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Colon luminal content and epithelial cell morphology are markedly modified in rats fed with
a high-protein diet
Mireille Andriamihaja, Anne-Marie Davila, Mamy Eklou-Lawson, Nathalie Petit, Serge Delpal,
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Amount and fate of egg protein escaping
assimilation in the small intestine of humans
PIETER EVENEPOEL, DIRK CLAUS, BENNY GEYPENS, MARTIN HIELE,
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- is highly efficient. Recent studies in healthy volunteers
tin Hiele, Karen Geboes, Paul Rutgeerts, and Yvo Ghoos. using intubation techniques or in ‘‘healthy’’ ileostomy
Amount and fate of egg protein escaping assimilation in the patients have, however, shown that the assimilation of
small intestine of humans. Am. J. Physiol. 277 (Gastrointest. even easily digestible protein is incomplete (8, 24). This
Liver Physiol. 40): G935–G943, 1999.—Studies attempting to
finding has led to a renewed interest in the process of
evaluate protein assimilation in humans have hitherto relied
on either ileostomy subjects or intubation techniques. The protein fermentation.
availability of stable isotope-labeled protein allowed us to Nonabsorbed dietary protein enters the human large
determine the amount and fate of dietary protein escaping intestine through the ileocecal valve in the form of a
digestion and absorption in the small intestine of healthy complex mixture of proteins and peptides. The majority
volunteers using noninvasive tracer techniques. Ten healthy of these substances are degraded to amino acids by both
volunteers were studied once after ingestion of a cooked test bacterial and pancreatic enzymes (23) and are subse-
The costs of publication of this article were defrayed in part by the Ten volunteers (5 females and 5 males, mean age 27 yr,
payment of page charges. The article must therefore be hereby range of 21–37 yr) participated. None of the subjects had a
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 history of gastrointestinal or metabolic disease or previous
solely to indicate this fact. surgery (apart from appendectomy). The subjects had no
gastrointestinal complaints and were free of antibiotics or analyzer isotope ratio mass spectrometer (ANCA-2020, Eu-
any other medical treatment for at least 3 mo before the start ropa Scientific, Crewe, UK). With the exact amino acid
of the study. The study was approved by the Ethical Commit- composition and the isotopic enrichment of the egg white, the
tee of the University of Leuven, and all subjects gave in- amount of [1-13C]leucine (99 mol%) incorporated could be
formed consent. calculated (9). Because ‘‘redistribution’’ of the 15N label is
likely to occur in the hen via transamination, the egg protein
Experimental Design can be assumed to be uniformly 15N labeled. Egg protein
The study was conducted over a 21-day period including a labeled with L-[ring-2H4]tyrosine was obtained by giving
7-day baseline period and two study periods, separated by a laying hens free access to a food containing 20% of the
washout period of 7 days. Each study period started with the (National Research Council required) phenylalanine content
ingestion of the labeled test meal and lasted 3 days. The two as free L-[ring-2H5]phenylalanine (98 mol%, Euriso-top). Due
study periods were identical, apart from the test meal, which to hydroxylation of L-[ring-2H5]phenylalanine by the hen,
had to be ingested once cooked and once raw. The two con- both L-[ring-2H5]phenylalanine and L-[ring-2H4]tyrosine are
secutive study periods were allocated in a randomized order. incorporated in the egg protein. The L-[ring-2H4]tyrosine
All subjects were studied after an overnight fast of at least content of the egg protein was determined by gas chromatog-
12 h. At 0845 on the first day of the study period, the raphy-mass spectrometry (GCQ, Finnigan, San José, CA) (14).
volunteers ingested the protein test meal together with 200
ml of water within 15 min. No further food was allowed until Sample Collection
1500, when the volunteers consumed a standard bread meal. Breath samples were collected in exetainers (Europa Scien-
Drinking of water was permitted from 1200 on. tific) before ingestion of the meal, every 15 min for the first
The experimental design is schematically represented in 6 h, and every 30 min up to 9 h after ingestion of the test meal.
