European Journal of Clinical Nutrition (2001) 55, 88±96
ß 2001 Nature Publishing Group All rights reserved 0954±3007/01 $15.00
www.nature.com/ejcn
A stearic acid-rich diet improves thrombogenic and atherogenic
risk factor pro®les in healthy males
FD Kelly1, AJ Sinclair1*, NJ Mann1, AH Turner2, L Abedin1 and D Li1
1
Department of Food Science, RMIT University, Melbourne, Australia; and 2Medical Laboratory Science, RMIT University, Melbourne,
Australia
Objective: To determine whether healthy males who consumed increased amounts of dietary stearic acid
compared with increased dietary palmitic acid exhibited any changes in their platelet aggregability, platelet
fatty acid pro®les, platelet morphology, or haemostatic factors.
Design: A randomized cross-over dietary intervention.
Subjects and interventions: Thirteen free-living healthy males consumed two experimental diets for 4 weeks
with a 7 week washout between the two dietary periods. The diets consisted of 30% of energy as fat (66% of
which was the treatment fat) providing 6.6% of energy as stearic acid (diet S) or 7.8% of energy as palmitic
acid (diet P). On days 0 and 28 of each dietary period, blood samples were collected and anthropometric and
physiological measurements were recorded.
Results: Stearic acid was increased signi®cantly in platelet phospholipids on diet S (by 22%), while on diet P
palmitic acid levels in platelet phospholipids also increased signi®cantly (8%). Mean platelet volume, coagulation
factor FVII activity and plasma lipid concentrations were signi®cantly decreased on diet S, while platelet
aggregation was signi®cantly increased on diet P.
Conclusion: Results from this study indicate that stearic acid (19 g=day) in the diet has bene®cial effects on
thrombogenic and atherogenic risk factors in males. The food industry might wish to consider the enrichment of
foods with stearic acid in place of palmitic acid and trans fatty acids.
Sponsorship: Grant from Meat Research Corporation, Australia and margarines donated by Meadow Lea Foods
Ltd, Australia.
Descriptors: stearic acid; palmitic acid; platelets; fatty acids; haemostatic factors; lipoproteins; platelet aggregation; mean platelet volume
European Journal of Clinical Nutrition (2001) 55, 88±96
Introduction
The relationship between dietary fatty acids and blood
cholesterol levels was reported in the late 1950s when
Keys et al (1957) found that saturated fatty acids (SFA)
from 12 to 18 carbon atoms were potent cholesterol-raising
agents. Subsequently, it was found that the predictive
equations for dietary fatty acids on plasma cholesterol
levels were inadequate when cocoa butter was used
*Correspondence: AJ Sinclair, Department of Food Science, RMIT
University, GPO Box 2476V, Melbourne, Victoria 3001, Australia.
E-mail: [email protected]
Guarantor: AJ Sinclair.
Contributors: FDK, AJS, NJM and AHT initiated the study. FDK prepared
the drafts of the paper, did dietary analysis, collected data, helped in
laboratory assays and did the statistical analysis. AJS selected the study site,
supervised the project and secured the funding. LA and DL helped in
laboratory assays. All authors contributed to the drafts of the paper.
Received 13 June 2000; revised 6 October 2000;
accepted 9 October 2000
(Hegsted et al, 1965; Keys et al, 1965). This was attributed
to the stearic acid in the cocoa butter. Since then, various
predictive equations incorporating individual saturated fatty
acid contributions to plasma cholesterol changes show
stearic acid as neutral (Kris-Etherton & Mustad, 1994).
The risk of coronary events however is not solely
dependent on plasma cholesterol levels. Carefully conducted autopsy studies have demonstrated that thrombotic
obstruction of the coronary artery is involved in most cases
of sudden cardiac death (Davies & Thomas, 1984). Arterial
thrombosis involves clot formation based around existing
plaque and arterial lesions with the ®rst step involving
platelet aggregation, followed by ®brin formation through
activation of the coagulation pathways. Any factor leading
to increased platelet activity or up-regulation of the coagulation cascade will thus increase the risk of thrombotic
events.
Early studies suggested that stearic acid was prothrombotic based on three levels of evidence. Firstly, Connor
Stearic acid-rich diet
FD Kelly et al
(1962) reported that stearic acid was particularly effective at
shortening thrombus formation time during in vitro studies
using citrated blood and sodium salts of fatty acids. Secondly, Renaud and Gautherton (1975) reported that diets
rich in stearic acid were associated with increased coagulation in New Zealand rabbits. This change was speculated to
result from an enhanced activity of platelet factor 3 (PF3)
due to an increase in stearic acid in the platelet phospholipids. Thirdly, Renaud et al (1978) found that saturated fat
and especially stearic acid were signi®cantly correlated with
clotting activity and platelet aggregation in several population comparisons in man. These data alerted researchers to
potentially negative effects of stearic acid on thrombotic
factors and resulted in several short-term studies being
conducted in humans (Tholstrup et al, 1994, 1996; Schoene
et al, 1992; Mutanen & Aro, 1997; Dougherty et al, 1995).
