Gastrojejunal kinetics and the digestion of [15N]f3-

lactoglobulin and casein in humans: the influence of the

nature and quantity of the 2

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

Sylvain Mah#{233},Nils Roos, Robert Benamouzig, Laurence Davin, Catherine Luengo, Louise Gagnon,
Nicolas Gausserg#{232}s, Jacques Rautureau, and Daniel Tome

ABSTRACT The evolution and luminal effects of different depends on both the liquid and solid composition and energy
quantities of casein and f3-lactoglobulin were investigated in the content of the meal (3, 4). Proteins, according to both their
upper jejunum of 35 volunteers who ingested 400 mL water with structure and the nature of the chyme, are degraded differently
either -lactoglobulin or casein in either low or high doses (72.6 by gastric and pancreatic proteases into peptides and amino
mmol N, Llg; 71.7 mmol N, LCas; 368.2 mmol N, HI3lg; 386.8 acids and taken up by the intestinal mucosa, where brush
mmol N, HCas). The flow rate of the liquid effluents as well as the border membrane peptidases and transport systems are respon-
nitrogen movements were measured and the exogenous ([15N]) sible for the ultimate transfer of amino acids to the blood (5).
and endogenous nitrogen fractions analyzed in the upper jejunum We previously showed that some dietary proteins or polypep-
after milk protein ingestion. The basal jejunal liquid flow rate tides may escape the luminal hydrolytic process and reach the
(mL/min) was 3.88 ± 1.84 and peaked in the 0-20 mm period for intestinal mucosa in significant amounts, where they are sub-
water (9.92 ± 3.72) and Llg (7.27 ± 3.08) and during the 20-40 sequently absorbed intact through transcytosis mechanisms (6).
mm period for LCas (5.69 ± 2.49), HCas (6.32 ± 1 .85), and H/31g In addition, dietary proteins are suspected to differently stim-
(6. 1 1 ± 2.3 1 ). One hour after water, LCas, Llg, Hlg, and HCas ulate gastrointestinal hormones (gastrin, cholecystokinin, Se-
ingestion, 100%, 95%, 85%, 71%, but only 38% of the liquid cretin, and gastrin inhibitory peptide), which modulate the
phase of the meal were passed through the jejunum, respectively. overall digestion process through a control in both gastric as
The flow rate of the endogenous nitrogen was 12.93 ± 5.22 mmol well as intestinal motility, and gastric as well as pancreatic
N/h before meal ingestion; remained unchanged after water, LCas, secretions (7). It has yet to be determined whether the stimu-
or Hf3lg ingestion: but increased significantly (P < 0.05) after lation originates only from the amino acids absorbed or
L3lg and HCas ingestion. The net disappearance of exogenous whether it is also due to the presence of specific bioactive
nitrogen in the upper jejunum 240 mm after HCas, Llg, LCas, peptides cleaved from dietary proteins during digestion and
and Hlg ingestion was 82.6 ± 9.5%, 61.6 ± 9.6%, 58.4 ± 14.7%, acting either at the luminal level or after absorption (8, 9).
and 44.7 ± 24.4%, respectively. The exogenous fraction of protein Bovine milk protein is made up of two fractions including
nitrogen recovered in the upper intestinal lumen represented 43.3% casein, which is a micellar fraction, and soluble whey protein,
of the ingested Hlg nitrogen. but only 4.9% of the ingested HCas in which f3-lactoglobulin is the major component. In previous
nitrogen. In conclusion, casein and f3-lactoglobulin present differ- studies in healthy human volunteers we showed that after milk
ences in both the intestinal kinetics of amino acid delivery and in ingestion a nitrogen fraction showed delayed gastric emptying
the nature of the products in the intestinal lumen. These differences (10). Further studies with a low intake of purified casein and
have to be taken into account from both nutritional and physiologic -lactoglobulin in healthy humans showed delayed gastric
points of view for the utilization of these proteins in humans. emptying of casein compared with 3-lactog1obulin but no
Am J C/in Nutr 1996;63:546-52. difference in either the stimulation of endogenous nitrogen
secretion or in the gastrojejunal absorption of nitrogen (1 1). In
KEY WORDS Milk proteins, stable isotope, intestine the same way, no significant increase in the endogenous nitro-
gen fraction was observed in the jejunum of healthy humans

