AN ABSTRACT OF THE THESIS OF
LISA SCHWEICKHARDT HOLDEN for the degree of Master of Science
in
Foods and Nutrition
Title:
presented on
March 1 8, 1976
LECITHIN SUPPLEMENTATION AND FREE AND
ESTERIFIED CHOLESTEROL IN PLASMA
Abstract approved:
Dr. Elisabeth/S. Yearick
The effect of oral lecithin supplementation on plasma lipids
was investigated.
Thirty-one healthy middle-aged men received 7.2
gm soy lecithin daily for two months.
Blood was drawn both before
and after supplementation and analyzed for total and esterified
cholesterol, total and individual phospholipids, total lipids, triglycerides, and lecithin:cholesterol acyltransferase (LCAT) activity.
Information on dietary intake, physical activity, smoking habits and
relative weight was also obtained.
The proportion of cholesterol ester increased significantly
(p < 0. 01) with supplementation, as did LCAT activity (p < 0. 025).
None of the other lipid parameters changed during the experimental
period.
A positive correlation was found between LCAT activity and
the ratio of lysophosphatidyl choline to phosphatidyl choline.
Triglycerides were positively correlated with smoking and percent
of desirable weight, and negatively correlated with activity level.
Higher proportions of cholesterol ester were associated with more
vigorous physical activity and with lower relative weights.
Lecithin Supplementation and Free and Esterified
Cholesterol in Plasma
by
Lisa Schweickhardt Holden
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed March 18, 1976
Commencement June 1976
APPROVED:
Professor of Foods and Nutrition
in charge of major
Head of ©qpartment of Foods and Nutrition
Dean of Graduate School
Date thesis is presented
March 1 8, 1976
Typed by A & S Bookkeeping/Typing for Lisa Schweickhardt Holden
ACKNOWLEDGEMENTS
As nay advisor, Dr. Elisabeth Yearick has offered me an
invaluable combination of challenge and encouragenaent.
Her untiring
support of my research, especially during the manuscript preparation, is deeply appreciated.
Many thanks are also due to Dr. Margy
Woodburn and Dr. Lorraine Miller for their encouragement and
interest in my studies.
I appreciate very much the cooperation of Joan Halvorson in
organizing and initiating the experiment and for the use of her
phospholipid data.
Thanks also to Gil Won Song for her LCAT data.
Mr. Robert Lowry was very helpful in giving insights into the
difficulties of cholesterol ester separation, and generously offered
the results of his personal experimentation with column chromatography.
Spencer Chester and Jason Johnston both helped greatly in
the statistical analysis of my data.
Mary Yocom has spent many hours critiquing and typing this
manuscript and many mo re hours being a loving and stabilizing
friend.
Special thanks to Robert Holden who has laughed and cried
and prayed with me, listened and suggested.
He has offered
scientific criticism and his bounding enthusiasm.
Without his
support, this research could not have been accomplished.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
Plasma Lipids
Plasma Lipids and Heart Disease
Normal Plasma Lipid Levels
Lecithin
Lecithin Supplementation
Interactions Between Lecithin and Plasma
Lipids
3
3
3
5
12
13
15
MATERIALS AND METHODS
Experimental Design
Subjects
Dietary Analysis
Additional Information Collected
Blood Lipid Determinations
Collection of Blood Samples
Analysis of Total Cholesterol
Analysis of Cholesterol Ester
Determination of Total Lipids
Phospholipid Determination
Analysis of Triglycerides
Assay of Lecithin:Cholesterol Acyltransferase
Statistical Treatment
19
19
20
20
21
22
22
23
23
25
25
26
26
26
RESULTS AND DISCUSSION
Plasma Lipid Concentrations
Mean Plasma Lipids
Changes in Plasma Lipids
Correlations Between Lipid Parameters
Dietary Information
Mean Dietary Intakes
Correlations Between Dietary and Lipid
Parameters
Plasma Lipids and Additional Variables
Conclusions
27
27
27
31
.34
38
38
39
40
42
Pase_
SUMMARY
44
BIBLIOGRAPHY
46
APPENDIX
55
LIST OF TABLES
Table
Page
1.
Plasma lipids in healthy populations.
2.
.Description of subjects.
28
3.
Plasma lipids and lipid ratios before and
after lecithin supplementation
29
Significant correlations among plasma lipids
and between plasma lipids and other factors.
35
Significant correlations between lipids and
percent change in plasma lipids.
37
Dietary factors most pertinent to lipid
metabolism.
39
4.
5.
6.
6
LIST OF APPENDIX TABLES
i.
ii.
iii.
iv.
v.
vi.
Complete description of subjects.
55
Plasma lipids and lipid ratios of individual
subjects, before and after supplementation
(mg/100 ml).
57
Correlation coefficients among plasma lipids
and between plasma lipids and other factors.
59
Correlation coefficients between lipids and
other factors, and percent change in plasma
lipids.
61
Paired_t-statistics to evaluate changes with
supplementation.
62
Dietary intake of subjects compared to
National Research Council Recommended
Dietary Allowances, 1974.
63
LECITHIN SUPPLEMENTATION AND FREE AND
ESTERIFIED CHOLESTEROL IN PLASMA
INTRODUCTION
The epidemiological association between hyperlipemia and
cardiovascular disease has prompted a search for means to lower
plasma lipids.
One agent which has received a great deal of
attention recently is lecithin.
Popular writers have claimed that
soy lecithin, when used as a food supplement, is effective in
reducing plasma lipids and even in dissolving cholesterol deposits
(Williams, 1971).
Recommended dosages in the popular literature
vary from 6 gm/day (Davis, 1965) to 24 gm/day (Morrison, 1971).
Results of the scientific research done on lecithin supplementation in humans have been contradictory.
Morrison (1958) obtained
a significant reduction in total cholesterol when he fed 36 gm of
lecithin daily to hypercholesteremic patients.
Other investigators,
using 0.6-2. 4 gm/day, were unable to observe any changes in the
plasma lipids of coronary patients (Butler et al. , I960; Enticknap,
1962) and type II hyperlipoproteinemic patients (ter Welle et al. ,
1974).
Takeuchi and Yamamura (1973) have suggested that supplemental lecithin might stimulate the enzyme responsible for plasma
cholesterol esterification, lecithinrcholesterol acyltransferase
(LCAT).
Higher proportions of cholesterol ester and increased
LCAT activity have been observed in healthy subjects compared to
coronary patients (Soloff, Rutenberg and Lacko, 1973).
Therefore, this study was undertaken to examine the effect of
lecithin supplementation on total cholesterol concentrations and on
cholesterol esterification.
Healthy subjects were used and a
lecithin dosage was chosen that avoided the extremes of previous
studies and was more typical of the dosages recommended to the lay
public.
This was part of a larger study in which total lipids, total
phospholipids, individual phospholipids, triglycerides and LCAT
activity were also measured.
In addition, information on dietary
intake, activity patterns, smoking habits and body weight was
obtained.
LITERATURE REVIEW
Plasma Lipids
Plasma Lipids and Heart Disease
Despite recent advances made in determining the pathogenesis
of cardiovascular disease, the underlying causes of the disease
remain poorly defined.
For this reason, epidemiological studies
which can help to identify risk factors are of great importance.
Elevated plasma lipids have been frequently correlated with heart
disease.
Plasma cholesterol is the lipid fraction most often associated
with heart disease.
Doyle (1966) found that the probability of
developing cardiovascular disease after the age of 40 increased as
the plasma cholesterol increased over 275 mg/100 ml.
In an
analysis of the Framingham study, cholesterol concentrations of
over 260 mg/100 ml were associated with an increasing risk of
heart disease (Dawber, Moore and Mann, 1957).
There is
evidence that even these limits may be too high.
Fredrickson et al.
(1973) suggested that patients under age 55 with cholesterol values
that exceed 220 mg/100 ml warrant a physician's closer scrutiny;
such patients may be predisposed to hyperlipoproteinemia, and
hence, to heart disease.
4
Some investigators have found triglycerides to be a more
important indicator of heart disease than cholesterol.
