Effect
of hydrogenated
cholesterol F. H.
Mattson,
and
triglyceride
E. .1. Hollenbach,
ABSTRACT
and
A group
their calories as fat.
The
the
80%
subjects.
latter
group
fatty
acid
consumed, triglyceride the this
effect effect
726-731,
acids were intakes the
trans two
were
the
to the
The
of man’
fed for 21 days of the diet
fat was
was
fat oii blood by the
replaced
one-half
a formula 25%
Except the
isomeric
with
of the
diet
saturates,
that
supplied
Over fat
consuming lipid
levels
form
of the
4-week
showed the
a hydrogenated
fat. acids
presence
the
no
unsaturated
Over of the
60% diet
of the of this
or absence
of trans
acids,
the
diets
change
of
The subjects In the diet of
period
unhydrogenated
is determined
38%
16% polyunsaturates,
polyunsaturated
for the
same.
hydrogenated
subjects
that
in plasma fat.
by its fatty acids.
It
the were
cholesterol
J.
or
concluded
that
composition
and
is
acid Am.
two
Chin.
Nutr.
28:
1975.
It is now generally accepted that the levels of blood lipids are influenced by the fatty acid composition of the diet. Thus, relative to monounsaturated acids, saturated fatty acids elevate and polyunsaturated acids depress blood cholesterol; the effects on triglycerides are directionally similar but of a much smaller magnitude. Based on these concepts, changes in the lipid composition of the diet have been recommended. A question arises when these proposals are applied to the usual American diet, because part of the unsaturated fatty acids from ruminant animals and of hydrogenated vegetable oils have a trans rather than a cis configuration. Should these acids be classified simply according to their unsaturation or does the presence of a trans double bond alter the effect of an acid? The answer to this is of particular importance because Call and Sanchez (1) have shown that over the past few decades hydrogenated fats have made an important contribution to the increase in polyunsaturated fatty acids and decrease in saturated fatty acids in the U.S. diet. As a result of the partial hydrogenation of a vegetable oil, trans double bonds may occur in either monounsaturated or diunsaturated acids; the predominant species is monounsat726
was
configuration. groups
receiving
altered
males
composition
approximately
in the
relative
levels Kligman
of the dietary
and
of a hydrogenated is not
acid
plasma
All of the unsaturated acids were in the cis configuration. groups. One group of 17 men continued on the same diet.
of the group
levels
M.
of 33 adult
two
remaining
A.
fatty
and 58% monounsaturates. were then divided into monounsaturated
fat on the
American
Journal
of Clinical
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/726/4716478 by University of Wyoming Libraries user on 19 June 2018
urated acids. For example, Table 1 lists the fatty acid composition that is typical of commercial shortenings prepared from hydrogenated vegetable oils: 95% of such products that are sold through retail stores in the United States today have this composition. Margarines and lightly hydrogenated salad and cooking oils also contain trans acids, and again the isomer is chiefly the monounsaturated acid. Dietary recommendations, such as those of the American Heart Association (2), besides calling for a decrease in saturated acids, are now placing greater emphasis on the total unsaturation of the dietary fat, rather than simply on polyunsaturated fatty acids. As a consequence, it is important to determine whether the isomeric structure of an unsaturated acid alters the effect of that acid on the blood lipid levels. The blood lipid levels of subjects who were consuming hydrogenated fats have been measured by a number of investigators. In some instances an elevation of blood cholesterol or triglyceride has been observed (3-10); in
‘From the Miami Valley Laboratories, Gamble Company, Cincinnati, Ohio 45257, University of Pennsylvania Medical School, phia. Pennsylvania 19174.
Nutrition
28:
JULY
1975,
pp.
726-731.
Printed
Procter & and the Philadel-
in U.S.A.
HYDROGENATED TABLE Fatty
I acid
composition
of vegetable
shortenings#{176}-
Saturated Monounsaturated cis trans
25 47
Polyunsaturated
28
33 14
23 5
cis,cis cis,
FAT AND
trans
trans.
trans
Values sold through 0. 1 to 0.3% #{176}
Trace are typical of 95% of such products retail stores in the United States. of the total fatty acids are conjugated
that are b From dienes.
others, the levels of these lipids were unchanged (5, 11-14). In many of these studies, any effect observed could have been due to fatty acid composition, rather than isomeric structure, because the levels of saturated and polyunsaturated acids were changed as well. In some, the level of dietary trans acids was quite low, and any effect could have been so small that it was not measurable. The present study was carried out for the purpose of establishing the effect on blood lipids of the trans acids that are normally consumed in the largest amount, namely, the monounsaturated acids. The use of experimental and control fats of the same fatty acid composition, except for the presence or absence of trans acids, and the inclusion of trans acids at a markedly elevated level overcame the shortcomings of the earlier studies. Materials
PLASMA
part ofTable 2 under the column headed High trans. On the basis of these values, appropriate portions of olive oil, safflower seed oil, and completely hydrogenated soybean oil were blended together and transesterified randomly. The fatty acid composition of this mixture of fats together with the added egg yolk lipids, as recovered from the diet dry mix, is shown in Table 2 under the column marked, High cis. The fatty acid compositions of the two diets were quite similar. The level of geometrical isomers in the high trans fat was determined by analysis of the partially hydrogenated soybean oil component. Absorptivity at 232 nm showed it to contain no more than 0.3% conjugated diene. Lipoxigenase assay (IS) showed the complete absence of cis, cis methylene interrupted double bonds. The contents of cis and trans monounsaturated fatty acids and cis, trans and trans, trans diunsaturated fatty acids were measured by absorption spectroscopy. The method of Allen (16) was used for infrared absorption,, except that the samples were scanned as liquid smears between CsBr plates rather than as solutions in CS2. Raman spectroscopy was carried out according to the method of Bailey and Horvat (17). The infrared and Raman spectroscopy were performed on the partially hydrogenated soybean oil and on the monoand diunsaturated fatty acids isolated from it by preparative gas-liquid chromatography. From these values and from the portion of partially hydrogenated soybean oil that was added to the diet, the isomeric fatty acid content of the high trans fat was calculated; these values are given in the lower part of Table 2. As can be seen from this
TABLE
2 acid composition given in percent Fatty
Subjects The subjects were male inmates County Prison at Holmesburg, ranged in age from 24 to 41 years; 31 years. All subjects were examined found to be in good health with disorders.
