DFA: Conjugated Linoleic Acid (CLA) Supplementation and Intermediate Health Outcomes (2011)

Study Design:
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Quality Rating:
Research Purpose:

The purpose of this study is to examine the effect of dietary supplementation of a combination of conjugated linoleic acid (CLA) (50:50 c9t11:t10c12) and n-3 fatty acids in a 12-week randomized crossover study on body composition, regional adiposity and adiposity-related circulating hormone levels in healthy human volunteers.

Inclusion Criteria:
  • Healthy
  • Non-smoker
  • No regular medications
  • Normal physical exam and electrocardiogram.
Exclusion Criteria:
  • Recent use of aspirin
  • Recent use dietary antioxidant supplements
  • Change in body weight (>3kg) within past three months.
Description of Study Protocol:


Not specified.


Subjects were randomly assigned to two intervention groups in a double-blind, placebo-controlled crossover study. Supplements given were in the form of 1-g capsules and subjects ingested six capsules daily, containing either CLA or n-3 long-chain polyunsaturated fatty acids (LC-PUFA) (three capsules each) or control oil for 12 weeks. The daily intake of CLA from the capsules was 2.28g and that of eicosapentaenoic acid and docosahexaenoic acid was 1.53g.

Blinding used

Double-blind, placebo-controlled.


  • For the CLA plus n-3 LC-PUFA intervention group, subjects received three 1-g capsules of CLA (Clarinol G-80 containing 760mg CLA per gram, consisting of 50:50 mixture of cis-9, trans-11: trans-10, cis-12 CLA; Loders Croklaan, Wormerveer, Holland) and three 1-g capsules of n-3 PUFA (EPAX 5500TG; containing 300mg eicosapentaenoic acid and 210mg docosahexaenoic acid per gram; Pronova Biocare AS, Lysaker, Norway).
  • For the control fat, subjects received six 1-g capsules each consisting of 800mg palm oil and 200mg soya bean oil per gram (Cardinal Health, Swindon UK). The fatty acid composition of the control fat was specifically chosen to represent the fatty acid profile present in the average UK diet [41% saturated fatty acid (SFA):36% monounsaturated fatty acid (MUFA):21% PUFA in the control fat vs. 38% SFA:33% MUFA:21% PUFA in the average diet]
  • Before and after each 12-week supplementation period, subjects visited the Human Nutrition Unit at the Rowett Research Institute between 0800 and 0900 hours after an overnight fast (approximately 12 hours). Upon arrival, anthropometric and body composition measurements were taken.
    • Body weight and height were measured using a scale and a stadiometer to the nearest 0.5kg and 0.5cm, respectively
    • Waist and hip circumferences were measured and the waist-to-hip ratio calculated
    • Standard procedures were followed for the anthropometric measurements and the mean of three measurements was taken
    • Whole-body dual energy X-ray absorptiometry (DXA) (Norland XR-46, Mark II highspeed pencil beam scanner equipped with dynamic filtration, with version 2.5.2 of the Norland software; Norland, Fort Atkinson, WI) was used to measure body composition
    • Venous blood samples were collected in EDTA Vacutainers and plasma was separated and stored at −20°C.

Statistical Analysis

  • Data are expressed as mean±SEM, (except in Table 1 in the article, which are means±SD)
  • Calculations were done with GenStat (VSN International; version 8) with a P value of <0.05
  • Statistical analysis of plasma leptin concentrations was performed using a log scale
  • Baseline measurements between groups were compared using one-way analysis of variance (ANOVA) and unpaired T-tests on the changes in absolute values
  • Statistical analysis was done to determine whether there were any effects of treatment sequence as a result of the crossover design
  • No treatment-by-period interactions were observed (data not shown)
  • All data were treated as paired samples from a crossover study and the differences between control fat and CLA plus n-3 LC-PUFA treatments were examined using paired T-tests on the change between 0- and 12-week measurements
  • The significance of the association between measurements was examined using Pearson’s correlation coefficient.
Data Collection Summary:

Timing of Measurements

  • Subjects were asked to take two capsules with each of the three main meals in the day
  • Each supplementation period lasted 12 weeks with a 12-week washout period in between the crossover, with approximately equal numbers of subjects assigned to the CLA plus n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) or control fat supplementation group in each period.