However, few of these have examined more than one
measure of thrombosis tendency. Because there are often
interactions involving more than one component of the
haemostatic system, there is no single screening test
which can be used to identify a thrombotic tendency.
Therefore a range of screening tests should be adopted to
detect markers known to be associated with cardiovascular
risk (Giddings & Yamamoto, 1995).
The aim of this study was to compare the thrombosis
potential of diets rich in stearic acid with palmitic acid-rich
diets by measuring platelet aggregation, platelet volume and
other key components of the haemostatic pathways.
Methods
Subjects
Thirteen free-living healthy males were recruited following
advertisement of the study throughout RMIT University.
Subjects were aged 35 12 y (mean s.d.), of normal body
weight (body mass index (BMI) ˆ 26.0 3.3 kg=m2. Subjects had no known metabolic, endocrine or haematological
diseases, were non-smokers, not on any form of medication
and had a moderate activity level. Subjects were advised not
to take any form of non-steroidal anti-in¯ammatory drugs
14 days prior to commencement and during the study.
Subjects were counselled to maintain their usual intake of
alcohol and to abstain from consuming alcohol 24 h prior to
blood sampling. All subjects were required to give written
consent to participate and were free to withdraw at any time.
The study protocol was approved by the Human Research
Ethics Committee of RMIT University (approval number
17=96).
Experimental design
The dietary intervention consisted of subjects consuming
both a high stearic (diet S) and a high palmitic acid diet (diet
P) for 4 weeks in a random crossover design, with a 7 week
wash-out (habitual diet) period between the two phases.
Subjects consumed the equivalent of approximately twothirds of their habitual fat intake, as the test fats, during the
intervention periods.
Subjects were counselled prior to commencement of the
®rst intervention period on which foods were to be excluded
from the diet (included in a detailed information booklet).
Advice was provided on suitable low-fat alternatives to
substitute for foods normally eaten, such as regular fat
dairy products, pastries and cakes etc. Subjects were given
training in recording their food intakes and completed a 7
day weighed food record of their habitual diet prior to the
study. Seven day weighed food records were also obtained
during each intervention period and washout phase. Dietary
composition was determined using Diet 1 (Version 4,
NUTTAB 95, Xyris Software Pty Ltd, Queensland, Australia) based on Composition of Foods, Australia (National
Food Authority, 1995). The database was modi®ed by the
inclusion of fatty acid data, for a wider variety of foods, for
C12:0 to C18:0 (lauric, myristic, palmitic and stearic acids),
palmitoleic acid (C16:ln-7) and oleic acid (C18:ln-9), linoleic acid (18:2n-6) and alpha linolenic acid (18:3n-3)
obtained from Dr M James, University of Adelaide.
During the two intervention stages, the percentage
energy derived from fat, carbohydrate and protein was
kept constant for each individual, based on habitual food
records. The high stearic and high palmitic acid test fats
were supplied by Meadow Lea Foods (Sydney, Australia).
Test fats were supplied to subjects in the form of baking and
spreading margarines and incorporated into biscuits, cakes
and muf®ns given as snacks and in salad rolls supplied for
weekday lunches. Breakfasts, dinners and weekend lunches
were left to the subjects' discretion following dietary
modi®cation advice. The remaining fat intake was obtained
from commercially available foods low in fat.
89
Test fats
The stearic acid-enriched baking margarine was produced
by an interesteri®ed blend of 35% hardened canola (100%
hydrogenated) and 65% Sunola1 (a high oleic acid variety
of sun¯ower seed oil), and the stearic acid-enriched spreading margarine was a blend of 30% hardened canola and 70%
Sunola1. The palmitic acid-enriched baking margarine was
produced by interesterifying a blend of 55% palm stearin,
20% palm olein and 25% Sunola1. The palmitic acidenriched spreading margarine was produced as a mixture
of interesteri®ed palm stearine (50%) and Sunola1 (50%).
Both stearic acid and palmitic acid levels in their respective
margarines were maximized within the limits of physical
property characteristics conducive to normal use. The oleic
acid content in the stearic acid-rich fats and biscuits was
higher than for the palmitic acid-rich fats and biscuits in
order for stearic acid products to be spreadable at room
temperature. The fatty acid pro®le of the margarines used is
shown in Table 1.
Physiological measurements
Subjects had their weight, height and body mass index
(BMI, kg=m2) determined, and percentage body fat measured using a bioimpedence fat analyser=scale (TBF-501
Tanita Corporation, Illinois, USA). Systolic and diastolic
blood pressures and pulse were also measured at each
European Journal of Clinical Nutrition
Stearic acid-rich diet
FD Kelly et al
90
Table 1
Fatty acid composition and fat content of test fats and biscuits
Fatty acid content (as percentage total fatty acids)
Stearic acid-enriched
Palmitic acid-enriched
Spreading
Baking
Spreading
Baking
margarine margarine Biscuit margarine margarine Biscuit
C16:0
C18:0
C18:1
C18:2
C18:3
4.3
29.1
56.2
7.1
0.7
4.4
33.8
51.8
6.6
0.6
4.7
31.1
50.9
7.7
0.8
31.3
4.5
53.5
7.8
0.5
42.1
4.5
43.1
7.6
0.3
41.7
4.5
42.4
8.6
0.5
Fata
82
82
24
79
79
22
a
g=100 g edible fat or biscuit.
appointment on a Lumiscope Digital Auto In¯ate Blood
Pressure Monitor (Lumiscope Company Inc., NJ, USA).