INTRODUCTION i From the Unite INRA de Nutrition Humaine et de Physiologic Intes-

tinale, Facult#{233}des Sciences Pharmaceutiques et Biologiques, Paris; the
The delivery of amino acids and nitrogen from the gut to the
Institut f#{252}r
Physiologic und Biochemie der Ern#{228}hrung, Bundesanstalt f#{252}r
organism depends on the different intestinal processes, includ-
Milchforschung, Kid, Germany; and the Service de Gastroent#{233}rologie,
ing gastric and intestinal motility, luminal digestion, and mu-
H#{244}pitalAvicenne, Bobigny, France.
cosal absorption. These different steps are affected by the 2 Address reprint requests to S Mah#{233},Unite de Nutrition Humaine et de
nature of the meal, ie, the liquid or solid composition of the Physiologic Intestinale (INRA), Facult#{233}des Sciences Pharmaceutiques et
meal, as well as by the type of meal components (eg, protein, Biologiques, 4 avenue de l’Observatoire, 75270 Paris C#{233}dex06, France.
lipid, and carbohydrate) (1 , 2). The gastric emptying rate, Received July 19, 1995.
which controls the transfer of nutrients to the small intestine, Accepted for publication December 5, 1995.

546 Am J Cliii Nutr l996;63:546-52. Printed in USA. tO 1996 American Society for Clinical Nutrition
 JEJUNAL DIGESTION OF [‘5NIMILK PROTEINS IN HUMANS 547

after they ingested a low amount of [‘5Nlcasein (12). These Perfusion technique
observations at a low protein intake agree with the current idea
A tube was passed through the nasal cavity to the small
that the delayed gastric emptying of casein originates, at least intestine at the level of the jejunum as described previously
in part, from its clotting in the stomach whereas whey protein (10). The intestinal tube had a double lumen, one of which was
remains soluble and is more rapidly emptied. The consequence
used to aspirate intestinal contents in the upperjejunum and the
of these different gastric emptying rates on subsequent intes-
other to perfuse phenol red at the Treitz angle 20 cm upstream,
tinal and systemic processes remains to be clearly established which allowed us to calculate the effluent flow rate by using
at both low and high protein intakes. Because bioactive pep-
the slow-marker technique.
tides have been shown in the sequence of both proteins, but
especially in the sequence of the casein in which opioid and gut Experimental design
hormone-stimulating peptides were suspected, an additional
The first day the subjects were fitted with the intestinal tube
specific effect of casein on gastric and intestinal processes
and fasted overnight. The morning of the experiment the po-
cannot be excluded (8, 13).

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

sition of the tube was checked under radioscopic control.
The aim of the present experiment was to further determine
Twenty minutes before meal ingestion, a first sample of intes-
the evolution and luminal effects of different quantities of
tinal contents, which corresponded to the basal endogenous
casein and j3-lactoglobulin in the upper jejunum in human
nitrogen secretion of the subject, was collected. After this
volunteers. For this purpose healthy human volunteers ingested
moment and during the whole test period (4 h), a saline
either a low or a high dose of the protein [‘5N]-lactoglobu1in
solution containing phenol red was perfused into the intestine
or casein to differentiate between endogenous and exogenous
at the rate of 1 mL/min. Intestinal samples were collected
intestinal nitrogen as described previously (12).
continuously through the distal opening of the jejunal tube.
Aspirates were collected on ice and pooled in 20-mm intervals.
MATERIALS AND METHODS At the end of the test, the gastric content was completely
aspirated by using a gastric tube (single lumen sump tube) and
Diets the stomach was washed with 200 mL normal saline solution.
Water (Vittel, 03270; Saint-Yorre, France) was used as the Subjects were not allowed to ingest food or fluids during the
control. [‘5N]milk was obtained from a cow whose rumen was remainder of the collection period.
perfused continuously with a solution of ([15N1H4)2SO4. Purified
Analytical methods
milk proteins were prepared from [15N]milk by membrane micro-
filtration as described previously (14) and was made up of [‘5N1/3- The volume and pH of the digesta were measured after
lactoglobulin, whose enrichment was 0.4532 atom percent and homogenization. The digesta were treated with 0. 1 mmollL of
native phosphocaseinate ([‘5NJcasein), whose enrichment was the protease inhibitor diisopropylfluorophosphate, frozen at
0.5070 atom percent. The [i5N]proteins were suspended in water - 20 #{176}C,
and then freeze-dried. The concentration of PEG-4000
and polyethylene glycol (PEG-4000, 37.5 gIL) was added to the in the digesta, which corresponded to the liquid phase of the
meals as a nonabsorbable marker of the liquid phase. meal, was measured by a turbidimetric method (15) and those
of phenol red by colorimetry (16).
Subjects

