supplemented with daily dose of 3 gm. sulphadimethylpyrimidine (' sulphamezathine' of Imperial Chemical Pharmaceutical, Ltd.) in

Transcription

1 EFFECT OF SULPHADIMETHYLPYRl- MIDINE ON THE MICROBIAL SYNTHESIS OF THIAMINE AND NICOTINIC ACID IN HUMAN INTESTINE By H. N. DE M. C. MALAKER nd A. Iv. PAUL (Nutrition Reserch Unit, I. R. F. A., Biochemi0^ Lborry, Dcc University, Est Pkistn) Introduction In recent yers it hs been relized tht tn micro-orgnisms intestine possess mount s01i\ synsize cpcity lrge essentil vitmins which re bsorbed.nutilized by host niml nd tht bcterio~ sttic gents like sulphonmides cn effectively inhibit this intestinl synsis vitminswith se drugs s new ols nd with improve technique estimtion, it hs been shown Wf some nutrition experts nd bcteriologists th rts cn synsize some members vitmins K nd E in intestine. Njr et o. (1943, 1944), Ellinger et l. (1944, Ellinger nd Coulson (1944) nd Oppel by conducting experiments on humn subjec hve reported tht similr synsis thimin riblvin, nicotinic cid nd biotin tkes lso in humn intestine. They hve shown tht insoluble sulphonmides s sulph gunidine nd sulphsuxidine, becuse l poor bsorption through intestine, indue mximum inhibition in biosynsis *n bove vitmins B group. Kornberg et (1944) in course ir study bl0~ synsis vitmin K succeeded in producing deficiency this vitmin in rts by using soluble sulphonmides s sulphpyrzine, su phdizine nd sulphthizole. By compr tive study with or insoluble sulphonmiue3 B-comple^ 194% P^c furtne^ ^

2 dec., 1949] INTESTINAL SYNTHESIS OF VITAMINS effectiveness follows sulphpyrzine most effective, sulphdizine nd sulphthizole next nd sulphnilmide sulphsuxidine nd sulphgunidine lest effective. From survey it is evident tht y hve found order s bove works insoluble sulphgunidine nd sulphsuxidine "which produce mrked inhibition on microbil ribosynsis thimine, nicotinic cid nd flvin fil produce ny distinct effect on t le intestine., vitmin K in inhibition ppers tht.synsis ny prticulr vitmin in due is intestine due ny sulphonmide ction specificity bcteriosttic intestin different sl\lphonmides wrds in synsis tke tht prt micro-orgnisms vitmins. Investigtion hs, iefore, oeen begun in this lborry study how different sulphonmides vilble in miket behve in inhibiting biosynsis thimine, nicotinic cid nd or members B-complex nd lso study ctul mechnism biosynsis refore, nihibition. For systemtic survey, sulphonmides so prepred my be clssed under three mjor lr groups (-4) Insoluble ~ ^nlphgunidine, 1 in wter, cid nd, lkli. nd succinylsulphthizole thlylsulphthizole. f-r) Soluble in cid nd lkli but springly soluble in wter.sulphdizine nd sulphmethylpyrimidine. (C) Soluble in wter, cid nd lkli. fnilmide, sulphthizole, sulphpyridine Sulnd sulphpyrizhe. much work hd been done with sul group A, our ttention ws B study those groups which nd C nd in communiction, present first series works begun Since phonmides dlrected were DE & OTHERS mesured. 543 After this bsl diet ws supplemented with dily dose 3 gm. sulphdimethylpyrimidine ( sulphmezthine Imperil Chemicl Phrmceuticl, Ltd.) in three equl doses (ech dose two tblets 0.5 gm.) fter n initil dose 3 gm. The urine on this supplement ws collected for period three dys fter preliminry period sme dys on this drug. To see wher bove sulph drug supplement interfered with bsorption nd srge thimine, test dose 5 mg. thimine ws n given long with bove drug nd urine on such double supplements ws collected for period three consecutive dys fter sme preliminry period s before. The recovery test dose bove vitmin under influence bove sulph drug ws n compred with tht when test dose ws given lone with bsl diet without sulph drug supplement s crried out in experiment no. 3. The influence bove sulphdimethylpyrimidine on biosynsis nicotinic cid hs been studied in nor set experiment no. 2 nd this ws crried out in mnner similr tht dopted in experiment no. 1 except tht in this cse test dose used ws 300 mg. nicotinic cid nd recovery this test dose with sulph drug ws compred with tht when it ws used without sulph drug s in experiment no. 4. Thimine nd nicotinic cid urine were mesured ccording