The Journal of Nutrition. First published ahead of print October 21, 2015 as doi: 10.3945/jn.115.216812.

The Journal of Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Casein Compared with Whey Proteins Affects

the Organization of Dietary Fat during Digestion
and Attenuates the Postprandial Triglyceride
Response to a Mixed High-Fat Meal in Healthy,
Overweight Men13
Francois Mariotti,4,5* Marion Valette,4,5 Christelle Lopez,6,7 Hele`ne Fouillet,4,5 Marie-Hele`ne Famelart,6,7
Veronique Mathe,4,5 Gheorghe Airinei,4,5 Robert Benamouzig,4,5 Claire Gaudichon,4,5 Daniel Tome,4,5
Dimitrios Tsikas,8 and Jean Francois Huneau4,5

Downloaded from jn.nutrition.org at UNIVERSITY OF NEBRASKA on October 23, 2015

4
AgroParisTech, CRNH-IdF, UMR914 Nutrition Physiology and Ingestive Behavior, Paris, France; 5INRA, CRNH-IdF, UMR914 Nutrition Physiology
and Ingestive Behavior, Paris, France; 6INRA, UMR1253 Science and Technology of Milk and Egg, Rennes, France; 7Agrocampus Ouest, UMR1253
Science and Technology of Milk and Egg, Rennes, France; and 8Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany

Abstract
Background: Postprandial lipemia is a risk factor for cardiovascular disease. The potential impacts of the type/nature of
dietary protein on postprandial lipemia and associated dysregulations have been insufficiently investigated.
Objective: We investigated the postprandial effect of including in a high-fat meal some milk protein fractions that
markedly differ in their physicochemical properties and composition [either casein (CAS), whey protein (WHE), or
a-lactalbumin-enriched whey protein (LAC)].
Methods: The protein fractions were incorporated as 15% energy in a high-fat meal in a 3-period, crossover postprandial study of 10
healthy overweight men with an elevated waist circumference (>94 cm). We measured postprandial changes in plasma lipids, amino
acids, glucose, and oxidative stress markers, vascular function (using pulse contour analysis), and low-grade inflammation (using plasma
markers). We also characterized in vitro the meal structures, including the size of the fat globule, and possible changes during digestion.
Results: The type of protein did not affect postprandial plasma glucose, amino acids, insulin, or nonesterified fatty acids,
but, compared with WHE and LAC, which did not differ, CAS markedly reduced postprandial triglycerides (TGs), achieving
a 22 6 10% reduction in the 6-h area under the curve (P < 0.05). Similar trends were shown for plasma chylomicrons
[apolipoprotein (apo)B-48; P < 0.05]. However, there were no significant differences between the meals regarding
postprandial oxidative stress (plasma hydroperoxides and malondialdehyde), endothelial dysfunction (salbutamol-induced
changes in pulse contour analysis), or low-grade inflammation. In vitro studies showed that when the pH of the meal
decreased to stomach pH values, the reduction in the solubility of casein resulted in a phase separation between fat and
protein, whereas the proteins in the other meals remained suspended with fat globules.
Conclusion: In healthy overweight men, casein has specific physical interactions with fat that affect postprandial TGs,
leading to the formation of fewer chylomicrons or an increase in chylomicron clearance. This trial was registered at
clinicaltrials.gov as NCT00931151. J Nutr doi: 10.3945/jn.115.216812.

Keywords: high-fat meal, postprandial period, dietary protein, triglycerides, humans, metabolic dysregulation,
meal structure, milk protein

Introduction
In the postprandial state, a sudden influx of energy and nutrients
challenges the metabolism and normal postprandial allostasis,
1
Supported in part by a grant from the National Interprofessional Centre of the
Dairy Industry and the French Agency for Research and Technology (National
Program for Research in Food and Human Nutrition 2006, project SURPROL).
* To whom correspondence should be addressed. E-mail: francois.mariotti@
agroparistech.fr.
resulting in an increase in the metabolic allostatic load (1). A high-
fat meal has been shown to induce oxidative stress, low-grade
2
Author disclosures: F Mariotti, M Valette, C Lopez, H Fouillet, M-H Famelart,
V Mathe, G Airinei, R Benamouzig, C Gaudichon, D Tome, D Tsikas, and
JF Huneau, no conflicts of interest.
3
Supplemental Figures 1 and 2 and Supplemental Table 1 are available from the
Online Supporting Material link in the online posting of the article and from the
same link in the online table of contents at http://jn.nutrition.org.