of the Philadelphia Pennsylvania. They with the median being before the study and no known metabolic
Diet For this study two diets, one containing a high level of trans monounsaturated fatty acids and the other a comparable level of cis monounsaturated fatty acids, were desired. Aside from this difference in the content of geometrical isomers, the diets were designed to be the same in fatty acid composition and caloric density and in their content of cholesterol, protein, fat, and carbohydrate. The high level of trans monounsaturated fatty acids was provided by soybean oil that had been hy-
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/726/4716478 by University of Wyoming Libraries user on 19 June 2018
727
drogenated under conditions that maximize the level of this isomer. Since this fat had an undesirable waxy feel in the mouth, nine parts of it was transesterified randomly with one part of safflower seed oil. The dried egg yolk, which was added to the diet as a source of cholesterol, contributed 1 1% of the total fatty acids in the diet. The fatty acid composition of the mixture of these three fats, as isolated from the dry mix that was used in the preparation of the formula diet, is shown in the upper
Fatty weight
and methods
LIPIDS
acid
of diets,
High
cis#{176}
Total 12.0 13.0 58.4 16.3 Isomeric 58
16:0 18:0 18:1 18:2 l8:lcis
cis,
acids 10.9 15.5 53.6 20.0 fatty acids 20 34 9 9 I
16 trans
trans. a
Consisted
trans of:
65
olive
oil,
trans6’
fatty
trans l8:2cis,cis
High
12
safflower
oil.
12
completely hydrogenated soybean oil, Il egg yolk lipids. 6Consisted of 81 partially hydrogenated soybean oil, 9 safflower oil, II egg yolk lipids. ‘Conjugated diene constituted 0.3% of the total fatty acids.
728 table,
MATTSON the
chief difference between the two fats in the diets was the presence (44% of the total acids) or absence of trans double bonds. This was most marked
formula
the
monounsaturated fatty acids; in the high trans fat these acids were of the trans configuration, whereas in the high cis fat the monounsaturated acids were only of the cis configuration. Table 3 shows the composition of the dry mix used for the preparation of the formula diets. The test fat (containing trans isomers) or the control fat (free of trans isomers) constituted 18% of the dry mix, or 35% of the calories. The remaining fat was contributed by the dried egg yolk. The cholesterol in the dry mix resulted in an intake of approximately 500 mg of cholesterol per subject per day. The men also received vitamin-mineral capsules, salt tablets, methyl cellulose wafers, and one small serving of a low fat, low calorie food item per day, such as a raw fruit or vegetable. More detailed descriptions of the components and of the preparation of the formula diets from the dry mixes have been presented (8, 10).
ET AL. TABLE
for the
63%
Group
PEO-I4
All
61
All
37
TDO-2l TD
HC
TD22-49
HT
Number
a
TD-21
of
subjects
the HC group of 16 subjects.
Institution
diet
Changeover to high cis emulsion diet 33 Emulsion diet, high cis fat 17#{176}Emulsion diet, high cis fat 13#{176}Emulsion diet, high trans fat
HC+HT
22-49
Treatment
completing consisted
the
study;
of 17 subjects
and
at
day
the
HT
procedure
The sequence in which the diets were consumed by the subjects is shown in Table 4. During an initial 14-day preexperimental period (period PE), 61 subjects received the normal institution diet. Blood samples were taken on days 2, 3, 7, 10, and 14. A number of individuals chose to drop out of the study and those whose plasma cholesterol level was less than 150 mg/100 ml were discontinued. The 37 men who qualified had an average plasma cholesterol level of 187.8 and triglyceride level of 108.4 mg/ 100 ml. During the subsequent 7 days (period TP), the participants consumed gradually increasing amounts of the formula diet at the expense of the institution diet; the diet containing the high cis fat was used during this period. All 37 subjects consumed solely this high cis formula diet for the subsequent 21 days (period TD 0-21). During the final 4 weeks of the study (period TD 22-49), 17 of the subjects continued to receive the formula diet containing the high cis fat; the remaining 16 participants received the formula diet containing the high trans fat. All of the subjects assigned to the high cis group completed the study, three of the subjects in the high trans group dropped out. The loss of participants subsequent to day TP-0 was for reasons unrelated to the study. Only the measurements obtained on subjects completing the study were used. At each of the four feedings on days TD 0-49, each subject received one-fourth of his daily allotment of the
Composition
No. of subj
TPO-7
group
TABLE
design
Period and days
of
Feeding
4
Experimental
3 of dry
mixes.#{176}g/ 100 g dry
Dried egg whiteb Dried egg yolk Dextrose Test fat Sodium chloridec Flavoring
mix 19.6 3.8 56.7 18.4 0.5 0.4
#{176} Diets contained per 100 g; 18.0 g protein, 20.1 g fat, 54.3 g carbohydrate. 104 mg cholesterol, 477 kcal. One-tenth milligram biotin added to each 100 g of dry mix to inactive avidin. Contained 0.01% potassium iodide.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/726/4716478 by University of Wyoming Libraries user on 19 June 2018
formula. Initially, body wt per day. ment
in energy
body weight. each individual weight. Over average gain Blood
sampling
the diet was Subsequently,
intake
was
fed at a level of 33 kcal/kg some individual adjust-
necessary
to maintain
constant
This
continuous adjustment of the intake of essentially maintained their initial body the course of the experiment there was an of0.3 lb (range + 13 to -6). and
anal vses
In all
of the experimental periods, blood samples were drawn twice weekly after an overnight fast. Collections were by Vacutainer tubes containing EDTA. The recovered plasma was analyzed for total cholesterol and triglycerides on the Technicon AutoAnalyzer (18). Besides the usual chemically pure cholesterol and triglyceride standards, a desiccated serum sample (Desiccated Serum, Difco Laboratories, Detroit, Michigan) was used as a reference. Replicate samples of this were analyzed with each set of blood plasma. Over the course of the study, a total of 325 determinations were made on this reference serum; the standard deviation of the analytical methods in mg/ 100 ml was 4.69 for cholesterol and 4.29 for triglyceride.
