Effects of milk supplementation with conjugated linoleic acid (isomers cis-9, trans-11 and trans-10, cis-12) on body composition and metabolic syndrome components

Abstract

The effects of conjugated linoleic acid (CLA) on body weight and body composition in man are controversial. 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. This was a randomised, double-blind, placebo-controlled trial. Subjects were randomised to a daily intake of 500 ml milk supplemented with 3 g CLA (using a mixture of the bioactive isomers cis-9, trans-11 and trans-10, cis-12, marketed as Tonalin, Naturlinea; Central Lechera Asturiana) or placebo for 12 weeks. Sixty healthy men and women (aged 35-65 years) with signs of the metabolic syndrome participated (BMI 25-35 kg/m2). Dual-energy X-ray absorptiometry was used to measure body composition (week 0 baseline and week 12). Total fat mass in the CLA-milk subgroup with a BMI < or = 30 kg/m2 decreased significantly while no changes were detected in the placebo group (approximately 2 %, P = 0.01). Trunk fat mass showed a trend towards reduction (approximately 3 %, P = 0.05). CLA supplementation had no significant effect on the parameters of the metabolic syndrome, nor was it associated with changes in haematological parameters or renal function. The supplementation of milk with 3 g CLA over 12 weeks results in a significant reduction of fat mass in overweight but not in obese subjects. CLA supplementation was not associated with any adverse effects or biological changes.

Full text

Effects of milk supplementation with conjugated linoleic acid (isomers cis-9,
trans-11 and trans-10, cis-12) on body composition and metabolic syndrome
components
Nuria Laso
1
, Emma Brugue
´
2
, Josep Vidal
2
, Emilio Ros
2
, Joan Albert Arnaiz
3
, Xavier Carne
´
3
, Sergi Vidal
4
,
Sergi Mas
1
, Ramon Deulofeu
5
and Amalia Lafuente
1
*
1
Dep. Farmacologı
´
a y Quı
´
mica Terape
´
utica, Universidad de Barcelona, IDIBAPS, Casanova 143, E-08036 Barcelona, Spain
2
Endocrinology Service, Hospital Clı
´
nico Universitario de Barcelona, Villarroel 170, 08036 Barcelona, Spain
3
Clinical Pharmacology Service, Hospital Clı
´
nico Universitario de Barcelona, Villarroel 170, 08036 Barcelona, Spain
4
Nuclear Medicine Service, Hospital Clı
´
nico Universitario de Barcelona, Villarroel 170, 08036 Barcelona, Spain
5
Clinical Biochemistry Service, Hospital Clı
´
nico Universitario de Barcelona, Villarroel 170, 08036 Barcelona, Spain
(Received 20 December 2006 – Revised 12 March 2007 – Accepted 27 March 2007)
The effects of conjugated linoleic acid (CLA) on body weight and body composition in man are controversial. The aim of this study was to inves-
tigate the effects of milk supplementation with CLA on body composition and on the biochemical parameters of the metabolic syndrome. This was
a randomised, double-blind, placebo-controlled trial. Subjects were randomised to a daily intake of 500 ml milk supplemented with 3 g CLA (using
a mixture of the bioactive isomers cis-9, trans-11 and trans-10, cis-12, marketed as Tonalin
w
, Naturlinea; Central Lechera Asturiana) or placebo
for 12 weeks. Sixty healthy men and women (aged 35–65 years) with signs of the metabolic syndrome participated (BMI 25–35 kg/m
2
). Dual-
energy X-ray absorptiometry was used to measure body composition (week 0 baseline and week 12). Total fat mass in the CLA-milk subgroup
with a BMI # 30 kg/m
2
decreased significantly while no changes were detected in the placebo group (, 2%, P¼ 0·01). Trunk fat mass showed a
trend towards reduction (, 3%, P¼ 0·05). CLA supplementation had no significant effect on the parameters of the metabolic syndrome, nor was
it associated with changes in haematological parameters or renal function. The supplementation of milk with 3 g CLA over 12 weeks results in a
significant reduction of fat mass in overweight but not in obese subjects. CLA supplementation was not associated with any adverse effects
or biological changes.
