Amount and fate of egg protei n escapi ng

assi mi l ati on i n the smal l i ntesti ne of humans

PI ETER EVENEPOEL, DI RK CLAUS, BENNY GEYPENS, MARTI N HI ELE,
KAREN GEBOES, PAUL RUTGEERTS, AND YVO GHOOS
Department of Medicine, Division of Gastroenterology and Gastrointestinal Research Centre,
University Hospital Leuven, B-3000 Louvain, Belgium
Evenepoel, Pieter, Dirk Claus, Benny Geypens, Mar-
tinHiele,KarenGeboes,Paul Rutgeerts,andYvoGhoos.
Amount and fate of egg protei n escapi ng assi mi l ati on i n the
smal l i ntesti ne of humans. Am. J . Physiol. 277 (Gastrointest.
Liver Physiol. 40): G935G943, 1999.Studi es attempti ng to
eval uate protei n assi mi l ati on i n humans have hi therto rel i ed
on ei ther i l eostomy subjects or i ntubati on techni ques. The
avai l abi l i ty of stabl e i sotope-l abel ed protei n al l owed us to
determi ne the amount and fate of di etary protei n escapi ng
di gesti on and absorpti on i n the smal l i ntesti ne of heal thy
vol unteers usi ng noni nvasi ve tracer techni ques. Ten heal thy
vol unteers were studi ed once after i ngesti on of a cooked test
meal , consi sti ng of 25 g of
13
C-,
15
N-, and
2
H-l abel ed egg
protei n, and once after i ngesti on of the same but raw meal .
Amounts of 5.73% and 35.10% (P 0.005) of cooked and raw
test meal , respecti vel y, escaped di gesti on and absorpti on i n
the smal l i ntesti ne. A si gni cantl y hi gher percentage of the
mal absorbed raw egg protei n was recovered i n uri ne as
fermentati on metabol i tes. These resul ts 1) conrm that sub-
stanti al amounts of even easi l y di gesti bl e protei ns may
escape assi mi l ati on i n heal thy vol unteers and 2) further
support the hypothesi s that the metabol i c fate of protei n i n
the col on i s affected by the amount of protei n made avai l abl e.
protei n fermentati on; protei n assi mi l ati on; stabl e i sotopes;
phenol s
THE MOST I MPORTANT FUNCTI ON of the col on i s to absorb
sal t and water and provi de a mechani sm for the orderl y
di sposal of waste products of di gesti on. Recentl y, i t has
become cl ear that the col on may al so pl ay a rol e i n the
sal vage of energy from carbohydrate and ni trogen from
protei n not di gested i n the upper gut. Thi s i s achi eved
through the metabol i sm of the bacteri a and i s known as
fermentati on. Thi s process obvi ousl y i nuences col oni c
functi on and may have heal th consequences for the
host. The knowl edge of fermentati on may be the key to
understandi ng the normal physi ol ogy of the col on and
the eti ol ogy of i ts di seases (1, 19, 23, 29, 31, 33, 35).
Most research has been focused on the fermentati on
of carbohydrates. The end products formed, l i ke hydro-
gen, methane, and especi al l y short-chai n fatty aci ds
(SCFAs) (23), have al ready been i nvesti gated i n depth.
SCFAs are general l y accepted to be beneci al to the
host (23, 31).
Protei n fermentati on, on the other hand, has been
i nvesti gated l ess i ntensi vel y, most probabl y because i t
was general l y bel i eved that the assi mi l ati on of protei n
i s hi ghl y effi ci ent. Recent studi es i n heal thy vol unteers
usi ng i ntubati on techni ques or i n heal thy i l eostomy
pati ents have, however, shown that the assi mi l ati on of
even easi l y di gesti bl e protei n i s i ncompl ete (8, 24). Thi s
ndi ng has l ed to a renewed i nterest i n the process of
protei n fermentati on.
Nonabsorbed di etary protei n enters the human l arge
i ntesti ne through the i l eocecal val ve i n the form of a
compl ex mi xture of protei ns and pepti des. The majori ty
of these substances are degraded to ami no aci ds by both
bacteri al and pancreati c enzymes (23) and are subse-
quentl y fermented (23). Some of the fermentati on me-
tabol i tes produced i ncl ude thi ol s, phenol s, ammoni a,
i ndol es, and ami nes, whi ch are potenti al l y toxi c (3, 23,
27, 29, 39).
I ncorporati on i nto the bacteri al mass and the subse-
quent fecal excreti on and l umi nal accumul ati on as free
fermentati on metabol i tes and then the subsequent
absorpti on i nto the portal bl ood and excreti on i n uri ne
are major fates of protei n made avai l abl e to the col on.
Al though the regul ati ng mechani sms are onl y parti al l y
understood to date, there i s substanti al , al bei t i ndi rect,
evi dence i n the l i terature that the rati o of carbohydrate
to ni trogen i s cruci al (2, 6, 7, 25, 38).
Several studi es have i nvesti gated the i nuence of an
i ncreased avai l abi l i ty of fermentabl e carbohydrates on
the handl i ng of ni trogen i n the col on. Fermentabl e
carbohydrates sti mul ate bacteri al growth, whi ch re-
sul ts i n an enhanced i ncorporati on of ni trogen i nto the
bacteri al protopl asm (2, 17, 37).
The i mpact of an i ncreased avai l abi l i ty of protei n on
bacteri al metabol i sm, on the other hand, remai ns
l argel y unknown. Several protei n fermentati on metabo-
l i tes were recentl y shown to be i ncreased after i nges-
ti on of a suppl ementary l oad of di etary protei n (12).
The ai ms of the present study were 1) to quanti fy the
amount of di etary protei n escapi ng di gesti on and ab-
sorpti on i n heal thy vol unteers i n physi ol ogi cal condi -
ti ons and 2) to eval uate to what extent the bacteri al
metabol i sm of di etary protei n i n the col on i s affected by
the amount of protei n made avai l abl e. Noni nvasi ve
tracer techni ques usi ng protei n l abel ed wi th di fferent
stabl e i sotopes (
13
C,
15
N,
2
H) were used to achi eve these
goal s.