Results Only the data from subjects who completed the entire study are reported. The average plasma cholesterol and triglyceride levels of the subjects for each of the days that blood was collected are shown in Table 5. For statistical treatment, the values obtained on days TD 2-21, when a-li of the subjects received the formula diet containing the high cis fat, and days TD 3 1-49, when they were receiving either the high cis or the high trans fat, were used. Table 6 shows the average plasma triglyceride and cholesterol values, according to the group to which the subjects were assigned during the final 4 weeks of the
HYDROGENATED TABLE
FAT
AND
PLASMA
729
LIPIDS
S
Average plasma cholesterol and of the mean of subjects receiving
triglyceride an emulsion
levels and standard diet,#{176}’ b mg/ 100
error ml
Cholesterol
Triglyceride high trans group
high cis group
Day
All
a
0 3 7 10 14 17 21
185.4 182.4 179.4 179.2 181.3 183.5 194.9
±
24 28 31
187.1 194.8 190.5
±
35 38 42 45 49
186.5 188.2 191.6 183.5 189.1
±
Group
designation
the mean = S/Vn n = 13 for high
TABLE
6
Average
plasma
± ± ± ±
6.6 6.7 ±6.2 ±
± ± ± ±
5.8 6.2 8.3 6.0 6.2
196.1 190.5 189.8 190.9 184.4
determined
by dietary number 17 for
triglyceride
cholesterol
HC HT HC HT Standard error averages.
#{176}
±
where, n is the trans group and
Group
subject
±
191.6 187.5 182.2 179.6 182.2 187.8 198.1 Subjects 197.7 197.7 190.9±
7.7 7.0 6.6 6.4 6.8 6.0 7.0
and
mean
± ± ± ± ±
± ±
8.4 7.3 8.1 8.1 6.4
± ± ± ±
fat consumed
110.1 107.1 104.0 109.1 101.1 during
days
S is the standard
and
standard
error
Dietary fat
TD2-21 TD 2-21 TD3I-49 TD3I-49 of the
±
of subjects and high cis group.
Period and days
high cis high cis highcis high trans =
S/.,fli
where
n
is
the
study. Separate values are given for the High Trans (HT) and High Cis (HC) groups during days TD 2-21, even though all subjects were receiving the high cis fat at that time. The data were examined in four different comparisons by one-way analysis of variance with no significant differences being found. The similar values for the two groups during period TD 2-21, when they were receiving the same diet, show that the two groups of subjects were comparable. The absence of a time effect is shown by the almost identical values exhibited by the group receiving the high cis fat during both periods TD 0-21 and TD 2 1-49. The introduction of the high trans fat during period TD 2 1-49 did not result in a
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-
subjects fed high cis fat 6.2 110.6 ± 10.1 6.5 107.4 ± 11.6 6.0 105.4 ± 9.4 5.0 106.8 ± 9.2 5.5 lOO.7 ± 10.1 5.3 98.6 ± 11.0 7.9 109.9 ± 9.7 fed high cis or high trans fat 7.4 108.1 ± 11.6 9.4 117.5 ± 9.7 7.1 118.1 ±9.8
±
±
levels
high trans group
high cis group
number
8.0 8.8 7.2 10.6 8.6
± ± ± ± ±
22 to 49. deviation
of the
See
Table
4.
of the daily
mean,#{176}mg/
117.8 95.8 96.6 98.4 94.7 89.0 103.5
±
98.2 121.5 124.4
±
121.2 98.6 115.5 95.8 94.8
±
± ± ± ± ± ±
± ±
± ± ± ±
8.8 7.2 7.9 8.0 7.1 6.8 8.7 10.7 12.4 15.4 9.8 7.7 15.3 11.4 7.3
6Standard values
error
100 ml
Plasma triglyceride
Plasma cholesterol
104.6 ± 9.4 96.2 ± 6.8 108.2±8.0 108.2 ± 9.2
184.1 ± 6.7 186.2 ± 5.6 188.6±6.2 189.4 ± 6.9
of subjects
and
S is the
of
for the subjects,
standard
deviation
of the
change in the plasma lipid levels relative to that attained by the same group (HT) of subjects while they were on the high cis fat (period TC 0-21). Moreover, the levels realized on the high trans fat did not differ from those of the subjects who were receiving the high cis fat during the same period (TD 2 1-49).
