pubs.acs.org/JAFCPublished on Web 01/25/2010
©2010 American Chemical Society
J. Agric. Food Chem. 2010, 58, 2157–2163
Dietary Milk Fat Globule Membrane Reduces the
Incidence of Aberrant Crypt Foci in Fischer-344 Rats
DALLIN R. SNOW,†RAFAEL JIMENEZ-FLORES,‡ROBERT E. WARD,†,§JESSE CAMBELL,†
MICHAEL J. YOUNG,†ILKA NEMERE,†AND KORRY J. HINTZE*,†,§
†Department of Nutrition, Dietetics and Food Sciences, Utah State University, 750 N 1200 E, Logan,
Utah 84322-8700, and‡Dairy Products Technology Center, Department of Agriculture,
California Polytechnic State University, San Luis Obispo, California 93407.
§These authors contributed equally to this work
Milk fat globule membrane (MFGM) is a biopolymer composed primarily of membrane proteins and
lipids that surround the fat globules in milk. Although it is considered to have potential as a
bioactive ingredient, few feeding studies have been conducted to measure its potential benefits. The
aim of this investigation was to determine if dietary MFGM confers protection against colon
carcinogenesis compared to diets containing corn oil (CO) or anhydrous milk fat (AMF). Male,
weanling Fischer-344 rats were randomly assigned to one of three dietary treatments that differed
only in the fat source: (1) AIN-76A diet, corn oil; (2) AIN-76A diet, AMF; and (3) AIN-76A diet, 50%
MFGM, 50% AMF. Each diet contained 50 g/kg diet of fat. With the exception of the fat source, diets
were formulated to be identical in macro and micro nutrient content. Animals were injected with 1,2-
dimethylhydrazine once per week at weeks 3 and 4, and fed experimental diets for a total of 13
weeks. Over the course of the study dietary treatment did not affect food consumption, weight gain
or body composition. After 13 weeks animals were sacrificed, colons were removed and aberrant
crypt foci (ACF) were counted by microscopy. Rats fed the MFGM diet (n = 16) had significantly
fewer ACF (20.9 ( 5.7) compared to rats fed corn oil (n = 17) or AMF (n = 16) diets (31.3 ( 9.5 and
29.8 ( 11.4 respectively; P < 0.05). Gene expression analysis of colonic mucosa did not reveal
differential expression of candidate colon cancer genes, and the sphingolipid profile of the colonic
mucosa was not affected by diet. While there were notable and significant differences in plasma and
red blood cell lipids, there was no relationship to the cancer protection. These results support
previous findings that dietary sphingolipids are protective against colon carcinogenesis yet extend
this finding to MFGM, a milk fat fraction available as a food ingredient.
KEYWORDS: Milk fat globular membrane (MFGM); anhydrous milk fat (AMF); colon cancer; sphingo-
lipid; sphingomyelin; aberrant crypt foci (ACF)
Colon cancer is the third most commonly diagnosed cancer in
the United States and the second most common cause of cancer-
incidence of colon cancer (2). Although various carcinogens are
present in foods, their effects may be minor when compared with
dietary components that inhibit the cancer process (2). As a
consequence, many dietary treatments have been tested specifi-
cally for their ability to inhibit colon cancer.
Previous studies have demonstrated that purified sphingo-
lipids, such as sphingomyelin, are protective against colon
cancer in animal models (3, 4). Sphingolipids are composed of
a ceramide core, which, in turn, is composed of a sphingosine
backbone with a fatty acid covalently bonded via an amide
linkage. Several different head groups may be covalently attached
to the ceramide, each resulting in a different class of sphingolipid.
Examples include sphingomyelin, with a phosphocholine head-
of sphingolipids is primarily associated with their metabolites,
ceramide and sphingosine (5). Ceramide is an important second
messenger implicated in various physiological functions, like
apoptosis, and has recently been associated with targeting mito-
chondrial activity in colon cancer cells (6, 7). Sphingolipid diges-
tion is slow and occurs along the entire length of both the small
sphingosine in the lumen producing the potentially beneficial
effects (8). Dietary sphingomyelin and glycosphingolipids isolated
from milk have been shown to inhibit chemically induced colon
cancer in a mouse model (9,10), and administration either before
or after tumor initiation reduced tumor formation (11).
*Corresponding author. Phone: 435-797-2124. Fax: 435-797-2379.
2158J. Agric. Food Chem., Vol. 58, No. 4, 2010Snow et al.