2015 American Society for Nutrition.

Manuscript received May 11, 2015. Initial review completed June 23, 2015. Revision accepted September 25, 2015. 1 of 8
doi: 10.3945/jn.115.216812.
Copyright (C) 2015 by the American Society for Nutrition
inflammation, and endothelial dysfunction, and these effects are
very closely associated with the postprandial increase in TGs (2
8). It should be noted that these postprandial effects are also more
pronounced when subjects present risk factors at baseline relative
to the metabolic syndrome, such as being overweight/obese and
having an elevated waist circumference (912). Indeed, the post-
prandial TG response to a high-fat challenge is considered to
be a cardiovascular risk factor (13, 14). Several studies have
investigated the importance of the type of dietary fats and the role
of added carbohydrates, particularly as simple sugars, and the
findings from these studies have contributed to our understanding
of the potential pathogenic roles of dietary fats and carbohydrates
based on their postprandial effects (1418). However, only a few
studies have investigated the role of dietary proteins, although there
are many mechanisms by which dietary proteins might modulate
postprandial lipemia and its associated adverse effects. For instance,
some proteins and amino acids have been reported to alleviate
postprandial oxidative stress and endothelial dysfunction (19,
20). Dietary proteins may also exert a more direct effect on
postprandial allostatic stress by modulating the postprandial
TG response (21). Indeed, a seminal study by Westphal et al.
(22) reported that adding protein to a high-fat meal decreased
postprandial TGs and alleviated postprandial endothelial
dysfunction. This effect could have been mediated by a change
in the gastric emptying of fat and an increase in postprandial
insulin induced by adding considerable amounts of protein
(;50 g). The addition of protein in smaller amounts, or the
presence of specific proteins in a high-fat meal, may also
interact with the fatty acid absorption rate and chylomicron
formation, ultimately affecting the TG response and allostatic
load. In this regard, some dietary proteins vary in terms of their
physicochemical characteristics and digestion rates, and casein
in particular is known to have a specific gastrojejunal digestion
rate because of its dramatic decrease in solubility when the
pH decreases to values found in the stomach (2325). The
objective of this study was therefore to determine whether
dietary proteins that differ in terms of their physicochemical
properties could affect the TG response and ensuing postpran-
dial adverse effects after a high-fat meal. We studied casein
compared with 2 different whey protein fractions in a mixed
high-fat meal using a crossover design in healthy overweight
men with an enlarged waist circumference.
Methods
Study population and ethical aspects
Eleven healthy young men who were overweight (BMI >25 kg/m2) and had
an enlarged waist circumference (>94 cm) were recruited for the study and
randomized. The noninclusion criteria included any established disease or
regular use of medication, excessive alcohol intake (>2 drinks per day),
moderate or high use of tobacco products ($9 cigarettes per day), the use
of any nutritional supplement, and moderate or high levels of physical
activity (taken as >4 h of high/moderate physical activity per week), blood
hemoglobin <13 g/dL, and the presence of hypertension. The subjects were
considered to be in generally good health based on their self-reported
medical history, an examination by a physician, and standard blood
chemistry at screening. One subject chose to withdraw from the study
during the first session because he felt nauseated after the meal. Therefore,
10 subjects received all treatments and were analyzed. The characteris-
tics of these subjects were as follows (means 6 SDs): age, 34 6 9 y; height,
178 6 3 cm; weight, 96 6 6 kg; BMI, 30.2 6 1.5; body fat, 24.3 6 2.0%;
and waist circumference, 96.3 6 3.2 cm. All participants gave their written
informed consent before enrolling. The study was approved by the
Institutional Review Board for Saint-Germain-en-Laye Hospital and
authorized by the French Ministry of Health.
2 of 8 Mariotti et al.
Design and intervention
We used a 3-period randomized crossover design according to a Latin
square. Each period consisted of a postprandial study that was separated
by at least 2 wk, and the volunteers were not aware of the nature or order
of the meals being tested.
The participants were instructed to maintain their usual diet (partic-
ularly on the day before the visit), to eat their dinner at the usual time,
and to refrain from unusual physical activity on the day before the visit
and before their arrival at the research center in the morning. They were
asked to arrive at the center after a 912-h overnight fast. The partic-
ipants then underwent a standard examination to assess weight, body
composition (determined by bioelectrical impedance analysis), and blood
pressure, and then an intravenous catheter was inserted for blood
sampling. During the day of experiment, the participants relaxed on a
bed in a quiet room and stayed calm before and after the meal.
The participants were allowed 15 min to ingest the meal and were
then studied for 6 h after they had started eating. Blood was sampled
before the meal and at 0.5, 1, 1.5, 2, 3, 4, and 6 h after the meal. Their
digital volume pulse (DVP)9 was analyzed before and at 2, 4, and 6 h