Discussion The study reported here was designed so that the only dietary variable was the presence or absence of trans acids. Grasso et al. (9) and Erickson et al. (10) used a similar experimental design in that the hydrogenated and unhydrogenated fats in each of their
730
MATTSON
studies had a similar total fatty acid composition. However, the fats fed in those earlier studies were of the type that are customarily used in the preparation of margarines, shortenings, and salad oils, and consequently did not contain as high a level of trans acids. In the present investigation, the level of trans acids, particularly the monounsaturated trans acids, was greatly magnified by hydrogenating soybean oil under conditions that would maximize their formation. The use of this as the principal dietary fat resulted in over 60% of the monounsaturated fatty acids having a trans configuration. Even at this greatly magnified level of feeding, we, like Grasso et al. (9) and Erickson et al. (10), found the level of plasma cholesterol’ and triglyceride to be the same regardless of the isomeric structure of the dietary fatty acids. In most of the other reported studies, the process of hydrogenation was used either to decrease the polyunsaturated fatty acid or to increase the satuiated fatty acid levels of a fat. The hydrogenated and unhydrogenated fats then were fed to the subjects to determine the effects of such changes in the composition of dietary fatty acids on blood lipid levels. Since it is now recognized that saturated fatty acids elevate plasma lipids and polyunsaturated fatty acids decrease them, it is not possible to attribute any specific effect to the trans acids of the fats that were fed in those early studies. An example of such a difficulty can be seen in two of three experiments reported by Anderson, Grande, and Keys (5). In these, the subjects received either an unhydrogenated fat or the same fat after partial hydrogenation. As a consequence, the control and experimental diets differed in their fatty acid compositions. They attempted to compensate for this variable by applying their equation (19) that predicts the blood cholesterol levels that should have been attained as the result of consuming diets with those fatty acid compositions. In one experiment, the blood cholesterol levels that were observed were essentially identical with those that were calculated. Thus, trans isomers evidently had the same effect as did the cis isomers. In another experiment, the hydrogenated fat gave a
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ET AL. lower blood cholesterol level than that predicted by the fatty acid composition of the diet; a result that could be interpreted as showing trans isomers to be more effective than cis isomers in lowering this blood lipid component. In the third experiment described in the same report (5), the fatty acid compositions ofthe hydrogenated and unhydrogenated fats were the same except for the presence or absence of trans acids. In this instance, the hydrogenated fat resulted in a relative elevation of the blood cholesterol level. It is significant that the hydrogenated fat used in the third experiment had an unusual fatty acid composition. As in our study hydrogenation had been carried out under special conditions to maximize the content of trans fatty acids, but the particular conditions employed by them also resulted in relatively high contents of conjugated dienes and of trans, trans diunsaturated fatty acids; the sum of these two was 10% of the total fatty acids. The levels of both of these materials in the fat of their third experiment were considerably higher than they were in the high trans fat used for our study (Table 2) or that found in the hydrogenated fats used for margarines, salad oils, or shortenings (Table 1). The possibility exists then that the singular effects reported by Anderson, Grande, and Keys (5) may have been the result of either the trans. trans diunsaturated acids, or the conjugated dienes. A preliminary report by Vergroesen (20) also indicated that specific fatty acids may have an unique effect. This brief paper stated that the consumption of elaidic acid, trans9-octadecenoic acid, resulted in an increase in the level of blood cholesterol. Since this was a preliminary report, few experimental details are given making it difficult to relate Vergroesen’s observations to ours. The effects of particular fatty acid may be reflected also in the studies of Mishkel and Spritz (21). The fat used by those investigators was not hydrogenated, but rather it was linoleate that had been isomerized with SO2 and subsequently fractionated. As a consequence of these treatments, the predominant fatty acid was trans, trans-dienoic acid. Consumption of this fat as 20 or 40% of the
HYDROGENATED
FAT
calories resulted in a marked elevation of plasma triglycerides. The high trans fat that we used was obtained by hydrogenation and contained only 1 % of the trans. trans isomer of linoleic acid. We did not see an elevation of the plasma triglyceride level. On the other hand, the hydrogenated fat that contained appreciable amounts of trans, trans acids and that was fed in the third experiment of Anderson, Grande, and Keys (5) resulted in a small increase in plasma triglycerides. Thus there is the possibility that certain particular isomers of fatty acids may alter plasma lipid levels. However, this does not appear to apply to the hydrogenated fats that are customarily consumed in the United States such as margarines, shortenings, and salad oils (7-10).
ci
Our thanks to the following individuals for their assistance-C. Theuring for the preparation of the fat, E. Anderson and W. Tettenhorst for analysis of the plasma, A. J. FehI and R. P. Oertel for determination of the isomer levels, and S. E. Michaels for statistical analysis of the data.
References I. CALL, D. L., AND A. M. SANCHEZ. Trends in fat disappearance in the United States l909-65. J. Nutr. 93: Suppl. I; 1, 1967. 2. Primary Prevention of the Atherosclerotic Diseases. Report of Inter-Society Commission for Heart Disease Resources. Circulation 42: ASS, 1970. 3. BEVERIDGE, J. M. R., W. F. CONNELL, 0. A. MAYER AND H. L. HAUST. Plant sterols, degree of unsaturation, and hypocholesterolemic action of certain fats. Can. J. Biochem. Physiol. 36: 895, 1958. 4. WILCOX, E. B., AND L. S. GALLOWAY. Serum cholesterol and different dietary fats. J. Am. Dietet. Assoc. 38: 227, 1961. 5. ANDERSON, J. T., F. GRANDE AND A. KEYS. Hydrogenated fats in the diet and lipids in the serum of man. J. Nutr. 75: 388, 1961. 6. MORSE, E. H., E. BICKNELL, E. P. LEWIS, S. B. MERROW AND C. A. NEWHALL. Relation of dietary fats to blood lipids in young men. J. Am. Dietet. Assoc. 41: 323, 1962. 7. BEVERIDGE, J. M. R., AND W. F. CONNELL. The
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AND
8.
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effect of commercial terol levels in man. MCOSKER, D. E., GEN
margarines on plasma cholesAm. J. Clin. Nutr. 10: 391, 1962. F. H. MATTSON, H. B. SwERINA. M. KLIGMAN. The influence of partially
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MICHAELS and hy-
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IMAICHI,
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F. BROCK. serum-cholesterol 12.
