are advised for cholesterol reduction, but their combined effect
on plant sterol absorption has never been tested. We assessed
their combined action on serum sterols in hyperlipidemic sub-
jects who were following low-saturated fat diets before starting
the study and who returned to these diets post-test. The 1-mon
test (combination) diet was high in plant sterols (1 g/1,000 kcal),
soy protein (23 g/1,000 kcal), viscous fiber (9 g/1,000 kcal), and
almonds (14 g/1000 kcal). Fasting blood was obtained for serum
lipids and sterols, and erythrocytes were obtained for fragility
prior to and at 2-wk intervals during the study. The combination
diet raised serum campesterol concentrations by 50% and β-sitos-
terol by 27%, although these changes were not significant after
Bonferroni correction; near-maximal rises were found by the end
of the first week, but no change was found in red cell fragility de-
spite a 29% reduction in the LDL cholesterol level. No significant
associations were observed between changes in red cell fragility
and blood lipids or sterols. We conclude that plant sterols had a
minimal impact on serum sterol concentrations or red cell
fragility in hyperlipidemic subjects on diets that greatly reduced
their serum lipids.
Paper no. L9398 in Lipids 40, 169–174 (February 2005).
Plant sterols, soy proteins, viscous fibers, and nuts
Plant sterols have been shown to lower serum LDL cholesterol
(1–3) by 8–12% in the absence of other dietary modifications
(e.g., vs. low-fat diets) in a meta-analysis by Law (4). Despite
broad acceptance of the safety of plant sterols by most Western
countries, concerns have been raised that absorption of plant
sterols may have adverse consequences, possibly by modifying
cell membrane fragility resulting from the displacement of cho-
lesterol (5). Few data are available from human studies concern-
ing the influence of dietary sterols on membrane fragility and
serum sterols, especially using diets that result in marked reduc-
tions in serum cholesterol levels (6,7). We therefore assessed the
effect on serum plant sterols of diets high in other cholesterol-
lowering dietary components. These components, which in-
cluded viscous fiber (8–11), soy protein (12,13), and almonds in
combination (dietary portfolio), have been shown to produce a
marked reduction in serum cholesterol (6,7). The specific objec-
tive was to assess whether a plant sterol-enriched diet, combined
with other agents known to reduce circulating cholesterol levels,
would result in a change in cell membrane fragility and circulat-
ing plant sterol and lipid concentrations in hyperlipidemic indi-
Subjects. Thirteen subjects (7 men and 6 postmenopausal
women), aged (mean ± SE) 65 ± 3 yr (median 64 yr; range
43–84 yr); body mass index 25.6 ± 0.9 kg/m2(median 26.1
kg/m2; range 20.6–30.7 kg/m2); baseline LDL cholesterol 4.22
± 0.11 mmol/L (median 4.27 mmol/L; range 3.51–4.99
mmol/L) were recruited from patients attending the Risk Fac-
tor Modification Center, St. Michael’s Hospital. All subjects
had taken part in previous dietary studies, were experienced in
following dietary protocols and previously had had raised LDL
cholesterol levels (>4.1 mmol/L) (14). At the time of the study,
5 subjects had raised LDL cholesterol levels, one subject had
raised TG levels (>2.30 mmol/L, range 0.7–5.1 mmol/L), 3
subjects had both raised LDL cholesterol and TG levels, one
subject had a low HDL cholesterol concentration (<0.9
mmol/L), and 3 subjects had blood lipids in the normal range
(14). None of the subjects had a history of diabetes, renal dis-
ease, or liver disease, and none were taking medications known
to influence serum lipids. One subject completed only 3 wk and
withdrew because of dyspepsia associated with a Helicobacter
pylori infection requiring antibiotic therapy.
Dietary advice on low-saturated fat (<7% dietary calories) and
low-cholesterol diets (<200 mg/d) had been reinforced on at least
two occasions over the previous year, and at entry to the study, 6
subjects had recorded diets with <7% (total energy) saturated fat
and 9 subjects had followed diets with <200 mg/d cholesterol.