after the meal, and their endothelial function (measured as salbutamol-
induced changes in DVP) was assessed before the meal and 4 h thereafter.
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The composition of the meals was as follows: energy, 1200 kcal; fat,
93.3 g (70% energy); carbohydrates, 45 g (15% energy); and crude pro-
tein, 45 g (15% energy). The test meals consisted of a mixture of 233 g
of 40%-fat milk cream, 45 g of sucrose, 160 mL of water, and protein
isolates of either casein (CAS), whey protein (WHE), or a-lactalbumin-
enriched whey protein (LAC) in quantities of 54, 55, and 49 g, respec-
tively, adjusted to yield the same amount of protein (i.e., 45 g). Cream
(traditional, thick, and acidic) and regular white table sugar were stan-
dard commercially available products. Ingredia provided CAS and WHE,
and Armor Protein provided LAC. Because the amounts of remaining
carbohydrates and minerals were not the same in these protein-rich
powders, the meals were balanced by adding lactose, calcium phos-
phate, potassium phosphate, and magnesium phosphate (Supplemental
Table 1).
Assay methods
Standard analytical methods. We used enzymatic colorimetric methods
according to commercial kits to assay plasma glucose (bioMerieux),
TGs, and nonesterified fatty acids (Randox Laboratories), with Unitrol
(bioMerieux) as a quality control standard. We measured the total free
a-amino nitrogen in plasma as a measure of total plasma amino acids
according to a technique developed in-house and based on a fluori-
metric method using o-phthaldialdehyde as the derivatization reagent
(26, 27).
Immunochemical assays. ApoB-48 was assayed using an ELISA assay
(BioVendor). Plasma concentrations of insulin, IL-6, monocyte chemo-
attractant protein-1, TNF-a, vascular cell adhesion molecule-1, intercel-
lular adhesion molecule-1, and plasminogen activator inhibitor-1 were
determined using 2 customized mixed-assay kits with antibody-coated
beads (MILLIPLEX HADK2-61K-B and HCVD1-67AK-05; Millipore)
using the Luminex xMAP technology platform for multiplexing immuno-
chemical bioassays (Bio-Plex 200 with high-throughput fluidics system;
Bio-Rad Laboratories).
Other assays. Hydrogen peroxide concentrations were measured in
heparinized plasma using the ferrous ion oxidation in xylenol orange
assay (28). Malondialdehyde (MDA) concentrations were measured by
a gas chromatography-tandem mass spectrometry method after the
derivatization of MDA and its internal standard 1,3-dideutero-MDA
with pentafluorobenzyl bromide to their dipentafluorobenzyl deriva-
tives, as described in full elsewhere (29). Plasma concentrations of the
F2-isoprostane 15(S)-8-iso-prostaglandin F2a (PGF2a) were determined
using chromatography-tandem mass spectrometry after alkaline hydrolysis,
9
Abbreviations used: CAS, casein; DVP, digital volume pulse; LAC, a-lactalbumi-
n-enriched whey protein; MDA, malondialdehyde; PGF2a, prostaglandin F2a;
RI, reflection index; WHE, whey protein.
immunoaffinity chromatography column extraction, and derivatization
of 15(S)-8-iso-PGF2a and its internal standard tetradeutero-15(S)-8-iso-
PGF2a, as described in detail elsewhere (30).
Vascular measurements
Subjects rested in a supine position for 10 min before each measurement.
DVP was recorded on the forefinger of the nondominant arm with 35
repeated measurements of estimates of 20-s averages of waveform
geometry, as assessed by photoplethysmography (PulseTrace; Micro
Medical); it was then used to calculate the stiffness index (m/s) as the
inverse of the time between the first and second peaks of the pulse wave
multiplied by the height of the subject and the reflection index (RI; %) as
the height of the second peak divided by that of the first peak (31, 32).
Endothelial function was assessed by monitoring the decrease in DVP-RI
every 5 min for 35 min after administering 400 mg of salbutamol by
inhalation (31, 33). Because the DVP-RI value peaked mostly between
20 and 30 min after salbutamol administration, the DVP-RI response to
salbutamol was taken as the average of the 2030-min values. Then, as
a control, endothelium-independent vascular function was assessed by
the decrease in DVP-RI 3 min after administering 300 mg of glyceryl
trinitrate sublingually (31).
oscillation rheology (maximum strain, 0.1%; frequency, 0.5 Hz). A
fraction of the sample was also placed in a water bath at 37C to
monitor macroscopic and pH changes over time. Pictures of the
acidified meals were taken.
The size of the fat globules in the acidified nonhomogenized cream
and in the test meals was determined using laser light scattering
(Mastersizer 2000; Malvern Instruments). The refractive indexes used
were 1.458 and 1.460 for milk fat at 633 and 466 nm, respectively,
and 1.33 for water. The samples were dispersed in 1% sodium dodecyl
sulfate under gentle stirring for 10 min before the measurements to
dissociate the protein network. The casein micelles were dissociated
by adding 1 mL of 35 mM EDTA/NaOH buffer (pH 7) to the CAS
samples.
Statistics
Data are reported as means 6 SEs. All statistical analyses were
performed using SAS version 9.1 (SAS Institute). Significance was set
at P < 0.05. Kinetic data were analyzed using mixed models for repeated
measurements with covariance structure modeling, with the protein type
in the meal and the time since ingestion as independent fixed (repeated)
factors and the subject as a random factor. The interaction between the

