phosphorus, and magnesium

Transcription

1 Gut, 1964, 5, 274 Experiences with the use of interrupted crmine red nd continuous chromium sesquioxide mrking of humn feces with reference to clcium, phosphorus, nd mgnesium G. ALAN ROSE From the Metbolic Unit, Deprtment of Medicine, The Generl nfirmry Leeds The norml dult in clcium blnce excretes some 8% of the intke in the stools. This is in contrst to the excretion of sodium, potssium, nd nitrogen which is predominntly in the urine. t follows tht wheres inccurcies in mesurement of fecl sodium, potssium, nd nitrogen my not seriously ffect overll blnce results, similr inccurcies re not permissible in clcium blnce studies. rregulrities in bowel hbits mke 24-hour collections of stools lmost meningless in the mjority of ptients, nd to overcome this difficulty Albright nd his collbortors therefore utilized crmine red fecl mrkers given orlly every few dys so s to permit timing of the feces (Reifenstein, Albright, nd Wells, 1945). Since then, most investigtors hve used this principle, lthough occsionlly different mrker dyes hve been used nd periods hve vried from three to seven dys. However, this technique is subject to certin prcticl difficulties. First, collections of feces re known never to be complete becuse of loss on toilet pper nd in generl mnipultion. Secondly, other unknown losses my occur through ccidentl rejection of stools (n event which my occur even in the best regulted of metbolic units). Thirdly, the mrker dye is not lwys clerly visible nd is not lwys discretely loclized. n ddition there re theoreticl objections to the use of these mrkers t ll. Humphreys nd Scott (1962) showed tht nylon blls nd chromium sesquioxide seprted on pssge through the limentry trct of the ct. Wiggins nd Dwson (1961) showed tht stndrd test mel, which contined phenol red, dissocited in the stomch into solid phse rich in ft nd poor in mrker, nd liquid phse poor in ft nd rich in mrker. The liquid phse ws preferentilly emptied from the stomch. McSwiney (personl communi- 274 ction) gve crmine red nd chrcol mrkers 12 hours prt nd observed them in the stools in the reverse order on ech of the three occsions when this ws tried. Most investigtors when discussing the use of crmine red mrking of stools will dmit tht fecl results so obtined often vry remrkbly from period to period. Severl groups hve ttempted to improve the ccurcy of fecl collections by using continuous fecl mrking with chromium sesquioxide. The most extensive study in humns yet reported ws by Whitby nd Lng (196) who studied 4 individuls, using three-dy fecl periods without the use of intermittent dye mrkers. They observed no side effects from the use of the chromium sesquioxide nd found tht they could correct for irregulrities in bowel ction nd for incomplete fecl collections. The pprent dvntges of continuous chromium mrkings re impressive nd this procedure ws therefore dopted in this unit in Jnury The interrupted crmine red technique offers one cler dvntge however. When there is sudden chnge in diet (or therpy) it is desirble to divide the stools into pools corresponding to the old nd the new diets (or therpy) respectively, s otherwise the results my be 'blurred'. We hve therefore used both continuous chromium nd interrupted dye mrkers in combintion. Over 1 six-dy blnce periods hve now been completed nd the first 13 results from 21 studies on 18 ptients re reported here. Exmintion of the results revels for the first time to wht extent the ' six-dy' fecl collection of the crmine red technique vries from true six-dy fecl collection. n three ptients, ech individul stool ws nlysed for chromium, clcium, nd phosphorus (results for Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.