Blood sampling and preparation
Fasting venous blood samples ( 10 h) were taken from the
subjects before the commencement (day 0) and at the end
(day 28) of each study period by a quali®ed nurse at the
RMIT University Health Service using standard vacutainer
collection tubes. Blood for lipid analysis and cell counts was
collected into EDTA containing vacutainers (Greiner Labortechnik, Austria). Blood for platelet fatty acid analysis was
collected into CTAD (citric acid, theophylline, adenosine,
dipyridamole) vacutainers (Becton Dickinson Ltd, Cowley,
UK). Blood for whole blood platelet aggregation and the
coagulation and ®brinolytic factors was collected into separate sodium citrate vacutainers (Greiner Labortechnik).
EDTA and citrated plasma were obtained from blood spun
at 3000 rpm at 4 C for 15 min. Plasma phospholipid (PL)
fatty acids, total cholesterol, HDL cholesterol and triacylglycerols were determined from EDTA plasma stored at
ÿ20 C. Citrated plasma was stored at ÿ70 C for subsequent
analysis of coagulation and ®brinolytic factors. Following
blood collection, CTAD tubes were maintained at 37 C to
prevent platelet activation and used within 1 h for platelet
aggregation by the method of Castaldi and Smith (1980).
Plasma lipoprotein lipids
Total cholesterol (TC) and triacylglycerol (TAG) concentrations in plasma were determined with a centrifugal
autoanalyser (Hitachi Autoanalyser System 705, Japan)
using commercially available enzymatic kits, CHOD-PAP
and GPO-PAP for TC and TAG, respectively (Boehringer
Mannheim, Germany). High-density lipoprotein cholesterol
(HDL-C) was measured after all other plasma lipoproteins
were precipitated with polyethylene glycol 6000 (PEG6000, BDH Chemicals, Kilsyth, Victoria). Low density
lipoprotein cholesterol (LDL-C) was calculated from the
values obtained for TC, HDL-C and TAG for each subject
by using the Friedewald formula as developed by DeLong
et al (1986).
European Journal of Clinical Nutrition
Platelet and plasma fatty acids
Platelet and plasma lipids were extracted with chloroform:
methanol 1:1 (C:M, v=v) containing 10 mg=l of butylated
hydroxytoluene (Labco, Vic, Australia), and 10 mg=l of
C17:0 phospholipid (La-phosphatidylcholine diheptadecanoyl, Sigma-Aldrich, Pty. Limited, NSW, Australia) as
internal standard, as reported previously (Sinclair et al,
1987). The total platelet PL and total plasma PL fractions
were separated by thin-layer chromatography. The methyl
esters of the fatty acids were prepared by saponi®cation
using KOH (0.68 mol=l in methanol) followed by transesteri®cation with 20% boron tri¯uoride (BF3) in methanol
and the fatty acid compositions were determined by gas ±
liquid chromatography as previously described (Sinclair
et al, 1987).
Full blood examination
A full blood cell count (including platelet count (Plt) and
mean platelet volume (MPV)) was performed on an automated haematological analyser (Coulter Counter STKR,
Coulter Electronics Inc., Hialeah, USA) within 2 h of
blood collection.
Plasma haemostatic factors
Prothrombin time (PT), activated partial thromboplastin
time (APTT), ®brinogen, factor VII coagulant activity
(FVII:C), plasminogen and antithrombin III (ATIII) levels
were determined using a centrifugal analyser (ACL 200,
Coulter IL, Ltd) with commercially available kits (Coulter,
Instrumentation Laboratory, Milano, Italy).
Agonist induced ex vivo whole blood platelet aggregation
Platelet aggregation was determined in whole blood using
a two-channel whole blood impedance aggregometer
(Chrono-Log Aggregometer, Model 540V5, Chrono-Log
Corporation, Havertown, Philadelphia, USA). Agonists
used were collagen (2 mg=l), arachidonic acid (AA,
1.0 mmol=l) and adenosine diphosphate (ADP) (8 and
17 mmol=l). Aggregation was measured as rate of aggregation (slope, O=min) as reported by Ingerman-Wojenski and
Silver (1984).
Statistical analyses
Statistical analysis was performed using Statistical Packages
for Social Scientists (SPSS version 9.0, 1998, Chicago, IL).
Comparisons between the dietary groups involved a threeway repeated measures analysis of variance using a general
linear model (GLM), where diet type (palmitic or stearic
rich) and time were within-subject factors and dietary order
was the between-subject factor (P < 0.05 was regarded as
signi®cant). Comparisons within individual diets (between
days 0 and 28) were conducted on parameters where a
signi®cant time treatment interaction or time effect was
observed using paired t-tests and Bonferroni correction
(P < 0.025 was regarded as signi®cant).
Stearic acid-rich diet
FD Kelly et al
Results
All subjects stayed within 2 kg of their initial body weights
throughout the dietary phases of the study. There were no
signi®cant changes in BMI, waist-to-hip ratio, or blood
pressure during either dietary period.