Thirty-five healthy volunteers (15 males and 20 females) were

Nitrogen analysis
selected according to the following criteria: no history of gastro- Precipitable and soluble nitrogen were measured after etha-
intestinal problems; no disorders of the gastrointestinal system; nol precipitation of the dry samples as described previously
not pregnant; a stable, satisfactory nutritional status; as well as a (12). Briefly, ethanol (70%, by vol) and hexane were added to
stable body weight. The subjects were divided into five groups: 1) the aliquots of the dry matter of the digesta and allowed to
four female volunteers ranging in age from 23 to 32 y (28 ± 4 y) flocculate at 4 #{176}C
for 1 h. Then the digesta were centrifuged at
and in weight from 56 to 60 kg (58 ± 2 kg) were provided with 2500 x g at 4 #{176}C
for 25 mm. The upper hexane layer, which
400 mL water; 2) seven volunteers (four males and three females) contained lipids, was discarded and the ethanol fraction col-
ranging in age from 20 to 45 y (29 ± 8 y) and in weight from 56 lected. The pellet was washed once more by using the same
to 82 kg (67 ± 8.4 kg) were given 71.7 mmol [i5Nlcasein in 400 procedure. The pellet and the supernate were dried under
mL water (a low amount ofcasein, LCas), 3) eight volunteers (two reduced pressure with a vacuum concentrator. The pellet was
males and six females) ranging in age from 21 to 35 y (28 ± 5 y) believed to be made up of proteins (protein nitrogen, PN) and
and in weight from 45 to 104 kg (63 ± 18 kg) were given 386.8 the dried supernates to contain peptides and free amino acids
mmol [‘5N]casein in 400 mL water (a high amount of casein, (nonprotein nitrogen, NPN). The protein, nonprotein, and total
HCas), 4) seven volunteers (four males and three females) ranging nitrogen contents as well as the isotopic ratio of ‘5N to i4N
in age from 20 to 34 y (26 ± 5 y) and in weight from 60 to 72 kg were determined by mass spectrometry. An aliquot of the
(67 ± 7 kg) were given 72.6 mmol [t5NJ(3-lactoglobulin in 400 freeze-dried samples was burned in the presence of purified
mL water (a low amount of -lactoglobulin, Llg), and 5) nine oxygen in the combustion unit of an elemental analyzer (NA
volunteers (five males and four females) ranging in age from 19 to 1500; Fisons, Manchester, United Kingdom) at 950 #{176}C.
The
31 y (26 ± 4 y) and in weight from 52 to 82 kg (65 ± 10 kg) were combustion unit was coupled with an isotope ratio mass spec-
given 368.2 mmol [‘5N]3-lactoglobu1in in 400 mL water (a high trometer (Optima; Fisons). The isotope ratio of N2 was mea-
amount of f3-lactoglobulin, H/31g). The protocol was previously sured in reference to a calibrated ‘5N: i4N nitrogen tank.
approved by the Ethical Committee of the hospital. All subjects The samples from jejunal effluents were analyzed by using
gave their consent to participate in the study. sodium dodecyl sulfate-polyacrylamide gel electrophoresis
548 MAHE ET AL

(SDS-PAGE) with 15% polyacrylamide gels in the presence of 280

0. 1 % SDS. Standard proteins were used as internal standards: 240
casein, -lactoglobulin, and a-lactalbumin. 200
160 Water
Calculations and statistical analysis
120
The intestinal flow rate (F; mL/20 mm) was calculated 80
according to the following formula:
40
F = (Fm X Cm)/Cd, (1) 0
240
where Fm 5 the flow rate of the nonabsorbable marker (phenol
200
red), Cm 5 the concentration of phenol red in the perfusion, and
160 LB1g
Cd S the concentration of phenol red in the digesta. This F
( 120

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

value was minored by the perfusion volume 1 mL/min). The
differentiation between the exogenous and endogenous nitro- 80
gen fractions was determined by the isotopic ratio of i5N to i4N 40
according to the following formula: 0
240
Nexo N10 X (APEd APE0)/(APEm APE0) (2)
. 200
a) 160 LCas
where N101 is total nitrogen, and APEd, APE0, and APEm are the
enrichment of the digesta, the basal secretions, and the ingested 120
meal, respectively. The endogenous nitrogen in the digesta z 80
(Nendo) was calculated as N101 - Nexo. The results were ex- .
pressed as mean ± SD. .a5, 0

