EAL

DLM: Plant Stanols and Sterols (2010)

Citation:

Study Design:

Class:

- Click here for explanation of classification scheme.

Quality Rating:

Research Purpose:

To determine the efficacy on plasma cholesterol-lowering of plant sterol esters or non-esterified stanols eaten within low-fat foods as well as margarine.

Inclusion Criteria:

Known hypercholesterolemia untreated with lipid-lowering drugs for preceding six months or longer
Total cholesterol greater than 5.5mmol per L
Triglycerides (TG) less than 3mmol per L
Informed consent.

Exclusion Criteria:

Hormone replacement therapy (HRT)
Smoking
Alcohol exceeding four drinks per day (men), two drinks per day (women)
Medication likely to affect plasma lipids
Bowel, liver and kidney disorders
Thyroid dysfunction
Diabetes mellitus.

Description of Study Protocol:

Recruitment

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Design

Randomized, crossover.

Blinding

Subjects were blinded to diet assignment.

Intervention

Study 1:
- Two-week run-in
  - Habitual diet
- Four-week control period
  - Habitual diet plus test foods minus supplementation were provided
- Two four-week test periods:
  - Randomization to one of the following, followed by the other dietary intervention:
    - Habitual diet plus 2.4g plant sterol ester
      - 50% sitosterol
      - 20% stigmasterol
      - 20% campesterol
    - Habitual diet plus similar amount of non-esterified plant stanols
      - 70% sitostanol
      - 27% campestanol
      - ~1% free sterols
  - Test substances provided in three different foods: Cereal, bread, margarine
  - Equal amounts of tocopherol added to all margarines
Study 2: Initiated several weeks after the end of Study 1
- Control period: Habitual diet
- Two four-week test periods
  - Randomization to one of the following, followed by the other dietary intervention:
    - Habitual diet plus 2.4g plant sterol ester-containing spread (50% fat)
      - 40% butter fat and included canola oil, soybean sterol ester and trans-free palm oil blend
      - 46% saturated fatty acids, 28% oleic, 17% linoleic, 3% alpha linolenic acid
      - 2% trans fatty acids
    - Habitual diet plus 50% fat control spread
      - 40% butter fat, 32% canola oil, 8% hardened trans-free palm oil blend
      - 43% saturated fatty acids, 47% mono- and polyunsaturated fatty acids
      - 2% trans fatty acids
    - Equal amounts of tocopherols added to margarine.

Statistical Analysis

Repeated measures ANOVA on ranks (Freedman), followed by pairwise multiple comparison procedures, due to absence of normal distributions for plasma total and LDL cholesterol. Data provided as medians.
For normally distributed parameters, values are given as means
Plasma sterols and carotenoids analyzed by paired T-test.

Data Collection Summary:

Timing of Measurements

Study 1
- Baseline
  - Three-day food frequency questionnaire (FFQ) focusing on fats, cholesterol and fiber
  - Plasma lipids
- Beginning of each intervention
  - Plasma lipids
- After each intervention
  - Three-day FFQ focusing on fats, cholesterol and fiber
  - Plasma lipids
  - Plasma sterols
  - Plasma carotenoids
  - Plasma tocopherols
Study 2
- Plasma lipids were drawn at baseline and at the beginning and end of each intervention.

Dependent Variables

Plasma lipids (Study 1 and 2)
- Total cholesterol
- Triglyceride
- HDL cholesterol
- LDL cholesterol
Plant sterols(Study 1)
- Campesterol
- Sitosterol
- Lathosterol: index of cholesterol synthesis (precursor of cholesterol)
Carotenoids(Study 1)
- Lycopene
- α-carotene
- β-carotene
- β-cryptoxanthin
Tocopherols (Study 1)
- α-tocopherol
- y-tocopherol.

Independent Variables

Study 1
- 2.4g esterified plant sterols
- 2.4g non-esterified stanols
Study 2
- 2.4g esterified plant sterols (dairy spread)
- Control dairy spread.

