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Department of Experimental Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263;
*
Dipartimento di Biologia Sperimentale, Universita degli Studi di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy;
Division of Laboratory Research, AMC Cancer Research Center, Denver, CO 80214;
**
Department of Food Science and
Department of Animal Science, Cornell University, Ithaca, NY 14853
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9-desaturase reaction.
Further studies will be conducted to investigate other attributes of this novel dairy product.
KEY WORDS: • conjugated linoleic acid • butter fat • mammary gland morphogenesis • mammary cancer prevention • tissue CLA isomers • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) because of the biohydrogenation of dietary unsaturated
fatty acids by rumen bacteria. CLA is an intermediate in the biohydrogenation of linoleic acid, and a portion of the CLA in milk fat
arises from CLA that has escaped complete rumen biohydrogenation. The other portion of CLA in milk fat is synthesized in the tissues by
9-desaturase from trans-11 18:1, another intermediate in rumen biohydrogenation (Griinari and Bauman 1999
).
Recent studies have shown that the milk fat content of CLA can be markedly enhanced by several dietary manipulations, especially those
involving dietary additions of plant oils, which are high in unsaturated fatty acids (Griinari and Bauman 1999
,
Kelly et al. 1998
).
CLA is a potent cancer preventive agent (Ip et al. 1994
). In animal models of chemical carcinogenesis, CLA has
been shown to inhibit skin papillomas (Belury et al. 1996
), forestomach neoplasia (Ha et al. 1990
),
mammary tumors (Ip et al. 1996
) and colon aberrant crypt
foci (Liew et al. 1995
). Moreover, CLA is also effective
in reducing the size and metastasis of transplanted human breast cancer
cells and prostate cancer cells in SCID mice (Cesano et al. 1998
, Visonneau et al. 1997
). In the rat model,
Ip et al. (1995)
also demonstrated that when CLA feeding
was limited to only the period of pubescent mammary gland development,
it was able to confer a lasting protection against the subsequent
induction of mammary tumors. An evaluation of the digitized image of
the mammary tree in whole mounts suggested that there was diminished
epithelial branching as a result of CLA treatment (Thompson et al. 1997
). This change in morphogenesis could account in part
for the reduced susceptibility to cancer induction because of a
decrease in the population of terminal end buds (Banni et al. 1999
), which are the target sites of mammary carcinogenesis.
To date, all of the in vivo work with CLA has been done with a
commercial free fatty acid preparation containing a mixture of 8,10-,
9,11-, 10,12- and 11,13-isomers, although CLA in food is predominantly
(80–90%) the 9,11-isomer present in triacylglycerols. To date, there
has been no information on whether CLA delivered as a constituent of
food has biological activities similar to those of the mixture of CLA
isomers delivered as free fatty acids. This was one of the objectives;
thus, we fed dairy cows in a manner that allowed the production of high
CLA butter. In addition to feeding a group of rats the high CLA butter,
the design included two other groups that were given either a mixture
of CLA isomers (obtained from Nu-Chek-Prep, Elysian, MN, hence
designated as Nu-Chek CLA for convenience) or a synthetic CLA
preparation consisting predominantly of the 9,11-isomer (obtained from
Matreya, Pleasant Gap, PA, designated as Matreya CLA); both
preparations provide CLA in the free fatty acid form. A major goal of
this investigation therefore was to address the question whether
different CLA isomers have different biological activities. The
prepubertal rat model alluded to above (Ip et al. 1995
,
Thompson et al. 1997
) was employed for this research.
The following endpoints were used to assess the activities of the
different sources of CLA: 1) image analysis of mammary gland
development; 2) terminal end bud (TEB) density;
3) proliferative activity of TEB cells; and 4)
mammary cancer prevention bioassay. Measurements of CLA isomer
incorporation into tissues were also made to provide insight regarding
the bioavailability of individual isomers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Holstein cows were used to produce the milk fat that was used to make
butter. Cows were located at the Cornell University Teaching and
Research Center; this portion of the study was approved by the Cornell
University Institutional Animal Care and Use Committee. Control cows
(n = 10) were fed a total mixed diet composed of
concentrates plus corn silage as the roughage source. Cows
(n = 20) used to provide the high CLA butter were
fed a similar diet with the addition of 5.3% sunflower oil
(Kelly et al. 1998
). After 1 wk of consuming the
sunflower oil diet, milk samples were obtained and the CLA content of
the milk fat was determined. There was substantial individual variation
in CLA concentration as reported previously (Kelly et al. 1998
). Cows (n = 9) with the highest
concentrations of CLA continued to consume the diet for a second week,
and their milk was collected to make the high CLA butter.
