- Click here for explanation of classification scheme.

To assess the combined effect of plant sterols, soy proteins, viscous fibers and nuts on cell membrane fragility and circulating plant sterol and lipid concentrations in hyperlipidemic individuals.

• Attending the Risk Factor Modification Center, St. Michael's Hospital, Ontario, Canada
• Taken part in previous dietary studies
• Experienced in following dietary protocols
• Previously had raised LDL cholesterol levels (>4.1mmol/L)
• No history of diabetes, renal disease or liver disease
• Not taking medications known to influence serum lipids.

No subjects had a history of diabetes, renal or liver disease and none were taking medications known to influence serum lipids.

 

Recruitment

Subjects were recruited from patients attending the Risk Factor Modification Center at St. Michael's Hospital in Ontario, Canada. 

Intervention

Subjects were followed on their own low-saturated fat therapeutic diets (followed National Cholesterol Education Program Step two guidelines; <7% energy from saturated fat and <200 milligrams per day dietary cholesterol) for one week prior to the start of the study, and for an additional two weeks after the study on return to their low-saturated fat therapeutic diets. Dietary advice on low-saturated fat (<7% dietary calories) and low-cholesterol diets (<200 milligrams per day) had been reinforced on at least two occasions over the previous year and at entry to the study. During the middle four weeks, subjects followed a combination diet in which all foods were provided at weekly clinic visits with the exception of fresh 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 seven-day rotating menu plan, including specified fruits and vegetables which they checked off each item as it was eaten and confirmed the weight of the foods. The same menu plan was used for all subjects but was modified to suit individual preferences, providing the goals for viscous fiber, soy protein, plant sterols and almond consumption were met. Subjects were provided with self-taring electronic scales and asked to weigh all food items consumed during the study period. 

The aim of the combination diet was to provide 1g of plant sterols per 1,000kcals as an enriched margarine. The Unilever margarine contained approximately 46% sitosterol, 26% campesterol, 19% stigmasterol, 2.7% brassicasterol, 1.3% sitostanol, 0.8% campestanol, and 0.8% avenasterol, 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 25g of margarine, for 82kcal. In addition, the diet supplied 8.2g viscous fiber per 1,000kcal from oats, barley, and psyllium and 22.7g of soy protein per 1,000kcal as soy milk or meat analogs. Raw or unblanched almonds also provided vegetable protein (2.9g/1,000kcal). Emphasis was placed on eggplant and okra as additional sources of viscous fiber (0.55g/1,000kcal and 0.67g/1,000kcal, respectively). Thus, 200g of eggplant and 100g of okra were prescribed to be eaten on a 2,000kcal diet each day. Diets were provided at a targeted intake to maintain body weight based on estimated caloric requirements.

Compliance was assessed from the completed weekly checklists and from the return of uneaten food items.

Statistical Analysis

The results were expressed as means ± SE. The significance of the differences between the pre-treatment-diet, combination diet, and post-treatment diet was assessed by the least squares means test with the Tukey-Kramer adjustment for multiplicity of simultaneous comparisons (PROC MIXED/SAS 8.2). The model used had the treatment value as the response variable and the week and interaction term diet by sex as main effects and a random term corresponding to subject nested within sex. Student's paired T-test (two-tailed) was used to assess the significance of the percentage change from pretreatment.

With the present subject numbers for red cell fragility, assuming 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 0.015g/100mL, a difference of 0.013g/100mL should be detected as significant.

 

Timing of Measurements

Blood samples and body weights were obtained after 12-hour overnight fasts at weekly intervals and at week two of the washout. Seven-day weighed diet histories were obtained for the week prior to and for two weeks following the combination diet. Completed menu checklists were returned at weekly intervals during the four-week combination diet period.

Dependent Variables

• Variable 1: Change in red cell fragility (assessed on fresh red cells collected in vacutainer tubes containing EDTA)
• Variable 2: Serum campesterol and ß-sitosterol (In serum and membrane, measured by GLC [HP 5890 Series II; Hewlett-Packard, Palo Alto, CA])
• Variable 3: LDL cholesterol (calculated)
• Variable 4: HDL cholesterol (In serum and membrane, measured by GLC [HP 5890 Series II; Hewlett-Packard, Palo Alto, CA] using Lipid Research Clinic's protocol for TC, TG, and HDL cholesterol)
• Variable 5: Triglyceride (In serum and membrane, measured by GLC [HP 5890 Series II; Hewlett-Packard, Palo Alto, CA] using Lipid Research Clinic's protocol for TC, TG, and HDL cholesterol). 

Independent Variables

• Combination diet as described earlier (measured by seven-day weighed histories, menu checklists, food returned at visits)
• Low-saturated fat (<7% dietary calories) and low-cholesterol diets (<200 milligrams per day) (recorded diets).

