Human Nutrition and Metabolism

Digestibility of Cooked and Raw Egg Protein in Humans as Assessed by

Stable Isotope Techniques1,2,3
Pieter Evenepoel, Benny Geypens, Anja Luypaerts, Martin Hiele, Yvo Ghoos4
and Paul Rutgeerts
Department of Medicine, Division of Gastroenterology and Gastrointestinal Research Centre, University
Hospital Leuven, B-3000 Leuven, Belgium

KEY WORDS: egg protein digestibility stable isotopes food processing humans

The nutritional value of dietary proteins depends on both

the concentration and distribution pattern of their constituent
amino acids and their bioavailability, i.e., the proportion of
amino acids available for metabolic utilization (Young and
Pellett 1991). The latter is determined by digestion and absorption processes in the small intestine. In vitro enzymic assays
as well as animal assays have been used to predict protein
digestibility in humans. The lack of accuracy and species differences, however, are important drawbacks (Bodwell et al.
1980). Protein digestibility values in humans are often abstracted from fecal balance studies (FAO/WHO 1990). These
studies, however, do not take into account the intense intraluminal bacterial metabolism, and absorption and secretion of
nitrogen taking place in the colon (Moran and Jackson 1990).
The results thus obtained are therefore devoid of any physiologic significance. Direct determination of the ileal protein
digestibility in vivo can be achieved only by balance studies
in ileostomy patients (further referred to as the ileostomy

model) and in healthy volunteers by using intubation techniques. A great difficulty in the assessment of protein digestibility in humans arises from the fact that after ingestion, dietary
proteins are mixed with endogenous proteins secreted in the
lumen, e.g., gastric, biliopancreatic and intestinal secretions,
and sloughed epithelial cells (Alpers 1994, Matthews 1990).
Endogenous protein is generally accepted to be digested at
slower rates than dietary protein (Matthews 1990). A proteinfree diet has been used to estimate the endogenous nitrogen
loss in intestinal contents. This value has been applied in
other studies (using other diets) to calculate the exogenous
fraction (Chako and Cummings 1988, Fuller et al. 1994, Mahe
et al. 1992 and 1994a). The amount of endogenous protein
delivered into the intestinal lumen, however, is not fixed, but
is influenced by the nature of the meal, including the dry
matter, energy, protein and dietary fiber contents (Chako and
Cummings 1988, Corring et al. 1989).
Other approaches therefore have been developed to distinguish the exogenous from the endogenous protein fraction in
the intestinal contents including the use of dietary protein
labeled with homoarginine (Siriwan et al. 1994) and the evaluation of the endogenous/exogenous ratio of effluents, which is
extrapolated by comparing the amino acid composition of both
the chyme and the meal (Baglieri et al. 1995, Mahe et al.
1994b). Primarily for safety reasons, however, the tracer dilution technique using stable isotopes is the most appropriate
for studies involving human subjects. The widespread use of
this technique for the study of protein digestibility has been

1
Supported by a grant from Biomed PL93-2139, Vlaamse Executive and
Nutricia Chair in gastrointestinal microenvironment.
2
Presented in part at the Digestive Disease Week, 1997, San Francisco,
CA [Evenepoel, P., Geypens, B., Maes, B. & Ghoos, Y. (1997) Endogenous and
exogenous protein, escaping digestion and absorption in the small intestine after
ingestion of a physiological load of egg protein. Gastroenterology 112: A873
(abs.)].
3
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 USC section 1734 solely to indicate this fact.
4
To whom correspondence should be addressed.

0022-3166/98 $3.00 q 1998 American Society for Nutritional Sciences.