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180:
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J. T. ANDERSON AND F. GRANDE. Serum in man: Diet fat and intrinsic responsiveness. Circulation 19: 201, 1959. VERGROESEN, A. J. Dietary fat and cardiovascular disease: possible modes of action of linoleic acid. Proc. Nutr. Soc. 31: 323, 1972. MISHKEL, M. A., AND N. SPRITZ. The effects of trans isomerized trilinolein on plasma lipids of man. Drugs Effecting Lipid Metabolism, edited by W. L. Holmes, L. A. Carlson, and R. Paoletti. New York: Plenum, 1969, p. 355. KEYS,
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of hydrogenated
cholesterol F. H.
Mattson,
and
triglyceride
E. .1. Hollenbach,
ABSTRACT
and
A group
their calories as fat.
The
the
80%
subjects.
latter
group
fatty
acid
consumed, triglyceride the this
effect effect
726-731,
acids were intakes the
trans two
were
the
to the
The
of man’
fed for 21 days of the diet
fat was
was
fat oii blood by the
replaced
one-half
a formula 25%
Except the
isomeric
with
of the
diet
saturates,
that
supplied
Over fat
consuming lipid
levels
form
of the
4-week
showed the
a hydrogenated
fat. acids
presence
the
no
unsaturated
Over of the
60% diet
of the of this
or absence
of trans
acids,
the
diets
change
of
The subjects In the diet of
period
unhydrogenated
is determined
38%
16% polyunsaturates,
polyunsaturated
for the
same.
hydrogenated
subjects
that
in plasma fat.
by its fatty acids.
It
the were
cholesterol
J.
or
concluded
that
composition
and
is
acid Am.
two
Chin.
Nutr.
28:
1975.
It is now generally accepted that the levels of blood lipids are influenced by the fatty acid composition of the diet. Thus, relative to monounsaturated acids, saturated fatty acids elevate and polyunsaturated acids depress blood cholesterol; the effects on triglycerides are directionally similar but of a much smaller magnitude. Based on these concepts, changes in the lipid composition of the diet have been recommended. A question arises when these proposals are applied to the usual American diet, because part of the unsaturated fatty acids from ruminant animals and of hydrogenated vegetable oils have a trans rather than a cis configuration. Should these acids be classified simply according to their unsaturation or does the presence of a trans double bond alter the effect of an acid? The answer to this is of particular importance because Call and Sanchez (1) have shown that over the past few decades hydrogenated fats have made an important contribution to the increase in polyunsaturated fatty acids and decrease in saturated fatty acids in the U.S. diet. As a result of the partial hydrogenation of a vegetable oil, trans double bonds may occur in either monounsaturated or diunsaturated acids; the predominant species is monounsat726
was
configuration. groups
receiving
altered
males
composition
approximately
in the
relative
levels Kligman
of the dietary
and
of a hydrogenated is not
acid
plasma
All of the unsaturated acids were in the cis configuration. groups. One group of 17 men continued on the same diet.
of the group
levels
M.
of 33 adult
two
remaining
A.
fatty
and 58% monounsaturates. were then divided into monounsaturated
fat on the
American
Journal
of Clinical
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/726/4716478 by University of Wyoming Libraries user on 19 June 2018
urated acids. For example, Table 1 lists the fatty acid composition that is typical of commercial shortenings prepared from hydrogenated vegetable oils: 95% of such products that are sold through retail stores in the United States today have this composition. Margarines and lightly hydrogenated salad and cooking oils also contain trans acids, and again the isomer is chiefly the monounsaturated acid. Dietary recommendations, such as those of the American Heart Association (2), besides calling for a decrease in saturated acids, are now placing greater emphasis on the total unsaturation of the dietary fat, rather than simply on polyunsaturated fatty acids. As a consequence, it is important to determine whether the isomeric structure of an unsaturated acid alters the effect of that acid on the blood lipid levels. The blood lipid levels of subjects who were consuming hydrogenated fats have been measured by a number of investigators. In some instances an elevation of blood cholesterol or triglyceride has been observed (3-10); in
‘From the Miami Valley Laboratories, Gamble Company, Cincinnati, Ohio 45257, University of Pennsylvania Medical School, phia. Pennsylvania 19174.
Nutrition
28:
JULY
1975,
pp.
726-731.
Printed
Procter & and the Philadel-
in U.S.A.
HYDROGENATED TABLE Fatty
I acid
composition
of vegetable
shortenings#{176}-
Saturated Monounsaturated cis trans
25 47
Polyunsaturated
28
33 14
23 5
cis,cis cis,
FAT AND
trans
trans.
trans
Values sold through 0. 1 to 0.3% #{176}
Trace are typical of 95% of such products retail stores in the United States. of the total fatty acids are conjugated
that are b From dienes.
