egg protein assimilation

8/12/2019 Egg Protein assimilation

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Amount and fate of egg protein escaping

assimilat ion in the small intest ine of humans

P I E TE R E V E N E P O E L , D I R K C L AU S , B E N N Y G E Y P E N S , M AR TI N H I E L E ,

K A REN G EB OES , PA UL RUT G EERT S , A N D YV O G HOOS

Departm ent of M edi cin e, Di vision of Gastr oent er ology and Gastroin testina l Resear ch Cent r e,

Universi ty Hospital Leuven, B-3000 Louvain, Belgium

Evenepoel, Pieter, Dirk Claus, Benny Geypens, Mar-tin Hiele,Karen Geboes,Paul Rutgeerts,andYvoGhoos.Amount and fate of egg protein escaping assimilation in thes ma ll int es t ine of huma ns . A m . J . P h y si o l .277 (Gastrointest.Liver Physiol.40): G 935G 943, 1999.St udies a tt empting t oevaluate protein assimilation in humans have hitherto reliedon either ileostomy subjects or intubation techniques. Thea va ila bil i t y of s t a ble is ot ope-la beled prot ein a llow ed us t odet ermine t he a mount a nd fa t e of diet a ry prot ein es ca pingdiges t ion a nd a bs orpt ion in t he s ma ll int es t ine of hea lt hyvolunteers using noninvasive tr acer techniques. Ten healt hyvolunteers were stu died once after ingestion of a cooked t estmea l, cons is t ing of 25 g of 13C -, 15N-, a nd 2H-labeled eggprot ein, a nd once a f t er ingest ion of t he s a me but ra w mea l.Amounts of 5.73%an d 35.10%(P 0.005) of cooked a nd ra wtest meal, respectively, escaped digestion and absorption int he s ma ll int es t ine. A significa nt ly higher percent a ge of t hem a l a b s or b ed r a w e gg p r ot e in w a s r e cov er e d i n u r in e a sfermenta tion meta bolites. These results 1) confi rm t ha t s ub-s t a n t i a l a m o u nt s of e ve n e a s il y d i ge st i b le p r ot e in s m a ye sc a pe a s s i m il a t i on i n h e a l t h y v ol u n t ee r s a n d 2) furt hers upport t h e hypot hesis t ha t t he met a bolic fa t e of prot ein inthe colon is affected by t he am ount of protein mad e ava ilable.

prot ein ferment a t ion; prot ein a s s imila t ion; s t a ble is ot opes ;phenols

THE MOST IMP ORTANT FU NCTION of the colon is to absorbsa lt a nd wa ter a nd provide a mechanism for the orderlydisposal of waste products of digestion. Recently, it hasbecome clear tha t t he colon may a lso play a role in thesa lvage of energy from ca rbohydra te a nd nitr ogen fromprotein not digested in the upper gut. This is achievedthr ough the meta bolism of the bacteria a nd is known a sferment a tion. This process obviously infl uences colonicfu n ctio n an d m ay h ave h e al th co n se qu e n ce s fo r th ehost. The knowledge of fermenta tion ma y be t he key toundersta nding t he norma l physiology of the colon a ndt he et iology of its disea ses (1, 19, 23, 29, 31, 33, 35).

Most research ha s been focused on th e fermentat ionof ca rbohydr a tes. The end products formed, like hydro-

g e n , m e th an e , an d e sp e cial ly sh o rt- ch ain fat ty acid s(SCFAs) (23), have already been investigated in depth.S CF A s are g e n e ral ly acce p te d to b e b e n e fi cial to th ehost (23, 31).

Pro te in fe rm e n tatio n , o n th e o th e r h an d , h as b e e ninvestigat ed less intensively, most probably beca use itwas generally believed that the assimilation of protein

is highly efficient. Recent studies in healthy volunteersusing intuba tion t echniques or in healt hy i leostomypatients have, however, shown that the assimilation ofeven ea sily d igest ible protein is in complete (8, 24). Thisfinding has led to a renewed interest in the process ofprotein fermenta tion.

Nonabsorbed dieta ry protein enters the huma n lar geintestine through the ileocecal valve in the form of acomplex mixtur e of proteins a nd peptides. The ma jorit yof these substa nces a re degraded to a mino acids by bothb acte rial an d p an creat ic e n zym e s (23) an d ar e su b se-quently fermented (23). S ome of t he fermenta tion me-

tabolites produced include thiols, phenols, ammonia,indoles, and a mines, wh ich a re potentia lly t oxic (3, 23,27, 29, 39).

Incorporation into the bacterial mass and the subse-quent fecal excretion and lumina l accumulation as freef er m en t a t i on m et a b ol it e s a n d t h e n t h e s u bs eq u en ta bsorption into th e porta l blood an d excretion in ur ineare major fates of protein made available to the colon.Although the regula ting mechanisms a re only part iallyunderstood to dat e, there is substa ntia l, albeit indirect,evidence in the literat ure tha t t he rat io of carbohydra teto nit rogen is crucia l (2, 6, 7, 25, 38).

Several st udies have investiga ted the infl uence of anincreased availability of fermentable carbohydrates on

th e h an d lin g o f n i tro g en in t h e colon . F e rm en ta b leca r b oh y d r a t e s s t i m ul a t e b a ct e r ia l g r ow t h , w h i ch r e-sults in a n enha nced incorpora tion of nitrogen into theba cteria l protoplasm (2, 17, 37).

The impact of an increa sed a vaila bility of protein onb a ct e r ia l m et a b ol is m , on t h e ot h e r h a n d , r em a i n slargely unknown. S evera l protein fermentat ion meta bo-lites were recently shown to be increased after inges-tion of a supplementa ry loa d of dieta ry protein (12).

The aims of the present study were 1) to qu an t i fy th eam o u n t of d ie tary p rote in e scap in g d ig e stion an d ab -sorption in healthy volunteers in physiological condi-t io n s an d 2) t o ev a l u a t e t o w h a t e xt e n t t h e b a c t er i a lmeta bolism of dieta ry protein in t he colon is affected by

th e a m ou n t of p ro tein m a d e a vai lab le. N o n in vasivetracer techniques using protein labeled with differentsta ble isotopes (13C , 15N, 2H) were used to a chieve thesegoals.

MATERI ALS AND METHODS

Subjects

Ten volunt eers (5 fema les a nd 5 ma les , mea n a ge 27 yr ,ra nge of 2137 yr) pa rt icipa t ed. None of t he s ubject s ha d ahis t ory of ga s t roint es t ina l or met a bolic dis ea s e or previouss urgery (a pa rt from a ppendect omy). The s ubject s ha d no

The costs of publication of this article were defrayed in part by thep a y m e n t of p a g e c h a r g es . Th e a r t ic le m u s t t h e r ef or e b e h e r e b ymarked advertisement in a ccordance with 18 U.S.C. Sect ion 1734solely to indicate this fact.

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ga s t roint es t ina l compla int s a nd w ere free of a nt ibiot ics oran y other medical trea tment for at lea st 3 mo before the star tof the study. The study w as a pproved by the E thical Commit -t e e o f t h e U n i v e r s i t y o f L e u v e n , a n d a l l s u b j e c t s g a v e i n -formed consent.