Albrink,
Meigs and Man (1961) reported that the incidence of heart disease
was much higher with elevated plasma triglycerides than with elevated
cholesterol.
Hatch et al„ (1966) also found high triglyceride levels
associated with heart disease.
It has been pointed out, however,
that persons with the highest concentrations of individual lipids
often have multiple lipid elevations.
When triglycerides, cholesterol
and lipoprotein levels were examined in a twelve-year follow-up of
the Framingham study, no single lipid fraction appeared to increase
the risk of heart disease.
The highest morbidity was found with
multiple lipid abnormalities (Kannel, Castelli, and McNamara, 1967).
Abnormalities of lipid ratios have also been observed in heart
disease.
Morrison, in 1952, found that the phospholipid to choles-
terol ratio was higher in normal subjects than in coronary patients.
The '. lysophosphatidyl choline (LPC) to phosphatidyl choline (PC)
ratio was lower than normal in the plasma of patients with type IV
hyperlipoproteinemia (Kunz, Matt and Hackl, 1970) and atherosclerosis (Gillett and Besterman, 1975).
Many other risk factors for cardiovascular disease have been
identified.
Smoking more than 20 cigarettes a day (Hammond, 1966;
Kahn, 1966), being more than 20% overweight, having an abnormal
electrocardiogram, having increased blood pressure, or having a
family history of heart disease are all factors associated with an
increased probability of developing heart disease (Doyle, 1966;
Kannel et al. , 1967).
Normal Plasma Lipid Levels
Plasma lipid concentrations vary a great deal between
individuals comprising a healthy population.
Table 1 summarizes
values that have been reported by several different investigators.
Some of the observed variability can be attributed to differences in
methodology and technique, or to day-to-day fluctuations.
However,
factors such as sex, age, exercise, smoking habits, stress, obesity
and diet have also been found to affect the lipid levels of normal
people.
Before the age of 20, the cholesterol concentrations of boys and
girls are similar.
Men between the ages of 20 and 50 tend to have
higher cholesterol values than women.
surpass men.
After 50, however, women
This is apparently due to the hyperlipemia associated
with menopause (Lopez-S, Krehl and Hodges, 1967).
Adlersberg
et al. (1956) reported a similar pattern in phospholipid values.
In
contrast, Bdttiger (1973) found no significant difference between
total phospholipid or PC values of men and women.
LPC, however,
was considerably lower in women at all ages than in men.
Table 1.
Plasma lipids in healthy populations.
Total
Cholesterol
(mg/100 ml)
Reference
Ahrens and Kunkel, 1949
Cholesterol
Esters
(% of total)
Phospholipids
(mg/100 ml)
Triglycerides
(mg/100 ml)
Total
Lipids
(mg/100 ml)
25 men and women
185
70
219
636
Deuell, 1955
118 men and women
152
70
165
530
Lindholm, 1956
men, aged 40-59
180
63
250
1101
Adlersberg et al.,
1956
230 men, aged 38-57
240
Okey et al., 1960
30 men. aged 50f
201
71
252
Svanborg and
Svennerholm, 1961
62 men, aged 16-35
192
67
208
84
609
Bleileret al., 1963
11 men, aged 39-59
272
72
126 men, aged 40-55
(low exercise)
224
201
114
660
Marumoto, 1970
19 men, aged 18-19
138
163
73
464
MacDonald, 1972
10 men, aged 18-21
181
185
104
Bottiger, 1973
30 men, aged 38-63
263
278
122
Miller, 1973
11 men and women
186
73
Billimoria et al.,
1975
58 men and women aged
30-70 (non-smokers)
215
80
215
82
Hoffman, Nelson
and Goss, 1967
285
64
788
O
7
The plasma lipids of healthy men tend to increase with age.
Cholesterol and phospholipids begin to increase dramatically at age
20 and may stabilize by age 33 (Adlersberg et al. , 1956).
Lindholm
(1956) reported that the rise in total lipids, phospholipids and
cholesterol continued into the middle years.
This was confirmed
by Lopez-S et aL (1967) who found that cholesterol may continue to
rise until age 45 or 50.
Lipid levels have stabilized by 50 years and
may decrease after 60 (Lopez-S et al. , 1967).
This decrease
probably reflects the greater longevity of individuals with lower lipid
levels.
There is some disagreement concerning the response of the
cholesterol ester fraction to age.
The general consensus is that the
proportion of cholesterol ester is quite stable in healthy people,
ranging between 66 and 75% of the total (Cook, 195 8; Oser, 1965).
The results of Adlersberg et al. (1956) agree with this position;
no variation was found in cholesterol esters with age.
On the other
hand, Lopez-S et al. (1967) observed a trend towards a decreased
proportion of esterified cholesterol with increasing age.
Since total
cholesterol tends to increase with age, it is possible that the
phenomenon which Lopez-S attributed to age could really be a result
of the increased total cholesterol which often accompanies aging.
This was in fact the case in a study done on African subjects whose
total cholesterol varied "widely.
As the total cholesterol increased,
8
the proportion of esterified cholesterol decreased.
Total cholesterol
had a much greater influence on the percent ester than did age
(Leonard, Shaper and Jones, 1965).
Exercise has also been found to affect lipid levels.
In a study
on age-matched men, in which activity was classified by occupation,
a highly significant inverse relationship was found between the
intensity of exercise and serum cholesterol (Stulb et al. , 1965).
Similar results were reported when a dynamic exercise program was
introduced into a sedentary life-style.
Campbell (1965) found that
exercise involving fast, continuous muscular movement was effective
in lowering cholesterol in male freshmen participating in a ten-week
program.
Medical students experienced a significant decrease in
triglycerides and pre- (3-lipoproteins and a moderate decrease in
cholesterol on a seven-week exercise program (Lopez-S, 1974).
Air Force officers, 40 to 55 years old, who exercised daily for a
year had lowered triglyceride, cholesterol and total lipids (Hoffman,
1967).
Cigarette smoking does not appear to have an immediate effect
on blood cholesterol.
Page et al. (1959) had 20 adults smoke two
cigarettes, inhaling deeply.
There were no differences in cholesterol,
triglyceride or phospholipid concentrations in blood drawn before and
after inhalation.
There was also no difference between smokers and
non-smokers in their response to the trial.
Very different results
were found when the lipid levels of chronic smokers and non-smokers
were compared.
Significant elevations in phospholipids, triglycerides
and cholesterol were found in men who smoked over 20 cigarettes
per day.
These differences were not observed between smoking and
non-smoking women (Billimoria et al. , 1975).
Dietary composition has been found to profoundly affect plasma
lipid levels.
Dietary fat increases the amount of exogenous
cholesterol that will be absorbed.
This is because the secretion of
bile acids, necessary for cholesterol absorption, is stimulated by fat
in the diet (Goodhart and Shils, 1973).
In one study, serum choles-
terol increased in rats when fat was included in the diet, despite a
constant cholesterol intake (Swell et al. , 1955).
The nature of the dietary fat is also important.
When saturated
fats (S) in synthetic diets were replaced with polyunsaturated fats
(P), significant decreases in total lipids, total cholesterol, triglycerides and phospholipids were found (Okey et al. , I960; MacDonald,
1972).
The proportion of cholesterol ester was largely unaffected
by manipulation of the P/S ratio (Okey et al. , I960).
Some inves-
tigators have been able to quantify changes in lipid levels.
Changes
in serum cholesterol were correlated with the square root of the
iodine value of the new fat (Keys, Anderson and Grande, 1965).
Ahrens et al. , (1957) found that serum levels of cholesterol and
phospholipids varied inversely with the iodine value of the dietary
10
fat.
Results like these, obtained using synthetic diets, must be
interpreted with caution, but studies using natural foods confirm
these general trends.
People who adhered to the American Heart
Association fat-controlled diet experienced a significant decrease in
cholesterol, triglycerides and p-lipoproteins.
Although the total
fat content of the new diet was the same as that of previous diets1
(41% of calories), the change to a diet with polyunsaturated fat
resulted in a change of P/S ratio from 0. 4 to 1.6 (Wilson et al. ,
1971).
Some investigators have been concerned about the fate of the
cholesterol which disappears from the serum as a result of a polyunsaturated diet.
