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MINI REVIEW
The role of casein in the development of hypercholesterolemia
Olivia Hanna Koury &Celena Scheede-Bergdahl &
Andreas Bergdahl
Received: 27 February 2014 /Accepted: 6 October 2014
#University of Navarra 2014
Abstract Atherosclerosis remains the leading cause of
severe cardiovascular complications such as cardio- and
cerebrovascular events. Given that prevention and early
intervention play important roles in the reduction of
cardiovascular complications associated with athero-
sclerosis, it is critical to better understand how to target
the modifiable risk factors, such as diet, in order to best
minimize their contributions to the development of the
disease. Studies have shown that various dietary sources
of protein can affect blood lipid levels, a modifiable risk
factor for atherosclerosis, either positively or negatively.
This clearly highlights that not all proteins are “created
equal.”For example, consumption of diets high in either
animal- or vegetable-based sources of protein have re-
sulted in varied and inconsistent effects on blood cho-
lesterol levels, often depending on the amino acid com-
position of the protein and the species investigated.
Careful consideration of the source of dietary protein
may play an important role in the prevention of athero-
sclerosis and subsequent cardiovascular complications.
Given the recent focus on high protein diets, an empha-
sis on controlled studies in the area is warranted. The
goal of this review is to present the current state of the
literature that examines the effects of casein, a common-
ly utilized animal-based protein, on blood cholesterol
levels and the varying effects noted in both animals and
humans.
Keywords Casein protein .Hypercholesterolemia .Soy
protein .Lipoprotein .Cardiovascular disease
Cholesterol and the development of atherosclerosis
Atherosclerosis lies at the root of many serious cardio-
vascular complications such as myocardial infarction,
stroke, gangrene, intermittent claudication, and limb
amputation [39]. The initiation of the atherosclerotic
process depends mainly on the state and function of
the endothelial layer, which represents the demarcation
between the vessel wall and the blood [15]. Endothelial
dysfunction is characterized by two aspects: a reduction
of the bioavailability of nitric oxide, which leads to
impaired vasoreactivity, and the activation of the endo-
thelial cells [7,51]. Taken together, these features induce
a pro-inflammatory, proliferative, and pro-coagulatory
state, all of which contribute to the progression of ath-
erogenesis [1]. Factors associated with endothelial dys-
function include smoking, oxidative stress, diabetes,
metabolic dysfunction, obesity, hypercholesterolemia,
and hypertension [20](Fig.1).
High-density lipoproteins (HDL) are considered to
be a negative risk factor for the development of cardio-
vascular disease and have been casually referred to as
“good”cholesterol. The cardioprotective effects of HDL
J Physiol Biochem
DOI 10.1007/s13105-014-0365-9
O. H. Koury:A. Bergdahl (*)
Department of Exercise Science, Concordia University,
7141 Sherbrooke Street West, Montreal, QC H4B 1R6,
Canada
e-mail: andreas.bergdahl@concordia.ca
C. Scheede-Bergdahl
Department of Kinesiology & Physical Education,
McGill University,
475 Pine Avenue West, Montreal, QC H2W 1S4, Canada
are due to its role in reverse cholesterol transport (RCT),
a key process that regulates cholesterol clearance from
the systemic circulation. The purpose of RCT is the
removal of excess (free) cholesterol from peripheral
cells and reuptake by the liver for eventual bile salt
synthesis and excretion [8]. In the early stages of ath-
erosclerosis, a higher than normal concentration of cir-
culating low-density lipoproteins (LDL) results in their
penetration into the subendothelial layer of the blood
vessel. LDL, transported by apolipoprotein B (ApoB)
into the vascular wall, becomes biochemically modified
and, subsequently, triggers an inflammatory response
[21]. There are three histological features of the unstable
atheroma: (1) the large lipid core which is present when
the abovementioned RCT is incapable of regulating
blood cholesterol levels, (2) the abundance of inflam-
matory cells, and (3) a thin fibrous cap [17,22]. The
atheroma is not only a collection of cholesterol, waste,
and fibrotic tissue but is also a lesion composed of
endothelial and smooth muscle cells with infiltrating
leukocytes and other inflammatory cells [32]. Essential-
ly, the initial endothelial dysfunction leads to the fatty
streak formation and, ultimately, fibrous cap formation
[39].