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In vitro characterization of meal structures
The dynamic viscosity of the meals was measured with the AR2000
rheometer (TA Instruments) with coaxial cylinders. After 5 min of
storage at 37C, the shear rate was increased from 0.01 to 100 s21 (steps
of 5 s, logarithmic series, 10 points per decade).
To characterize the changes that acidification caused during
digestion, the meals were prepared exactly as they were during the
experimental sessions (see Design and intervention), supplemented
with 15 mM NaCl (1 volume per 9 volumes of meal), heated to 37C,
and supplemented with glucono-d-lactone to reach pH 3 in 2 h. The
meals were placed in the cone-plate geometry of a rheometer (diameter,
6 cm; angle, 4) during acidification and applied small-amplitude
protein type and time, the order of treatment (i.e., which meal was
received first), and the number of the visit (i.e., first, second, or third)
were also introduced into the model. When the protein type or the
interaction between the protein type and time were significant (P < 0.05),
comparisons between the protein types at each time point were made as
multiple comparisons under the mixed model with Hochberg-Bonferroni
tests. The effect of protein type on the AUC was analyzed using a similar
approach, i.e., the same model without any time-related factor. For the
statistical analysis of TG kinetics, we also tested the effect of adding
apoB-48 as a covariate in the model to determine whether and to what
extent it may explain the effect of the meal protein on postprandial
changes in TG. We also tested whether TG and apoB-48 concentrations
were correlated and reported Pearsons r.

FIGURE 1 Postprandial kinetics of plasma TGs (A), apoB-48 (B), glucose (C), insulin (D), amino acids (E), and nonesterified fatty acids (F) in
healthy overweight men after a high-fat meal that included CAS, WHE, or LAC. Data are presented as means 6 SEs (n = 10). Labeled means at a
time and AUC means without a common letter differ, P , 0.05, as tested with Hochberg-Bonferroni correction when the time-protein interaction
was significant. CAS, casein; LAC, a-lactalbumin-enriched whey protein; WHE, whey protein.