2 ft re to be reported elsewhere), nd in two of these cses rndom smples of ech stool were lso nlysed for the sme components. t is therefore possible to ssess, first, to wht extent fecl losses cn be ssessed by recovery of chromium, nd secondly whether the smll rndom smple is representtive of the homogenized stool with regrd to chromium, clcium, nd phosphorus (nd ft). METHODS Methods nd techniques All the ptients were in metbolic unit nd the generl principles of Reifenstein et l. (1945) were observed. Any given ptient te the sme norml diet every dy throughout his study period. The clcium content of this diet mtched, s closely s could be scertined by dietitin, the ptient's own norml intke of clcium. When the diet ws commenced, cpsules contining 5 g. chromium sesquioxide (1-25 g. per dy of chromium) were strted, nd given thrice dily until the study ended. These cpsules were prepred nd dministered exctly s described by Whitby nd Lng (196) except tht two children of 11 yers (cses 16 nd 18 of Fig. 3) were given only hlf doses of chromium. After five or six dys of equilibrtion on the diet, the first crmine red mrker ws given t brekfst time, nd this ws repeted every six dys until the study ended. Stools were homogenized in six-dy periods s indicted by the beginning of the crmine mrkers, except when individul stools were nlysed, when these were homogenized individully (fter removl of smll rndom smples in some cses). Homogenized nd rndom smples of stool were dried t 1 C. nd shed t 6 C. for 12 to 16 hours. The chromium sesquioxide ws oxidized with perchloric cid nd ssyed by the flme photometric method of Anderson nd Weinbren (1961). Clcium ws determined on the sme oxidized shings using the flme photometric technique of Mclntyre (1957) with only minor modifictions. The Zeiss PMQ spectrophotometer with flme ttchment ws used throughout for the clcium nd chromium estimtions. Phosphorus ws mesured in the sme shings on the Technicon Auto- Anlyzer by the method of Fiske nd Subbrow (1925). Mgnesium ws mesured on seprte smple of homogente by Dr. F. W. Heton of the Deprtment of Urology, Leeds Generl nfirmry, using tomic bsorption spectrophotometry s described by Dwson nd Heton (1961). n order to see whether results were more consistent by correcting for chromium content of the 'six-dy' fecl collection, ll other results were multiplied by the fctor: chromium given in six dys/chromium found in 'six-dy' fecl collection. RESULTS When chromium sesquioxide ws dded to stools lredy in the homogenizer nd immeditely before homogeniztion so s to exclude losses due to.1 z / recovery of Cr FG. 1. Histogrm of overll recoveries of chromium in the 21 studies. mnipultion, the recoveries of chromium on four seprte dditions were respectively 976%, %, nd 98.4%. The totl chromium recovery in the 21 studies ws 578 g. or 93.% of the 621 g. dministered. The minimum totl recovery in ny study ws 8 % nd the mximum totl recovery in ny study ws 13 %. The distribution of these totl recoveries is shown in Figure 1. The chromium recoveries in ech of the 13 six-dy fecl collections re shown in Figures 2 nd 3. Figure 2 shows the overll distribution of the recoveries nd Fig. 3 shows recoveries in the 21 individul studies on the 18 ptients. Figure 4 shows the fecl results from one ptient both with nd without correction (see bove) by the Z A 6-dy Cr recovery Cg.) 275 FG. 2. Histogrm of chromium recoveries in 13 consecutive six-dy periods. n ech six-dy period 616 g. of chromium ws given, except tht the two children (nos. 16 nd 18) received hlf this dose. Results from these children hve been multiplied by 2 to mke them comprble with the other dt. Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.