Conjugated linoleic acid: Body fat composition: Metabolic syndrome: Man: BMI
Conjugated linoleic acid (CLA) is a mixture of linoleic acid
isomers with conjugated double bonds. This fatty acid is
produced naturally in the rumen of ruminant animals by
biohydrogenation of linoleic acid or synthetically
1
. CLA was
first identified when extracts from fried beef were found to
be anticarcinogenic
2
. This effect was confirmed in animal
and in vitro models of carcinogenesis
3–8
. Further studies
showed other beneficial effects of this CLA, including protec-
tion against arteriosclerosis, immune stimulation and modu-
lation of body composition
9–12
.
The main isomer of CLA in natural foods is cis-9, trans-11,
but trans-10, cis-12 is the isomer that affects energy metab-
olism and body fat deposition and composition in mice
11,12
.
The main dietary source of CLA for man is ruminant
meats, such as beef and lamb, and dairy products, such as
milk and cheese. The mean estimated daily CLA intake is
0·3–2·6 g/d
13,14
, but in recent years, dietary changes have
resulted in a decrease of CLA consumption.
It has been proposed that supplementing food with CLA could
benefit health
15
. The effects of CLA on body weight and body
composition in man are documented, although some clinical
studies in this field have been short term or have included a
small sample of subjects
16
. Moreover, the type of CLA isomer
and the dose used varied between studies which may account
for the discordant results. While some studies using a mixture
of the bioactive isomers cis-9, trans-11 and trans-10, cis-12,
reported reductions in body fat mass (BFM)
17 – 20
or an increase
in lean body mass
17,18,21
, other short-term studies showed no
effect on body composition
22 – 25
.
Here we examined the effect of a 50 % mixture of the two
active CLA isomers (cis-9, trans-11 and trans-10, cis-12;
Tonalin
w
, Naturlinea; Central Lechera Asturiana, Spain)
added to a skimmed milk, on BFM, lean body mass, BMI
and biochemical parameters related to the metabolic
syndrome. Although no serious toxic effects of CLA have
been reported, we also evaluated alterations in the hepatic
* Corresponding author: Dr Amalia Lafuente, fax þ 34 934035881, email amalialafuente@ub.edu
Abbreviations: BFM, body fat mass; CLA, conjugated linoleic acid; DEXA, dual-energy X-ray absorptiometry.
British Journal of Nutrition (2007), page 1 of 8 doi: 10.1017/S0007114507750882
q The Authors 2007

and renal function in addition to changes in haematological
parameters.
Methods
We performed a randomised, double-blind, placebo-controlled
dietary intervention trial. Participants in the study were men
and women (n 60), aged 35– 65 years, with a BMI of 25 –35
(kg/m
2
) and with a waist diameter . 102 cm in men and
. 88 cm in women. Moreover, to be included in the study,
subjects had to fulfil two additional National Cholesterol Edu-
cation Program criteria for the metabolic syndrome, namely
(1) systolic pressure . 130 mmHg or diastolic blood pressure
. 85 mmHg; (2) fasting plasma glucose . 110 mg/dl; (3)
HDL-cholesterol , 50 mg/dl in women and , 40 mg/dl in
men; (4) TAG . 150 mg/dl. Otherwise participants were
apparently healthy. Finally, participants had to show a stable
weight defined as a body weight variation of , 5 % in the 3
months previous to the study. Exclusion criteria included:
alcoholism, active thyroid disease, diabetes mellitus treated
with insulin or drugs, renal or liver dysfunction, and malignant
tumours or other serious diseases. Moreover, subjects on anti-
obesity drugs, with dietary variations of over 10 % in kJ/d
during the study, subjects taking adrenergic agonists (a, b
or both), pregnant or lactating women and subjects participat-
ing in another dietetic study were also excluded. The study
was approved by the Ethical Research Board of the Hospital
Clinic. Patients gave their signed informed consent. All sub-
jects were recruited either from a primary care centre or an
outpatient clinic dealing with cardiovascular risk. Subjects
were randomised to receive 3 g CLA (using a mixture of the
bioactive isomers cis-9, trans-11 and trans-10, cis-12; Tona-
lin
w
; n 30) in 500 ml skimmed milk (0·3 % total fat mass;
Central Lechera Asturiana) or placebo (500 ml of the same
skimmed milk; n 30) every day for 12 weeks. Packages of
milk were delivered monthly by a home delivery service.