Study protocol. Subjects were followed on their own low-
saturated fat therapeutic diets for 1 wk prior to the start of the
study, and for an additional 2 wk after the study on return to
their low-saturated fat therapeutic diets. During the middle 4
Copyright © 2005 by AOCS Press169 Lipids, Vol. 40, no. 2 (2005)
*To whom correspondence should be addressed at Clinical Nutrition and
Risk Factor Modification Center, St. Michael’s Hospital, 61 Queen St. East,
Toronto, Ontario, Canada, M5C 2T2. E-mail: firstname.lastname@example.org
Effects of a Diet High in Plant Sterols, Vegetable
Proteins, and Viscous Fibers (Dietary Portfolio)
on Circulating Sterol Levels and Red Cell Fragility
in Hypercholesterolemic Subjects
Peter J. Jonesa, Mahmoud Raeini-Sarjaza, David J.A. Jenkinsb,c,d,e,*, Cyril W.C. Kendallb,d,
Edward Vidgenb,d, Elke A. Trautweinh, Karen G. Lapsleyi, Augustine Marchieb,d,
Stephen C. Cunnaneb, and Philip W. Connellyc,f,g
aSchool of Dietetics and Human Nutrition, McGill University, Montréal, Qúebec; bClinical Nutrition & Risk Factor
Modification Center and cDepartment of Medicine, Division of Endocrinology and Metabolism, St. Michael’s Hospital,
Toronto, Ontario; Departments of dNutritional Sciences, eMedicine, fBiochemistry, and gLaboratory Medicine and Pathobiology,
Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 3E2; hUnilever Health Institute, Unilever R&D
Vlaardingen, The Netherlands; and iThe Almond Board of California, Modesto, California, 95354
wk, subjects followed a combination diet in which all foods
were provided with the exception of fresh fruit and most veg-
etables. Blood samples and body weights were obtained after
12-h overnight fasts at weekly intervals and at week 2 of the
washout. Seven-day weighed diet histories were obtained for
the week prior to and for 2 wk following the combination diet.
Completed menu checklists were returned at weekly intervals
during the 4-wk combination diet period.
The study was approved by the Ethics Committee of the
University of Toronto and St. Michael’s Hospital, and informed
consent was obtained from the subjects.
Diets. Diets eaten before and after the 4-wk combination diet
were the subjects’ routine therapeutic low-fat diets, which ap-
proximated National Cholesterol Education Program Step 2
guidelines (<7% energy from saturated fat and <200 mg/d dietary
cholesterol) (Table 1) (14). Subjects were provided with self-tar-
ing electronic scales and asked to weigh all food items consumed
during the study period. During the combination diet period, all
foods consumed by the subjects were provided at weekly clinic
visits with the exception of fruit and low-calorie vegetables (i.e.,
non-starch-containing vegetables), which subjects were instructed
to obtain from their local stores. Subjects were provided with a 7-
d rotating menu plan, including specified fruits and vegetables,
on which they checked off each item as it was eaten and con-
firmed the weight of the foods. The same menu plan was used for
all subjects but was modified to suit individual preferences, pro-
viding the goals for viscous fiber, soy protein, plant sterols, and
almond consumption were met. For ease of consumption, where
possible, items were prescribed in whole units.
The aim of the combination diet (dietary portfolio) was to
provide 1 g of plant sterols per 1,000 kcal as an enriched mar-
garine. The Unilever margarine contained approximately 46%
sitosterol, 26% campesterol, 19% stigmasterol, 2.7% brassi-
casterol, 1.3% sitostanol, 0.8% campestanol, and 0.8% avenas-
terol, with the remainder made up of various other plant sterols.
The Unilever margarine provided 12% plant sterol (w/w). Two
grams of plant sterol was contained in 25 g of margarine, for
82 kcal. In addition, the diet supplied 8.2 g of viscous fiber per
1,000 kcal from oats, barley, and psyllium and 22.7 g of soy
protein per 1,000 kcal as soy milk or meat analogs. Raw, un-
blanched almonds also provided vegetable protein (2.9 g/1,000
kcal). Emphasis was placed on eggplant and okra as additional
sources of viscous fiber (0.55 g/1,000 kcal and 0.67 g/1,000
kcal, respectively). Thus, 200 g of eggplant and 100 g of okra
were prescribed to be eaten on a 2,000-kcal diet each day. Diets
were provided at a targeted intake to maintain body weight
based on estimated caloric requirements (15).
