- Click here for explanation of classification scheme.

To examine the effect of two doses of CLA supplementation for 12 weeks on body composition, weight, resting energy expenditure (REE) and safety measures in obese individuals free of chronic disease.

• Healthy
• Non-smoking
• BMI between 30 and 35
• Between 18 and 50 years of age.

• Chronic illness, including history of cancer, cardiovascular disease, diabetes or gastrointestinal disorder
• Anemia
• HIV positive
• Significant abnormal clinical laboratory (including unacceptable hematopoietic, hepatic and renal function)
• Food allergies and intolerances
• Current drug therapy for a diagnosed disease, including medications known to alter lipid metabolism
• Current use of weight-lowering medications or diets
• Use of tobacco products
• Use of CLA-containing dietary supplements during the previous three months
• Consuming a medically prescribed diet that may interfere with the intervention
• Current or planned pregnancy.

Recruitment

Recruited from the population living in the Durham, Chapel Hill, Carrboro and surrounding areas of North Carolina.

Design

• Single-center, randomized, double-blind, placebo-controlled clinical trial
• Because 24 hours were needed for complete laboratory results to be obtained after the baseline visit, participants were randomized into the study on the day of their baseline visit and were instructed not to begin taking the study pills until the next day, following confirmation of eligibility
• The randomization list was kept secure by the General Clinical Research Center Pharmacy and was opened only after participants had completed the study protocol.

Blinding Used

Double-blind.

Intervention

• Placebo
• CLA: 3.2 or 6.4g per day
• Participants were instructed to maintain their normal diet and exercise routine throughout the study protocol.

Statistical Analysis

• The authors assumed a difference of 2.8kg between the change in body fat mass of the placebo group and the 6.4g per day CLA group with an SD of 2.4kg, based on results from a previous study. An estimated 16 participants were required in each group for 90% statistical test power and 5% significance level.
• Means ±SD or SEM for continuous variables and frequencies for categorical variables were calculated for each variable by treatment group
• One-way ANOVA on continuous variables and chi-square test on categorical variables were used to determine whether baseline demographic characteristics and adverse event reporting rates differed between intervention groups
• Log-transformation was performed on variables not normally distributed
• Pre- and post-group means were compared by T-test. A one-way ANOVA was used to examine whether changes from week zero to week 12 were different between treatment groups. If ANOVA revealed that the overall test for treatment was significant, T-tests were used for post-hoc comparisons.
• All statistical tests were assessed at a level of significance α=0.05; each test was two-sided (specified a null hypothesis of equality)
• Subjects with only a baseline visit (week zero) were not included in the analyses
• Because there was a limited number of subject visits, the last-value-carried-forward method was not used. 

Timing of Measurements

• Body composition and REE (indirect calorimetry) were measured at baseline and the final 12-week visit
• Clinical assessments were made at baseline, week six and a final visit at week 12
• Compliance was measured at six weeks and 12 weeks by comparing the number of unused pills with the number expected to be taken and by measuring plasma CLA values from baseline to week 12 in each group
• Five 24-hour recalls were administered over the phone to check dietary compliance
• A brief physical activity questionnaire was administered at baseline, six weeks and 12 weeks. 

Dependent Variables

• Body composition: Measured by dual-energy X-ray absorptiometry (Hologic Delphi W model)
• REE and RQ: Medgraphics Cardiorespiratory Diagnostic Systems CPX D Series Indirect Calorimeter was used. Each participant fasted overnight and then rested in the supine position for 10 to 30 minutes prior to being measured to allow time for breathing to become regular and to avoid the effects of previous voluntary activity on REE. Each participant was fitted with a noseclip and flexible disposable mouthpiece that contained the pneumotach and gas detection line. Measurements were taken for 20 minutes in a dark, quiet, thermoneutral environment.
• Clinical assessment: Heart rate, blood pressure, lipid profile, alanine transaminase, aspartate transaminase, alkaline phosphatase, hemoglobin, hematocrit, bilirubin, calcium, chloride, creatinine, erythrocytes, white blood cells, platelets, gamma-glutamytransferase, lactate dehydrogenase, leukocytes, potassium, sodium, glucose, C-reactive protein and IL-6. 