The mean nutrient intakes for all subjects at baseline and
during the two dietary phases are presented in Table 2.
There were no signi®cant differences in total energy intake
during either dietary period compared with baseline or
between the two dietary phases. The changes in the percentage of total energy (%TE) of protein, fat and carbohydrate
during diet S were not signi®cantly different to those during
diet P. There was a signi®cant decrease in fat intake (%TE,
P < 0.025) compared with baseline during diet P. Compared
with baseline, there was a signi®cant decrease (P < 0.025) in
the percentage of total energy (%TE) from saturated and
polyunsaturated fatty acids (PUFA) during diet S and a
signi®cant decrease (P < 0.025) in PUFA during diet P.
Compared with their respective baseline intakes, there was
a signi®cant increase (P < 0.025) in MUFA (%TE) during
both diet S and diet P.
There was a 12 g=day increase in stearic acid
(P < 0.025) and a 9 g=day decrease (P < 0.025) in palmitic acid intake on diet S. On diet P, there was a
7 g=day increase (P < 0.025) in palmitic acid and a
91
Table 2 Daily nutrient intakes of subjects at baseline (habitual diet) and during the 4 week stearic acid (diet S) and palmitic acid
(diet P) intervention periods (n ˆ 13)
Diet S a
Nutrient
Total energy (kJ)
Protein (%TE)
Fat (%TE)
SFA (%TE)
MUFA (%TE)
PUFA (%TE)
Carbohydrate (%TE)
Alcohol (%TE)
Dietary ®bre (g)
Cholesterol (mg)
Diet P a
Pb
Day 0 c
Day 28 d
Day 0 c
Day 28 d
T
TD
10259 1676
17.1 1.5
29.3 5.1
11.5 2.5
10.3 1.5
4.7 1.5
50.4 5.4
3.2 2.9
25 7
244 59
11001 1680
16.0 1.6
27.8 3.8
10.0 1.5*
13.0 2.1*
2.9 0.3*
54.4 4.1*
2.1 2.1
30 8
172 42*
10335 1537
16.8 2.2
29.9 5.3
11.5 2.5
10.5 2.1
4.6 1.8
50.7 4.9
2.6 2.3
27 8
236 72
10694 1561
16.9 1.3
27.4 4.1*
10.3 1.3
12.3 2.2*
2.9 0.4*
53.5 4.0
2.2 2.6
29 9
185 50
NS
NS
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
NS
< 0.05
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
a
Values are mean s.d., based on 7 day weighed food records.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in dietary intake between diets,
P < 0.05 (time treatment interaction); GLM Ð Repeated Measures ANOVA.
c
Baseline dietary intake.
d
Dietary intake during the study.
%TE ˆ percentage of total energy intake.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
b
Table 3 Daily fatty acid intakes of subjects at baseline (habitual diet) and during the 4 week stearic acid (diet S) and palmitic acid
(diet P) intervention periods (n ˆ 13)
Diet S a
Dietary fatty acid
Total fat (g)
C12:0 (g)
C14:0 (g)
C16:0 (g)
C18:0 (g)
C16:1 (g)
C18:1 (g)
C18:2 (g)
C18:3 (g)
Diet P a
Pb
Day 0 c
Day 28 d
Day 0 c
Day 28 d
T
TD
79 16
1.3 0.8
29. 1.0
15.4 3.3
7.3 1.9
1.3 0.3
25.2 5.1
11.1 3.9
1.1 0.5
81 15
0.2 0.1*
0.7 0.3*
6.6 1.4*
19.4 4.5*
0.4 0.2*
37.3 7.9*
7.3 1.3*
0.8 0.3*
81 17
1.4 0.8
3.0 1.0
15.5 3.3
7.6 2.1
1.3 0.3
26.0 6.5
11.7 5.1
1.1 0.5
77 14
0.2 0.1*
1.1 0.2*
22.5 5.3*
4.4 0.8*
0.6 0.2*
33.1 6.7*
7.7 1.3*
0.7 0.2*
NS
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
< 0.05
NS
NS
NS
< 0.05
< 0.05
NS
< 0.05
NS
NS
a
Values are mean s.d., based on 7 day weighed food records.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in dietary intake between diets,
P < 0.05 (time treatment interaction); GLM Ð repeated measures ANOVA.
c
Baseline dietary intake.
d
Dietary intake during the study.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
b
European Journal of Clinical Nutrition
Stearic acid-rich diet
FD Kelly et al
92
Table 4 Lipoprotein concentration of subjects at baseline (habitual diet) and during the 4 week stearic acid (diet S) and palmitic acid
(diet P) intervention periods (n ˆ 13)
Diet S a
Parameter
Diet P a
Day 0
TC (mmol=l)
HDL-C (mmol=l)
LDL-C (mmol=l)
TAG (mmol=l)
4.67 1.03
1.22 0.32
3.03 0.90
1.15 0.64
Day 28
4.21 0.94*
1.09 0.24*
2.67 0.79*
1.20 0.62
Day 0
Pb
Day 28
{
4.71 1.04
1.20 0.28
3.13 0.98
1.02 0.43
4.44 1.00
1.13 0.31{
2.84 0.90
1.15 0.64
T
TD
< 0.05
< 0.05
< 0.05
NS
NS
NS
NS
NS
a
Values are mean s.d.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in dietary intake between diets,
P < 0.05 (time treatment interaction); GLM Ð repeated Measures ANOVA.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
{
Signi®cant dietary order effect, P < 0.05 (GLM Ð repeated measures ANOVA).
