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The influence of myristic acid of dietary fats on serum cholesterol
Journal of A therosclerosis Research
51
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
T H E I N F L U E N C E OF MYRISTIC ACID OF D I E T A R Y FATS ON SERUM CHOLESTEROL
S. M U K H E R J E E ,
R. D U T T A AND C. B A N D Y O P A D H Y A Y
Laboratories of Lipid Research, Department of Applied Chemistry, University of Calcutta, Calcutta (India) (Revised, received February 6th, 1969)
SUMMARY
The effect of dietary myristic acid on serum cholesterol of rats was studied using mixtures of tfimyristin and groundnut oil. Incorporation of trimyristin in the oil resulted in marked rise in serum cholesterol of rats over the control group fed groundnut oil. A direct relationship between serum cholesterol and the myristic acid content of dietary fat was not apparent. Thus the cholesterol response to a diet containing an interesterified fat, prepared from a mixture of trimyristin and groundnut oil, was lower than that obtained b y administering the same fat mixture without treatment.
Key words: S e r u m cholesterol - Glyceride structure - M y r i s t i c acid content of dietary f a t s - Trisaturated glyceride - Interesterified f a t - F a t t y acid randomization
INTRODUCTION
The interrelationship of dietary f a t t y acids and the serum cholesterol level in man has been extensively studied 1-3. I t has been accepted that saturated f a t t y acids in general tend to increase serum cholesterol while polyenoic acids oppose this effect. KEYS et al. 2 have established that the cholesterol effect of saturated (C12-Cls) acids is almost twice as great per g as those of the polyunsaturated acids. More recently HEGSTED et al. 4 demonstrated that the myristic acid content of dietary fats is the major factor contributing to nearly 70 % of the change in serum cholesterol level in man.
The purpose of the present investigation is to determine whether the hypercholesterolaemic response to myristic acid is related to its actual content in the dietary fat or whether it depends on the distribution pattern of this acid within the glyceride molecule. This preliminary study tends to show that dietary fats containing identical J. Atheroscler. Res., 1969, 10:51-54
52
S. MUKHERJEE, R. DUTTA, C. BANDYOPADHYAY
quantities of myristic acid m a y produce different effects on rat's serum cholesterol, and a possible correlationship between serum cholesterol and the trisaturated glyceride content of dietary fat is indicated. MATERIALS AND METHODS G r o u p s of 12 male albino r a t s of C D R I Colony (Lucknow), weighing 60-65 g, were fed s e m i s y n t h e t i c r a t i o n s c o n t a i n i n g 20 % casein, 12 % glucose, 50 % starch, 4 % W e s s o n salt m i x t u r e a n d 10 % of d i e t a r y fat. All diets were fortified w i t h v i t a mins a n d p r o d u c e d consistent gain in weight. D i e t 1 c o n t a i n i n g 10 % g r o u n d n u t oil, served as t h e control. Three m y r i s t i c acid regimens were e m p l o y e d which c o n t a i n e d m i x t u r e s of g r o u n d n u t oil a n d t r i m y r i s t i n as i n d i c a t e d in T a b l e 1. A f o u r t h m y r i s t i c a c i d regimen (Diet 5) c o n t a i n e d an interesterified fat p r e p a r e d from a m i x t u r e of 85 % g r o u n d n u t oil a n d 15 % t r i m y r i s t i n according to ECKEY 5. T h e a n i m a l s were m a i n t a i n e d on t h e i r respective diets for a p e r i o d of 12 weeks. T h e d i e t a r y i n t a k e of each g r o u p of r a t s was measured; it d i d n o t differ to a n y a p p r e c i a b l e e x t e n t a m o n g the v a r i o u s e x p e r i m e n t a l groups. A t t h e end of the feeding period r a t s were sacrificed u n d e r N e m b u t a l anaesthesia, b l o o d was w i t h d r a w n b y c a r d i a c p u n c t u r e a n d s e r u m was s e p a r a t e d . T o t a l lipids were e x t r a c t e d b y a modified SPERRY--BRAND procedure6; s e r u m cholesterol was d e t e r m i n e d b y a modified GPERRY--WEBB p r o c e d u r e 7. T h e c o n t e n t of t r i s a t u r a t e d glyceride (GS3) of d i e t a r y fats was d e t e r m i n e d a c c o r d i n g to BANDYOPADHYAY 8.