Experi mental Design

The stud y w as conducted over a 21-da y period including a

7-day baseline period and two study periods, separated by aw a s hout period of 7 da ys . Ea ch s t udy period s t a r t ed w it h t heingestion of the labeled test meal and lasted 3 days. The twostudy periods were identical, apart from the test meal, whichhad to be ingested once cooked and once raw. The two con-secutive study periods were allocated in a randomized order.

All subjects were studied after a n overnight fast of at least12 h . At 0845 o n t h e fi r s t d a y of t h e s t u d y p er i od , t h evolunteers ingested the protein test meal together with 200ml of water within 15 min. No further food was allowed until1500, when the volunteers consumed a standard bread meal.Drin king of wa ter w as permitt ed from 1200 on.

The experimental design is schematically represented inFig. 1.

Diet

The volunt eers w ere given no s t a nda rd diet s . How ever,they w ere asked to weigh and record all food and drinks ta kenfrom 3 days before until the end of each study period. Thesed a t a w e r e a n a l y ze d u s i n g a com p ut e r p r og r a m t o o bt a i nenergy an d nutrient int ake results (Nederlands voedingsstof-fenbestand 198990, Voorlichtingsbureau voor de voeding,Den Haag, The Netherlands).

Test M eal

The protein test mea l consist ed of 100 g of egg w hit e (i.e., 11g of egg w hit e prot ein) la beled w it h 15N, 100 g of egg w hit ela beled w it h L-[1-13C]leucine and L-[r i n g -2H 4]t yros ine, a ndthe yolk of one egg. Five m icrocuries [3H]polyethylene glycol([3H]PE G ) 4000 were add ed to the test mea l as a ra diolabeled

t r a n s i t m a r k e r. A ll t h e con s t i t ue n t s o f t h e t e s t m e a l w e r emixed before ingestion. Total caloric content of the t est m ealwas 150 kcal (25 g of protein, 5.56 g of fat , and a negligibleam ount of car bohydra tes).

The met hodology for obt a ining la rge a mount s of highlyenriched egg proteins labeled with stable isotopes has beendescribed elsewhere (9). B riefly, 13C- or 15N-labeled proteinsw ere produced by giving la ying hens free a cces s t o a foodcontaining 25%of the (National Research Council required)leucine content as free [1-13C]leucin e (99 mol%, E ur iso-top,Sa int-Aubin, Fra nce) an d [15N]leucine (99 mol%, E ur iso-top),respectively. The yolk a nd egg wh ite fra ctions of th e enrichedeggs were separated and pooled. The isotopic enrichment ofboth pools was determ ined using a continuous flow elementa l

analyzer isotope ratio mass spectrometer (ANCA-2020, Eu-r op a S c ie n t ifi c, C r e w e , U K ). Wi t h t h e e xa c t a m i n o a ci dcomposition a nd t he isotopic enrichment of the egg w hite, th ea mount of [1-13C]leucine (99 mol%) incorporated could becalculated (9). Because redistribution of the 15N l a b e l i slikely to occur in the hen via t ra nsa mina tion, the egg proteinc a n b e a s s u m e d t o b e u n i f o r m l y 15N la beled. Egg prot einl a b el ed w i t h L-[r i n g -2H 4]t y r os i ne w a s ob t a i n ed b y g i vi n gl a y i n g h e n s f r e e a c ce ss t o a f ood c on t a i n i n g 2 0% of t h e(National Research Council required) phenylalanine contenta s free L -[r i n g -2H 5]pheny lala nine (98 mol%, E uriso-top). D uet o h ydroxyla t ion of L-[r i n g -2H 5]p h en y l a l a n i ne b y t h e h e n ,both L-[r i n g -2H 5]phenyla la nine a nd L-[r i n g -2H 4]tyrosine areincorpora t ed in t he egg prot ein. The L-[r i n g -2H 4]tyrosinecontent of the egg protein wa s determined by ga s chromatog-ra phy-ma ss spectrometry (GC Q, Finn igan , Sa n J ose, CA) (14).

SampleCollection

B reat h sa mples were collected in exetainers (Europa S cien-t ifi c) before inges t ion of t he mea l, every 15 min for t he fi rs t6 h, a nd every 30 min up to 9 h a fter ing estion of the test mea l.

During ea ch s t udy period, urine w a s collect ed in pla s t icbottles for t he following periods: 03 h, 36 h, 6 9 h, 924 h,

2448 h, 4872 h. M oreover, a 24-h urine collect ion w a sobtained th e day preceding each stud y period. Neomycin wa sa dded t o t he pla s t ic cont a iners us ed for t he collect ions t oprevent bacterial growth. After measurement of the volume,s a mples w ere t a ken a nd s t ored a t 20 C unt il a na lys is .

A ll s t ools voided during ea ch of t he s t udy periods w erecollected as well. Date and time of voiding of stools were notedin a diary. The stools were frozen immediately after voidinga nd s t ored a t 20 C unt il a n a lys is .

Analytical Procedures and Calculations

Breath samples.The breat h sam ples were a na lyzed for 13Ccont ent by mea ns of a cont inuous-flow is ot ope ra t io ma s sspectrometry (AB CA, Eur opa Scientifi c). The values givenby isotope rat io mass spectrometry w ere converted t o percent-

a ge of 13C recovery per hour of t he init ia l a mount a dminis -ter ed (%dose 13C/h) a ccording to calculations previously de-scribed in detail (8, 15). Cumulative percentages of recoveredlab el (cumula tive %dose 13C) were calculat ed by means of thet ra pezoida l rule. From t hes e da t a , t he follow ing pa ra m et ersof protein assimilation were derived: the maximum percent-a ge of a dminis t ered dos e of 13C excret ed per hour a nd t hecumulative percentage of ad ministered dose of 13C recoveredin breat h over 6 h.

Fecal sampl es. Af t er t h a w i n g , t h e s t ool s a m p le s w e r eweighed a nd homogenized for each d ay of collection. Sa mplesof know n w eight w ere t a ken a nd freeze-dried. The driedm a t e ri a l w a s w e ig h ed , a n d a l iq u ot s w e r e t a k e n f or t h ea n a l y s i s of t o t a l n i t r og e n con t e n t , 15N e n r ich m e n t , a n d[3H]PE G 4000 content .

The [3H]PEG 4000 content was measured by the oxidationmethod (Pa ckard sa mple oxidizer, model 306, Pa ckard In stru-ment , D ow ners G rove, I L ), w it h s ubs equent l iquid s cint i l la -tion counting (model 2450, Packard Instrument) and correc-t ion for quenching. R es ult s w ere expres s ed in cumula t ivepercenta ge of the adm inistered dose of 3H recovered over 72 h

(further r eferred to as 0 h72 h%dos e [3H]PEG 4000).

Tot a l nit rogen cont ent a nd 15N e n r ich m e n t w e r e d e t er -mined us ing a cont inuous fl ow element a l a na lyzer-is ot opera tio m ass spectrometer (ANCA-2020, E uropa Scientifi c).B riefly, a n a liquot of know n w eight of t he lyophilized feca lsam ple was combusted in the presence of oxygen at 1,000 C.The combustion products therea fter pa ssed thr ough a second

Fig. 1. Experimental design. 1st s ta nds for first s tool voided on thef ou r t h d a y a f t e r i n g e st i on of t h e t e s t m e a l . d 1, Day preceding thestudy period.