Independent Variables

• Placebo: 8g safflower oil
• CLA: 3.2 or 6.4g per day; Tonalin from Cognis. Pills were composed of C18:2 cis-9, trans-11 and the C18:2 trans-10, cis-12 isomers in a 50:50 ratio.
• The placebo and CLA pills were identical in appearance.

Control Variables

• Plasma CLA concentrations as a measure of compliance
• 24-hour dietary recalls to assess maintenance of current diet
• Physical activity questionnaire to assess maintenance of exercise routines.

• Initial N: 55 participants randomized
• Attrition (final N):
  • Three participants were determined to be ineligible after randomization at the end of the baseline visit (one from placebo, two from 3.2g CLA)
  • Three participants withdrew within the intervention time period for personal reasons and one withdrew due to pregnancy (zero from placebo; two from 3.2g CLA; two from 6.4g CLA)
  • Final N=48; 16 participants per group
  • Placebo 25% male, 3.2g CLA 31% male, 6.4g CLA 25% male
• Age: Means ranged from 34.1 to 36.3 years
• Ethnicity:
  • African American: Placebo 31%, 3.2g CLA 31%, 6.4g CLA 38%
  • White: Placebo 69%, 3.2g CLA 63%, 6.4g CLA 62%
  • Other: Placebo 0%, 3.2g CLA 6%, 6.4g CLA 0%
• Anthropometrics:
  • There were no differences in baseline age, BMI or body fat mass among participants who withdrew and those who completed the study
  • LBM was significantly higher in those who withdrew from the study than in those who completed the study, which may be explained by the fact that a higher proportion of males did not complete the study (71% of non-completers were male) compared with those who completed the study (2.5% of completers were male)
  • Treatment groups did not differ in demographic characteristics or in weight, BMI, body fat mass, LBM or physical activity at baseline
  • BMI (kg/m²): Placebo 32.7±1.9; 3.2g CLA 32.7±1.8; 6.4g CLA 32.7±1.7
  • Body fat mass (kg): Placebo 35.6±4.9; 3.2g CLA 34.8±5.4; 6.4g CLA 34.8±5.3
  • Lean body mass (kg): Placebo 50.7±7.9; 3.2g CLA 54.1±9.0; 6.4g CLA 50.9±9.1
  • Resting energy expenditure (kJ per day): Placebo 7,312.1±1,592.6; 3.2g CLA 6,944.5±1,761; 6.4g CLA 7,052.8±1,085.9
  • Respiratory quotient: Placebo 0.9±0.2; 3.2g CLA 0.8±0.1; 6.4g CLA 0.8±0.1
  • Physical activity (MET hours per week): Placebo 31.4±19.2; 3.2g CLA 26.9±20.2; 6.4g CLA 24.4±13.1 
• Location: Durham, Chapel Hill, Carrboro and surrounding areas of North Carolina.