Raw milk was pasteurized by the high temperature–short time method (model #3919, Alfa-Laval Type-P13-RCF 1982, Kenosha,WI) at 175°F for 18 s, then separated into cream and skim milk. The cream was vat pasteurized at 162°F for 30 min and stored in the cooler for 24 h. It was then churned (Zane Butter Churn Model #A, General Dairy Equipment, Minneapolis, MN) for 30 min at 50°F until butter was the size of popcorn kernels; then the buttermilk was drained off. The butter was rinsed and washed with 4°C water, and the unsalted butter was transferred to 0.5-kg plastic containers and kept at -20°C until use.
Fatty acid methyl esters for butter fat analysis were prepared by the
procedure of Christie (1982)
and determined by gas
chromatography with the use of a Hewlett-Packard GCD system
(Palo Alto, CA) equipped with HP G107A GCD software for peak
integration (Kelly et al. 1998
). Control butter and high
CLA butter contained 5.1 and 41.0 mg CLA/g of fat, respectively. The
fatty acid composition of these two types of butter is shown in
Table 1
. Saturated fatty acids (4:0–18:0) constituted ~67.7% of the total
in control butter fat, but only 48.1% in high CLA butter fat. A major
difference was accounted for by palmitic acid, 28.6% vs. 17.8%. The
concentration of oleic acid was quite similar, i.e., 18.5% in control
butter fat vs. 16.0% in high CLA butter fat. However, there was much
more trans-octadecenoic acid in the high CLA butter fat
(25.0%) than in the control butter fat (3.8%); a major
trans-isomer was vaccenic acid
(t11-C18:1). Despite the differences in their fatty acid
composition, we decided to use the two kinds of butter fat as is,
without making any further adjustment to the fatty acid composition of
the rat diet. The argument is that the most important fatty acid for
modulation of mammary carcinogenesis in the rodent model is linoleic
acid (Ip 1987
and Ip 1997
), and the level of this fatty
acid was equivalent in the two kinds of butter fat.
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Pathogen-free female Sprague-Dawley rats were purchased from
Charles River Breeding Laboratories (Raleigh, NC) at weaning. Upon
arrival, they were divided equally into four dietary groups:
1) control butter fat, which provided 0.1% CLA in the
diet; 2) high CLA butter fat, which provided 0.8% CLA
in the diet; 3) Matreya CLA; and 4)
Nu-Chek CLA. Matreya CLA and Nu-Chek CLA, purchased from their
namesake vendors located in Pleasant Gap, PA, and Elysian, MN,
respectively, were added as free fatty acids to the diet to match the
total level of butter CLA present in the second group. Table 2
summarizes the butter fat content of the different diets, the
composition of the remaining ingredients, the total CLA level in each
diet and the major CLA isomers in the various sources of CLA. It should
be noted that the analysis of CLA isomers in the commercial CLA
preparations was done by one of the authors (S.B.), and is not based on
information provided by the vendors. All rats were fed these four
different diets for 1 mo (i.e., from 23 to 55 d of age) to prepare
them for the following four sets of experiments: 1)
digitized image analysis of mammary epithelium in whole mounts,
n = 9; 2) quantitation of TEB
density in the mammary gland and assessment of proliferative activity
in TEB cells, n = 6; 3) mammary
carcinogenesis bioassay, n = 30; and
4) measurements of CLA isomers in tissues,
n = 6.
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The abdominal-inguinal mammary gland chain was excised in one piece and stretched onto a 75 x 50 mm2 microscope slide. The whole mount was fixed in methacarn for 12–18 h and rinsed in 70% ethanol. It was then dehydrated using a series of ethanol solutions (70, 95 and 100%) for 1 h each and cleared in xylene for 2 h. The tissue was rehydrated with descending grades of ethanol and immersed in fresh 0.4% alum carmine stain for 3 d. Once staining was completed, the whole mount was dehydrated using ethanol as described above and cleared with one change of xylene for 2 h. Each whole mount was then placed in a 10 x 15 cm2 heat-sealable pouch and filled with 20 mL of methyl salicylate. Methyl salicylate was chosen as the clearing agent because its refractory index is very close to that of tissue. This resulted in superior photographic resolution with a clean background. The pouch was left overnight; on the next day, it was pressed flat to remove excess methyl salicylate and air.