Dependent Variables

  • Body composition (measured by DXA)
  • Adiposity (abdominal fat and percentage fat-free mass)
  • Hormone levels (fasting adiponectin, leptin, glucose, insulin concentration and insulin resistance).

Independent Variables

Combined 3-g fat supplement containing CLA plus n-3 LC-PUFA.

Control Variables

  • Non-consumed capsules were counted and analysis of n-3 LC-PUFA content in blood cell membranes was done to confirm compliance
  • All participants were asked to maintain their usual dietary and lifestyle habits.
Description of Actual Data Sample:

Initial N

Not stated.

Attrition (final N)

  • Young lean: n=13
  • Young obese: n=12
  • Older lean: n=20
  • Older obese: n=14
  • Total n=59.

Age (y±SD)

  • Young lean: 30.5±4.9
  • Young obese: 32.4±2.3
  • Older lean: 56.3±4.2
  • Older obese: 56.9±5.4.


Not stated.


  • The subject groups were well matched for age and BMI
  • The lean subjects had lower adiposity indices as determined by DXA compared to the obese subjects
  • Obese subjects, both young and older, had higher levels of fasting glucose (P<0.001), fasting insulin
    (P<0.001) and greater insulin resistance as determined by homeostasis model assessment insulin resistance index (HOMA-IR) (P<0.001) than did lean subjects
  • The older obese subjects had the highest fasting glucose levels (P<0.01)
  • HOMA-IR values positively correlated with leptin, abdominal fat mass, body weight, BMI, total and percentage fat mass (P<0.001) and negatively correlated with adiponectin (P<0.001)
  • Obese subjects also had lower levels of adiponectin (P<0.01) and higher levels of leptin (P<0.001) than leaner subjects
  • Fasting plasma adiponectin concentrations grouped from all subjects in the study were negatively correlated with HOMA-IR (r=−0.44, P<0.01), fasting plasma insulin (r=−0.45, P<0.01), fasting plasma leptin (r=−0.29, P<0.05), BMI (r=−0.44, P<0.01), total percentage body fat (r=−0.33, P<0.001), abdominal fat mass (r=−0.32, P<0.001) and waist circumference (r=−0.42, P<0.001)
  • Baseline levels of fasting leptin were negatively correlated with fasting adiponectin (P<0.05) and positively correlated with abdominal fat mass, total fat mass, percentage fat mass, body weight, BMI, and fasting insulin (P<0.001).


Rowett Research Institute, Aberdeen, UK.


Summary of Results:

Effect of CLA plus n-3 supplementation on body composition:

  • Both body weight and BMI were increased in the young obese group after the CLA plus n-3 LC-PUFA supplementation relative to baseline (both P=0.002)
  • In the older lean group with the control fat treatment, body weight, BMI and waist were all increased (P=0.049, P=0.047, P=0.049, respectively)
  • The total fat mass tended to increase in young obese subjects during the control fat supplementation phase of the study (P=0.052), whereas percentage fat mass increased (by 3%, P=0.038) along with abdominal fat mass (by 10%, P=0.008) relative to baseline
  • Supplementation with CLA plus n-3 LC-PUFA did not increase abdominal fat mass (−1%, P=0.55) in the young obese group, but fat-free mass was increased relative to control fat (by 2.4%, P=0.04) 
  • No significant effect was observed in any of the other groups on either abdominal fat mass or fat-free mass when comparing CLA plus n-3 LC-PUFA supplementation with control
  • There was no other significant effect in any subject group comparing the CLA plus n-3 LC-PUFA supplementation with control fat on total body weight, BMI, waist circumference, total or percentage fat mass, or total bone mass (data not shown) as assessed by DXA.