 

 

Initial N

13 subjects (seven men and six post-menopausal women)

Attrition (final N)

12 (one subject completed only three weeks and withdrew because of dyspepsia associated with Helicobacter pylori infection requiring antibiotic therapy)

Age

65± three years (median 64 years; range 43-84)

Other relevant demographics

Anthropometrics

BMI 25.6±0.9kg/m² (median 26.1kg/m²; range 20.6-30.7kg/m²); baseline LDL cholesterol 4.22±0.11mmol/L (median 4.27mmol/L; range 3.51-4.99mmol/L)

At the time of the study three subjects had blood lipids in the normal range, one subject had a low HDL-C (<0.0mmol/L), one subject had raised TG level, five subjects had raised LDL-C levels, three subjects had a raised LDL-C and TG level

Location

St. Michael's Hospital, Ontario, Canada

 

Sera for plant sterol analysis were unavailable for one subject, and an additional three subjects were missing one or both week one and week three samples.

Table 1. Blood lipids and apolipoproteins at baseline and on the combination diet.

 

Variables	Baseline (week 0)	Mean Treatment A (weeks two to four)	pb
Cholesterol
Total-C	6.46±0.21	5.01±0.20	<0.001
LDL-C	4.22±0.11	3.02±0.17	<0.001
HDL-C	1.37±0.11	1.34±0.11	<0.992
TGc (mmol/L)	1.92±0.35	1.45±0.18	<0.984
Apolipoproteinsd (g/L)
ApoA-1	1.70±0.07	1.61±0.08	0.334
ApoB	1.32±0.05	1.01±0.05	<0.001
Ratios
Total-C/HDL-C	5.06±0.41	4.00±0.30	0.004
LDL-C/HDL-C	3.31±0.26	2.45±0.24	<0.001
ApoB/apoA-1	0.80±0.05	0.64±0.05	0.004

• Treatment values represent the mean of weeks two, three, and four
• P-values after Bonferroni correction
• To convert cholesterol and TG to mg/dL, multiply by 38.67 and 88.57, respectively
• To convert apolipoprotein A-1 and ß values to mg/dL, multiply by 100.

Other Findings

Table 2. Calculated macronutrient intakes (mean + SE) during the run-in, test, and run-out phases of the portfolio study

 

	Run-in (N=12)	Portfolio Diet (mean weeks two to four, N=13)	Run-out (week six, N=12)
Energy (kcal per day)	1,703±120	1,999±118	1,703±104
Total protein (percentage of protein)	17.3±0.8	22.4±0.5	18.1±0.8
Vegetable protein	48.7±3.5	96.8±0.2	39.1±2.8
Available carbohydrate (percentage of energy)	52.9±2.8	50.6±0.6	58.2±1.3
Total dietary fiber (grams per 1,000kcal)	17.1±1.9	30.7±1.0	17.8±1.8
Total fat (percentage of energy)	28.3±2.5	27.0±0.8	22.7±1.5
SFA	7.7±0.7	4.3±0.1	6.2±0.7
MUFA	11.9±1.6	11.8±0.5	9.0±0.7
PUFA	6.0±0.4	9.9±0.2	5.3±0.5
Dietary cholesterol (mg per 1,000kcal)	99±13	10±3	79±9
Alcohol (percentage of energy)	1.5±0.5	0.2±0.1	1.0±0.4
Satiety (-3 to +3)a	1.3±0.2	2.9±0.2	1.3±0.3

 ^aSatiety: -3 extreme hunger, +3 extremely full

Demographics and compliance: Throughout the period of observation, subjects tended to lose weight: [-0.10±0.05 kilograms per week (P=0.127) over the combination diet, and -0.2±0.05 kilograms per week (P=0.001) during the run-out phase]. In the majority of subjects, compliance in terms of caloric intake was good, with 92.5±2.9% of calories prescribed being consumed.

Serum plan sterols: Serum plant sterol concentrations tended to increase over the one-month combination diet. Serum campesterol concentrations increased by 50±15% from baseline for weeks two to four; the respective increase for serum sitosterol was 27±14%. For campesterol, the unadjusted rise was significant (P=0.007) but disappeared after Bonferroni correction (P=0.139).

Serum lipids: Significant reductions in blood lipids were seen at the end of the combination diet compared with the run-in and run-out periods. 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 four of the combination diet. No significant differences between mean pre- and post-combination diet values were seen at any time point when expressed as unadjusted O.D. readings at 540nm 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 significant 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 associations were significant.

For serum sterols, an association was seen at baseline between plasma sitosterol and total cholesterol (r=0.72, P=0.008), LDL cholesterol (r=0.83, P=0.001), and apolipoprotein B (r=0.91, P<0.001). No relation was seen between changes in serum sterols and blood lipids.

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.

Government:	Federa; Gov. of Canada, Canadian Natural Sciences and Research Council
Industry:	Loblaw Brands, Almond Board of Canada, Unilever (Canada), Unilever Research & Development (Netherlands), Natural Temptations (Canada) Food Company: Commodity Group:

Results do not isolate effect of almonds on dependent variables