The study protocol is summarised in Fig. 1. In brief, demo-
graphic data were recorded when subjects began the study
(at week 2 6). Weight, waist circumference, BMI, blood
pressure, dietary control and adverse effects were recorded
on each monthly visit (week 2 6, week 0, week 4, week 8,
week 12). Body composition was analysed by dual-energy
X-ray absorptiometry (DEXA; with a Lunar Prodigy and soft-
ware version 5.6). BFM (trunk and total) and lean body mass
(trunk and total) were assessed at each time-point.
Blood samples were obtained from fasting subjects in week
2 6 to check whether they met the inclusion criteria and again
at week 0 and week 12. All samples were analysed in the same
laboratory. The following analytic variables were studied:
metabolic syndrome parameters (HDL-cholesterol, TAG, fast-
ing plasma glucose and fasting immunoreactive insulin) and
security parameters (haematological profile, renal and hepatic
function). Insulin sensitivity was estimated from the HOMA-
IR index [HOMA-IR ¼ fasting glucose (mmol/l) £ fasting
immunoreactive insulin (mU/ml)/22·5].
Compliance and changes in the current diet were assessed
through 3 d diet records on each visit. Each subject also com-
pleted a FFQ at weeks 2 6, 0 and 12. A dietitian provided gen-
eral counselling 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.
A software program, Dietsource
w
(Novartis, Switzerland)
was used to calculate energy intake and macro- and micro-
nutrient distribution. The dietitian also gave recommendations
about exercise which was monitored throughout the study
using the International Physical Activity Questionnaire
26
.
Three exercise categories were established (low, medium
and intensive). Since the main aim of the study was to test
the effects of CLA on body composition, 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 . 5 % during the study.
Statistical analyses were performed using the SPSS soft-
ware package version 11.0 (SPSS Inc., Chicago, IL, USA).
Changes in anthropometric and analytical variables from base-
line to week 12 in each study group were assessed by paired
Student’s t -test. An unpaired Student’s t-test was used to com-
pare 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 x
2
; Student’s t-test was used to assess differences in
continuous variables (age). P, 0·05 was considered statisti-
cally significant.
Results
In our trial, ten participants from the CLA group and seven
from the control group were excluded from analysis because
of protocol violations: because of loss of follow-up (one par-
ticipant from each group), dietary energy intake variations
$ 10 % during the study (seven participants from the CLA
group and four participants from the placebo group), weight
reduction $ 5 % (one participant from the CLA group and
two participants from the control group), both dietary and
weight variations (one participant from the CLA group)
(Fig. 2). There were no differences in exclusion percentages
in the two groups (x
2
NS). Primary DEXA and analysis
were performed in the ‘per protocol’ population. Patients
lost to follow-up and patients with major protocol violations
(those with dietary or weight changes outside the pre-defined
boundaries) were excluded from the study.
At baseline, mean age (CLA 54·79 (
SE 7·55) years; placebo
52·92 (
SE 7·92) years), sex (CLA 75 % male; placebo 78 %
male) or tobacco consumption (CLA 31·5 % smokers; placebo
41·6 % smokers) showed no significant differences between
the two groups. Alcohol consumption and exercise categories
were also similar in the two groups. Likewise, at this time-
point, metabolic syndrome variables (Table 1) did not differ
Weeks –6 0–4 4 8 12
Dietetic and
clinical control
Analytic
control
Densitometry
Fig. 1. Study design.
N. Laso et al.2

Patients randomised
(
n
60)
CLA
(
n
30)
Placebo
(
n
30)
Lost to follow-up
(
n
1)
Lost to follow-up
(
n
1)
Protocol violation
Dietary: 7
Weight: 1
D+W: 1
Protocol violation
Dietary: 4
Weight: 2
D+W: 0
Protocol compliant
(
n
20)
Overweight (
n
10); male/female 6/4; mean age 55.5±6
Obese (
n
10); male/female 9/1; mean age 54.1±8
Protocol compliant
(
n
23)
Overweight (
n
11); male/female 10/1; mean age 49.9±8
Obese (
n
13); male/female 8/5; mean age 55.4±7
Data available
(
n
29)
Data available
(
n
29)
Fig. 2. Study participant data. CLA, conjugated linoleic acid.
