Studies on the Relation Between Intake and Urinary Excretion of Thiamin in Normal Man Citation Heegaard, Erik Vilhelm (1940) Studies on the Relation Between Intake and Urinary Excretion of Thiamin in Normal Man. Master's thesis, California Institute of Technology. doi:10.7907/23wg-vy77. https://resolver.caltech.edu/CaltechTHESIS:01212025-223602853 Abstract No abstract. Item Type: Thesis (Master's thesis) Subject Keywords: (Chemistry) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Unknown, Unknown Thesis Committee: Unknown, Unknown Defense Date: 1 January 1940 Record Number: CaltechTHESIS:01212025-223602853 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:01212025-223602853 DOI: 10.7907/23wg-vy77 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 16956 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 23 Jan 2025 17:17 Last Modified: 23 Jan 2025 17:17 Thesis Files PDF - Final Version See Usage Policy. 10MB Repository Staff Only: item control page

Studies on the Relation Between by In Partial Fu.lf'illment of the Requirement for the Degt,ee of Master of S.cienee CAL.tFOR'!HA I HSTlTU'ffl OF. ncr.moLOGY Intro&uction. • • a • • • • • • • ll • • f' • • • • • • It •- • • • • • • • • • • • • • • • • • .. - l E!eerim$ll~al L 1- Hethod.s It 3 2. Materials. . . . . . . . . . . . . . . . . . . . . • 16 3. Individtw,ls participating in ex:perimf}nts . . . . . . 16 h. Experiments with oral in t ake . . . ~ . . . . . . . • 17 5. Jllxperiments wi t h i ntramuscular in,jections . . . . . . 24 6. Detf)rmination of pyrimidine in urine . . . . . . . . 28 Di~1 cussion . . . . . . . . . . . . . . . . . . . . . . . 30 Literature cited . . . . . . . . . . . . . . . . . . . . 36 INTRO:OOCTION The development of the Th:l.ochreme Method for determining 1ihiamin by Jansen (5) gave rise to a number of investigations of i.he cpntent of t h iamin in a.if:f erent organs and i n body :flu.id.a • ' •.',. , .~:-:.··A of 'both humruis and animals, ?..nd both on normal a.ntl on thiamin deficient diets (See Westenlrrink and Goudsmit,. 15). Another me- thod for determining ~h4arntnr the Ph;},·cgnw:<;,es. method of Sehopter and Ju:ag ( 9), also contributed considerably to the progress of similar work ( Sae Sinelar, 10 and. Me!klejohn, 7) . A series of investigations ef the t.hiamin content of d.if'i'er-- en.t organs by applioation o:f' the well.known had a.lreaey beon carried out before the curvative p igeon test, above two method.~ were developed; due, however, to the unsystematic nature of these in... vest iga.Hons the results mu.st be taken with considerable raser... va.tion ( See Ylilliams and Spies. 16). ~a present wo:rlt waa an attempt to investigate t he relation between t h iamin intake and. its urinary excretion in normal man. !fue method applied h the 'lhiochrome Method modified by Westen... brink and Goudamit (14) for application to deter.m i. nation in urine. From t he ex_perimentn.l data an attempt will be made to deduce some i:rifomation conee,m ing the storage and destruction of thia. min in the normal human body. lt i.s to 'be hoped, t hat t his knowledge ma.y 'be helpful in l ater studies of the met abolism of t hi a.min in pathological eases. The Thiochrome Met hod depends on t he f act t hat t hiami n oen be oxidized with ferrieyanic1e in alkaline solution to a strongly fluorescent sub-stance, thlochrome. '!'his m1bstanee Cai). be extracted wit.h iaobutanol from the a.queous oxidation mixtu..ts. and its fluorescence m~,y be observed !'l,nd qlll!l.llti ta.vely detenni11ed with ultraviolet light . !Phis method haa al:l"eady been worked out in great deta.U by Jan.sen and Weaten'b:rink as mentioned above, and it wal:i not con- sidered necessary to try to i mprove their methodt but it waa necessary to adept t h e method. to the limited facili tiea of this laboratory. ...3 ... EXPERI MlilNT.AL PAR'l': -• 1\.d~t-::-"?.t~on of the Thiochron1e leffetho~-~to .t he f£~il_:1 ties of t~ ;La.,boratocy . 'l'he Thiochrome Method as described 'by Jansen (5) and in Us modification by Westen'brink and Go1.1.dsmit (llJ,), could not be ap... plied with.out modification, sin ce ne photoeleetric fluo:rometer Kaner (6) nae was available . reported. the c>pplication of the Thiochrome Method with out such a, photoelectric fluorometer, Ee d.ete:rmined the :relaU ve fluorescence of thiochrome solutions by direct visua l observation . This simplification hm.s ce rtain ad.- vantages be.cause it omit$ the use 0£ an expensive and sensitive galve.nometer . As a source of ultraviolet light Karre:r used a Analyse Qp.arh Lamp . Hanauer This exp ensive instrument not being avail- able, a satisfactory source of u.1 traviolet light wa.s f ou.no. in t he 11.Argon Bulb" ma.nufaetured by the General Eleetric Va.porlamp Company . This lamp eroi ts some visible light in the violet as well as ultraviolet and to obtain :pure ultraviolet light it was necessary to tre.nemit the light from the bulb t hreugh Ffo . 