b
3 g=day decrease (P < 0.025) in stearic acid. The changes
in the amount of stearic, palmitic and oleic acid intake
on diet S were signi®cantly different (P < 0.05) to those
on diet P. On both diets there were signi®cant changes
(relative to baseline) for other fatty acids, particularly an
increase in oleic acid and decrease in linoleic acid (Table
3). While these changes were not planned, they were the
result of efforts to maintain energy intake constant while
removing certain fat sources from the diet and replacing
them with the test fats.
The results of lipoprotein concentrations are reported in
Table 4. Diet S resulted in a signi®cant decrease in totalcholesterol (TC), HDL-cholesterol (HDL-C) and LDLcholesterol (LDL-C, P < 0.025) compared with baseline,
however, there were no signi®cant changes in plasma
lipoprotein lipids on diet P. A diet order effect was present
in the changes in TC and HDL-C (ie affected by whether
the subjects started on diet S or diet P ®rst). TC and HDL-C
fell in both groups on diet P but fell signi®cantly more in
one group than the other on diet S.
The full blood examination and haemostatic parameters
are reported in Table 5. There was a signi®cant difference
(P < 0.05) in the change in MPV on diet S compared with
diet P. The MPV decreased signi®cantly following diet S
(P < 0.025) while there was no change in MPV on diet P.
The diet order effect on diet P was due to one group
showing an increased MPV value. The FVII:C activity
was signi®cantly decreased (P < 0.025) during diet S compared with baseline. The FVII:C activity and ®brinogen
levels of two samples taken during diet S were excluded
from calculations due to suspected pre-activation of clotting
(related to venipuncture technique). There were no signi®cant changes in PT, APTT, plasminogen, ®brinogen or
ATIII on either diet.
Fatty acid compositions of platelet PL are shown in Table
6. There were signi®cant differences in the changes in
platelet stearic and palmitic acid levels on diet S compared
with diet P (P < 0.05). Diet S resulted in signi®cantly
increased stearic-acid levels and signi®cantly decreased
palmitic acid levels in platelet PL compared with baseline
Table 5 Full blood examination and haematological parameters of subjects at baseline and the end of the 4 week stearic acid (diet S)
and palmitic acid (diet P) intervention periods (n ˆ 13)
Diet S a
Parameter
9
WBC (10 =l)
RBC (1012=l)
Hgb (g=l)
PLT (109=l)
MPV (¯)
Fibrinogen (g=l)b
FVII (% activity)
PT (s)
APTT (s)
ATIII (%)
Plasminogen (%)
a
b
Diet P a
Pc
Day 0
Day 28
Day 0
Day 28
T
TD
6.6 1.0
5.1 0.3
153 9
223 40
8.9 0.6
2.4 0.4
94 24
14.2 0.8
33.9 3.2
90 17
88 10
6.5 1.2
5.0 0.2
152 7
225 33
8.4 0.5*
2.5 0.3
85 18*
14.6 0.8
33.8 3.8
93 13
85 8
6.3 0.9
5.0 0.2
151 9
228 38
8.8 0.6
2.4 0.4
96 21
14.2 1.0
33.8 5.2
92 11
85 11
6.7 1.1
5.0 0.3
152 12
226 35
9.0 0.6{
2.4 0.5
92 24
14.9 1.6
35.6 6.4
92 11
86 9
NS
NS
NS
NS
< 0.05
NS
< 0.05
< 0.05
NS
NS
NS
NS
NS
NS
NS
< 0.05
NS
NS
NS
NS
NS
NS
Values are mean s.d., bn ˆ 11.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in parameter between diets, P < 0.05
(time treatment interaction); GLM Ð repeated measures ANOVA.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
{
Signi®cant dietary order effect, P < 0.05 (GLM Ð repeated measures ANOVA).
c
European Journal of Clinical Nutrition
Stearic acid-rich diet
FD Kelly et al
(P < 0.025). Diet P resulted in a signi®cant increase
(P < 0.025) in the palmitic acid level in platelet PL. These
changes were of a lesser magnitude compared with the
change on diet S. Both dietary interventions resulted in a
signi®cant decrease in linoleic acid and a signi®cant
increase in oleic acid (P < 0.025) compared with baseline.
On diet S there was a signi®cant increase (P < 0.025) in
the stearic acid proportion in the plasma PL (13.6 1.2% to
16.5 2.3% of plasma PL fatty acids) and a signi®cant
decrease (P < 0.025) in the level of palmitic acid
(28.1 1.7% to 25.9 2.4%). Diet P led to a signi®cant
increase (P < 0.025) in the level of palmitic acid
(28.3 1.7% to 29.9 1.2%). The changes in the proportion
of stearic acid and palmitic acids on diet S were signi®cantly
different (P < 0.05) to those on diet P (data not shown).