Table 1. Clinical values of metabolic syndrome of subjects participating in the conjugated linoleic acid (CLA) and placebo groups, at week
0 (pre-value) and week 12 (post-value) and stratified by BMI at the beginning of the study
BMI # 30 BMI . 30
CLA Placebo CLA Placebo
Mean
SE Mean SE Mean SE Mean SE
BMI (kg/m
2
) Pre 28·1 2 27·1 1 32·8 2 33·4 1
Post 27·9 2 27·1 1 32·7 2 33·1 2
Systolic blood pressure (mmHg) Pre 146·1 15 145·0 19 150·5 13 146·9 17
Post 142·2 20 148·1 25 155·5 12 150·3 18
Diastolic blood pressure (mmHg) Pre 83·8 9 81·3 8 87·0 10 85·3 11
Post 81·6 9 80·9 11 88·5 9 83·8 7
Waist circumference (cm) Pre 101·4 9 99·4 6 111·9 7 109·5 9
Post 101·3 9 99·0 6 111·3 8 109·4 10
Total intake
in kJ Pre 9506·9 2456 10 177·6 1879 9580·9 2079 8563·8 2289
Post 9614·8 2418 10 391·0 2301 10 139·9 1925 9207·7 1456
in kcal Pre 2272·2 587 2432·5 449 2289·9 497 2046·8 547
Post 2298·1 578 2483·5 550 2423·5 460 2200·6 348
Glycaemia (mg/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
HDL-cholesterol (mg/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
LDL-cholesterol (mg/dl) Pre 159·2 37 134·0 37 153·7 29 161·4 38
Post 175·6 42 127·0 38 155·8 32 155·1 31
Total cholesterol (mg/dl) Pre 253·3 69 205·5 37 235·2 39 241·4 42
Post 262·5 61 204·9 43 244·4 41 234·6 38
TAG (mg/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
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
Mean values were significantly different from the pre-values when comparing paired data within groups: *P¼ 0·02; **P¼ 0·009.
Mean value was significantly different from that of the placebo group: †P¼ 0·04.
§ HOMA-IR ¼ fasting glucose (mmol/l) £ fasting immunoreactive insulin (mU/ml)/22·5.
Milk supplemented with CLA and fat-mass reduction 3

significantly between the two groups. In the overweight sub-
group, total fat tissue was significantly higher in the CLA
than in the placebo group at week 0 (Table 2).
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.
When all subjects were considered, neither BFM (total fat
tissue 30·6 to 30·5 kg in the CLA group; 30·3 to 30·0 kg in
the placebo group) nor lean body mass (52·0 to 52·2 kg in
the CLA group; 52·4 to 52·9 kg in the placebo group) differed
significantly from baseline values. Gender did not affect these
parameters. However, when subjects were stratified by BMI at
week 0 (BMI . 30 or # 30 kg/m
2
), we observed a slight but
significant decrease in total fat mass and a trend towards a
decrease in trunk fat mass in the CLA subgroup when compar-
ing paired data within cases (pre v. post values), while no
changes in the placebo group were detected (Table 2). The
differences were statistically significant when the change (D)
in BFM parameters were compared between the two groups,
CLA v. placebo. When ANOVA was used to test the overall
effect of the CLA supplementation (F ¼ 0·02; P¼ 0·8), BMI
(F ¼ 0·001; P¼ 0·9) and interaction between the two, the
former interaction was statistically significant (F ¼ 8·3;
P¼ 0·006).
CLA supplementation was not associated with a significant
change in any of the components of the metabolic syndrome
compared with the control (Table 1). At week 12, there was
no significant change in waist circumference, fasting plasma
glucose, plasma TAG, total cholesterol, or systolic or diastolic
blood pressure. LDL-cholesterol increased slightly in both
CLA groups but these changes were not statistically signifi-
cant. In contrast, in the subgroup of overweight patients,
HDL increased in both groups. Insulin sensitivity at the end
of the study period, as assessed from the HOMA-IR index,
did not differ significantly from baseline levels. We did not
observe a worsening in either hepatic or renal function mar-
kers, nor in haematological parameters in the CLA group
(Table 3). We detected a significant reduction of alanine ami-
notransferase, a marker of liver disease in the CLA overall
group (P¼ 0·01; results not shown). The statistical significance
disappeared when the groups were stratified by BMI. Eight
subjects in the control group (26·6 %) and one case (3·5 %)
in the CLA group presented mild intestinal adverse effects
(laxative effects and flatulence).
Discussion
Milk supplementation with CLA (3 g/d) significantly reduces
BFM after 12 weeks of treatment in overweight patients but
not in grade I obesity subjects. Reductions affected both
total and trunk values. We used DEXA technology to deter-
mine changes in body composition. This method has been
validated by other studies and it can detect even small changes
in body weight or composition.