MATERIALS AND METHODS
Subjects
Ten vol unteers (5 femal es and 5 mal es, mean age 27 yr,
range of 2137 yr) parti ci pated. None of the subjects had a
hi story of gastroi ntesti nal or metabol i c di sease or previ ous
surgery (apart from appendectomy). The subjects had no
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1999 the Ameri can Physi ol ogi cal Soci ety G935
gastroi ntesti nal compl ai nts and were free of anti bi oti cs or
any other medi cal treatment for at l east 3 mo before the start
of the study. The study was approved by the Ethi cal Commi t-
tee of the Uni versi ty of Leuven, and al l subjects gave i n-
formed consent.
Experimental Design
The study was conducted over a 21-day peri od i ncl udi ng a
7-day basel i ne peri od and two study peri ods, separated by a
washout peri od of 7 days. Each study peri od started wi th the
i ngesti on of the l abel ed test meal and l asted 3 days. The two
study peri ods were i denti cal , apart from the test meal , whi ch
had to be ingested once cooked and once raw. The two con-
secuti ve study peri ods were al l ocated i n a randomi zed order.
Al l subjects were studi ed after an overni ght fast of at l east
12 h. At 0845 on the rst day of the study peri od, the
vol unteers i ngested the protei n test meal together wi th 200
ml of water wi thi n 15 mi n. No further food was al l owed unti l
1500, when the vol unteers consumed a standard bread meal .
Dri nki ng of water was permi tted from 1200 on.
The experi mental desi gn i s schemati cal l y represented i n
Fi g. 1.
Diet
The vol unteers were gi ven no standard di ets. However,
they were asked to wei gh and record al l food and dri nks taken
from 3 days before unti l the end of each study peri od. These
data were anal yzed usi ng a computer program to obtai n
energy and nutri ent i ntake resul ts (Nederl ands voedi ngsstof-
fenbestand 198990, Voorl i chti ngsbureau voor de voedi ng,
Den Haag, The Netherl ands).
Test Meal
The protei n test meal consi sted of 100 g of egg whi te (i .e., 11
g of egg whi te protei n) l abel ed wi th
15
N, 100 g of egg whi te
l abel ed wi th L-[1-
13
C]l euci ne and L-[ring-
2
H
4
]tyrosi ne, and
the yol k of one egg. Fi ve mi crocuri es [
3
H]pol yethyl ene gl ycol
([
3
H]PEG) 4000 were added to the test meal as a radi ol abel ed
transi t marker. Al l the consti tuents of the test meal were
mi xed before i ngesti on. Total cal ori c content of the test meal
was 150 kcal (25 g of protei n, 5.56 g of fat, and a negl i gi bl e
amount of carbohydrates).
The methodol ogy for obtai ni ng l arge amounts of hi ghl y
enri ched egg protei ns l abel ed wi th stabl e i sotopes has been
descri bed el sewhere (9). Bri ey,
13
C- or
15
N-l abel ed protei ns
were produced by gi vi ng l ayi ng hens free access to a food
contai ni ng 25% of the (Nati onal Research Counci l requi red)
l euci ne content as free [1-
13
C]l euci ne (99 mol %, Euri so-top,
Sai nt-Aubi n, France) and [
15
N]l euci ne (99 mol %, Euri so-top),
respecti vel y. The yol k and egg whi te fracti ons of the enri ched
eggs were separated and pool ed. The i sotopi c enri chment of
both pool s was determi ned usi ng a conti nuous ow el emental
anal yzer i sotope rati o mass spectrometer (ANCA-2020, Eu-
ropa Sci enti c, Crewe, UK). Wi th the exact ami no aci d
composi ti on and the i sotopi c enri chment of the egg whi te, the
amount of [1-
13
C]l euci ne (99 mol %) i ncorporated coul d be
cal cul ated (9). Because redi stri buti on of the
15
N l abel i s
l i kel y to occur i n the hen vi a transami nati on, the egg protei n
can be assumed to be uni forml y
15
N l abel ed. Egg protei n
l abel ed wi th L-[ring-
2
H
4
]tyrosi ne was obtai ned by gi vi ng
l ayi ng hens free access to a food contai ni ng 20% of the
(Nati onal Research Counci l requi red) phenyl al ani ne content
as free L-[ring-
2
H
5
]phenyl al ani ne (98 mol %, Euri so-top). Due
to hydroxyl ati on of L-[ring-
2
H
5
]phenyl al ani ne by the hen,
both L-[ring-
2
H
5
]phenyl al ani ne and L-[ring-
2
H
4
]tyrosi ne are
i ncorporated i n the egg protei n. The L-[ring-
2
H
4
]tyrosi ne
content of the egg protei n was determi ned by gas chromatog-
raphy-mass spectrometry (GCQ, Finnigan, San Jose, CA) (14).
SampleCollection
Breath sampl es were col l ected i n exetai ners (Europa Sci en-
ti c) before i ngesti on of the meal , every 15 mi n for the rst
6 h, and every 30 mi n up to 9 h after i ngesti on of the test meal .
Duri ng each study peri od, uri ne was col l ected i n pl asti c
bottl es for the fol l owi ng peri ods: 03 h, 36 h, 69 h, 924 h,
2448 h, 4872 h. Moreover, a 24-h uri ne col l ecti on was
obtai ned the day precedi ng each study peri od. Neomyci n was
added to the pl asti c contai ners used for the col l ecti ons to
prevent bacteri al growth. After measurement of the vol ume,
sampl es were taken and stored at 20C unti l anal ysi s.
Al l stool s voi ded duri ng each of the study peri ods were
col l ected as wel l . Date and ti me of voi di ng of stool s were noted
i n a di ary. The stool s were frozen i mmedi atel y after voi di ng
and stored at 20C unti l anal ysi s.
