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Postprandial lipid and carbohydrate responses after the ingestion of
a casein-enriched mixed meal
1–3
Sabine Westphal, Stephanie Ka¨stner, Elina Taneva, Andreas Leodolter, Jutta Dierkes, and Claus Luley
ABSTRACT
Background: Postprandial lipemia is markedly modulated when
carbohydrates are added to a fatty meal. The effect of added protein
is less known, however, and the data are controversial.
Objective: We investigated the effects of casein added to various
fat-rich meals in the absence and presence of oligosaccharides.
Design: Four different test meals were given to 24 healthy volun-
teers:1) fatalone, consistingof 3g cream/kgbody wt;2) fatplus 75g
oligosaccharides; 3) fat plus 50 g sodium caseinate; and 4) a com-
bination of all 3 components. Blood samples were taken before the
mealsand 1,2,3, 4,5, 6,7, and8h thereafter.The variablesmeasured
wereserum freefatty acids,arginine, glucose, insulin,and C-peptide
as well as triacylglycerol in serum, in chylomicrons, and in VLDL.
Gastric emptying was monitored with the use of a
13
C breath test.
Results:Addition of oligosaccharides resulted in the known delay
and reduction in postprandial lipemia. Casein caused additional ef-
fects: chylomicrons were further reduced and delayed, indepen-
dently of gastric emptying. C-peptide and insulin, as expressed by
their areas under the curves, were raised not only during the early
response to the glucose load but also in the postabsorptive state.
Concentrations of free fatty acids, which were markedly suppressed
by 24% after oligosaccharides alone, were lowered a further 20%
after the addition of casein.
Conclusions:Casein added to a fatty meal lowers free fatty acids
markedly in the postprandial and postabsorption phases, probably
via its insulinotropic activity. Postprandial lipemia is also moder-
ately reduced. Am J Clin Nutr 2004;80:284–90.
KEY WORDS Protein, triacylglycerol, postprandial lipemia,
insulin
INTRODUCTION
Single meals usually contain all 3 macronutrients: fat, carbo-
hydrate, and protein. The digestion and metabolism of these 3
main constituents are known to interact directly, eg, during the
absorptionstage, andindirectly, eg,via theactivation ofenzymes
or the secretion of hormones. Fat metabolism has been studied
repeatedly after the consumption both of fat alone and of fat
combinedwithcarbohydrates. It has been shown byothers(1–3)
and by ourselves (4) that carbohydrates added to a fatty meal
cause pronounced alterations in gastric emptying and in lipopro-
tein metabolism. Postprandial lipemia is both delayed and re-
duced.The loweringof thepostprandiallipemia ismost probably
caused by insulin acting via several mechanisms. One mecha-
nismconcerns the releaseof free fattyacids (FFAs) fromadipose
tissue, which is markedly suppressed by insulin through its in-
hibitory activity on the intracellular hormone-sensitive lipase.
Because the rate of hepatic VLDL production is strongly depen-
dentonthe FFA supply (5),hepatictriacylglycerol synthesis and
VLDL secretion are consequently slowed down (6–8). Finally,
insulin might stimulate lipoprotein lipase (9), resulting in an
accelerated clearance of triacylglycerol-rich lipoproteins (10).
Much less work has been done on the interactions caused by
the protein component in a meal. The results are conflicting, in
that either an increase in postprandial lipemia or no effect at all
isreported (11, 12).However,it couldbepostulated that proteins
such as casein interact with lipid metabolism as a result of their
insulinotropiceffects (13, 14).Wetherefore aimed toinvestigate
theeffect of caseinadded to variousfat-rich meals tosee whether
effects could be observed in the presence or absence of oligo-
saccharides in the meal. Furthermore, both the postprandial
phase and the postabsorption phase were studied for up to 8 h
after the meal.
SUBJECTS AND METHODS
Subjects
Twenty-fourhealthy students (12males, 12 females)tookpart
in the study. Inclusion criteria were as follows: normal weight
(body mass index, in kg/m
2
,쏜18.5 but 쏝25.0); nonsmoking;
normalalcohol habits;no historyof obesity,diabetes, orliver and
kidney diseases; normal blood pressure; and no regular use of
medications (Table 1). The subjects were instructed to refrain
from any unusual changes in their habits concerning physical
activity and nutritional behavior 4 wk before and throughout the
study. Their habitual dietary intake was recorded by self-report;
averageenergy intakes fromcarbohydrates, fat, andprotein were
49.2%, 37.4%, and 13.2% of energy, respectively.