TABLE 1 SERUM
CHOLESTEROL
OF RATS ON MYRISTIC
ACID DIETS
Figures in parentheses indicate number of rats. Dietary fat
Serum cholesterol total • S E M b (mg/lO0 ml)
% free
Series 1
Diet Diet Diet Diet Diet
1 2 3 4 5
groundnut oil (12) 48.5 :i: 2.5 5 % trimyristin + 95 % groundnut oil (12) 50.6 i 1.5 10 % trimyristin + 90 % groundnut oil (12) 68.7 -4- 3.3 15 % trimyristin + 85 % groundnut oil (12) 104.8 :t- 4.8 interesterified fat a (12) 70.0 J: 3.2
17.5 17.6 19.1 25.0 17.8
groundnut oil (8)
17.6
Series 2
Diet 1 Diet 4 Diet 5
50.6 -4- 2.0 (P < 0.001) 15 % trimyristin + 85 % groundnut oil (10) 108.0 • 4.7
24.0
(P < o.os) interesterified fat (8)
68.0 i 1.1
18.3
a Prepared from a mixture of 15 % trimyristin and 85 % groundnut oil according to ECKEY5. b Standard error of the mean. j . Atheroscler. Res., 1969, 10:51 54
MYRISTIC ACID AND SERUM CHOLESTEROL
53
TABLE 2 FATTY
ACID AND
GSa C O N T E N T
OF ]~XPER1MENTAL
DII~TS
S = saturated; D = dienoic; M = myristic; GSs = trisaturated glyceride.
Diet Diet Diet Diet Diet
1 2 3 4 5
Total S (%)
M (%)
D (%)
GS3 (%)
19.7 23.2 26.7 31.9 32.0
0.75 5.10 9.61 14.10 14.10
26.2 24.8 23.8 22.4 22.3
0.0 --15.0 3.8
RESULTS AND DISCUSSION The serum cholesterol responses to dietary myristic acid, fed as trimyristin either mixed or interesterified with groundnut oil, are shown in Table 1. The addition of 5 % trimyristin to groundnut oil (Diet 2) caused no significant change in the serum cholesterol level over that in the controls (0.3 > P > 0.2), but when the myristic acid content of the dietary fat is raised to approximately 10 % (Diet 3) the serum cholesterol is statistically significantly increased, the increase being mainly in the esterified fraction (P < 0.05). A pronounced hypercholesterolaemic effect was noted when 15 % of the dietary fatty acids were myristic acid (Diet 4; P < 0.001). At low trimyristin levels any effect of myristic acid on the serum cholesterol level was masked by the high content of dienoic acid (Diet 2; vide Table 2). Although appreciable quantities of dienoic acid were present in Diet 4 (2.05 g), rats received on average 1.4 g of myristic acid per day from this diet as against only 0.08 g from the control diet and 0.5 g from Diet 2. The most striking effect of dietary myristic acid on the serum cholesterol level was obtained when fed as trimyristin at the 15 % level; in this case both ester and free cholesterol levels were elevated. While these results are in general agreement with the observations of HEGSTED et al.4, a discrepancy was noticed when tr~myristin was fed after interesterification with groundnut oil. From the differences in the serum cholesterol responses to Diets 4 and 5, which contained identical quantities of myristic and dienoic acids, it is difficult to visualize that the myristic acid content of the dietary fat was alone responsible for increasing the serum cholesterol level. The lower serum cholesterol levels obtained with Diet 5 (containing interesterified fat) indicate that the structural composition of the glyceride that contains the myristic acid may have an important influence on these levels. The actual content of trisaturated glycerides (GSa) in the interesterified fat (Diet 5) is 3.8 % resulting from randomization of the glyceride structure, compared with 15 % in the fat mixture employed in Diet 4. Although a strict correlation between the GS3 content of dietary fats containing myristic acid and serum cholesterol values has not been obtained, this preliminary study tends to show that the effect of dietary myristic acid on the serum cholesterol level depends on the distribution j . Atheroscler. Res., 1969, 10:51-54
54
s. MUKHERJEE, R. DUTYA, C. BANDYOPADHYAY
p a t t e r n of this f a t t y acid w i t h i n the glyceride molecule. E x p e r i m e n t s on the rates of absorption of fat m i x t u r e s c o n t a i n i n g v a r y i n g concentrations of t r i m y r i s t i n a n d interesterified fat (prepared from m i x t u r e s of g r o u n d n u t oil a n d trimyristin) are i n progress: it is a n t i c i p a t e d t h a t the observed differences m a y be related to the rates of a b s o r p t i o n of the s y n t h e t i c glycerides, a l t h o u g h the digestibility of fats in the various e x p e r i m e n t a l diets was n o t significantly different. ACKNOWLEDGEMENTS The work was supported b y a g r a n t from the U.S. D e p a r t m e n t of Agriculture (PL-480). The a u t h o r s are i n d e b t e d to Dr. B. Mukherjee for the p r e p a r a t i o n of the interesterified fat used in this investigation.