Manuscript received 6 October 1997. Initial review completed 18 November 1997. Revision accepted 3 June 1998.
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ABSTRACT Egg proteins contribute substantially to the daily nitrogen allowances in Western countries and are
generally considered to be highly digestible. However, information is lacking on the true ileal digestibility of either
raw or cooked egg protein. The recent availability of stable isotopelabeled egg protein allowed determination of
the true ileal digestibility of egg protein by means of noninvasive tracer techniques. Five ileostomy patients were
studied, once after ingestion of a test meal consisting of 25 g of cooked 13C- and 15N-labeled egg protein, and
once after ingestion of the same test meal in raw form. Ileal effluents and breath samples were collected at regular
intervals after consumption of the test meal and analyzed for 15N- and 13C-content, respectively. The true ileal
digestibility of cooked and raw egg protein amounted to 90.9 { 0.8 and 51.3 { 9.8%, respectively. A significant
negative correlation (r 00.92, P 0.001) was found between the 13C-recovery in breath and the recovery of
exogenous N in the ileal effluents. In summary, using the 15N-dilution technique we demonstrated that the assimilation of cooked egg protein is efficient, albeit incomplete, and that the true ileal digestibility of egg protein is
significantly enhanced by heat-pretreatment. A simple 13C-breath test technique furthermore proved to be a
suitable alternative for the evaluation of the true ileal digestibility of egg protein. J. Nutr. 128: 17161722, 1998.

EGG PROTEIN DIGESTIBILITY IN HUMANS

MATERIAL AND METHODS

Subjects. Studies were conducted on five ileostomy patients (4
female, 1 male; aged between 28 and 76 y). The subjects had total
proctocolectomy with terminal ileostomy carried out for ulcerative
colitis (n 4) or polyposis coli (n 1) 1 y before the study. All
were otherwise healthy and had normally functioning ileostomies.
The study was approved by the Ethical Committee of the University
of Leuven and all subjects gave informed consent.
Protein test meal. The protein test meal consisted of 100 g of
13
C-labeled egg white, 100 g of 15N-labeled egg white (markers of
protein assimilation) and the yolk of one egg doped with 74 kBq of
[1-14C]octanoic acid (Dupont, NEN Research, Boston, MA) (marker
of gastric emptying) (Ghoos et al. 1993). Five microcurie 3H-polyethylene glycol (3H-PEC) 4000 was added to the test meal as a nonabsorbable radiolabeled transit marker. All constituents were homogenized before ingestion. The test meal had to be consumed either raw
or after being cooked in a microwave oven (see Experimental design).
The methodology for obtaining large amounts of highly enriched
egg proteins labeled with stable isotopes was extensively described
elsewhere (Evenepoel et al. 1997). Briefly, 13C- or 15N-labeled proteins were produced by giving laying hens free access to a food containing 25% of the (NRC required) leucine content as free [1-13C]leucine (99 mol%, Euriso-top, Saint-Aubin, France) and [15N]leucine
(99 mol%, Euriso-top), respectively. The yolk and egg white fractions
of the enriched eggs were separated and pooled. The isotopic enrichment of both pools was determined using a continuous flow elemental
analyzer isotope ratio mass spectrometer (IRMS)5 (ANCA-SL, Europa Scientific, Crewe, UK). Knowing the exact amino acid composition and the isotopic enrichment of the egg white, the amount of
[1-13C]leucine (99 mol%) incorporated could be calculated. This
value was taken into account in the calculation procedures, outlined
below. Because redistribution of the 15N-label is likely to occur in
the hen via transamination, the 15N-labeled egg protein can be assumed to be uniformly labeled. Total caloric content of the test meal
was 624 kJ (25 g protein, 5.56 g fat and a negligible amount of
carbohydrate).
Experimental design. Each subject was studied in two different
randomly applied test situations as follows: 1) after ingestion of the
cooked egg protein test meal (cooked), and 2) after ingestion of

5
Abbreviations used: AP, atom percent; BSA, body surface area; GEC, gastric
emptying coefficient; IRMS, isotope ratio mass spectrometer; PEG, polyethylene
glycol; % dose/h, percentage of administered dose excreted per hour; % dose
cum 6 h, cumulative percentage of administered dose excreted in 6 h; t1/2, half
emptying time; tlag, lag phase.