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furna ce contain ing copper a t 600 C, w here excess oxygen w asa b s or b ed a n d n i t r og e n ox id e s w e r e r e d uc ed t o e le m en t a lnitrogen. Total nit rogen content w as m easured by m eans of atherm al conductivity detector, and 15N enrichment w as deter-m i n ed b y m e a n s o f a n i sot o pe r a t i o m a s s s pe ct r om e t er,coupled to the combustion unit of the elemental analyzer. The15N-to-14N isotope ratio of N 2w a s mea s ured w it h reference t oa ca libra t ed la bora t ory s t a nda rd (i . e. , a s t a nda rd a m monium

sulfat e solution). The va lues were expressed in at om percent(AP). The intraassay variability of this method, assessed byth e coefficient of va ria tion (CV), am ounted t o 2.6 and 0.3%fort o t a l n i t r og e n con t e n t a n d 15N enrichment, respectively.Results w ere expressed in percentage of a dministered d ose of15N recovered per da y a nd in cumula t ive percent a ge recov-ered over 72 h.

The percentage of administered dose of 15N recovered perday was calculated as follows

%15N dose/da y mg excess 15N t(a)

mg excess 15N a dminis t ered(b) 100

w h e r eai s

1AP

s 0.368

100 2 N f totw here A P s i s t h e m e a s u r e d 15N enrichment of t he s t oolscollected on da y t, expressed in AP; 0.368 is the natural 15Ncont ent of s t ools , expres s ed in A P ; a nd N f t ot i s t h e t o t a lnitrogen content of the st ools collected on da y t, expressed inmill igra ms .

The calcula tion of bi s

1AP m 0.368

100 2 N mwh ere AP m is t he w eighed a vera ge 15N enrichment of the testmeal (calculated for each test meal separately), expressed in

AP, and N m is the nitrogen content of the protein test meal,i.e., 4,000 mg.The cumula t ive percent a ge of a dminis t ered dos e of 15N

recovered in feces over 72 h (further referred to as 0 h72 h%dose

15N f a d m ) w a s obt a ined by s umma t ion.A cor r e ct i on w a s m a d e f or g a s t r oi n t es t i na l t r a n s i t b y

dividing 0 h72 h%dose 15N f a d m by t he cumula t ive percent a ge of

ad ministered dose of 3H r ecovered over 72 h

0 h

72 h

%dos e of 15N f a d m corrected

0 h

72 h

%dos e of 15N f a d m

0 h

72 h

%dose of [3H]PEG 4000

U r i n a r y s a mp l e s . U R I N AR Y P H E N O L A N D P -CR E SO L. P henol,[r i n g -2H 4]phenol, p-cresol, and p-[r i n g -2H 4]cresol were mea-s u r ed b y g a s ch r om a t o g r a ph y -i on t r a p t e ch n ol og y a s d e -s c r i b e d b y G e y p e n s e t a l . ( 1 3 ) . B r i e fl y , 1 m l o f u r i n e w a sdiluted w ith 3 ml of distilled wa ter. Seventy-fi ve microliters of2,6-dimethy lphenol solution (20 mg/100 ml) w ere add ed asi n t er n a l s t a n d a r d . Th e p H w a s a d j us t ed t o 1 w i t h c on ce n -t r a t e d H 2S O4, a n d t h e s o lu t i on w a s r e fl u xe d f or 7 5 m in t ohydrolyze the conjugated phenols. After a cooling-down pe-riod t o a mbient t empera t ure, phenols w ere ext ra ct ed w it h 2ml of diet hylet her. One microlit er w a s inject ed int o t he ga schromatography-mass spectrometer (GCQ, Finnigan). Afters ep a r a t i on on t h e a n a l y t i ca l col u m n , a 2 5-m 0.25-mm

CP -Sil 5 CB-MS wit h a fi lm thickness of 0.25 m (Chrompa ck,Middelburg, the Netherlands), the phenolic compunds wereidentifi ed by ion t ra p technology (ITD 700, Finnigan ).

R es ult s for t he unla beled compounds w ere expres s ed ina m o u nt s e xc re t ed p er h ou r a n d i n cu m u la t i v e a m ou n t sexcreted over 72 h. Because phenol and p-cresol are qua ntit a-t ively t he ma in phenolic compounds found in urine, t ot a lphenols w ere mea s ured a s t he combina t ion of phenol a nd

p-cres ol (3, 23).R e su l t s f or t h e l a b el ed com p ou n d s w e r e e xp r es s ed i n

percent administered dose of L-[r i n g -2H 4]tyrosine recoveredper hour and in cumulative a mounts excreted over 72 h.

The percent a dminist ered dose ofL -[r i n g -2H 4]tyrosine recov-ered per hour a s [r i n g -2H 4]phenol was ca lculated a s follows

%dos e of [r i n g -2H 4]phe nol /h

100 [r i n g -2H 4]phenol r ect

d L-[r i n g -2H 4]tyrosine administered

w h e r e [r i n g -2H 4]phenol rect i s t h e t o t a l a m ou n t o f [r i n g -2H 4]phenol recovered in the urine fraction of period t (ex-p re ss ed i n m ol ) (t h e n a t u r a l u r in a r y con t e nt of [r i n g -

2H 4]phenols is zero); L-[r i n g -2H 4]tyrosinea d m i n i st ered i s t h ea mount of L-[r i n g -2H 4]t yros ine a dminis t ered, expres s ed inmoles (calculated for each test meal separately); and d is t hedura tion of the collection period, expressed in hour s.

Th e p er ce n t a g e of t h e a d m i n is t er e d d os e of L-[r i n g -2H 4]t yros ine recovered per hour a s p-[r i n g -2H 4]cresol wa sca lcula t ed in t he s a me ma nner. B eca us e phenol a nd p-cresolar e qua ntit at ively the major bacterial metabolites of tyrosine,t he percent a ge of a dminis t ered dose of L-[r i n g -2H 4]tyrosinefermented in the large gut per hour is obtained by summa tion(%dos e of [r i n g -2H 4]phe no l/h %dose ofp-[r i n g -2H 4]cres ol/h ).

The cumula tive percentag e of dose of L-[r i n g -2H 4]tyrosineadm inistered recovered in urine over 72 h (further referred to

a s 0 h72 h%dose 2 H 4 a d m ) was calculated by summation.

The cumulative values were corrected for gastrointestinal

transit as follows

0 h

72 h

%dos e of 2H 4 a d m corrected

0 h

72 h

%dos e of 2H 4 a d m

0 h

72 h

%dos e of [3H]PEG 4000

U R I N AR Y Nt ot AND 15N. Af t er t h a w i n g , a k n ow n a m o u n t o f

urine (15 l) wa s a bsorbed on chromosorb in a t in capsule.Tot a l nit rogen cont ent a nd 15N e n r ich m e n t o f u r i n e w e r edet ermined us ing a cont inuous fl ow element a l a na lyzer is o-tope rat io ma ss spectrometer (ANC A-2020, Eu ropa S cientifi c)as described previously.

Results for 15N were expressed in percentage of the a dmin-is t ered dose of 15N recovered per hour a nd in cumula t ivepercentages recovered over 72 h.