240
Statistical analysis
200 HB1g
Statistical analysis was performed by using one-way analysis 160
of variance (ANOVA) and Tukey’s Studentized range test
120
(SAS version 6.03; SAS Institute, Inc, Cary, NC).
80
40
RESULTS 0
240
Flow rates of liquid effluents HCas
200
The liquid flow rates of jejunal effluents were calculated 160
from the phenol red concentrations and corrected for perfusion 120
( 1 mL/min)
I Al
volume every 20 mm before and after meal inges-
80
tion (Figure 1). In the 20 mm preceding meal ingestion, the
40
flow rate was 3.88 ± 1 .84 mL/min (n = 23) and differed
0
significantly from 0 (P < 0.05). After ingestion of 400 mL
200 240
water,
mm period
the flow rate peaked
and returned
at 9.92 ± 3.72 mL/min
to the basal value
in the 0-20
after 40 mm. After
#{176}D
40 80 120 160
Time (mm)
ingestion of a low amount of f3-lactoglobulin (Lj31g), the jeju- MEAL
nal flow rate peaked at 7.27 ± 3.08 mL/min during the 0-20
FIGURE 1. iejunal effluent flow rate after the ingestion of water, a low
mm period and returned to the basal value during the 40-60
amount of Ei5N]3..lactog1obulin (L3lg) and [iSN]casein (LCas), and a high
mm period. After ingestion of LCas, the jejunal flow rate amount of [i5Nl(34actoglobulin (H(3lg) and [iSN]casein (HCas). Each
peaked at 5.69 ± 2.49 mL/min during the 20-40 mm period value represents the mean (± SD) of four subjects in the water group,
and returned to the basal value after 80 mm. After ingestion of seven subjects in the L(31g and LCas groups, and eight subjects in the H3lg
H3lg, the jejunal flow rate peaked at 6.1 1 ± 2.31 mL/min and HCas groups.
during the 20-40 mm period and returned to the basal value
after 60 mm. After ingestion of HCas, the jejunal flow rate
peaked at 6.32 ± 1.85 mL/min during the 20-40 mm period
and returned to the basal value after 140 mm. after Lf3lg and Hf3lg ingestion, 85% and 71% of the liquid
The transit of the liquid phase of the meals was calculated phase of the meal were passed through the jejunum, respec-
from the appearance of PEG-4000 at the collection site, ie, tively. After ingestion of LCas, the liquid-meal flow rate
jejunum (Figure 2). The liquid phase of the meal was peaked during the 0-20 mm period and 95% of the liquid
rapidly emptied from the stomach after the ingestion of phase of the meal was passed through the jejunum during the
water because “80% of the PEG was recovered during the first hour. In contrast, the liquid-meal flow rate peaked at
0-20 mm period. The liquid-meal flow rate peaked during 20-40 mm after HCas ingestion but only 38% of the liquid
the 0-20 mm period after L3lg ingestion, whereas it peaked phase of the meal was passed through the jejunum during the
during the 20-40 mm period after Hj31g ingestion. One hour first hour.
 JFJUNAL DIGESTION OF [‘5N]MILK PROTEINS IN HUMANS 549

100 . tion; but increased significantly (P < 0.05) compared with the
basal value during the 0-20-mm and 20-40-mm test periods
80
after Lj3lg and HCas ingestion, respectively (Figure 3). The
60 Water values of endogenous nitrogen (mmol N) recovered during the

I
0-120-mn period after meal ingestion were 13.85 ± 3.64, 15.9
40
± 4.66, 18.35 ± 7.93, 13.39 ± 7.82 and 27.63 ± 5.62 for
20 water, Lf3lg, LCas, H/31g and HCas, respectively. These values

0 T1T i i I were not statistically different except for HCas, which was
significantly higher (P < 0.05)
80

c’ 60

a, 40 20 .
Q

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

01
16 . .
.E 20
0-
T I 12 . Water
0

‘- 80
E
60 . LCas

? 40

.E 20 1
0
0 12 LB1g
iJirar I I 1
>
0 80
0

L 60 . . HBIg
C,
w
Q_

16
20
C

0 ) 12#{149} LCas
0

80
HCas
8-

!
E
60

40
. 16
20
p?1 I I P’A ri -. -. 8) 12
0
0 40 80 120 160 200 240
Time (mm)

MEAL

FIGURE 2. iejunal effluent flow rate of the liquid phase of the meal 1. HCas
after the ingestion of water, a low amount of [‘5N]3-Iactoglobulin (L(31g)
12 .
and [15N}casein (LCas) and a high amount of [‘5N]13-lactoglobulin (H(31g)
and [‘5N]casein (HCas). Meals were adjusted to 37.5 g/L with polyethylene
glycol 4000 (PEG-4000) as a nonabsorbable liquid-phase marker. Each
value represents the mean (± SD) of four subjects in the water group,
seven subjects in the L(3lg and LCas groups, and eight subjects in the H(31g
and HCas groups.
ofI\ 40 80 120 160 200 240
Time (mm)
MEAL
Nitrogen movements