The literature on the effects of CLA on body composition
gives discordant results. Some of the factors that may account
for disparity between studies are the type of CLA isomers
used, the length of the treatment period and the type
of patients enrolled. Table 4 shows some of the most relevant
studies in this field. Most of the short-term assays (3 months)
have been done with overweight patients. We believe that the
lack of effect of CLA on BFM in obese subjects could be
related to the short duration of the study. A 3- or 4-month
study may not be long enough to detect all the changes
caused by CLA. Only the study of Riseru
´
s et al.
23
showed sig-
nificant changes in obese subjects (BMI $ 30) after 3 months
of treatment. However, anthropometric measurements may not
be reliable. The study of Gaullier et al.
27,28
, the only long-term
one (12 months and 24-month follow-up), was performed on
overweight subjects (average BMI between 27 and 28).
These subjects showed reductions of 7·5 % in BFM from 6
to 12 months of treatment with CLA. If these changes in
BFM are extrapolated to 3 months (Fig. 3), they would
coincide with the 3 % decrease observed in the present
Table 2. Results of dual-energy X-ray absorptiometry, at week 0 (pre-value) and week 12 (post-value) and stratified
by BMI at the beginning of the study
BMI # 30 BMI . 30
CLA (n 10) Placebo (n 11) CLA (n 10) Placebo (n 13)
Mean
SE Mean SE Mean SE Mean SE
Trunk fat tissue (kg) Pre 16·7 4 13·7 2 18·7 2 21·4 5
Post 16·2 4 13·7 2 19·0 2 21·1 5
D 2 0·50‡ 0·05 0·26 2 0·33
Total fat tissue (kg) Pre 29·1† 7 22·6 3 32·1 5 36·7 7
Post 28·5* 6 22·9 4 32·4 5 36·1 8
D 2 0·61‡‡ 0·28 0·32 2 0·67
Trunk lean tissue (kg) Pre 22·5 4 23·8 2 26·9 4 25·6 5
Post 22·3 4 23·5 2 27·0 4 25·9 5
D 2 0·15 2 0·33 0·08 0·33
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
D 0·32 0·42 0·06 0·55
CLA, conjugated linoleic acid; D, change.
Mean value was significantly different from the pre-value when comparing paired data within groups: *P¼ 0·02.
Mean value was significantly different from those of the placebo group: †P¼ 0·03.
D Mean values were significantly different (CLA v. placebo): ‡P¼ 0·05; ‡‡P¼ 0·01.
N. Laso et al.4

study. In the present study most of the fat mass was lost from
the trunk area, which is not surprising since individuals suffer-
ing from the metabolic syndrome usually present an accumu-
lation of abdominal fat. The use of the DEXA technique
allows not only the study of body composition but also the
descriptive study of such modifications in the different areas
of the body.
It should be emphasized that this is the first study to be
reported in which the mixture of two isomers was assessed
in a large number of subjects and also the first in which the
effects on body composition were measured using DEXA in
both overweight and obese subjects.
It has been claimed that cis-9, trans-11, the main isomer
found in the diet, modulates growth
29
, and that trans-10,
cis-12 has a beneficial effect on body composition and dia-
betes in mice
30
. Furthermore, trans-10, cis-12 also has been
found to increase insulin resistance in a low percentage of
human subjects with metabolic syndrome
23
.
The mechanisms by which CLA may affect body compo-
sition are unclear. CLA is believed to accumulate in tissues
of animals and man, where it is readily metabolised. In vitro
and in vivo studies report that the capacity of CLA to reduce
adipose tissue can be explained by one or more of the following
mechanisms: the induction of adipocyte apoptosis
31
; reduced
accumulation of fatty acids in adipocytes because of an
inhibition of lipoprotein lipase and an increase in carnitine
palmitoyltransferase in serum
32
; the binding to peroxisome
proliferator-activated receptor present in fat tissue and modifi-
cation of the signalling cascade to down-regulate leptin
expression
33
; the prevention of the TAG accumulation in adipo-
cytes
34
; and the modification of the metabolic rate
11,35
.
In the present study, energy intake slightly increased
throughout the study in both experimental groups, although
differences were not significant. This observation indicates
that the effects of CLA on fat mass are diet-independent.
Exercise and use of tobacco did not differ significantly
between groups, and therefore neither factor can explain the
changes in body composition observed in the CLA group.