others, the levels of these lipids were unchanged (5, 11-14). In many of these studies, any effect observed could have been due to fatty acid composition, rather than isomeric structure, because the levels of saturated and polyunsaturated acids were changed as well. In some, the level of dietary trans acids was quite low, and any effect could have been so small that it was not measurable. The present study was carried out for the purpose of establishing the effect on blood lipids of the trans acids that are normally consumed in the largest amount, namely, the monounsaturated acids. The use of experimental and control fats of the same fatty acid composition, except for the presence or absence of trans acids, and the inclusion of trans acids at a markedly elevated level overcame the shortcomings of the earlier studies. Materials
PLASMA
part ofTable 2 under the column headed High trans. On the basis of these values, appropriate portions of olive oil, safflower seed oil, and completely hydrogenated soybean oil were blended together and transesterified randomly. The fatty acid composition of this mixture of fats together with the added egg yolk lipids, as recovered from the diet dry mix, is shown in Table 2 under the column marked, High cis. The fatty acid compositions of the two diets were quite similar. The level of geometrical isomers in the high trans fat was determined by analysis of the partially hydrogenated soybean oil component. Absorptivity at 232 nm showed it to contain no more than 0.3% conjugated diene. Lipoxigenase assay (IS) showed the complete absence of cis, cis methylene interrupted double bonds. The contents of cis and trans monounsaturated fatty acids and cis, trans and trans, trans diunsaturated fatty acids were measured by absorption spectroscopy. The method of Allen (16) was used for infrared absorption,, except that the samples were scanned as liquid smears between CsBr plates rather than as solutions in CS2. Raman spectroscopy was carried out according to the method of Bailey and Horvat (17). The infrared and Raman spectroscopy were performed on the partially hydrogenated soybean oil and on the monoand diunsaturated fatty acids isolated from it by preparative gas-liquid chromatography. From these values and from the portion of partially hydrogenated soybean oil that was added to the diet, the isomeric fatty acid content of the high trans fat was calculated; these values are given in the lower part of Table 2. As can be seen from this
TABLE
2 acid composition given in percent Fatty
Subjects The subjects were male inmates County Prison at Holmesburg, ranged in age from 24 to 41 years; 31 years. All subjects were examined found to be in good health with disorders.
of the Philadelphia Pennsylvania. They with the median being before the study and no known metabolic
Diet For this study two diets, one containing a high level of trans monounsaturated fatty acids and the other a comparable level of cis monounsaturated fatty acids, were desired. Aside from this difference in the content of geometrical isomers, the diets were designed to be the same in fatty acid composition and caloric density and in their content of cholesterol, protein, fat, and carbohydrate. The high level of trans monounsaturated fatty acids was provided by soybean oil that had been hy-
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727
drogenated under conditions that maximize the level of this isomer. Since this fat had an undesirable waxy feel in the mouth, nine parts of it was transesterified randomly with one part of safflower seed oil. The dried egg yolk, which was added to the diet as a source of cholesterol, contributed 1 1% of the total fatty acids in the diet. The fatty acid composition of the mixture of these three fats, as isolated from the dry mix that was used in the preparation of the formula diet, is shown in the upper
Fatty weight
and methods
LIPIDS
acid
of diets,
High
cis#{176}
Total 12.0 13.0 58.4 16.3 Isomeric 58
16:0 18:0 18:1 18:2 l8:lcis
cis,
acids 10.9 15.5 53.6 20.0 fatty acids 20 34 9 9 I
16 trans
trans. a
Consisted
trans of:
65
olive
oil,
trans6’
fatty
trans l8:2cis,cis
High
12
safflower
oil.
12
completely hydrogenated soybean oil, Il egg yolk lipids. 6Consisted of 81 partially hydrogenated soybean oil, 9 safflower oil, II egg yolk lipids. ‘Conjugated diene constituted 0.3% of the total fatty acids.
728 table,
MATTSON the
chief difference between the two fats in the diets was the presence (44% of the total acids) or absence of trans double bonds. This was most marked
formula
the
monounsaturated fatty acids; in the high trans fat these acids were of the trans configuration, whereas in the high cis fat the monounsaturated acids were only of the cis configuration. Table 3 shows the composition of the dry mix used for the preparation of the formula diets. The test fat (containing trans isomers) or the control fat (free of trans isomers) constituted 18% of the dry mix, or 35% of the calories. The remaining fat was contributed by the dried egg yolk. The cholesterol in the dry mix resulted in an intake of approximately 500 mg of cholesterol per subject per day. The men also received vitamin-mineral capsules, salt tablets, methyl cellulose wafers, and one small serving of a low fat, low calorie food item per day, such as a raw fruit or vegetable. More detailed descriptions of the components and of the preparation of the formula diets from the dry mixes have been presented (8, 10).
ET AL. TABLE
for the
63%
Group
PEO-I4
All
61
All
37
TDO-2l TD
HC
TD22-49
HT
Number
a
TD-21
of
subjects
the HC group of 16 subjects.
Institution
diet
Changeover to high cis emulsion diet 33 Emulsion diet, high cis fat 17#{176}Emulsion diet, high cis fat 13#{176}Emulsion diet, high trans fat
HC+HT
22-49
Treatment
completing consisted
the
study;
of 17 subjects
and
at
day
the
HT
procedure
The sequence in which the diets were consumed by the subjects is shown in Table 4. During an initial 14-day preexperimental period (period PE), 61 subjects received the normal institution diet. Blood samples were taken on days 2, 3, 7, 10, and 14. A number of individuals chose to drop out of the study and those whose plasma cholesterol level was less than 150 mg/100 ml were discontinued. The 37 men who qualified had an average plasma cholesterol level of 187.8 and triglyceride level of 108.4 mg/ 100 ml. During the subsequent 7 days (period TP), the participants consumed gradually increasing amounts of the formula diet at the expense of the institution diet; the diet containing the high cis fat was used during this period. All 37 subjects consumed solely this high cis formula diet for the subsequent 21 days (period TD 0-21). During the final 4 weeks of the study (period TD 22-49), 17 of the subjects continued to receive the formula diet containing the high cis fat; the remaining 16 participants received the formula diet containing the high trans fat. All of the subjects assigned to the high cis group completed the study, three of the subjects in the high trans group dropped out. The loss of participants subsequent to day TP-0 was for reasons unrelated to the study. Only the measurements obtained on subjects completing the study were used. At each of the four feedings on days TD 0-49, each subject received one-fourth of his daily allotment of the
Composition
No. of subj
TPO-7
group
TABLE
design
Period and days
of
Feeding
4
Experimental
3 of dry
mixes.#{176}g/ 100 g dry
Dried egg whiteb Dried egg yolk Dextrose Test fat Sodium chloridec Flavoring
mix 19.6 3.8 56.7 18.4 0.5 0.4
#{176} Diets contained per 100 g; 18.0 g protein, 20.1 g fat, 54.3 g carbohydrate. 104 mg cholesterol, 477 kcal. One-tenth milligram biotin added to each 100 g of dry mix to inactive avidin. Contained 0.01% potassium iodide.