Total nitrogen was measured for each sample collected every FIGURE 3. The endogenous nitrogen profiles ofthe digesta in the jejunum
after the ingestion of water, a low amount of [‘5Nlf3-lactoglobulin (Llg) and
20 mm before and after meal ingestion. Both the exogenous
[‘5N}casein (LCas) and a high amount of f’5N](3-lactoglobulin (Hf3lg) and
and the endogenous nitrogen stemming from the meals con-
[‘5N]casein (HCas). After [‘5N]protein ingestion, the endogenous nitrogen
taming [‘5N] or endogenous secretion were calculated from the
fractions were from the ratio of 5N to 4N in the digesta
calculated except after
ratio of ‘5N to ‘4N in jejunal effluents every 20 mm after [‘5N]
water ingestionall the nitrogen
where
recovered was assumed to be the basal
ingestion. The flow rate of the endogenous nitrogen was endogenous protein secretion. Each value represents the mean ( ± SD) of four
12.93 ± 5.22 mmol N/h (n 30) before meal ingestion; subjects in the water group, seven subjects in the L3lg and LCa.s groups. and
remained unchanged after either water, LCas, or Hf3lg inges- eight subjects in the Hlg and HCa.s groups.
550 MAHE ET AL

The exogenous nitrogen recovered in the upper intestinal lumen with ethanol to separate PN (pellet) and NPN (supernate)
was different according to the ingested test meal (Figure 4). After fractions. The total amount as well as the PN and NPN frac-
L3lg ingestion, exogenous nitrogen peaked significantly (P < tions of the exogenous nitrogen recovered in the jejunal efflu-
0.05) in the first 20-mm period and then disappeared after 60 mm. ents were calculated during the 240 mm after ingestion of the
The exogenous nitrogen appeared in the first 20 mm after inges- different meals (Table 1). The PN fraction was the main part of
tion of LCas, peaked in the 20-40-mn period, and then rapidly the exogenous nitrogen recovered after ingestion of Hlg,
decreased in the 60-80-mm period. In the same way, the exoge- whereas it was dramatically reduced after HCas. After inges-
nous nitrogen appeared in the first 20 mm after ingestion of HCas tion of a low amount of protein, the net disappearance of
but remained stable during the 40-100-mm period and then slowly exogenous nitrogen in the upper intestine was 61 .6 ± 9.6%
decreased in the 100-240-mm period. In contrast, a large amount (44.9 ± 7.0 mmol N) and 58.4 ± 14.7% (41.9 ± 10.5 mmol N)
of exogenous nitrogen peaked significantly (P < 0.05) in the in the 240 mm after -lactoglobulin and casein ingestion,
20-40-mm period after ingestion of H(31g and decreased during respectively, and was not significantly different. However,
the 40-l60-min period. after ingestion of a high amount of protein, a dramatic differ-

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

ence was noticed between the two dietary proteins. In this case
Characterization of the exogenous nitrogen after
the net disappearance of exogenous nitrogen in the upper
ingestion of a large amount of protein
intestine was 82.6 ± 9.5% (338.7 ± 20.0 mmol N) for casein
To more precisely characterize the exogenous nitrogen frac- but was 44.7 ± 24.4% (155.0 ± 105.9 mmol N) for 3-lacto-
tion, the effluents collected after meal ingestion were treated globulin. Moreover, the exogenous PN fraction recovered in
the upper intestinal lumen represented 43.3% of the ingested
140 j3-lactoglobulin nitrogen, whereas it was only 4.9% of the
120 ingested casein nitrogen. The presence of intact f3-lactoglobu-
100 lin in the upper intestinal lumen after ingestion of a high
80 amount of -lactoglobulin was confirmed by electrophoresis
LB1g
60 (Figure 5). In this case, a band corresponding to the intact
40 protein was detected in the jejunum digesta during the 0-100-

20 mm period after f3-lactoglobulin ingestion.

0 I -- F T I I
0
E 120
E 100 DISCUSSION
. LCas
C
80
01
Dietary protein quality for humans is usually assessed in
0 60
terms of digestibility and amino acid composition but neither of
40
C these factors are still clearly understood. There are controver-
a
20 sies about amino acid requirement values in humans because
0 0 I - I I I amino acids are required to replace metabolic oxidation that is
C
0) 120 adaptative (17, 18). Digestibility as simple hydrolysis and
01

x
0 100 . . T , HBlg protein absorption is only part of a complex series of meta-
0)
80 bolic, physiologic processes and transactions in the gastroin-
0
C

0)
60
40
20
H#{224} testinal
utilization
part of the small
tract
of
that

intestine,
proteins
probably
(5).
influence
During
dietary
the
proteins
systemic
digestion
are mixed
distribution

with
in the upper
and

_____ ______________________________ many endogenous secretions that represent a flux of endoge-

0 Ii11I I
nous nitrogen entering the lumen. The differentiation between
120
the fractions of endogenous and exogenous nitrogen is essential
100
to determine both the real luminal concentration of the ab-
80
sorbed proteins and their digestion products as well as the
60
effects of the meal on the nitrogenous biliary and pancreatic
40 secretion in the lumen. We proposed different methods to
20 estimate the endogenous and exogenous nitrogen fluxes in
0 . .. humans (12, 19). In the present study ‘5N-labeled milk proteins
0 40 80 120 160 200 240 were used to demonstrate that I) f3-lactoglobulin was rapidly