Analytical Procedures and Calculations
Breath samples. The breath sampl es were anal yzed for
13
C
content by means of a conti nuous-ow i sotope rati o mass
spectrometry (ABCA, Europa Sci enti c). The val ues gi ven
by i sotope rati o mass spectrometry were converted to percent-
age of
13
C recovery per hour of the i ni ti al amount admi ni s-
tered (%dose
13
C/h) accordi ng to cal cul ati ons previ ousl y de-
scri bed i n detai l (8, 15). Cumul ati ve percentages of recovered
l abel (cumul ati ve %dose
13
C) were cal cul ated by means of the
trapezoi dal rul e. From these data, the fol l owi ng parameters
of protei n assi mi l ati on were deri ved: the maxi mum percent-
age of admi ni stered dose of
13
C excreted per hour and the
cumul ati ve percentage of admi ni stered dose of
13
C recovered
i n breath over 6 h.
Fecal samples. After thawi ng, the stool sampl es were
wei ghed and homogeni zed for each day of col l ecti on. Sampl es
of known wei ght were taken and freeze-dri ed. The dri ed
materi al was wei ghed, and al i quots were taken for the
anal ysi s of total ni trogen content,
15
N enri chment, and
[
3
H]PEG 4000 content.
The [
3
H]PEG 4000 content was measured by the oxi dati on
method (Packard sampl e oxi di zer, model 306, Packard I nstru-
ment, Downers Grove, I L), wi th subsequent l i qui d sci nti l l a-
ti on counti ng (model 2450, Packard I nstrument) and correc-
ti on for quenchi ng. Resul ts were expressed i n cumul ati ve
percentage of the admi ni stered dose of
3
H recovered over 72 h
(further referred to as
0h
72h
%dose [
3
H]PEG 4000).
Total ni trogen content and
15
N enri chment were deter-
mi ned usi ng a conti nuous ow el emental anal yzer-i sotope
rati o mass spectrometer (ANCA-2020, Europa Sci enti c).
Bri ey, an al i quot of known wei ght of the l yophi l i zed fecal
sampl e was combusted i n the presence of oxygen at 1,000C.
The combusti on products thereafter passed through a second
Fi g. 1. Experi mental desi gn. 1st stands for rst stool voi ded on the
fourth day after i ngesti on of the test meal . d
1
, Day precedi ng the
study peri od.
G936 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
furnace contai ni ng copper at 600C, where excess oxygen was
absorbed and ni trogen oxi des were reduced to el emental
ni trogen. Total ni trogen content was measured by means of a
thermal conducti vi ty detector, and
15
N enri chment was deter-
mi ned by means of an i sotope rati o mass spectrometer,
coupl ed to the combusti on uni t of the el emental anal yzer. The
15
N-to-
14
N i sotope rati o of N
2
was measured wi th reference to
a cal i brated l aboratory standard (i .e., a standard ammoni um
sul fate sol uti on). The val ues were expressed i n atom percent
(AP). The i ntraassay vari abi l i ty of thi s method, assessed by
the coeffi ci ent of vari ati on (CV), amounted to 2.6 and 0.3%for
total ni trogen content and
15
N enri chment, respecti vel y.
Resul ts were expressed i n percentage of admi ni stered dose of
15
N recovered per day and i n cumul ati ve percentage recov-
ered over 72 h.
The percentage of admi ni stered dose of
15
N recovered per
day was cal cul ated as fol l ows
%
15
N dose/day
mg excess
15
N
t
(a)
mg excess
15
N admi ni stered(b)
100
where a i s
1
AP
s
0.368
100
2
N
f tot
where AP
s
i s the measured
15
N enri chment of the stool s
col l ected on day t, expressed i n AP; 0.368 i s the natural
15
N
content of stool s, expressed i n AP; and N
f tot
i s the total
ni trogen content of the stool s col l ected on day t, expressed i n
mi l l i grams.
The cal cul ati on of bi s
1
AP
m
0.368
100
2
N
m
where AP
m
i s the wei ghed average
15
N enri chment of the test
meal (cal cul ated for each test meal separatel y), expressed i n
AP, and N
m
i s the ni trogen content of the protei n test meal ,
i .e., 4,000 mg.
The cumul ati ve percentage of admi ni stered dose of
15
N
recovered i n feces over 72 h (further referred to as
0h
72h
%dose
15
N
f adm
) was obtai ned by summati on.
A correcti on was made for gastroi ntesti nal transi t by
di vi di ng
0h
72h
%dose
15
N
f adm
by the cumul ati ve percentage of
admi ni stered dose of
3
H recovered over 72 h

0 h
72 h
%dose of
15
N
f adm
corrected

0 h
72 h
%dose of
15
N
f adm

0 h
72 h
%dose of [
3
H]PEG 4000
Urinary samples. URI NARY PHENOL AND P-CRESOL. Phenol ,
[ring-
2
H
4
]phenol , p-cresol , and p-[ring-
2
H
4
]cresol were mea-
sured by gas chromatography-i on trap technol ogy as de-
scri bed by Geypens et al . (13). Bri ey, 1 ml of uri ne was
di l uted wi th 3 ml of di sti l l ed water. Seventy-ve mi crol i ters of
2,6-di methyl phenol sol uti on (20 mg/100 ml ) were added as
i nternal standard. The pH was adjusted to 1 wi th concen-
trated H
2
SO
4
, and the sol uti on was reuxed for 75 mi n to
hydrol yze the conjugated phenol s. After a cool i ng-down pe-
ri od to ambi ent temperature, phenol s were extracted wi th 2
ml of di ethyl ether. One mi crol i ter was i njected i nto the gas
chromatography-mass spectrometer (GCQ, Fi nni gan). After
separati on on the anal yti cal col umn, a 25-m 0.25-mm
CP-Si l 5 CB-MS wi th a l m thi ckness of 0.25 m (Chrompack,
Mi ddel burg, the Netherl ands), the phenol i c compunds were
i denti ed by i on trap technol ogy (I TD 700, Fi nni gan).