The risk of developing atherosclerosis and conse-
quent ischemic heart disease increases with the presence
of pro-atherogenic substances such as intermediate den-
sity lipoproteins (IDL), low-density lipoproteins (LDL),
and very low-density lipoproteins (VLDL) [33]. Evi-
dence in the literature has suggested that certain proteins
appear to exert a greater effect on blood cholesterol
levels than others [50]. Given that the recent trend
towards high protein diets in the pursuit of weight loss
and reduction of chronic disease risk, it remains imper-
ative to fully appreciate the associations between certain
protein types and the potential for increased cardiovas-
cular disease risk. This increased risk may occur despite
successful weight loss. The goal of this review paper is
to examine whether casein, a dietary source of protein,
has an effect on blood cholesterol and whether it can be
considered a positive risk factor for the development of
atherosclerosis.
What is casein and does it have a role
in the atherosclerotic process?
Over the last five decades, there has been a steady
interest in the abilities of certain proteins to promote
either a pro- or anti-atherogenic effect. In particular,
casein has often been included in studies as an animal
source of protein [50]. Milk products contain two main
protein components: whey and casein. Whey protein
represents approximately 18 to 20 % of mammalian
milk protein, while casein represents the remaining
80–82 % [4]. Casein is regarded as one of the most
Fig. 1 Schematic view of the arterial wall and the steps in ather-
oma formation. Risk factors such as a biochemical imbalance
(high LDL) trigger an endothelial activation beyond the normal
noxious stimuli. LDL enters the subendothelial layer and become
oxidized to allow attraction of monocytes, cytokines, and other
inflammatory cells to support the inflammatory process
O.H. Koury et al.
nutritive milk proteins, as it contains all common amino
acids and is rich in essential amino acids (EAA) [45].
Purified casein is produced from skim milk by a pro-
cessing technique where the protein is separated from
the whey, dried, and then resolubilized [9]. Isolated milk
casein forms micelle complexes when dispersed in the
water phase of milk. The micelle structures have five
different subunits of the casein subtype: α-casein, α-2
casein, β-casein, κ-casein, and γ-casein. Common
among these five structures are the calcium-phosphate
bonds that hold them together and that they all contain
salt and water [9].
The primary difference between casein, whey pro-
tein, and other high-quality proteins is the rate of digest-
ibility. Casein is considered to be a slow-digesting pro-
tein because it curdles or gels in the stomach, thus
delaying release in the intestines. This results in a grad-
ual but steady rise in blood amino acid concentration
following ingestion [9]. Since the blood amino acid
concentrations are kept relatively low, it slows but ex-
tends the rate of protein synthesis [16]. Casein also
demonstrates anti-catabolic properties, which simulta-
neously inhibits protein breakdown [4,6]. In situations
where weight loss is desired, the anti-catabolic proper-
ties of casein result in it being the preferred source of
protein for hypocaloric diets [5]. In light of these char-
acteristics, casein is attractive for many weight loss
programs that include high protein intake.
As early as the 1970s and 1980s, animal studies
reported that casein increased serum cholesterol levels,
thus playing a role in the development of atherosclero-
sis. Previously, it was believed that cardiovascular dis-
ease and atherosclerosis were a result of the amount of
fat in the diet and, in particular, the cholesterol and
saturated fats. In light of results seen in animal studies,
a high casein diet may also be considered a risk factor in
the development of atherosclerotic plaque. Casein-
mediated hypercholesterolemia has been shown to de-
velop independently of exogenous cholesterol and sat-
urated fat consumption [28]. Studies have been per-
formed in order to compare lipid profiles upon adher-
ence to diets that vary in amount of cholesterol,
cholesterol-free semi-purified diets, and various protein
sources. Early studies have shown that soy protein, a
source of vegetable protein, appears to play a protective
role in the vasculature and reduces the concentration of
total cholesterol and LDL, contrary to the detrimental
effects of casein [10]. A negative association between
soy protein intake and the development of coronary
heart disease and nonfatal myocardial infarctions was
also shown in a group of middle-aged and older Chinese
women [52], although these protective effects of soy are
not detected in all studies [24]. The association between
animal-based protein and cardiovascular disease is also
supported by a meta-analysis of five prospective studies
that compared mortality in vegetarians and non-vegetar-
ians. This analysis showed that subjects who consumed
animal protein had a 24 % higher mortality from ische-
mic heart disease, even after controlling for potential
confounding factors such as age, sex, smoking status,
alcohol, habitual exercise, education, and body mass
index [29]. Given the potential detrimental effects of
casein and other animal-based proteins in the develop-
ment of vascular disease, as suggested in studies such as
these, it is important to consider dietary protein source
when recommending high protein diets rather than con-
sidering all proteins equal.