‘1 Time (mm) recovered in the upper intestine mostly in the form of intact
protein that needs to be degraded before being absorbed more
MEAL distally, 2) casein was slowly recovered in the jejunum mainly
in the form of degraded peptides efficiently absorbed in the
FIGURE 4. The exogenous nitrogen profiles of the digesta in the
upper part of the intestine, and 3) a high amount of casein both
jejunum after ingestion of a low amount of [‘5N13-lactoglobulin (Lf3lg)
stimulated pancreatic secretion and reduced gastrojejunal tram-
and [‘5N]casein (LCas) and a high amount of [‘5N]13-lactoglobulin (Hf3lg)
and [‘5N]casein (HCas). After I’Nlprotein ingestion, the exogenous ni-
sit more efficiently than did -lactoglobulin.
trogen fractions were calculated from the ratio of ‘5N to 4N in the digesta. The nature and the composition of the chyme is known to
Each value represents the mean ( ± SD) of seven subjects in the Llg and modify the gastric emptying rate, intestinal motility, and gastric as
LCas groups and eight subjects in the HJ3lg and HCas groups. well as biliopancreatic secretions. Moreover, the liquid and solid
 JEJUNAL DIGESTION OF 1’5NIMILK PROTEINS IN HUMANS 551

TABLE 1
Exogenous nitrogen in the human jejunum 240 mm after j3-lactoglobulin and casein ingestion’

Net disappearance
Exogenous nitrogen recovered in the jeju nal effluents’
of exogenous
Nitrogen ingested2 PN4 NPN4 Total nitrogen nitrogen

iiitnol N %
L3lg (72.6 mmol N: n = 7) - - 27.7 ± 7.0 61.6 ± 9.6
LCas (71.7 mmol N; n = 7) - - 29.8 ± 10.5 58.4 ± 14.7
H/JIg (368.2 mmol N; n - 8) 159.5 ± 95.8 53.8 ± 15.5 213.2 ± 105.9 44.7 ± 24.4
HCas (386.8 mmol N; n = 8) 19.2 ± 6.8 27.9 ± 13.9 48.1 ± 20.0 82.6 ± 9.5
/: ± SD.

2 J3-lactoglobulin and casein were ingested in low (Llg and LCas) and in high (Hlg and HCas) amounts.

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

3 Exogenous and endogenous nitrogen fractions stemming from [‘5N] protein or endogenous secretion were calculated from the ratio of ‘5N to ‘4N in
jejunal effluents after [‘5N] protein ingestion.
4 Ethanol precipitation of the jejunum effluents after protein ingestion gave a pellet fraction made up of proteins (protein nitrogen. PN) and a supernatant
fraction made up of peptides and free amino acids (nonprotein nitrogen, NPN).

composition of the meal modifies gastric emptying because solids casein could originate from the remaining fraction of clotted
are emptied more slowly than liquids. In the present study it was casein within the stomach. However, a gastric washing at the end
demonstrated that 3-lactoglobulin transits more rapidly than does of the experiments indicated no significant quantity of I ‘5N]casein
casein to the upper jejunum. Although each protein test meal was in the stomach 4 h after ingestion.
not balanced by other solutes, the resulting difference in osmolar- Dietary proteins, and especially casein, are believed to stim-
ity among the groups was low, ie, a difference of < 3-4 mOsmol, ulate gastrointestinal hormones acting on both gastric and
and would not presumably affect gastric emptying. The difference intestinal motility and on exocrine pancreatic secretion (7, 9).
between f3-lactoglobulin and casein was previously observed in Casein, ingested at a high amount, appeared to stimulate en-
humans and is probably due in large part to the clotting and/or dogenous nitrogen secretions more efficiently than did f3-lac-
precipitation of casein in the acidic media of the stomach (10, 1 1). toglobulin. The nature of the compounds responsible for these
f3-lactoglobulin remains soluble in the stomach and empties rap- different effects has yet to be clearly established. Integrated
idly as an intact protein that needs further hydrolysis by pancreatic mechanisms are probably implicated in these actions and more
proteases and is then more distally absorbed than is casein. In than one compound is involved in each effect. It could origi-
contrast, clotted casein is more exposed to gastric peptic hydro- nate from the highest flux of amino acid absorption in the
lysis and empties slowly from the stomach in the form of degraded duodenum after ingestion of a high amount of casein. A puta-
products, which are subsequently hydrolyzed by pancreatic pro- tive hormone-modulation effect could also explain, in part, the
teases and absorbed in the upper part of the intestine. In this study, more pronounced effect of casein on gastric and pancreatic
the absorption of the proteins was calculated from their net dis- digestion. Like endogenous mediator precursors, casein is es-
appearance (ingested minus recovered exogenous nitrogen) in the pecially considered to be an exogenous prohormone with ac-
lumen. Under these conditions, the apparent high absorption of tions including protective functions, regulation of digestion and