Resul ts for the unl abel ed compounds were expressed i n
amounts excreted per hour and i n cumul ati ve amounts
excreted over 72 h. Because phenol and p-cresol are quanti ta-
ti vel y the mai n phenol i c compounds found i n uri ne, total
phenol s were measured as the combi nati on of phenol and
p-cresol (3, 23).
Resul ts for the l abel ed compounds were expressed i n
percent admi ni stered dose of L-[ring-
2
H
4
]tyrosi ne recovered
per hour and i n cumul ati ve amounts excreted over 72 h.
The percent admi ni stered dose of L-[ring-
2
H
4
]tyrosi ne recov-
ered per hour as [ring-
2
H
4
]phenol was cal cul ated as fol l ows
%dose of [ring-
2
H
4
]phenol /h
100
[ring-
2
H
4
]phenol rec
t
d L-[ring-
2
H
4
]tyrosi ne
admi ni stered
where [ring-
2
H
4
]phenol rec
t
i s the total amount of [ring-
2
H
4
]phenol recovered i n the uri ne fracti on of peri od t (ex-
pr essed i n mol ) (the natur al ur i nar y content of [ring-
2
H
4
]phenol s i s zer o); L-[ring-
2
H
4
]tyr osi ne
admi ni ster ed
i s the
amount of L-[ring-
2
H
4
]tyrosi ne admi ni stered, expressed i n
mol es (cal cul ated for each test meal separatel y); and d i s the
durati on of the col l ecti on peri od, expressed i n hours.
The per centage of the admi ni ster ed dose of L-[ring-
2
H
4
]tyrosi ne recovered per hour as p-[ring-
2
H
4
]cresol was
cal cul ated i n the same manner. Because phenol and p-cresol
are quanti tati vel y the major bacteri al metabol i tes of tyrosi ne,
the percentage of admi ni stered dose of L-[ring-
2
H
4
]tyrosi ne
fermented i n the l arge gut per hour i s obtai ned by summati on
(%dose of [ring-
2
H
4
]phenol /h %dose of p-[ring-
2
H
4
]cresol /h).
The cumul ati ve percentage of dose of L-[ring-
2
H
4
]tyrosi ne
admi ni stered recovered i n uri ne over 72 h (further referred to
as
0h
72h
%dose
2
H
4adm
) was cal cul ated by summati on.
The cumul ati ve val ues were corrected for gastroi ntesti nal
transi t as fol l ows

0 h
72 h
%dose of
2
H
4 adm
corrected

0 h
72 h
%dose of
2
H
4 adm

0 h
72 h
%dose of [
3
H]PEG 4000
URI NARY N
tot
AND
15
N. After thawi ng, a known amount of
uri ne (15 l ) was absorbed on chromosorb i n a ti n capsul e.
Total ni trogen content and
15
N enri chment of uri ne were
determi ned usi ng a conti nuous ow el emental anal yzer i so-
tope rati o mass spectrometer (ANCA-2020, Europa Sci enti c)
as descri bed previ ousl y.
Resul ts for
15
N were expressed i n percentage of the admi n-
i stered dose of
15
N recovered per hour and i n cumul ati ve
percentages recovered over 72 h.
The percentage of admi ni stered dose of
15
N recovered per
hour was cal cul ated as fol l ows
%
15
N dose/h
mg excess
15
N
t
(a)
mg excess
15
N admi ni stered (b) d
100
where a i s
1
AP
s
AP
d 1
100
2
N
u tot
G937 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
where AP
s
i s the measured
15
N enri chment of the uri ne
col l ected i n peri od t, expressed i n AP; AP
d 1
i s the
15
N
enri chment of the uri ne of peri od d 1, i .e., the natural
15
N
content of uri ne before i ngesti on of the l abel ed meal , ex-
pressed i n AP; and N
u tot
i s the total ni trogen content of the
uri ne, col l ected i n peri od t, expressed i n mi l l i grams.
The cal cul ati on of bwas as fol l ows
1
AP
m
AP
d 1
100
2
N
m
where AP
m
i s the wei ghted average
15
N enri chment of the test
meal , expressed i n AP (cal cul ated for each test meal sepa-
ratel y), and N
m
i s the ni trogen content of the meal , i .e.,
4,000 mg.
The cumul ati ve percentage of admi ni stered dose of
15
N
recovered i n uri ne over 72 h (
0h
72h
%dose of
15
N
u adm
) was
cal cul ated by summati on.
15
N RETENTI ON. The percentage of the admi ni stered dose of
15
N, retai ned i n the body after 72 h, was cal cul ated as fol l ows
%
15
N retenti on 100

0 h
72 h
%dose of
15
N
f adm
corrected

0 h
72 h
%dose of
15
N
u adm
)
Statistical Methods
Resul ts are expressed as means SE. Stati sti cal anal ysi s
was performed wi th SAS software package. Parameters
obtai ned after i ngesti on of the raw test meal were pai rwi se
compared wi th val ues obtai ned i n the control study usi ng the
pai red t-test. Correl ati ons were obtai ned by Pearsons test.
RESULTS
Diet
No si gni cant di fferences were found between the
two study peri ods ei ther i n the i ntake of protei n, fat,
and carbohydrates or i n the i ntake of di etary ber
(Tabl e 1).
Breath Tests
Fi gure 2 shows the mean
13
CO
2
excreti on rate i n
breath after i ngesti on of the raw and cooked test meal .
Di fferences between both test si tuati ons are obvi ous.
The curve obtai ned after i ngesti on of the cooked test
meal i s characteri zed by a steep ascendi ng sl ope, a hi gh
peak excreti on rate, and an i ni ti al steep descendi ng
sl ope that smoothes down consi derabl y after 6 h. After
i ngesti on of the raw test meal , the
13
CO
2
excreti on rate
i ncreased more sl owl y, di d not reach the hi gh val ues
obtai ned after i ngesti on of the cooked test meal , and
remai ned on a rather constant l evel after the maxi mum
was reached. The cumul ati ve percent
13
C of admi ni s-
tered dose, recovered i n breath over ti me after i nges-
ti on of the l abel ed cooked and raw test meal , i s shown
i n Fi g. 3.