Digitization of whole mounts and assessment of optical density of mammary epithelium.
All whole-mount images were captured by digital photography (Kodak
DCS 420, Kodak Digital Science, Rochester, NY) with the light source
passing through the sample from underneath the slides. The Kodak DCS
420 is a digital camera that has a spatial resolution of 1.5 x 106 pixels (1012 x 1524) per image. The images were
downloaded to Adobe Photoshop (Adobe Systems, San Jose, CA) using a
Kodak DCS TWAIN driver. The digitized color images were converted to
gray scale (256 shades) images and analyzed by the Image-Pro Plus
software (Media Cybernetics, Silver Spring, MD). An image-filtering
technique was introduced to reduce the intensity variations in the
background pixels. Measurements of the mammary fat pad area and the
mass of mammary epithelium were performed on the digitized image of the
entire abdominal-inguinal mammary gland chain. Mammary fat pad area
is defined as the area within the perimeter bounded by circumscribing
the outermost terminal end buds of the mammary tree. The mass of
mammary epithelium was determined based on the calculation of 
(a
defined area containing mammary epithelial elements x optical
density of the assigned area). This integrated value is expressed in
arbitrary units of optical density (AUOD). Statistical analysis among
groups was done by ANOVA with post-hoc comparisons using Tukey’s
multiple range test (Zhu et al. 1998
).
Quantitation of TEB density.
The procedure of preparing mammary whole mounts and staining with alum
carmine was similar to that described in the above section. The outer
5-mm margin of the mammary whole mount was examined by light microscopy
using the criteria of Russo and Russo (1978)
. This area
represents the location of most of the actively proliferating TEB
structures of the mammary gland for a young virgin rat. Images from a
minimum of eight fields were transferred from the microscope to a Kodak
8650 PS color printer. Hard-copy pictures were printed out with the
use of Adobe Photoshop (Adobe Systems, San Jose, CA). The density of
TEB in each field was determined by adjusting the micrometer bar to the
power of magnification (Banni et al. 1999
). Statistical
analysis was done by ANOVA with post-hoc comparisons as described
above.
Immunohistochemical staining of PCNA in TEB cells.
The proliferating cell nuclear antigen (PCNA) is expressed in early G1 and S phases of the cell cycle and serves as a good marker for proliferating cells. Methacarn-fixed mammary tissues were processed in a Tissue-Tek Vacuum Infiltration Processor (Miles Scientific, Elkhart, IN) and embedded in paraffin blocks. Ribbons of 5-µm thickness were cut and placed on slides that had been treated with 3-aminopropyl-triethoxysilane. The sections were heat immobilized, deparaffinized in xylene, rehydrated in descending grades of ethanol and rinsed in deionized water and then PBS.
Mouse monoclonal PCNA antibody, purchased from Santa Cruz
Biotechnology, (Santa Cruz, CA), was used at a dilution of 1:20,000.
Tissue sections were exposed to the primary antibody for 1 h at
room temperature in a humid chamber. They were then treated with a
biotinylated rabbit secondary antibody against mouse immunoglobulin.
This was followed by the addition of streptavidin horseradish
peroxidase, which binds to biotin. Diaminobenzidine was used as the
chromogen to generate a brown precipitate due to its reaction with
peroxidase. All slides were counterstained with hematoxylin, rinsed,
dehydrated and mounted with Permount (Fisher Scientific, Fair Lawn,
NJ). Cells expressing the antigen were identified by a brown stain over
the nucleus. Because immunohistochemical staining for a protein is not
stoichiometric relative to the amount of protein present, differences
in staining were analyzed by using a Kruskall-Wallis rank test as
described in detail previously (Zhu et al. 1998
).
Mammary carcinogenesis bioassay.
Rats in the four different dietary groups were given a single dose (50
mg/kg body weight) of methylnitrosourea (MNU) intraperitoneally at
55 d of age for the induction of mammary tumors. Immediately after
carcinogen treatment, all rats were switched to a basal 5% corn oil
diet (Ip et al. 1995
) without CLA. This diet consisted
of 5% corn oil, 20% casein, 65% dextrose, 3.5% AIN-76 mineral mix,
1% AIN-76A vitamin mix, 5% alphacel, 0.3% methionine and 0.2%
choline bitartrate. Rats were palpated for mammary gland tumors once a
week. The experiment was continued for 24 wk before termination. By
this time, the development of palpable tumors had reached a plateau for
several weeks across all groups. At necropsy, all tumors were excised
and fixed for histological evaluation. Only confirmed adenocarcinomas
are reported in the results. Tumor incidences at the final time point
were compared by 
-squared analysis, and the total tumor yield was
compared by frequency distribution analysis (Horvath and Ip 1983
).