1
From the Institute of Clinical Chemistry (SW, SK, ET, JD, and CL) and
the Department of Gastroenterology (AL), Magdeburg University Hospital,
Magdeburg, Germany.
2
Supported by grants from the Ministry for Education and Science Ger-
many (BMBF-Netzwerk, Molekulare Erna¨hrungsforschung, Förderkenn-
zeichen 0312750B).
3
Reprints not available. Address correspondence to S Westphal, Institute
ofClinical Chemistry,Leipziger Strasse44,D-39120 Magdeburg,Germany.
E-mail: sabine.westphal@medizin.uni-magdeburg.de.
Received October 27, 2003.
Accepted for publication February 2, 2004.
284 Am J Clin Nutr 2004;80:284–90. Printed in USA. © 2004 American Society for Clinical Nutrition
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Design
Each individual was studied 4 times, with intervals of 욷1wk
between the 4 oral fat loads. The liquid test meals consisted of
30% whipping cream, 3 mL (1 g fat) being given per kg body
weight. The cream (per 100 mL) contained 30 g fat (18.2 g
saturated and 9.04 g monounsaturated fatty acids), 3.5 g carbo-
hydrates,and 2.5gprotein. Thecream was mixed,in randomized
order, with 300 mL water containing or not containing 75 g of a
monosaccharide-oligosaccharide mixture (Dextro O.G-T;
Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany). The
protein-rich meal consisted of 30% whipping cream and 50 g
sodiumcaseinate (NumicoResearch,Wageningen, Netherlands;
Table 2) dissolved in water, with or without the oligosaccharide
mixture. The meals were drunk within 15 min between 0730 and
0800. All of the test meals were well tolerated, and no gastroin-
testinalsymptomswere reported. An ethics committeeapproved
theprotocol, andall subjectsgave theirwritten informedconsent.
Thefirstblood sample was withdrawn aftera fasting period of
12 h. Further blood samples were then taken immediately before
and 1, 2, 3, 4, 5, 6, 7, and 8 h after the oral fat tolerance test. No
other source of energy was provided, but water was allowed ad
libitum. The participants did not engage in any physical activity
duringthetest and had avoided exercising duringthe24 h before
the tests. Venous blood samples were collected under standardized
conditions, and serum was separated from the blood cells by cen-
trifugationfor10 min at 3000 ҂g.Analyses of the lipoproteins and
the metabolic variables were carried out within 24 h.
Determination of lipids and conventional lipoproteins
Theseparation of chylomicronsby ultracentrifugationandrepro-
ducibility issues are described in detail elsewhere (15). To isolate
chylomicrons, 1 mL plasma was layered under 2 mL saline (9 g
NaCl/L, d҃1.006 g/mL) and ultracentrifuged in polycarbonate
tubes (BeckmanInstruments, Krefeld, Germany)at 20 000rpm in a
50.3 Ti rotor for 20 min at 10 °C. Chylomicrons were carefully
isolated from the supernatant fluid. To determine triacylglycerol in
VLDL, the serum was ultracentrifuged for 18 h under the same
conditions, and the supernatant fluid containing VLDL plus chylo-
microns was aspirated off. The triacylglycerol in VLDL was calcu-
lated by subtracting chylomicron triacylglycerol from total triacyl-
glycerol in this fraction. All values were corrected for different
yields by weighing the tubes before ultracentrifugation and after
removal of the supernatant fluid.
Triacylglycerolconcentrationswere measured by use of com-
mercial enzymatic methods in a random-access analyzer (Hita-
chi911;Roche Diagnostics, Mannheim, Germany). Allreagents
and calibrators were from Roche Diagnostics. Plasma glucose
was measured by use of a commercial enzymatic method (GOD;
RocheDiagnostics), insulin bya commercial radioimmunoassay
(BI-InsulinIRMA;BIO-RAD, Munich, Germany),andFFAs by
a commercial enzymatic colorimetric method (Wako Chemicals
GmbH, Neuss, Germany). C-peptide was analyzed by use of the
Immulite system (DPC Diagnostic Products Corporation, Los
Angeles, distributed in Germany by DPC Biermann GmbH, Bad
Nauheim, Germany), which is a fully automatic random-access
chemiluminescence-enhanced enzyme immunoassay system
(16). Arginine was measured by HPLC.