The effects of casein in the animal model
Research conducted in male New Zealand white rabbits
by Huff and colleagues clearly demonstrated that chang-
es in body weight, plasma cholesterol, triglyceride
levels, as well as liver cholesterol occurred upon varying
the dietary protein source (i.e., either animal or plant
protein) [26]. The diet used in the Huff study was termed
a“low-fat semi-purified diet”,consistingofeither27%
casein or soy isolate, along with 60 % dextrose, 5 %
celluflour, 4 % salt mix, 3 % molasses, and 1 % corn oil.
The 16 animals were divided into 2 groups: 8 received a
diet rich in casein and 8 received a diet rich in soy
protein isolate. This low-fat semi-purified diet was giv-
en to both groups for 10 months, resulting in signifi-
cantly lower levels of mean plasma cholesterol in the
soy isolate group as compared to the casein semi-
purified group. After 10 months, the casein-fed animals
ended up with higher mean triglyceride levels, higher
liver cholesterol (Table 1), and developed atherosclerot-
ic lesions, particularly in the aortic arch region. Despite
the negative effects on plasma lipids and cholesterol,
casein-fed animals gained less weight than the soy-fed
animals (2.9 and 3.3 kg, respectively).
Plasma VLDL, LDL, and IDL levels were all signif-
icantly higher in the casein-fed versus the soy-fed ani-
mals (Table 2). Huff and colleagues concluded that this
hypercholesterolemic state must be the result of choles-
terol that is endogenously produced by the liver or
Effects of casein on blood lipid profile
intestine in response to the manipulation of dietary
protein source [26]. This significant rise in IDL, the
main transporter of cholesterol, has been observed under
similar conditions since although the underlying cause
remains speculative [44].
In the Huff study, it is interesting that both the casein
and soy diets lacked exogenous cholesterol and were
low in saturated fats. From these, it is evident that the
source of protein directly affects the distribution pattern
and concentration of cholesterol being transported in the
blood [26]. The amino acid compositionof the protein is
as important as the protein source [11]. It is known that
amino acids vary in their effects on serum cholesterol
concentrations, as well as further variations when
ingested as proteins [11]. Illustrating this concept, the
amino acids lysine and methionine contribute to the
development of hypercholesterolemia, whereas arginine
is able to counteract this effect [30,31]. The essential
amino acids found in casein have been hypothesized to
be responsible for the observed increase of total choles-
terol and LDL [11]. By replacing casein with isolated
soy protein, which has a different amino acid composi-
tion, the increases in total and LDL cholesterol content
in the serum associated with casein can be avoided [3].
Terpstra and colleagues reported similar results, but
their data also demonstrated the dose effect of casein fed
to Zucker strain rats [46]. Their study included six
groups of animals fed with either a commercial diet with
no cholesterol, a commercial diet with 1.2 % cholester-
ol, or four types of semi-purified cholesterol enriched
diets (20 % casein, 50 % casein, 20 % soybean, or 50 %
soybean (g/100 g of feed)). The results revealed a more
prominent hypercholesterolemic effect occurring in di-
ets with a higher percentage of casein. The same effect
was observed in rabbits [25,46], as well as in pigeons
[34,35].
Evidence in the literature supports the notion that the
casein-mediated cholesterol increases may be biphasic
in nature: extremely low and extremely high amounts of
casein in the diet appear to have the greatest impact on
blood cholesterol levels [27,40]. Increases in cholester-
ol were most apparent with either diets containing rela-
tively small amounts (5 %) or large amounts (40 to
60 %) of casein. Diets consisting of moderate amounts
of casein (i.e., 20 %) appeared to produce the smallest
effects [19]. Gender also appears to play a role: Female
rats were more predisposed to developing hypercholes-
terolemia in response to casein ingestion [47]. This
observation had also been previously reported by Filios
and colleagues [19] with blood cholesterol levels dou-
bling in magnitude in female versus male rats.