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FIGURE 1 Experimental study design.

the same but raw protein test meal (raw). The test situations were
separated from each other by a time span of at least 1 wk. Measurements of gastric emptying (breath test technique), small intestinal
transit time (nonabsorbable transit marker) and protein assimilation
(both by breath test technique and analysis of ileostomy effluents)
were performed on both occasions.
All subjects were studied after an overnight fast of at least 12 h.
At 0845 h on the test day, they were asked to consume the meal
together with 200 mL of water within 15 min. Sodium chloride
seasoning was allowed. No further food was eaten until 1500 h when
the patients ingested 200 mL of a formula food, based on whey, casein
and lactalbumin (Nutridrink, Nutricia, Zoetermeer, The Netherlands). Five milligrams of phenol red was added to this formula food
as a nonabsorbable transit marker. Drinking of water was permitted
from 1200 h on.
The patients emptied their ileostomy bag immediately after ingestion of the meal (i.e., at 0900 h), at 1-h intervals throughout the
day until 1900 h and at 0900 h the next morning. Breath samples
for 13CO2 and 14CO2 were collected before ingestion of the meal
and every 15 min thereafter for 6 h. The experimental design is
schematically represented in Figure 1.

Analytical procedures
Breath samples. 14CO2 (gastric emptying). 14CO2 recovered in
breath is derived from the oxidation of [1-14C]octanoic acid. The
analytical methods and calculations used for the determination of the
breath testderived parameters of gastric emptying, i.e., the gastric
emptying coefficient (GEC), the half excretion time (t1/2) and the
lag phase (tlag), have been described elsewhere (Ghoos et al. 1993).
13
CO2 (protein assimilation). 13CO2 recovered in breath is derived
from the oxidation of L-[1-13C]leucine, made available to the body
through assimilation of the 13C-labeled egg white protein.
Sampling and analysis. The samples for detection of 13CO2 were
collected by blowing air through a straw into a small tube, called
exetainer (Europa Scientific). The breath samples were analyzed for
13
C-content by means of a continuous flow IRMS (ABCA, Europa
Scientific).
Calculations of results. The d-values given by the IRMS were
converted to percentage 13C recovery per hour of the initial amount
administered (% 13C dose/h); this calculation is given by the following
equation:
% 13C dose/h

mmol excess 13Ct(a)

1 100
mmol C excess administered (b)
13

where (a) is

% 13Ct 0 % 13Ct0/
1 CO2 production
100

r % 13C

(dt/1000 / 1) 1 0.0112372
,
((dt/1000 / 1) 1 0.0112372) / 1

where % 13Ct is the relative concentration, expressed as percentage

13
C of total C in breath CO2, on time t after ingestion of the substrate;
% 13Ct0 is the relative concentration, expressed as percentage 13C of
total C in breath CO2, on time t zero, i.e., before ingestion of the
substrate; dt is d on time t; and 0.0113272 is the 13C/12C of Pee Dee
Belemnite reference.

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hindered for a long time by the lack of an adequate substrate,

i.e., stable isotopelabeled protein. In recent years, different
proteins labeled with 15N have become available (e.g., milk
protein, soy protein or pea protein) (Gaussere`s et al. 1996,
Kayser et al. 1992, Mahe et al. 1994b). Very recently, we
succeeded in producing both 15N- and 13C-labeled egg protein
in an easily reproducible and highly efficient manner (Evenepoel et al. 1997).
The digestibility of protein is affected by its state of processing before ingestion. Both an enhanced and a reduced
protein digestibility have been observed after food processing
(e.g., boiling, drying, deep-freezing or microwave-heating)
ste 1991). Using the ileostomy model, this study at(O
tempted the following: 1) to determine the amount of nitrogen
escaping digestion and absorption after ingestion of a physiologic load of egg protein and to distinguish the exogenous from
the endogenous fraction; 2) to validate the 13C-egg protein
breath test as a tool for the evaluation of protein digestibility
in vivo; and 3) to evaluate the influence of heat-pretreatment
on the efficiency of egg protein assimilation. Protein assimilation is defined as the overall process of digestion and absorption finally resulting in the appearance of free amino acids in
the portal circulation.

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EVENEPOEL ET AL.

