weeks of balance in one patient taking PEG. In six was made. During this period the administration of

Transcription

1 Gut, 1971, 12, Polyethylene glycol 4 s continuously dministered non-bsorbble fecl mrker for metbolic blnce studies in humn subjects R. WILKINSON From the Medicl Reserch Council Minerl Metbolism nit, The Generl Infirmry, Leeds SMMARY The use of polyethylene glycol 4 s non-bsorbble, continuously fed fecl mrker is described. The intestinl hndling nd. trnsit of polyethylene glycol is compred with tht of chromium sesquioxide nd brium sulphte. Polyethylene glycol is n cceptble non-bsorbble fecl mrker for clcium, phosphorus, nd ftty cids, nd hs severl fetures which commend its use in preference to insoluble chromium sesquioxide nd brium sulphte prticulrly when mrking wter-soluble dietry constituents. Kreul (1947) ws the first to crry out continuous fecl mrking of metbolic blnce investigtions with chromium sesquioxide (Cr23) nd he hs been followed by mny workers including Whitby nd Lng (196) nd Rose (1964). Brium sulphte (BSO4) hs been used s non-bsorbble mrker (Dick, 1967; Figuero, Jordn, nd Bssett, 1968) nd more recently Dick (1969) hs used cuprous thiocynte. All these uthors hve shown n increse in the reproducibility of blnce dt due to more ccurte ssessment of the fecl component using continuously fed non-bsorbble mrker. Polyethylene glycol 4 (PEG) hs been employed s non-bsorbble fecl mrker in niml studies (Hyden, 1956; Corbett, Greenhlgh, Gwynn, nd Wlker, 1958; Mnners nd Kidder, 1968). Only one report of its use in mn for this purpose cn be found in the literture but no ttempt ws mde to vlidte the use of PEG, since totl fecl recoveries were not reported (Beeken, 1967). The present pper reports the results of using PEG s continuously fed non-bsorbble fecl mrker for clcium nd phosphorus blnces. A comprison of PEG with Cr23 nd BSOg is presented. The use of PEG to correct fecl ftty cid output is lso described. Procedure ptient te the sme norml diet every dy throughout the study. The study ws designed to give n equilibrtion period of five or six dys on diet nd mrkers followed by two consecutive seven-dy periods of blnce. The ptients defected directly into stinless steel continers held in suitble commode. The feces pssed in the equilibrtion period were pooled nd then two seven-dy fecl collections of the blnce were mde. Toilet pper ws not collected. Dily fecl collections were mde for the two weeks of blnce in one ptient tking PEG. In six ptients tking PEG post-blnce fecl collection ws mde. During this period the dministrtion of mrkers ws discontinued nd the ptients te free diet. Feces were collected until the PEG ws completely excreted. Eleven ptients were given both Cr2O, nd PEG throughout their study nd nother 11 ptients were given both Cr23 nd BSO4. One dditionl ptient took ll three mrkers simultneously. In the second prt of the study the recoveries of PEG in seven-dy period of blnce following seven-dy period of equilibrtion on diet nd PEG re reported. There re 165 observtions on 64 ptients in this prt of the study. In ll cses 5 mg of the pproprite mrkers, in cpsules, ws dministered three times dy with All the ptients were in metbolic wrd nd the the min mels during the equilibrtion nd blnce generl principles of Reifenstein, Albright, nd periods. Wells (1945) were observed. Diets were prepred Polyethylene glycol 4 (Hopkin nd Willims under the supervision of dietitin nd ny given Ltd, Chdwell Heth, Essex) ws powdered nd Received for publiction 27 My filled into cpsules by hnd to 5 mg ± 5 mg. For '54 Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.