Analysis of CLA isomers in tissues.
Liver, mammary fat pad, peritoneal fat and plasma were stored at
-80°C until they were ready for analysis. Total lipid was extracted
by the method of Folch et al. (1957)
. Free fatty acids
were obtained by a mild saponification procedure described by
Banni et al. (1994)
. Methyl esters were prepared by the
addition of 14% BF3/CH3OH at room temperature
and immediately extracted into a solvent consisting of
n-hexane/water (4:3). After centrifugation at
900 x g for 10 min to separate the two phases, the hexane
phase was saved and the aqueous phase was further extracted by another
round of hexane. The two hexane collections were combined, dried and
redissolved in 500 µL n-hexane.
Although the BF3 method causes isomerization of CLA,
especially at high temperature (Banni and Martin 1998
),
we verified the lack of isomerization by BF3 under the
present condition on the basis of the equal analysis of free fatty acid
t,t-CLA isomers measured in a C-18 column and the
methylated t,t-CLA isomers measured in a silver-ion
column (Banni et al. 1994
). Separation of CLA isomers
was carried out with a Hewlett-Packard 1050 HPLC system
(Hewlett-Packard, Palo Alto, CA) equipped with a diode array detector
1040M. A silver-ion ChromSpher 5 lipid Chrompack column (Chrompack
International BV, Middelburg, The Netherlands), 5-µm
particle size, 250 x 4.6 mm, was used with a mobile phase of
n-hexane with 0.0375% of CH3CN at a flow
rate of 1 mL/min. This technique separates the positional and geometric
(cis and trans) isomers of CLA
(Sehat et al. 1998
). Conjugated diene unsaturated fatty
acids were detected at 234 nm. Spectra (195–315 nm) of the eluate were
obtained every 1.28 s and were stored electronically.
Second-derivative UV spectra of the conjugated diene fatty acids
were generated using the Phoenix 3D HP Chemstation (Hewlett-Packard)
software. These spectra were taken to confirm the identification of the
HPLC peaks. Details of the methodology regarding the characterization
of conjugated diene unsaturated fatty acids in both reference and
biological samples have been published by Banni and co-workers (1996)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CLA feeding had no effect on the growth of the rats. After 1 mo of
consuming the different diets starting from weaning, their body weights
were: control group, 180 ± 2 g; butter CLA group, 181
± 2 g; Matreya CLA group, 180 ± 2 g; and
Nu-Chek CLA group, 179 ± 2 g. Figure 1
shows a representative mammary gland whole mount from a rat fed either
the control diet or the 0.8% butter CLA diet. Because rats fed a high
CLA diet during the 1st mo after weaning developed a less complex
network of ductal-alveolar branching regardless of the source of
CLA (i.e., butter CLA, Matreya CLA or Nu-Chek CLA), only one
example from a high CLA group is presented as an illustration. CLA
feeding reduced the development of the mammary tree within the mammary
fat pad. The data on epithelial mass of the entire
abdominal-inguinal mammary gland chain, as presented in Table 3
, are expressed in arbitrary unit of optical density (AUOD). There was
~25% lower total mass of mammary epithelium and amount of epithelium
per unit area of mammary fat pad in all of the CLA-treated groups
compared with the control group (P < 0.01). We did not
detect any differences in the area of the mammary fat pad among the CLA
treatment groups. The results suggest that CLA suppressed mammary
branching morphogenesis but did not interfere with the ability of the
secondary or subtending ducts to invade up to the boundary of the fat
pad.
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). CLA feeding resulted in a 30% decrease in TEB density (P
< 0.05). This magnitude of response was uniform across all of the
CLA treatment groups. The proliferative activity of TEB cells was
assessed by PCNA immunohistochemistry. Treatment with CLA caused a
25–30% reduction (P < 0.05) in the proportion of TEB
cells expressing the PCNA antigen.