Although CLA supplementation was associated with a
change in body composition, it was not related to significant
alterations of metabolic parameters. Previous studies on
CLA report contradictory effects on blood lipids, which
have recently been reviewed
36
. According to Tricon et al.
37
,
trans-10, cis-12-CLA could increase both total and LDL-
cholesterol concentrations more than the cis-9, trans-11-
CLA isomer. However, none of the studies reviewed in this
report
36
described significant effects attributable to CLA
intake on the concentrations of total cholesterol or LDL-
cholesterol. Smedman & Vessby
18
observed, as we did in
the present study, increases in LDL-cholesterol levels in the
group receiving CLA, although the changes observed were
not significantly different from the control. In principle, non-
significant changes should be considered as fluctuations
within the normal physiological range. Moreover, in the
present study they were not reproduced in the other BMI
subgroup (. 30). As to other possible modifications of the
lipid profile, the literature contains references to HDL
reduction
16,15
, VLDL reduction
38
or any effect on choles-
terol
21
. In the present study, no significant variation in total
cholesterol or TAG variations were observed. In a similar
study, Basu et al.
39,40
showed that men with metabolic syn-
drome increased F
2
-isoprostane excretion after supplemen-
tation with a CLA isomer mixture. This increased excretion
returned to baseline levels 2 weeks after withdrawing CLA
treatment. These findings indicate that CLA may induce
lipid peroxidation; however, we did not test these possible
effects in the present study.
We did not observe changes in fasting plasma glucose,
blood pressure or insulin sensitivity estimates. Riseru
´
s et al.
20
showed an increased insulin resistance after 12 weeks of treat-
ment with trans-10, cis-12-CLA isomer, in a male population
with metabolic syndrome. Notably this effect was not
observed when the mixture of isomers (Tonalin
w
mixture
used in the present study) was used
20
.
We detected a significant reduction of alanine aminotrans-
ferase in the overall CLA group but the statistical significance
Table 3. Safety analysis, at week 0 (pre-value) and week 12 (post-value) and stratified by BMI at the beginning of the study
BMI # 30 BMI . 30
CLA Placebo CLA Placebo
Intervals reference Mean
SE Mean SE Mean SE Mean SE
Creatinine (mg/dl) 0·3–1·3 Pre 1·0 0 1·2 0 1·1 0 1·2 0
Post 1·0 0 1·2 0 1·1 0 1·1 0
Insulin (mU/l) 12·4 ^ 3 Pre 13·6 4 13·9 3 15·3 10 25·8 7
Post 13·9 3 15·4 6 17·4 9 25·7 13
AST (U/l) 5–40 Pre 29·0 10 24·6 3 25·0 8 26·6 7
Post 25·0 2 27·0 6 23·0 4 25·9 7
ALAT (U/l) 5–40 Pre 31·9 10 29·9 6 33·1 21 32·8 15
Post 27·3 10 33·4 10 27·2 12 33·2 12
Leucocytes ( £ 10
9
/l) 4–11 Pre 7·9 2 7·0 1 7·8 1 7·5 1
Post 7·2 1 6·9 1 7·9 1 7·6 2
Hb (g/dl) 120–170 Pre 150·3 11 149·6 9 153·6 11 146·9 12
Post 147·2 9 149·5 10 152·3 11 146·1 11
Haematocrit (l/l) 0·36 –0·51 Pre 0·4 0 0·4 0 0·5† 0 0·4 0
Post 0·4 0 0·4 0 0·4 0 0·4 0
Platelet ( £ 10
9
/l) 150–400 Pre 280·0†† 51 216·8 30 248·3 51 228·8 54
Post 261·4 45 219·8 26 239·1 46 220·1 46
ALAT, alanine aminotransferase; AST, aspartate aminotransferase; CLA, conjugated linoleic acid.
Mean values were significantly different from those of the placebo group: †P¼ 0·03; ††P¼ 0·01.