The percentage of administered dose of 15N recovered perhour wa s calculat ed as follows

%15N dos e/h mg excess 15N t(a)

mg excess 15N a dminis t ered (b) d 100

w here ai s

1AP s AP d 1

100 2 N u t o t

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w here A P s i s t h e m e a s u re d 15N e n ri ch m e nt of t h e u r in e

collected in period t, expres s ed in A P ; A P d 1 i s t h e 15Nenrichment of t he urine of period d 1, i .e . , t he na t ura l 15Ncont ent of urine before inges t ion of t he la beled mea l, ex-press ed in AP ; a nd N u t o t is t he t ot a l nit rogen cont ent of t heurin e, collected in period t, expressed in milligram s.

The calcula tion of bwa s as follows

1AP m AP d 1

100 2 N mw here AP mis t he w eight ed a vera ge 1 5N enrichment of the test

mea l, expres s ed in A P (ca lcula t ed for ea ch t es t mea l s epa -r a t e l y ) , a n d N m i s t h e n i t r og e n c on t e n t of t h e m e a l , i . e. ,4,000 mg .

The cumula t ive percent a ge of a dminis t ered dos e of 15N

r e cov er e d i n u r i ne ov er 7 2 h (0 h72 h%dose of 15N u a d m ) w a s

calculated by summation.15N RETENTION. The percenta ge of the a dministered d ose of

15N, reta ined in the body after 72 h, wa s calculat ed as follows

%15N retention 100

(0 h

72 h

%dos e of 15N f a d m corrected 0 h

72 h

%dos e of 15N u a d m)Stati stical M ethods

Results are expressed as means S E . S t a t i s t ic a l a n a l y s i sw a s p er f or m ed w i t h S AS s of t w a r e p a ck a g e. P a r a m e t er sobt a ined a f t er inges t ion of t he ra w t es t mea l w ere pa irw is ecompar ed with va lues obta ined in the control study using thepaired t-test . Correla tions were obtained by P earsons test .

RESULTS

Diet

N o sig n ifi can t d i f fe re n ce s we re fo u n d b e twe e n th etw o stu d y p eriod s e i th e r in th e in ta k e of p rote in , fat ,a n d c a r b o h y d r a t e s o r i n t h e i n t a k e o f d i e t a r y fi b e r(Ta ble 1).

Breath Tests

F i g u r e 2 s h o w s t h e m e a n 13C O2 e xcre tio n rate inbreat h a fter ingestion of the ra w a nd cooked test meal.Differences between both test situations are obvious.The curve obtained after ingestion of the cooked testmeal is characterized by a steep ascending slope, a highp e ak e xcre tio n rate , an d an in i t ial s te e p d e sce n d in gslope tha t smoothes down considerably aft er 6 h. Afteringestion of the ra w t est meal, the 13C O2excretion rat eincreased more slowly, did not reach the high valuesob ta in ed af te r in g estion of th e cook ed te st m e al , a n drema ined on a ra ther consta nt level after the maximum

was reached. The cumulative percent 13C o f ad m in is-tered dose, recovered in breath over time after inges-tion of the la beled cooked and ra w test meal, is shownin Fig. 3.

Ta ble 2 summa rizes the par a meters of protein a ssimi-l a t i on a s d er i ve d f r om t h e b r ea t h t e st d a t a . B ot h t h emaximum percentage of administered dose of 13C e x-cr e t ed p er h ou r a n d t h e cu m u la t i v e p er ce nt a g e ofad ministered dose of 13C, recovered in breath over 6 h,were significantly higher after ingestion of the cookedtest meal compared with the raw test meal.

F eces

F eca l ou t pu t v a r ia b les s uch a s w e t w e ig h t , d r yweight, t otal nitr ogen content , and n itrogen density didnot differ significantly after ingestion of the cooked andra w test mea l (Ta ble 3). The cumula tive fecal r ecoveryof 15N, however, wa s significan tly higher a fter ingestion

of t h e r a w t es t m e a l [0 h72 h%dos e of 15N f a d m corrected:

4.16 0.27% (cooked) vs. 14.16 1.70% (raw); P 0.001] (Ta ble 4).

U r i n e

Nitrogen.Figure 4 shows the mea n 15N excretion ratein urine aft er ingestion of the ra w a nd cooked test mea l.Although signifi cant differences in the 15N excretionra te w ere noted, the cumulat ive percenta ge of a dminis-

Ta ble 1. Di etar y int ake of protein , fat, carbohydr ates, and d ietary fiber d ur in g th e cooked and r aw stud y peri od

C ooked Mea l Ra w Mea l

In t a ke, g/da y E n er gy % I nt a ke, g/da y E n er gy %

P r ot ein 77.88 5.36 13.15 0.93 71.89 4.09 13.16 0.88F a t 111.98 11.13 40.63 1.92 99.89 8.91 39.81 1.61C a r boh y dr a t es 274.51 28.61 44.79 1.89 249.46 13.78 44.89 1.35D iet a r y fi ber 21.76 1.86 18.97 1.41

Values are means S E .

Fig. 2. Mean 1 3C O2excretion rate in breath, expressed as percentageof t h e a d m in is t e r e d d os e of 13C e x c r e t e d p e r h ou r in 1 0 h e a l t h yvolunteers aft er ingestion of a cooked and ra w test m eal, consistin g of25 g of 13 C -,2 H-, and 1 5N-labeled egg protein. Values a re mea ns S E .



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tered dose of 15N , re co ve re d in u rin e o ve r 72 h was

alm o st th e sam e in b o th te st s i tu atio n s [0 h72 h%dos e of

15Nu a d m: 35.91 2.12% (cooked) vs. 32.68 1.35%(raw);P 0.12] (Ta ble 3).

Phenols. F i g ur e 5 s h ow s t h e e xcr e t ion p a t t e r n ofphenol, p-cresol, a nd tota l phenols in u rine. The excre-t i o n r a t e w a s h i g h e r a f t e r i n g e s t i o n o f t h e r a w m e a lth an af te r in g e stio n o f th e co o k e d m e al . S ig n ifi can cew a s rea ched in the 9-t o 24-h period.

The excretion pat ter n of [r i n g -2H 4]phenol and p-[r i n g -2H 4]cresol w as sim ilar to t h e e xcre tion p att e rn o f t h eunlabeled components (Fig. 6). p-[r i n g -2H 4]cresol ap-p ear e d in u rin e som e wh at late r t h an [r i n g -2H

4]phenol.

Overall , the maxima l excretion rat e wa s reached in the9- to 24-h period. The cumula tive percenta ge of adm in-istered dose of L-[r i n g -2H 4]tyrosine, recovered in urinea s t o t a l [r i n g -2H 4]p h en ols , wa s sig n ifi can tly h ig h erafter ingestion of the raw test meal compared with thecooked test meal (20.63 5.59%vs. 1.60 0.44%, P 0.005) (Ta ble 3).

15N Retention

The percent a ge of adm inist ered dose of 15N, retainedi n t h e n it r og en pool of t h e b od y a f t er 72 h , w a s

sign ifi can tly lowe r a f te r in g estion o f th e ra w te st m e alcompa red w ith th e cooked test mea l (49.63 2.69%vs.63.16 1.36%, P 0.0002) (Ta ble 3).