Fig. 1. During each study period, urine was collected in plastic
furnace containing copper at 600°C, where excess oxygen was CP-Sil 5 CB-MS with a film thickness of 0.25 µm (Chrompack,
absorbed and nitrogen oxides were reduced to elemental Middelburg, the Netherlands), the phenolic compunds were
nitrogen. Total nitrogen content was measured by means of a identified by ion trap technology (ITD 700, Finnigan).
thermal conductivity detector, and 15N enrichment was deter- Results for the unlabeled compounds were expressed in
mined by means of an isotope ratio mass spectrometer, amounts excreted per hour and in cumulative amounts
coupled to the combustion unit of the elemental analyzer. The excreted over 72 h. Because phenol and p-cresol are quantita-
15N-to-14N isotope ratio of N was measured with reference to
2 tively the main phenolic compounds found in urine, total
a calibrated laboratory standard (i.e., a standard ammonium phenols were measured as the combination of phenol and
sulfate solution). The values were expressed in atom percent p-cresol (3, 23).
(AP). The intraassay variability of this method, assessed by Results for the labeled compounds were expressed in
the coefficient of variation (CV), amounted to 2.6 and 0.3% for percent administered dose of L-[ring-2H4]tyrosine recovered
total nitrogen content and 15N enrichment, respectively. per hour and in cumulative amounts excreted over 72 h.
Results were expressed in percentage of administered dose of The percent administered dose of L-[ring-2H4]tyrosine recov-
15N recovered per day and in cumulative percentage recov-
ered per hour as [ring-2H4]phenol was calculated as follows
ered over 72 h.
The percentage of administered dose of 15N recovered per %dose of [ring-2H4]phenol/h
day was calculated as follows
[ring-2H4]phenol rect
mg excess 15Nt(a) 5 100 3
%15N dose/day 5 3 100 d 3 L-[ring-2H4]tyrosineadministered
mg excess 15N administered(b)
where [ring-2H4]phenol rect is the total amount of [ring-
where a is 2H ]phenol recovered in the urine fraction of period t (ex-
1 23N
APs 2 0.368 2H ]phenols is zero); L-[ring-2H ]tyrosine
4 4 administered is the
f tot
100 amount of L-[ring-2H4]tyrosine administered, expressed in
moles (calculated for each test meal separately); and d is the
where APs is the measured 15N enrichment of the stools duration of the collection period, expressed in hours.
collected on day t, expressed in AP; 0.368 is the natural 15N The percentage of the administered dose of L-[ring-
content of stools, expressed in AP; and Nf tot is the total 2H ]tyrosine recovered per hour as p-[ring-2H ]cresol was
4 4
nitrogen content of the stools collected on day t, expressed in calculated in the same manner. Because phenol and p-cresol
milligrams. are quantitatively the major bacterial metabolites of tyrosine,
The calculation of b is the percentage of administered dose of L-[ring-2H4]tyrosine
1 23N
fermented in the large gut per hour is obtained by summation
APm 2 0.368
(%dose of [ring-2H4]phenol/h 1 %dose of p-[ring-2H4]cresol/h).
m
100 The cumulative percentage of dose of L-[ring-2H4]tyrosine
administered recovered in urine over 72 h (further referred to
where APm is the weighed average 15N enrichment of the test as o72 h 2
0 h %dose H4 adm ) was calculated by summation.
meal (calculated for each test meal separately), expressed in The cumulative values were corrected for gastrointestinal
AP, and Nm is the nitrogen content of the protein test meal,
transit as follows
i.e., 4,000 mg.
The cumulative percentage of administered dose of 15N 72 h
recovered in feces over 72 h (further referred to as o72
15N
h
0 h %dose
72 h o %dose of 2
H4 adm
o %dose of
f adm ) was obtained by summation. 0h
A correction was made for gastrointestinal transit by
2
H4 adm corrected 5 72 h
Table 1. Dietary intake of protein, fat, carbohydrates, and dietary fiber during the cooked and raw study period
Cooked Meal Raw Meal
where APs is the measured 15N enrichment of the urine was reached. The cumulative percent 13C of adminis-
collected in period t, expressed in AP; APd 2 1 is the 15N tered dose, recovered in breath over time after inges-
enrichment of the urine of period d 2 1, i.e., the natural 15N tion of the labeled cooked and raw test meal, is shown
content of urine before ingestion of the labeled meal, ex- in Fig. 3.
pressed in AP; and Nu tot is the total nitrogen content of the
Table 2 summarizes the parameters of protein assimi-
urine, collected in period t, expressed in milligrams.