Description of Actual Data Sample:

Initial N:
- Study 1: 22 (18 men; four women)
- Study 2: 15 (gender breakdown not provided)
  - Some advised to seek more active lipid lowering medication
  - Some unavailable for a further eight weeks
Attrition:
- Study 1: 22
- Study 2: 15
Age: 60±9 years (range 34 to 70 years)
Ethnicity: Not described
Other relevant demographics:
- Normotensive: None higher than145/95mmHg
- Cholesterol: 21 of 22 had cholesterol value greater than 5.5mmol per L
- Triglyeride level between 2.0 and 2.6mmol per L: Five subjects
- HDL cholesterol: None less than 1.0mmol per L
Anthropometrics: 24±1.9kg per m² (range 18.3 to 26.9kg per m²)
Location: Australia.

Summary of Results:

Study 1:

Plasma Lipids Following Plant Sterol- or Stanol-Enriched Foods

Intervention	Cholesterol (mmol/L) median (25-75%)	LDL cholesterol (mmol/L) median (25-75%)
Baseline	Not reported	Not reported
Control	7.0 (6.25-7.40)	4.77 (4.0-5.35)
Sterol esters	6.40 (5.85-7.15)	4.12 (3.45-5.10)
Stanols	6.75 (5.80-7.30)	4.37 (3.6-4.95)

Median total cholesterol fell significantly by 8.5% with sterol esters and by 3.5% with the non-esterified stanol (P<0.001 by ANOVA and P<0.05 by pairwise comparison of each test intervention vs. control diet). The response with sterol esters was possibly significantly greater than with non-esterified stanol (P=0.05).
Median LDL-cholesterol was reduced by the sterol esters (-13.6%, P<0.001 by ANOVA on ranks, P<0.05 by pairwise comparison) and by non-esterified stanols (-8.3%, P=0.003, ANOVA and P<0.05 pairwise comparison)
The minor changes in HDL-cholesterol and TG were not significant
With sterol esters plasma plant sterol levels rose (35% for sitosterol (P<0.01), 51% for campesterol, (P<0.001), plasma lathosterol rose 20% (P=0.03), indicating compensatory increased cholesterol synthesis
With stanols, plasma sitosterol fell 22% (P=0.004), indicating less cholesterol absorption
None of the four carotenoids measured in plasma changed significantly
α- and y-tocopherols both increased in plasma with the sterol supplement: α-topherol 35.2 and 40.1µM per L; y-tocopherol 11.1 and 17.7 µM per L (61%, P=0.044).

Study 2:

Plasma Lipids Following Dairy Spread

Intervention	Total cholesterol (mmol/L) median (25-75%)	LDL cholesterol (mmol/L) median (25-75%)	Triglyceride (mmol/L) mean±sd
Baseline	6.65 (5.70-6.98)	4.60 (3.67-4.85)	1.11±0.29
Control dairy spread	7.05 (6.14-7.42)	4.90 (4.12-5.35)	1.37±0.59
Sterol ester dairy spread	6.40 (6.05-7.15)	4.30 (3.62-4.97)	1.36±0.50

Median total cholesterol fell 8.5% on the sterol-ester fortified spread (P<0.001 by ANOVA and P<0.05 by pairwise comparison)
Median LDL-cholesterol rose 6.5% with dairy spread and fell 12.2% with the sitosterol ester fortified spread (P=0.03 ANOVA and P<0.05 by pairwise comparison)
HDL-cholesterol did not change between dietary changes
Plasma triglyceride rose 23% with the dairy fat spread (P=0.017).

Other findings:

Background diet info (Study 1) comparing beginning to end of study
- Small increase in fat consumption as energy (32 to 34%, P=0.02).
- Maintained saturated fat as energy (11.5±2%)
- Maintained cholesterol intake (164±47mg)
- Fiber (19g vs. 18g)
Four to eight weeks after conclusion of Study 2, 13 of 15 subjects returned for re-measurement of lipids
- Mean total cholesterol: 6.69±1.1 mmol per L
- Mean LDL-cholesterol: 4.71±1.0 mmol per L.

Author Conclusion:

This study has confirmed the substantial LDL-cholesterol-lowering effect achievable with sterol esters in moderately hypercholesterolemic subjects
Only one-third of the sterol was in a fat spread, the remaining two-thirds having been eaten as bread and cereal. This extends the range of foods within which such cholesterol-lowering compounds may be delivered.
The LDL-cholesterol raising effect of butter fat could be countered by including sterol esters
Plasma carotenoids and tocopherols were not reduced in this study. This may relate to the generally high consumption of fruit and vegetables by Australians, especially those on cholesterol-lowering diets.
This report is to our knowledge the first demonstration of the LDL-cholesterol lowering efficacy of either phytosterol esters or non-esterified phytostanols delivered in bread and cereal.