Correlations

Significant correlations were found between severalfecal, urina ry, an d breat h var iables (Ta ble 5). Therewa s a negat ive correlat ion betw een t he recovery of 13Cin b re ath an d th e r e covery of e i th e r 15N in feces (r

F ig . 3 . M e a n c um u la t i v e p e r ce n t a g e of a d m in is t er e d d os e of 13C ,recovered in brea th over tim e (%dose cumul/h) in hea lthy volunteersafter ingest ion of a cooked and raw test meal, consist ing of 25 g of13C -, 2H - , a n d 15N-labeled egg protein. Values a re m eans S E ;n 10.

Ta ble 2. Parameters of protein assimilation in healthy

volunteers after ingestion of a cooked and raw testmeal, consisting of 25 g of 13C-, 2H - , a n d 15N -la bel ed egg pr otein

Test C onditi on

PC ooked Ra w

%D os e cu m 6 h 17. 23 0.69 8.20 0.94 0.0001%ma x 5.25 0.26 1.91 0.18 0.0001

V a lu e s a r e m e a n s S E , n 10. %Dose cum 6 h , cumulat ivepercentage of administered dose of 1 3C, recovered in breath over 6 h;%ma x , ma ximum percenta ge of administered dose of

1 3C excreted perhour.P values obtained by paired t-test.

Ta ble 3. M ajor uri nar y and fecal vari ables in th etwo test situations

Variable

Test Situa tion

PC ooked R a w

F eces Wet w eigh t,g/da y

149.2 22.3 152.0 24.9 0.92

D r y w e ig h t ,

g/da y

25.3 2.7 27.1 1. 7 0. 31

D r y m a t t er, % 37. 7 5.1 41.2 6. 7 0. 31N excretion,

g/da y1.89 0. 18 2. 10 0.29 0.49

N den sit y, %drym a t t e r

5.02 0. 47 5. 09 0.55 1.00

%Cum ulat ive15N f(c)

4.16 0.27 14.50 1.70 0.00020

R (d) 67.7 3.7 74.0 4. 8 0. 32U r in e Tot a l ph en ol s,

mg /da y32.14 3.50 47.10 5.06 0.0040

N excretion,g/da y

10.38 0.67 10.68 0.57 0.47

%Cum ulat ive2H 4(e)

1.60 0.44 20.63 5.59 0.00010

%Cum ulat ive15N u ( f)

32.68 1.35 35.91 2.12 0.12

Calculatedvalues

%Ma la bsorbed(c c)

%Accumul a tion(e/c e)

5.73 0.5024.14 6.07

35.10 6.78

50.62 6.10

0.0020

0.0030

%Incorpor a tion(c/c c)

75.86 6.07 49.38 6.10 0.0030

%Reta ined in Npool(100% c f)

63.16 1.36 49.63 2.69 0.00020

Values are means S E ; n 10. %Cum ulat ive 15N f 0h72h %dose

15N f ad m corrected (see text). R 0h72h %dos e [3H]PE G 4000 (see text).

%Cum ula tive 2H 4 0h72h %dose 2 H 4 a d m corrected (see text ). %Cu mu-

lat ive 15Nu 0h72h %dose 1 5N u a d m (see text). %Incorporat ion a ssumes

that 100%of 15N recovered in feces is incorporated into the bacterialm a s s .Pvalues w ere obtained by paired t-test.

Ta ble 4. Fecal 15N excr eti on in 10 healt hy volun teer safter ingestion of a cooked and raw test meal consisting

of 25 g of 13C- , 2H - , a n d 15N-labeled egg protein

Fecal 15N Excretion

Excretion perda y, %dose/da y

Cumulativeexcretion

024 h 2448 h 4872 h0h

72h %dose adm

72 0h72h %

dose admcorrected

Cooked 0.340.27 0.960.31 1.570.29 2.870.30 4.160.27Ra w 1.811.37 4.481.37 4.200.94 10.491.26 14.51.7P 0.10 0.005 0.020 0.001 0.001

Values are means S E ; P values were obtained by paired t-test.adm, Administered.



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0.71, P 0.005) or [r i n g -2H 4]phenols in urine (r 0.62,P 0.005). The recovery of [r i n g -2H 4]phenols i nurine correlat ed positively w ith the recovery of 15N infeces (r 0.77,P 0.005) (Fi g. 7).

No correla tion wa s found between the fecal nitr ogenou t p ut a n d t h e r e cov er y of e it h e r 15N i n f ece s or[r i n g -2H 4]phenols in urine.

DISCUSSION

The efficacy of protein assimilation has been studiedt o d a t e b y s ev er a l r e sea r c h er s ei t h er i n i le os t om ysubjects (4, 8, 11, 22, 32, 34) or in healthy volunteersusing intubation techniques (24). I t was demonstratedt h a t t h e a m o un t of p rot e in es ca p i ng d ig es t i on a n da bsorption in the sma ll intestine is affected by the typeand amount of protein (8, 11, 16, 22, 34) as well as thepresence of other constituents (e.g. , resistant starch)(32) in th e d ie t . L e ss is k n o wn ab o u t th e p ro ce ss o fp r ot e in f er m en t a t i on i n v iv o i n h u m a n s , w h i ch i slargely due to the physiological inaccessibility of thecolon.

T h e avai lab i l i ty o f p ro te in lab e le d with stab le iso -topes a llowed us to stud y protein (ma l)a bsorption a nd

fermentat ion in hea lthy volunteers by means of nonin-v a s i ve t r a c er t e ch n iq u es . A n i n he re nt a d v a n t a g e oftrace r te ch n iqu e s is th at th e y d o n o t d istu rb n o rm alphysiology. All volunteers w ere st udied in t w o differentra ndomly applied test situa tions:1) a fter ing estion of acooked egg protein mea l la beled w ith L-[1-13C]leucine,[15N]amino acids, and L-[r i n g -2H 4]tyr osine a nd 2) afteri n ge st i on of t h e s a m e b u t r a w m ea l . B ot h t e st s it u a -tions were separated by a 1-wk washout period. Thisperiod was sufficiently long to return isotope enrich-

ment t o baseline (da ta not shown).P rotein (ma l)a bsorption and fermenta tion were eva lu-

ated quantitatively through the analysis of metabolitesexcreted in brea th, urine, or feces. Brea th w a s an a lyzedfor 13C O2, feces for 15N, and urine for [r i n g -2H 4]phenol,p-[r i n g -2H 4]cresol, and

15N.The breat h test results obtained in th e present st udy

we re in accord an ce with th o se ob ta in ed in a re ce n tstu d y p e rfo rm e d in i le o sto m y p atie n ts u n d e r sim ilartest conditions (8). In t he lat ter st udy, a highly signifi-can t n e g ative co rre lat io n was d e m o n strate d b e twe e nt h e 13C re co ve ry in b re ath an d th e re co ve ry o f e xo g -enous protein in the ileal effluents. In t he extr a pola tionof th is fi n d in g to su bje cts w ith a n in ta ct g ast roin te sti-

nal system, the low recovery of 13C O2 i n b r ea t h a f t e rin g estion of t h e ra w p rote in t e st m e al su g g ests ove rtmalabsorption.