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The mammary carcinogenesis data in rats fed different sources of CLA
for 1 mo from weaning and then given a single dose of MNU are
summarized in Table 5
. There was a significant inhibition of both tumor incidence and yield
due to pre-MNU feeding of CLA. Overall, CLA treatment decreased
mammary cancer risk by ~50%. The different sources of CLA showed
similar efficacies as determined by the two variables (tumor incidence
and total number of tumors) of the bioassay. Thus the data in Tables 3
4
5
collectively suggest that CLA feeding during pubescent mammary
gland development down-regulates -mammary epithelial growth,
decreases the population and proliferative activity of the target TEB
cells, and therefore reduces mammary cancer risk when the rats are
challenged with a carcinogen.
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The total CLA content in tissues of rats fed different sources of CLA
for 1 mo from weaning is reported in Table 6
. As expected, rats fed a high CLA diet had significantly more total CLA
in liver, mammary fat, peritoneal fat and plasma. It is noteworthy to
point out that rats given butter CLA generally accumulated more CLA in
their tissues compared with those given either Matreya CLA or
Nu-Chek CLA. The extra load was particularly marked in mammary and
peritoneal fat pads. For example, the increase in total CLA content in
the mammary fat pad was fourfold by butter CLA, 2.6-fold by Matreya CLA
and 2.9-fold by Nu-Chek CLA. In peritoneal fat pad, it was 6.5-fold
by butter CLA, 3.8-fold by Matreya CLA and 2.8-fold by Nu-Chek CLA.
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, 8
, 9
, 10
. In all three CLA-treated groups, the major CLA isomer found in the
tissues was the c9,t11-isomer. A significant
greater percentage of this isomer was found in tissues from the butter
CLA and the Matreya CLA groups than in tissues from the Nu-Chek CLA
group. The pattern essentially reflected the proportion of the
c9,t11-CLA in the various CLA sources, i.e., 92%
in butter CLA, 81% in Matreya CLA and 25.3% in Nu-Chek CLA (see
Table 2
). The t,t-isomers represented only a
small fraction. The c,c-isomers were also very
low (or sometimes undetectable) in the butter CLA and Nu-Chek CLA
groups, but were considerably higher in the Matreya CLA group. This was
most likely due to the relatively abundant supply of
c,c-isomer in the Matreya CLA (~18%, see Table 2
).
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Because the mammary gland is the target tissue of interest, the data in
Table 8
will be highlighted in an attempt to gain new insight from the
analytical information. The emphasis again will be placed on the
c9,t11-CLA because it is the predominant form.
The concentrations of this isomer in the mammary tissue were ~34.7
µg/mg lipid in the butter CLA group and 15.1
µg/mg lipid in the Nu-Chek CLA group. However, the
cancer protection benefit was similar in the two groups (Table 5)
. This
is an interesting observation and its implication will be discussed
below.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the high
CLA butter contains more medium-chain fatty acids than the control
butter. We do not believe that this is an important contributing factor
to the cancer inhibitory effect of CLA-enriched butter because
these fatty acids are not known to modulate carcinogenesis in animal
models.
Naturally produced CLA consists predominantly of the 9,11-isomer,
whereas the synthetic CLA preparation that is most commonly used in
laboratory research contains a mixture of positional isomers. On the
basis of a number of endpoints, including mammary morphology,
proliferative activity and susceptibility to mammary carcinogenesis, we
conclude that the 9,11-CLA isomer is at least as potent as the mixture
of isomers in modulating these biological responses at the
concentration of dietary CLA provided in this study. There is growing
information in the literature on the relationship of CLA isomer
specificity and biological or biochemical effects. At the
whole-animal level, the 10,12-CLA isomer has been reported to be
responsible for the CLA-induced reduction in body fat accretion in
growing mice (Park et al. 1999
) and in milk fat
synthesis in lactating cows (Baumgard et al. 1999
).
Park et al. (1999)
also showed that in cultured 3T3-L1
adipocytes, t10,c12-CLA depressed lipoprotein
lipase and enhanced glycerol release into the medium. These biochemical
responses, however, were not elicited by c9,t11-
or t9,t11-CLA. On the other hand, recent evidence
has indicated that Nu-Chek CLA and Matreya CLA were equally
effective in inhibiting growth and inducing apoptosis of rat mammary
epithelial cells in primary culture (M. Ip et al. 1999
).