One significant source of dietary sphingolipids is the milk fat
globule membrane (MFGM), a protein-lipid complex that is
derived from the apical surface of mammary epithelial cells and
surrounds the fat globules in milk. During the synthesis of milk,
fat droplets originate in the endoplasmic reticulum and transit
directly to the apical surface of the cell. As they transit out of the
epithelial cells and into the alveolar lumen, they pass throughthe
apical membrane and are encapsulated in the plasma membrane
is that milk fat is present as discrete globules, which range from
a nonpolar lipid core (mainly triglycerides) surrounded by the
MFGM, which is composed of phospholipids and membrane
glycoproteins (see Table 1 for major components of MFGM).
Triglycerides are the dominant lipid class, and account for
approximately 98% of milk fat, while the balance is composed
of phospholipids (0.8%), diglycerides (0.3%), monoglycerides
(0.03%), cholesterol (0.3%), and free fatty acids (0.1%) (14).
While there is some MFGM in all dairy fats, it is especially
When cream is churned to make butter, the MFGM is released
from the surface of milk fat globules, and it is recovered in the
aqueous phase. Because of its unique lipid profile, relative
sphingolipid enrichment, and widespread availability, MFGM
is a good candidate for a colon chemopreventive, bioactive
ingredient. MFGM has an interesting profile of carbohydrates,
lipids and proteins, as has recently been demonstrated in several
is the most diverse fraction of milk. The unique compositional
feature has led to the suggestion that MFGM may have interest-
ing nutraceutical properties (18), and several research groups
have conducted studies to facilitate its recovery from butter-
Although very few studies have been conducted to determine
A recent study concluded that digestion products of MFGM
may have antimicrobial activity (31). Because of its unique
biochemical nature, sphingolipid enrichment, and resulting puta-
tive chemoprotective properties, we investigated whether diets
containing MFGM are protective against colon cancer in
Fischer-344 rats using the aberrant crypt foci (ACF) model.
MATERIALS AND METHODS
Isolation of Milk Fat Globule Membrane. Sweet cream was
obtained from Cal Poly Dairy Farm milk by centrifugation after pasteur-
ization. It was churned after a waiting period of 16 h at 4 ?C using a
continuous pilot scale butter churn (Egli AG, B€ utschwil, Switzerland).
Buttermilk was recovered, and butter fines were removed by filtration
through cheese cloth.
buttermilk concentration. The process was carried out at 25 ?C, the
transmembrane pressure was 6 bar, and the linear velocity was approxi-
mately 1 m/s. The ultrafiltration was conducted until a 10-fold volumetric
concentration factor was reached. Diafiltration was completed by adding
tap water continuously at 25 ?C to the feed tank to replace the removed
permeate until reaching a 5-fold diafiltration factor. In each step of the
filtration, samplesof retentateswere collected for composition analysis and
Filterlab Spray-drier, Hudson, WI) to obtain whey buttermilk powders.
Diet Formulation. Diets were formulated to differ only in fat
composition. This was achieved by analyzing the composition of the
MFGM isolate, selecting an appropriate amount to add to the diets to
other nutrients accordingly. The measurement of protein, total fat, ash,
and lactose were conducted as previously described (21). Ash was further
(ICP-AES) at a core facility on the Utah State University campus. The
composition of the three experimental diets is shown in Table 2.
Animals and Diets. Sixty-three male, weanling Fischer-344 rats
(Charles River Laboratories) were randomly assigned to one of three
dietary treatments that differed only in the fat source as previously
described. The diets were based on the AIN-76A rodent diet, and the fat
sourceswere(a)cornoil,(b) anhydrous milk fat,and(c) acombinationof
AMF and MFGM. After a 7-day acclimation period on standard chow
diets, the rats were individually housed in a room controlled for tempera-
ture, humidity, and light cycle and were given free access to experimental
and the animals were monitored for signs of disease. All experimental
protocols involving animals were approved by the Utah State University
Institutional Animal Care and Use Committee.
Table 1. Major Components of MFGM (Adapted from Ref12)
lipids% polar lipids% proteins
PAS III monoacyl-
free fatty acids
Table 2. Composition of Dietary Treatments
Protein (g/kg diet)
Fat (g/kg diet)
corn oil (control)
Vitamins and Mineralsd(mg/kg diet)
Sphingolipid Content (% by Weight)
aDiets were prepared by Dyets.com.bFour grams of lactose was added to the
control and AMF diets to balance lactose in MFGM isolate.cMFGM isolate is 68%
protein, 20% fat, 4% ash, and 4% lactose. The casein to whey ratio is 80:20. The
triglyceride to polar lipid ratio is 3:1.dMineral composition of MFGM isolate was
determined by ICP-AAS. Minerals were adjusted in MFGM diet accordingly.
eAmount derived from MFGM portion of the diet in parentheses (mg/kg diet).