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r CO2 production 300 mmol 1 BSA/h

Body surface area (BSA) is calculated from the height-weight formula
of Haycock et al. (1978). BSA is expressed in m2.
BSA W0.5378 1 H0.3964 1 0.024265
where W is the weight (kg), H is the length (cm) and (b) is

m
% 13Cs 0 % 13Ct0
1
,
M
100

Nexo Ntot 1 (APd 0 AP0)/(APm 0 AP0)

where Ntot is the amount of total N in the sample, APm is the 15N
enrichment of the test meal, APd is the measured 15N enrichment of
the ileostomy effluent sample (digesta), and AP0 is the (natural) 15N
enrichment of the ileostomy effluent as measured at the start of the
test. The endogenous N in the digesta (Nend) was calculated from
the difference Nend Ntot 0 Nexo.
Any samples that were stained with phenol red from the second
liquid meal were discarded for Nend calculation. The cumulative Nend
delivery over 24 h, related to the experimental protein meal, was

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FIGURE 2 Mean 13CO2 (A, representing protein assimilation) and

CO2 (B, representing gastric emptying) excretion rates, expressed as
percentage of the administered dose of 13/14C excreted per h (% dose/
h) in ileostomy patients after ingestion of a test meal consisting of 25
g of either cooked or raw egg protein. Values are means { SEM, n
5.
14

calculated by dividing the cumulative Nend in the unstained samples

by the normalized cumulative percentage radioactivity recovered in
these samples. This correction assumed that 3H-PEG is an accurate
marker of the rate of delivery of the protein meal.
Statistical analysis. Relations between different parameters were
calculated by regression analysis. Differences between sample means
were calculated using the paired Wilcoxon test, because the results
were not normally distributed. All values are expressed as means
{ SEM.

RESULTS
Breath test results. As shown in Figure 2, the course of
both the 13CO2 (panel A) and 14CO2 (panel B) excretion rate
was influenced by the state of the test meal, whether cooked
or raw. The parameters of protein assimilation and gastric
emptying obtained in both test situations are given in Table
1. Significant differences between the two test situations were
found for several variables.
Ileostomy effluents. Validation of use of 3H-PEG as transit
marker. There was a significant correlation (r2 0.97, P

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where % 13Cs is the relative concentration 13C in position C1 of the

administered substrate (i.e., L-[1-13C]leucine), expressed in percent,
which is 99; % 13Ct0 is the relative concentration 13C of total C in
breath CO2 on time zero, i.e., before ingestion of the meal; M is the
molar mass of the substrate, 132 g/mol; and m is the amount of
substrate administered (mg), calculated separately for each test meal
(Evenepoel et al. 1997).
Cumulative percentages of label recovery were calculated by
means of the trapezoidal rule. From these data, the following parameters of protein assimilation were derived: the maximum percentage
of administered dose of 13C, excreted per hour (%max) and the cumulative percentage of administered dose of 13C, recovered in breath over
6 h (% dose cum 6 h).
Ileostomy effluents. The ileal effluents were collected in individual, labeled plastic containers and stored in the refrigerator (at
0207C) until further analysis. After thawing, each individual collected ileostomy effluent was homogenized. Samples of known weight
were taken for analysis of phenol red, nitrogen and radioactivity (3HPEG 4000). Phenol red in ileostomy effluents was analyzed by means
of a spectrophotometer (Scheld 1966). For analysis of the radioactivity and nitrogen, the samples were freeze-dried. The dried material
was weighed and aliquots were removed for further analysis. The 3HPEG 4000 content was measured by the oxidation method (Packard
Sample Oxidizer, model 306, Packard Instrument Company, Downers
Grove, IL), with subsequent liquid-scintillation counting (Packard,
model 2450) and correction for quenching. Results were expressed
as cumulative percentage administered dose of 3H recovered over
time. The individual results were normalized by setting the cumulative amount of 3H excreted over 24 h equal to 100%. Values for the
time taken for 50% of the meal to be emptied from the ileostomy
(ileal t1/2) were read from the actual curves.
Total N content and 15N-enrichment in the lyophilized aliquots
of the ileostomy effluents were determined by using a continuous
flow elemental analyzer-isotope ratio mass spectrometer (ANCA-SL,
Europa Scientific). Briefly, an aliquot of known weight of the lyophilized ileostomy sample was combusted in the presence of oxygen at
10007C. The combustion products thereafter passed through a second
furnace containing copper at 6007C where excess oxygen was absorbed and nitrogen oxides were reduced to elemental nitrogen. Total
nitrogen content was measured by means of a thermal conductivity
detector, whereas the 15N enrichment was determined by means of
an IRMS detector coupled to the combustion unit of the elemental
analyzer. The isotope ratio 15N/14N of N2 was measured with reference
to the laboratory standard (i.e., an ammonium sulfate solution). The
values were expressed in atom percent (AP). The intra-assay variability of this method, assessed by the CV amounted to 2.6 and 0.3%
for total nitrogen content and 15N-enrichment, respectively. From
these data, both the exogenous and endogenous contribution to the
nitrogen loss in the ileostomy effluents was derived. The exogenous
nitrogen (Nexo) was calculated as follows:

EGG PROTEIN DIGESTIBILITY IN HUMANS

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TABLE 1
Protein assimilation, gastric emptying and ileal emptying and
the small bowel transit time in ileostomy patients after
ingestion of cooked and raw test meals consisting
of 25 g egg protein1
Test condition

Protein assimilation
% dose cum 6 h,2 %
% dose/hmax,2 %
Gastric emptying
gastric t1/2 ,2 min
tlag,3 min
GEC
Ileal emptying
ileal t1/2 , h
Small bowel transit
time,4 h

Cooked

Raw

16.04 { 1.00
4.95 { 0.49

6.04 { 1.52
1.71 { 0.32

68 { 6
9{3
3.02 { 0.05

25 { 9
2.2 { 2.2
3.68 { 0.11

5.33 { 0.76

5.29 { 0.50

4.19 { 0.81

4.84 { 0.49

0.0001) between the delivery of the radioactive marker in

the ileostomy effluent and the delivery of exogenous protein
in each test condition (Fig. 3).
Profile of delivery of meal residues from the ileum. Ileal emptying of the test meal began in the first hour after ingestion of
the cooked and raw test meals (Fig. 4). The emptying rate
accelerated temporarily in the 1- to 2-h period and clearly
peaked in the 6- to 7-h period. Fifty percent of the meal had
emptied from the ileostomy (ileal t1/2) by 5.33 { 0.76 and 5.29
{ 0.50 h after the ingestion of the cooked meal and raw test
meal, respectively (Table 1).
Exogenous and endogenous nitrogen in ileostomy effluents. Figure 5 shows the endogenous and exogenous nitrogen fraction profiles after 15N-egg protein ingestion in the two test
conditions. The cumulative quantities of exogenous and endogenous nitrogen, recovered in the ileostomy effluent over
24 h are given in Table 2. A significantly greater amount
of exogenous nitrogen was recovered in the ileal effluent
over 24 h after ingestion of the raw test meal compared with
the cooked test meal (1949.4 { 390.3 m vs. 360.6 { 30.6
mg, P 0.05). Taking into account these data and the
amount of N ingested, it was possible to calculate the true
ileal digestibility of egg protein, i.e., the percentage of exogenous egg protein assimilated in the small intestine. The
true ileal digestibility of raw egg protein was significantly
impaired compared with that of cooked egg protein (51.3
{ 9.8 vs. 90.9 { 0.8%, P 0.05).
Correlations. A significant negative correlation was found
between the amount of exogenous N recovered in the ileostomy effluent and the cumulative percentage administered
dose of 13C recovered in breath over 6 h (Fig. 6). There were
no significant correlations between the gastric emptying half
time (gastric t1/2) and the small bowel transit time (defined as
ileal t1/2 0 gastric t1/2), nor between the small bowel transit
time and the true ileal digestibility of egg protein in each of
the test conditions.