Connor et al. (1969), using a sterol balance
technique, found that the fecal sterols increased when the iodine
value of formula diets was increased.
These results were confirmed
by Nestel et al. (1975) who reported that the increased sterol
excretion occurred during the first three weeks after the change to a
polyunsaturated diet; after three weeks, a steady state was reached.
Hellstrom and Lindstedt (1966) found no increased sterol excretion
when they examined the bile of subjects on a polyunsaturated diet.
However, they waited three weeks before collecting bile and may
therefore have missed the excretion stage.
There is general agreement that dietary cholesterol affects
plasma cholesterol, but there is some disagreement about whether
11
this is a significant phenomenon.
Keys, Anderson and Grande (1965)
found that plasma cholesterol responded minimally to changes in
cholesterol levels in synthetic diets.
In contrast, Connor, Stone
and Hodges (1964) found a significant decrease in cholesterol when
the cholesterol content of a natural diet was reduced.
Adherents to
the American Heart Association diet experienced a decrease in serum
cholesterol proportional to the decrease in dietary cholesterol
(Wilson et al. , 1971).
The more dramatic results found by Connor
and Wilson and their colleagues may be due to their use of natural
diets.
The cholesterol of egg yolk was found to be more hyper-
cholesteremic than equal amounts of pure cholesterol dissolved in
oil, even when the quantity and quality of fat was controlled in the
diets (Connor, Stone and Hodges, 1964).
Although the effect of obesity on the incidence of heart disease
is well documented (Doyle, 1966; Kannel et al. , 1967), the effect on
lipid concentrations is less clear.
Subscapular skin-fold thickness
was highly correlated to plasma cholesterol in young men (Tanner,
1951).
However, Thomas (I960) found no difference in serum
cholesterol levels of young males when compared with weight,
weight corrected for build, or radiographic outer-fat shadow
measurements.
An additional factor which may affect lipid levels is stress.
Thomas and Murphy (1958) examined first-year medical students
12
immediately after an examination considered to be the most stressful
one in medical school, and again during a less stressful period.
They found the mean cholesterol level at exam time to be 10%
higher than when less academic pressure was present.
Some workers have suggested that seasonal change may be
responsible for some of the variability of blood lipids.
Thomas,
Holljes and Eisenberg (1961) studied cholesterol concentrations in
young male prisoners over a year's time.
Cholesterol tended to be
higher in November, December and January and lower in the
warmer months.
No differences in activity patterns or stress
seemed to be responsible.
However, the possibility that fat intake
had increased during the winter months was suggested.
In another
study, the cholestdrol levels of men were highest from March to
May (Bleiler et al. , 1963).
Possible changes in dietary intake with
seasonal change were not examined in the study.
No significant
differences were found when total lipids, cholesterol, phospholipids
and triglycerides were measured in middle aged men at different
times of year (Hoffman et al. , 1967).
It seems likely that seasonal
differences which have been observed are secondary to other changes.
Lecithin
The term "lecithin" is commonly used in two different ways.
It is used to refer to the specific phospholipid, phosphatidyl choline,
13
and also to the general class of phospholipids which are soluble in
most organic solvents except acetone.
In the following pages,
lecithin will refer to the extractable class of phospholipids, while
phosphatidyl choline (PC) will be referred to by name.
It is
interesting to note that the most abundant phospholipid in lecithin is
PC, composing 29-38% (Erdahl, Stolyhwo and Privett, 1973;
Halvorson, 1976).
Lecithin is found naturally in many foodstuffs and is extracted
commercially from soybeans.
Many claims have been made about
its effectiveness as a dietary supplement.
It is purported to dissolve
cholesterol deposits (Williams, 1971), to lower cholesterol by
enhancing fat metabolism and transport, and to promote feelings of
well-being (Morrison, 1971).
Adelle Davis (1965) describes lecithin
as helpful in dealing with such varied conditions as obesity, multiple
sclerosis and nephritis.
Dosages recommended to the lay public
vary from 6 gm/day (Davis, 1965) to 24 gm/day (Morrison, 1971).
Lecithin Supplementation
When lecithin has been used to treat animals and humans,
varied results have been reported in the scientific literature.
Intravenous infusions of lecithin given to rats caused an increase in
both plasma cholesterol and plasma phospholipids (Friedman and
Byers, 1956).
The increase in cholesterol occurred even when the
14
liver was absent, implying that cholesterol was diffusing out of other
parts of the body (Friedman, Byers and Rosenman, 1957).
However,
Howard and Patelski (1974) examined the aortas of atherosclerotic
baboons after an infusion of lecithin and found no regression of
aortic lesions as judged by cholesterol content.
Since these studies
were all done on animals, their direct applicability to human beings
is questionable.
One of the early lecithin studies using humans was done by
Morrison in 1958.
Twenty-one hypercholesteremic patients were
given 36 gm lecithin daily for three months.
They had all been on a
low fat (less than 25 gm/day) diet for a year or more and had been
treated unsuccessfully with other hypocholesteremic agents.
Six of
the patients quit the study because of intolerance to the lecithin
dosage.
The 15 who remained experienced significant decreases in
plasma cholesterol and total lipids and an increase in phospholipids.
On the other hand, a commercial phospholipid preparation
(lipostabil ) was unsuccessful in changing 10 blood parameters when
given to 114 coronary patients for a period of more than two years
(Enticknap, 1962).
However, the dosage was considerably lower
than that given by Morrison (1958), 0. 6-1. 8 gm/day.
Butler et ah
(I960) also reported no change in plasma cholesterol or phospholipids
Aspro-Nicholas, Slough, Bucks, England.
15
when lipostabil was given to 50 coronary patients.
Unfortunately
these investigators did not report the amount of phospholipid given.
Most recently, ter Welle et al. (1974) have given lecithin supplements
to 12 persons with type II hyperlipoproteinemia. After 8 months of
supplementation with 1. 2-2.4 grams lecithin per day, no changes in
blood lipids were found.
different studies.
day.
It is difficult to draw conclusions from these
The dosages varied from 0.6 gm to 36 gm per
The subjects' previous medical history and diets are additional
variables.
Since these studies were done on persons with abnormal
lipid conditions, it is impossible to know what effect lecithin
supplementation would have on normal subjects.
Interactions Between Lecithin and Plasma Lipids
Other research has suggested possible mechanisms by which
lecithin might affect lipid metabolism.
One of the earliest theories
relating to lecithin supplementation was based on the phospholipid/
cholesterol ratio in serum.
It was found that serum turbidity was
related to the low phospholipid content of serum and not to total
lipid content (Boyd, 1937).
Coronary patients had a greater frequency
of serum turbidity than did controls (Horlick, 1954) and a lower
phospholipid/cholesterol ratio (Morrison, 1952).
Morrison (1958)
was able to significantly increase the phospholipid/cholesterol ratio
of 15 hypercholesteremic patients by feeding them 36 gm of lecithin
16
daily.
Studies using smaller doses, however, have been unsuccessful
in modifying the phospholipid/cholesterol ratio (Butler et al. , I960;
Enticknap, 1962; ter "Welle et al. , 1974).
It has been theorized that lecithin may stimulate the lecithin:
cholesterol acyltransferase (LCAT) reaction in plasma.
LCAT is
an enzyme which catalyzes the transfer of a fatty acid from the C-2
position of phosphatidyl choline (PC) to the hydroxyl group of free
cholesterol, forming lysophosphatidyl choline (LPC) and esterified
cholesterol.
Cholesterol is esterified preferentially from high
density lipoproteins compared with other lipoproteins (Glomset,
1968).
The LCAT reaction is responsible for the formation of
almost all the cholesterol ester in plasma (Glomset, 1970).
Coronary
patients have been found to have a lesser ability to esterify
cholesterol than controls.
This was shown by a lower rate of serum
esterification during in vitro incubation and a lower fraction of
cholesterol ester in the serum (Soloff, Rutenberg and Lacko, 197 3).
Animal studies have indicated that feeding lecithin causes an increase
in serum cholesterol esterification (Takeuchi and Yamamura, 1973).