597 Corning filter.. The la.'tlp and filter was built into a practical unit by mounting them in a wooden box, whioh is pictured on The main difficru.lty in applying t he Thiochrome Method. to urln~ is, as Wes tenbrink and Gou.dsmi t a.l read,y have observed, tha. t urine contains a conside rable amount of :f'luore!lcent mat erial which tends to prod.uce a very high blank value for t he determin.ation, a.nd. t hereby decreases th~ aecu.racy of the determ:i.na:bion , -4MOUHTING OF ARQOJ{ LAMP AND FILTER IJf WOOD1E~ BOX. Filter Scale 0 I I l 2 I 3 4 ~ inches pa.rticu.18.f!ly when Ollly small amounts of th1~nin are present . To dimi.n ish this .so"tU"ee of uncertainty in the determination. the method of Westenbrink ana. Goudsmit , (14) was adopted. after other methods had been tried unsueoessf'ully~ ',,· In particular it was attempted to extra.et t he disturbing :tluoreJcent mibstan-ces ~,ith different solvents! and by adsorbing with activated Ch?...r- ooal, but :in both eases only negative re:sults ware obtained. Westen'bl"ink and Gou.demit us eel the following technique to make a preHm1nar,v separation of the thia.-nit,1 i'irom d.istutbing fluorescent $Ubstances: ''Urine was diluted with 3 to 10 times its volume o.f water ?..nd brout~ht to pH ... about 3 by adding hydrochloric Uid. 100 mg. Franconite r.'.J; per 30 ml. urine are added and the mixture is stirred for 3 minutes. The Fra.noonite the liquid by eentr:ifuging is then separated from washing successively with aeidulated water pH=J and 96% aloohol and dried at 100° C. tt The a dsorption of' thiamin on :f'ranoonite 1a so strong that U can be e.ccomplished even if th0 urine i$ first diluted 10 times, but the disturbing~ substl/U'lCes are not absorbed. nearly as well u..t1der t he conds.tions clea•e ribed. '!'his results in a, quite effective qua,nti tative s~al"a.tion of the thiamin present in the u.ri.ne from the disturbing substances. When the inveatige,tion was sta.z-ted no Jrranconi te Kt Wa.$ avail... able t and before it eould be obtained. from Ge:nna.n..v , a.nother applfo... able adsorption material \!fas obtained. It we,s found that ll\tlle:r•s ea.:rt h could be used; and it was applied throughout the investi- gation. With this material a pH of 4 was better than a pH of 3, a.nd ins tee.d of 100 mg • per 30 ml. urine ( 8.-fa with li':raneon He) ~ 4 grams of Mler's earth per tot~.l ind.ivid11,,,_l daily e.x eretion o.t -6VNIT OF SHARPLES CENTRIFUGE AND ADSORPTION JAR. A urine was used . The use of' so l a.rge an amount of" urine as the total daily e~cretion of a man was due to t he f a ct tlw.t in cases of very low thiami n exeretion , it wa.® neat,sse.ey to ha.Ve 811 the excret·ed urine to be able to determine the thiam:i.n . Since it was desired to standardize t he method througl.1ou.t; the same teehni{tUe wns a.p... plied for urine with high thiamin content~ The fact that the experimente,1 persons excreted a.bout 1000...1500 ml . urine in 24 hours. and that this urine had to be diluted to 10 times ita own volume, gave rise to clif:ficulties eoneerni11g the oentri:fu.gation of the Fu.J.ler• s earth t r om t he dilute tU'ine . It was undesirable to leave the urine anti lf'ul l er'a earth i n oonte.ct with ea.oh other more than a short time , partly becaua~ this would prolong the p rocedure; and. also because it would interfere with the results since the disturbing subatancea are a.c1sor'bed i:f given a su:ff ic• ;Ai':. S.ently long time . A Sharples centrifuge was found very useft\l fo:r the purpose of aepare:hing F.uller ia earth :from t he dilute u,:;... ine after adsorption . The centrifuge was equipped. with two 20 Liters jars (see p . 6) . With the method. of J ansen (5) it is poss i ble to st.a..ndard:l.ze the galvanometer readings against kliown amounts of thiamin, and is unnecessary to compare t he unJ.cnown solutions w'.i. th fresh sta.n.- dard.s a.s described by Karrer . The fact that the thioehrome solu• ti ons did not s eem to be s table ( if not mad.a u:p by dissolving pure thiochrome , which was not available at this time) ma.de it impossible to make U¾) a know·.11 series of standards with which the unknown th:i.ochrome solutions could be compared. To a.void the re- peated mDki ng of standards an atte!I\Pt was made to f'ind a. compound other th...tm thioehrome poseessing a similar fluorescence, but -8- RBLATION BETWEEN FLUORESENCE or qU1~INE ST.A.rfDARDS AND THIOCHROME SOLUTIONS. microgram thiochroru/15 ml ieobutanol 8 ~ '7 I 6 / ~ ~ 5 I V II, J 4 V II 3 V' 2 I I ,~I Iii 1 ) I /1 2 '4 s £ 1 2 '1 8 9 l D 11 12 1~ 14 1~ 1( 17 (j 4 microg~a,m Q~i"1ne sulfate 1.rr. 10 ntl 0. 2N sulfuric acid quinine sulfa:te in O. 2M H2SO}.~ wer e mru'l.e up , and thei:r nthioc'hrome value1:1 11 det ermined by comparing them und.er ultraviolet light wit-h known amounts of oxidized t.hiarn:ln . page 10. 