Compliance was assessed from the completed weekly
checklists and from the return of uneaten food items.
Analyses. Serum lipid data were reported previously (6). All
samples were stored at –70°C prior to analysis. Sera for plant
sterol analysis were unavailable for one subject, and an addi-
tional 3 subjects were missing one or both week 1 and week 3
samples. As also mentioned earlier, one subject dropped out at
week 3. Plant sterols and cholesterol in serum and membrane
were measured by GLC (HP 5890 Series II; Hewlett-Packard,
Palo Alto, CA). Briefly, 5α-cholestane was added to each sam-
ple as an internal standard. Samples were saponified with 0.5 M
methanol-KOH for 1 h at 100°C, and sterols were extracted
using petroleum ether and injected into the gas–liquid chromato-
graph. The column temperature was 285°C. Isothermal running
conditions (oven temperature 285°C) were maintained for 42
min. The injector and detector were set at 300 and 310°C, re-
spectively. The carrier gas (helium) flow rate was 1.2 mL/min
with the inlet splitter set at 100:1. Individual plant sterols and
cholesterol were identified using authentic standards (Sigma-
Aldrich Canada Ltd., Oakville, Ontario). The CV in the plant
sterol measurement was 4% (16). Serum was analyzed accord-
ing to the Lipid Research Clinics’ protocol (17) for total choles-
terol, TG, and HDL cholesterol, after dextran sulfate–magne-
sium chloride precipitation (18). Levels of LDL cholesterol were
calculated (19). Serum apolipoprotein A-l and B were measured
170P.J. JONES ET AL.
Lipids, Vol. 40, no. 2 (2005)
Calculated Macronutrient Intakes (mean + SE) During the Run-in, Test, and Run-out Phases
of the Portfolio Study
2–4, n = 13)
(n = 12)
(week 6, n = 12)
Total protein (% of protein)
Available carbohydrate (% of energy)
Total dietary fiber (g/1,000 kcal)
Total fat (% of energy)a
Dietary cholesterol (mg/1,000 kcal)
Alcohol (% of energy)
Satiety (–3 to +3)b
aSFA, saturated FA; MUFA, monounsaturated FA; PUFA, polyunsaturated FA.
bSatiety: −3, extreme hunger; +3, extremely full.
1,703 ± 120
17.3 ± 0.8
48.7 ± 3.5
52.9 ± 2.8
17.1 ± 1.9
28.3 ± 2.5
7.7 ± 0.7
11.9 ± 1.6
6.0 ± 0.4
99 ± 13
1.5 ± 0.5
1.3 ± 0.2
1,999 ± 118
22.4 ± 0.5
96.8 ± 0.2
50.6 ± 0.6
30.7 ± 1.0
27.0 ± 0.8
4.3 ± 0.1
11.8 ± 0.5
9.9 ± 0.2
10 ± 3
0.2 ± 0.1
2.9 ± 0.2
1,703 ± 104
18.1 ± 0.8
39.1 ± 2.8
58.2 ± 1.3
17.8 ± 1.8
22.7 ± 1.5
6.2 ± 0.7
9.0 ± 0.7
5.3 ± 0.5
79 ± 9
1.0 ± 0.4
1.3 ± 0.3
by nephelometry (20). All samples from a given individual wer-
analyzed in the same batch.
Red cell fragility was assessed on fresh red cells collected
in vacutainer tubes containing EDTA (Becton Dickinson, Mis-
sissauga, Ontario). Packed red cells (0.02 mL) were added to 2
mL unbuffered saline covering the range of sodium chloride
concentrations from 0.20 to 0.70 g/L in 0.05 g/L increments.
After 1 h, the cells were centrifuged at 1,000 × g at room tem-
perature for 5 min and the supernatant was read at 540 nm (21).
Data are presented as unadjusted O.D. readings and as adjusted
percentages of the maximum O.D. obtained for both tests com-
bined. The adjusted values were used to calculate the saline
concentration that corresponded to the 50% hemolysis value.