ArticleJ. Agric. Food Chem., Vol. 58, No. 4, 20102159
Animals were fed experimental diets for three weeks and then injected
(intraperitoneal) with 1,2-dimethylhydrazine (25 mg/kg of body weight,
Sigma) in phosphate-buffered saline with 1 mmol/L EDTA (Sigma) once
per week for two consecutive weeks. Seventeen or sixteen rats per
treatment were injected with the carcinogen, and four rats per treatment
were injected with a saline vehicle control. The colonic mucosa, red blood
and fatty acid analysis. Following injections, animals were fed experi-
mental diets for nine additional weeks. After MRI analysis of body
composition (EchoMRI-900), rats were sacrificed by cardiac puncture
following ketamine/xylazine anesthesia. Tissues and organs (except lower
open, laid flat, and stored in 70% ethanol at 4 ?C. To avoid bias, colons
were randomized and ACF in the entire colon were counted by light
microscopy following staining with methylene blue.
RNA Isolation and Gene Expression. Mucosal cells from animals
injected with the vehicle control were obtainedby scraping saline-washed,
split colons with a glass slide, and then flash frozen in liquid nitrogen.
and total RNA was extracted using the RNAqueous kit (Ambion)
according to the manufacturer’s protocol. Total RNA was frozen and
Inc.). Results were analyzed using a software analysis program, Flex-
Array, developed by Genome Quebec to normalize data. Data was then
explain fewer aberrant crypts in MFGM fed animals compared to the
control and AMF treatments.
Lipid Profiling. Lipids were quantified in diets, plasma, red blood
cells, and mucosa using a combination of thin layer chromatography
separation and gas chromatographic analysis of fatty acid methyl ester
derivatives (FAMEs). Lipids were extracted using the method of
Folch (32), and separated on silica TLC plates according to the method
of Watkins (33). Bands were visualized using the fluorescent dye primu-
caps. FAMEs were prepared according to the method of Curtis (35) and
analyzed using a Shimadzu GC-2010 equipped with a BPX-70 capillary
column (10 m ? 0.1 mm i.d. ? 0.2 μm film thickness, SGE Inc., Austin,
injector was maintained at 250 ?C and all samples were injected in split
mode (triglyceride samples at a 250:1 ratio and all other samples 25:1).
Hydrogen was used as the carrier gas at a linear velocity of 56.4 cm/s, the
was as follows: initial temperature of 50 ?C for 0.29 min, 82 ?C/min ramp
35.54 ?C/min, hold for 2 min. The detector was maintained at 250 ?C; the
Total run time was 9.04 min. Fatty acids were identified according to
retention time to authentic standards, and quantified using surrogate
spikes and experimentally determined response factors.
Statistical Analysis. Differences in feed intake, body weight, body
phospholipids, mucosa sphingomyelin and RBCs were determined by
Table 3. Fatty Acid Composition of Diets (%)
C18:2 9c, t11
Figure 1. Effectofexperimentaldietsonconsumption(A),totalweightgain(B)
and body fat composition (C). Values are means (n = 17 control diet, n = 16
anhydrous milk fat diet, n = 16 milk fat globular membrane diet) ( standard
deviation. Significant differences were determined by ANOVA. Experimental
Figure 2. Effect of experimental diets on (A) 1,2-dimethylhydrazine
induced aberrant crypt foci (ACF) and (B) ACF size. Values are means
(n = 17 control diet, n = 16 anhydrous milkfat diet, n = 16 milk fat globular
membrane diet) ( standard deviation. Differently lettered columns are
ANOVA and Fisher’s LSD test.
2160 J. Agric. Food Chem., Vol. 58, No. 4, 2010Snow et al.
ANOVA and Fisher’s LSD test. Significant differences in geneexpression
were determined by FlexArray 1.3 analysis software. Expression was
normalized using the lumi method (a pipeline for processing Illumina
microarray) and differences in relative gene expression were determined
using the EB (Wright and Simon) method (36).
RESULTS AND DISCUSSION
isolated MFGM was 68% protein, 20% fat, 4.5% ash and 4%
of the diet (w/w) would supply approximately 1/2 of the fat in the
10% of the fat as membrane lipids with about 0.1% (w/w) as
sphingomyelin. As the MFGM extract also contains a significant
8:2), addition of 12.5% MFGM also contributed 1.7% whey
protein. To prevent potential confounding effects from different
contain 1.7% whey protein. Additionally, some of the proteins of
the MFGM powder are membrane proteins associated with the
material itself. Compositional analyses of highly purified MFGM
indicate the material is approximately 50% protein and 50%
lipid (37). Therefore, the contribution of MFGM membrane
proteins to the MFGM diet may be as high as 12.5% of protein,
and were unique to this diet. We also adjusted the carbohydrate
content of the control and AMF diets to accommodate a small
amount of lactose in the MFGM powder. Finally, we balanced
diets to account for the mineral profile of MFGM powder, see
Table 2 for diet compositions.
