- Click here for explanation of classification scheme.

The aim of this study was to investigate the effects of milk supplementation with CLA on body composition and on the biochemical parameters of the metabolic syndrome.

• Aged 35 to 65 years
• BMI of 25 to 35kg/m²
• Waist diameter higher than 102cm in men and higher than 88cm in women
• Subjects had to fulfill two additional National Cholesterol Education program criteria for the metabolic syndrome:
  • Systolic pressure higher than 130mm Hg or diastolic blood pressure higher than 85mm Hg
  • Fasting plasma glucose higher than 110mg per dL
  • HDL-cholesterol lower than 50mg per dL in women and less than 40mg per dL in men
  • TAG higher than 150mg per dL
• Stable weight defined as a body weight variation of less than 5% in the three months previous to the study.

• Alcoholism
• Active thyroid disease
• Diabetes mellitus treated with insulin or drugs
• Renal or liver dysfunction
• Malignant tumors
• Other serious diseases
• Anti-obesity drugs
• Dietary variations over 10% in kJ per day during the study
• Subjects taking adrenergic agonists (alpha, beta or both)
• Pregnant or lactating women
• Participation in another dietetic study
• Subjects were excluded from the study when their total daily energy intake varied more than 10% or when the variation in energy intake had resulted in a weight change more than 5% during the study.

Recruitment

All subjects were recruited either from a primary care center or an outpatient clinic dealing with cardiovascular risk.

Design

Randomized, double-blind, placebo-controlled.

Blinding used

Double blind.

Intervention

• Placebo
• CLA
• BMI 30 or higher than 30.

Statistical Analysis

• Changes in anthropometric and analytical variables from baseline to week 12 in each study group were assessed by paired Student's T-test
• An unpaired Student's T-test was used to compare the mean change in each study variable between the two treatment groups
• ANOVA was used to test the overall effect of the CLA supplementation and the interaction between CLA and other parameters
• At baseline, differences in qualitative variables (such as sex or tobacco consumption) were tested through Chi-square; student's T-test was used to assess differences in continuous variables (age)
• P<0.05 was considered statistically significant. 

Timing of Measurements

• Study was 12 weeks long
• Demographic data were recorded when subjects began the study at week minus six
• Weight, waist circumference, BMI, blood pressure, dietary control and adverse effects were recorded on each monthly visit (week minus six, week zero, week four, week eight and week 12)
• Body composition was measured at week zero and week 12
• Blood samples were taken in week minus six to check whether they met the inclusion criteria and again at week zero and week 12.

Dependent Variables

• Weight
• Waist circumference
• BMI
• Blood pressure
• Body composition via DEXA (with a Lunar Prodigy and software version 5.6). Body fat mass (trunk and total) and lean body mass (trunk and total) were assess at each time-point
• Blood samples were analyzed for metabolic syndrome parameters (HDL-cholesterol, TAG, fasting plasma glucose and fasting immunoreactive insulin) and security parameters (hematological profile, renal and hepatic function)
• Insulin sensitivity was estimated from the HOMA-IR index [HOMA-IR = fasting glucose (mmol per L times fasting immunoreactive insulin (uU per ml) over 22.5]

Independent Variables

• Placebo: 500ml of the same skimmed milk used for CLA
• CLA: 3g CLA (using a mixture of the bioactive isomers cis-9, trans-11 and trans-10, cis-12; Tonalin) in 500ml skimmed milk (0.3% total fat mass)
• Subjects grouped according to BMI: 30.

Control Variables

• Compliance and changes in the current diet were assessed through three-day diet records on each visit
• Each subject also completed a FFQ at weeks minus six, zero and 12. A dietitian provided general counseling at the beginning of the study with detailed instruction on how to complete the FFQ, but no specific limits were set regarding energy intake or dietary habits.
• The dietitian also gave recommendations about exercise that was monitored throughout the study using the International Physical Activity Questionnaire. Three exercise categories were established (low, medium and intensive).

• Initial N: 60
• Attrition (final N):
  • Figure two shows a schematic of the participant data in terms of number at follow-up
  • Two subjects were lost to follow-up: one each from placebo and CLA
  • Protocol violations for the placebo group were: Dietary, four; weight, two; for a loss of six subjects
  • Protocol violations for the CLA group were: Dietary, seven; weight, one; dietary plus weight, one; for a loss of nine subjects
  • Protocol compliant for placebo N=23:
    • Overweight = 11: Males, 10; female, one
    • Obese = 13: Males, eight; females, five
  • Protocol compliant for CLA N=20:
    • Overweight = 10: Males, six; females, four
    • Obese = 10: Males, nine; females, one
  • There were no differences in exclusion percentages in the two groups (chi-square NS).
• Age: Mean ages ranged from 49.9±8 to 55.5±6
• Other relevant demographics:
  • CLA 75% male; placebo 78% male
  • Tobacco consumption: CLA 31.5% smokers; placebo 41.6% smokers
  • Alcohol consumption and exercise categories were also similar in the two groups
  • At baseline, metabolic syndrome variables did not differ significantly between the two groups
• Anthropometrics:
  • At baseline, mean BMI ranged from 27.1 to 33.4kg/m²
  • Waist circumference ranged from 99.4 to 111.9cm
  • In the overweight group, total fat tissue was significantly higher in the CLA (29.1kg) than in the placebo group (22.6kg) at week zero
  • In the obese group, total fat tissue was 32.1kg in the CLA group and 36.7kg in the placebo group.

