gradual return of the serum-cholesterol levels from lqw-fat basal levels to control levels in a 2-week test period. With stock eggs, the serum-cholesterol rose more abruptly and reached hohef levels. 1. Messinger, W. J., Prosowska, Y., Steele, J. M. Arch. intern. Med. 1950, 86, 189. 2. Bronte-Stewart, B., Antonis, A., Eales, L., Brock, J. F. Lancet, 1956,
i, 521. 3. Deuel, H. J., Jr. The Lipids: Their Chemistry and Biochemistry; vol. 2. London, 1951. 4. Ahrens, E. H., Jr., Hirsch, J., Insult, W., Tsaltas, T. T., Blomstrand, R., Peterson, M. L. Lancet, 1957, i, 943. 5. Cruickshank, E. M. Biochem. J. 1934, 28, 965. 6. Sinclair, H. M. Lancet, 1956, i, 381. 7. Kinsell, L. W., Michaels, G. D., Friskey, R. W., Splitter, S. ibid. 1958, i, 334.
weekly. The yolk lipids from the hens on the experimental feed showed a progressive increase in iodine value, and by the end of 3 weeks this had reached 100, at which level it remained for the duration of the experiment. The iodine value of the yolk lipids from the control hens did not change and at 3 weeks averaged 73 (standard deviation 1-5). At this stage all the eggs from the control and the experimental hens were collected and used for human feeding trials. The experimental egg yolks were a deeper yellow than the controls, but when they were cooked they could not be distinguished by taste. The experimental hens throve on their diet, and their eggproduction was in no way inferior to that of the controls. It interested us to find that, after the 6 weeks, the mean serumcholesterol level of the ten experimental hens was 117 mg. per 100 ml. (S.D. 23), compared with 166 mg. (s.D. 34): these mean values were significantly different (p less than 002). By this time the body fats of the experimental hens had become relatively unsaturated: the values for the control and experimental hens respectively were: abdominal fat, 76 and 94; subcutaneous fat, 76 and 85; muscle fat, 77 and 91; liver fat, 77 and 90. Too few analyses were performed, however, to permit of any statistical comparison. The effect of feeding these eggs was studied in 2 healthy Bantu male volunteers, each 34 years old. During the
experiments they lived in the metabolism ward and received the basal diet, very low in fat (11 g. daily), which 8 we have previously described, Except as indicated their diets were constant with respect to below, kept calories (2500 daily) and protein (88 g. daily). Experiment 1 (fig. 1).-On the basal diet, the mean serumcholesterol level was 75 mg. per 100 ml. Introduction of ten boiled " control " egg-whites into the daily diet for 10 days did not alter it. When they were replaced by ten boiled control egg yolks daily for 10 days, it rose to 100 mg. These were then replaced by ten boiled " unsaturated " yolks, and during the next 10 days the serum-cholesterol level fell to 95 mg. per 100 ml.-a trivial change. In the next period, ten whole " unsaturated " eggs fried in 100 g. of sunflower-seed
245
oil were eaten daily (3100 calories, 112 9th day the serum-cholesterol level practically to basal level. On returning diet there was a temporary rise in the
protein), and by the fell to 77 mg.-i.e., the subject to his basal serum-cholesterol, but
g.
returned to the basal level. In this first experiment there was no difference in the effect on the serum-cholesterol level of " control " and " It remained possible, however, unsaturated " eggs. that if the latter had been eaten for more than 10 days the cholesterogenic effect of the control eggs might have been reversed. In the next experiment, therefore, the unsaturated eggs were fed without a preliminary period on control eggs. Experiment 2 (fig. 2).-During an initial 6-day basal period, the serum-cholesterol level averaged 188 mg. per 100 ml. Then ten boiled unsaturated egg yolks daily were introduced into the diet and fed for 7 days; the serum-cholesterol level promptly rose to 218 mg. The subject was then returned to his basal diet and 3 g. of cholesterol daily (i.e., the amount in ten yolks) was mixed with his food for the remaining 8 days of the experiment. The serum-cholesterol quickly returned to the basal level, and this showed again that the serum-cholesterol-increasing action of eggs is not due to their cholesterol
it
quickly
content.