'flhrJ eonCel'ltrations of the It was found t hat t he relative change in fluorescence nin-e sulfate istilu tions wi t h:th ee:L"'ta.1n eoneent:ra.tion limits . In oth®r wo?'ds, i f the ooneentra.tion is plotted against fi uoreseence a s on page 8~ we n.nc'L t hat within the :plied, t he reh,ti on is a s traight line . conce:ntration limits &p-!' The eonofJntraMon of the d.iffeTenee in ooneent:ration it was possible to recognize with oertatnt~r by the f luorescence method. A test for the u.s e:fuluess standards in the clal"l::: by thei:r fJ.uorescenee . with t he chosen eoneentra,tions . Th:ls was p ossible FtU'themore~ the dtf:ference in with i neTeas:lng number of t he standards, but the ma..~imum uncertainty on the det ermination is constantly 10;;&. (The standards ht,~ e, belo,,, Ne . 7 has- 157& ,liffsrence in concentration) . J'\fter t hl'll n1e·chod had. been ariplied f or some time it was :p0s\pl£..tt\ e l.f' sfble to determine not only -1£'· the u..11lknown tube ha,cl. a fluorescence ~-'/o(i(M wM.oh l ay between t wo st~idards, but also to d eterm.ine /2:f it we,~ Standard number ----1 'Y Qµ.inine sulfate per 10 ml. 0.2N ~S°4 - 0.95 mi Cli'Og • 'Y ~ta.min per ..........,. mL __ ____ 5.-. !u.OR .,.- O.OiB 2 1,08 ft 0.093 3 1. 24 ti 0 .107 4 1.42 tt 0 .125 5 L68 ,. 0 , 12:µi 6 1.95 tt 0 . 1.66 7 2 .30 n 0 ,189 g 2.50 n 0 . 206 9 2.71 It 0 . 223 10 2.96 ti 0 . 24o 11 3.22 fl 0.265 12 3.51 fl 0.2$9 13 3.84 (f, 0.315 14 4.20 fl 0.345 15 4.60 n 0.378 16 5.04 .If o.41; 17 5.54 II o.455 equally far from both staJ1d.ards or nea rer to one o:f the two stan- dards . Thus it was p ossible to increase the certaint y of the st~ d.a:r.d. deter..nination to 5'% of the measured amount . FineJ.ly one more question has t o be d.iscu.ssed., namely , the amoim.t of ferricyanide appl iec1 i t he o:ddation of the thiamin . :Both tTBnsen and Karre r iound t hat if the ainount of :fet>:t'ieyan.ide user.l to o:ddi:r.e a given amount of t h iamin was varied , the fluot>es- e-ence of the :resu:tiug t hfoehrom.e sol:u.tions w-ould al.so vary somewha.t . T'.nis led 0-aeh o±' tham to oo determinaM.ons wit h a series of differ-e nt r,,..mounh of ferr:l.oye.nid.e •. and. t hen seleet the thiochrome solution with t he la.rgest fluo:res-eence as the one with the ¢-orrect a.mount (>f ferrieya.nide . This method was quite laborious and it was at temp ted to s;void rna.iting mo-r e th:m one oxidation for ea.eh c-mal ysis. '!"his wa s p ossible b ecause J::,nsen ( 5 ) had. found , t ha.t the ad.di tion of methyl H.leohel to t he e:xidat:lon mixture ttould cause t he dec-rea!"S.e in fluortH3CEm-ce with su£f'ic'.i. ant e~eass: ef f -e rrieyan:ld.0 t-o be almost zero . :Ely alweys using a. :rat her large e2:eess of f er;ri- cy a,nide it waa p~ss fhl e t o r efu1e® t.-0 ~- minummi t he fl.uct"tiat:ion of fluorescence . '.the f a.ct t lk-'1.t t he sta.-1de..rds w:e1·e determine.t with. a. simila r excess of :ferricyanide made t he results CO!¥Ja:!.'8,ble ~ even if t he ;naximum f'luor~scence wa/43 n-0t ob tained b:y t hte method . :F,,u-... th.ennore, when $UGh an excess ef fenicyanide used~ no effect of using a ir as a stirrlng agemt i n tho e.."!tre;ction with isob\ttanol a£ t he oxidized t hia.'llin solutions oould be observed . Suoh an eff ect wa-s observed by Wes t enbrink and Oou.d.smi t and led them to use rU tro-. gen u a stirrtng ~.gent . With these preliminary remB,rka . it will now be pos s ible to describe t he eowJlet e metho(l of a.neJ.ygis , as applied t o all the determinations in the :lnveJtrHgation : 1 ..1:ha volume of the specimen of n.rin.e to be u.sed for t hiamin determina tion. consisting of the total excretion in 21!. hours by one of the persons cooperating in the experiment~ was first meas .... ured. The pH was than determined with a Becr,:man PR- inetor~ and. the uri.ne ad.justed. to pH ti by eel.ding 2N sulfmiie a.Cid. The u:eine wee poured :i.nto t he t op jar ( see pe~e 6) e.nd t ap water was added until the total volume was l.8 liters 1 as determined by a ni-c'.rk in- side tho Jar . Fo"tU" grams of l!'uller' s earth were t hen added., and the eleetrie stirrer started. .After five mimites the Sharples centr:i:fuge ,,,as started., and t he suspensi on :run thrm.1.e.,h the eent-ri.. :fuge by ope:niri..g valve A (i=H3 c1 page 6) . The eer1tri:fugation took tle a oid:u.lated w&ter, f il te!'ed on a :Stfohnor funnel, w-a.ehed with 96% alcohol and :fin~.lly d:ried a.t 100° C. ~1J / , One gram 0£ the dry !<'uller 's earth was noj p laced 1n era.eh. ef two eantr:U'uge tu.bes . metr.cy-l alcohol. To ea.ch was added 2 ml. 10% Na.OH and 1 ml .. After a gentle shaking, 1 ml . 1% f errieyanide solu·Uon waa now added to one of tht~ tubes and l ml. distilled wa.ter to t he other. To both was added 15 ml . isobu.tanol t F..ast- man) from .:1 burette and a stre2..Jl~ of air passed through the tu.be,s The tubes were finally Gientrifuge8. to s~ara.te t he two layore We have n.ow two test tubes. -0ne oonte.ining the oxid.hed thiamin plus t he l'ema.1.ning all'mll a."