Casein and postprandial lipemia 3 of 8

Results
Postprandial kinetics of energy nutrients. The postprandial
increase in plasma TGs depended upon the protein type included
in the meal (Figure 1A). The 6-h AUC values were significantly
lower after the high-fat meal made with CAS than after the high-
fat meal made with WHE and LAC. Compared with the other
meals taken together, CAS lowered the AUC by 22 6 10%. ApoB-
48 data showed similar trends (protein-time interaction, P =
0.06), and the apoB-48 AUC for CAS tended to be lower than that
of WHE (P = 0.09). When the apoB-48 data were included as a
covariate to analyze the TG data, the protein-time interaction was
no longer significant, indicating that the effect of CAS on TGs
could be, at least in part, a result of its effect on chylomicron
kinetics. As expected, postprandial TGs and apoB-48 were corre-
lated (r = 0.38, P < 0.001) when considering all meals together,
and the correlation was stronger with the CAS meal (r = 0.67,
P < 0.001). In contrast to the TG kinetics, the protein type in the
meal did not affect postprandial plasma amino acids, insulin, or

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nonesterified fatty acids and had only a small effect on plasma
glucose kinetics (protein-time interaction, P < 0.05; Figure 1).

Postprandial oxidative stress, low-grade inflammation, and

vascular function. Plasma hydroperoxide and MDA concen-
trations significantly increased after the high-fat meals were
ingested (Figure 2). None of the parameters for systemic or vas-
cular proinflammatory status and oxidative stress [i.e., IL-6,
TNF-a, monocyte chemoattractant protein-1, intercellular ad-
hesion molecule-1, vascular cell adhesion molecule-1, plasmin-
ogen activator inhibitor-1, or plasma 15(S)-8-iso-PGF2a] increased
after the meal (Table 1). The DVP-RI decreased significantly af-
ter meals, as did the vascular endothelial-dependent reactivity
(based on the salbutamol-induced decrease in DVP-RI; Table 1).
DVP-SI and endothelium-independent vascular reactivity (based
on the glyceryl trinitrate-induced decrease in DVP-RI) did not
vary after the meal was ingested (Table 1). The protein type in
the meal affected neither of these measurements during the post-
prandial period (Table 1).
In vitro characterization of meal structures. The pH of the
cream used for preparing the high-fat meals was 4.4 6 0.3,
whereas those of the meals were 5.4 6 0.1 for CAS, 5.4 6 0.1 for
WHE, and 6.0 6 0.1 for LAC. The cream was not homogenized
(mean diameter of fat globules, 3.9 6 0.5 mm), and the fat was
dispersed in the meals in the form of milk fat globules with a
modal diameter of ;1020 mm (Figure 3). We observed a phase
separation in the CAS meal with the sedimentation of proteins
and the formation of a fat layer as the upper phase after the
meals were acidified. Such phase separation was not observed
for the WHE or LAC meals (Figure 4).
The high viscosity of the WHE meal (Supplemental Figure 1)
and the formation of an early and firm gel of proteins in the LAC
meal at pH ;5.2 (Supplemental Figure 2) prevented phase separa-
tion between the fat and proteins after these meals were acidified.
Whereas the milk fat globules stayed dispersed in the high-viscous
continuous protein phase of the WHE meal or were physically
entrapped in the hard protein gel of the LAC meal, the proteins in
the CAS meals were already organized in a very soft viscoelastic
solid with a low G# value of 10 Pa at pH 5. This very soft gel was
reorganized at around pH 4.2 (i.e., slight increase in the loss angle
value and then decrease) and the low G# value at ;100 Pa did not
prevent settling of the protein. This behavior explained the phase
separation observed in Figure 4, with fat globules at the top of the
sample and the protein network at the bottom.
4 of 8 Mariotti et al.
FIGURE 2 Postprandial kinetics of plasma hydroperoxides (A) and
malondialdehyde (B) in healthy overweight men after a high-fat meal
that included CAS, WHE, or LAC. Data are presented as means 6 SEs
(n = 10). CAS, casein; LAC, a-lactalbumin-enriched whey protein;
WHE, whey protein.
Discussion
The principal finding of this study is that depending on their
physicochemical properties during digestion, dietary proteins
can modulate the postprandial increase in TGs. We found that in
a high-fat meal, casein, because of its low solubility at lower
pHs, induced a phase separation with the lipids and reduced the
postprandial increase in TGs.
Dietary protein, insulin, and postprandial lipemia. Few
studies have examined the effects of the quantity and type of
dietary protein on postprandial lipids. The addition of casein to
a mixed high-fat meal was shown to decrease postprandial TGs
in healthy and diabetic subjects (22, 34, 35). Such a protein
effect is usually ascribed to an insulin-mediated effect (34, 35),
and different amino acids and their rates of appearance are
considered to be associated with varied insulinotropic effects
(36, 37). It should be noted in this respect that whey protein has
been shown to be insulinotropic when compared with casein
(38). However, in mixed meals that contain high energy levels,
there is a lack of evidence of a difference in the insulinemic
response between dietary proteins in nondiabetic subjects (39).
During this study and regardless of the protein fraction, we
showed an early insulin peak and an early decrease in free fatty
acids, evidencing an early secretion and action of insulin,
whereas the rise in plasma amino acids was much more even and
spread out over the 6-h postprandial period. The similar in-
sulinemic response to the different protein meals could be re-
lated to the high energy content of the meals, which resulted in
a slow gastric emptying rate and a similarly slow rate of amino
 acid absorption (40). These considerations are noteworthy when