following methods Thimine.This ws estimted exchnge technique using Declso bsorbent ccording method by * s bse selective Hennessy nd Cerecedo (1939) s modified by Brown et l. (1943). Declso ws used for dsorption nd purifiction thimine. Nicotinic cid.this ws estimted fter hydrolysis with cid, nd with lkli nd ure ccording method Swminthn (1942) with some ltertions bsed on technique ^presents typicl mem- Wng nd Kodicek (1943) nd vlues ^eie,fsulphdimethylpyrimidine )e,r group Bhs been represent nicotinic cid, nicotinmide nd nicosulphonmides Elected investigte its effect on microbil tinuric cid, nd lso trigonelline in which Nsynsis thimine nd nicotinic cid in methyl-nicotinmiden importnt product Umn intestine nd lso see wher nicotinic cid metbolism in urineis lso ppliction this drug interferes with included nd ll se hve been expressed s tl nicotinic cid. Sorption nd srge bove vitmins. The chronologicl order experimentl Experimentl periods is denoted by rithmeticl numbers Experiments were crried out on five dult s,, etc., nd periods which do nnnn subjects ges vrying from not figure in tbles must be ssumed be nd weights vrying from kilos. preliminry periods for succeeding ones. r^rs ley were kept under proper cre nd strict The results bove four experiments hve, observtion in hospitl nd were given dily been summrized nd presented in tble.,.ig,weighed iole Irm for mount bsl diet which lsted eighteen experimentl period In experiment twenty-four dys. no. 1 subjects were first fed Results Effect sulphdimethylpyrimidine on biosynsis thimine.it is observed from results experiment no. 1 tht urinry diet nd fter preliminry penod collected * Declso F used for estimtion thimine, -vys dys 24 hours urine ws 01 from Messrs. Permutit Co., Ltd., Englnd, dys received consecutive furr period three whom our thnks re due. 011 tht bsl diet nd ir thimine contents nf ii^e ^sl three

3 544 THE INDIAN MEDICAL GAZETTE [Dec., 1949 Tble Effect suiphdimethylpyrimidine on thimine nd nicotinic cid excretion in five norml humn subjects. The figures indicte dily verge vlues n individul. Bsl diet composed gm. rice, 70 gin. pulse, 70 gm. fish, 200 gm. vegetbles, 30 gm. mustrd oil, nd 4 oz. milk twice week Period Diet nd supplement Urinry elimintion Rnge Men Experiment no. 1. Experiment with sulph drug nd sulph ding + test dose thimine. P-VI + 3 gm. sulph drug. + 3 gin. sulph drug + 5 mg. test dose thimine. Totl Bi in /ig. per dy Per cent reduction elimintion due sulph drug. Totl B, in ng. per dy Per cent recovery test dose Experiment no. 2. Experiment with sulph drug nd sulph drug + test dose nicotinic cid. P-VI +3 gm. sulph drug. 4-3 gm. sulph drug mg. test dose nicotinic cid. Totl N.A. in mg. per dy. Per cent reduction elimintion due sulph drug. Totl N.A. in mg. per dy. Per cent recovery test dose S S Experiment no. 3. Test dose experiment with thimine. -f 5 mg. thimine s test dose. Totl Bi in /ig. per dy Per cent recovery test dose Experiment no. 4. Test dose experiment with nicotinic cid mg. nicotinic cid s test dose. Totl N.A. in mg. per dy. Per cent recovery test dose The subjects employed were B. S.body-weight 49 kilos, ge 29 yers. M. R A. D B. M S. T., thimine excretion five subjects on bsl diet rnged from /*g. with men vlue 120 ju,g. After orl dministrtion 3 gm. sulphdimethylpyrimidine for six dys dily excretion bove vitmin in urine decresed limit fig. with men vlue 25 /xg. The verge per cent reduction in elimintion thimine under influence bove sulphonmides ws found be 78.8 when subjects were n given test dose 5 mg. thimine long with bove sulph drug in succeeding periods P-Y nd P-YI so s determine s wher ingestion drug in previous periods long with bsl diet o11 ^ ffected bsorption nd srge thimin it ws found tht per cent ingested test dose ws recovered in urine n se vlues lmost corresponded thos obtined in nor experiment no. 3 ingestion similr test dose thimine wit, out bove sulphonmide supplement. ^ e bove sulph drug lso does not interfel with estimtion thimine by fluorirnetr1^ method is evident from fct tht tn thimine contents smples norin urine did not show ny vrition even when tn sulphonmide ws dded m.