!lotmts of i nte r feri ng fhtorescent su1,stances which we shaH desit,'1late as the aiw.J;rti~~ 1.."Ube, th.e other with t he unox:td:i.zed. thiamin (non-fluorescent) 'but with t he same amount of interfering subs t anees . the blank tu.be. This we shall de111ignate as The two tubes were now eOtl".ipared. ~i t h t h e (!tlinine stan.o.ard.s t n t he box ( see page )4-) until t uo tdil'.ncl.a:rds were found 'hetween which lay t he fluorescen ce 0£ t he m1know11 tube. U' the coneent:rr. tion of the thieehrome solution in the anal yti cal tube was too g reat to fit with the standai't'.ls, t he thioohrome solution was dilut.ed wit h isobutan.ol until a s.atisfac·~ory cont'H:rnt:ration we.s obt ained . The a;ppr. 11 1.l?hioehrone value" of the u..-riknown t1;1bes cou.1d now be f ou.ntl fr.om t he graph on page $ . % su.btra.et1.ng tihe value of t he blaJ1.k, from t hat of t he- WIB.lytical tube, a..ri.d ~Jd.ng i nt e consiclex-ation t he amount of earth used. i'o:u t he ads orption a.n.d. a,na,.. ly-sia . and. t he v-olu,me oi isobutanol of t he t h:loohrome SC)luM.ons , the thitimin content of t he urine could. be computed . However, it is ne eeasa,ry t o eJtt.i11\s.ct t he water l a.var a.g~:dn with isobut anol if large amounts of' thiamin are prosent i n t he urine• becauae in this oa.se t he f r H,et ion of thi a.1i i n l ef t beM.nd .a.:fter the :first e:xt:r·a,c... tion is eonsi d.er a..b1@ . Jf this we.a the ease both t he analytical tube e.ni.i the bhn..1< t ,:i.bB were e::ttra.eted re.pe!!?.ted.ly with. 15 ml. iso- buta.nol an.cl t heir thtoeh:t:>ome valuet"l determined. as 8-bove . The $'l1l:ll of the thiochro:ne values f or t he analytical tu.bes minus the !!um of the values from t he blank tubes gives the thian1 in eon tent . On work . the next :p<il.,g-e :i.f; given a. data sheet as used during t he ihis eontain,s in IR. convenient wey al l t he neeNH'>ar s, de.ta for later ref'e:retioe . As a check on '.the met hod it wa.s trh . d ttl a scertain if it is possible to recover to 100/ thirunin added to u rine . The f'Qll.ow.... i.ng e~eriment shows that t his is p o.ssiblEH In pe.1:·son ~•s u.rine from the tenth da,y of the second per-iod of the oral e:x:periments, was foun d 3li5 microgram t hiamin in the analyt:teal tu.be . !oh.elf. o±' t he urine , 'ba:fot'e adserptton, w~ . ft} added 0 . 05 mg . t h iamin , and. t he anc":l.1;.t'bical t ube oe.nto.ined. 450 microgram t h iamin . The bla.nlt tubeB we:re t he same in both c aees- . The d iff erence is 105 mie:rog:rom t h.i amin. is inside t he Qx_pe :t"imental error . To half Gf the urine -14a- VITAMIN BJ DETERMINATION ·ies no: Analysis n o : _ I Date: - 193 ,ject: ,al amount of fullers earth: ---g. Used for analysis: _ _...;g. .ume of urine : ___ cc. pH: .uted to: Total time for adsorbtion: liter. - - - - min. .lysis: .rac,n no. Volume cc Quinine standard B1 per cc . i-BuOH micro;ng B1 ! ii i I I i 1 J I ! 2 i ; ' 3 I -·i 4 I I 5 I I I I Total: mrng tnk: ;rac>n no. Volume cc Quinine standard B1 per cc. i-BUOH micro/4g Bl l I _J i ~ i -t! 3 ~ 4 I l i 5 iI Total: tlysis: mk: ~fer. 1arks: mmg ... mmg = I.u. per day mmg ~ p!izeo_myces Het11_~ Stoel cu.1 tu.res of :F'hJ1 com;vees Blf~.kesleee.nua were me,int aine.d on malt agar . l!'he e~>per iment al 0t1:l turea were ma.de up with med-. ium cont aining: fJ!,gS0>+ , 7H20 . t:S:2P04. . 1.5 » Aspara.gln . 4.o tt . 100 . 0 11 De~t:rolil e f.1ad.e u:p to l liter with d istilled water . !fo 10 ml. of th:i.s med- i um was added t he des ired a.r.1ount of lll'ine to be min. The experimente,1 media were t hen tested. for t hia,.,. autoclaved for l ~i minutes a t 15 p ouncls pressure . and inoculated wi t h with equt>J. volu..rnes of a sterile $pore aus;penlil io:n . 25° C.. , t h.e mycel:1.u111 t he,n :f:llte:i'."ed. c,ff 10 days at weighe d , All cn.1 tures were allowed to remain O dl'iect e.n-d An e~erime.""l.ts were cp.rried out i n tri:pl icate. T"ne weights of t he m;;ceHur,1 is proportional to t he amoun t of thiamin present in tl'>..e sample. i f t he wei ght of t he mycelium i s not mo-re t han 40 llllt • Th1.1.s by know:tng the wei ght of t he myeelium grotm on a l<:-aown a:mou.nt of t h i a.'llin {0 . 1 microgr f.\Jn t hiamin gives 30 mg . myeel iu.m ) i t is p ossible to evaluate the mnoUt'"lt of t himnin p re... s ent i n the s ample. The me t hod is :not as Specif ic a.s t he t h iochron.10 method. be- cause Phycomyees i s able to utilize s,lso ooca r bo:icyli~se t?.n a. :free ;pyrimicline + t h i a zQl . lfowevex • t his is wha.t in pa.rtteular makes it us~:ful f or the pu.rpos-e of de t e rm:tning fre e py:r-S.m:l.dine or t h ia- zol, because if u:rine contains one of the t wo . and. t he other is a.d.ded, n.n increased g rowt h of t he myeeHain will b e observed . Th.e respon.se of Phycomyces t o coearbozylase is p r obably not of ar.,y i mp ortance tn tha case of urine. since t he latter is reported to It was atte.'T',pted to utilhe Fuller ' s ewth ad.so1·'bates as source o:f t h:i.amin for Phycomyees. but the mould seems not to b$ able t o utili~e 1JJ,ll of t he thiamin :pT8$en.t , when it is a.deor'bed on l!u.1.ler •a earth . Urine was, t herefore, used directly i n the tests . Since it was desired to ~preach as closely as poesible to a natural diet, Galell :8 a co11.oen·tr ~.te o:f rice bran containing 150 micrograms thiamin. per ml . . was used i v.steed o:f synthetic •t hiamin fo r t i e oral experiments . ,!hiall1in...gv:~o_ghloricle je-e tions . was ·ased for t he iutraxm.1scu.la:r i n- It was obtained in ster ile solution at a ooncaut.ra- t i on o:f 10 mg . p er ml. The t hfamin. was synthes ized by tho Both preparations were kindly supplied by t he Galen Compa.rcy· . :Berkeley, California . The three per -sons, desig,nated as ~. !!