It appears from these two experiments that increase of the iodine value of egg yolks from 72 to 100 does not materially affect their cholesterogenic action. Although egg-yolk lipids with iodine value of 100 markedly raise the serum-cholesterol level, we have frequently observed that certain other more saturated fats, such as hydrogenated sunflower seed oil (iodine value 74), do not raise the serum-cholesterol, and that some, such as olive oil (iodine value 88) and peanut oil (iodine value 89), sometimes lower it. Thus we conclude that the effect of all dietary fats on the serum-cholesterol level is not simply a function of their degree of saturation. The suggestion of Keys et al.8-that, to offset the cholesterogenic effect of saturated fatty acids, at least twice as much polyethenoid fatty acid is required-might appear to be a satisfactory explanation of our findings; thus, the additional linoleic acid introduced into the unsaturated eggs may not have sufficed to neutralise their saturated-fatty-acid In Cruickshank’s experiments,5 the polyethenoidcontent. fatty-acid content of her unsaturated eggs was more than double their saturated-fatty-acid content; had we been able " to unsaturate " our eggs beyond iodine value 100, it is conceivable that we might have produced a " cholesterol-decreas8. Keys,
A., Anderson, J. T., Grande, F. ibid. p. 742.
ing " egg. It must be noted, however, that in several other dietary experiments in man, a fall in serum-cholesterol has been produced with far less than a 2 : 1 ratio of polyethenoid to saturated fatty acids. For example, fig. 8 of our paper last December,9 shows that the cholesterogenic effect of 100 g. of hydrogenated coconut oil (almost all saturated fatty acids) was abolished by 100 g. of sunflower-seed oil-i.e., by only 65 g. of linoleic acid. In a number of long-term studies of active men consuming their ordinary diets, which were often rich in saturated fats, we found that by simply administering 50 g. of sunflower-seed oil (about 33 g. of linoleic acid) daily, substantial decrease in the serum-cholesterol level
a
was
usually produced and maintained.10 It may be that the rule of Keys et al.8 is valid when applied to their type of group " experiment but we agree with Kinsell 11 that the individual response to a dietary fat is variable and cannot be predicted by a mathematical formula. "
We have no data on the individual fatty-acid components of our control and unsaturated egg yolks, but on the basis of Cruickshank’s findings 5 we have assumed that the unsaturation of the latter is achieved by an increase in the proportion of linoleic acid at the expense of the monoethenoid and saturated fatty acids. Failure of this presumed rise in the linoleic-acid content of our eggs to inhibit the cholesterogenic effect seems to be at variance with the " essential fatty acid deficiency " hypothesis. We must point out, however, that it is not known whether the " linoleic acid " of egg yolks is the biologically active It is known that the " linoleic cis-cis isomer or not. acid " of a number of animal fats is in the inactive form,12 isomerisation apparently having taken place in the process of digestion, absorption, or deposition of the active linoleic acid of the diet. Thus, Hilditch and Longenecker 13 found that, despite an abundance of natural linoleic acid in pasture grasses, the depot fats of ruminants contain very little of it; most of the octadecadienoic acids of these fats are in the trans-cis or other isomeric forms. It is possible that the failure of our unsaturated egg yolks to decrease the serum-cholesterol level is due to isomerisation of the linoleic acid of the dietary sunflower seeds in the course of its incorporation into the egg yolk. The above speculation implies acceptance of the " essential fatty acid deficiency " hypothesis. This hypothesis, however, is far from proven: hypercholesterolxmia does not occur in animals fed on a diet deficient in essential fatty acids,14 nor is there any direct evidence of such deficiency in hypercholesterolaemic human beings. It is true that Kinsell et al. have shown that 95% pure linoleic acid can lower the, serum-cholesterol level of man, but the effect of its " non-essential " isomers has not been studied. We must conclude that the factor or factors in dietary fats which regulate the serumcholesterol level are still unknown. These experiments were performed with the cooperation of Dr. M. Finlayson, the owner, and Mr. J. Mirvish, the manager, of the farm Kelvin Grove in the Constantia valley. We are grateful to
them for their kindness.
H. GORDON M.D.,
Department of Medicine, University of Cape Town, and the Clinical
Nutrition Research Unit of the Council for Scientific and Industrial Research, Union of South Africa 9. 10. 11. 12. 13. 14.
B.SC.
Cape
Town
J. WILKENS B.SC.
W’srand
J. F. BROCK D.M. Oxon., F.R.C.P.
Gordon, H., Lewis, B., Eales, L., Brock, J. F. ibid. 1957, ii, 1299. Gordon, H., Brock, J. F. S. Afr. med. J. 1958, 32, 397. Kinsell, L. W. Lancet, 1958, i, 857. Hilditch, T. P. Chemical Constitution of Natural Fats. London, 1956. Hilditch, T. P., Longenecker, H. E. Biochem. J. 1937, 31, 1805. Deuel, H. J., Jr., Alfin-Slater, R. B., Wells, A. F., Kryder, G. D., Aftergood, L. J. Nutr. 1955, 55, 337.