(0) (1) (2) (3) (4) (5) (6) (7)

FIGURE 5. Typical Coomassie blue stain of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ofjejunal digesta after ingestion
of a high amount of [‘5N]J3-lactoglobulin (HJ3lg). The digesta were recovered 0-20 (column I), 20-40 (column 2). 40-60 (column 3), 60-80 (column 4),
80-100 (column 5), 100-120 (column 6), and 120-140 mm (column 7) after ingestion of Hf3lg. The standard proteins casein, f3-lactoglobulin, and
a-lactalbumin appear on the far left. H(31g and (0) correspond to the ingested protein and the endogenous basal nitrogen secretion, respectively.
552 MAHE ET AL

nutrient uptake, metabolic or physiologic regulation, or immu- emptying regulates the kinetics of nitrogen absorption from ‘5N-
nomodulatory effects (20). An important group of casein- labeled milk and ‘5N-labeled yogurt in miniature pigs. J Nutr 1994;

derived peptides could act at different levels, including anal- 124:1970-7.

5. Alpers DH. Digestion and absorption ofcarbohydrate and proteins. In:
gesia, gastric and intestinal motility, intestinal ion transport,
Johnson LR, ed. Physiology of the gastrointestinal tract. New York:
systemic and/or central effects (13, 2 1 , 22).
Raven Press, l987;2:l469-87.
The kinetic difference between /3-lactoglobulin and casein in
6. Caillard I, Tome D. Modulation of 3-lactoglobulin transport in rabbit
the upper intestine probably has important nutritional and meta-
ileum. Am J Physiol l994;266:Gl053-9.
bolic implications because the optimal utilization of dietary pro- 7. Hara H, Fujibayashi A, Kiriyama S. Pancreatic protease secretion
tein for human foods needs to take into account the kinetics of profiles after spontaneous feeding of casein or soybean protein diet in
intestinal delivery of amino acids to the organism. The gastrodu- unrestrained conscious rats. J Nutr Biochem l992;3:l76-8l.
odenal steps, which mainly consist of gastric emptying and duo- 8. Morley JE, Levine AS, Yamada T, et al. Effects of exorphins on
denal digestion, represent the major processes that control the gastrointestinal functions, hormonal release and appetite. Gastroenter-
kinetics of protein digestion and absorption. We demonstrated ology l983;84: I517-23.

Downloaded from https://academic.oup.com/ajcn/article-abstract/63/4/546/4651235 by guest on 21 May 2020

previously that the gastric emptying and absorption rates of milk 9. Daniel H, Vohwinkel M, Rehner G. Effect of casein and j3-casomor-
phin on gastrointestinal motility in rats. J Nutr 1990:120:252-7.
proteins were directly correlated (4). In these conditions, casein
10. Mah#{233}S. Huneau J-F, Marteau P. Thuillier F, Tome D. Gastroileal
and 3-lactoglobulin, although each has a high amino acid equi-
nitrogen and electrolyte movements after bovine milk ingestion in
librium and digestibility, could differ in the kinetics of amino acid humans. Am J Clin Nutr l992;56:4l0-6.
delivery. This aspect has to be further measured with respect to 1 1. Mah#{233}
5, Benamouzig R, Gaudichon C, Huneau J-F, De Cruz I, Tome
both the appearance of labeled amino acids in the blood and the D. Nitrogen and electrolyte movements in the upperjejunum lumen in
associated metabolic and hormonal changes. For instance, protein humans fed low amounts of casein or -lactoglobulin. Gastroent#{233}rol
with fast amino acid uptake must be associated with rapidly Clin Biol 1995:19:20-6.
absorbed carbohydrates whereas protein with slow amino acid I 2. Mah#{233}5, Roos N, Benamouzig R, et al. True exogenous and endoge-
uptake associated with slowly absorbed carbohydrates can help in nous nitrogen fractions in the human jejunum after ingestion of small
amounts of ‘5N-labeled casein. I Nutr l994;l24:548-55.
reducing the length of the postabsorptive phase and maintain
13. Zioudriou C, Streaty RA, Klee WA. Opioid peptides derived from
nitrogen balance. Another important aspect is the presence of
food proteins: the exorphins. J Biol Chem 1979;254:2446-9.
intact 3-lactog1obulin in the intestinal lumen, which should also be
14. Mah#{233}S. Fauquant J, Gaudichon C, Roos N, Maubois JL, Tome D.
taken into account in the formulation of foods with milk proteins. ‘5N-labelling and preparation of milk, casein and whey proteins. Le
It was observed that macromolecules such as f3-lactoglobulin are Lait 1994;74:307-l 2.
able to cross the epitheliaJ barrier, but the role of the different 15. Hyden S. A turbidimetric method for the determination of higher
pathways in the hypothetical functions, ie, immune surveillance, polyethylene glycols in biological materials. Kungl Lantbrukshogsko-
metabolic regulation, or gastrointestinal disease, still remains un- lans Ann l955;22:l39-45.
known (6, 23-26). The absorption of large molecules in antigenic 16. Scheld HP. Use of polyethylene glycol and phenol red as unabsorbed
and biologically active quantities is especially believed to play a indicators for intestinal absorption studies in man. Gut l966;7: 159-63.