_ an r:l £ ;i,n t.b.e :report. A We ight 177 l'bs . .... 28 II II ti 160 (1 0 25 II II 1l 155 u The par,sons fl. and ! pa:rticipa.ted. i n ·th~) e:rperiment wh.ieb. l asted 48 days . The experiment consisted of six period.s w:l. th di.ffe,rent intake of Galen :B . after each other. The pe:t·iods f ollor1ed The diet was 1;-1hat eould be immedia·t ely designatecl a.a a normal. diet, probs,bl y col'.lta ining about 1 mg . t hlamin per d8W . However, t he diot wa.s seleoted. with speda.1 a ttant:ton ·to uni- f orm.i tr from dr.-w t o d.a,,v, 'by i?~voiding <'m.Y food :partieul.arly high i n t hia.min . The ~'?'n 33 when a cl.ose was givrm . Wf! S t a.ken in one do-se on t he deys WHh t he hi gh doses of 40 and 60 ml., this eausoed a, p ronounced fe eling of hunger m1d some d:i.seom-- f ort~ The urine was eoUected t u b-ottles t'r1om 8 A. M. of one rlay until the s ame time. t he ne:x:t da.y . Tempera tu,re changes i n. th~ e.tmosrJhere had ;3- pronounee.d effect on the volume of urine e:ic• creted . 'l1he pH o'f t he urine was quite c~r.1.s·tant at about 6 . 2 . Detennination of t he this.m:tn content were ti.a.de daily as far ,Ml possli.bla, ~nd i n p :roo·t:teally eJ.l Ctisea t he G.v.:tsorption on lro.11e:r 1 s earth wa.a done the day t;i.f·t er t he urine had been collect ed . First Period: - ~ :a Pel's.on J. - 150 60 m:wrog. :a~ J. 2.10 2 22; 75 fl ti 3 210 .,. 150 = 60 11· II 4 215 r.: ? 7 - 150 ::.125 205 - 140 = 65 200 - 150 50 8 220 ;o: - 150 e !-'ere-0n ..... 199 ... l4o = 50 mi croe . B·1 - 160 80 210 - 150 6o 24o e II 1J ::: II n <:O 50 !I ii II 11 200 - 150 If !l 200 - 150 = 50 ti i1 ti fl 17r.: ) - - 115 "' Go ti n 11 II 1$0 - 140 tio 10:: fl f; ii it 185 ;::. 60 ll n 9 215 ·- 111-0 "' 75 n II 175 10 2;~ . ... 1'50 "' zo H " 1JQ. - 130 .:;, 11,0 " 71 ti If i<-verr:-,ge - 150 = 65 27i~.) 6 ,; :: - 150 70 == - • • •. Avera.'1:,;i , ;:: 8 . - 125 - 100 75 ·= ;:: It -~ 56 ll ·~ 11 11 Second. Period: ---··- lnta.k~ll':· iiormal J>iet , plus 10 ml . Galen :B daily . E~eretion;. ...-..-.,.-.,.__..~ - Person :,8 Pe1•eon !_ 220 - 14-1 :;, $0 mier0-g. Bi 370 - 160 = 190 mie3'.'og .:B 1 2 3so - 160 3 360 l~ :no - 130 ;.: .J 360 - 160 - 150 == 270 220 fl II 420 l't ti - 16o = 200 II II 1~05 .,. 200 == 205 It It ;:: 21¾) u It l~50 ... 200 = 250 It ti ;:; 200 II 'II ;f!-0 - 200 = 1>-io » u 6 170 - 150 :z 20 ?" H hlO .,. 200 :':: 210 11 t, 7 liJ.1.0 !I t1 555 8 3so - 150 = 230 II ·11 9 10 i;:: - 150 .::: 290 Aver age ,:;:; 191 ll n 200 "' 355 II 5t~5 ... 200 = 3li5 n 385 ~ ... 100 ,;:: 2~!__~ - II ·" tt ff Q.g5_;:___20Q_,: _ 425 ff n = 255 u If 200 :::: 185 Averoge - -1 6oo ... 160 "" 44o m:i.c:tog . :B :i. 1010 2 570 .,. 150 6$0 ... 200 :;; 4$0 tt 3 580 ... 175 41: l¼)5 4 :Pay Persoi1. ,;ii lt20 ll ,~ Pera.on i ! If - 200 IC 810 mic:rog . - 200 ,.,, f.l tt 695 lt95 « tt 630 - 195 ;;; l,1,35 u fl 685 .,.. 200 r:;;: l~l!Si:;.., ti JI 15 56ti ff f.l 715 ... 200 = 515 n (I, 6 690 ... 200 II' 490 ti # 700 ... 200 C!:: 500 (I II 7 .. 1000 ... aoo ... 800 u n .. 150 41; ::, > > ... g 740 9 - - -· ~ Ave',l'.'age -t:: 455 II t:, ti - 200 = 5tio tt II fil. .. 200 . 675 ll - ;o ~ Aver~e, , = 590 H JI n P·e:rson ..... l3 2 1510 ... 3t~o =1170 It 3 1310 ... 330 4 805 ... 250 ;:, 555 H ; 6 - 220 1005 .. 150 7 1040 ~ n 980 n 1075 ,.,.. 275 = 800 " Ii 1150,... 260 o:: $90 II rt {I if ;,;> t')l5 l1 ,;,:: s50 ll 1020 - 195 ~ 825 H il - 210 $30 " 1305"' 195 =1110 tt u 1,035 = g --Average lat 870 " 11 Fifth -pe;riod:: !ntakeJ. lforrnal Diet, plus 60 ml . C.}al$1 ;a dail.v Person A 3 Persou i 11.,.00 ... 215 = 11$5 ml crog . Bi 16o0 - 225 la' 1375 microg . B.2, 1550 .. 315 1225 n 11 1460 - 235 ~ 1225 11 If 1680 .. 290 c; 1390 11 .11 1900 - 245 = 1655" n 2105 - 235 = uqo 11 l'I 2010 ,.. 320 = 1690 ll f1 i:: 4 It 5 6 llB5 ~ 315 = 870" n 7 1450'"' 235:.: 12:J.5 II II s l4>40 ... 350 ~ }090 ·-"_ _ _" c:: 1215 II tl - Averag e Person A Dal Person B 585 ... 185 = 400 Microg . B:;. 1 695 .. 300 2 350 ... 190 = 160 11 tt 505 .. 205 3 4 250 ... l4o =105 n II 245 II 255 ... 1:;5 12D II u. 5 235 6o. fl It 210 = 165 c; 45 It " 6 260 .. 210 = 5() u II 180 "" 180 = 0 n II ~ 395 miorog . Bi 320 ... 210 "" 110 It - 175 ,,.. - 300 ll II 210 = 35 II ll ¢ t;: -cl- RELATION BETWEEN ORAL INTAK8 A~D URINARY EXCRETION OF THIALVlIN~ (Person A) micro :t-r'Am Bil 1600 ,- - 1400 .. r .. 1200 .,.I .. 1000 .. Jl- 800 . 600 - J 400 nn11- 200 - ~ .. 1D i 20 -no---L1oi20 40 30 40 - 60 - 50 days no-. ml Galen. .B. -22RELATION BETWEEN ORAL INTAKE AND URINARY EXCRETION OF THIA.MIN. (Person B) micrograllt .B 1 1800 . 