Research conducted by Hermus and colleagues fo-
cused on demonstrating the different combinations of
protein with gelatin on serum cholesterol levels and
body weight gain in rabbits [23]. The team set up four
experimental groups: (1) semi-purified diet containing
strictly casein as the protein source; (2) casein and
gelatin; (3) casein, gelatin, and fish protein; and (4)
casein, gelatin, fish protein, and soy protein. After
58 weeks of diet adherence, the group fed the diet
consisting of casein only demonstrated a growth-
retarding effect compared to the other groups who
achieved normal growth. Additionally, the casein group
Tabl e 1 The condition of the rabbits following the observance of
a casein- or soy-rich diet. Cholesterol and triglyceride contents in
the plasma and liver were all significantly elevated, excluding the
liver triglyceride (adapted from [26]withpermission)
Casein
(mg/dl)
Soy protein
(mg/dl)
Mean plasma cholesterol
a
(mg/dl) 247±12 66± 3
b
Liver cholesterol (mg/g wet wt) 6.6± 1 3.3± 0.2
b
Mean plasma triglyceride
a
(mg/dl) 95 ±4 58± 3
b
Liver triglyceride (mg/g wet wt) 7.0±0.7 6.6± 0.3
Lipid profile in rabbits fed either casein or soy protein diet for
10 months. Results are expressed as a mean ± SE for 8 rabbits in
each dietary group
a
The overall mean ± SE for the entire 10-month period
b
Significantly different from the casein-fed group P<0.01 from
Student’sttest
Tabl e 2 The varying distribution of plasma cholesterol that de-
veloped among the four lipoprotein classes: VLDL, IDL, LDL,
and HDL, as well as the total plasma concentration. Differences
with both diet groups in all lipoprotein classes are significant,
excluding the HDL (adapted from [26] with permission)
Density class Casein (mg/dl) Soy protein (mg/dl)
VLDL d< 1.006 76± 6 11±3
a
IDL 1.006<d > 1.019 132±14 25± 6
a
LDL 1.019<d > 1.063 46± 6 10±2
a
HDL 1.063<d>1.21 19±4 12±3
Total plasma concentration 275±25 58± 6
a
Plasma cholesterol distribution among lipoprotein classes. Results
are expressed as mean ± SE for 6 rabbits in each dietary group
a
Significantly different from the casein-fed rabbits (P<0.01) from
Student’sttest
O.H. Koury et al.
also achieved a hypercholesterolemic state that was
unseen in the other three experimental groups. This
study demonstrated that the addition of alternate protein
sources to the diet was able to blunt the hypercholester-
olemic effects of casein.
Overall, it is understood that the atherosclerotic pro-
cess can be initiated by hypercholesterolemia, with se-
rum total cholesterol and lipoproteins being well-
established risk factors for the disease [21]. Although
elevated concentrations of serum cholesterol contribute
to the development of fatty streaks, this is not the sole
reason for disease susceptibility [28]. Other known risk
factors, such as family history, obesity, chronic hyper-
glycemia, and physical and/or biochemical injuries,
have also been implicated [21,28]. To fully appreciate
the origins and mechanisms involved in the progression
of atherosclerosis, what has been learned from studies
involving possible dietary sources of cholesterol must
also be considered. When certain animals are fed with a
casein-rich diet, there is a higher correlation with lipo-
philic plaques and high serum cholesterol content than a
diet consisting of plant protein [28]. The lipoprotein
density concentration is also altered, depending on
whether a plant or animal protein source is considered
[37]. Theories suggest that soybean protein contain sa-
ponins, which are protective against hypercholesterol-
emia [42]. It is also thought that dietary fiber increases
absorption of bile acids in the intestine. This will result
in loss of bile acid through fecal excretion which will be
compensated by stimulation of hepatic conversion of
cholesterol into bile acids [41]. The main question is
how casein protein causes an elevation in cholesterol,
such that the protein source is as detrimental as the
source of fat [26]. What provokes this mechanism to
increase cholesterol to such a level as to induce athero-
genic plaque?