Funding Source:

Industry:

Meadow Lea Foods (Australia), Uncle Toby’s Company (Victoria), Milling & Baking (Sydney), COGNIS Australia Pty Ltd

Food Company:

In-Kind support reported by Industry:

Yes

Reviewer Comments:

Well-controlled study. Author notes that a limitation of Study 1 is that the control foods were eaten initially by all subjects and only the two test diets were randomized.
Per authors, washout period not considered necessary since sterols and stanols are minimally absorbed, and three to four weeks is considered adequate to reach new steady-state values for plasma cholesterol, triglyceride and HDL cholesterol.
One subject did not meet cholesterol eligibility criterion
Baseline plasma lipid levels not provided for Study 1. Authors reported that these were similar to those at the end of the initial control dietary period.
Although authors reported that subjects were required to return all food packages, compliance was not reported.

Quality Criteria Checklist: Primary Research
Relevance Questions
	1.	Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)	Yes
	2.	Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?	Yes
	3.	Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?	Yes
	4.	Is the intervention or procedure feasible? (NA for some epidemiological studies)	Yes

Validity Questions
1.	Was the research question clearly stated?		Yes
	1.1.	Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?	Yes
	1.2.	Was (were) the outcome(s) [dependent variable(s)] clearly indicated?	Yes
	1.3.	Were the target population and setting specified?	Yes
2.	Was the selection of study subjects/patients free from bias?		Yes
	2.1.	Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?	Yes
	2.2.	Were criteria applied equally to all study groups?	Yes
	2.3.	Were health, demographics, and other characteristics of subjects described?	Yes
	2.4.	Were the subjects/patients a representative sample of the relevant population?	Yes
3.	Were study groups comparable?		Yes
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	No
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	N/A
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	Yes
	3.4.	If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?	N/A
	3.5.	If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)	N/A
	3.6.	If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?	N/A
4.	Was method of handling withdrawals described?		Yes
	4.1.	Were follow-up methods described and the same for all groups?	Yes
	4.2.	Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)	Yes
	4.3.	Were all enrolled subjects/patients (in the original sample) accounted for?	Yes
	4.4.	Were reasons for withdrawals similar across groups?	N/A
	4.5.	If diagnostic test, was decision to perform reference test not dependent on results of test under study?	N/A
5.	Was blinding used to prevent introduction of bias?		Yes
	5.1.	In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?	Yes
	5.2.	Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)	Yes
	5.3.	In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?	N/A
	5.4.	In case control study, was case definition explicit and case ascertainment not influenced by exposure status?	N/A
	5.5.	In diagnostic study, were test results blinded to patient history and other test results?	N/A
6.	Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?		Yes
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	Yes
	6.2.	In observational study, were interventions, study settings, and clinicians/provider described?	N/A
	6.3.	Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?	Yes
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	???
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	N/A
	6.6.	Were extra or unplanned treatments described?	N/A
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	N/A
	6.8.	In diagnostic study, were details of test administration and replication sufficient?	N/A
7.	Were outcomes clearly defined and the measurements valid and reliable?		Yes
	7.1.	Were primary and secondary endpoints described and relevant to the question?	Yes
	7.2.	Were nutrition measures appropriate to question and outcomes of concern?	Yes
	7.3.	Was the period of follow-up long enough for important outcome(s) to occur?	Yes
	7.4.	Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?	Yes
	7.5.	Was the measurement of effect at an appropriate level of precision?	Yes
	7.6.	Were other factors accounted for (measured) that could affect outcomes?	Yes
	7.7.	Were the measurements conducted consistently across groups?	Yes
8.	Was the statistical analysis appropriate for the study design and type of outcome indicators?		Yes
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	Yes
	8.4.	Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?	Yes
	8.5.	Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?	No
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	8.7.	If negative findings, was a power calculation reported to address type 2 error?	No
9.	Are conclusions supported by results with biases and limitations taken into consideration?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
10.	Is bias due to study's funding or sponsorship unlikely?		Yes
	10.1.	Were sources of funding and investigators' affiliations described?	Yes
	10.2.	Was the study free from apparent conflict of interest?	Yes