1600 1400 n. ,.. 1200 1000 . -r .. 800 LJ .. i 600 I l.,-J'- . 400 . ,r - ru- - . 200 1b i---- no - l . ,. o 3o : 40 1 , 10----1- 2 o - L 40160-~ no - 5o .Days ml Galen B, -23RELATION .BETWEEN ORA.L ltfTAK.E AND URINARY EXCRETION OF TRIAM.IN. (Averages af A and B) microgram B 1600 lf-00 1200 1000· 800 60Q, 400 200 tnl Clalen 10 2.0 40 50 60 tho persons !J:. and .Q. participated i n t he expEiriroen.t which. l a sted for 30 de~s . The eJtperimen.t eonsisted of f0~ periods vii t h dii'fe.rent afl'l ounts cf t h.fa.min in.jected intramuscularly i n t h e uppe:r half of. the left i:1rm. ly after ea.ch othe r . oxpel"iment was The periods foll owed. immediate... A d.iet simila.l' to t hat used during t he ora l maintain.ad . inj.ect'.lon of a. high dose of t hia.min i ntramuscula r was n-ot fol.l owed by ~,. discomfort . The injected thiamin seemed to have a good i nfluence on the genera.l well be tng lectl3d as for t he orRl experiments . Th,e fitst peri?d cons isted of 10 days where only a :normal diet was g:tven, t he excret i on i:n t h:i.s period aver-aged. 60 miel"ograra of t h iami:n per day f' or 'both :p~rsons . Intake : ~~ lformal Diet , p lus 3 mg . t h iami11 intramuseuiarly . Pereon A - ~ - Pers-0n. 0 1 o.6lw mg. t h i amh1 2 1.200 II 11 0.930 u II 3 1.130 n If 0.940 u 11 4 l.200 11 H 1 . 110 11 fl 5 1.270 If 0 . 990 n If G 1 .090 It l'I ~--- 1 .090 n 14 0 .890 n Average = " 0 .730 mg . thirunin ll -25- Third ieriod, ~~: lifo:rmal Diet. :plus 10 mg . t h iamin intramuscularly . - P& l? erson ! Person C 1 4.26 mg . t h is.min 3 .15 mg . thiatlli.n 2 h.79 II II 4.n Ii II 3 5.71 II H 5.03 ff ti Lt 8. 60 ti n 5.33 11 It 5 11 .95 n IIH 6.34 11 " 6 8.~5 .II II 5.12 II 11· 7.28 ti 11 4.85 II JI .Average == Fourt h oer:i.od: ~.ke,: Ho i njections . irorrnal Diet. :Excretion; - - Ji"srson A Person C l 2.41~ mg . t hiomin 1.99 mg . t hia.11in 2 1.02 fl If 0.63 u fl 3 0 .80 ·11 0. 3.4 n u 4 0.65 If 11 0 .30 fl " 5 0 .22 It ll o.42 II ff 6 0 . 21 II ll 0 .20 11 n 7 0 .15 II II 0 .07 It fl 8 0 . 11 II n 0 . 05 II II 9 0 .09 II fl 0 DP~ ,, -26- RELATION BETWEE~ I~TRAMUSCULARLI INJECTED AND URINARY EXCRETED lll ~ THTAMTN (Person. A) .B11 :J 11.b .R 8 7 6 ,- ,- 4 2 r-r1l 1 ... l p ~no ~ ... 20 ;3 --14-10 · ,c 40 n o - injected mg 50 B1 daya ·-27- RELATION BETWEEH INTRAMUSCULARLY INJECTED AijD URINARY EXCRETE]) TH 1AM1.tf (Person C) . mg B, 8 1 6 I- 5 - 4- .. 3 - 2 n 1 r1 lD 0 - 20 --10 ~ . ;. ,. 0 11.0------ 40 injected mg .B l 50 days t he second. periods second day i n t he i nj ection e.:i::pe r inent se:r1es.. min t)l' l . 79 mic:rogr0zn per ml. Z:~!'S01:l. _J31 excr eted. 490 ml. urine eon taini ng 0 .93 mg . t h i amin or 1. 90 mi crog:ram per ml. The e ontf>,nt s of t h e f l as ks and t he aver~ weights of t he myeeUum has 't)een stated below: Flask ,.._ ·•' tfo . . Content l 2 31 mg . 3 5 6 7 d.iff' . 17 mg _. O.05 w,L of ! 1 a a:rine + 10-?mol . t hi azol 8 9 0 . 05 ml. o-:f O' s urine 10 11 12 0 . 05 ml . of C' s UTine + 10-5mol . thiazol Controls :;: 0 29 mg . d.i:tf . 13 mg . 0 . 1 mi e!"ogram t h ia..m:ln ;;: 30 mg . my c el. riinitliae f ow.•1<l bo-u.ncl in t h i amin . - 11he mnou.nt found in Ct s urine was e quivalent to 29% of the pyrimidine found bound fo t,h irunin i n ....0 1 s urine . The r esults of t he experiments with ot>a l adminfo-tration of th:lamin he-Ve b l'-}en plott ed m1 pagm21 a.11d 22 ., I t is to be noted t ha.t t here is a. consider;"'b le vari r.tio-n in ea eh pe-r iod both for persons ! and !· This can be e:xplained. partly by e:itperime:nt al errors in t he analys i s , but t he main reason for t he variation is p robably to be f ound tn the sons _participating i n t he physiological eondi ti ons of t he p er- experiment . exerci se on certe,:tn d 1~rs give for thie.min due to an For example, a:ugruented rise to an inereased requirement increased. carbohydrate metabolism. Fu.:rther- more, i n the f.'01.u-t h e,ncl fifth p er't ods. where t he in take of Galen B was very high , the laxative effoct of t he t hi amin eauae<l. in some cases Di arrhef.l, whieh :mi ght have prevented t he t hiam i n from being completely o,bsorbed by the i nt e stine . On page 23 t he average va.1ues :for ea.ch period have been plotted a.ga.inst t he intake of t hi~;,min in. the form of Galen :B . The feur p oints on. t he curve :fit fl straigh t line going thr ough t he o.r ig in :reasonably well in bo·th cases . Th is indica t es t h..ti,t under t he conditions of the exper iment a const ant pr opo:rtfon of the oraly adminis tered t hiamin is exeretea. in t he urinB, even when the :i.ntE>.ke is varied over verJ broad limits. HowEl!Ve:r, t he experi ment t.l oes not p$rmi t of generalizing on t hi s observation 1 beQattse the preceding perlods mi t;ht he.ve hr-,d en effect on t he following periods. It i s reasonable to assume that t he hodie~ relative saturatfon with t hiamin in the b egim1ing o:f t h0. exp eriment me.y als·o be a fa.etor in t he p ror:) ortion o:f t hiamin exerete(l in the urine~ T"a.e difference in t he slop es of t he lines for J)e rsons A and !, ean possibly be accounted for by t he difference in bocy weights ~- of the individuals ...... A a.nd ._ :B, since it ie known that th1am:in rem1:1re.