I t m a y b e a r g u e d t h a t i n s u b j e c t s w i t h a n i n t a c tg astro in te stin al syste m th e 13C O2 recovered in br eat hmay be derived from the fermentation of malabsorbedL-[1-13C]leucine in the colon as well. However, becauseit was observed in the ileostomy study formerly men-tioned (8) that 50%of the malabsorbed cooked and rawp ro te in h ad e m p tie d fro m th e i le o sto m y b y 5.33 an d5.29 h, respectively, it can be assum ed tha t most of the13C O2 a p p e a r i n g i n b r e a t h w i t h i n 6 h f o l l o w i n g t h e

F ig . 4 . U r in a r y 15N excret ion pat tern in 10 healthy volunteers afteringest ion of a cooked (open bars) and raw (closed bars) test meal,consisting of 25 g of 13 C -,2 H-, and 1 5N-labeled egg protein. Values ar em e a n s S E. * P 0.05, pa ired t-test.

Fig. 5. Ur ina ry excretion patt ern of phenol (A ),p-cresol (B), and tota lphenols (C) in 10 healthy volunteers a fter ingest ion of a cooked a ndraw test mea l, consist ing of 25 g of egg protein. Values a re mean s S E. * P 0.05, pa ired t-test.

F ig . 6 . U r in a r y e xc r et ion p a t t e r n of [r i n g -2H 4]phenol (A ), p-[r i n g -2H 4]cresol (B) , a n d t ot a l [r i n g -2H 4]phenols (C) a fter ingest ion of a

cooked (open ba rs) and ra w (closed bar s) test mea l, consistin g of 25 gof 13C -, 2H -, a n d 15N-labeled egg protein. Values are means S E .* P 0.05, pa ired t-test.



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ingestion of the labeled test meal is derived from them e tab o lism o f p rote in a ssim ilate d in t h e sm a ll in te s-tine.

T h e m e a n d a i l y f e c a l w e t w e i g h t , d r y w e i g h t , a n dnitrogen content were consistent with previous investi-ga tions in hum a ns (2, 7, 36, 37). None of t hese para m-

eters wa s affected significan tly by th e nat ure of the testmeal (Table 3).

The cumulative percentage of administered dose of15N r ecovered in feces over 72 h aft er ingestion of thecooked egg protein meal amounted to 4.16 0.27%.T h is valu e is co m p arab le with fi g u re s p re vio u sly re -port ed for y east , egg, a nd soya protein (20, 40). Thecumula tive recovery of 15 N in feces aft er ingestion of th era w m e al w as sig n ifi can tly h ig h e r (14.50 1.70%). I thas previously been demonstrated that at least 60%ofth e feca l nitr ogen content is of bacteria l origin (36). Forpractical purposes, however, it is assumed in the pre-

se n t s tu d y th at al l 15N recovered in feces is of bacteria lorigin (i.e., incorpora ted in t he bact eria l ma ss).

The cumulative percentage of administered dose of2H recovered in urine over 72 h amounted to 1.60 0.44%a nd 20.63 5.59%, respecti vely, aft er ing est iono f th e co o k e d an d raw te st m e al . T h e p e rce n tag e o f

a dministered dose of 2H recovered in ur ine representsthe portion of consumed egg protein that is accumu-lated in the colonic lumen as free fermentation metabo-lites, subsequently absorbed into the portal blood andfi na lly excreted in urine.

The excretion pattern of 2H 4-labeled total phenolsaf te r in g estion of t h e ra w te st m e al coin cid ed alm o stcompletely w ith the excretion pat tern of the unla beledfraction. This indicates that the observed increase ofthe unla beled tota l phenols is relat ed to mala bsorptiono f t h e t e s t m e a l . [r i n g -2H 4]phenol appeared in urineslightly earlier than p-[r i n g -2H 4]cresol. This is in a ccor-da nce wit h previous studies suggesting tha t phenol andp-cresol are p red om in an tly form e d in th e te rm in al

ileum (an d cecum) a nd left colon, respectively (3). Thedelayed a ppeara nce ofp-cresol might a lso be expla inedby a slower production rate.

Assuming that the fate of both the 15N a n d 2H trace ris id e n tical in th e larg e in te st in e , th e p e rce n tag e o fingested protein t ha t escaped digestion a nd a bsorptioncou ld b e ca l cu la t e d a p pr ox im a t e l y. I t a m o un t e d t o5.73 0.50%a nd 35.10 6.78%after ingestion of thecooked a nd r a w test meal, r espectively (Fig. 8). Ma lab-sorption might be overestimated in the present study

Ta ble 5. Corr elat ions betw een br eath , fecal, and ur in ary var iabl es

F eca l Va r ia bles U r in a r y Va ria bles

Tota l N,g/da y

Wet weight,g/da y

%Cum ula tive15N

Total phenol,mg /da y

%Cumula tive2H 4

B r e a t h v a r ia b le%D ose 13 C 6 h 0.10 0.049 0.71 0.67 0.62

Fecal variables

Tot a l N, g/da y 0.75 0.19 0.22 0.045Wet w eight , g/da y 0.032 0.22 0.11%Cum ulat ive 15 N f 0.46* 0.77

U r in a r y v a r i a b l eTot a l ph en ols, m g/da y 0.62%Cum ula tive 2 H 4

n 20 (10 subjects 2 test situt ions). * P 0.05; P 0.005.

Fig. 7. Correla tions betw een para meters of protein assimila tion (i.e.,%cumu la tiv e 13C) a n d p a r a m e t e r s of p r ot ein m a la b s or p t ion (i . e. ,%cumu la tiv e 15N and %cumulat ive 2H 4). %cum

13C, c um u la t iv epercentage of administered dose of 13C recovered in breath over 6 h.

%cum 15N, 0h72h %dos e of 15N f a d m correct ed. %cum

2H 4, 0h72h %dos e of

2H 4 a d mcorrected (see text for complete explan at ion of abbrevia tions).Fig. 8. Amount a nd fa te of 25 g of egg protein escaping a ssimilat ionin the small intest ine of humans.



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due to t ra cer r ecycling. Tra cer r ecycling occurs t hroughd e squ a m at ion of in te st in al m u cosa an d se cre tion ofd ig estive en z ym e s an d u re a in th e sm a ll in te st in e an dcolon, respectively (20, 26). Little information is avail-ab le o n th e m ag n itu d e o f th is b ias , w h ich , m o st p rob -ably, is due to methodological problems. Kayser et al .(20) quantified 15N trace r re cycl in g b y m e asu rin g th eappearance of 15N in stools after an intravenous injec-tio n o f 250 m g o f 15N-enriched glycine (99 AP). Thefra ctiona l feca l loss (i .e. , t ra cer recycling) amounted to1.43 0.64%(means SD ). Although it is reasona bleth a t th e m a g n itu d e of tra cer re cycl in g is n o t fi xed b u tin flu e n ce d b y d ie tary facto rs , th e lat te r valu e m ay b eindicative.

Despite possible overestimation due to tracer recy-cling, the malabsorption percentages observed in thep rese n t a re st i l l som e wh at lowe r t h an th o se we p revi-ously reported in healthy ileostomy patients in identi-cal test condit ions (8). This difference can be explainedeither by differences in t he efficiency of protein a ssimi-lat io n b e twe e n h e al th y vo lu n te e rs an d i le o sto m y p a-

tients or by sa lvage of n itrogen in t he colon (18). Thelat te r p o ssib i l i ty is su p p o rte d b y h u m an an d an im aldata (17, 21).