Upon receipt, the diets were analyzed for total fatty acid
composition (Table 3), phospholipid composition, and sphingo-
myelin content (Table 2). As was planned, the MFGM powder
contributed approximately 10% phospholipids (0.53% w/w of
diet) to the lipid fraction, of which sphingomyelin made up 20%
was that the sphingomyelin concentration of the control and
AMF diets was not insignificant (0.03% w/w for both diets). As
the values are the same and as the fatty acid profiles of the
sphingomyelin are the same despite the different fat sources, we
conclude that sphingomyelin is likely contributed by either the
casein or the whey, or a combination of both. Nonetheless,
previous studies have found significant effects with such low
levels of sphingomyelin, and therefore the protective effects
provided by MFGM in this study may be somewhat under-
tion. Dietary treatment did not significantly affect consumption
or weight gain. MRI analysis of whole animals indicated no
significant effect on body fat percentage (Figure 1). Given that
feed intake can affect carcinogenesis, confounding factors are
possible when feeding different diets even though the diets are
isocaloric. Since the difference in fat source between the three
body fat, feeding behaviors in each group cannot account for
differences in ACF.
Figure 4. Effect of experimental diets on fatty acid profile of (A) red blood cells and (B) mucosal sphingomyelin. Samples were taken from saline injected
control animals (n = 4 control diet, n = 3 anhydrous milk fat diet, n = 4 milk fat globular membrane diet). Values are the mean percent of total fatty acids (
Red blood cell fatty acids that comprise less 1% of total fatty acids are not shown.
Figure 3. Venn diagram of differentially regulated genes in the mucosa
between dietary treatments. Out of 21,792 genes, a total of 417, 450, and
321 genes were differentially regulated with significance in the mucosa
treatments, respectively. See Supplementary Table 1 in the Supporting
Information for a listing of the most differentially regulated genes between
Article J. Agric. Food Chem., Vol. 58, No. 4, 20102161
Effect of Dietary Treatment on Appearance of ACF. The ACF
model is a well-established model of colon cancer and has been
(P < 0.005) compared to the control (n = 17) (20.9 ( 5.7 vs
31.3( 9.5) and AMF (n = 16) diets (29.8( 11.4), and ACF was
not significantly different between control and AMF treatments
(Figure 2A). Dietary treatment had no effect on ACF size
(Figure 2B) nor was there a significant difference in the number
suggesting that MFGM treatment is more relevant to preventing
ACF initiation as opposed to ACF growth progression.
a total of 417, 450, and 321 genes were differentially regulated
AMF and control, and MFGM and AMF dietary treatments,
respectively (Figure 3). Despite the observation that MFGM
treatment decreased ACF compared to both control and AMF;
no common gene regulation or change in cancer pathways were
observed between MFGM vs control and AMF.
Although dietary treatment did not influence the expression
of common colon cancer genes, sphingomyelin and MFGM’s
ability to regulate post-transcriptional gene expression cannot be
completely ruled out. A recent study demonstrated that sphingo-
a significant effect on protein levels of genes critical to the early
stages of colon cancer, such as beta-catenin, connexin-43 and
Bcl-2 (41). Our results together with these recent findings suggest
that the sphingolipids present in the MFGM may not be regulat-
ing transcription but may be regulating specific post-transcrip-
involved in colon carcinogenesis. Because many of the phospho-
lipids found in MFGM are common second messengers, future
MFGM and relevant metabolic pathways are needed.
Effect of Diet on Tissue and Plasma Lipids. The fatty acid
composition of the AMF and MFGM diets were very similar at
thefattyacidlevel andverydifferent fromthatofthecontrol diet
(Table 3). To determine how the fatty acids in the three diets
affected tissue levels, we measured the fatty acid compositions of
the red blood cells (RBCs) as well as the mucosa (Figure 4).
reflected that of the diet, while the mucosa did not. Several
notable differences are apparent in the RBCs. For example, the
animals fed the control diets had a lower percentage of oleic acid
(C18:1n9) in RBCs despite the fact that there was more of this
Figure 5. Effectof diet on fattyacid profileof (A) plasma triglycerides, (B) plasma phospholipids,(C) sum total ofplasma triglycerides,and (D) sumtotal of
phospholipid fatty acids. Samples were taken from saline injected control animals (n = 4 control diet, n = 3 anhydrous milk fat diet, n = 4 milk fat globular
values less than 10 μg/mL are not shown.