The p50 value for red cell hemolysis (50% hemolysis value)
was calculated, assuming a linear response between the two
consecutive O.D. readings spanning the half-point of the maxi-
mum hemolysis recorded for the subject. For each subject, the
maximum O.D. (maximum hemolysis) obtained from both
tests combined represented the 100% hemolysis value for that
subject. Preliminary data on red cell fragility expressed as 50%
hemolysis were reported previously (6).
Diets were analyzed using a program based on USDA data
(22) with additional data on foods analyzed in the laboratory for
protein, total fat, and dietary fiber using AOAC methods (23).
FA were analyzed by GLC (24). Additional dietary fiber values
were obtained from the tables of Anderson and Bridges (25).
Statistical analysis. The results were expressed as means ±
SE. The significance of the differences between the pretreat-
ment diet, combination diet, and post-treatment diet was as-
sessed by the least squares means test with the Tukey–Kramer
adjustment for multiplicity of simultaneous comparisons
(PROC MIXED/SAS 8.2) (26). The model used had the treat-
ment value as the response variable and the week and interac-
tion term diet by sex as main effects and a random term corre-
sponding to subject nested within sex. Student’s paired t-test
(two-tailed) was used to assess the significance of the percent-
age change from pretreatment.
With the present subject numbers for red cell fragility, assum-
ing a SD of effect of 3.4%, a 3% difference could be detected as
significant (P < 0.05). Likewise, assuming a SD of effect of
0.015 g/100 mL, a difference of 0.013 g/100 mL should be de-
tected as significant.
The concentration required to obtain 50% hemolysis was
determined assuming a linear response between consecutive
observations. For each subject, the maximal O.D. obtained
from both tests combined represented the 100% hemolysis
value for that subject. A Pearson correlation analysis was used
to assess relations between plant sterol measurements and other
measurements. A Bonferroni adjustment was also made to the
significance levels to allow for the multiple comparisons (26).
Six largely independent primary measures were recognized:
change in red cell fragility, serum campesterol and β-sitosterol,
and LDL cholesterol, HDL cholesterol, and TG.
Demographics and compliance. Throughout the period of ob-
servation, subjects tended to lose weight: [–0.10 ± 0.05 kg/wk
(P = 0.127) over the combination diet, and –0.2 ± 0.05 kg/wk
(P = 0.001) during the run-out phase]. In the majority of sub-
jects, compliance in terms of caloric intake was good, with 92.5
± 2.9% of the calories prescribed being consumed.
Serum plant sterols. Serum plant sterol concentrations
tended to increase over the 1-mon combination diet (Fig. 1).
Serum campesterol concentrations increased by 50 ±15% from
baseline for weeks 2–4; the respective increase for serum sitos-
terol was 27 ± 14% (Table 2). For campesterol, the unadjusted
rise was significant (P = 0.007) but disappeared after Bonferroni
correction (P = 0.139).
Serum lipids. Full details of the blood lipid responses were
reported previously (6). Significant reductions in blood lipids
were seen at the end of the combination diet compared with the
run-in and run-out periods (Table 2). From baseline, reductions
were seen in LDL cholesterol (29.0 ± 2.7%, P < 0.001),
apolipoprotein B (24.3 ± 2.0%, P < 0.001), and the total/HDL
cholesterol ratio (19.8 ± 2.9%, P = 0.004).
Red cell fragility. No significant difference was seen in red
cell fragility between the pretreatment and week 4 of the com-
CHOLESTEROL-LOWERING DIET AND MEMBRANE STEROLS171
Lipids, Vol. 40, no. 2 (2005)
FIG. 1. Mean plasma values for campesterol and sitosterol over 6 wk in 7 subjects. (hatched bar) Administration pe-
riod of sterols. Plant sterol data were unavailable for one subject, an additional subject dropped out prior to the
week 4 sample, and 3 subjects were lacking one or both week 1 and week 3 samples.
bination diet (saline concentration for 50% hemolysis, 0.436 ±
0.007 g/100 mL vs. 0.441 ± 0.006 g/100 mL, P = 0.223, unad-
justed, P = 0.734 adjusted). No significant differences between
mean pre- and postcombination diet values were seen at any
time point when expressed as unadjusted O.D. readings at 540
nm (Fig. 2A) or as percent hemolysis, where the highest O.D.
value for each individual was taken as 100% for that individual
Relation of plant sterols to other measurements. No signifi-
cant associations were seen between change in serum sterols
and red cell fragility (serum campesterol, r = –0.09, P = 0.803;
sitosterol r = 0.12, P = 0.717). The negative values indicate the
tendency of a reduced fragility (reduced saline concentration
for hemolysis) with increasing plant sterols, although no asso-
ciations were significant.