REFERENCES 1 AHRENS, E. H. JR., W. INSOLL JR., R. BLOMSTRAND, J. HIRSCH, T. T. TSALTAS AND M. L. PETERSON, The influence of dietary fats on serum lipid levels in man, Lancet, 1957, i: 943.
2 KEYS, A., J. T. ANDERSONAND F. GRANDE, Prediction of serum cholesterol responses of man to changes in fats in the diet, Lancet, 1957, ii: 959. 3 I~INSELL, L. W., G. D. MICHAELS, R. W. FRISKEY AND S. D. SPLITTER, Essential fatty acid, lipid metabolism and atherosclerosis, Lancet, 1958, i: 334. 4 HEGSTED, D. M., R. ]3. MCGANDY, M. L. MEYERS AND F. J. STARE, Quantitative aspects of dietary fat on serum cholesterol in man, Amer..[. clin. Nutr., 1965, 17: 281. 5 ECKEY, E. W . , Directed interesterification in glycerides, Ind. Eng. Chem., 1948, 40: 1183. 6 SPERRY, W. M. AND F. C. BRAND, The determination of total lipids in blood serum, J. biol. Chem., 1955, 213: 69. 7 SPERRY, W. M. AND M. WEBB, A revison of the Schoenheimer-Sperry method for cholesterol determination, J. biol. Chem., 1950, 187: 97. S BANDYOPADHYAY, C., Estimation of trisaturated glycerides in fats by argentation thin layer chromatography, J. Chromatog., 1968, 37: 123.
J. Atheroscler. Res., 1969, 10:51-54
51
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
T H E I N F L U E N C E OF MYRISTIC ACID OF D I E T A R Y FATS ON SERUM CHOLESTEROL
S. M U K H E R J E E ,
R. D U T T A AND C. B A N D Y O P A D H Y A Y
Laboratories of Lipid Research, Department of Applied Chemistry, University of Calcutta, Calcutta (India) (Revised, received February 6th, 1969)
SUMMARY
The effect of dietary myristic acid on serum cholesterol of rats was studied using mixtures of tfimyristin and groundnut oil. Incorporation of trimyristin in the oil resulted in marked rise in serum cholesterol of rats over the control group fed groundnut oil. A direct relationship between serum cholesterol and the myristic acid content of dietary fat was not apparent. Thus the cholesterol response to a diet containing an interesterified fat, prepared from a mixture of trimyristin and groundnut oil, was lower than that obtained b y administering the same fat mixture without treatment.
Key words: S e r u m cholesterol - Glyceride structure - M y r i s t i c acid content of dietary f a t s - Trisaturated glyceride - Interesterified f a t - F a t t y acid randomization
INTRODUCTION
The interrelationship of dietary f a t t y acids and the serum cholesterol level in man has been extensively studied 1-3. I t has been accepted that saturated f a t t y acids in general tend to increase serum cholesterol while polyenoic acids oppose this effect. KEYS et al. 2 have established that the cholesterol effect of saturated (C12-Cls) acids is almost twice as great per g as those of the polyunsaturated acids. More recently HEGSTED et al. 4 demonstrated that the myristic acid content of dietary fats is the major factor contributing to nearly 70 % of the change in serum cholesterol level in man.