2 Polyethylene glycol 4 s continuously dministered non-bsorbble fecl mrker in humn subjects 655 the second prt of the study specil btch of PEG cpsules ws obtined from Sndoz Products Ltd (London). Since PEG is not definite chemicl compound but mixture of polymers with slightly different moleculr weights, the reltive proportions vrying with the btch mnufctured, the stndrds were mde from the sme btch of PEG tht ws used to prepre the ptients' cpsules. Cre ws tken over the mediction of ptients on blnce becuse PEG is used in certin orl phrmceuticl formultions. Brium sulphte BP ws lso hnd-filled into cpsules to the sme tolernce limits s PEG. Chromium sesquioxide cpsules were obtined from Sndoz Products Ltd (London). Anlyticl Methods Feces were homogenized in the collection continer with three times their weight of deionized distilled wter using Silverson AXR homogenizer. Whilst the homogenizer ws still running liquots of the homogente (weighed on Slter top-pn blnce.1 g) were tken for the pproprite nlyses. The digestion nd oxidtion steps in the estimtion of chromium sesquioxide were crried out, with suitble sfety precutions, using the method of Dly nd Anstll (1964) The resulting dichromte solution ws determined colorimetriclly ccording to the method described by Clrkson (1967). The grvimetric method of Dick (1967) ws used to estimte BSO4. Filtrtion of the BSO4, dissolved in lkline disodium ethylenedimine tetr-cetic cid solution through two sheets of Whtmn no. 115 filter pper, removed Cr23 if it ws lso present in the feces. The turbidimetric method of Mlwer nd Powell (1967) ws modified to estimte PEG on 1. g liquots of fecl homogente. The use of Whtmn no. 542 filter pper for the filtrtion stge fter protein precipittion removed ny Cr23 or BSO4 present in the feces nd produced cler, colourless filtrte. Since there ws no difference between the estimte of PEG stndrds in feces nd PEG stndrds in wter the ltter were used routinely. The removl of the fine BSO4 precipitte from the stndrd solutions fter the protein precipittion stge often required the use of two filter ppers. Clcium nd phosphorus were determined by AutoAnlyzer techniques on shed liquots of fecl homogente tken up in cid. Totl fecl ftty cids were estimted ccording to modifiction of the method of vn der Kmer, Huinik, nd Weyers (1949) s reported by Vrley (1962). Anlyses of ll mrkers, clcium, inorgnic phosphorus, nd ftty cids were the men of duplicte estimtions. Results RECOVERIES OF MARKERS FROM FAECES The recoveries of Cr2,O, BSO4, nd PEG dded directly to mrker free feces re shown in Tble I. None of the mrkers interfered with the recovery of ny of the other two mrkers nd zero recoveries of ll three mrkers were obtined from six fecl smples contining no mrkers. Mrker Men SD (%) No. of Recovery Estimtions Chromium sesquoxide Brium sulphte Polyethylene glycol Tble I Recoveries ofmrkers dded directly to feces The error of the nlyticl method, for ech of the three mrkers, is shown in Tble II. If x1 nd x2 Mrker Error of No. of Error of Single Smple Mrker Estimte of Estimtes in Method when Mrker Duplicte Men of Duplicte Estimtions sed Chromium sesquioxide Brium sulphte Polyethylene glycol Tble II Error of mrker determintions represent duplicte estimtions of the percentge of the dministered dose of mrker in given fecl collection then the error of single estimte of the mrker,, is given by L7(Xl - x2)2 V2n where n is the number of collections estimted in duplicte. The error of the mrker determintion when the men of duplicte estimtions is used is 1V2. RECOVERIES OF MARKERS FROM PATIENTS Tble III shows the totl fecl recoveries of ll the PEG given throughout the study from the six ptients on whom post-blnce fecl collections were mde. There is men fecl recovery of 98.65% of the dministered PEG, the rnge being 95.5 %-11 5 %. A histogrm distribution of the individul seven-dy fecl recoveries of PEG in the second prt of the study is shown in Figure 1. The recovery of PEG is normlly distributed nd hs men of 99.29% of the dministered dose, which is Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.