One theory, therefore, is that supplemental lecithin could protect
against heart disease by increasing the serura LCAT activity.
Another theory is that LCAT may promote clearance of free
cholesterol from peripheral tissues.
Murphy (1962) showed that
when serum and erythrocytes were incubated together, there was a
17
flux of free cholesterol out of the red blood cells.
The amount of
free cholesterol lost from the cells was related to the amount of
cholesterol ester that appeared in the serum.
This flux is presumably
an equilibrium response, the free cholesterol from erythrocytes
replacing that which is esterified by the LCAT reaction.
If a
similar equilibrium existed between serum and arterial tissues,
then the increased proportion of cholesterol esters formed by LCAT
in serum could help remove free cholesterol from arteries.
Ruten-
berg and Soloff (1971) tested this hypothesis by incubating healthy
arterial segments in serum.
into the serum.
They observed a flux of free cholesterol
This theory could explain the studies in which
intravenous infusions of lecithin caused an increase in plasma
cholesterol and apparently a diffusion of cholesterol out of peripheral
tissues (Friedman and Byers, 1956; Friedman, Byers and Rosenman,
1957).
Increased LCAT activity in plasma would have the effect of
increasing the lysophosphatidyl choline (LPC) and decreasing the
phosphatidyl choline (PC).
Some recent research has focused on
changes in the LPC/PC ratio in atherosclerosis.
Portman et al.
(1970) found that the LPC/PC ratio and LCAT activity were higher
in the plasma of monkeys with nutritionally-induced atherosclerosis
than in the controls.
However, Mohan and Chakravarti (1975)
compared the phospholipid profiles of rhesus monkeys with
18
spontaneous and with diet-induced1 atherosclerosis.
They found the
LPC/PC ratio of monkeys with spontaneous atherosclerosis to be
much lower than the non-diseased group, whereas induced atherosclerosis caused only a slight decrease.
The work of Gillett and
Besterman (I 975) confirms these findings.
Their data indicate that
the LPC/PC ratios of humans with chronic or acute ischemic heart
disease, peripheral arterial disease or myocardial infarctions were
considerably lower than normal controls.
The differences between
the Portman et al. (I 970) study and the ones done by Mohan and
Chakravarti (1975) and Gillett and Besterman (I 975) seem to be the
result of differences between natural and induced atherosclerosis.
19
MATERIALS AND METHODS
Experimental Design
Dietary supplements of lecithin were given to a group of
middle-aged men for a two-month period beginning in January, 1975,,
Each subject received 7. 2 gm of lecithin per day in the form of
gelatin capsules.
2
Two capsules containing 1.2 gm soy lecithin in
soybean oil were taken three times a day.
This dosage was selected
because it is typical of recommended supplemental dosages.
Blood samples were drawn both before and after the period of
supplementation to determine the effects of lecithin on blood lipids.
Duplicate sampling was done to minimize the effects of normal daily
fluctuations in plasma lipids; two blood samples were taken within
three days of each other before supplementation began and two more
were taken at the end of the study.
Each sample was analyzed
separately, but values were reported as averages of the two samples
obtained in the same week.
The experimental plan was approved by the Committee for the
Protection of Human Subjects in accordance with the DHEW guidelines (DHEW, 1971).
2
"Natural Needs1' Soya Lecithin, Western Wholesalers, Portland,
Oregon.
20
Subjects
Thirty-one men between the ages of 38 and 56 participated i n
the study.
By limiting the subjects to men of one age group, it was
hoped that differences in lipid patterns due to age and sex would not
mask possible changes attributable to lecithin.
The men were
healthy, with no history of heart disease and no known abnormality
of lipid metabolism.
lipid metabolism.
They were not taking any drugs which alter
The subjects agreed to maintain constant patterns
of dietary intake and physical activity.
Participants were not chosen
by random selection; instead, men volunteered who were interested
in the research topic.
Dietary Analysis
To obtain information about their usual dietary habits, the
subjects were asked to record three-day dietary intakes, both at
the beginning and at the end of the study.
Each three-day period
consisted of two weekdays and one weekend day, since food consumption is often very different on weekends.
The diets were
analyzed by computer, using a nutrient data bank compiled at Ohio
State University, essentially from information in Watt and Merrill
(1963).
The following nutrients were computed in the diets: Calories,
protein, total fat, saturated fat, linoleic and oleic acids, calcium,
21
Vitamin A, thiamin, riboflavin, niacin, ascorbic acid, and iron.
In addition, the cholesterol content of the diets^was calculated from
the table compiled by Feeley, Criner and Watt (1972).
Dietary
information was reported as an average of the two three-day records.
Additional Information Collected
Activity levels were determined from information supplied by
the subjects about the nature of their work, their planned exercise
and sports, and their mode of travel to and from work.
The subjects
were assigned to one of the activity groupings defined by Bogert,
Briggs and Galloway (1973): 1) sedentary, 2) light activity, 3)
moderate activity, and 4) strenuous activity.
The energy demands
of a subject's occupation determined the group into which he was
initially placed.
In addition, if he undertook 45 minutes of strenuous
exercise at least three times a week, he was placed in the next
higher category.
Percent of ideal weight was calculated from the subjects'
height and weight according to the Metropolitan Life Insurance
Company tables (Keys and Grande, 1973).
assumed to be of medium frame.
The subjects were
If a subject was overweight, the
percentage of ideal weight was calculated from the highest number of
the suggested range.
used.
If he was underweight, the lowest number was
22
Subjects were classified according to their smoking habits.
If a person smoked 20 cigarettes or more per day, he was called a
"smoker".
Those who smoked infrequently or not at all were "non-
smokers".
Blood Lipid Determinations
Collection of Blood Samples
Blood samples were collected in the morning after an overnight
fast.
Twenty milliliters of venous blood were drawn into Vacutainers
containing ethylenediaminetetraacetic acid (EDTA) as anti-coagulant.
The blood was stored on ice until it could be returned to the laboratory.
Hemoglobin and hematocrit were determined once at the
beginning of the study and once at the end to give an indication of
overall nutritional status.
Hemoglobin was measured by the
cyanomethemoglobin method (Oser, 1965), and hematocrit was
measured according to the method described by Richterich (1969).
The blood was centrifuged at 35, 000 RPM for 35-40 minutes.
The plasma was removed and stored in capped vials at -10 C until
further analyses could be carried out.
23
Analysis of Total Cholesterol
Total cholesterol was determined on the Technicon AutoAnalyzer according to the method of Block, Jarrett and Levine
(1966).
This method is based on the reaction of steroids having the
5-ene, 3(3-ol grouping with concentrated sulfuric acid and ferric
chloride in glacial acetic acid (color reagent).
(1:20 in isopropanol) were prepared in advance.
Plasma extracts
The extracts were
mixed on the AutoAnalyzer with preheated color reagent (95 C) and
the absorbance measured at 550 nm in a tubular flow cell.
Total
cholesterol was calculated from the regression equation determined
from standards.
Analysis of Cholesterol Ester
Esterified cholesterol was first separated chromatographically
from free cholesterol and then measured on the AutoAnalyzer.
In
preparation for chromatographic separation, 0. 5 ml of plasma was
extracted into 10 ml of isopropanol.
Two 3-ml aliquots of the extract
■were dried under purified nitrogen gas in a 50 C water bath.
were used for duplicate cholesterol ester analyses.
These
In addition, 2-ml
of the same extract were stored in an AutoAnalyzer sampling cup
for total cholesterol analysis.
24
Lowry's
3
adaption of the column chromatography method of
Fillerup and Mead (1953) was modified for use in our laboratory.
A
glass blower was asked to construct glass columns having a twenty
milliliter reservoir above a 10x70 mm tube.
The columns were dried
thoroughly and filled with silicic acid (100-200 meshBio-Sil A) which
had been heated overnight at 120 C
The columns were tapped and
tamped to insure uniform packing and then rinsed with hexane.
The
dried isopropanol extracts were dissolved in hexane and transferred
quantitatively to the column.
The cholesterol ester was eluted, first
with 10 ml of 2. 5% diethyl ether in hexane, followed by 5 ml of 10%
diethyl ether in hexane.