All values are means 6 SEs (n = 10) for each parameter measured before (0 h) and after (2, 4, and 6 h) the ingestion of high-fat meals that included either CAS, WHE, or LAC in a crossover design in healthy overweight men. NS, P . 0.05. CAS,
casein; DVP, digital volume pulse; GTN, glyceryl trinitrate; ICAM, intercellular adhesion molecule; LAC, a-lactalbumin-enriched milk soluble protein; MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1; PGF2a,
Meal Time 3 meal
interpreting the results. Our data therefore show that in a high-

NS
NS

NS
fat meal, a specific effect of a protein type cannot be explained

NS

NS
NS
NS

NS
NS

NS

NS
by mechanisms related to insulin.
Statistics

NS
NS

NS
NS

NS
NS
NS

NS
NS

NS

NS
A specific effect of casein. In contrast to our results, 2 studies
Postprandial markers of low-grade inflammation and vascular function in healthy overweight men after the high-fat meals that included CAS, WHE, or LAC1

previously reported that whey lowered postprandial TGs com-

P , 0.001
6 0.06 P , 0.01

P , 0.01
pared with casein (21, 39). There are different possible reasons
Time

NS
NS

NS
NS

NS

NS

NS

NS
for this discrepancy. First, the study population, which consisted
of either postmenopausal women (39) or patients treated for
6 0.91
6 0.62

6 0.14
6 0.93

6 10.5

6.74 6 0.16 6.63 6 0.14

67.2 6 1.0
type-2 diabetes (21), was different. The latter subjects were
6 28
6h

treated with lipid-lowering drugs and expected to display

4.37

283

119
4.58

2.47
16.1
0.45
marked differences in lipoprotein and fatty acid metabolism
compared with our healthy subjects. This would explain why
1.19
0.96

0.10
1.06
0.06
9.2

69.9 6 0.7
25.1 6 1.7

225.1 6 1.1
43

these 2 studies did not find a differential effect of the same meals
4.56 6
5.70 6

2.41 6
334 6

15.8 6
0.45 6
85.7 6
4h
LAC high fat meal

when given to obese nondiabetic subjects (41). However, more

importantly, the meals in these studies were made from protein
ingredients that were not, or not closely, premixed with the rest
0.98
0.84

0.10
34.1

1.06
0.05
10.2

6.70 6 0.19 6.60 6 0.17

66.8 6 1.2

of the meal. Furthermore, in 1 study the fat load was given as

Downloaded from jn.nutrition.org at UNIVERSITY OF NEBRASKA on October 23, 2015

2h

6
6

6
6

6
6
6

butter added to a soup; in the other study it was given as

4.05

325
4.91

2.54
16.0

102
0.46

margarine spread on a bread roll (21, 39). The meals therefore

6 1.16
6 1.00

6 0.13
6 34.6

6 1.22
6 0.10

did not involve a close association of the different energy nutri-

6 8.8

78.1 6 0.7
213.6 6 2.1

222.3 6 1.0

ents and may have precluded mechanisms that imply nutrient

0h

interactions during digestion.