3 656 Ptient Men SD 268 Percentge oj Totl Administered Dose of PEG Recovered from Feces Tble III Totl fecl recovery of polyethylene glycol 4 dministered to ptients not significntly different from 1% (t = 386). PEG, therefore, is completely recoverble in the feces nd is being estimted ccurtely. The reltionship of the fecl recoveries of Cr23 nd PEG for the 12 ptients given these two mrkers should be evenly distributed round the 45 line of equlity if they re excreted t the sme rte. However, in the equilibrtion period more PEG is excreted thn Cr23 the points ll lying below the 45 line (Fig. 2). In week 1 the points still tend to be slightly below the expected line nd it is not until week 2 tht fecl Cr23 output equls fecl PEG output. Figure 3 shows the corresponding dt for Cr23 nd BSO4. Slightly more BSO4 thn Cr23 is excreted in the equilibrtion period, but probbly by week 1 nd definitely by week 2 the fecl outputs of Cr23 nd BSO4 re equl. The interreltionship of fecl output nd trnsit time of the mrkers is well illustrted by the results from the ptient who took ll three mrkers simultneously (Fig. 4). In the equilibrtion period more PEG is excreted thn BSO4, nd more BSO4 is excreted thn Cr23. In week 2 of the blnce the fecl excretions of ll the mrkers re the sme, the lines in Fig. 4 being lmost prllel. Five dys fter discontinuing mrker dministrtion ll the PEG is completely recovered. However, two dys fter this BSO4 nd Cr23 re still being excreted in the feces nd hve not yet been completely recovered. PEG CORRECTION OF FAECAL CALCIM, PHOS- PHORS, AND FATTY ACID OTPTS Figure 5 shows the dily fecl clcium nd phosphorus outputs of the one ptient on whom dily fecl collections were mde throughout the two weeks of blnce. Knowing the PEG content of ech smple, PEG-corrected 24-hour fecl clcium nd phosphorus vlues were clculted. In contrst to the uncorrected vlues the PEG-corrected dily fecl clcium nd phosphorus vlues re virtully constnt. Similrly the dily fecl ftty cid outputs of ptient on eight consecutive dys of blnce re 2 = S r m MEAN 99,29% 7 DAY RECOVERY OF PEG (C' of Administered Dose) R. Wilkinson Fig. 1. Histogrm showing distribution of seven-dy fecl recoveries ofpeg expressed s percentge of the dose dministered in seven dys. The ptients were equilibrted before the fecl collection ws begttn. Men recovery ofpeg = % (SD = 23S57%, SEM = 1.84 %).." LW cm z v. hi O. 1 V* 1 EQILIBRATION PERIOD WEEK 1 WEEK 2 PERCENTAGE OF ADMINISTERED DOSE OF PEG IN FAECES. Fig. 2. Comprison of the fecl recoveries of Cr2O, nd PEG in the equilibrtion period nd weeks 1 nd 2 of blnce. (The dotted line is the 45 line of equlity.) Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.

4 Polyethylene glycol 4 s continuously dministered non-bsorbble fecl mrker in humn subjects 657 W ui z CV ' o C In ox 4 b- z u. 11.., ~~~ 1 PERCENTAGE OF ADMINISTERED DOSE OF BSO4 IN FAECES. Fig. 3. Comprison of the fecl recoveries nd BSO4 in the equilibrtion period nd wi of blnce. (The dotted line is the 45 line oj EQILIBRATION PERIOD shown in Figure 6. There is much less vrition in the PEG corrected thn in the uncorrected fecl ftty cid vlues. Discussion All three mrkers were well tolerted by the ptients nd no toxic effects were observed. None of the 76 ptients tking PEG showed ny side effects ttribut- WEEK I ble to the dministrtion of this mrker. The men recovery of 99.6% of PEG dded directly to feces is comprble to tht of Cr23 nd BSO4 (Tble I). The errors of ll the methods of nlysis (Tble II) re cceptble nd similr to those published (Whitby nd Lng, 196). The turbidimetric estimtion of PEG is ccomplished esily nd ccurtely with commonly vilble equipment nd there is no dnger of n explosion in the estimtion of PEG s there is in the nlysis of Cr23 (Anlyticl Methods Committee, 1959). The PEG method is less lborious nd excting thn the WEEK 2 grvimetric determintion of BSO4, though recently Dick (1968) hs reported n emission flme photometric method which reduces the time tken for BSO4 estimtions. Schffer nd Critchfield (1947) concluded tht PEG ws not bsorbed from the gstrointestinl trct of rt or mn nd PEG is the most populr inert mrker used for intubtion studies (Soergel, 1968). Men fecl recoveries of between 96.7 % nd 99. % hve been reported fter the ingestion of lrge single doses of PEG by humn subjects (Hyden, 1956; Borgstrom, Dhlqvist, Lundh, nd Sjovll, v of Cr2O3 1957; Soergel nd Hogn, 1967; Shields, Hrris, nd eeks 1 nd 2 Dvies, 1968). requlity.) The men totl fecl recovery of 98.65% of the Gut: first published s gut on 1 August Downloded from ir. E V _ EQILIRATION M BWKALANCE BALANCE POST BALANCE usperiod WEEK 1 WEEK 2 PERIOD ~E MARKER ADMINISTRATION ~ ~ ovc >*1.5G T.D.S. ] 2 25 Fig. 4. Cumultive fecl recoveries of mrkers from the ptient given ll three mrkers simultneously. on 16 Jnury 219 by guest. Protected by copyright.