Gastric emptying
Allgastricemptying tests were done incombinationwith the test
meals,150 mg[
13
C]sodiumacetate beingdissolved inthe fattymeal
(17). Breath samples were collected before and then every 15 min
for 240 min after the test meal and were analyzed for isotopic en-
richmentby using anisotope ratio massspectrometer with anonline
gas-chromatographic purification system. The half-time of gastric
emptying was calculated after curve fitting of the
13
C exhalation to
a modified power exponential function.
Statistics
Data are presented as means 앐SEMs. The gastric emptying
data are presented as medians and 25th and 75th percentiles. To
evaluatethe overall responseof total triacylglycerol,triacylglyc-
erol in VLDL, triacylglycerol in chylomicrons, FFAs, glucose,
insulin, and C-peptide during the 8-h postprandial period, the
areas under the postprandial curve (AUCs) were calculated by
the trapezoid rule. Statistical analysis of the data was performed
by using a two-factor repeated-measures analysis of variance.
Differences between the test meals were tested for significance
by using Tukey’s post hoc test. Differences in the gastric emptying
between the different test meals were checked for significance by
TABLE 1
Characteristics of the subjects
1
Value
Age (y) 21 앐2
BMI (kg/m
2
)22 앐1
Height (cm) 1.74 앐0.10
Weight (kg) 68.6 앐13.1
Glucose (mmol/L) 5.15 앐0.30
Insulin (pmol/L) 27 앐14
Triacylglycerol (mmol/L) 1.07 앐0.27
Cholesterol (mmol/L) 5.10 앐0.37
LDL cholesterol (mmol/L) 3.16 앐0.36
HDL cholesterol (mmol/L) 1.5 앐0.36
Free fatty acids (mmol/L) 0.50 앐0.15
1
All values are x앐SEM; n҃12 men, 12 women.
TABLE 2
Amino acid composition of the intact casein protein
Amino acid Value
g/100 g
L-Alanine 2.77
L-Arginine 3.52
L-Aspartate 6.11
L-Cysteine 0.45
L-Glutamate 20.56
L-Glycine 1.74
L-Histidine 3.45
L-Isoleucine 5.42
L-Leucine 8.70
L-Lysine 7.36
L-Methionine 2.60
L-Phenylalanine 4.72
L-Proline 10.26
L-Serine 5.44
L-Threonine 4.46
L-Tryptophan 1.16
L-Tyrosine 5.02
L-Valine 6.24
POSTPRANDIAL RESPONSES TO A CASEIN MEAL 285
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use of Wilcoxon’s signed-rank test with Bonferroni correction.
SPSS for WINDOWS (version 7.5; SPSS Inc, Chicago) was used
for the analyses. Significance was set at P쏝0.05.
RESULTS
Gastric emptying
The effects on gastric emptying of the addition of oligosac-
charidesorcasein to fat, separately orin combination, are shown
inFigure 1. Thehalf-timeof gastricemptyingafter fat alonewas
127 min and was substantially prolonged to 193 min after the
additionofoligosaccharides (P쏝0.01). Whencaseinwas added
to fat alone or in combination with oligosaccharides, no further
significant effect was detectable by use of the
13
C breath test.
Postprandial lipemia
The kinetics of total serum triacylglycerol after the ingestion
of the 4 different meals is shown in Figure 2A. Compared with
fat alone, oligosaccharides exerted 2 pronounced effects: they
delayed the triacylglycerol peak from 3 to 5 h and lowered the
triacylglycerol concentrations, particularly at early time points.
Casein had a similar but considerably weaker action; the triac-
ylglycerol peak was delayed by 앒1h.
The triacylglycerol concentrations in chylomicrons and in
VLDL are shown in Figure 2, B and C. As in serum, a delay and
a reduction were observed for each density fraction. A strong
effect was exerted by oligosaccharides, a somewhat weaker one
by casein, and the greatest by the combination of the 2. The
reductions were more pronounced in chylomicrons than in
VLDL, in which delay was the predominant effect.