CLA is a high affinity ligand and activator of peroxisome
proliferator–activated receptors (PPAR), a family of transcription
factors known to affect gene expression in a tissue-specific manner
(Moya-Camarena et al. 1999a
). Using a scintillation
proximity assay with bacterially expressed human PPAR
ligand binding
domain, Moya-Camarena and co-workers (1999b)
showed
a hierarchy of binding affinity for certain CLA isomers in the order of
c9,t11 > t10,c12
> t9,t11. In agreement with its high
binding affinity, c9,t11-CLA was also the most
efficacious PPAR activator, as determined in a PPAR-reporter gene
assay system. This is probably the first study in which the specificity
of a CLA isomer is distinguished with the help of molecular tools.
Depending on the complexity of the model system, the ability to control
for confounding variables and the nature of the assay endpoints, it is
not surprising that different conclusions are obtained from the
different studies. It does not mean that these conclusions are
conflicting. The lesson to be learned is that the data have to be put
in the proper perspective so that they can be interpreted
appropriately.
On the basis of the analytical data from four different tissues in this
study, there appears to be some selectivity in the uptake or
incorporation of the c9,t11-isomer over the
t10,c12-isomer. Studies in lactating dairy cows
have also found a similar difference in that the transfer efficiency of
a dietary supplement of the 10,12-CLA isomer was only about one half of
that observed for the 9,11-CLA isomer (Chouinard et al. 1999
). Similarly, in pigs fed a commercial CLA mixture,
Kramer et al. (1998)
showed that there was a
preferential incorporation of the c9,t11-isomer
into liver phospholipids, and of the
c11,t13-isomer into heart lipids. Little
information is currently available regarding the biochemical mechanism
that regulates the metabolism of the different CLA isomers. Previous
research has shown that CLA can be further desaturated and elongated
(Banni et al. 1996
and 1999
, Sebedio et al. 1997
). Some of the ingested CLA is likely oxidized for the
production of energy. Future studies should be designed to provide
information concerning whether individual CLA isomers are utilized
differently via various metabolic pathways.
We have shown that the reduction in mammary cancer risk by CLA under the present experimental conditions is likely due to a decrease in the target cell population, coupled with a lower level of proliferative activity of the target cells. The diminution in TEB density by CLA feeding is consistent with the down-regulation of mammary epithelial branching as determined by digitized image analysis of the whole mount. All of the above variables responded with the same magnitude of change to the different sources of CLA, even though there was more total CLA in the mammary tissue of rats fed butter CLA compared with those fed either Matreya CLA or Nu-Chek CLA. Clearly the 9,11-CLA isomer is biologically active as an anticarcinogen, given the results of the butter CLA. The similarity of the effects of butter CLA and the other two CLA sources suggests that other isomers of CLA may also possess anticarcinogenic activity. However, we cannot rule out the possibility that the 9,11-CLA isomer is already reaching a maximal effect at the tissue level of 9,11-CLA achieved by feeding the Nu-Chek preparation. It would be instructive to carry out dose-response studies with different CLA isomers.
There is one potential advantage to the high CLA milk fat (or
butter fat) that merits further attention. Rats consuming butter CLA
consistently accumulated more total CLA in their tissues compared with
those consuming either Matreya CLA or Nu-Chek CLA. We suspect that
this was probably due to the consumption of vaccenic acid
(t11–18:1) provided by the high CLA butter fat (see Table 1
). Vaccenic acid is converted to 9,11-CLA by the 9-desaturase
enzyme (Griinari and Bauman 1999
). The efficiency of
this reaction in rodents will be examined in future experiments. Thus,
in addition to providing CLA as is, butter fat may also supply the
precursor for the endogenous synthesis of CLA. If the rate of
endogenous synthesis of CLA is adequate, this pathway may play an
important role, which could translate into a potential difference in
dose response to CLA-enriched milk (butter) fat vs. synthetically
prepared CLA. This represents a new dimension of the project that was
not anticipated at its inception. Further studies will be conducted to
investigate other attributes of this novel dairy product.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AUOD, arbitrary unit of optical density; CLA, conjugated linoleic acid; MNU, methylnitrosourea;
PCNA, proliferating cell nuclear antigen; PPAR, peroxisome proliferator–activated receptor; TEB, terminal end bud.
Manuscript received June 1, 1999. Initial review completed July 29, 1999. Revision accepted September 3, 1999.
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REFERENCES |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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4. Banni S., Martin J. C. Conjugated linoleic acid and metabolites. Sebedio J. L. Christie W. W. eds. Trans Fatty Acids in Human Nutrition 1998:261-302 Oily Press Aberdeen, UK.
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