5 658 I. 9v NCORRECTED DAILY FAECAL CALCIM OTPT 3, PEG CORRECTED DAILY FAECAL CALCIM OTPT fz * 1 4. v4 Fig. 5. 4K I- 9-. I.i -X Fig. 6. F! 1n R. Wilkinson NCORRECTED DAILY FAECAL PHOSPHORS OTPT DAILY FAECAL PHOSPHORS OTPT Dily fecl clcium nd phosphorus excretion for 14 dys ofblnce with nd without PEG correction. I NCORRECTED FAECAL FATTY ACIDS 4C 3-2.C 1* j v, g^ c _i_. ~.1K 21@ 3 b PEG CORRECTED FAECAL FATTY ACIDS Dily feclft excretionfor eight consecutive dys of blnce with nd without PEG correction. b Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.

6 Polyethylene glycol 4 s continuously dministered non-bsorbble fecl mrker in humn subjects 659 dministered PEG (Tble III) is comprble to the totl recovery of 93 % of the dministered Cr23 by Rose (1964) nd 97% of the dministered BSO4 by Dick (1967). In the second prt of the study the norml distribution of PEG recovery hs men of 99.29% of the dministered dose (Fig. 1). These results substntite the previous observtions tht PEG is not bsorbed from the humn gstrointestinl trct. In view of the bove findings it is perhps surprising tht Beeken (1967) reported stedy-stte recoveries of PEG of only 7%. Beeken suggests s one of the possible resons for this low recovery tht his subjects my hve filed to tke the PEG. Also some of the equilibrtion periods in the study were s short s three dys. It is pprent from Figs. 2, 3, nd 4 tht PEG hs more rpid intestinl trnsit time thn BSO4 which in turn hs slightly fster trnsit time thn Cr,3. These differences re most esily observed in the equilibrtion nd post-blnce collection periods. However, in week 2 of the blnce the excretions of PEG, Cr23, nd BSOg re the sme. The results could be explined if there is lrger ded spce in the intestinl trct for Cr23 nd BSO4 thn for PEG tht must be filled before stedy-stte excretion is ttined. The difference between ded spces my be due to the fct tht PEG is wter soluble wheres BSO4 nd Cr23 re insoluble nd considerbly more dense thn food (the density of Cr23 ws 5.21 nd of BSO4 4.5). PEG will move with the wter-soluble phse of food nd this hs n dvntge in mrking clcium nd phosphorus since both re wter soluble. However, PEG my be lbelling wter-soluble component of the ded spce which is smller thn the true ded spce for food in the intestine. Alterntively the ded spce for Cr23 nd BSO4 my be greter thn the true ded spce for food. The very fine, dense, insoluble powders of Cr23 nd BSO4 tend to dhere to ny surfce on which they re spred. The lrge surfce re nd mny smll villous folds of the intestinl trct my trp the insoluble mrkers producing lrger ded spce for Cr23 nd BSO4 thn for either food or wtersoluble PEG. Dvignon, Simmonds, nd Ahrens (1968) hve shown tht 2% of their ptients on prolonged blnce studies filed to ttin idel stedy-stte excretion of Cr23. They demonstrted tht some of these ptients hd sequestrtion nd ccumultion of Cr23 in the lrge bowel, supporting the hypothesis tht Cr2O3 is not lwys hndled in the sme wy s food residues by the intestine. Fifteen of 52 blnces reported by Whitby nd Lng (196) were not cceptble becuse the ptients hd not reched stedy stte excretion with respect to Cr23. Whitby nd Lng lso wrned of the possibility of streming of the intestinl contents occurring nd under these conditions the density of Cr23 might cuse it to sediment nd be crried irregulrly long the intestinl lumen. Sequestrtion is less lible