Tabl e 2 summari zes the parameters of protei n assi mi -
l ati on as deri ved from the breath test data. Both the
maxi mum percentage of admi ni stered dose of
13
C ex-
creted per hour and the cumul ati ve percentage of
admi ni stered dose of
13
C, recovered i n breath over 6 h,
were si gni cantl y hi gher after i ngesti on of the cooked
test meal compared wi th the raw test meal .
Feces
Fecal output vari abl es such as wet wei ght, dry
wei ght, total ni trogen content, and ni trogen densi ty di d
not di ffer si gni cantl y after i ngesti on of the cooked and
raw test meal (Tabl e 3). The cumul ati ve fecal recovery
of
15
N, however, was si gni cantl y hi gher after i ngesti on
of the raw test meal [
0h
72h
%dose of
15
N
f adm
corrected:
4.16 0.27% (cooked) vs. 14.16 1.70% (raw); P
0.001] (Tabl e 4).
Urine
Nitrogen. Fi gure 4 shows the mean
15
N excreti on rate
i n uri ne after i ngesti on of the raw and cooked test meal .
Al though si gni cant di fferences i n the
15
N excreti on
rate were noted, the cumul ati ve percentage of admi ni s-
Tabl e 1. Dietaryintakeof protein, fat, carbohydrates, and dietaryber duringthecooked and rawstudyperiod
Cooked Meal Raw Meal
I ntake, g/day Energy % I ntake, g/day Energy %
Protei n 77.885.36 13.150.93 71.894.09 13.160.88
Fat 111.9811.13 40.631.92 99.898.91 39.811.61
Carbohydrates 274.5128.61 44.791.89 249.4613.78 44.891.35
Di etary ber 21.761.86 18.971.41
Val ues are means SE.
Fi g. 2. Mean
13
CO
2
excreti on rate i n breath, expressed as percentage
of the admi ni stered dose of
13
C excreted per hour i n 10 heal thy
vol unteers after i ngesti on of a cooked and raw test meal , consi sti ng of
25 g of
13
C-,
2
H-, and
15
N-l abel ed egg protei n. Val ues are means SE.
G938 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
tered dose of
15
N, recovered i n uri ne over 72 h was
al most the same i n both test si tuati ons [
0h
72h
%dose of
15
N
u adm
: 35.91 2.12% (cooked) vs. 32.68 1.35%
(raw); P 0.12] (Tabl e 3).
Phenols. Fi gure 5 shows the excreti on pattern of
phenol , p-cresol , and total phenol s i n uri ne. The excre-
ti on rate was hi gher after i ngesti on of the raw meal
than after i ngesti on of the cooked meal . Si gni cance
was reached i n the 9- to 24-h peri od.
The excreti on pattern of [ring-
2
H
4
]phenol and p-[ring-
2
H
4
]cresol was si mi l ar to the excreti on pattern of the
unl abel ed components (Fi g. 6). p-[ring-
2
H
4
]cresol ap-
peared i n uri ne somewhat l ater than [ring-
2
H
4
]phenol .
Overal l , the maxi mal excreti on rate was reached i n the
9- to 24-h peri od. The cumul ati ve percentage of admi n-
i stered dose of L-[ring-
2
H
4
]tyrosi ne, recovered i n uri ne
as total [ring-
2
H
4
]phenol s, was si gni cantl y hi gher
after i ngesti on of the raw test meal compared wi th the
cooked test meal (20.63 5.59% vs. 1.60 0.44%, P
0.005) (Tabl e 3).
15
N Retention
The percentage of admi ni stered dose of
15
N, retai ned
i n the ni trogen pool of the body after 72 h, was
si gni cantl y l ower after i ngesti on of the raw test meal
compared wi th the cooked test meal (49.63 2.69% vs.
63.16 1.36%, P 0.0002) (Tabl e 3).
Correlations
Si gni cant correl ati ons were found between several
fecal , uri nary, and breath vari abl es (Tabl e 5). There
was a negati ve correl ati on between the recovery of
13
C
i n breath and the recovery of ei ther
15
N i n feces (r
Fi g. 3. Mean cumul ati ve percentage of admi ni stered dose of
13
C,
recovered i n breath over ti me (%dose cumul /h) i n heal thy vol unteers
after i ngesti on of a cooked and raw test meal , consi sti ng of 25 g of
13
C-,
2
H-, and
15
N-l abel ed egg protei n. Val ues are means SE;
n 10.
Tabl e 2. Parameters of protein assimilation in healthy
volunteers after ingestion of a cooked and rawtest
meal, consistingof 25gof
13
C-,
2
H-, and
15
N-labeled eggprotein
Test Condi ti on
P Cooked Raw
%Dose cum 6 h 17.230.69 8.200.94 0.0001
%
max
5.250.26 1.910.18 0.0001
Val ues are means SE, n 10. %Dose cum 6 h, cumul ati ve
percentage of admi ni stered dose of
13
C, recovered i n breath over 6 h;
%
max
, maxi mum percentage of admi ni stered dose of
13
C excreted per
hour. P val ues obtai ned by pai red t-test.