New solvent was added only after the
previous solvent had passed through the column.
Eluates were
collected in tubes and dried under nitrogen gas in a 50 C water bath.
For analysis on the AutoAnalyzer, the cholesterol ester was
redissolved in two milliliters of isopropanol and transferred to
sampler cups.
Each cholesterol ester sample (in duplicate) was run
on the AutoAnalyzer in juxtaposition to the corresponding total
cholesterol sample.
This eliminated any slight variability that would
arise from differences in standards or in instrument behavior.
Concentrations were calculated in the same way as total cholesterol,
and the cholesterol ester was calculated as percent of total.
3
Robert Lowry, personal communication.
25
Precision and accuracy were examined by running several
consecutive assays of Hyland control serum.
The assigned choles-
terol ester value of this lot of Hyland serum was 78% of total
cholesterol.
The modified method described above yielded a mean
of 78% + 1.6% (p < 0.05).
Determination of Total Lipids
Total lipids were extracted with chloroform and methanol
(2:1 v/v) according to the method of Chiu (1969).
The extract was
reduced under purified nitrogen gas in a 50 C water bath, transferred quantitatively to a tared one-milliliter volumetric flask, and
dried under nitrogen.
Total lipids were determined gravimetrically.
The lipid extracts were stored at -10 C.
Phospholipid Determination
The chloroform/methanol lipid extract was used for phospholipid analysis.
Total phosphorus was measured according to the
method of Lowry et al. (1954) as modified by Hawthorne, Smith
and Pescadore (1963).
Individual phospholipids were separated by
thin-layer chromatography, the spots removed and the phospholipid fractions quantified by the total phosphorus method.
4
Analysis done by Ms. Joan Halvorson.
4
26
Analysis of Triglycerides
Triglycerides were analyzed by the micromethod of Van Handel
5
and Zilversmit (1957), using the chloroform/methanol lipid extract.
Assay of LecithimCholesterol Acyltransferase
Plasma LCAT activity was determined according to the method
of Stokke and Norum (1971).
Statistical Treatment
The paired t-test was used to compare the means of plasma
lipid concentrations before and after supplementation.
A one-tailed,
unpaired_t-test was used to determine if the change in LCAT activity
was significantly greater than zero.
In addition, correlation
coefficients were determined for all pertinent combinations of
variables (Snedecor and Cochran, 1973).
5
Analysis done by Dr. Elisabeth Yearick.
Analysis done by Ms. Gil Won Song.
27
RESULTS AND DISCUSSION
A description of the participants is summarized in Table 2.
In addition, the data for individual subjects appears in Appendix
Table i.
The subjects were, on the average, slightly overweight
(4% above ideal weight).
Three individuals were less than 100% of
their ideal weight for height while fourteen were more than 100%;
only one subject was 25% above ideal weight and could be considered
obese.
Twenty-one of the thirty-one subjects had light physical
activity patterns.
Four were sedentary, three were moderately
active, and three engaged in strenuous activity.
The predominance
of low activity levels is undoubtedly due to the fact that 26 of the
subjects were university professors and therefore confined to an
office most of the day.
All of the subjects had hemoglobin and
hematocrit values within the ranges found in healthy men (Oser,
1965; Wintrobe, 1961).
Plasma Lipid Concentrations
Mean Plasma Lipids
The means and ranges for plasma lipids are found in Table 3,
while individual data are detailed in Appendix Table ii.
Total
cholesterol averaged 206 mg/100 ml before and 210 mg/100 ml
28
Table 2.
Description of subjects.
Age
Relative Weight13
Activity Level
a
Mean
Range
46
38-56
104%
2
Hemoglobin gm/100 ml
15.9
Hematocrit %
45
94-129%
1-4
14.1-17.3
40-48
a.
Four out of thirty-one subjects were smokers; the rest, nonsmokers.
b.
Percent of desirable weight for height from the Metropolitan
Life Insurance Company tables (Keys and Grande, 1973).
c.
Classification of activity levels: 1 = sedentary, 2 = light activity,
3 = moderate activity, 4 = strenuous (Bogert, Briggs and
Galloway, 1973).
29
Table 3. Plasma lipids and lipid ratios before and after lecithin supplementation.
Variables
No.
Mean
S.D.
Range
Total Cholesterol
(mg/100 ml)
Before
31
206
36
122-266
After
31
210
37
102-288
Percent Cholesterol Ester
Before
31
66
2
60-70
After
31
67
3
59-74
Total Lipids
(mg/100 ml)
Before
31
655
164
287-1282
After
31
645
133
372-1146
Phospholipids
(mg/100 ml)
Before
31
181
32
89-268
After
31
187
27
132-263
Triglycerides
(mg/100 ml)
Before
31
91
43
22-209
After
31
106
59
40-285
Phospholipid/Cholesterol
Before
31
0.88
0.12
0.72-1.18
After
31
0.90
0.11
0.73-1.29
Before
12
0.14
0.05
0.06-0.23
After
12
0.12
0.03
0. 08-0. 18
(%)
LPC/PC
LCAT Activity
b
Change as percent of initial value.
Data supplied by Gil Won Song.
12
Percent
change3
2%
2%
(p<.01)
-2°/o
3%
16%
2%
-9%
11%
(p<0. 025)
30
after supplementation.
Other investigators have obtained similar
means for middle-aged men (Okey et al. , I960; Hoffman, Nelson
and Goss, 1967).
One subject had considerably lower cholesterol
concentrations than the other subjects: 122 and 102 mg/100 ml.
Eight of the subjects had concentrations greater than the 220 mg/100
ml limit proposed by Frederickson et al. (1973), although for only
two of them were the concentrations consistently above the 260 mg/
100 ml level associated with increased risk of heart disease (Dawber,
Moore and Mann, 1957).
Sixty-six percent of the cholesterol was in the ester fraction
prior to the study and 67% at the conclusion.
Although these values
are at the lower end of the normal range given by Oser (1965), other
studies report similar means (Lindholm, 1956; Svanborg and
Svennerholm, 1961).
The mean concentrations of total lipids, phospholipids and
triglycerides are comparable to those reported in the literature
(Hoffman, Nelson and Goss, 1967; Billimoria et al. , 1975).
It is
interesting to note that the subject with the lowest cholesterol concentrations also had extremely low levels of the other lipid fractions.
The eight subjects who had high cholesterol also tended to have
elevated concentrations of the other lipids.
Pertinent ratios were calculated from the raw data.
The
LPC/PC ratio decreased from an average of 0. 137 to 0. 124 during
31
the experimental period (Table 3).
Both of these ratios are similar
to those found in healthy, middle-aged men (B'ottiger,-, 1973;
Gillett and Besterman, 1975).
averaged 0. 88 and 0. 90.
Phospholipid/cholesterol ratios
When phospholipid/cholesterol ratios are
calculated from other studies, a ratio greater than one is almost
always obtained (Okey et al. , I960; Marumoto, 1970; BSttiger,'.
1973).
In addition, Morrison (1952) found that healthy controls had
phospholipid/cholesterol ratios greater than one, whereas coronary
patients had ratios less than one.
However, a ratio of 0. 90 can be
calculated from the data of Hoffman, Nelson and Goss (1967).
Hoffman's study involved middle-aged Air Force officers with
sedentary jobs in the Pentagon.
These subjects may be more com-
parable to the subjects in the present study, most of whom also have
sedentary and prestigious positions.
Since all the men, both in the
Hoffman study and in the present study, are apparently healthy it
is possible that the calculated phospholipid/cholesterol ratio is
normal when variables of sex, age and occupation are held constant.
Changes in Plasma Lipids
The degree that each lipid fraction changed during the period
of lecithin supplementation, expressed as the percent of the initial
concentration, is also found in Table 3.
Although most of the lipid
fractions increased during the experimental period, only one
32
increased significantly (the _t-statistics are found in Appendix Table
v).
The 2% increase in the proportion of cholesterol ester was
highly significant (p < 0.01).
This finding suggested that lecithin
supple mentation had stimulated the cholesterol esterifying
mechanism.
Soloff, Rutenberg and Lacko (1973) had demonstrated
an association between cholesterol esterification and LCAT activity
in men who had varying levels of these two factors.