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formula. Initially, body wt per day. ment
in energy
body weight. each individual weight. Over average gain Blood
sampling
the diet was Subsequently,
intake
was
fed at a level of 33 kcal/kg some individual adjust-
necessary
to maintain
constant
This
continuous adjustment of the intake of essentially maintained their initial body the course of the experiment there was an of0.3 lb (range + 13 to -6). and
anal vses
In all
of the experimental periods, blood samples were drawn twice weekly after an overnight fast. Collections were by Vacutainer tubes containing EDTA. The recovered plasma was analyzed for total cholesterol and triglycerides on the Technicon AutoAnalyzer (18). Besides the usual chemically pure cholesterol and triglyceride standards, a desiccated serum sample (Desiccated Serum, Difco Laboratories, Detroit, Michigan) was used as a reference. Replicate samples of this were analyzed with each set of blood plasma. Over the course of the study, a total of 325 determinations were made on this reference serum; the standard deviation of the analytical methods in mg/ 100 ml was 4.69 for cholesterol and 4.29 for triglyceride.
Results Only the data from subjects who completed the entire study are reported. The average plasma cholesterol and triglyceride levels of the subjects for each of the days that blood was collected are shown in Table 5. For statistical treatment, the values obtained on days TD 2-21, when a-li of the subjects received the formula diet containing the high cis fat, and days TD 3 1-49, when they were receiving either the high cis or the high trans fat, were used. Table 6 shows the average plasma triglyceride and cholesterol values, according to the group to which the subjects were assigned during the final 4 weeks of the
HYDROGENATED TABLE
FAT
AND
PLASMA
729
LIPIDS
S
Average plasma cholesterol and of the mean of subjects receiving
triglyceride an emulsion
levels and standard diet,#{176}’ b mg/ 100
error ml
Cholesterol
Triglyceride high trans group
high cis group
Day
All
a
0 3 7 10 14 17 21
185.4 182.4 179.4 179.2 181.3 183.5 194.9
±
24 28 31
187.1 194.8 190.5
±
35 38 42 45 49
186.5 188.2 191.6 183.5 189.1
±
Group
designation
the mean = S/Vn n = 13 for high
TABLE
6
Average
plasma
± ± ± ±
6.6 6.7 ±6.2 ±
± ± ± ±
5.8 6.2 8.3 6.0 6.2
196.1 190.5 189.8 190.9 184.4
determined
by dietary number 17 for
triglyceride
cholesterol
HC HT HC HT Standard error averages.
#{176}
±
where, n is the trans group and
Group
subject
±
191.6 187.5 182.2 179.6 182.2 187.8 198.1 Subjects 197.7 197.7 190.9±
7.7 7.0 6.6 6.4 6.8 6.0 7.0
and
mean
± ± ± ± ±
± ±
8.4 7.3 8.1 8.1 6.4
± ± ± ±
fat consumed
110.1 107.1 104.0 109.1 101.1 during
days
S is the standard
and
standard
error
Dietary fat
TD2-21 TD 2-21 TD3I-49 TD3I-49 of the
±
of subjects and high cis group.
Period and days
high cis high cis highcis high trans =
S/.,fli
where
n
is
the
study. Separate values are given for the High Trans (HT) and High Cis (HC) groups during days TD 2-21, even though all subjects were receiving the high cis fat at that time. The data were examined in four different comparisons by one-way analysis of variance with no significant differences being found. The similar values for the two groups during period TD 2-21, when they were receiving the same diet, show that the two groups of subjects were comparable. The absence of a time effect is shown by the almost identical values exhibited by the group receiving the high cis fat during both periods TD 0-21 and TD 2 1-49. The introduction of the high trans fat during period TD 2 1-49 did not result in a
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-
subjects fed high cis fat 6.2 110.6 ± 10.1 6.5 107.4 ± 11.6 6.0 105.4 ± 9.4 5.0 106.8 ± 9.2 5.5 lOO.7 ± 10.1 5.3 98.6 ± 11.0 7.9 109.9 ± 9.7 fed high cis or high trans fat 7.4 108.1 ± 11.6 9.4 117.5 ± 9.7 7.1 118.1 ±9.8
±
±
levels
high trans group
high cis group
number
8.0 8.8 7.2 10.6 8.6
± ± ± ± ±
22 to 49. deviation
of the
See
Table
4.
of the daily
mean,#{176}mg/
117.8 95.8 96.6 98.4 94.7 89.0 103.5
±
98.2 121.5 124.4
±
121.2 98.6 115.5 95.8 94.8
±
± ± ± ± ± ±
± ±
± ± ± ±
8.8 7.2 7.9 8.0 7.1 6.8 8.7 10.7 12.4 15.4 9.8 7.7 15.3 11.4 7.3
6Standard values
error
100 ml
Plasma triglyceride
Plasma cholesterol
104.6 ± 9.4 96.2 ± 6.8 108.2±8.0 108.2 ± 9.2
184.1 ± 6.7 186.2 ± 5.6 188.6±6.2 189.4 ± 6.9
of subjects
and
S is the
of
for the subjects,
standard
deviation
of the
change in the plasma lipid levels relative to that attained by the same group (HT) of subjects while they were on the high cis fat (period TC 0-21). Moreover, the levels realized on the high trans fat did not differ from those of the subjects who were receiving the high cis fat during the same period (TD 2 1-49).