The AUCs are shown in Table 3. When fat alone was compared
with fat plus oligosaccharides, the reductions in triacylglycerol
FIGURE1. Half-time of gastric emptying in 24 normolipidemic subjects
after 4 different meals: 1) 1 g fat/kg body wt, 2) fat combined with 50 g
sodium caseinate, 3) fat combined with 75 g oligosaccharides, and 4) fat
combined with oligosaccharides and caseinate. Results are shown as box
plots, in which the parts of the plot are the median, the box (which indicates
the 25th and 75th percentiles), and the error bars (which indicate the 5th and
95th percentiles).
*
Significantly different from fat alone, P쏝0.05 (Wilcox-
on’s signed-rank test with Bonferroni correction).
FIGURE 2. Mean (앐SEM) postprandial triacylglycerol concentrations
in serum (A), chylomicrons (B), and VLDL (C) in 24 normolipidemic sub-
jects after 4 different meals in each case: 1) 1 g fat/kg body wt (continuous
line), 2) fat combined with 75 g oligosaccharides (broken line), 3) fat com-
bined with 50 g sodium caseinate (dotted line), and 4) fat combined with
oligosaccharides and caseinate (dot-dash line). For all panels, there were
significanteffects oftime (P쏝0.001)and meal (P쏝0.001) and asignificant
meal ҂time interaction (P쏝0.001), ANOVA for repeated measures.
286 WESTPHAL ET AL
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AUCs in serum, chylomicrons, and VLDL were 10%, 21%, and
6%, respectively. The effects of casein on the triacylglycerol
AUCs were significant only when casein was added together
with oligosaccharides. This additional lowering caused by ca-
sein was 8%, 17%, and 6% for triacylglycerol in serum, chylo-
microns, and VLDL, respectively.
Carbohydrate metabolism
As was to be expected, fat alone did not significantly alter
concentrations of C-peptide, insulin, or glucose (Figure 3).
When oligosaccharides and oligosaccharides plus casein were
added, however, these variables were affected in 2 phases: in an
early phase up to 3 h, which was dominated by glucose regula-
tion, and in a second phase, between 4 and 8 h, when the specific
effect of casein was observed. In the early phase, C-peptide and
insulin increased sharply and glucose returned to baseline.
In the second phase, concentrations of C-peptide and insulin
were elevated by between 30% and 92% after the casein-
containingmeals.These delayed elevations were independentof
the prior addition of oligosaccharides.
Free fatty acids
Although FFAs are known to be closely linked with the me-
tabolismoftriacylglycerol and carbohydrates, their postprandial
kinetics do not quite run in parallel with those of the above-
mentioned variables (Figure 4). After fat alone, there was an
early increase up to 3 h, similar to the increase in triacylglycerol
but with a subsequent plateau. When oligosaccharides were
addedtothe fat drink, there wasapronounced suppression of the
FFAs for 3 h, after which there was an increase similar to that
after fat alone but delayed by 3 h. Casein added to fat alone
caused significant reductions in FFAs at almost all time points,
which resulted in a curve parallel to that given by fat alone but
with the concentrations being lower by 13–36%. When both
oligosaccharides and casein were added to the fat, the effects on
FFA were additive. Compared with fat alone, the suppression
reached maximum values of 18–72% at time points of 0.5–6h.
The AUCs of C-peptide, insulin, and FFAs reflect these
changes (Table 3). Note that the casein-induced changes in
C-peptide and insulin, expressed as percentages, were more pro-
nouncedduring the postabsorptionphase (4–8 h)thanduring the
early phase (0 and 3 h), particularly when casein had been given
together with oligosaccharides. Because the FFA AUC was
markedly lowered by oligosaccharides and by casein, there was
a pronounced lowering by combination of the 2, by 46% com-
pared with the AUC of FFAs after fat alone.
DISCUSSION
The effects of additions of oligosaccharides to a fatty meal
have been investigated repeatedly (2–4). The effect of protein
added to fat has been studied much less and with conflicting
results.Sullivan (11) studied6 subjectsover3 handreported that
casein caused an increase in postprandial lipemia. Cohen (12)
added sodium caseinate to fat and did not observe any change in
postprandial lipemia over a period of 7 h. Neither investigator
studiedthe effect ofcasein plus oligosaccharideson postprandial
lipemia and, consequently, neither looked at the variables of
carbohydratemetabolism. Our studyconsideredfor thefirsttime
the interaction of all 3 nutritional components. We found that
casein1) moderatelyreducesand delaysthe postprandiallipemia
and2) markedly lowerspostprandialand postabsorptive concen-
trations of FFAs. We postulate that the insulinotropic action of
casein is responsible for both of these effects.