4 Dec., 1949] INTESTINAL SYNTHESIS OF VITAMINS DE & OTHERS 545 All bove observtions clerly indicte tion sulphgunidine nd sulphsuxidine s tht reduction thimine excietion s observed by Njr et l. (loc. cit.) nd Ellinger obtined fter orl dministrtion sulph- et l. (loc. cit.) represents only portion dimethylpyrimidine in period IV experiment tl synsized cid left unutilized by or n. 1 is not due interference with bsorp- species intestinl micro-orgnisms. From tion nd present investigtion it is observed srge thimine but my be scribed results orl s due tht fter dministrtion sulphdime this decrese in biosynsis dult humn subjects re increse in elimintion nicotinic cid species which led synsis o by bout 6 mg. nd this vlue represents lrge mount this vitmin in norml mount synsized cid which is destroyed or utilized by some micro-orgnisms in conditions. intestine in norml conditions. So Effect on nicotinic cid synsis.-in expeiiment no. 2 it hs been found tht subjects minimum quntity nicotinic cid synsized dily in humn intestine is between 9 0I} bsl diet excreted from mg. or with men vlue 6.95 mg. nicotinic 10 mg. cid dily in urine. Unlike previous Discussion experiment, orl dministrtion sulphin dily dose 3 gm. in Micro-orgnisms which tke prt in this cse led increse urini y vitmin Bx do not require it s synsis elimintion nicotinic cid n enoimous fcr in ir medium nd vitmin B, growth extent rnge being mg. with synsized by m functions s n essentil men vlue mg. 1 he per dy. metbolite tking prt in vrious enzymtic percentge increse nicotinic cid excietion rections required for ir growth. Since both due dministrtion bove sulphonmide thimine nd sulphdimethylpyrimidine, used in ws found vry from with present investigtion, contin common men vlue When test dose 300 mg. nicotinic cid ws given subjects pyrimidine ring it seems tht decrese in long with this sulphonmide percentge elimintion thimine fter orl dose bove is probbly due inhibition recovery dded test dose ws found sulphonmide thimine synsizing growth between 19.1 nd 24.0 with men vlue intestinl micrlor s result diffusion 21-2 nd this vlue corresponds with tht this sulphonmide within ir cells nd obtined in experiment no. 4 by dministering interference with essentil enzymtic recsimilr test dose without ny sulphonmide. tions by competition with enzyme constructed this shows clerly tht orl dministrtion from vitmin B,. The competition with does not interfere with nor essentil metbolite p-mino-benzoic bsorption nd utiliztion nicotinic cid in cid s postulted by Fildes (1940) my lso be body. It hs furr been observed tht ddition this sulphonmide urine does prtly responsible for bove inhibition growth thimine synsizing bcteri. not ffect colour rection in estimtion cid The incresed nicotinic elimintion nicotinic cid by cynogen bromide 1 egent. So it seems evident tht incresed observed fter orl dministrtion bove elimintion nicotinic cid fter orl dminis- sulphonmide my be explined in following bove sulphdimethylpyrimidine is wys one increse in vilbility tl 1. It is not unlikely tht micro-orgnisms nicotinic cid synsized by intestinl flor. which synsize thimine require for ir tt is suggested tht under norml conditions growth nicotinic cid which is vilble from competition exists between micrlor lrge sre synsized by or species. As n Which synsize nd those which or destroy exmple it my be cited tht propionic nd lctic utilize nicotinic cid nd or vitmins for cid bcteri (some species nd strins), dysenir growth. Most probbly bove sulphotery bcilli nd C. diphri re ble synnmide inhibits growth ltter species size thimine but require nicotinic cid s () micro-orgnisms intestine nd thus growth fcr in ir medium. So in present eses fir mount nicotinic cid, which investigtion when sulphdimethylpyrimiwould orwise hve been utilized bv se dine is dministered growth thiminefor ir metbolic ctivities, for synsizing micro-orgnisms is inhibited nd bsorption intestine nd for utilizlrge mount