2162 J. Agric. Food Chem., Vol. 58, No. 4, 2010 Snow et al.
fattyacids.For example,the RBCs oftheanimals fed thecontrol
diets were enriched in linoleic acid (C18:2n6) and its elongation
and desaturation products arachadonic acid (C20:4n6) and
docosatetraenoic acid (C22:4n6). The animals fed the AMF
and MFGM diets, on the other hand, had greater RBC fatty
acid proportions of docosapentaenoic acid (C22:5n3) and
docosahexaenoic acid (C22:6n3) despite the fact that the overall
percentage of n3 fatty acid contribution to the diet was lower
It has been hypothesized that high tissue levels of arachadonic
acid may affect susceptibility to cancer via inflammatory signal-
ing (43). However, our results do not indicate that there is any
effect as the animals fed the AMF and the control diets had
similar levels of ACF.
As sphingomyelin is thought to be slowly digested along the
length of the digestive tract (8), it stands to reason that it may be
available in the gut lumen for absorption by the epithelia.
Alternatively, as was noted with RBCs, if absorbed, sphingo-
myelin might also be provided to the mucosa via the systemic
circulation. However, unlike RBCs, the sphingomyelin profile of
the colonic mucosa was not significantly affected by the differ-
ences in dietary fatty acids (Figure 4). According to the diet
analysis, the sphingomyelin fatty acids most abundant in the
MFGM diet, compared to the control and AMF diets, were
C16:0, C22:0, C23:0 and C24:0 (data not shown). Yet, the
sphingomyelin of the MFGM diets does not seem to be enriched
in the longer chain species, although there is a small effect with
C16:0 (Figure 4).
Plasma triglycerides differed significantly only between ani-
mals fed the AMF (1,634 μg/mL plasma ( 477) and MFGM
(3,666 μg/mL plasma ( 703; P < 0.02) dietary treatments
(Figure 5). Plasma phospholipids were significantly different in
animals fed the AMF (640 μg/mL plasma ( 35) diet from those
fed the control (1,025 μg/mL plasma ( 194; P < 0.04) and
MFGM (1,069 μg/mL plasma ( 194; P < 0.04) diets; however,
no significant difference was seen between the animals fed the
profiles of MFGM and AMF diets, they have very different
effects on plasma triglycerides and phospholipids that are not
related to differences seen in cancer protection.
Our results support the hypothesis that diets containing
perhaps because of MFGM’s high polar lipid content, namely,
sphingomyelin. By incorporating MFGM into the diet, ani-
mals were provided (0.11% w/w) sphingomyelin. Previous
studies using sphingomyelin concentrations ranging from
sphingomyelin’s role in the prevention of colon carcino-
genesis (9-11, 38, 42); however many of those studies used a
very pure form of isolated sphingomyelin. In this study, the
sphingomyelin is in a more practical form or more similar to
how it would be incorporated into human diets, and the
MFGM contains other important phospholipids such as phos-
phatidylcholine and phosphatidylethanolamine. One other
major difference in the MFGM diet, compared to the control
and AMF diets, was the contribution of MFGM proteins.
Several of these membrane proteins have been hypothesized to
provide biological effects, and we cannot rule out the fact that
they may have contributed to the protective effect of the diet.
In pilot studies conducted in vitro, we noted that these proteins
are extensively proteolyzed by a combination of stomach and
pancreatic proteases, but this does not rule out bioactivity
mediated by the peptide fragments. Thus, the contribution of
these proteins remains unknown. The results of this study
demonstrate that MFGM, a readily available byproduct from
dairy processing, can be incorporated into diets and is protec-
tive against colon cancer.
MFGM, milk fat globule membrane; AMF, anhydrous milk
fat; TLC, thin layer chromatography; ACF, aberrant crypt foci;
FAMEs, fatty acid methyl esters; RBCs, red blood cells.
We thank Dr. Aaron Olsen and Kent Udy for assistance with
Supporting Information Available: Tables containing gene
expression from microarray including the five most up-regulated
and down-regulated genes between MFGM diet vs control
diet, MFGM diet vs AMF diet, and AMF diet vs control diet.
This material is available free of charge via the Internet at http://
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Received for review October 14, 2009. Revised manuscript received
January 4, 2010. Accepted January 11, 2010. Financial support for
BioSystems Seed Grant and by the Utah Agricultural Experiment
Station. This paper was approved by the Utah Agricultural Experi-
ment Station as paper #8315.