For serum sterols, an association was seen at baseline be-
tween plasma sitosterol and total cholesterol (r = 0.72, P =
0.008), LDL cholesterol (r = 0.83, P = 0.001), and apolipopro-
tein B (r = 0.91, P < 0.001). No relation was seen between
changes in serum sterols and blood lipids.
Supplementation with plant sterols in a diet that also contained
high levels of viscous fibers, soy protein, and almonds pro-
duced large reductions in serum lipids but was associated with
only relatively small increases in plasma sterol concentrations.
There has been concern that significant increases in plasma
sterols may result in modification of membrane lipids with the
exclusion of cholesterol and that this change would increase
membrane fragility (5). The present data indicate that the small
changes in serum lipids do not relate significantly to red cell
The absence of effect on red cell fragility indices examined
opposes recent findings in rats suggesting that diets high in
172 P.J. JONES ET AL.
Lipids, Vol. 40, no. 2 (2005)
Blood Lipidsaand Apolipoproteins at Baseline and on the Combination Diet
aBlood lipid results have been published in full elsewhere (6). Total-C, total cholesterol; LDL-C, LDL
cholesterol; HDL-C, HDL cholesterol.
bTreatment values represent the mean of weeks 2, 3, and 4.
cP values after Bonferroni correction.
dTo convert cholesterol and TG to mg/dL, multiply by 38.67 and 88.57, respectively.
eTo convert apolipoprotein A-1 and B values to mg/dL, multiply by 100.
6.46 ± 0.21
4.22 ± 0.11
1.37 ± 0.11
1.92 ± 0.35
5.01 ± 0.20
3.01 ± 0.17
1.34 ± 0.11
1.45 ± 0.18
1.70 ± 0.07
1.32 ± 0.05
1.61 ± 0.08
1.01 ± 0.05
5.06 ± 0.41
3.31 ± 0.26
0.80 ± 0.05
4.00 ± 0.30
2.45 ± 0.24
0.64 ± 0.05
FIG. 2. (A) Osmotic fragility of red blood cells from subjects at baseline
(week 0) and week 4 of the combination diet after 6 h of exposure to
varying saline concentrations. Data are presented as unadjusted O.D.
readings. (B) Osmotic fragility of red blood cells from subjects at base-
line (week 0) and week 4 of the combination diet after 6 h of exposure
to varying saline concentrations. Data are presented as the percentage
of hemolysis, with the maximal O.D. reading obtained for each subject
representing 100% hemolysis.
plant sterols may increase the predisposition to hemorrhagic
events (5). Clearly, more effort is required to define the real risk
of hemorrhagic sequelae in the use of plant sterols as choles-
terol-lowering agents at the levels provided in functional foods.
Components used in this combination diet were based on
the hypothesis that during the course of human evolution, the
diet was likely to have been high in vegetable proteins, dietary
fiber, and plant sterols. All these components have now been
recognized to lower serum cholesterol (1–3,8–13, 27, 28). Veg-
etable protein as soy (13), viscous dietary fiber, oats (27), psyl-
lium (10), and, most recently, plant sterols (4) have received
FDA approval for health claims for coronary heart disease risk
reduction. These components, together with almonds (29,30),
which may combine several cholesterol-lowering components,
were the focus of the present diet.
In a previous study an attempt was made to reconstruct diets
that may have been eaten at an earlier stage in human evolu-
tion by using plant foods readily available in the supermarket
(31). The plant sterol intake on this “Myocene,” or 4–7 million-
year-old, diet was 1 g/d for men, indicating that historically
high plant sterol intakes may have been associated with diets
high in fiber and vegetable protein (31). The cholesterol lower-
ing achieved with this evolutionary model diet was of a magni-
tude similar to that seen in the present study, in which the diet
was high in the same active ingredients.