Many speculations and theories have been put forth
over the past 30 years as to how and why casein inges-
tion raises cholesterol levels and why certain species are
more susceptible to the effects of casein. The activity
and concentration of enzyme alkaline phosphatase play
an important role since it has the potential to dephos-
phorylate casein and prevent accumulation of
phosphopeptides [36]. In 1988, Van Der Meer and col-
leagues conducted studies that involved feeding both
rabbits and rats a similar diet that included elevated
casein content in order to investigate intestinal absorp-
tion and bile acid excretion. They reported that casein
induces a hypercholesterolemic effect in rabbits due to
low intestinal phosphatase activity and with a high
glycine conjugation of bile acids, whereas in the rat,
where little effect of casein was noted, the conjugation
of bile acids occurs primarily via other amino acids,
such as taurine [48].
Another potential factor that may contribute to the
effects of casein is the LDL receptors. It has been
reported that animals fed casein-enriched diet had a
downregulation of hepatic LDL receptors preceded by
an increase in plasma cholesterol [12]. Other studies
have also shown that casein stimulates LDL ApoB
synthesis, therefore increasing the circulating LDL [11].
What effect does casein have in a human model?
Contrary to the observations seen in animals, the major-
ity of intervention studies that have investigated the
effects of plant and animal protein on serum cholesterol
levels in humans have reported inconsistent effects. In
1983, Sacks and colleagues tested whether dairy protein
(casein) or soy protein would have an effect on plasma
cholesterol in 13 strict vegetarians. The study design
consisted of a 1-week pre-intervention period, during
which, baseline measurements such as body weight,
cholesterol profile, triglycerides, and VLDL-c/TG ratio
were measured. Following the baseline period, all 13
subjects were split into groups that followed two phases:
a diet enriched in casein for 20 days and then soy for
20 days or vice versa. Results yielded no significant
changes in LDL or protective HDL cholesterol from
baseline. Aswell, there was no difference oflipid profile
or lipoproteins in the soy and casein groups during their
40-day intervention [43].
Another study conducted by Van Raaij and col-
leagues looked at similar aspects, but in healthy non-
vegetarian subjects eating a “western”simulated diet.
All 69 participants began by eating a casein-soy or
“cassoy”diet for a control period of 10 days in order
to establish baseline measurements for the remainder of
the experimental design. In this cassoy diet, 65 % of the
protein content was a 2:1 mixture of casein and soy,
respectively. Following the 10-day control period, indi-
viduals were divided into three groups for 28 days:
maintenance cassoy diet, casein diet, or soy diet. Inter-
estingly, there were barely any changes seen between
the experimental casein and soy groups in regard to total
cholesterol. Subjects adhering to the casein-enriched
diet did not demonstrate any significant changes in
Effects of casein on blood lipid profile
lipoprotein fractions. On the other hand, the soy group
demonstrated improvements in the LDL and HDL con-
centrations as compared to the casein group, and the
reductions in LDL were also significant within the soy
group from their baseline measurements [49]. The im-
provements in LDL-c and HDL-c were also seen in
another study with participants eating a cholesterol-
enriched casein or soy diet [38]. VLDL concentrations
remained unchanged in both study groups.
Crouse and colleagues conducted a similar study
with healthy individuals having elevated LDL concen-
trations between 3.62 and 5.17 mmol/L. Subjects main-
tained a casein-rich diet or a soy-rich diet with varying
amounts of isoflavones for 9 weeks. Following this
dietary intervention, LDL-c and total cholesterol
lowered significantly from baseline in individuals in
the soy group, with a dose-dependent relationship be-
tween cholesterol improvements and isoflavone
amount. There was no significant improvement in
HDL-c or TG concentration between the groups [14],
suggesting that casein supplementation is not an effec-
tive intervention for individuals with pre-existing high
cholesterol.