Agonist-induced whole blood platelet aggregation results
are reported in Table 7. On diet S, there were no signi®cant
changes in the rate of aggregation while during diet P, the
rate of aggregation was signi®cantly increased (P < 0.025)
in response to collagen and ADP (8 and 17 mm) compared to
baseline.
93
Discussion
This study examined the effect of diets rich in stearic acid
relative to palmitic acid on thrombotic risk factors associated with platelets and haemostasis, with platelet aggregation as an important outcome. The test fats were prepared by
interesterifying blends of particular fats as described, which
Table 6 Fatty acid composition of total platelet phospholipids (percentage fatty acid) of subjects at baseline and the end of the 4
week stearic acid (diet S) and palmitic acid (diet P) intervention periods (n ˆ 13)
Diet S a
Diet P a
Pb
Nutrient
Day 0 c
Day 28 d
Day 0 c
Day 28 d
T
TD
C14:0
C16:0
C17:0
C18:0
C16:ln-9
C16:ln-7
C18:ln-9
C18:ln-7
C18:2n-6
C20:0
C20:1
C20:3n-6
C20:4n-6
C20:5n-3
C22:4n-6
C22:5n-6
C22:5n-3
C22:6n-3
0.2 0.1
15.4 0.9
0.5 0.1
20.1 1.6
0.3 0.1
0.2 0.1
14.0 0.8
1.0 0.2
5.1 0.5
0.9 0.1
0.6 0.1
1.6 0.4
25.1 1.3
0.4 0.2
2.4 0.4
0.3 0.1
1.9 0.3
1.7 0.4
0.1 0.0*
13.0 1.4*
0.4 0.1
24.6 2.2*
0.3 0.1
0.2 0.1*
14.9 0.8*
1.0 0.1
4.5 0.4*
1.0 0.2*
0.6 0.1
1.5 0.4
25.8 0.9
0.3 0.1
2.5 0.4
0.3 0.1
1.8 0.2
1.7 0.4
0.2 0.1
15.5 0.9
0.4 0.1
21.4 1.1
0.3 0.0
0.2 0.1
14.2 0.9
1.0 0.2
5.1 0.5
0.9 0.1
0.6 0.1
1.5 0.3
25.1 1.2
0.4 0.2
2.4 0.4
0.3 0.1
1.9 0.4
1.7 0.4
0.1 0.0*
16.7 1.1*
0.4 0.0
20.1 1.7
0.3 0.1
0.2 0.1
15.2 0.7*
1.0 0.2
4.5 0.4*
0.8 0.1*
0.7 0.1*
1.5 0.4
24.9 1.0
0.3 0.1
2.4 0.4
0.2 0.1
1.7 0.2*
1.7 0.4
< 0.05
< 0.05
< 0.05
< 0.05
NS
< 0.05
< 0.05
NS
< 0.05
< 0.05
NS
NS
NS
NS
NS
NS
< 0.05
NS
NS
< 0.05
NS
< 0.05
NS
< 0.05
NS
< 0.05
NS
< 0.05
< 0.05
NS
NS
NS
NS
NS
NS
NS
a
Values are mean s.d.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in parameter between diets, P < 0.05
(time treatment interaction); GLM Ð repeated measures ANOVA.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
b
Table 7 Agonist-induced whole blood platelet aggregation in subjects at baseline and the end of the 4 week stearic acid (diet S) and
palmitic acid (diet P) intervention periods
Diet S a
Nutrient
c
Collagen (2 mg=ml)
Arachidonate (1.0 mM)b
ADP (8 mM)c
ADP (17 mM)c
Diet P a
Pd
Day 0
Day 28
Day 0
Day 28
T
TD
6.6 1.1
7.4 2.3
3.8 1.5
4.7 1.6
7.0 1.5
7.0 3.0
4.0 2.2
4.9 1.9
7.4 2.0
7.5 1.5
3.8 1.4
5.1 1.6
8.4 1.5*
8.3 1.4
5.2 2.3*
6.4 2.4*
< 0.05
NS
< 0.05
< 0.05
NS
NS
NS
NS
a
Values are mean s.d.; bn ˆ 11; cn ˆ 12.
P, level of signi®cance; T, signi®cant time effect, P < 0.05; T D, signi®cantly different change in dietary intake between diets,
P < 0.05 (time treatment interaction); GLM Ð repeated measures ANOVA.
*Signi®cantly different from baseline within diet, P < 0.025 (paired t-test Bonferroni correction), applied to parameters where a
signi®cant time treatment interaction or time effect was observed.
d
European Journal of Clinical Nutrition
Stearic acid-rich diet
FD Kelly et al
94
gave stearic acid or palmitic acid-rich products; however, in
order to make the stearic products spreadable, the stearic
acid products contained a higher level of oleic acid than the
palmitic acid products. Thus, it is conceivable that some of
the results found could be in¯uenced by the stearic acid
content as well as the oleic acid content. In reality, the oleic
acid intake=day=subject on the stearic acid diet was
37 g=day compared with 33 g=day on the palmitic acid
diet and the difference was probably not of biological
signi®cance. We do not know of any evidence which
shows that this small difference in oleic acid between the
two diets would produce the changes in platelet aggregation,
MPV or Factor VII observed.