Discussion The study reported here was designed so that the only dietary variable was the presence or absence of trans acids. Grasso et al. (9) and Erickson et al. (10) used a similar experimental design in that the hydrogenated and unhydrogenated fats in each of their
730
MATTSON
studies had a similar total fatty acid composition. However, the fats fed in those earlier studies were of the type that are customarily used in the preparation of margarines, shortenings, and salad oils, and consequently did not contain as high a level of trans acids. In the present investigation, the level of trans acids, particularly the monounsaturated trans acids, was greatly magnified by hydrogenating soybean oil under conditions that would maximize their formation. The use of this as the principal dietary fat resulted in over 60% of the monounsaturated fatty acids having a trans configuration. Even at this greatly magnified level of feeding, we, like Grasso et al. (9) and Erickson et al. (10), found the level of plasma cholesterol’ and triglyceride to be the same regardless of the isomeric structure of the dietary fatty acids. In most of the other reported studies, the process of hydrogenation was used either to decrease the polyunsaturated fatty acid or to increase the satuiated fatty acid levels of a fat. The hydrogenated and unhydrogenated fats then were fed to the subjects to determine the effects of such changes in the composition of dietary fatty acids on blood lipid levels. Since it is now recognized that saturated fatty acids elevate plasma lipids and polyunsaturated fatty acids decrease them, it is not possible to attribute any specific effect to the trans acids of the fats that were fed in those early studies. An example of such a difficulty can be seen in two of three experiments reported by Anderson, Grande, and Keys (5). In these, the subjects received either an unhydrogenated fat or the same fat after partial hydrogenation. As a consequence, the control and experimental diets differed in their fatty acid compositions. They attempted to compensate for this variable by applying their equation (19) that predicts the blood cholesterol levels that should have been attained as the result of consuming diets with those fatty acid compositions. In one experiment, the blood cholesterol levels that were observed were essentially identical with those that were calculated. Thus, trans isomers evidently had the same effect as did the cis isomers. In another experiment, the hydrogenated fat gave a
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ET AL. lower blood cholesterol level than that predicted by the fatty acid composition of the diet; a result that could be interpreted as showing trans isomers to be more effective than cis isomers in lowering this blood lipid component. In the third experiment described in the same report (5), the fatty acid compositions ofthe hydrogenated and unhydrogenated fats were the same except for the presence or absence of trans acids. In this instance, the hydrogenated fat resulted in a relative elevation of the blood cholesterol level. It is significant that the hydrogenated fat used in the third experiment had an unusual fatty acid composition. As in our study hydrogenation had been carried out under special conditions to maximize the content of trans fatty acids, but the particular conditions employed by them also resulted in relatively high contents of conjugated dienes and of trans, trans diunsaturated fatty acids; the sum of these two was 10% of the total fatty acids. The levels of both of these materials in the fat of their third experiment were considerably higher than they were in the high trans fat used for our study (Table 2) or that found in the hydrogenated fats used for margarines, salad oils, or shortenings (Table 1). The possibility exists then that the singular effects reported by Anderson, Grande, and Keys (5) may have been the result of either the trans. trans diunsaturated acids, or the conjugated dienes. A preliminary report by Vergroesen (20) also indicated that specific fatty acids may have an unique effect. This brief paper stated that the consumption of elaidic acid, trans9-octadecenoic acid, resulted in an increase in the level of blood cholesterol. Since this was a preliminary report, few experimental details are given making it difficult to relate Vergroesen’s observations to ours. The effects of particular fatty acid may be reflected also in the studies of Mishkel and Spritz (21). The fat used by those investigators was not hydrogenated, but rather it was linoleate that had been isomerized with SO2 and subsequently fractionated. As a consequence of these treatments, the predominant fatty acid was trans, trans-dienoic acid. Consumption of this fat as 20 or 40% of the
HYDROGENATED
FAT
calories resulted in a marked elevation of plasma triglycerides. The high trans fat that we used was obtained by hydrogenation and contained only 1 % of the trans. trans isomer of linoleic acid. We did not see an elevation of the plasma triglyceride level. On the other hand, the hydrogenated fat that contained appreciable amounts of trans, trans acids and that was fed in the third experiment of Anderson, Grande, and Keys (5) resulted in a small increase in plasma triglycerides. Thus there is the possibility that certain particular isomers of fatty acids may alter plasma lipid levels. However, this does not appear to apply to the hydrogenated fats that are customarily consumed in the United States such as margarines, shortenings, and salad oils (7-10).
ci
Our thanks to the following individuals for their assistance-C. Theuring for the preparation of the fat, E. Anderson and W. Tettenhorst for analysis of the plasma, A. J. FehI and R. P. Oertel for determination of the isomer levels, and S. E. Michaels for statistical analysis of the data.
References I. CALL, D. L., AND A. M. SANCHEZ. Trends in fat disappearance in the United States l909-65. J. Nutr. 93: Suppl. I; 1, 1967. 2. Primary Prevention of the Atherosclerotic Diseases. Report of Inter-Society Commission for Heart Disease Resources. Circulation 42: ASS, 1970. 3. BEVERIDGE, J. M. R., W. F. CONNELL, 0. A. MAYER AND H. L. HAUST. Plant sterols, degree of unsaturation, and hypocholesterolemic action of certain fats. Can. J. Biochem. Physiol. 36: 895, 1958. 4. WILCOX, E. B., AND L. S. GALLOWAY. Serum cholesterol and different dietary fats. J. Am. Dietet. Assoc. 38: 227, 1961. 5. ANDERSON, J. T., F. GRANDE AND A. KEYS. Hydrogenated fats in the diet and lipids in the serum of man. J. Nutr. 75: 388, 1961. 6. MORSE, E. H., E. BICKNELL, E. P. LEWIS, S. B. MERROW AND C. A. NEWHALL. Relation of dietary fats to blood lipids in young men. J. Am. Dietet. Assoc. 41: 323, 1962. 7. BEVERIDGE, J. M. R., AND W. F. CONNELL. The
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