Data were
available on LCAT activity in plasmas of 12 of the subjects in the
present study.
As shown in Table 3, there was a significant
(p < 0.025) increase of 11% in LCAT activity.
This suggested that
the phosphatidyl choline contained in soy lecithin was absorbed in
sufficient quantities to stimulate the enzyme for which it is a
substrate.
Takeuchi and Yamamura (1973) also found increased
LCAT activity in rats after lecithin feeding.
It is interesting to
note in the present study that there is a range of 10% in cholesterol
ester values before supplementation but a range of 15% afterwards.
This increased variability implies that individuals respond differently
to the stimulus of lecithin supplementation.
Although most of them
experienced an increase in cholesterol ester, eight showed a decided
decrease.
The concentrations of total cholesterol, total lipids, phospholipids and triglycerides did not change significantly with supplementation.
Morrison (1958) was able to demonstrate significant changes
33
in cholesterol, phospholipids and total lipids by feeding his subjects
36 gm of lecithin daily.
On the other hand, other investigators have
found no change in these lipids after lew-level (< 2 gna/day) lecithin
supplementation (Enticknap, 1962; ter Welle et al. , 1974).
An
intermediate dose of 7.2 gm/day was used in the present study.
It
seems likely, therefore, that the effect of lecithin on these lipids is
dose-related and that 7. 2 gm/day was not sufficient to elicit the
response Morrison obtained using 36 gm/day.
Neither LCAT nor
cholesterol ester, the two parameters which changed significantly
with supplementation, have been measured in human lecithin studies
(Morrison, 1958; Butler et al. , I960; Enticknap, 1962; ter Welle
et al. , 1974).
The results of the present study suggest that LCAT
and cholesterol ester measurements should be included in future
investigations.
There are other factors which may account for the lack of
significant change in total cholesterol, phospholipids and triglycerides.
The hypercholesteremic effect of stress (Thomas and
Murphy, 1958) may have counteracted the possible cholesterollowering effect of lecithin.
The beginning of the study coincided with
the beginning of the school term, a relatively calm time for university
professors.
The final blood samples were taken during the last two
weeks of the term, when the academic staff is under a great deal of
pressure.
The stress factor was therefore increasing throughout the
34
course of the study.
Although the effect of seasonal variation on
lipids was inconclusive in previous studies (Thomas et al. , 1961;
Bleiler et al., 1963; Hoffman et al. , 1967), the possibility that the
change in season during this study affected the lipid patterns cannot
be excluded.
Correlations Between Lipid Parameters
Significant correlations among plasma lipids are presented in
Table 4.
All of the correlation coefficients appear in Appendix
Tables iii and iv.
There were strong positive correlations between
total lipids, total cholesterol, phospholipids and triglycerides, both
before and after supplementation.
Other investigators have also
noted the tendency for elevations in one lipid to be associated with
increases in other lipids (Kannel, Castelli and McNamara, 1967;
Lindholm, 1956).
The cholesterol ester fraction was negatively correlated with
triglycerides and total lipids.
This supports the work of Lopez-S
et al. (1967) who observed that the percent cholesterol ester tended
to decrease as other lipids increased with age.
Another study
indicated that the proportion of cholesterol ester varied inversely
with total cholesterol concentration (Leonard, Shaper and Jones,
1965).
Although high concentrations of cholesterol tended to be
associated with lower proportions of cholesterol ester, this
Table 4.
Significant correlations among plasma lipids and between plasma lipids and other factors.
Total
cholesterol
Before After
Total
Cholesterol
^Cholesterol
Ester
Before After
Total
Lipids
Before
After
Phospholipids
Before After
Triglycerides
Before
After
Phospholipid/
Cholesterol
Before
After
Dietary
Cholesterol
Before
After
% Cholesterol Before
Ester
After
Total
Lipids
Before
After
Phospholipids
Before
After
Triglycerides
Before
Dietary P/S
Activity Level
% Desirable Weight
Smoking
Correlation coefficients were significant at the p <0.05 level and positive (+) or negative (-), or they were significant at the p < 0. 01 level
and positive (++) or negative (—).
36
relationship was not statistically significant.
Perhaps more distinct
patterns would have emerged with a larger sample size.
Significant correlations between the plasma lipids and the
percent change following lecithin supplementation are shown in Table
5.
Increases in triglycerides correlated with increases in total
lipids and phospholipidso
The change in phospholipids also cor-
related with the change in total lipids.
This further supports the
observation that lipid fractions tend to rise and fall together.
Higher
initial concentrations of total lipids, triglycerides, total cholesterol
and phospholipids were associated with a greater decline in total
lipids as a result of supplementation.
Initial phospholipid concen-
trations were also negatively correlated with the change in phospholipids and in triglycerides.
This indicates that the initial concen-
tration of these lipids influenced the direction of change undergone
by certain other lipids.
The change in LCAT enzyme activity was positively correlated
with the change in LPC/PC ratio (p < 0.05).
This correlation
occurred despite the fact that mean LCAT activity increased significantly with supplementation whereas the mean LPC/PC ratio
decreased slightly.
The direction of LPC/PC change was not con-
sistent, perhaps because of the small number of samples used for
this analysis (n = 12).
Nevertheless, when a change did occur, it
was associated with a change in LCAT activity in the same direction.
Table 5.
Significant correlations between plasma lipids and percent change in plasma lipids.
Total
Cholesterol
Total
Cholesterol
% Cholesterol
Ester
Before
After
Percent Change
Total
Phospholipids
lipids
—
++
Total Lipids
Before
—
Phospholipids
Before
~
Triglycerides
Before
-
LPC/PC
Before
LPC/PC
-
% Cholesterol
Ester
After
Phospholipid / Before
Cholesterol
After
Triglycerides
~
++
-
+
++
++
-
% Change in
Phospholipids
% Change in
Triglycerides
% Change in LPC/PC
Correlation coefficients were significant at the p <0.05 level and positive (+) or negative (-), or they were significant at the p <0. 01 level
and positive (++) or negative (--).
LCAT
38
It is to be expected that LCAT activity and the LPC/PC ratio would
be related since an increase in LCAT activity would be followed by
an increase in LPC and a decrease in PC concentrations.
Indeed,
parallel changes in LCAT and LPC/PC ratios have been reported
previously (Portman et al. , 1970).
Dietary Information
Mean Dietary Intakes
Average dietary intakes for individual subjects are presented
in Appendix Table vi.
Twenty-two of the thirty-one subjects com-
pleted dietary records both at the beginning and end of the study.
Six participants returned only one diet record.
The reported intakes
were compared to the National Research Council Recommended
Dietary Allowances (1974) for middle-aged men.
All of the diets
were adequate in Calories, protein, iron, niacin and ascorbic acid.
Calcium and vitamin A intakes were low (less than 67% of the RDA)
in three cases,
thiamine was low in four cases and one subject had
a low intake of riboflavin.
A qualitative examination of the diets
suggested that a limited consumption of milk and vegetables was
responsible for these low-nutrient intake's.
The means and ranges of the dietary factors most pertinent to
lipid metabolism are found in Table 6.
Fat as percent of Calories
averaged 38% and 418 mg cholesterol were consumed per day.
The
average American eats slightly more fat and cholesterol than these
subjects, with 40% of the Calories as fat and 533 mg dietary
39
cholesterol per day (National Diet-Heart Study Final Report, 1968;
Friend, 1967).
The P/S ratio in the typical American diet is 0.25
(National Diet-Heart Study, 1968) compared to a mean of 0. 37 in
the present study.
Because high cholesterol and saturated fat
intakes have been linked with elevated plasma cholesterol, the Food
and Nutrition Board (1972) has issued a statement advising that
persons in coronary risk groups increase the P/S ratio and decrease
the cholesterol content of their diets.
It seems, therefore, that the
subjects in this study had diets slightly less atherogenic than the
American norm.
Table 6.
Dietary factors most pertinent to lipid metabolism.