There were no effects of diet S on collagen or ADP
agonist-induced in vitro platelet aggregation, despite the
fact that the stearic acid level in platelets increased signi®cantly (P < 0.025). In contrast, diet P led to a signi®cant
increase in the level of palmitic acid in the platelets which
was associated with a signi®cant increase in agonistinduced platelet aggregation in response to collagen and
ADP. This ®nding is novel since no previous studies have
compared platelet aggregation on diets rich in stearic acid vs
palmitic acid.
In vitro platelet aggregation is based on the assumption
that an increased response indicates an increased tendency
for thrombogenesis in vivo. Interpretation of results, however, is dif®cult due to many factors affecting in vitro
measurements (Schoene, 1997). To date, the data reported
by various authors on the effect of dietary stearic acid on
platelet aggregation are con¯icting. It has been demonstrated that, for a particular dietary fatty acid, the aggregatory response to the same agonist can give opposing results
(Turpeinen et al, 1998; Kwon et al, 1991), due partly to the
medium in which platelets have been assessed. There is
argument as to whether whole blood or platelet-rich plasma
(PRP) samples are more appropriate for determination of ex
vivo aggregation, or as a model to represent the in vivo
situation. Whole blood aggregation testing used in the
present study may represent more closely physiological in
vivo conditions compared with PRP used in other studies
(Renaud et al, 1981; 1986a, b). It has been demonstrated,
using whole blood in vitro aggregation techniques, that
platelet aggregates contain both leukocytes and erythrocytes
(Joseph et al, 1989). Leukocytes can down-regulate platelet
reactivity via prostacyclin synthesis while erythrocytes can
enhance platelet activation via ADP release, the active takeup of adenine nucleotides and the preferential binding of
prostacylin. These factors which can in¯uence platelet
function are excluded in the process of aggregation in
PRP as opposed to whole blood techniques which allow
the study of platelets in their natural milieu (Joseph et al,
1989; Marcus & Sa®er, 1993; Homstra, 1989). Despite this,
some researchers still believe that PRP is more appropriate
since it removes such factors giving a better picture of the
changes in platelets (Turpeinen et al, 1998).
Turpeinen et al (1998) demonstrated using PRP that a
high stearic acid containing diet (9.3% total energy) for 5
weeks in a group of 80 volunteers led to a signi®cantly
European Journal of Clinical Nutrition
enhanced collagen-induced platelet aggregation (but not
with ADP) compared with a high trans fatty acid diet
(8.7% total energy). In comparison, a study by Kwon et al
(1991) incorporating a 3 week controlled SFA diet, demonstrated using whole blood that stearic acid in platelet
phospholipids was associated (r ˆ ÿ0.69) with decreased
collagen-induced platelet aggregation, suggesting that
stearic acid may not be prothrombotic.
Tholstrup et al (1996) studied the acute effects of stearic
and myristic acids on ADP- and collagen-induced platelet
aggregation. Both diets reduced platelet aggregation postprandially after a high-fat meal compared with fasting
levels, with signi®cantly lower platelet aggregation at 24 h
after consumption of stearic acid than myristic acid. It was
hypothesized that a mechanism for this decrease in aggregation was the coating of the platelets with chylomicrons,
which may interfere with the platelet ± collagen interaction in the initial stage of platelet aggregation (platelet
activation).
In the present study, MPV signi®cantly decreased on diet
S by 6% relative to baseline and by 7% relative to diet P.
This ®nding is supported by data from Schoene et al (1992),
who reported a similar decrease in MPV in 10 subjects fed
in excess of 20 g stearic acid=day from shea butter compared
with a high palmitic acid diet. The MPV is regarded as an
index of platelet activation and has been shown to be an
independent risk factor for recurrent myocardial infarction
(Martin et al, 1991; Schultheiss et al, 1994). Platelets
normally circulate as thin discs and smaller platelets are
thought to be less active and, thus, in a quiescent state are
less likely to be involved in thrombotic conditions (Schoene
et al, 1992). Activated platelets produce changes in the
platelet microtubules, causing them to undergo a disc-tosphere shape transformation (Laufer et al, 1979), resulting
in an increase in volume, and these larger platelets have
been shown to have enhanced activity (Abbate & Boddi,
1987; Sharp et al, 1994). Flow cytometry techniques have
demonstrated that this greater reactivity of larger platelets is
associated with more ®brinogen receptors being exposed,
initiating arterial thrombi formation to a greater extent than
smaller platelets (Giles et al, 1994; Michelson, 1996).
Further studies on the biological signi®cance in the reduction of the MPV following stearic acid-rich diets are
warranted.
Prospective studies have demonstrated that coagulation
factor VII is an independent predictor of total and fatal CHD
in the ®rst 5 y of follow up of the Northwick Park Heart
Study (NPHS) (Meade et al, 1980, 1986). This observation
has also been found in fatal CHD in longer follow-up
periods of the NPHS (Meade et al, 1993) and The Prospective Cardiovascular Munster (PROCAM) study (Assmann et
al, 1996). It has also been suggested that decreasing the total
dietary fat content reduces factor VII coagulant activity
(Marckmann et al, 1990, 1993, 1994; Miller et al, 1989).