The purpose of the present investigation is to determine whether the hypercholesterolaemic response to myristic acid is related to its actual content in the dietary fat or whether it depends on the distribution pattern of this acid within the glyceride molecule. This preliminary study tends to show that dietary fats containing identical J. Atheroscler. Res., 1969, 10:51-54
52
S. MUKHERJEE, R. DUTTA, C. BANDYOPADHYAY
quantities of myristic acid m a y produce different effects on rat's serum cholesterol, and a possible correlationship between serum cholesterol and the trisaturated glyceride content of dietary fat is indicated. MATERIALS AND METHODS G r o u p s of 12 male albino r a t s of C D R I Colony (Lucknow), weighing 60-65 g, were fed s e m i s y n t h e t i c r a t i o n s c o n t a i n i n g 20 % casein, 12 % glucose, 50 % starch, 4 % W e s s o n salt m i x t u r e a n d 10 % of d i e t a r y fat. All diets were fortified w i t h v i t a mins a n d p r o d u c e d consistent gain in weight. D i e t 1 c o n t a i n i n g 10 % g r o u n d n u t oil, served as t h e control. Three m y r i s t i c acid regimens were e m p l o y e d which c o n t a i n e d m i x t u r e s of g r o u n d n u t oil a n d t r i m y r i s t i n as i n d i c a t e d in T a b l e 1. A f o u r t h m y r i s t i c a c i d regimen (Diet 5) c o n t a i n e d an interesterified fat p r e p a r e d from a m i x t u r e of 85 % g r o u n d n u t oil a n d 15 % t r i m y r i s t i n according to ECKEY 5. T h e a n i m a l s were m a i n t a i n e d on t h e i r respective diets for a p e r i o d of 12 weeks. T h e d i e t a r y i n t a k e of each g r o u p of r a t s was measured; it d i d n o t differ to a n y a p p r e c i a b l e e x t e n t a m o n g the v a r i o u s e x p e r i m e n t a l groups. A t t h e end of the feeding period r a t s were sacrificed u n d e r N e m b u t a l anaesthesia, b l o o d was w i t h d r a w n b y c a r d i a c p u n c t u r e a n d s e r u m was s e p a r a t e d . T o t a l lipids were e x t r a c t e d b y a modified SPERRY--BRAND procedure6; s e r u m cholesterol was d e t e r m i n e d b y a modified GPERRY--WEBB p r o c e d u r e 7. T h e c o n t e n t of t r i s a t u r a t e d glyceride (GS3) of d i e t a r y fats was d e t e r m i n e d a c c o r d i n g to BANDYOPADHYAY 8.
TABLE 1 SERUM
CHOLESTEROL
OF RATS ON MYRISTIC
ACID DIETS
Figures in parentheses indicate number of rats. Dietary fat
Serum cholesterol total • S E M b (mg/lO0 ml)
% free
Series 1
Diet Diet Diet Diet Diet
1 2 3 4 5
groundnut oil (12) 48.5 :i: 2.5 5 % trimyristin + 95 % groundnut oil (12) 50.6 i 1.5 10 % trimyristin + 90 % groundnut oil (12) 68.7 -4- 3.3 15 % trimyristin + 85 % groundnut oil (12) 104.8 :t- 4.8 interesterified fat a (12) 70.0 J: 3.2
17.5 17.6 19.1 25.0 17.8
groundnut oil (8)
17.6
Series 2
Diet 1 Diet 4 Diet 5
50.6 -4- 2.0 (P < 0.001) 15 % trimyristin + 85 % groundnut oil (10) 108.0 • 4.7
24.0
(P < o.os) interesterified fat (8)
68.0 i 1.1
18.3
a Prepared from a mixture of 15 % trimyristin and 85 % groundnut oil according to ECKEY5. b Standard error of the mean. j . Atheroscler. Res., 1969, 10:51 54
MYRISTIC ACID AND SERUM CHOLESTEROL
53
TABLE 2 FATTY
ACID AND
GSa C O N T E N T
OF ]~XPER1MENTAL
DII~TS
S = saturated; D = dienoic; M = myristic; GSs = trisaturated glyceride.