- ment is dependent on 'bod.y weight . Person ! t,Jeighed. l~J less than A. The resul t!:! of the experiments with i ntramuscularly injected t.hia.mi u h ;.i.v e bErnn p lotted on pi.'l(~fH:l 26 and 27 . The e:r.par:iments ,;1ere $tarted by first exnmining the thiamin excretion in the urine f~r to days on a normal diet. 'The exeretion. in this perio~1 a:verai.ged about 60 g "'.:m·aa cla ily fo!' both persons . thi.amin were g iven ln the next pe:i.·tod 3 mg . dr~Hy intramuscularly. ;i.nr1 it will b e noted that on t he first day or£ly 20",h -o f t he inje.c ted we.r e e.xereted.. After this fir~t day t he excretion M HU..l'!le(l a r ela.tive1.y- oonst~nt level with ~.ppro:dmatel y l~o-% of the daily i njectea. thia."Tlin excreted. in t h e ur- ine. In the next p eriod. where 10 mg. t h il!ll'n1.n were giv en daily :l.nt:ra- muaoula.rJ.y, the s~..me phenolllenon was observed. Aft er a period of in... creasing u:rin,'.iry thiamin out put (firs t day --42~t, seeond day -43% , thir<:1 da.:7 ...57%), a reb,ti,rely construit level we,s rea4'hed. ea se .o f In the ! practicE11ly all of t he t h iamin given is e:xcreted (aver. age of fourth, fifth and sixth day for! was 98%). age was only 5S"'rS during the same d.eys. differently in this re91:ieet. - - A and C seems to behave . On the twent2r---seeoncl d.a_y of the e~eri.,.. ment the injections 'W'ere stopped, and the urinary outpu.t of thiamin deereased da ily, until it regained the original level. This period l asted 9 d.£Ws. In totally 78 mg. thh.min were injeeted. . - A exerete<l. a total of 50 mg. of thia.min or 65%of the adminhtered amount during the period of injeetion. of that administered .. - 0 excreted in the smne :period 34 mg. or 44% In the after-period of nine days l excreted a total of 5.7 mg. and O a total of 4.o mg . Coinparing t he results of t he inject:ton. experiments •.11. th t h ose whe:to an oral <lose of th.t~ $?. me me..gnitud.e was given , we ;?ind that t he excretion in the cas e of injected t hiamin is much highev. This ma.y poss.:i'bly be du0 to mt ineomplete absorption of thiamin in the intestine . !n t h i s ea se ~,itamln should. be :found in the feces . There is eVio.enee for the oceurenee feces . of large amou.."t.ts of thiamin in Jto:t even i f t h e fecal thi am in is t aken into consideration , it is not possibln to account for all of that admin:lstered. . ll"'urthe rmore, in the an:se o:f injec·ted thiamin, it is unlikely tha t feCf!.1 excreti on c an aocowit for the 00101.mt not excreted in the urine . / Hatcher and Borsook (2) h~., s- found only very smal l amounts of thiam:Ln in the feces when the th:lamin wa.g ad.ministered by i n,jeetion. The f ,;,ct th~Ji; it is impo-ssibl e to a.ccount fo:r the total amount of t hi.:-unin alhll:'i.n:tstc.;! :r.ed by urinary a nd feca l excretion , suggest that t he body may be able to destroy thiamin, enzym?.tieall.r or otherwise . Such n possibl e breakdown is ob s cu.re at pres ent , and ha s nt)t been clemonstrnted in ma.rmnalian t:tsS,u es. iiowev,,n,, t .e clinicts1l ano. theo- 1"etical inipor~-m:c-e v1h ie1.1 mi ght be cm11nect ed, with t h e elucids.tion of the :prwpertie s of such an enzyme sho1.u.cl 'bP- ex.pectect to be eonsider~:ble, so th,'.!!.t furth.e r work along thi kl line should, be worth while. In t his connection t he work of :Bon..'1er c,nd Bucl:mnn (1) on the de struction of thiemin b y t h e ruovJ.d Phycomyces sh.ould be mentioned . 13or.m er and Bu~hnm.n fou.n,l, the.t the thiazol part of th0. t h iamin molea1..~.le i s 'broken dmm by tion in t he mycelium . J3lakesleaa.1nls The.y we:re able to is e. result of t his breakdown. a..ll enz;;rmatic r 't1 ac-- show t hat fr ee_p~rimicUne The reaction products of the b;rea}:!;d.own .o f 'the t hiazel part are not yet known bu,\ their experi:1nents SU€gest that the in:$. t1a1 ' , l •' :'' rupture of the thia.zol ring . :· occurs in the 2-3-positi.011. They found. that the vitamin P..nalog r, which hns a OII3.:.g:roup in the 2-:position of ?Vy {<-'i.J./.J{,A the thiazol rlng Hi~not b}oken down enz;rmatically. J!h0 2-CH -e;roup 1 3 is able to pro'tect t.~e vi ta.min against destruction and it can., therefore , be assumed that the des tru.etion of the thiazol ring be... gins with the o.ouble bond beirween ni t:rogen D.nd. carbon. A pre1:1rninarJ *~er:i.ment t o :t'inct if urine eonta.ine any un.... combined vi t rm.iin- :pyrim:l cline hn.s been reported en p::i,ge It was fmmd that th8 t litO sar1ples of u:rine ex2mined oontS:ii ned. an amount of fre"l :pyrimidine equal to about 30% of th~ py1"imidi11e combined with ' })yrim~dine in the b&sic d:i.et rathot· t!1a.n to t he d,.estruetio.n of in• ,jeet ed. vi t rni-1in,. since it is lmo1;m tlm t mfl.ny plan.ts con to.in up to :five timos as mwh :free a.s co:n~oined pyrimidine. ne.c~essary to pe:rform It iG, therefore, similar ex_pe:rimentg with a diet known to be free of u.noom'bi.ned :pyrim:ldine. The injection e:11peritle:ats show th.