Tabl e 3. Major urinaryand fecal variables in the
twotest situations
Vari abl e
Test Si tuati on
P Cooked Raw
Feces Wet wei ght,
g/day
149.222.3 152.024.9 0.92
Dry wei ght,
g/day
25.32.7 27.11.7 0.31
Dry matter, % 37.75.1 41.26.7 0.31
N excreti on,
g/day
1.890.18 2.100.29 0.49
N densi ty, %dry
matter
5.020.47 5.090.55 1.00
%Cumul ati ve
15
N
f
(c)
4.160.27 14.501.70 0.00020
R (d) 67.73.7 74.04.8 0.32
Uri ne Total phenol s,
mg/day
32.143.50 47.105.06 0.0040
N excreti on,
g/day
10.380.67 10.680.57 0.47
%Cumul ati ve
2
H
4
(e)
1.600.44 20.635.59 0.00010
%Cumul ati ve
15
N
u
(f )
32.681.35 35.912.12 0.12
Cal cul ated
val ues
%Mal absorbed
(cc)
%Accumul ati on
(e/ce)
5.730.50
24.146.07
35.106.78
50.626.10
0.0020
0.0030
%I ncorporati on
(c/cc)
75.866.07 49.386.10 0.0030
%Retai ned i n N
pool
(100%cf )
63.161.36 49.632.69 0.00020
Val ues are means SE; n 10. %Cumul ati ve
15
N
f

0h
72h
%dose
15
N
f adm
corrected (see text). R
0h
72h
%dose [
3
H]PEG 4000 (see text).
%Cumul ati ve
2
H
4

0h
72h
%dose
2
H
4adm
corrected (see text). %Cumu-
l ati ve
15
N
u

0h
72h
%dose
15
N
u adm
(see text). %I ncorporati on assumes
that 100% of
15
N recovered i n feces i s i ncorporated i nto the bacteri al
mass. P val ues were obtai ned by pai red t-test.
Tabl e 4. Fecal
15
N excretion in 10healthyvolunteers
after ingestion of a cooked and rawtest meal consisting
of 25gof
13
C-,
2
H-, and
15
N-labeled eggprotein
Fecal
15
N Excretion
Excretion per
day, %dose/day
Cumulative
excretion
024 h 2448 h 4872 h

0h
72h
%
dose adm
72
0h
72h
%
dose adm
corrected
Cooked 0.340.27 0.960.31 1.570.29 2.870.30 4.160.27
Raw 1.811.37 4.481.37 4.200.94 10.491.26 14.51.7
P 0.10 0.005 0.020 0.001 0.001
Val ues are means SE; P val ues were obtai ned by pai red t-test.
adm, Admi ni stered.
G939 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
0.71, P 0.005) or [ring-
2
H
4
]phenol s i n uri ne (r
0.62, P 0.005). The recovery of [ring-
2
H
4
]phenol s i n
uri ne correl ated posi ti vel y wi th the recovery of
15
N i n
feces (r 0.77, P 0.005) (Fi g. 7).
No correl ati on was found between the fecal ni trogen
output and the recovery of ei ther
15
N i n feces or
[ring-
2
H
4
]phenol s i n uri ne.
DISCUSSION
The effi cacy of protei n assi mi l ati on has been studi ed
to date by several researchers ei ther i n i l eostomy
subjects (4, 8, 11, 22, 32, 34) or i n heal thy vol unteers
usi ng i ntubati on techni ques (24). I t was demonstrated
that the amount of protei n escapi ng di gesti on and
absorpti on i n the smal l i ntesti ne i s affected by the type
and amount of protei n (8, 11, 16, 22, 34) as wel l as the
presence of other consti tuents (e.g., resi stant starch)
(32) i n the di et. Less i s known about the process of
protei n fermentati on i n vi vo i n humans, whi ch i s
l argel y due to the physi ol ogi cal i naccessi bi l i ty of the
col on.
The avai l abi l i ty of protei n l abel ed wi th stabl e i so-
topes al l owed us to study protei n (mal )absorpti on and
fermentati on i n heal thy vol unteers by means of noni n-
vasi ve tracer techni ques. An i nherent advantage of
tracer techni ques i s that they do not di sturb normal
physi ol ogy. Al l vol unteers were studi ed i n two di fferent
randoml y appl i ed test si tuati ons: 1) after i ngesti on of a
cooked egg protei n meal l abel ed wi th L-[1-
13
C]l euci ne,
[
15
N]ami no aci ds, and L-[ring-
2
H
4
]tyrosi ne and 2) after
i ngesti on of the same but raw meal . Both test si tua-
ti ons were separated by a 1-wk washout peri od. Thi s
peri od was suffi ci entl y l ong to return i sotope enri ch-
ment to basel i ne (data not shown).
Protei n (mal )absorpti on and fermentati on were eval u-
ated quanti tati vel y through the anal ysi s of metabol i tes
excreted i n breath, uri ne, or feces. Breath was anal yzed
for
13
CO
2
, feces for
15
N, and uri ne for [ring-
2
H
4
]phenol ,
p-[ring-
2
H
4
]cresol , and
15
N.
The breath test resul ts obtai ned i n the present study
were i n accordance wi th those obtai ned i n a recent
study performed i n i l eostomy pati ents under si mi l ar
test condi ti ons (8). I n the l atter study, a hi ghl y si gni -
cant negati ve correl ati on was demonstrated between
the
13
C recovery i n breath and the recovery of exog-
enous protei n i n the i l eal effl uents. I n the extrapol ati on
of thi s ndi ng to subjects wi th an i ntact gastroi ntesti -
nal system, the l ow recovery of
13
CO
2
i n breath after
i ngesti on of the raw protei n test meal suggests overt
mal absorpti on.
I t may be argued that i n subjects wi th an i ntact
gastroi ntesti nal system the
13
CO
2
recovered i n breath
may be deri ved from the fermentati on of mal absorbed
L-[1-
13
C]l euci ne i n the col on as wel l . However, because
i t was observed i n the i l eostomy study formerl y men-
ti oned (8) that 50% of the mal absorbed cooked and raw
protei n had empti ed from the i l eostomy by 5.33 and
5.29 h, respecti vel y, i t can be assumed that most of the
13
CO
2
appeari ng i n breath wi thi n 6 h fol l owi ng the
Fi g. 4. Uri nary
15
N excreti on pattern i n 10 heal thy vol unteers after
i ngesti on of a cooked (open bars) and raw (cl osed bars) test meal ,
consi sti ng of 25 g of
13
C-,
2
H-, and
15
N-l abel ed egg protei n. Val ues are
means SE. *P 0.05, pai red t-test.