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FIGURE 3 Mean recovery of 3H-polyethylene glycol (3H-PEG)

(normalized) and exogenous nitrogen (Nexo) in the ileal effluents of ileostomy patients after ingestion of the cooked (A) and raw (B) test meals
containing 25 g egg protein. Values are means, n 5. The indices
letters denote the collection periods to which the values are related: a:
01 h; b: 02 h; c: 03 h; d: 04 h; e: 05 h; f: 06 h; g: 07 h; h:
08 h; i: 09 h; j: 010 h; k: 024 h.

DISCUSSION
Dietary protein provides 1015% of the total energy intake
of healthy subjects consuming a standard Western diet and
supplies the essential amino acids required for protein synthesis. The nutritional value of dietary protein for humans is

FIGURE 4 Ileal emptying profile of the cooked and raw protein

meal, expressed in percentage of administered dose of 3H-polyethyle
glycol (PEG) recovered in the effluents of ileostomy patients per hour
(% dose 3H-PEG/h). Values are means { SEM, n 5.

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1 Values are means { SEM, n 5. Abbreviations: % dose cum 6 h,

cumulative percentage of administered dose excreted in 6 h; % dose/
hmax , maximal percentage of administered dose excreted per hour;
gastric t1/2 , gastric half emptying time; tlag, lag phase; GEC, gastric
emptying coefficient; ileal t1/2 , ileal half-emptying time.
2 Pairs Wilcoxon P 0.005; 3 paired Wilcoxon P 0.05.
4 Small bowel transit time is defined as ileal t1/2 0 gastric t1/2 .

EVENEPOEL ET AL.

1720

of different protein sources has already been assessed in healthy

volunteers with the use of an intestinal perfusion technique
(Chung et al. 1979, Gaussere`s et al. 1996, Mahe et al. 1994a).
This technique, however, is time-consuming and invasive and,
moreover, appears to delay gastric emptying and to shorten
transit time in the small intestine (Read et al. 1983). Shortening of the transit time may compromise the assimilation of
macronutrients (Chapman et al. 1985, Holgate and Read
1993). Because ileal effluents are easily collectible in ileostomy
patients, nitrogen balance studies in these subjects are an attractive alternative for the evaluation of ileal digestibility
(Chapman et al. 1985, Fuller et al. 1994, Gibson et al. 1976,
Rowan et al. 1994, Sandstrom et al. 1986). A common problem encountered in studies designed to determine the ileal
digestibility is the differentiation between the exogenous and
endogenous origin of nitrogen recovered in ileal effluents. Because we recently obtained large amounts of highly enriched
15
N-labeled egg proteins (Evenepoel et al. 1997), we were able
to apply the isotope dilution technique for the assessment of
the true ileal digestibility of egg protein.
Five healthy ileostomy patients were studied after ingestion
of a physiologic load (25 g) of egg protein, labeled with 13C

FIGURE 5 Delivery of endogenous (Nendo), exogenous (Nexo)

and total (Ntot) nitrogen in ileal effluents of ileostomy patients after
ingestion of cooked and raw test meals consisting of 25 g egg protein
(4000 mg N). Values are means { SEM, n 5.

usually determined from a chemical score or more recently

from the protein digestibility-corrected amino acid score in
which digestibility represents an important aspect (Young and
Pellett 1991). According to results obtained from nitrogen
balance studies in healthy subjects, the true (fecal) digestibility
of (spray dried whole) egg protein has been estimated to be
as high as 9297% (Bodwell et al. 1980). However, because
nitrogen is intensely metabolized, absorbed and secreted in the
colon, fecal digestibility values do not necessarily equal ileal
digestibility values. This has been demonstrated in an animal
study, in which fecal and ileal digestibility were measured simultaneously (Mosenthin et al. 1994). Because the ileal digestibility represents the efficiency of protein assimilation, ileal
digestibility values are more relevant from a nutritional point
of view than are fecal digestibility values. The ileal digestibility

TABLE 2
Exogenous and endogenous nitrogen yield over 24 h in ileal effluents of ileostomy patients after ingestion
of 25 g of either cooked or raw 15N-labeled egg protein1,2
N, recovered over 24 h

Ingested N

Endogenous3

Exogenous4

mg

Test condition
Cooked
Raw

4000
4000

400.2 { 31.5
199.3 { 67.2

360.6 { 30.6
1949.4 { 390.3

1 Values are means { SEM, n 5.

2 25 g protein 4000 mg N.
3 Paired Wilcoxon P 0.01.
4 Paired Wilcoxon P 0.05.