3 276 ' CP E (E 1 * 2 1? CX2) (X2) CASE NUMBER FG. 3. The sme dt s in Fig. 2 but re-rrnged to show the vritions in chromium recoveries in ech ptient. chromium method. These results re selected for illustrtion becuse this ptient (no. 8) pssed very constipted nd drk stools so tht crmine red mrkers were difficult to see, nd the chromium contents of the 'six-dy' stools were the most irregulr tht we encountered (see Fig. 3). Results of individul stool nlysis on ptient E.Wi. re shown in Figure 5. Results of nlysis of frctions of stools from ptients A.R. nd E.McC. re shown in Figs. 6 nd 7 respectively. DSCUSSON The chromium sesquioxide cpsules were tolerted well by the ptients, whether dults or children, nd no side effects hve been observed. The stools were generlly olive green in colour, ginst which bckground the crmine red could usully be redily identified. Since some feces is lwys lost on the toilet pper, nd some is lso lost in trnsfer from the cellophne wrpper to the homogenizer, chromium recoveries of 1 re not to be expected. The overll chromium recovery of 93 must therefore be considered stisfctory. On most ptients the overll chromium recovery ws 9 to 1% (see Fig. 1) but on few ptients the recovery dropped to s low s 8%. n some of these cses, the low recoveries could be ttributed to the short durtion of the studies (12 dys only in some cses) but in some other cses this explntion ws not pplicble nd it is believed tht there must hve been fecl losses which would not otherwise hve been recognized. Tken with the other evidence reveled below, n verge loss of 7 % of chromium must indicte corresponding 7 % loss of fecl clcium, phosphorus, nd mgnesium. This mens tht fecl mesurements of clcium on norml clcium intke my be 5 to 1 mg. dy too low when uncorrected for chromium. On high i -- G. Aln Rose c 2n t. 1s v T r r 8 t 2, o ^ -B _ O Totl chromium Totl chromium given 67-8g. recovered 64-1g. or 94-6% o * ' FEB MARCH APRL 'o sin z o ' 6 oo1 o' 2 o %fi 1-4 % l 11 vb -41 therpy A *. * therpy B ,&A 1±i * t i di e lb Lgcl 1Q LJ cv c FEB MARCH APRL 1962 FG. 4. Results of 'six-dy' fecl collections from ptient no. 8 A with, nd B without, correction for chromium recoveries. Results on this constipted ptient re quite vlueless when uncorrected for chromium, but when the corrections re mde it is seen tht chnges in fecl clcium re referble to chnges in therpy. Note especilly the constncy of the fecl mgnesium, this being uninfluenced by the therpy. c Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.

4 E 1.5 %i ',, 2-- * 1. Methods nd techniques,jk NOV. DEC FG. 5. ndividul stool nlyses on ptient E. Wi. Note how during preliminry period without therpy the fecl clcium nd chromium rise nd fll together so tht the clcium per dy's chromium intke is nerly constnt. The rise in fecl clcium due to the intrvenous clcium therpy, lthough quite smll, cn be clerly seen. '. v ' u S * i~ :4-2 O- Totl fiiecl U Totl fccl /dily Cr.1 1O S Totl Totl fccl fccl / dily Cr =AU. Homogente Rndom smfcel / ple fccl / dily Cr dily Cr m_ STOOL NO. FG. 6. From ech stool rndom smple ws tken for nlysis for clcium, phosphorus, nd chromium. A lrger smple ws nlysed fter homogeniztion, nd the reminder lso nlysed fter homogeniztion. Totl results on ech stool were then obtined by pproprite dditions. Only six stools were pssed by this constipted ptient in 12 dys despite the liberl use ofpurgtives. No other therpy ws given throughout. is 5 STOOL NO. Homogente fecl / dily Cr FG. 7. The stools were treted substntilly s is described Figure 6. This ptient pssed 16 stools in 3 dys. i -i-j -L Rndom smple fccl / dily Cr in the legend to 277 Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.