to occur with PEG thn with insoluble Cr23 or BSO4 nd streming would not cuse wter-soluble PEG to sediment. The wide vritions of consecutive dily fecl clcium nd phosphorus outputs re virtully eliminted when corrected by their respective PEG content (Fig. 5). Since the clcium to PEG nd the phosphorus to PEG rtio in these fecl smples is constnt then PEG is evenly distributed in the feces with respect to clcium nd phosphorus. Wiggins nd Dwson (1961) showed tht PEG being wter soluble emptied more quickly from the stomch in the wter-soluble phse thn did the ft of test mel. Vvfinkov nd Krondl (1965) reported tht PEG, t concentrtions of 1 mgml or greter, inhibited lipse from hog pncres nd humn duodenl juice in vitro nd therefore PEG my not be suitble for the investigtion of ft bsorption. No increse in the fecl ftty cid excretion of ptients tking PEG hs been observed nd fter suitble equilibrtion period PEG cn be used to correct dily fecl ftty cid output with some success (Fig. 6). Thus PEG, lthough wter soluble, is reltively evenly distributed in the feces with respect to ftty cids. To reduce ny possible effect on lipse ctivity in vivo the dose of PEG cn be decresed to 25 mg three times dy. The lower concentrtion of PEG cn be estimted by reding the turbidity t 42 mu s suggested by Boulter nd McMichel (197). The criteri for the idel non-bsorbble mrker stted by Whitby nd Lng (196) nd Fordtrn (1966) re fulfilled eqully well by PEG s by the estblished mrkers Cr23 nd BSOQ. In fct PEG hs fetures which commend its use in certin circumstnces. In contrst to the estimtion of Cr23 the estimtion of PEG is not hzrdous. Becuse of its smller intestinl ded spce equilibrtion is chieved more quickly with PEG thn with the two insoluble mrkers nd this llows more rpid ssessment of the blnce. Being wter soluble PEG more closely resembles clcium, inorgnic phosphte, nd other ions thn do insoluble Cr23 nd BSO4. The lrge spred of the seven-dy fecl PEG outputs in Fig. 1 gin emphsizes the bsolute necessity for continuous fecl mrking in metbolic blnce investigtions. The 95 % limits for sevendy timed fecl collection vry from the equivlent of 3.7 dys to 1.3 dys excretion of food residue. PEG is recommended s non-bsorbble con- Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.

7 66 R. Wilkinson tinuously fed fecl mrker prticulrly for dietry constituents which re wter soluble. The uthor wishes to cknowledge the interest nd dvice of Professor B. E. C. Nordin nd to thnk him for permission to publish detils of studies on ptients under his cre. My thnks go to Dr Munro Pecock nd Dr Jon Znelli for dvice in the preprtion of the mnuscript, to L. Bulusu for dvice on sttisticl spects of the work, to the nursing nd dietetic stff of the metbolic units t Leeds Generl Infirmry, to the stff of the phrmcy for the hnd preprtion of the PEG nd BSO4 cpsules used in the initil stges of the work, nd to Miss S. Brker, Miss W. Oldfield, M. Youssf Khn, nd T. Thompson for vluble technicl ssistnce during prts of this work. I m lso indebted to Sndoz Products Ltd (London), for prepring the cpsules of PEG used in the second prt of the study. References Anlyticl Methods Committee (1959). Notes on perchloric cid nd its hndling in nlyticl work. Anlyst, 84, Beeken, W. L. (1967). Clernce of circulting rdiochromted lbumin nd erythrocytes by the gstrointestinl trct of norml subjects. Gstroenterology, 