Mean
Dietary fat as percent
of Calories
P/S ratio
Dietary cholesterol
(mg/day)
Range
38+6%
2 3-48%
0.37+0.22
0.12-1.15
418+156
200-783
Correlations Between Dietary and Lipid Parameters
Very few significant correlations were observed between
dietary intake and plasma lipids (Table 4).
Higher cholesterol
intakes were associated with lower phospholipid and total lipid concentrations after supplementation.
In contrast to previous studies
40
(Connor, Stone and Hodge, 1964; Wilson et aL , 1971), no relationship
between dietary cholesterol and plasma cholesterol was found.
It
was also expected, based on the findings of Okey et al. (I960) and
MacDonald (1972), that the amount and quality of dietary fat would
affect plasma lipids.
However, no significant correlations were
observed between plasma lipids and the fat as percent of Calories
or the P/S ratio.
This is in part due to the wide variation that
existed in the self-chosen diets, whereas the investigators cited
above used a metabolic ward or more rigidly controlled diets.
Other
variables, such as activity levels, body weight and stress, were not
controlled in the present study and may have influenced the plasma
lipid concentrations sufficiently to mask the effect of the dietary
parameters.
Plasma Lipids and Additional Variables
The average activity level and relative weight are given in
Table 2.
Significant correlations between these variables and plasma
lipids are presented in Table 4.
More vigorous physical activity
■was associated with lower initial triglyceride concentrations and
with higher proportions of cholesterol ester.
This confirms the
work of Lopez-S (1974) and Hoffman (1967) who have reported lower
triglycerides in subjects participating in daily exercise programs.
However, these investigators also found that exercise decreased
41
plasma cholesteroL
the present study.
No such hypocholesteremic effect was noted in
The significant relationship observed between
cholesterol ester and physical activity is not comparable to previous
studies since neither Lopez-S nor Hoffman measured cholesterol
ester.
Triglyceride concentrations and the final total lipid concentration correlated positively with relative body weight.
The initial
proportion of cholesterol ester, on the other hand, was lower in
those whose relative weight was high.
Other investigators have
measured only cholesterol when evaluating the effect of relative
weight on plasma lipids.
Tanner (1951) established a highly signi-
ficant relationship between cholesterol and the thickness of supscapular subcutaneous tissue.
On the other hand, Thomas (I960) found no
difference in the cholesterol concentrations of young men with different relative weights.
The present study shed no light on this
controversy; no correlation was observed between total cholesterol
and body weight.
Triglyceride was the only plasma lipid which correlated
significantly With smoking habits.
The four subjects who smoked all
had elevated triglyceride concentrations.
The hypertriglyceridemic
effect of smoking has been demonstrated by Billimoria et al. (1975).
These investigators also found significantly increased concentrations
of phospholipids and total cholesterol in chronic smokers.
Although
42
cholesterol and phospholipids tended to be higher in the smokers in
this study, the relationship was not statistically significant.
This study indicates that triglycerides and the proportion of
cholesterol ester are more closely associated with activity, body
weight and smoking than is the total cholesterol.
It has been demon-
strated that elevated triglycerides have predictive value for heart
disease (Albrink, Meigs and Man, 1961; Hatch et al. , 1966).
It has
also been suggested that a decreased proportion of cholesterol ester
raay be associated with atherosclerosis (Soloff, Rutenberg and Lacko,
1973).
For these reasons, it seems important that cholesterol ester
and triglyceride analyses be included in studies examining the salutory
effects of exercise, weight reduction and decreased smoking.
Conclusions
In this study, dietary supplementation with soy lecithin was
effective in increasing the proportion of cholesterol ester and the
LCAT activity in plasma.
It is impossible to know if these changes
improved latent atherosclerotic conditions since there is no way to
examine the arteries of human subjects.
There was also no way of
knowing if the changes brought about by lecithin supplementation
decreased the likelihood of developing heart disease, since this was
a short-term study with only healthy subjects.
Nevertheless, the
results of other studies which have compared healthy and diseased
43
subjects or have been done with animals suggest that an increase in
cholesterol ester and LCAT activity may indeed be beneficial.
More research needs to be done to clarify the interaction
between lecithin and plasma lipids.
Since the action of lecithin
appears to be a dose-related phenomenon, it might be helpful to use
different dosages of lecithin. A larger sample size, stricter controls
on dietary intake, physical activity and smoking habits could help to
decrease the variability of the data and make it easier to identify
which effects can be attributed to lecithin.
The results of this study
demonstrate that cholesterol ester analysis should be carried out
routinely in investigations dealing with interactions of plasma lipids
with dietary and supplementary components or with descriptive
parameters.
44
SUMMARY
The effect of lecithin supplementation on free and esterified
cholesterol in plasma was studied.
Thirty-one apparently healthy
middle-aged men were given 7.2 gm/day "Natural Needs" soy
lecithin.
Blood samples were drawn both before and after the two-
month period of supplementation.
The blood was analyzed for total
cholesterol, cholesterol ester, total lipids, total phospholipids,
individual phospholipids, triglycerides and LCAT enzyme activity.
Information was also collected about the subjects' diets, physical
activity, smoking habits and relative body weight.
Changes in plasma lipids with supplementation were evaluated.
Significant increases were observed in the proportion of cholesterol
ester (p < 0. 01) and in the LCAT activity (p < 0. 025).
It was sug-
gested that the increase in plasma enzyme activity might be due
to the increase of one of the substrates for the enzyme, lecithin, in
the diet.
Since LCAT is largely responsible for cholesterol esteri-
fication in plasma, the increased proportion of cholesterol ester
was explained by the increased LCAT activity.
None of the other
lipid parameters changed significantly during the experimental
period.
This may be because lecithin has no effect on these para-
meters or may be due to the contradictory influences of uncontrolled
variables.
45
Correlation coefficients were calculated on all pertinent combinations of variables.
Strong positive correlations were observed
among all lipid parameters, excepting cholesterol ester as percent
of total.
The cholesterol ester fraction varied inversely with most
other lipids.
The change in LCAT activity was accompanied by
parallel changes in the LPC/PC ratio.
Physical activity was
negatively correlated with the initial triglyceride concentration and
positively correlated with percent cholesterol ester.
Triglycerides
were higher with greater body weight, whereas the initial proportion
of cholesterol ester was lower.
with triglycerides.
Smoking was positively correlated
46
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APPENDIX
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Appendix Table i (continued).
Subject
Number
Age
Relative
Weight3
Activity
Levelb
Smoking
Habits
Hemoglobin
(gm/lOOml)
non-smoker
non-smoker
non-smoker
15.6
16.5
15.3
45
48
44
15.9
45
35
56
100
4
36
37
48
45
100
100
2
3
Mean
46
104
2
Hematocrit
(%)
Percent of desirable weight for height (from tables prepared by Metropolitan Life Insurance Company, Keys and Grande, 1973).
b
Activity ratings: 1 = sedentary, 2 = light activity, 3 = moderately active, 4 = strenuous activity (Bogert, Briggs and Calloway, 1973).
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57
Appendix Table ii (continued).
Subject
Number
Total
CholeiSterol
Before
After
Chole:sterol
Ester (%)
Before
After
Total
Lipiids
Before
After
Total
P-lipi ds
Before After
Tri-»
glycerides
Before After
P-lipid/
Chole;sterol
Before
After
LPC/PC
Before After
LCAT
Percent Change
35
36
37
195
255
166
203
263
160
66
66
66
70
72
69
550
702
564
645
796
606
182
192
163
172
218
154
56
62
60
56
65
84
0.93
0.75
0.98
0.85
0.83
0.96
0.227
0.135
0. 148
0. 184
0. 119
0.075
- 4
Mean
S.D.
206
36
210
37
66
2
67
3
655
164
645
170
181
32
187
27
91
43
105
59
0.88
0. 12
0.90
0. 11
0. 137
0.05
0. 123
0.03
—
10
21
11
CO
Table iii. Correlation coefficients among plasma lipids and between plasma lipids and other factors.
Total
Cholesterol
Before
After
.889
Total Cholesterol Before
After
% Cholesterol
Ester
Before
After
Total Lipids
Before
After
Phospholipids
Before
After
Triglycerides
Before
After
LPC/PC
Before
After3
% Cholesterol
Ester
Before After.