5 278 clcium intke of 3 g. dy, s recommended by some for osteoporosis (see Lncet, ,538), the error would be 21 mg. dy, giving flse hopes of cure. Figure 3 shows tht while the mjority of the sixdy fecl collections contined weight ofchromium resonbly ner to the six-dy intke, some six-dy collections contined s little s two-dy intke, nd others the equivlent of up to n 1-dy intke. n some cses (s in no. 8) such lrge vritions could hve been nticipted becuse of constipted, infrequent, nd irregulr bowel ctions, but in other cses surprising vritions occsionlly occurred despite regulr dily bowel ctions with well-formed stools in which the crmine mrkers were clerly visible. When such vritions occurred, there were lso irregulrities in the fecl clcium, phosphorus, nd mgnesium levels (see Fig. 4) nd these irregulrities lrgely disppered when results were corrected for chromium content. t is concluded tht while the crmine mrking of stools gives nerly constnt chromium content in few ptients, definite nd significnt vritions occur in most ptients nd very lrge vritions in smll number of ptients. Figure 5 shows how when individul stools were nlysed for clcium nd chromium, the two elements pprently followed one nother remrkbly closely so tht when clcium in ech stool is expressed per dily chromium intke, there is remrkbly little vrition from stool to stool. Agreement is so close in fct tht smll rise in fecl clcium following dily intrvenous clcium infusions cn be redily identified. Similrly, the speed of the effect of vitmin D on fecl clcium is redily observed. t is therefore concluded, first, tht ny one stool nlysed for clcium nd chromium will give n ccurte indiction of the dily fecl clcium, nd secondly, tht the ccidentl loss of complete stool during 'six-dy' collection would be precisely indicted by corresponding fll in chromium, the system of correcting clcium results for chromium recovery being vlid one. Two other ptients were studied in similr wy but in greter detil. The results in ptient Alfred R. re shown in Figure 6. t is pprent tht the clcium nd phosphorus hve followed the chromium so closely tht consistent results re obtined from nlysis of smll rndom smple of ech stool. The smple need not even hve been weighed, but merely shed nd nlysed for clcium, phosphorus, nd chromium. nspection of Fig. 6 shows the remrkble fct tht this procedure gve even more consistent results thn nlysis on homogenized reminders of stools. This must men tht the stools were homogeneous before tretment in the G. Aln Rose homogenizer, nd tht the homogeniztion procedure ws in fct centrifugl seprtion. The results in ptient E.McC. shown in Fig. 7 were slightly different. First, the results, corrected for chromium, were not quite s consistent from dy to dy, either in the rndom smples of stools or in nlyses of whole stools. Secondly, the dy-to-dy vritions were smller in the nlyses of whole stools thn in the rndom smple nlyses. Nevertheless, the nlysis of rndom smple of stool for clcium nd chromium seems to hve provided wy of clculting the dily fecl clcium to within 1 mg. While occsionl nlysis of rndom smple of stool for chromium, clcium, nd phosphorus would provide very rpid nd lboursving method of clculting fecl clcium nd phosphorus, it is not suggested here tht this is the technique of choice for ccurte blnce work. For such work it still seems desirble to collect ll the stools nd pool severl dys' stools. t is strongly suggested on the other hnd, tht the loss of stools, whether on toilet pper, or in mnipultion, or from ccidentl rejection of stool, will led to no significnt error provided the results re corrected for the chromium content of the stools. Aprt from the gin in ccurcy nd consistency using the continuous chromium mrking technique, other dvntges cn be derived. First, the six-dy blnce period used here could be shortened to four-dy period without significnt loss of ccurcy. Since it hs been our prctice generlly to crry out two or three six-dy blnce periods before therpy, ech ptient cn now be sved four to 1 dys of blnce study. Secondly, when it is desired to mesure the fecl content of substnce which is not dependent upon intke, this cn be done without metbolic unit nd indeed on n out-ptient bsis. A ptient need only be given the chromium sesquioxide cpsules for few dys nd told to bring ny single convenient stool t ny time fter the second dy. This cn then be homogenized nd nlysed for chromium nd the pproprite substnce under investigtion. We hve found this technique to be useful for mesuring fecl led in ptient with led poisoning who ws investigted nd treted in non-metbolic wrd. Fecl ft would lso seem to be redily mesureble by use of chromium sesquioxide mrker provided tht the chromium follows the ft sufficiently closely. This problem is under investigtion t the present time. ndividul stools or frctions of stools were not nlysed for mgnesium nd therefore it cn neither be climed nor denied tht the chromium follows mgnesium s closely s clcium. Since both clcium nd mgnesium re lrgely insoluble in the stools, however, it seems resonble to suppose tht Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.