role in the different physiologic and immunologic responses that 17. Fuller MF, Garlick P1. Human amino acid requirements: can the
controversy be resolved? Annu Rev Nutr l994;l4:217-4l.
contribute to the oral tolerance and its regulation (27, 28).
18. Millward DJ, Bowtell JL, Pacy P. Rennie Mi. Physical activity, protein
In conclusion, casein and 3-lactoglobulin represent differ-
metabolism and protein requirements. Proc Nutr Soc 1994:53:223-40.
ences in the nature of the products present in the intestinal 19. Gaudichon C, Laurent C, Mah#{233}
S. Marks L, Tome D, Krempf M. Rate
lumen, mainly because of their slow or fast gastric emptying of I 5Nl-leucine incorporation and determination of nitrogenous frac-
rate, respectively. Casein empties from the stomach in the form tions from gastro-jejunal secretion in fasting humans. Reprod Nutr
of degraded peptides, among which biologically active pep- Dcv I994;34:349-59.
tides could be present, whereas J3-lactoglobulin mainly empties 20. Coste M, Tome D. Milk peptides with physiological activities. II.
as intact protein with also putative physiologic functions. Opioid and immunostimulating peptides derived from milk proteins.
These differences should probably to be taken into account Le Lait 199l;7l:24l-7.
21. Tani F, Lio K, Chiba H, Yoshikawa M. Isolation and characterization
from both a nutritional and a physiologic point of view and
of opioid antagonist peptides derived from human lactofemn. Agric
must be further investigated to determine the optimal condi-
Biol Chem l990;54:I803-l0.
tions for the utilization of these proteins in humans. El
22. Beucher 5, Levenez F, Yvon M, Coning T. Effects of gastric digestive
We gratefully acknowledge the staff at the Avicenne Hospital, espe- products from casein on CCK release by intestinal cells in rat. J Nutr
cially M Deyra, for taking care of volunteers as well as for their technical Biochem l994;5:578-84.

assistance. We thank Suzanne Salter for her English assistance. 23. Sanderson IR, Walker A. Uptake and transport of macromolecules by
the intestine: possible role in clinical disorders (an update). Gastroen-
terology 1993:104:622-39.

REFERENCES 24. Gardner MLG. Gastrointestinal absorption of intact proteins. Annu

Rev Nutr l988;8:329-50.
I. Lurie B. Novis BH, Brom B. Bank 5, Marks IN. Pancreatic exocrine 25. Caillard I, Tome D. Transport of f3-lactoglobulin and a-lactalbumin in
responses to test meals of varying compositions in man. Dig Dis enterocyte-like caco-2 cells. Reprod Nutr Dcv 1995;35: 179-88.
1973; 18:847-50. 26. Smith PL, Wall DA, Gochoco CH, Wilson G. Routes of delivery: case
2. Malagelada JR. Go VLW, Summerskil WHL. Different gastric. pan- studies. Oral absorption of peptides and proteins. Adv Drug Dcliv Res
creatic and biliary responses of solid-liquid or homogenized meals. l992;8:253-90.
Dig Dis Sci 1979:24:101-10. 27. Bahna SL. Pathogenesis of milk hypersensitivity. Immunol Today
3. Low AG. Nutritional regulation of gastric secretion, digestion and l985;6: 153-4.
emptying. Nutr Res Rev 1990:3:229-52. 28. Bland PW, Kambarage DM. Antigen processing by isolated rat intes-
4. Gaudichon C, Roos N, Mah#{233}
S. Sick H. Bouley C, Tome D. Gastric tinal villus enterocytes. Gastroenterol Clin North Am l991;20:577-96.