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True ileal
digestibility4

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90.9 { 0.8
51.3 { 9.8

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FIGURE 6 Correlation between the cumulative amount of exogenous nitrogen, recovered in the ileal effluents over 24 h (Nexo 24 h cum)
and the cumulative percentage of administered dose of 13C, recovered
in breath over 6 h (% dose 13C cum 6 h) in ileostomy patients. (n:
individual values obtained after ingestion of 25 g of raw egg protein;
s: individual values obtained after ingestion of 25 g of cooked egg
protein). Twenty-five grams of protein corresponds to 4 g of nitrogen.

EGG PROTEIN DIGESTIBILITY IN HUMANS

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cantly increased ileocolonic transit was demonstrated postprandially (Camilleri et al. 1989). Further studies are required
to confirm this finding and to define the controlling factors as
well as the physiologic mechanism underlying the increased
postprandial colonic inflow. The mean ileal half emptying time
in this study was 5.3 h, which is almost identical to the
value encountered in healthy volunteers using a gamma camera (Read et al. 1986).
Labeling of the test meal with 14C-octanoic acid allowed
us to follow gastric emptying simultaneously. The raw test
meal was emptied significantly more quickly than the cooked
test meal, most probably because of its liquid consistency.
Small intestinal transit of the raw test meal, on the other
hand, tended to be slower than that of the cooked test meal.
A shortened transit time therefore cannot account for the
observed decreased digestibility of raw egg protein.
In this study, a significant negative correlation was observed
between the percentage of administered dose 13C recovered in
breath over 6 h and the amount of exogenous nitrogen recovered in the ileostomy effluent over 24 h. This finding implies
that the 13C-egg protein breath test may be regarded as an
accurate technique for the evaluation of protein digestibility.
Although the 13C-egg protein breath test is only semiquantitative (i.e., it reveals adequacy of function compared with a
normal standard), it has many advantages (i.e., it is noninvasive, simple and reproducible), by which it yet might be an
attractive alternative for the evaluation of the efficiency of
protein assimilation in healthy volunteers in different experimental conditions as well as in patients with digestive diseases.
Moreover, the information obtained with breath tests represents a dynamic evaluation, rather than a static estimation.
No significant correlation was found between the gastric
t1/2 and the small bowel transit time, which is in agreement
with other studies (Read et al. 1982 and 1986). These results,
however, should be interpreted with caution because of the
small number of subjects studied.
In conclusion, with the use of stable isotope techniques,
we were able to determine the amounts of egg protein escaping
digestion and absorption in the small intestine after ingestion
of a physiologic load. Native egg protein is malabsorbed to an
important extent. The assimilation of egg protein is facilitated
by heat-pretreatment, but remains incomplete. The excellent
correlation found between digestibility values and 13C-breath
test data validated the 13C-egg protein breath test as an accurate alternative for the evaluation of protein digestibility. Because of its many advantages, the 13C-egg protein breath test
might be very interesting for the study of protein digestibility
under different experimental conditions.
ACKNOWLEDGMENTS
D. Claus, N. Gorris, S. Rutten and L. Swinnen are acknowledged
for excellent technical assistance.