6 the chromium does follow the mgnesium sufficiently closely for present purposes. When in fct the 'sixdy' fecl mgnesium results were corrected for chromium content, the results becme very much more consistent s is shown in Figure 4. The dditionl lbour required for the nlysis of the chromium in stools by present techniques is remrkbly smll. The rewrds on the other hnd re very gret, with sving of time of investigtion, with gretly improved ccurcy nd consistency of results, nd with incresed confidence in the results. The min disdvntge hs been the lbour of ccurtely filling the chromium sesquioxide cpsules by hnd. (Messrs. Sndoz Products Ltd. of Horsforth, Yorkshire, hve now kindly greed to undertke this for us with the id of pproprite mchinery, thus removing this min disdvntge.) t is intended in this unit to continue indefinitely with the combined interrupted crmine red nd continuous chromium mrking of stools for ll clcium blnce work. Further, it is suggested tht this technique should be considered essentil for ll clcium blnce work when high degree of ccurcy is required. Such ccurcy is required, especilly when the intke of clcium is high. SUMMARY Results of continuous chromium sesquioxide nd interrupted crmine red mrking of feces re reported on the first 13 six-dy blnce periods. The errors of the crmine mrking method re exposed. Using correction fctor bsed upon the chromium recovery in the 'six-dy' fecl collections, the ccurcy nd consistency of fecl clcium, mgnesium, nd phosphorus results re gretly improved. n three cses, ech individul stool ws nlysed for clcium, phosphorus, nd chromium. The dily fecl clcium nd phosphorus results clculted Methods nd techniques 279 from ech stool were resonbly consistent from stool to stool. n two cses rndom smples of ech stool were nlysed for clcium, phosphorus, nd chromium. n one of these cses, the clculted dily clcium nd phosphorus levels were extremely consistent from dy to dy: in the other cse consistency ws not quite s impressive, but still good. The dvntges of continuous chromium sesquioxide mrking re considerble nd re stressed. The dditionl lbour cn be mde very smll. t is plesure to be ble to cknowledge the vluble ssistnce given by Sister Wlker, Sister Orson, nd the nursing nd dietetic stff of the metbolic unit. The flme photometric work ws crried out by Mr. D. Newton, nd the phosphorus nd nitrogen determintions by Miss V. Gryson. m grteful to Dr. F. W. Heton for the mgnesium determintions. Mr. Blcow, the nfirmry phrmcist, kindly rrnged for the hndfilling of the chromium sesquioxide cpsules. REFERENCES Anderson, J., nd Weinbren,. (1961). The determintion of chromium nd clcium in the feces by flme spectrophotometer. Clin. chim. Act, 6, Dwson, J. B., nd Heton, F. W. (1961). The determintion of mgnesium in biologicl mterils by tomic bsorption spectrophotometry. Biochem. J., 8, Fiske, C. H., nd Subbrow, Y. (1925). The colorimetric determintion of phosphorus. J. biol. Chem., 66, Humphreys, E. R., nd Scott, P. P. (1962). Observtions on the use of mrkers in determining food trnsit times in the ct. J. Physiol. (Lond.), 162, 28-29P. Mclntyre,. (1957). The flme-spectrophotometric determintion of clcium in biologicl fluids nd on isotopic nlysis of the errors in the Krmer-Tisdll procedure. Biochem. J., 67, Reifenstein, E. C., 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, Whitby, L. G., nd Lng, D. (196). Experience with the chromic oxide method of fecl mrking in metbolic blnce investigtions on humns. J. clin. nvest., 39, Wiggins, H. S., nd Dwson, A. M. (1961). An evlution of unbsorbble mrkers in the study of ft bsorption. Gut, 2, Gut: first published s /gut on 1 June Downloded from on 26 August 218 by guest. Protected by copyright.