 Key Findings

• When all subjects were considered, neither BFM (total fat tissue 30.6 to 30.5kg in the CLA group; 30.3 to 30.0kg in the placebo group) nor lean body mass (52.0 to 52.2kg in the CLA group; 52.4 to 52.9kg in the placebo group) differed significantly from baseline values
• When subjects were stratified by BMI at week zero, a slight but significant decrease in total fat mass and a trend towards a decrease in trunk fat mass in the CLA subgroup was observed when comparing paired data within cases (pre- vs. post-values), while no changes in the placebo group were detected
• The differences were statistically significant when the change in BFM parameters were compared between the two groups, CLA vs. placebo
• When ANOVA was used to test the overall effect of CLA supplementation (F=0.02, P=0.8), BMI (F=0.001, P=0.9) and interaction between the two, the former interaction was significant (F=8.3, P=0.006)
• CLA supplementation was not associated with a significant change in any of the components of the metabolic syndrome compared with the control
• At week 12, there was no significant change in waist circumference, fasting plasma glucose, plasma TAG, total cholesterol or systolic of diastolic blood pressure
• LDL-cholesterol increased slightly in both CLA groups but these changes were not statistically significant
• In the subgroup of overweight patients, HDL increased in both groups
• Insulin sensitivity at week 12, as assessed from the HOMA-IR index, did not differ significantly from baseline levels
• No worsening in either hepatic or renal function markers, nor in hematological parameters was observed in the CLA group
• A significant (P=0.01) reduction in alanine aminotransferase (a marker of liver disease) was observed in the CLA overall group; however, the data was not shown. However, no difference was obtained when the groups were stratified by BMI.
• Eight subjects in the placebo group (26.6%) and one subject (3.5%) in the CLA group presented mild intestinal adverse effects (laxative effects and flatulence).

Table 1 and 2. Selected results on clinical values of metabolic syndrome and body composition.

d	BMI CLA	BMI Placebo	BMI Higher than 30 CLA	BMI Higher than 30 Placebo
BMI (kg/m2)
Pre	28.1±2	27.1±1	32.8±2	334±1
Post	27.9±2	27.1±1	32.7±2	33.1±2
Glycemia (mg per dL)
Pre	96.4±19	87.2±18	91.4±18	103.4±32
Post	90.2±11	83.7±20	87.8±23	102.9±41
TAG (mg per dL)
Pre	170.0±107	161.0±88	183.9±141	183.7±51
Post	167.6±72	214.9±197	194.3±102	238.0±178
HDL-cholesterol (mg per dL)
Pre	47.2±8	39.3±7	46.0±11	43.2±7
Post	53.2±10*	45.1±6**	49.0±11	45.0±9
HOMA-IR index
Pre	3.2±1	3.0±1	3.7±3&	6.6±3
Post	3.1±0	3.2±1	3.8±2	6.7±4
Total fat tissue (kg)
Pre	29.1±7a	22.6±3	32.1±5	36.7±7
Post	28.5±6*	22.9±4	32.4±5	36.1±8
Change	-0.61b	0.28	0.32	-0.67
Total lean tissue (kg)
Pre	47.4±10	51.3±6	56.1±8	53.2±12
Post	47.7±10	51.7±6	56.2±8	53.8±13

Mean values were significantly different from the pre-values when comparing paired data within groups: ^*P=0.02; ^**P=0.009.

^&Mean value was significantly different from that of the placebo group: P=0.04. 

^aMean value was significantly different from those of the placebo group: P=0.03.

^bChange mean values were significantly different (CLA vs Placebo): P=0.01.

Other Findings

• Compliance with treatment was over 99%. At week 12, daily energy intake, body weight and BMI had not altered from baseline values in either group. [Reviewer note: This statement is misleading because it does not include those excluded due to protocol violations.]
• Gender did not affect body composition values.

• 3g CLA supplementation over a 12-week period produced a significant reduction of fat mass in overweight subjects with a BMI of 30 or less
• CLA supplementation was not associated with any adverse effects of biological changes
• Further long-term studies are needed to evaluate more important reductions or to assess the effects in obese subjects.

Government:	Spanish Ministry of Health, Institute Carlos III
Industry:	Corporacion Alimentaria Penasanta Other:

• Method of randomization was not given
• Fasting glucose in the BMI higher than 30 placebo group was greater than 100 at week zero, whereas it was 87 in the CLA group. This suggests that maybe fasting glucose concentrations should have been considered as well when stratification was performed.
• The authors stated '... this is the first study to be reported in which the mixture of two isomers was assessed in a large number of subjects....'. However, later in the discussion they state they used a 'per protocol' analysis on compliant patients in order to quantify the effect of the intervention with a limited sample size
• It is also restated in the discussion that compliance was high in the present study. This only refers to the consumption of the CLA because 15 subjects were excluded from data analysis due to protocol violations.
• There were three exercise categories and it was stated that the exercise categories were similar between the CLA and placebo groups. However, no other mention of exercise occurs. Does CLA have differing effects depending on the amount and intensity of exercise?