Diet Diet Diet Diet Diet
1 2 3 4 5
Total S (%)
M (%)
D (%)
GS3 (%)
19.7 23.2 26.7 31.9 32.0
0.75 5.10 9.61 14.10 14.10
26.2 24.8 23.8 22.4 22.3
0.0 --15.0 3.8
RESULTS AND DISCUSSION The serum cholesterol responses to dietary myristic acid, fed as trimyristin either mixed or interesterified with groundnut oil, are shown in Table 1. The addition of 5 % trimyristin to groundnut oil (Diet 2) caused no significant change in the serum cholesterol level over that in the controls (0.3 > P > 0.2), but when the myristic acid content of the dietary fat is raised to approximately 10 % (Diet 3) the serum cholesterol is statistically significantly increased, the increase being mainly in the esterified fraction (P < 0.05). A pronounced hypercholesterolaemic effect was noted when 15 % of the dietary fatty acids were myristic acid (Diet 4; P < 0.001). At low trimyristin levels any effect of myristic acid on the serum cholesterol level was masked by the high content of dienoic acid (Diet 2; vide Table 2). Although appreciable quantities of dienoic acid were present in Diet 4 (2.05 g), rats received on average 1.4 g of myristic acid per day from this diet as against only 0.08 g from the control diet and 0.5 g from Diet 2. The most striking effect of dietary myristic acid on the serum cholesterol level was obtained when fed as trimyristin at the 15 % level; in this case both ester and free cholesterol levels were elevated. While these results are in general agreement with the observations of HEGSTED et al.4, a discrepancy was noticed when tr~myristin was fed after interesterification with groundnut oil. From the differences in the serum cholesterol responses to Diets 4 and 5, which contained identical quantities of myristic and dienoic acids, it is difficult to visualize that the myristic acid content of the dietary fat was alone responsible for increasing the serum cholesterol level. The lower serum cholesterol levels obtained with Diet 5 (containing interesterified fat) indicate that the structural composition of the glyceride that contains the myristic acid may have an important influence on these levels. The actual content of trisaturated glycerides (GSa) in the interesterified fat (Diet 5) is 3.8 % resulting from randomization of the glyceride structure, compared with 15 % in the fat mixture employed in Diet 4. Although a strict correlation between the GS3 content of dietary fats containing myristic acid and serum cholesterol values has not been obtained, this preliminary study tends to show that the effect of dietary myristic acid on the serum cholesterol level depends on the distribution j . Atheroscler. Res., 1969, 10:51-54
54
s. MUKHERJEE, R. DUTYA, C. BANDYOPADHYAY
p a t t e r n of this f a t t y acid w i t h i n the glyceride molecule. E x p e r i m e n t s on the rates of absorption of fat m i x t u r e s c o n t a i n i n g v a r y i n g concentrations of t r i m y r i s t i n a n d interesterified fat (prepared from m i x t u r e s of g r o u n d n u t oil a n d trimyristin) are i n progress: it is a n t i c i p a t e d t h a t the observed differences m a y be related to the rates of a b s o r p t i o n of the s y n t h e t i c glycerides, a l t h o u g h the digestibility of fats in the various e x p e r i m e n t a l diets was n o t significantly different. ACKNOWLEDGEMENTS The work was supported b y a g r a n t from the U.S. D e p a r t m e n t of Agriculture (PL-480). The a u t h o r s are i n d e b t e d to Dr. B. Mukherjee for the p r e p a r a t i o n of the interesterified fat used in this investigation.
REFERENCES 1 AHRENS, E. H. JR., W. INSOLL JR., R. BLOMSTRAND, J. HIRSCH, T. T. TSALTAS AND M. L. PETERSON, The influence of dietary fats on serum lipid levels in man, Lancet, 1957, i: 943.
2 KEYS, A., J. T. ANDERSONAND F. GRANDE, Prediction of serum cholesterol responses of man to changes in fats in the diet, Lancet, 1957, ii: 959. 3 I~INSELL, L. W., G. D. MICHAELS, R. W. FRISKEY AND S. D. SPLITTER, Essential fatty acid, lipid metabolism and atherosclerosis, Lancet, 1958, i: 334. 4 HEGSTED, D. M., R. ]3. MCGANDY, M. L. MEYERS AND F. J. STARE, Quantitative aspects of dietary fat on serum cholesterol in man, Amer..[. clin. Nutr., 1965, 17: 281. 5 ECKEY, E. W . , Directed interesterification in glycerides, Ind. Eng. Chem., 1948, 40: 1183. 6 SPERRY, W. M. AND F. C. BRAND, The determination of total lipids in blood serum, J. biol. Chem., 1955, 213: 69. 7 SPERRY, W. M. AND M. WEBB, A revison of the Schoenheimer-Sperry method for cholesterol determination, J. biol. Chem., 1950, 187: 97. S BANDYOPADHYAY, C., Estimation of trisaturated glycerides in fats by argentation thin layer chromatography, J. Chromatog., 1968, 37: 123.
J. Atheroscler. Res., 1969, 10:51-54