Caseinwasshown to be insulinotropic as earlyasin 1966 (18)
and has since been studied on a cellular level and on a whole-
body level in vivo in humans. Sener and Malaisse (19) observed
that the addition of leucine to the incubation medium stimulates
insulin release by pancreatic

cells in vitro. Leucine activates
glutamate dehydrogenase activity. This subsequently leads to an
increase in tricarboxylic acid cycle activity and is attended by
increasedinsulin production. Byusing infusions, itwas shown in
humans that several amino acids lead to significant increases in
plasma insulin. Floyd et al (20, 21) investigated different combina-
tions of amino acids and found that the combined administration of
TABLE 3
Areas under the curves
1
Fat Fat ѿcasein Percentage
change
2
Fat ѿ
oligosaccharides
Fat ѿ
oligosaccharides
ѿcasein Percentage
change
3
Pfor meal
effect
(ANOVA)
%%
Triacylglycerol (mmol 䡠h/L)
Serum 13.3 앐2.1
b,4
13.5 앐1.8
b
1 12.0 앐1.4
a
11.1 앐2.3
a
Ҁ8 0.001
Chylomicron 3.8 앐0.8
c
3.9 앐0.9
c
3 3.0 앐0.7
a
2.5 앐0.8
b
Ҁ17 0.001
VLDL 5.5 앐0.5
a
5.8 앐0.4
a
5 5.1 앐0.7
b
4.8 앐0.6
b
Ҁ6 0.001
Free fatty acids (mmol 䡠h/L) 6.3 앐0.5
b
4.9 앐0.3
a
Ҁ20 4.5 앐0.7
a
3.4 앐0.2
c
Ҁ24 0.001
Glucose (mmol 䡠h/L) 49.6 앐9.7
b
50.5 앐8.5
b
253.9 앐7.6
a
52.5 앐7.8
a
Ҁ2 0.001
Insulin (pmol 䡠h/L)
0–3 h 176 앐54
a
227 앐0.42
a
29 844 앐49
b
931 앐51
b
10 0.001
4–8h 91앐118
a
118 앐0.42
b
30 126 앐40
c
182 앐20
d
44 0.001
C-peptide (pmol 䡠h/L)
0–3 h 3166 앐100
a
3314 앐101
a
5 8099 앐277
b
8717 앐326
b
8 0.001
4–8 h 2270 앐70
a
2711 앐65
b
19 2735 앐277
c
3758 앐326
d
37 0.001
1
n҃24. Values within a row with different superscript letters are significantly different, P쏝0.05 (post hoc tests with Tukey’s adjustment).
2
Percentage change from fat alone after the addition of casein.
3
Percentage change from fat ѿoligosaccharides after the addition of casein.
4
x앐SEM (all such values).
POSTPRANDIAL RESPONSES TO A CASEIN MEAL 287
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arginine-leucineandarginine-phenylalanine with glucose results in
thelargestincrease in plasma insulin concentrations.Thissynergis-
tic effect of the combined intake of carbohydrate and protein on
plasma insulin concentrations was later confirmed in both healthy
subjects(22) andthose withtype2 diabetes(23–25). Thiseffect was
considerably less pronounced in nondiabetic persons (26). Van
Loon et al (27–29) performed a series of studies in healthy young
subjectsinwhom he determined the invivoinsulinotropic potential
of various proteins, hydrolysates, and free amino acids in combina-
tionwith carbohydrates. Hefoundthe highestinsulinsecretion after
a mixture containing wheat protein hydrolysate, free leucine, and
phenylalanine. In contrast, arginine, in an amount likely to be in-
gested in a high-protein meal, does not stimulate insulin secretion
(30). Note that the above studies focused only on 2–3 h after the
oligosaccharidesintake.Our data, alsoobtainedin nondiabetic sub-
jects,showan increase in theinsulinAUC by 29% upto3 h; insulin
secretion,however, didnotaffect glucosewhen caseinwasadded to
thefatalone. This casein-inducedrisein insulin also occurredwhen
oligosaccharideswere givenin addition,leadingto alowering ofthe
peak glucose concentration by 10% after 60 min (P쏝0.05). Thus,
ourdata confirm earlyinsulinotropicaction of caseinin nondiabetic
persons.