 Key Findings

• Based on pill count, compliance was 88% for the placebo group, 92% for 3.2g per day CLA and 92% for 6.4g per day CLA
• Plasma CLA concentrations increased in both the 3.2g per day (+0.02±0.02mol per L, P=0.002; range = -3.4 to + 57.1mol per L) and 6.4g per day (+0.05±0.05mol per L, P=0.002; range = 0 to 99.5mol per L) groups, but did not change in the placebo group
• Body fat mass, weight, BMI and percentage of fat did not change in any of the groups after 12 weeks of intervention, and there was no effect of the intervention on the waist-to-hip ratio (data was not shown)
• LBM increased by 0.64kg in the 6.4g per day CLA group (P<0.05) and tended to increase by 0.65kg in the 3.2g per CLA group (P=0.18)
• There were no changed in REE or RQ in any of the groups
• Physical activity in MET hours per week reported throughout the 12-week intervention period for each group was, for the placebo group, 23.6±12.2; for the 3.2g per day CLA group, 22.4±11.9; and for the 6.4g per day CLA group, 20.5±13.8, which represented a significant decrease from baseline for the placebo and 6.4g per day CLA groups (P<0.05)
• Estimated energy intakes throughout the 12-week intervention period did not differ among the groups
• HDL-cholesterol decreased in both the placebo (-0.1mmol per L) and 6.4g per day CLA groups (-0.01mmol per L) following the intervention
• C-reactive protein (P=0.03; +3.1±5.1 to 10.3mg per L), IL-6 (P=0.04; +439±830.8 to 2,071pg per L), WBC (P=0.005; +1.2±1.5  to 7.4n per L x 10⁹), and alkaline phosphatase (P=0.002; +5.1±5.3 to 79.5U per L) increased, and hemoglobin (P=0.01; -3.9±5.4 to 134.7g per L), hematocrit (P=0.03; -0.01±0.02 to 0.39), and sodium (P=0.0008; -1.3±1.3 to 140.6mmol per L) decreased in the 6.4g per day CLA group from week zero to week 12, although the absolute mean values remained within normal limits. The changes in C-reactive protein, IL-6 and WBC differed significantly from those in the placebo group.
• 13 participants (27%) reported adverse events (four in the placebo group reported gas or bloating; two in the 3.2g per day CLA group reported gas or itching of the face; and seven in the 6.4g per day CLA group reported gas, bloating, indigestion, diarrhea or heartburn). There were no serious adverse events, and the majority of adverse events were mild gastrointestinal symptoms that may have been related to the study pills.

Selected Results on Changes from Week Zero to Week 12 (Unit/12 Weeks)

d	Placebo	3.2g per day CLA	6.4g per day CLA
Body weight, kg	+0.43±0.57	+0.40±0.30	+0.39±0.43
BMI, kg/m2	+0.01±0.27	+0.14±0.14	+0.07±0.19
Body fat mass, kg	+0.14±0.58	-0.09±0.03	-0.17±0.36
Lean body mass, kg	+0.33±0.32	+0.65±0.47	+0.64±0.29*
Fat, percentage	-0.06±0.44	-0.33±0.39	-0.43±0.30

^*Different from zero, P<0.05.

Other Findings

Mean glucose concentrations ranged from 5.1 to 5.2mmol per L.

• Supplementation with 6.4g per day CLA for 12 weeks increased LBM in healthy obese humans
• No significant effect on body fat mass, weight, BMI, REE or RQ
• Increases in C-reactive protein and IL-6 in the 6.4g per day CLA group suggest that the higher doses may increase markers of inflammation in healthy obese humans
• Limitation cited by authors: Study was not powered specifically to examine changes in clinical laboratory measures.

Government:	in part by NIH grants and GCRC program
Industry:	Cognis Corporation Pharmaceutical/Dietary Supplement Company:

• The randomization procedure is described as well as a power calculation. However, the numbers in the groups seem off if there was true randomization at the beginning. The power analysis indicated 16 participants were required for each group, but there is no description on how they then came up with the number of 55 participants to recruit. Then upon the randomization N equaled 17 for placebo, 20 for 3.2g CLA and 18 for 6.4g CLA. Why were there more participants in the 3.2g CLA group? This also raises the question, that since the exact number of participants was equal to the power calculation that this calculation may have been performed post-hoc. 
• If part of the primary goal was to evaluate safety measures of supplementation, then why were some of the clinical values not included in the power calculation?
• With regards to the plasma CLA concentrations, the apparent wide variation in the supplemented groups raises the question as to the confirmation of pill counts and compliance
• Even though participants were instructed to maintain normal exercise routines, physical activity decreases were noted in the placebo and 6.4g per day CLA groups. This is something that could have impacted the results obtained.
• Even though they remained within normal limits, the increases in C-reactive protein, IL-6 and WBC should be considered as possible determents to a dosage of 6.4g per day CLA. The increase in C-reactive protein to 10mg per L could be considered by some researchers as greatly increasing that individual's risk for cardiovascular disease
• Some of the adverse events reported in both CLA groups should also be considered further. Does the report of itching of the face suggest a possible allergic response?