Effect of CLA plus n-3 supplementation on fasting adiponectin, leptin, insulin and glucose levels

  • In the young obese group, adiponectin levels were increased (by 12%) with CLA plus n-3 LC-PUFA supplementation relative to control group (P=0.048)
  • Overall in the younger subjects, CLA plus n-3 LC-PUFA supplementation increased adiponectin levels (9%) relative to control group (P=0.011), whereas there was no significant effect in the older subjects
  • There were no effects of CLA plus n-3 LC-PUFA supplementation compared with control group on plasma leptin levels in any of the groups
  • Fasting glucose levels were increased in the older obese subjects after the CLA plus n-3 LC-PUFA supplementation compared with control group (P=0.027)
  • When comparing the difference in fasting glucose levels with CLA plus n-3 LC-PUFA supplementation vs. control group fat in the lean vs. older obese groups, fasting glucose levels were increased in the latter group [2% decrease vs. 6% increase, respectively (P=0.043)]
  • Fasting insulin levels tended to be increased by the control treatment in all groups, with a larger increase in obese compared with lean subjects (P=0.027)
  • CLA plus n-3 LC-PUFA supplementation, compared with control group, had no significant effect on fasting insulin levels in any group
  • However, in the older lean group, insulin levels were increased (by 18%) after the CLA plus n-3 LC-PUFA supplementation were compared to baseline (P=0.004), but when compared with control group, fat showed borderline significance (P=0.056)
  • Insulin resistance, as assessed by HOMA-IR, showed no significant changes when comparing values before and after supplementation with either control fat or CLA plus n-3 LC-PUFA treatment in any group except with control treatment in the young obese, for whom levels were increased by 18% (P=0.034)
  • When comparing the effects of the CLA plus n-3 LC-PUFA with control, there were no significant differences between HOMA-IR values in any of the subject groups.
Author Conclusion:

This study, based on groups matched for sex, BMI and age, suggests that supplementation with CLA plus n-3 PUFA is of potential benefit in preventing abdominal fat mass accretion and in improving adiponectin levels without causing any deleterious effect on insulin sensitivity, specifically in young obese individuals (23 to 38 years and BMI of 30 to 36). Conversely, these results also show that CLA plus n-3 LC-PUFA has no effect on fat mass in young and older lean and older obese individuals.

Twelve-week supplementation with a combination of CLA plus n-3 LC-PUFA prevents abdominal fat mass accrual in young obese men when supplemented with control fat and is associated with a smaller, but significant, increase in fat-free mass. There were no other significant effects of this supplementation on any measure of adiposity in any of the other groups.  Results from this study indicate that age, in addition to BMI, is an important factor in determining responses to fatty acid supplementation, with younger, obese individuals responding to a greater extent than either younger or older lean or older obese individuals.

CLA plus n-3 LC-PUFA treatment within the young obese group prevented the increase in abdominal fat with no deleterious effects on insulin sensitivity as assessed by HOMA-IR in any of the groups. However, the finding that fasting insulin levels were increased in the older lean group with the CLA/n-3 LC-PUFA treatment alone and that fasting glucose levels were increased slightly in the older obese group compared with the control fat, may indicate subtle reductions in insulin sensitivity in the older subjects; this to be examined in more detail.

Funding Source:
Government: European Union; Scottish Executive Environment and Rural Affairs Department
University/Hospital: Research Development Initiative of The Robert Gordon University, Aberdeen
Reviewer Comments:
  • It is unclear from the way the article's Methods section was written whether there were initially more subjects in the study. The Methods section states how many were recruited and gave informed consent. I assume that there was no attrition but it is unclear. There is no discussion of attrition in the results. 
  • Subjects were asked to maintain their usual dietary and lifestyle habits, but dietary analysis or lifestyle factors were never examined
  • There were only men this study.
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? ???
3. Were study groups comparable? N/A
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  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? N/A
  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? Yes
  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? Yes
  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? No
  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)? N/A
  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? No
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? No
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