Fi g. 5. Uri nary excreti on pattern of phenol (A), p-cresol (B), and total
phenol s (C) i n 10 heal thy vol unteers after i ngesti on of a cooked and
raw test meal , consi sti ng of 25 g of egg protei n. Val ues are means
SE. *P 0.05, pai red t-test.
Fi g. 6. Uri nary excreti on pattern of [ring-
2
H
4
]phenol (A), p-[ring-
2
H
4
]cresol (B), and total [ring-
2
H
4
]phenol s (C) after i ngesti on of a
cooked (open bars) and raw (cl osed bars) test meal , consi sti ng of 25 g
of
13
C-,
2
H-, and
15
N-l abel ed egg protei n. Val ues are means SE.
*P 0.05, pai red t-test.
G940 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
i ngesti on of the l abel ed test meal i s deri ved from the
metabol i sm of protei n assi mi l ated i n the smal l i ntes-
ti ne.
The mean dai l y fecal wet wei ght, dry wei ght, and
ni trogen content were consi stent wi th previ ous i nvesti -
gati ons i n humans (2, 7, 36, 37). None of these param-
eters was affected si gni cantl y by the nature of the test
meal (Tabl e 3).
The cumul ati ve percentage of admi ni stered dose of
15
N recovered i n feces over 72 h after i ngesti on of the
cooked egg protei n meal amounted to 4.16 0.27%.
Thi s val ue i s comparabl e wi th gures previ ousl y re-
ported for yeast, egg, and soya protei n (20, 40). The
cumul ati ve recovery of
15
N i n feces after i ngesti on of the
raw meal was si gni cantl y hi gher (14.50 1.70%). I t
has previ ousl y been demonstrated that at l east 60% of
the fecal ni trogen content i s of bacteri al ori gi n (36). For
practi cal purposes, however, i t i s assumed i n the pre-
sent study that al l
15
N recovered i n feces i s of bacteri al
ori gi n (i .e., i ncorporated i n the bacteri al mass).
The cumul ati ve percentage of admi ni stered dose of
2
H recovered i n uri ne over 72 h amounted to 1.60
0.44% and 20.63 5.59%, respecti vel y, after i ngesti on
of the cooked and raw test meal . The percentage of
admi ni stered dose of
2
H recovered i n uri ne represents
the porti on of consumed egg protei n that i s accumu-
l ated i n the col oni c l umen as free fermentati on metabo-
l i tes, subsequentl y absorbed i nto the portal bl ood and
nal l y excreted i n uri ne.
The excreti on pattern of
2
H
4
-l abel ed total phenol s
after i ngesti on of the raw test meal coi nci ded al most
compl etel y wi th the excreti on pattern of the unl abel ed
fracti on. Thi s i ndi cates that the observed i ncrease of
the unl abel ed total phenol s i s rel ated to mal absorpti on
of the test meal . [ring-
2
H
4
]phenol appeared i n uri ne
sl i ghtl y earl i er than p-[ring-
2
H
4
]cresol . Thi s i s i n accor-
dance wi th previ ous studi es suggesti ng that phenol and
p-cresol are predomi nantl y formed i n the termi nal
i l eum (and cecum) and l eft col on, respecti vel y (3). The
del ayed appearance of p-cresol mi ght al so be expl ai ned
by a sl ower producti on rate.
Assumi ng that the fate of both the
15
N and
2
H tracer
i s i denti cal i n the l arge i ntesti ne, the percentage of
i ngested protei n that escaped di gesti on and absorpti on
coul d be cal cul ated approxi matel y. I t amounted to
5.73 0.50% and 35.10 6.78% after i ngesti on of the
cooked and raw test meal , respecti vel y (Fi g. 8). Mal ab-
sorpti on mi ght be overesti mated i n the present study
Tabl e 5. Correlations between breath, fecal, and urinaryvariables
Fecal Vari abl es Uri nary Vari abl es
Total N,
g/day
Wet wei ght,
g/day
%Cumul ati ve
15
N
Total phenol ,
mg/day
%Cumul ati ve
2
H
4
Breath vari abl e
%Dose
13
C 6 h 0.10 0.049 0.71 0.67 0.62
Fecal vari abl es
Total N, g/day 0.75 0.19 0.22 0.045
Wet wei ght, g/day 0.032 0.22 0.11
%Cumul ati ve
15
N
f
0.46* 0.77
Uri nary vari abl e
Total phenol s, mg/day 0.62
%Cumul ati ve
2
H
4
n 20 (10 subjects 2 test si tuti ons). *P 0.05; P 0.005.
Fi g. 7. Correl ati ons between parameters of protei n assi mi l ati on (i .e.,
%cumul ati ve
13
C) and parameters of protei n mal absorpti on (i .e.,
%cumul ati ve
15
N and %cumul ati ve
2
H
4
). %cum
13
C, cumul ati ve
percentage of admi ni stered dose of
13
C recovered i n breath over 6 h.
%cum
15
N,
0h
72h
%dose of
15
N
f adm
corrected. %cum
2
H
4
,
0h
72h
%dose of
2
H
4adm
corrected (see text for compl ete expl anati on of abbrevi ati ons).
Fi g. 8. Amount and fate of 25 g of egg protei n escapi ng assi mi l ati on
i n the smal l i ntesti ne of humans.
G941 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N
due to tracer recycl i ng. Tracer recycl i ng occurs through
desquamati on of i ntesti nal mucosa and secreti on of
di gesti ve enzymes and urea i n the smal l i ntesti ne and
col on, respecti vel y (20, 26). Li ttl e i nformati on i s avai l -
abl e on the magni tude of thi s bi as, whi ch, most prob-
abl y, i s due to methodol ogi cal probl ems. Kayser et al .
(20) quanti ed
15
N tracer recycl i ng by measuri ng the
appearance of
15
N i n stool s after an i ntravenous i njec-
ti on of 250 mg of
15
N-enri ched gl yci ne (99 AP). The
fracti onal fecal l oss (i .e., tracer recycl i ng) amounted to
1.43 0.64% (means SD). Al though i t i s reasonabl e
that the magni tude of tracer recycl i ng i s not xed but
i nuenced by di etary factors, the l atter val ue may be
i ndi cati ve.