From these studies performed on humans, the results
suggest that casein protein may not have such a pro-
nounced effect on cholesterol levels as seen in certain
animal models, such as rabbits. The studies share mixed
results and conclusions. A soy diet would be more
beneficial for lowering cholesterol levels in hypercho-
lesterolemic patients since casein-enriched diets demon-
strate few advantages in lowering LDL-c or TG. In
healthy subjects, on the other hand, it seems as though
casein and soy diets lack influence on cholesterol levels
[43]. However, it should be pointed out that, in order to
better mimic the study design in animals, the human
experiments would need to be conducted for longer
periods of time and/or administered at earlier periods
in their lives [49]. The important point here is to con-
sider whether the lack of effect is indeed a phenomenon
in the human model or whether it is due to limitations in
study design. Further work is needed in order to objec-
tively determine the safety of diets high in casein.
Clinical implications of a high casein diet
Meal replacements (MR) containing elevated amounts
of plant or animal protein have proven successful in
promoting weight loss in obese patients. In a
randomized controlled trial by Anderson and colleagues
[2], patients consuming a MR with high casein for
16 weeks had a tendency towards more weight loss
and greater fat loss than individuals on high soy diets.
Although associated with only modest weight loss, the
individuals consuming soy MR showed promising im-
provements in their cardiovascular risk profile (i.e.,
LDL, TC, visceral fat, and systolic blood pressure).
The period following weight loss is crucial to success-
fully sustain weight reduction, and for this, high casein
diets have been found to be useful [13].
Attention must also be drawn to the popular use of
protein supplementation following exercise training.
Postprandial protein synthesis has been studied, and
the data indicates that amino acids dictate future protein
synthesis, breakdown, and oxidation [6]. Protein shakes
of whey and casein are frequently used to maintain and
increase muscle mass, hence protein synthesis or anab-
olism following exercise. As compared to whey protein,
ingestion of casein protein in a meal or drink may only
induce a small increase in protein synthesis, but is
associated with a substantial decrease in protein break-
down [6]. These physiological processes are possible
due to the retarded increase in plasma AA concentra-
tions. Without the pronounced hyperaminoacidemic
peak associated with whey, casein offers a “longer last-
ing”rise in AA levels and sustains protein breakdown
inhibition. Casein supplementation, compared to whey,
has been shown to improve body composition resulting
in decreased percent body fat; an increase of lean mass;
and greater muscle strength in legs, chest, and shoulders
[18]. These effects are most likely due to casein’santi-
catabolic property. Depending on individual aims or
motives, athletes may opt for casein as their choice of
protein supplementation for the aforementioned
reasons.
Conclusion
Casein is not only naturally present in foods containing
dairy such as milks and cheeses but is also extensively
utilized in its purified form as a powdered protein sup-
plement. For decades, saturated fats, and cholesterol
have been deemed responsible for the development of
cardiovascular disease and conditions such as athero-
sclerosis and the “clogging of arteries.”Studies have
also found that casein protein is just as aversive as fats
for some animal species. Casein can have a negative
O.H. Koury et al.
impact on the serum cholesterol concentration and raise
it to levels that pose a severe danger to the lipid profile.
There have been speculations that casein is responsible
for the disruption of bile acid binding in the small
intestine, leaving elevated levels of free bile acids to
be re-absorbed. It is key to clearly understand whether
hypercholesterolemia is enough to induce atherosclero-
sis and simulate endothelial dysfunction and to what
extent does itraise cholesterol and cause atherosclerosis.
Another important aspect that would require further
investigation is the possibility that humans may mani-
fest the same hypercholesterolemic dangers of casein if
given for longer periods of time comparable to animal
studies. Although healthy humans are assumed to be
less sensitive to dietary modifications than animals,
there may be changes noted if the human studies would
parallel animal studies in terms of time periods and
administration [49]. Would a diet low in fats but high
in animal protein be harmful to individuals and pose
cardiovascular risks? The potential dangers of casein
should serve as a red flag for dieters, trainers, physi-
cians, or nutritionists alike, especially when considered
for populations who may already be considered at risk
for cardiovascular complications. In regard to supple-
mentation, patients in a rehabilitation setting due to
conditions such as cancer cachexia or sarcopenia may
also be administered large doses of soy, whey, or casein.
Also of concern are people who exercise train and
consume exceptionally high concentrations of
whey or casein protein supplementation in order
to increase protein synthesis and muscle mass.
Understanding the implications of a high casein
protein diet is vital in order to assess the health
status and long-term lipid profile of an individual
adhering to such a diet.
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