FVII:C signi®cantly decreased on diet S relative to baseline,
suggesting favourable effects on haemostasis while no
signi®cant change was observed on diet P. Tholstrup et al,
(1994) also observed a 13% decrease in factor VII on a
Stearic acid-rich diet
FD Kelly et al
stearic acid-rich diet (from shea butter) compared with a
high palmitic acid diet. In contrast, Mutanen & Aro (1997)
demonstrated no signi®cant difference in the level of FVII:C
in 40 subjects fed stearic acid-rich diets (9.3% total energy)
for 5 weeks. Some authors have suggested that shea fat has a
unique ability in lowering FVII:C which may be unrelated
to its fatty acid composition, but more related to the nonglyceride components such as tocopherols and hydrocarbons
(Tholstrup et al, 1994; Mutanen & Aro, 1997).
Total cholesterol, LDL-C and HDL-C levels in the
plasma were all signi®cantly decreased on diet S. The
total cholesterol and LDL-C results observed are consistent
with data from Bonanome and Grundy (1988), Tholstrup
et al (1994) and Dougherty et al (1995), while signi®cant
decreases in HDL-C concentrations on high stearic acid
diets compared with baseline have been previously reported
by Tholstrup et al (1994) and Dougherty et al (1995). The
lack of effect on the plasma TAG concentration is consistent
with other data on stearic acid rich diets (Tholstrup et al,
1994; Dougherty et al, 1995; Bonanome & Grundy, 1988).
The mechanisms by which stearic acid lower plasma cholesterol levels are still uncertain; however, they may in part
be due to the regio-speci®c location of the saturated fatty
acids, which in turn in¯uences their absorption and further
metabolism. These properties have been attributed, ®rstly, to
the lower gastrointestinal absorption rates of stearate, especially when found in the sn-1 and sn-3 positions of the
triacylglycerol (Kritchevsky, 1994; Bracco, 1994). This is
largely due to its melting point being above body temperature and its ability to form calcium soaps (Small, 1991).
Secondly, it is possible that stearic acid is rapidly converted
to oleic acid in the liver (Grundy, 1994), thus reducing its
potential impact on saturated fatty acid levels in the body. In
natural lipid sources such as cocoa butter and shea butter
(Dougherty et al, 1995), the stearic acid is predominantly
located in the sn-1 and sn-3 positions with minimal amounts
in the sn-2 position (Padley et al, 1994). However, when fats
are interesteri®ed, as with the fats used in the present study,
a greater proportion of stearic acid is located in the sn-2
position, which is absorbed into the mucosal cells as a
monoacylglycerol. Further studies to show the absorption of
stearic acid from the sn-2 position could include chylomicron studies postprandially after stearic feeding and also
faecal studies to determine apparent absorption of these
formulated fats.
The use of stearic acid-rich lipid sources could be of
advantage to the food industry if it displaces other saturated
and trans fatty acids from the foods, giving them nutritional
and physiological advantages over natural lipid sources.
Furthermore, if the stearic acid-rich lipid source is interesteri®ed, a greater proportion of stearic acid could be
randomly distributed to the sn-2 position which in turn
could increase the amounts absorbed compared with natural
lipid sources.
In this study, the platelet stearic acid level increased by
22% on diet S, which is consistent with the extent of
increase reported by Turpeinen et al (1998). Plasma phospholipid stearic acid levels also increased by 21% in the
present study. It has been shown there is a high proportion
of stearic acid in platelet phosphatidylserine and phosphatidylinositol, which are important phospholipids for cell
membrane signalling events (Mori et al, 1987). Perhaps
the increased stearic acid in the platelet membranes is
speci®c for one of these two phospholipids or alternatively
the increased stearic acid might in¯uence the function of
cell membrane receptors through changes in membrane
¯uidity (Litman & Mitchell, 1996).
This study supports other studies which show that diets
enriched in stearic acid do not contribute to an increase in
classical cardiovascular risk factors and those related to
thrombosis. These data might appear to be in contradiction
with the recent report by Hu et al (1999), who suggest that
there is no basis for a distinction between stearic acid and
other saturated fatty acids in `normal' diets, because of the
high correlation between these two factors. Since stearic
acid levels in `normal' diets are half those of palmitic plus
myristic acid levels, it is not a major saturated fatty acid in
our diet. Our data on a modi®ed diet, containing approximately 2.5 times more stearic acid than usual, raises the
possibility of using an increased amount of stearic acid in
the food supply in place of those fats rich in palmitic and
myristic acids or trans fatty acids. The stearic acid could
satisfy the requirements for particular physical properties in
the food while appearing to have either neutral or bene®cial
effects in relation to risk factors for cardiovascular disease.
95
Acknowledgements ÐL Johnson and M Francis provided technical assistance. We gratefully acknowledge Dr Gerald R Elsworth from the Centre
for Program Evaluation, The University of Melbourne, Victoria, Australia
for advice on the statistical analysis of the data.
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