nicotinic cid required for ir tion withinthrough body humn subject. metbolic ctivities is thus relesed for bsorpthe results tion nd utiliztion by humn body. tble lso e presented in gi\ some indiction s tl mount 2. Or species micro-orgnisms not nicotinic cid synsized dily in humn synsizing thimine my lso be present in mtestine. Fifty 80 per cent reduction in intestine which require in ir medium, in nrinry elimintion nicotinic cid which ddition nicotinic cid, lso vitmin Bx which mounts bout 3 4 is vilble both from endogenous orl i.e. yitmin in intestine resulting inhibition growth those from 01 thylpyrimidine is n micro-orgnisms diniethylpyrimidine Jje ^olphdimethylpyrimidine trtion micro-orgnisms mg.due inges- source,

5 from micro-orgnisms which synsize this in intestine nd lso from exogenous dietry source." When bove sulphonmide is dministered endogenous source is spped s result inhibition growth vitmin Bx synsizing micro-orgnisms ccording mechnisms discussed bove nd exogenous dietry source seems be blocked extrcellulrly by drug nd thus when ll vilble sources thimine re spped, growth bove species orgnisms is inhibited rid lrge mount nicotinic cid is refter relesed for bsorption nd utiliztion by humn body. Similr blocking hs lso been suggested by Knight (1945) in explining inhibition growth Lcbcillus rbinosus. (requiring nicotinic cid for growth) fter ddition sulphpyridine in medium s observed by Tepley et l. (1943). From bove discussion it is evident tht sulphdimethylpyrimidine lthough highly soluble in cid nd lkli exerts its bcteriosttic ction on those micro-orgnisms which synsize thimine nd which utilize or destroy nicotinic cid in intestine. It my, refore, be postulted tht cpcity ny prticulr sulphonmide inhibiting microbil synsis vitmins in intestine depends not only on rte bsorption through intestine but lso on specificity bcteriosttic ction drug wrds micro-orgnisms involved in process biosynsis vitmins. This ntibcteril ction is dependent on its structurl reltionship with growth fcr or.essentil metbolite different micro-orgnisms tht tke prt in biosynsis. Summry dministrtion sulphdimethyldult humn five subjects decresed verge urinry elimintion thimine from /xg. The verge percentge reduction ws found be Orl pyrimidine nicotinic cid verge cse elimintion ws found increse from 6.95 mg mg. nd verge percentge increse ws clculted be [Dec., 1949 THE INDIAN MEDICAL GAZETTE 546 In urinry 3. The recovery test dose thimine nd nicotinic cid dministered with nd without bove sulphonmide supplement hs reveled tht this drug does not interfere with bsorption nd utiliztion bove vitmins. 4. The decresed elimintion thimine nd incresed elimintion nicotinic cid due ingestion bove sulphonmide re probbly due inhibition growth those micro-orgnisms which synsize thimine nd those which utilize or destroy nicotinic cid nd mechnise inhibition hs been discussed. 5. From dt obtined in present investigtion minimum quntity nicotinic cid synsized in humn intestine hs been clculted be 9 10 mg. REFERENCES Brown, E. B.. Hmm, J. C., nd Hrbison, H. E. (1943). Ellinger, P.. Benesch, R., nd Ky, W. W. J, Biol. Chcm., 151, 153. Lncet, i, 432. (1945). Ellinger, P., nd Coulson, R. A. (1944). Ellinger, P.. Cotjlson, R. A., nd Benesch, R. (1944). Fildes, P. (1940) Hennessy, D. J., nd Cerecedo, L. R. (1939). Knight, B. C. J. G. (1945)... Biochem. J., 38, 265. Nture, 154, J. Amer. Chem. Soc., 61, I- Lncet, i, Vitmins nd Hormones, 196. Acdemic Press, York. Kornberg, A., Dft, Pub. Helth Rep., 59, 832. F. S., nd Sebrell, W. H. (1944). Njr. V. A., nd Holt, J. Amer. Med. Assoc., L. E. (1943) Njr. V. A., Johns, Ibid., 126, 357. G. A., Mediry, G. C., Fleischmnn, G.. nd Holt, L. E. (1944). J. Clin. Invest., 21, 630. Oppel, T. W. (1942) M. (1942). Indin J. Med. Res., 30, 537Swminthn, Tepley, L. J., Axelrod, J. Phrmcol, nd Exp A. E., nd Elvehjem, Therp., 77, 207. C. A. (1943). nd Biochem. J., 37, 530. Y. L., Wng, Kodicek, E. (1943)....