3.99

311
5.60

16.5
2.62

0.61
92.9

The decrease in TGs with casein can be ascribed to a

6 1.57
6 1.03
6 55.0

6 1.34
6 0.19

6 0.06
6 10.0

6.52 6 0.13 6.87 6 0.11

67.1 6 0.8

phase separation of meal lipids. Postprandial insulin and

6h

nonesterified fatty acid levels were very similar with the different
5.19

312

103
5.14

15.1
2.32

0.42

protein meals. This similarity rules out the possibility that a

6 1.09
6 1.00
6 54.8

6 0.69
6 0.10

6 0.04
6 18.3

64.1 6 0.6
22.8 6 1.1

223.6 6 0.7

lower nonesterified fatty acid concentration might have limited

WHE high-fat meal

postprandial VLDL production (42). In contrast, and as dem-

4h

prostaglandin F2a; RI, reflection index; SI, stiffness index; VCAM, vascular cell adhesion molecule; WHE, milk soluble protein.
4.18

330
5.24

17.1

131
2.64

0.45

onstrated by the postprandial apoB-48 data, the postprandial

decrease in TGs with CAS can largely be ascribed to an effect on
1.10
0.96
44.3

0.59
0.07

0.07
22.2

6.46 6 0.14 6.36 6 0.11

plasma chylomicron levels. It is worth noting that because

61.9 6 0.8
2h

plasma TGs were still elevated at 6 h, we could not gather any

3.75 6
5.06 6
349 6

15.7 6
2.38 6

0.56 6
113 6

data regarding the effect the protein type has on TGs after 6 h
and thus cannot strictly exclude that CAS may have delayed
1.16
1.10
48.7

0.81
0.11

0.06
20.9

71.9 6 1.0
27.4 6 2.2

220.6 6 1.1

rather than lowered the postprandial TGs. However, because the

6
6
6

6
6

6
6
0h

apoB-48 levels decreased 6 h after CAS, reaching values similar

3.20

325
5.15

15.3

111
2.35

0.56

to the preprandial values (P = 0.34), we think that it is very

unlikely that CAS would have only delayed the elevation in TGs.
6 0.44
6 25.8

6 1.25
6 0.18

6 0.05
6 32.7

6.63 6 0.14 6.70 6 0.14

6 1.1

64.4 6 1.5

Whether the differences in TGs during the first 6 h after the meal
6h

resulted from less chylomicron formation, higher chylomicron

3.75

236
3.75

14.6

139
2.23

0.39

clearance, or both is unknown. Nonetheless, to analyze this

decrease in postprandial plasma chylomicrons levels, we studied
6 1.04
6 1.15
6 34.6

6 1.30
6 0.19

6 0.05
6 6.1

64.4 6 1.3
23.9 6 1.2

224.6 6 0.7

the interactions between protein and fat in the meals according

CAS high-fat meal
4h

to the expected changes in the pH as a result of acidification in

3.32

269
5.31

15.8
2.36

0.46
99.3

the stomach. In the mixed meals, dietary lipids were dispersed as

milk fat globules covered by their biological membrane, which is
6 1.11
6 0.69
6 32.8

6 1.40
6 0.22

6 0.05

0.12 6.51 6 0.14

6 9.6

61.5 6 1.0

rich in polar lipids. The mean size of fat globules was within the
2h

range of 1020 mm. When the pH was decreased to the values

3.49

306
4.82

14.6
2.24

0.43
97.1

prevailing in gastric juice, we showed that casein micelles were

1.46
1.04
35.5

0.86
0.14

0.09

not soluble at this pH and that the decrease resulted in a phase

8.6

0.8
2.1

1.1

separation of the lipids. This phenomenon was specific to CAS

3.66 6
5.44 6
310 6

16.2 6
2.42 6

0.65 6
101 6

71.3 6
22.9 6

220.8 6

6.50 6
0h

because it did not occur with the other 2 meals.

Compared with whey proteins, casein is known to precipitate
in the stomach and to display slow absorption kinetics that
change in DVP-RI, points

affect its further metabolism (24). Casein is a slow (i.e., slowly

GTN-induced change in
Physiological endpoints
8-iso-PGF2a, pmol/L

digested) protein, whereas other proteins such as whey are fast

Salbutamol-induced

DVP-RI, points

(rapidly digested). However, in a high-energy mixed meal, this

MCP-1, pg/mL
TNF-a, pg/mL
Plasma markers

VCAM, g/L

DVP-SI, m/s

difference in absorption kinetics is supposed to be much more

ICAM, g/L

PAI-1, g/L
TABLE 1

II-6, pg/mL

DVP-RI, %

limited because the high energy level dramatically restricts the

gastric emptying rate of protein (40, 43, 44). The global increase
in postprandial amino acids during this study did not differ
1