Despi te possi bl e overesti mati on due to tracer recy-
cl i ng, the mal absorpti on percentages observed i n the
present are sti l l somewhat l ower than those we previ -
ousl y reported i n heal thy i l eostomy pati ents i n i denti -
cal test condi ti ons (8). Thi s di fference can be expl ai ned
ei ther by di fferences i n the effi ci ency of protei n assi mi -
l ati on between heal thy vol unteers and i l eostomy pa-
ti ents or by sal vage of ni trogen i n the col on (18). The
l atter possi bi l i ty i s supported by human and ani mal
data (17, 21).
Amounts of 24.14 6.07% and 50.62 6.10% of the
mal absorbed cooked and raw egg protei n, respecti vel y,
were cal cul ated to accumul ate as free end products of
bacteri al metabol i sm i n the col oni c l umen (Fi g. 8).
Assumi ng that the two test condi ti ons onl y di ffered i n
the amount of protei n made avai l abl e to the col on, the
present resul ts support the hypothesi s that l umi nal
accumul ati on as free fermentati on metabol i tes be-
comes the preferenti al fate of mal absorbed protei n as
more protei n i s made avai l abl e to the col on.
Si gni cant di fferences were observed between both
test si tuati ons i n the pattern of excreti on rates of
15
N i n
uri ne. The
15
N excreti on rate was si gni cantl y l ower i n
the 0- to 6-h peri od and si gni cantl y hi gher i n the 9- to
72-h peri od after i ngesti on of the raw test meal com-
pared wi th the cooked test meal . Protei n assi mi l ati on
was suggested by the shape of the breath test curve to
be compl eted after 69 h. Bacteri al protei n metabo-
l i sm, reected by an i ncrease of the excreti on of [ring-
2
H
4
]phenol s i n uri ne, on the other hand, became appar-
ent from 9 h after i ngesti on of the test meal on.
Therefore,
15
N appeari ng soon after i ngesti on of the test
meal i s accepted to be deri ved uni quel y from the
metabol i sm of assi mi l ated protei n, whereas
15
N appear-
i ng l ater on i s accepted to be deri ved from bacteri al
metabol i sm of mal assi mi l ated protei n as wel l . As coul d
be predi cted, metabol i sm of assi mi l ated protei n was
more promi nent after i ngesti on of the cooked test meal ,
whereas bacteri al metabol i sm of mal assi mi l ated pro-
tei n was more promi nent after i ngesti on of the raw
protei n meal . Notwi thstandi ng these ki neti c di ffer-
ences, the cumul ati ve percentage of admi ni stered dose
of
15
N excreted i n uri ne over 72 h was si mi l ar i n both
test si tuati ons.
The cumul ati ve percentage of admi ni stered dose of
15
N, retai ned i n the ni trogen pool of the body, was
si gni cantl y l ower after i ngesti on of the raw protei n
meal compared wi th the cooked protei n meal . Neverthe-
l ess, the di fference between both test si tuati ons (13.50
2.20%) was l ess pronounced than was expected from
the di fference i n the percentage of mal absorpti on
(29.34 6.45%) (Tabl e 4). Thi s observati on mi ght
equal l y be expl ai ned by sal vage of ni trogen i n the col on.
Hi ghl y si gni cant negati ve correl ati ons were found
between parameters of protei n assi mi l ati on (i .e., cumu-
l ati ve percentage of admi ni stered dose of
13
C, recovered
i n breath over 6 h)and parameters of protei n mal absorp-
ti on (i .e., the amount of
15
N and [
2
H
4
]phenol s, recovered
i n feces and uri ne, respecti vel y). Thi s ndi ng supports
the val i di ty of the techni ques used. The l ack of a
si gni cant correl ati on between the fecal ni trogen out-
put and fecal
15
N recovery i ndi cates that the fecal
ni trogen output may not be regarded as a sensi ti ve
parameter of the effi ci ency of di etary protei n assi mi l a-
ti on i n the smal l i ntesti ne.
I n concl usi on, usi ng noni nvasi ve stabl e i sotope tech-
ni ques, we were abl e to eval uate protei n (mal )absorp-
ti on and fermentati on i n heal thy vol unteers i n a noni n-
vasi ve and quanti tati ve way. We deni ti vel y conrmed
mal absorpti on of even easi l y di gesti bl e protei n. Our
resul ts furthermore support the hypothesi s that an
i ncreased avai l abi l i ty of protei n i n the col on causes the
preferenti al fate of mal absorbed protei n to shi ft toward
l umi nal accumul ati on as free protei n fermentati on
metabol i tes. Thi s ndi ng may be i mportant from a
gastroi ntesti nal poi nt of vi ew, si nce several of these
metabol i tes (ammoni a, thi ol s, phenol s) are thought to
pl ay a rol e i n the eti opathogenesi s of, e.g., ul cerati ve
col i ti s and col oni c cancer (3, 5, 10, 23, 27, 28, 30, 39).
N. Gorri s, A. Luypaerts, S. Rutten, and L. Swi nnen are acknowl -
edged for excel l ent techni cal assi stance.
Thi s work was supported by a grant from Bi omed PL93-2139,
Vl aamse Executi eve and Nutri ci a Chai r i n gastroi ntesti nal mi croen-
vi ronment.
Address for repri nt requests and other correspondence: Y. Ghoos,
Uni ver si tai r Zi ekenhui s Gasthui sber g, Labor ator i um Di gesti e-
Absorpti e E 462, Herestraat 49, B-3000 Leuven, Bel gi um (E-mai l :
Yvo.Ghoos@uz.kul euven.ac.be).
Recei ved 25 June 1998; accepted i n nal form 2 August 1999.
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G943 AMOUNT AND FATE OF MALABSORBED EGG PROTEI N