In contrast with the above-mentioned studies, the present
studyinvestigated the postabsorptionphase upto8 h.During this
second phase, concentrations of C-peptide and insulin are ele-
vated almost during the entire period from 4 to 8 h and decline
only slowly. The reason for these late elevations is probably
again the release of insulinotropic amino acids from casein. The
kinetics of arginine as a representative amino acid are shown in
Figure 3. Interestingly enough, the variable reacting most sensi-
tively to this late and continuous insulin elevation was not glu-
cose but FFAs. Although glucose concentrations do not react at
all with the administration of casein, the FFA AUCs were re-
duced by 20% and 24% when casein was added to fat alone and
to fat plus oligosaccharides, respectively. The observation that
FFAsaremore sensitive to insulinvariationsthan is glucose isin
line with a clamp study by Boden et al (31) that showed a dose-
dependent lowering of FFAs by insulin before glucose was af-
fected.
FIGURE 3. Mean (앐SEM) postprandial concentrations of C-peptide in
serum, of insulin in serum, and of oligosaccharides and arginine in plasma in
24 normolipidemic subjects after 4 different meals in each case: 1) 1 g fat/kg
body wt (continuous line), 2) fat combined with 75 g oligosaccharides (bro-
kenline), 3) fatcombined with50 gsodium caseinate (dottedline), and4) fat
combinedwith oligosaccharidesand caseinate(dot-dash line). Forall panels,
there were significant effects of time (P쏝0.001) and meal (P쏝0.001) and
a significant meal ҂time interaction (P쏝0.001), ANOVA for repeated
measures.
FIGURE4. Mean (앐SEM) concentrations of free fatty acids in serum in
24 normolipidemic subjects after 4 different meals in each case: 1) 1 g fat/kg
body wt (continuous line), 2) fat combined with 75 g oligosaccharides (bro-
kenline), 3) fatcombined with50 gsodium caseinate (dottedline), and4) fat
combined with oligosaccharides and caseinate (dot-dash line). There were
significanteffects oftime (P쏝0.001)and meal (P쏝0.001) and asignificant
meal ҂time interaction (P쏝0.001), ANOVA for repeated measures.
288 WESTPHAL ET AL
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This lowering of FFAs may bring about certain beneficial
consequences. Because FFAs fuel hepatic triacylglycerol pro-
duction, a lowering of VLDL may be a long-term consequence
when fat-rich meals are regularly combined with arginine-rich
proteins such as casein. This assumption is in line with an ob-
servationby Hursonet al(32), whosupplemented17garginine/d
isocalorically to humans for 14 d and reported a significant 18%
lowering of the serum triacylglycerol. Further advantages of
reductionsinFFAs might relatetoa decrease intheLDL transfer
into endothelial cells (33), improvements of the antioxidative
protection of endothelial cells (34, 35), and inhibition of platelet
aggregation (36).
Regarding the delay and reduction of postprandial lipemia
after the addition of oligosaccharides to a fatty meal, another
mechanism is in operation besides the increase in insulin. The
pronounceddelays of chylomicronsand VLDL arecaused by the
well-known effects of glucose and insulin on gastric emptying
(37, 38). When casein was added, there was a further delay
without an accompanying delay in the
13
C breath test. We spec-
ulatethatthis additional postponementmayhave been caused by
gastric precipitation of casein (39, 40) slowing down the subse-
quent fat absorption in the intestine. In addition to these delays,
there were also reductions, mainly in chylomicrons. The chylo-
micron reduction may be secondary to the increased insulin con-
centrations, because insulin activates lipoprotein lipase (41, 42),
which might degrade chylomicron triacylglycerol more rapidly.
In conclusion, casein added to an oligosaccharide-containing
fatty meal reduces the chylomicron response. Casein also mark-
edly suppresses FFAs in the presence and absence of oligosac-
charidesin thefatty meal.The FFAsuppression occurringinboth
the postprandial and postabsorption phases may be beneficial.
We are indebted to Eva Maria Gittel, Katrin Deneser, and Marlies Kania
for excellent assistance with the laboratory work.
SW designed the study and wrote the manuscript, SK organized and
carried out the clinical study, ET organized the measurements and collection
ofdata, AD carried out the breath tests,JD performedthe statisticalanalyses,
and CL helped in study design, evaluation, and manuscript writing. None of
the authors had a personal or financial conflict of interest.
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