Amounts of 24.14 6.07%and 50.62 6.10%of th emalabsorbed cooked and raw egg protein, respectively,were calculated to accumulate as free end products ofb acte rial m e tab o lism in th e colon ic lu m e n (F ig . 8).Assuming that the two test conditions only differed inthe a mount of protein made a vaila ble to the colon, thep re se n t re su lts su p p o rt th e h yp o th e sis th at lu m in ala c cu m u la t i on a s f r ee f er m en t a t i on m et a b ol it e s b e-comes the preferential fate of malabsorbed protein asmore protein is ma de ava ilable to the colon.

Significant differences were observed between both

test sit ua tions in the pat tern of excretion rat es of 15

N inurine. The 15N excretion rat e wa s significant ly low er inthe 0- to 6-h period a nd significa ntly higher in th e 9- to72-h period after ingestion of the raw test meal com-pared with the cooked test meal. Protein assimilationwa s su g g este d b y th e sh a p e of th e b re ath te st cu rve tob e co m p le te d af te r 6 9 h . B acte rial p ro te in m e tab o -lism, reflected by a n increase of the excretion of [r i n g -2H 4]phenols in urine, on the other hand, became appar-en t f rom 9 h a f t er i ng es t ion of t h e t es t m ea l on .Therefore, 1 5N appear ing soon aft er ingestion of the testm e a l i s a c ce pt e d t o b e d e r iv ed u n iq u el y f r om t h emeta bolism of a ssimilated protein, whereas 15N appear -in g late r o n is acce p te d to b e d e rive d fro m b acte rial

meta bolism of mala ssimilated protein a s w ell. As couldb e p re d icte d , m e tab o lism o f assim ilate d p ro te in wasmore prominent a fter ingest ion of the cooked test m eal,wh e re as b acte rial m e tab o lism o f m alassim ilate d p ro -t e i n w a s m o r e p r o m i n e n t a f t e r i n g e s t i o n o f t h e r a w p rote in m e al . N otw ith sta n d in g th e se k in e tic d i ffer-ences, the cumulative percenta ge of adm inistered doseof 15N excreted in urine over 72 h was similar in bothtest situations.

The cumulative percentage of administered dose of15N , r e t a i n e d i n t h e n i t r o g e n p o o l o f t h e b o d y , w a ssig n ifi can tly lo we r af te r in g e stio n o f th e raw p ro te in

mea l compa red w ith t he cooked protein meal. Neverth e-less, the difference between both test situations (13.50 2.20%) w as less pronounced tha n w as expected fromt h e d i ff er e nce i n t h e p er ce n t a g e of m a l a b s or p t ion(29.34 6.45%) (Ta ble 4). This observat ion mightequa lly be explained by sa lva ge of nitr ogen in th e colon.

Hig h ly sig n ifi can t n e gat ive corre lat ion s we re fo u n dbetween parameters of protein assimilation (i.e., cumu-lative percentage of administered dose of 1 3C, recoveredin breath over 6 h) a nd para meters of protein ma labsorp-tion (i.e., the a mount of 15N an d [2H 4]phenols, recoveredin feces and urine, respectively). This finding supportst h e v a l id i t y of t h e t e ch n iq u es u s ed . Th e l a ck of asignificant correlation between the fecal nitrogen out-pu t a n d f eca l 15N r e cov er y i n di ca t es t h a t t h e f eca ln i t r og en ou t p ut m a y n ot b e r e ga r d e d a s a s en s it i v epara meter of the efficiency of dietar y protein a ssimila-tion in the small intestine.

In conclusion, using noninvasive stable isotope tech-niques, we were able to evaluate protein (mal)absorp-tion and fermenta tion in hea lthy volunteers in a nonin-

vasive and quantitative way. We definitively confirmedmalabsorption of even easily digestible protein. Ourr es u lt s f u rt h e r m or e s u pp or t t h e h y pot h e si s t h a t a nincreased a va ilability of protein in t he colon causes t hepreferential fa te of ma labsorbed protein to shift towa rdl um i na l a c cu m u la t i on a s f r ee p r ot e in f er m en t a t i onm et a b o li t es . Th i s fi n d in g m a y b e i m por t a n t f r om ag astro in te stin al p o in t o f vie w, s in ce se ve ral o f th e semetabolites (ammonia, thiols, phenols) are thought toplay a role in the etiopathogenesis of, e .g. , ulcerativecolitis and colonic cancer (3, 5, 10, 23, 27, 28, 30, 39).

N. Gorris , A. Luypaerts , S . Rutten, and L. Swinnen are acknowl-edged for excellent technical a ssista nce.

This w ork wa s supported by a grant from Biomed P L93-2139,

Vlaa mse Executieve and Nutricia Cha ir in gastr ointest inal microen-vironment.Address for reprint requests and other correspondence: Y. Ghoos,

U n i v e r si t a i r Z ie ke n h u is G a s t h u i sb er g , L a b o r a t o r iu m D i g es t i e-Absorptie E 462, Herestra at 49, B-3000 Leuven, Belgium (E-mail:[email protected]).

Received 25 J une 1998; accepted in fi na l form 2 Augu st 1999.

REFERENCES

1. Barker, H. A. Amino acid degradat ion by anaerobic bacteria .Ann u. Rev. Biochem. 50: 23 40, 1981.

2. Birkett, A., J . Muir, J . Phillips, G. J ones, and K . ODea.R e sis t a n t s t a r c h low e r s f ec a l c on ce n t r a t ion s of a m m on ia a n dphenols in huma ns. A m . J . C l i n . N u t r . 63: 766772, 1996.

3. Bone, E., A. Tamm, and M. Hill. The production of urina ryphenols by gut bacteria an d their possible role in the caus at ion of

large bowel cancer.A m . J . C l i n . N u t r . 29: 14481454, 1976.4. Chacko, A., and J . H. Cummings. N i t r og e n los s es in t h e

huma n sma ll bowel: obligatory losses a nd t he effect of physicalform of food. G u t29: 809815, 1988.

5. Corpet, D. E., Y. Yin, X. Zhang, C. Remesy, D. Stamp, A.Medline, L. Thompson, W. R. Bruce, and M. C. Archer.Colonic protein fermentation and promotion of colon carcinogen-esis and thermolyzed casein. N utr. Cancer 23: 271281, 1995.

6. Cummings, J . H., and S.A Bingham. Dietary fibre, fermenta-tion and large bowel cancer. Cancer Surv. 6: 601621, 1987

7. Cummings, J . H., M. J . Hill, E. S. Bone, W. J . Branch,andD.J .A. J enkins. The effect of meat protein and dietary fiber oncolonic function a nd metabolism. A m . J . C l i n . N u t r . 32: 20942101, 1979.



9/9

8. E venepoel, P., B. Geypens, A. Luypaerts, M. Hiele, Y.Ghoos, and P. Rutgeerts. Digest ibility of cooked and raw eggprotein, a ssessed by stable isotope techniques. J . N u t r . 128:17161722, 1998.

9. E venepoel, P., M. Hiele, A. Luypaerts, B. Geypens, J .Buyse, E. Decuypere, P. R utgeerts, and Y. Ghoos. Theproduction of egg proteins, enriched with L-leucine-13C 1, for thestudy of protein assimilat ion in huma ns by means of breath testtechnique. J . N u t r . 127: 327331, 1997.

10. Florin,T. H. J .,G. R. Gibson,G. Neale,andJ .H .C ummings.A role for sulfate reducing bacteria in ulcerat ive colit is (Ab-stract) . Gastroenterology98: A170, 1990.