52, Borgstrdm, B., Dhlqvist, A., Lundh, G., nd Sjovll, J. (1957). Studies of intestinl digestion nd bsorption in the humn. J. clin. Invest., 36, Boulter, J. M., nd McMichel, H. B. (197). Modifiction of polyethyleneglycol estimtion suitble for use with smllmmmls. Gut, 11, Clrkson, E. M. (1967). A rpid method for the determintion of chromium sesquioxide in fecl homogentes. Clin. chim. Act, 16, Corbett, J. L., Greenhlgh, J. F. D., Gwynn, P. E., nd Wlker, D. (1958). Excretion of chromium sesquioxide nd polyethyleneglycol by diry cows. Brit. J. Nutr., 12, Dly, J. R., nd Anstll, H. B. (1964). The determintion ofchromium sesquioxide in feces by flme spectrophotometry. Clin. chim. Act, 9, Dvignon, J., Simmonds, W. J., nd Ahrens, E. H., Jr. (1968). sefulness of chromic oxide s n internl stndrd for blnce studies in formul-fed ptients nd for ssessment of colonic function. J. clin. Invest., 47, Dick, M. (1967). se of brium sulphte s continuous mrker for feces. J. clin. Pth., 2, Dick, M. (1968). Estimtion of brium sulphte in feces by emission flme photometry. J. clin. Pth., 22, 378. Dick, M. (1969). se of cuprous thiocynte s short-term continuous mrker for feces. Gut, 1, Figuero, W. G., Jordn, T., nd Bssett, S. H. (1968). se of brium sulfte s n unbsorbble fecl mrker. Amer. J. clin. Nutr., 21, Fordtrn, J. S. (1966). Mrker perfusion techniques for mesuring intestinl bsorption in mn. Gstroenterology, 51, Hyden, S. (1956). The recovery of polyethylene glycol fter pssge through the digestivetrct.k. LntbrH6gsk. Annin., 22, Kreul, M. S. (1947). Absorption of crotene from crrots in mn nd the use of the quntittive chromic oxide indictor method in the bsorption experiments. Biochem. J., 41, Mlwer, S. J., nd Powell, D. W. (1967). An improved turbidimetric nlysis of polyethylene glycol utilizing n emulsifier. Gstroenterology, 53, Mnners, M. J., nd Kidder, D. E. (1968). Polyethylene glycol s mrker in piglet diets with high dry-mtter content. Brit. J. Nutr., 22, Reifenstein, E. C., Jr., Albright, F., nd Wells, S. L. (1945). The ccumultion, interprettion nd presenttion of dt pertining to metbolic blnces, notbly those of clcium, phosphorus nd nitrogen.j. clin. Endocr., 5, Rose, G. A. (1964). Experiences with the use of interrupted crmine red nd continuous chromium sesquioxide mrking of humn feces with reference to clcium, phosphorus nd mgnesium. Gut, 5, Schffer, C. B., nd Critchfield, F. H. (1947). The bsorption nd excretion ofthe solid polyethylene glycols. J. Amer. phrm. Ass. sci. Ed., 36, Shields, R., Hrris, J., nd Dvies, M. W. (1968). Suitbility of polyethylene glycol s dilution indictor in the humn colon. Gstroenterology, 52, Soergel, K. H. (1968). Inert mrkers. Gstroenterology, 54, Soergel, K. H., nd Hogn, W. J. (1967). On the suitbility of poorly bsorbed mrkers s dilution indictors in the gstrointestinl trct. Gstroenterology, 54, Vrley, H. (1962). Prcticl Clinicl Biochemistry, 3rd Ed., p Heinemnn, London. Vvfinkov, H., nd Krondl, A. (1965). se of polyethylene glycol in investigtion of bsorion of ft. Nture, 26, Whitby, L. G., nd Lng, D. (196). Experience with the chromic oxide method of fecl mrking in metbolic blnce investigtions on humns. J. clin. Invest., 39, Wiggins, H. S., nd Dwson, A. M. (1961). An evlution of unbsorbble mrkers in the study offt bsorption. Gut, 2, Gut: first published s gut on 1 August Downloded from on 16 Jnury 219 by guest. Protected by copyright.