Casein and postprandial lipemia 5 of 8

FIGURE 3 Size distribution of fat globules in the
acidified nonhomogenized cream used for the high-
fat meals and in the high-fat meals that included
CAS, WHE, or LAC. CAS, casein; LAC, a-lactalbumin-
enriched whey protein; WHE, whey protein.
between the meals. Therefore, whey proteins, like casein, re-
sulted in slow digestion kinetics, probably because of the high
energy content of the meals that we used. The particularly low
solubility of casein in the gastric juice did not affect its diges-
tion and absorption but probably did affect the digestion and
absorption of fat. The phase separation in the meal consists in
a regrouping of the fat droplets that are otherwise dispersed in
the protein aqueous phase, so that the fat tends to come up to
the surface of the meal in the gastric juice. It is very likely that the
digestion and absorption of fat were markedly slowed down
within the CAS meal. First, phase separation could have lowered
the gastric emptying rate of the fat (45) and slowed down gas-
troduodenal lipolysis. Indeed, even a moderately larger size of
droplets in a lipid emulsion has been shown to reduce the speed of
gastroduodenal lipolysis (46). A phase separation and change in
the rate of absorption could also have increased the clearance of
chylomicrons and the metabolism of fatty acids (47). Finally,
blunting of the postprandial TG excursion could result from both
the formation of fewer chylomicrons and their increased clearance
as a consequence of the low solubility of casein in the stomach.
However, further studies are necessary to ascertain the mechanism
underlying this change in chylomicron kinetics.
No evidence of an impact of the protein source on any
subsequent adverse postprandial effects. In line with previ-
ous reports, the high-fat meal that we used during this study
induced a postprandial increase in oxidative stress (as shown
by plasma hydroperoxides and MDA) and a reduction in phys-
iological markers of endothelial function (salbutamol-induced
changes to pulse contour analysis), although some other markers
FIGURE 4 Effects of acidification on the appear-
ance of the high-fat meal according to the dietary
protein included (CAS, left; WHE, middle; LAC,
right) at the pH values indicated. As annotated, after
acidification, which caused a pH close to the value
in the stomach with the CAS meal but not with the
other meals, there was a phase separation between
fat and proteins. CAS, casein; LAC, a-lactalbumin-
enriched whey protein; WHE, whey protein.
6 of 8 Mariotti et al.
Downloaded from jn.nutrition.org at UNIVERSITY OF NEBRASKA on October 23, 2015
of postprandial dysfunction remained unchanged. In this setting,
and given the clear effect of CAS on postprandial TGs, we would
have expected to find an effect of the protein type in the meal on
these markers (48). This was not the case, however. Whether there
was actually no difference in postprandial endothelial function
and inflammation between these meals or whether we did not
identify them in this setting and with these endpoints cannot be
determined. Holmer et al. (41) found no postprandial difference
between casein and whey in a high-fat meal with respect to a large
set of inflammatory markers [except for chemokine (C-C motif)
ligand 5/regulated upon activation, normal T-cell expressed and
secreted], but they also reported no difference in postprandial TG
values. Pal et al. (49) also observed no difference between pro-
tein meals regarding certain plasma inflammatory markers in the
postprandial state in overweight postmenopausal women de-
spite differences in their postprandial TG levels. To further study
how the dietary protein source could modulate the adverse post-
prandial adverse effects of high-fat meals, it would be interesting
to more thoroughly examine endothelial function and inflamma-
tion in future studies, notably relative to markers of low-grade
inflammatory activation in leukocytes (6, 50).
Conclusion
The low solubility of casein and its precipitation that forms a gel
in the stomach are known to influence its rate of absorption and
postprandial protein metabolism in the context of regular, low-
energy meals. We showed that with a high-fat, high-energy meal,
this characteristic of casein results rather in a marked effect on
chylomicron kinetics and a decrease in postprandial TGs, a risk
factor for cardiovascular disease.
Acknowledgments
We thank Marie-Claude Amard and Malika Jourdain for their
important contributions to the organization of the experimental
sessions at the clinical research center and for their practical
assistance. FM and JFH designed the research; FM, MV, GA,
and JFH conducted the research; FM, MV, CL, HF, M-HF, VM,
D Tome, D Tsikas, and JFH obtained and analyzed the data;
FM drafted the manuscript; and FM and JFH had primary
responsibility for the final content. All authors have read and
approved the final manuscript.
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