-.213
-.129
Total Lipids
After
Before
Phospholipids
Before
After
Triglycerides
Before After
LCAT3
Phospholipid/
Cholesterol
Before
After
-.133
-.032
.660
.625
.645
.632
.745
.803
.699
.778
.566
.544
.455
.316
-.197
-.072
-.305
-.055
-.534
-.634
.489
-.389
-.380
-.390
-.352
-.211
-.104
-.081
-.259
-.507
-.266
-.290
-.494
.096
.335
-.012
.122
.101
-.265
.915
.706
.638
.694
.726
.755
.723
.637
.692
.093
.007
-. 199
-.153
.522
.621
.526
.340
.628
.396
-.204
-.647
-.049
. 113
-.112
-.229
.220
-.139
-.194
.719
-.148
-.048
-.157
-.028
.569
.475
-.096
.125
N.C.
N.C.
N.C.
N.C.
-.284
-.159
.213
.069
N.C.
N.C.
N.C.
N.C.
-.334
.337
N.C.
N.C.
N.C.
N.C.
-.190
-.249
-.220
-,208
.016
.026
-.024
-.188
. 177
. 175
N.C.
.214
.119
-.141
-.107
-.097
.095
-.149
-.175
-.187
-. 136
-.259
-.288
N.C.
-.035
-.052
-.181
-.260
.001
.018
-.331
-.384
-.054
-.376
-.205
-. 199
N.C.
.220
-.059
-.276
-.272
.396
.362
-. 194
-. 144
-.127
-.197
-.457
-.294
N.C.
.078
. 144
Smoking
.331
.281
-.298
-.245
.238
.335
.346
.240
.477
.521
N.C.
.000
-.119
% Ideal Weight
.092
.047
-.440
-.240
.333
.358
. 177
.168
.570
.465
N.C.
.148
.070
Fat as % Calories
b
P/S Ratio
Diet Cholesterol
Activity Level
b
Number of subjects evaluated equaled 31 for each correlation except a (n= 12) and b(n == 28).
N. C. means Not Correlated.
60
Table iii (continued).
LPC/PC
LPC/PC Before3
.553
Fat as % Calories
P/S Ratio
b
Diet Cholesterol
Activity Level
Smoking
b
b
a
, b
P/S
Ratio
N.C.
-.267
b
Diet
Cholesterol
N.C.
Activity
Level
N.C.
Smoking
N.C.
% Ideal
Weight
N.C.
.361
-.226
.206
.258
-.436
.343
-.001
-.180
-.056
.097
-. 181
-.332
-.313
.312
Number of subjects evaluated equaled 31 for each correlation except a (n = 12) and b (n = 28).
N.C. means Not Correlated.
61
Table iv.
Correlation coefficients between lipids and other factors, and percent change in plasma
lipids.
Percent Change
Total
Cholesterol
% Cholesterol
Ester
Total
Lipids
Phospholipids
Triglycerides
LPC/PC3
Before
After
-.187
.272
.038
.075
-.392
-.368
-.270
-.285
-.294
-.468
.171
. 118
% Cholesterol Before
Ester
After
.117
.239
-.320
.670
. 154
. 118
.166
-. 146
.203
-.275
-.384
.134
Total
Cholesterol
Total
Lipids
Before
After
-.015
.004
-.087
-.057
-.577
-.225
-.246
-. 144
-.252
-.132
N.C.
N.C.
Phospholipids
Before
After
.184
. 178
.066
-.217
-.516
-.266
-.693
.216
-.492
-.038
.231
-. 181
Before
After
a
.011
-.209
. 146
-.290
-.416
-. 146
-.252
. 130
-.276
.337
N.C.
N.C.
.326
.242
N.C.
-.083
N.C.
.673
Before
After
.576
-.300
.138
-.376
-.245
.371
-.633
.765
-.372
.803
-.061
-.603
.010
.087
-.357
-. 114
N.C.
N.C.
.434
.200
N.C.
N.C.
-.679
. 179
-. 101
-.032
.039
-. 127
.052
N.C.
.083
.018
-.039
.090
.017
N.C.
-. 123
.025
.017
-.271
-.088
N.C.
.019
.051
. 184
.036
.125
N.C.
Smoking
-.090
-.005
.035
-. 140
.110
N.C.
% Ideal Weight
-.036
.121
-.132
-.099
-.057
N.C.
.154
-.055
-. 122
-.485
-.082
-.007
-.301
-.473
.307
.418
.504
N.C.
.615
-.362
Triglycerides
LCAT Activity
P-lipid/
Cholesterol
Befor|a
After*
b
Fat as % Calories
b
P/S Ratio
b
Diet Cholesterol
LPC/PC
Activity Level
Total Cholesterol
% Cholesterol Ester
Total Lipids
Phospholipids
Triglycerides
N.C.
Number of subjects evaluated equaled 31 for each correlation except a (n = 12) and b (n - 28).
N.C. means Not Correlated.
62
Table v.
Paired_t-statistics to evaluate changes with supplementation.
No.
_t-statistic
Significance
Total Cholesterol
31
-1. 185
N.S.
Percent Cholesterol Ester
31
-2.963
p<0„01
Total Lipids
31
1. 148
N.S.
Phospholipids
31
-0. 773
N.S.
Triglycerides
31
-1.828
N.S.
Phospholipid/Cholesterol
31
-0.512
N.S.
LPC/PC
12
1.244
N, S,
12
2.540
p< 0.025
LCAT Activity
*
Unpaired _t-test, one-tailed.
Table vi. Dietary intake of subjects compared to National Research Council recommended dietary allowances, 1974 (values are the average of
two three-day dietaries unless otherwise indicated).
Subject
Number
1
2
3'
4
5
6
8
9
10
12
13
14
15
a
16
17
20a
23
24
25a
26
28a
29a
30
31
33
35
36
37
Calories
% RDA
Protein
% RDA
72
94
145
149
127
170
160
190
128
175
164
169
192
152
168
169
162
111
154
168
187
244
184
226
184
156
172
236
138
150
63
102
83
122
80
77
88
84
92
76
106
84
86
74
80
128
121
128
89
130
116
101
115
139
93
74
Fat as %
Calories
Calcium
% RDA
Iron
% RDA
36
40
44
38
29
41
48
99
127
140
114
118
160
142
113
133
136
135
147
157
171
207
134
122
144
157
181
248
140
189
373b
138
152
250
185
99
37
27
37
41
38
42
38
32
37
23
42
46
41
35
43
40
45
34
48
32
32
42
82
172
126
146
100
42
104
96
191
105
80
139
159
121
90
104
137
234
167
149
104
66
69
142
140
158
values from these individuals are taken from only one dietary.
Vitamin A
% RDA
74
80
131
175
64
116
114
289
144
95
130
100
146
180
182
92
216
117
174
335
82
S3
230b
82
60
116
19 7b
112
Thaimin
% RDA
Riboflavin
% RDA
54
100
96
66
96
136
85
77
84
1325b
64
94
86
122
105
177
68
144
114
1383b
79
270
316b
106
108
216
344b
50
60
97
96
152
119
159
105
138
112
1116b
141
91
110
131
153
164
90
102
128
876b
161
232
317b
106
105
176
330b
128
Niacin
% RDA
103
139
92
109
141
130
112
142
122
754b
123
103
114
168
92
103
122
128
106
489b
104
176
304b
154
190
194
264b
120
Ascorbic
Acid % RDA
111
205
247
168
354
398
302
154
260
1536b
136
278
230
406
815b
522
251
434
279
2144b
172
446
802b
112
294
242
99 8b
170
Figures include dietary intake plus vitamin-sapplementation.
P/S
0.24
0.32
0.30
0.26
0.66
0.20
0.51
0.34
0.26
0.24
0.20
0.30
0.32
0.29
1. 15
0.85
0.28
0.37
0.27
0.24
0. 12
0.40
0.66
0.24
0.45
0.32
0.33
0.26
Cholesterol
mg'/day
310
410
200
320
208
640
342
309
548
591
361
332
478
244
264
296
466
652
615
724
783
356
397
331
360
466
328
371
00