11. Fuller, M. F., A. Milne, C. I . Harris, T. M. S. Reid, and R.Keenan. Amino acid losses in ileostomy fluid on a protein-freediet. A m . J . C l i n . N u t r . 59: 70 73, 1994.

12. Geypens, B., D. Claus, P. Evenepoel, M. Hiele, B. Maes, M.Peeters, P.Rutgeerts, and Y. Ghoos.The influence of dietaryprotein supplements on bacterial colonic metabolism. Gu t 41:7076, 1997.

13. Geypens, B., D. Claus, N. Gorris, P. Evenepoel, P. Rut-geerts, and Y. Ghoos. Determinat ion of total mult i-2H labeledphenol a nd p-cresol in urine to demonstra te protein fermenta tionin ma n (Abstra ct).Proc. Int. Symposium on Capil lary Chromatog-r aphy XX , Ri va del Gar da, I t al y, 1998.

14. Geypens, B., D. Claus, N. Gorris, A. Luypaerts, P. Evene-poel,P. Rutgeerts, andY. Ghoos.Determinat ion of deuteratedphenylalanine and tyrosine in egg protein by GCQ (Abstract) .Pr oc. I nt . Symposi um on Capi l l ar y Chr omat ogr aphy XX , Ri va del

Gar da, I t al y, 1998.15. Ghoos, Y., B. Geypens, B. Maes, M. Hiele, G. Vantrappen,

and P.Rutgeerts. B r e a t h t e s t s in g a s t r ic em p t y in g a n d t r a n s i tstudies: technical aspects of 13C O2-breath tests . In: Progress inU n d er s t a n d i n g a n d M a n a g em en t of G a st r o i n t est i n a l M ot i l i t y

Disorders, edited by J . J anssens. Leuven, Belgium: KU Leuven,1993, p. 169180.

16. Gibson, J . A., G. E. Sladen, and A. M. Dawson. Proteinabsorption and ammonia production: the effects of dietary pro-tein and removal of the colon. B r . J . N u t r . 35: 6165, 1976.

17. Heine,W., K. D. Wutzke, I. Richter,F. Walther,and C. Plath.Eviden ce of colonic absorption of protein nitrogen in infa nts. ActaPaedi atr. Scand. 76: 741744, 1987.

18. J ackson, A. A. Salvage of urea-nitrogen and protein require-ments. Pr oc. Nu tr. Soc. 54: 535547, 1995.

19. Kanawaza, K., F. Konishi, T. Mitsuoka, A. Terada, K. Itoh,S. Narushima, M. Kumemura, and H. Kimura. Factorsinfluencing the development of sigmoid colon cancer. Cancer 77:17011706, 1996.

20. Kayser, B ., K. Acheson, J . Decombaz, E. Fern, and P.Cerretelli. Protein absorption and energy digest ibility a t highalt itude. J . Appl . Physi ol . 73: 24252431, 1992.

21. Koishi, H. N u t r i t ion a l a d a p t a t ion of P a p u a N e w G u in ea h ig h -landers. E u r . J . C l i n . N u t r . 44: 853885, 1990.

22. Lien, K. A., M. I. McBurney, B. I. Beyde, A. B. R. Thomson,and W. C. Sauer. I le a l r e cove r y of n u t r ien t s a n d m u c in inhumans fed total enteral formulas supplemented with soy fiber.A m . J . C l i n . N u t r . 63: 584595, 1996.

23. Macfarlane, G. T., and J . H . C ummings. The colonic flora,fermentat ion, an d lar ge bowel digest ive function. In: T h e L a r g e In testin e: Physiology, Pathophysiology, an d Di sease, e d i t e d b yS. F. Phillips, H. Pemberton, and R. G. Shorter. New York: Raven,1991, p. 5192.

24. Mahe, S., J . Huneau, P.Marteau, F.Thuillier,and D. Tome.G a s t r oi lea l n i t r og e n a n d e le ct r oly t e m ove m en t s a f t e r b ov in emilk ingest ion in hum ans. A m . J . C l i n . N u t r . 56: 410416, 1992.

25. McBurney, M. I., P. J . Van Soest, and J . L. J eraci. Colonic

carcinogenesis : the microbial feast or fam ine mechanism. N u t r.Cancer10: 23 28, 1987.

26. Moran, B. J ., and A.A. J ackson. 15N-urea meta bolism in th efunctioning huma n colon: lumina l hydrolysis and mucosal perme-ability. G u t31: 454457, 1990.

27. Pitcher, M. C., and J . H. Cummings. H y d r og e n s u lfi d e : abacterial toxin in ulcerative colitis? G u t39: 1 4, 1996.

28. R ampton, D. S., N. I. McNeil, and M. Sarner. Analgesicingestion an d other factors preceding relapse in ulcerat ive colitis.

Gu t24: 187189, 1983.

29. Roberfroid, M. B., F. Bornet, C. Bouley, and J . H. C um-mings. Colonic microflora: nutrit ion and health. Nutr. Rev. 53:127130, 1995.

30. R oediger, W. E., A. Duncan, O. Kapaniris, and S. Millard.Reducing sulfur compounds of the colon impa ir colonocyte nut ri-t ion: implicat ions for ulcerat ive colit is . Gastroenterology 104:802809, 1993.

31. Royall, D., T. M. S. Wolever, and K. N. J eejeebhoy. Clinicalsignificance of colonic fermentat ion. Am. J. Gast r oent er ol . 85:13071312, 1990.

32. Sandberg,A.,H . Andersson, B. Hallgren, K. Hasselblad, B.Isaksson, and L. Hulten. Ex pe r im e n t a l m ode l f or in v ivodeterminat ion of dieta ry fi bre and its effects on th e absorption ofnutrients in th e small intest ine.B r . J . N u t r . 45: 283294, 1981.

33. Scheppach, W. Effects of short chain fatty acids on gut morphol-ogy and function.G u t1 ,Suppl .: S35S38, 1994.

34. Silvester, K. R., and J . H . C ummings.Does digest ibility ofmeat protein help explain large bowel cancer r isk?Nutr. Cancer24: 279288, 1995.

35. Simon, G. L., and S. L . Gorbach. Th e h u m a n in t e st in a lmicroflora.Dig. Dis. Sci. 31: 147S 162S, 1986.

36. Stephen,A. M., and J . H. Cummings. The microbial contribu-t ion to human fecal ma ss.J. M ed. M i cr obi ol . 13: 4556, 1980.

37. Stephen, A. M., and J . H. Cummings. Mechanism of action ofdietary fi bre in the human colon.N a t u r e 284: 283284, 1980.

38. Stephen, A. M., H. S.Wiggins, and J . H. Cummings. Effect ofchanging t ran sit t ime on colonic microbial metabolism. Gu t 28:601609, 1987.

39. Visek, W. J . D ie t a n d c e ll g r ow t h m od ula t ion b y a m m on ia .A m . J . C l i n . N u t r . 31: S216 S220, 1978.

40. Wutzke, K. D., W. Heine, M. Friedrich, F. Walther, M.Muller, and E. Martens. Excret ion of 15N and incorporat ionin t o p la s m a p r ot e in s a f t e r h ig h -d os a g e p u ls e la b el in g w i t hv a r iou s t r a c e r s u b s t a n c es in in f a n t s . C l i n . N u t r . ( P h i l a . ) 41:431439, 1987.


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