LITERATURE CITED
Alpers, D. H. (1994) Digestion and absorption of carbohydrates and proteins.
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Baglieri, A., Mahe, S., Benamouzig, R., Savoie, L. & Tome, D. (1995) Digestion
patterns of endogenous and different exogenous proteins affect the composition of intestinal effluents in humans. J. Nutr. 125: 18941903.
Bodwell, C. E., Satterlee, L. D. & Hackler, L. R. (1980) Protein digestibility of
the same protein preparations by human and rat assays and by in vitro enzymic digestion assays. Am. J. Clin. Nutr. 33: 677686.
Camilleri, M., Colemont, L. J., Phillips, S. F., Brown, M. L., Thomforde, G. M.,
Chapman, N. & Zinsmeister, A. R. (1989) Human gastric emptying and colonic filling of solids characterized by a new method. Am. J. Physiol. 257:
G284G290.
Chako, A. & Cummings, J. H. (1988) Nitrogen losses from the human small

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and 15N. The double labeling of the test meal allowed the
evaluation of egg protein assimilation by both 13C-breath test
technique and direct analysis of the ileostomy effluents (15Ndilution technique). The subjects were studied in different
experimental conditions to assess the influence of heat-pretreatment on the assimilation efficiency of egg protein.
After ingestion of the cooked egg protein meal, a substantial
quantity of nitrogen was recovered in the ileal effluent over
24 h. The calculated yield of endogenous nitrogen (i.e., 0.40
g N) was close to the yield of 0.55 g N obtained by other
researchers after ingestion of 17 g of pea protein (Gaussere`s
et al. 1994). The calculated true ileal digestibility of cooked
egg protein amounted to 91%. This finding demonstrates that
even cooked egg protein, which has generally been considered
to be easily digestible, is malabsorbed to some extent after
ingestion of a physiologic load. Incomplete assimilation of dietary protein may have important consequences not only from
a nutritional point of view, but also from a gastrointestinal
point of view. Indeed, some metabolites resulting from bacterial fermentation of malabsorbed proteins in the colon have
been implicated in the ethiopathogenesis of diseases such as
colonic cancer and ulcerative colitis (Macfarlane and Cummings 1991, Pitcher and Cummings 1994, Visek 1978). It has
already been reported extensively that food processing can
influence protein digestibility both beneficially and detrimen ste 1991). Egg white protein is generally considered
tally (O
to be less digestible than heat-pretreated egg white protein.
However, no data are available concerning the magnitude of
this impairment in vivo. In this study, it was shown that after
ingestion of 25 g of raw egg protein, almost 50% is malabsorbed
over 24 h. The higher digestibility of cooked egg protein presumably results from structural changes in the protein molecule
induced by heating, thereby enabling the digestive enzymes to
gain broader access to the peptide bonds. It has been suggested
that the reduced digestibility of raw egg white is at least partially related to the presence of trypsin inhibitors in raw egg
white (Matthews 1990). Ovomucoid is quantitatively the most
important trypsin inhibitor (Gilbert 1971, Kassell 1970). Ovomucoid, however, does not react with human trypsin and,
moreover, is relatively heat stable (Kasell 1970). Whether
other egg trypsin inhibitors (e.g., ovoinhibitor or papain inhibitor) interfere with the digestibility of unprocessed egg white
protein is unknown.
Interestingly, the yield of endogenous nitrogen after ingestion of the raw protein meal (i.e., 0.2 g N) was significantly
lower compared with the cooked protein meal. This finding is
in accordance with a recent study in which it was demonstrated that undigested protein, in contrast to digested protein,
only weakly stimulates gallbladder emptying and pancreatic
enzyme secretions (Thimister et al. 1996).
The incorporation of a nonabsorbable radioactive tracer
(3H-PEG) provided an accurate index of the rate of voiding
of the test meal. The delivery profiles of the cooked and raw
test meals in ileostomy effluents were comparable. Ileal emptying of the test meal started in the first hour, accelerated slightly
in the second hour after ingestion of the labeled test meal but
clearly peaked after ingestion of the second meal. The ileum
has been shown by previous transit studies using a gamma
camera to be a region of relative stasis (Read et al. 1986).
Exposure of the ileum to nutrients markedly inhibits upper
gastrointestinal motility. This negative feedback mechanism
is referred to as the ileal brake (Wen et al. 1995). The ileal
emptying pattern noted in this study suggests that transfer of
ileal digesta (into the ileostomy bag) is triggered by a subsequent meal. This interpretation is consistent with a scintigraphic study performed in intact humans, in which a signifi-

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