It is possible that almonds and viscous fiber in the present
diet may have protective effects on red cell fragility and that
adverse effects of plant sterols may have been masked. How-
ever, there are no data on the effects of nuts or viscous fiber on
red cell fragility. Equally, it could be argued that nuts, which
are good sources of plant sterols, might be expected to add to
any adverse effect of the sterol margarine if such existed and
that this might be compounded by viscous fiber, which further
reduces serum cholesterol levels, possibly increasing the stiff-
ness of the membrane and increasing the risk of hemolysis.
Nevertheless, these points are purely speculative since no sig-
nificant change in hemolysis was observed.
It is also possible that 4 wk was not long enough to demon-
strate more subtle changes in red cell hemolysis since the red
cell half-life in humans is 25–35 d for a mean life span of 120
d (32). In the rat the red cell half-life is of the order of 19 d with
a life span of 60 d (33). In this situation, 1-mon studies might
be more appropriate; however, this was the time frame used in
rat studies that showed increased hemolysis with plant sterols
by 4–5% (5). Based on studies of spontaneously hypertensive
rats (5), it has been suggested that increased plant sterol in-
takes, especially from canola oil, may increase the risk of hem-
orrhagic stroke (5). This effect is thought to be due to increased
cell membrane fragility (5,21) secondary to displacement of
cholesterol by plant sterols in the membrane. However, the
similar survival of olive oil-fed and corn-oil fed spontaneously
hypertensive rats leaves questions remaining as to the role of
phytosterol intakes in this process. Also, we found no relation
between serum plant sterol levels and red cell osmotic fragility.
A good relation, however, has been observed between plasma
and membrane plant sterols in a study involving hypercholes-
terolemic children (34). Furthermore, there was no significant
association between the change in concentration of plasma
sterols and red cell fragility, although our subject numbers are
small. These data add support to the general acceptance of plant
sterol-enriched margarine for use by the public. An additional
reason why the present study demonstrated no difference in red
cell fragility may relate to the lack of sensitivity of the test it-
self. Pre-incubated erythrocytes are more sensitive indicators
of red cell fragility than fresh erythrocytes, especially in situa-
tions of pathologically increased fragility (35). However, the
rat studies demonstrating the ill effects of plant sterols did not
use pre-incubated cells. Our study therefore endeavored to use
the same type of analysis to allow direct comparison.
Were plant sterol consumption to be restricted, then the
guidelines promoting increased intakes of fruit, vegetables,
whole-grain cereals, legumes, and unhydrogenated vegetable
oils would need to be revised. Such diets deliver appreciable
amounts of plant sterols, and it could be argued that, from the
evolutionary perspective, these are the diets to which we have
adapted in the context of high-fiber, vegetable protein, and
plant sterol intakes. The lack of these plant food components
results in unacceptable levels of serum cholesterol that will
qualify a significant proportion of the adult population for cho-
lesterol-lowering medications (14).
In conclusion, high plant sterol intakes in the context of
high-fiber vegetable protein diets have only a small effect on
serum plant sterol concentrations despite large reductions in
serum lipids. Moreover, high plant sterol-containing diets do
not alter red cell fragility, a finding that should be examined
further, given the recently reported promotion of hemorrhagic
events by plant sterols in rats.
The authors sincerely thank Loblaw Brands Limited (Toronto, On-
tario), Unilever Canada (Toronto, Ontario), the Almond Board of
California (Modesto, CA), and Procter & Gamble Canada (Toronto,
Ontario) for the generous donation of the foods used in this study.
The authors also wish to thank Robert Chenaux and Larry C. Griffin
of Loblaw Brands Ltd., Paul Schur of Unilever Canada, and Kathy
Galbraith of Natural Temptations Bakery (Burlington, Ontario) for
their assistance on this project. This research was supported by
Loblaw Brands Ltd., the Almond Board of California, the Canadian
Research Chair Endowment, and the Natural Sciences and Engineer-
ing Research Council of Canada.
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[Received October 23, 2003, and in revised form December 23, 2004;
revision accepted January 16, 2005]
174P.J. JONES ET AL.
Lipids, Vol. 40, no. 2 (2005)