The calculation of b was as follows lation as derived from the breath test data. Both the
maximum percentage of administered dose of 13C ex-
1 23N
APm 2 APd 2 1 creted per hour and the cumulative percentage of
100 m administered dose of 13C, recovered in breath over 6 h,
were significantly higher after ingestion of the cooked
Diet
No significant differences were found between the
two study periods either in the intake of protein, fat,
and carbohydrates or in the intake of dietary fiber
(Table 1).
Breath Tests
Figure 2 shows the mean 13CO2 excretion rate in
breath after ingestion of the raw and cooked test meal.
Differences between both test situations are obvious.
The curve obtained after ingestion of the cooked test
meal is characterized by a steep ascending slope, a high
peak excretion rate, and an initial steep descending
slope that smoothes down considerably after 6 h. After
ingestion of the raw test meal, the 13CO2 excretion rate
Fig. 2. Mean 13CO2 excretion rate in breath, expressed as percentage
increased more slowly, did not reach the high values of the administered dose of 13C excreted per hour in 10 healthy
obtained after ingestion of the cooked test meal, and volunteers after ingestion of a cooked and raw test meal, consisting of
remained on a rather constant level after the maximum 25 g of 13C-, 2H-, and 15N-labeled egg protein. Values are means 6 SE.
AMOUNT AND FATE OF MALABSORBED EGG PROTEIN G939
Breath variable
%Dose 13C 6 h 20.10 0.049 20.71† 20.67† 20.62†
Fecal variables
Total N, g/day 0.75† 0.19 20.22 20.045
Wet weight, g/day 20.032 20.22 20.11
%Cumulative 15Nf 0.46* 0.77†
Urinary variable
Total phenols, mg/day 0.62†
%Cumulative 2H4
n 5 20 (10 subjects 3 2 test situtions). * P , 0.05; † P , 0.005.
ingestion of the labeled test meal is derived from the sent study that all 15N recovered in feces is of bacterial
metabolism of protein assimilated in the small intes- origin (i.e., incorporated in the bacterial mass).
tine. The cumulative percentage of administered dose of
The mean daily fecal wet weight, dry weight, and 2H recovered in urine over 72 h amounted to 1.60 6
due to tracer recycling. Tracer recycling occurs through meal compared with the cooked protein meal. Neverthe-
desquamation of intestinal mucosa and secretion of less, the difference between both test situations (13.50 6
digestive enzymes and urea in the small intestine and 2.20%) was less pronounced than was expected from
colon, respectively (20, 26). Little information is avail- the difference in the percentage of malabsorption
able on the magnitude of this bias, which, most prob- (29.34 6 6.45%) (Table 4). This observation might
ably, is due to methodological problems. Kayser et al. equally be explained by salvage of nitrogen in the colon.
(20) quantified 15N tracer recycling by measuring the Highly significant negative correlations were found
appearance of 15N in stools after an intravenous injec- between parameters of protein assimilation (i.e., cumu-
tion of 250 mg of 15N-enriched glycine (99 AP). The lative percentage of administered dose of 13C, recovered
fractional fecal loss (i.e., tracer recycling) amounted to in breath over 6 h) and parameters of protein malabsorp-
1.43 6 0.64% (means 6 SD). Although it is reasonable tion (i.e., the amount of 15N and [2H4]phenols, recovered
that the magnitude of tracer recycling is not fixed but in feces and urine, respectively). This finding supports
influenced by dietary factors, the latter value may be the validity of the techniques used. The lack of a
indicative. significant correlation between the fecal nitrogen out-
Despite possible overestimation due to tracer recy- put and fecal 15N recovery indicates that the fecal
cling, the malabsorption percentages observed in the nitrogen output may not be regarded as a sensitive
present are still somewhat lower than those we previ- parameter of the efficiency of dietary protein assimila-
ously reported in healthy ileostomy patients in identi- tion in the small intestine.
cal test conditions (8). This difference can be explained In conclusion, using noninvasive stable isotope tech-
either by differences in the efficiency of protein assimi- niques, we were able to evaluate protein (mal)absorp-
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