Milk supplemented with CLA and fat-mass reduction 5

Table 4. Review comparing published results
Authors and year Journal and reference number
No. of cases/control
and sex Isomers, dosage and duration Method
BMI averages,
cases/control Results, BFM %
Thom et al. (2000) J Int Med Res
19
10/10 CLA c9,t11; CLA t10,c12; CLA
c11,t13; CLA t8,c10
Near IR light , 25 Reduction BFM
Men 1·8 g
3 months
Riseru
´
s et al. (2001) Int J Obes Relat Metab
Disord
23
14/10 CLA t10,c12; CLA c9,t11 SAD (anthropometry) 32·1/31·7 Reduction SAD (0·6 cm)
Men 4 g
Metabolic standard 4 weeks
Blankson et al. (2000) J Nutr
17
7/8 CLA t10,c12; CLA c9,t11 DEXA 27·7/28·2 Reduction, 6 %
Men and women 3·4 g
3 months
Riseru
´
s et al. (2002) Diabetes Care
20
19/19 1 group CLA t10,c12; SAD impedance 30·1/30·2 No reduction
Men 1 group CLA t10,c12; CLA c9,t11
Metabolic standard 3·4 g
3 months
Smedman & Vessby
(2001)
Lipids
18
25/25 CLA t10,c12; CLA c9,t11 Impedance 20– 25 Reduction, 3·8 %
Men and women 4·2 g
3 months
Kamphuis et al. (2003) Int J Obes Relat Metab
Disord
24
30/30 CLA t10,c12; CLA c9,t11 Hydrodensitometry 26·2/25·7 No reduction
Men and women 3·6 g
3 months
Gaullier et al.
(2004, 2005)
Am J Clin Nutr
27
and
J Nutr
28
60/59 CLA t10,c12; CLA c9,t11 DEXA 28·2/27·7 Reduction, 7·5 % (from 6th
to 12th month)
Men and women 4·5 g
12 months, follow-up 24 months
Malpuech-Brugere
et al. (2004)
Obes Res
25
45/45 1 group CLA t10,c12; DEXA 27·7/27·7 No reduction
Men and women 1 group CLA c9,t11
1·5 and 3 g
4 months
BFM, body fat mass; c, cis; CLA, conjugated linoleic acid; DEXA, dual-energy X-ray absorptiometry; SAD, sagital abdominal diameter; t, trans.
N. Laso et al.6

disappeared when the groups were stratified by BMI. Alanine
aminotransferase can be considered a marker for fat liver
degeneration, which can occur in obese subjects. Studies
such as that by Tsuboyama-Kasaoka et al.
41
show that high
doses of CLA induce liver enlargement and lipodystrophy in
experimental animals. The dose of CLA like the ones used
in the present study does not affect hepatic function, or may
even favour the normalisation of certain enzymes.
We performed a ‘per protocol’ analysis on compliant
patients with no major protocol violations, in order to quantify
the effect of the intervention with a limited sample size.
Patients were randomised when they entered the study, and
so any slight variations between the subgroup studied at
baseline are attributable to chance alone. For example, the
CLA group with the lowest weight (BMI , 30) showed greater
total fat mass in the DEXA compared with placebo group.
The degree of compliance was high in the present study,
over 99 %. This high compliance rate demonstrates good tol-
erance of CLA milk (Naturlinea with Tonalin
w
). Only 3·5 %
of the CLA group and 26·6 % of the control group showed
slight adverse effects in gastrointestinal symptoms such as
abdominal discomfort, laxative effects and flatulence. These
adverse effects have been described by other authors
17 – 20
.
Moreover, the lack of alterations in haematological parameters
and renal function further supports the security of milk sup-
plemented with CLA.
In conclusion, 3 g CLA supplementation (Naturlinea with
Tonalin
w
), over a 12-week period produced a significant
reduction of fat mass in overweight subjects with a BMI # 30.
CLA supplementation was not associated with any adverse
effects or biological changes. Further long-term studies are
needed to evaluate more important reductions or to assess
the effects in obese subjects. The present data indicate that
CLA supplementation may be a useful approach to reduce fat
mass, which is a prominent cardiovascular risk factor.
Acknowledgements
This study was supported by the Corporacio
´
n Alimentaria
Pen
˜
asanta, The Spanish Ministry of Health, Institute Carlos
III (Ciber CB06/03, Fisiopatologia, obesidad y Nutricio
´
n)
and DURSI GRC2005SGR00039. We especially wish to
thank Roser Mestres, Blanca Valero, Isidora Torralba and
Eva Sua
´
rez (Rossello
´
Primary Care Centre), Marı
´
a Mongay,
Mo
`
nica Domenech and Cristina Sierra (Hypertension Unit,
Hospital Clinic, Barcelona), and Ana Pe
´
rez, Berenice Corte
´
s
and Merce
`
Serra (Lipids Unit, Hospital Clinic, Barcelona)
for facilitating the recruitment of patients.
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