$.t there is H m~imum limit to the ammmt of thiami.n which the body will absorb. wnen the body ( ill the case of ~) has been "loa<led u:p 11 by injeetion of large doses of thia.min ovor some time, further adm:i.nisilration of thiamin leads be eata.blished if the blood :i.e constantly lo.<tded. wi th thiamin by in,Jeetion, e.,;nd a.s soon as the injection stops, the 'bor.1;, starts dis ... ch8,rgi:l(1 thbmin in the urine, ancl return-:, to its old Condition a.ftel" app roximately nine d.ays. 7 The rea son for th.is reject i on of excess t h i amin by the b,oq is not lmown, and t he roec..iie.nism will prooa.bly be difficult te eluci• date . )3;1,r t ak:i.ng i n.t o cons icler a tion 1 however, some of t he f a cts knmm a.bout t he reac t ion of t he vi t a.min i n ce:ctain mi cr oorganisms. as for example y ea.st, it is p ossible to offer a. partial e~pl anati.o n of the reason for and. t he meQh,r,tnism of this :rejecUon. li'rom t he equilibrium cons,M.t of the oxidat ive pyruvic a eid deca.rbo:r,ylati on it is kn.o\m t hat t his process £.e.vors the breakd.o.m p roduct s t o suo:i.'1. &n. extent t hat the reaotion ea"l be considered. as irreversible , in the boc'ly . The f act t hat all t he other reactions in"'tolved. in t he carbo hydr ate me t ab olism are r ev.,-,rs ible , m1=tkes it pO$Sible t hat if no o 'l:iher limiting factor stops t J,Jia reacti on , a high concentrf).-I;ion of c,irbox;irlase ~~ould t end to ma,ke the body de- plete itself rapidly- of ear'bohydra.te$. In this connection shou..ld 'be mentioned, that an increased amount of thiamin i n t)le boa;,,, really in• creases t he carbohydrate metabolism to a marked degree (see Hecht and Weese, J). It 5.s, therefore, U:.'l:ldez-standable that the 'body l3hould have a protective mechanism against excesdve doses of t hi e,min~ It is well known t hat t h:i.amin does not a.ct in the body as such ., but t hat it must first bi, pyrophoaphol:";11lated to eoearbo:x;;rla.se. The mechanism of this phosphorylation in mrurnnal:ta.n tis sues has llot yet 'been clearly es t ablished. it was reported that Exper iments of 'rauber {11 ) in which a cetone dried intestinal mueosa tissue is able to phosphorylate t n.rnmin have not 'be~m verifi ecl by other worke:rs ( see Ochoa, 8). However, Veil-}falherbe ( 12) demonstrated tha t washed yeast plus adenyl:pyrophos:phate in the presence of m~~esium was able te pyrophosphor;,rlate thi-?. ;nin, a.nd a similar me.c ha.nbm might very we:11 be possible in mammal ian tis sues. I t is known tha.t brain tissues can pyrophosphoryla te thiamiu. Weste.nbrink (13) found. t,.'1.,-1 t washed yeasrt can als o dephos:phoey... late ooea.rboxyla se, bu.t t ha·t l arge amount$ of free t hia.rnin are a ble to retard t h is reaction, possibly oy a mass a.ctiou effect . T'ne p/i ' J (.t:'(,?.,,v\·•·f"... ...c.J.J; same de-phosph.oryle.ti on of cocarboxyla.se was alise:rvee1 by Horowitz nc1 Ee c3gaa.rd (1:J.) in a,'l. enzyme pr eparation of pea roots~ 1:.... ]'rom a large number of cletermina.tions on t he proportion tween fre e -0!nc1. pyI'op}.1osphorylat0tl thiamin in be.. different ti$sues, Ochoa (S ) concluded. -t.b.at an equilibrium must exist between t he two compouncls in the tissues, aml t h is ean onl y be explained. by the pr esenc et::;f si;1il~t::0nz;,meir fri the tiss-ues~ to t hose found, by Weil• . . .. . . . . _ _ _¥ _ _ , · • .., _ •• .• ,,.--···- - • •. ,., . , Mal herbe i.:;••ncJ. Westenbrink. Finally t he p resence of t h,, phosphat e g roup i n the l:?,se proba,bly enabl es t his comp ound t o com11ine with t he ti ssue p roteins t han ctoes free the:t-efore, justified to assume that vol ved in t he bod.y 1 s coca.rboxy.... much more firmly thi amin. a..tld it seems, this phosphor.;lation is in.- mechanism t o absori) or r eject thia.'11111. One f a.ct '-'Ihioh emerges clear ly fro m t he pr esent experiments is; th.at it is not p ossible to protect t he body against future t liie.min defic i ent periods , by fill i ng it up with thiamin. because t he body e.utomatically '<lflll de·Jlete itself l"ftp idly. Th:h behavior of thi a.inin i s di:fferen·t from t he behavior of some of t h e othe-r vitamins, i n l)al't ieu..la:r :£'rota t h<:: 'behavior of t he fat-soluble vita,. min.s, which can 'be storecl i n t h e bocly for fu.tur e use . This is net surprising considering the clifferent chem5.cal na tures of these vi ta-, mins, but it constitutes an i rnportaxi. t f a ct in rela tion to the clinical use o:f thiamih. Litera.tur-e Cited 1. :Sonner, J. and ]l.,R. Buchman. Proc. Nat. Acad. S¢. 2. Hatcher, J.B. and IL Borsook . 3• Recht. G. aad H. Weeae. 4. Horo'Witz, N. II . a.nd :m.V. Heegaard. 25r 1611.. 1939. Unpublished results. Y..lin. Woohenschr. 16: 414~ 1937. Unpublished results. 5. Jansen., B.C.P. '.Ree .. trav. ohim. 55: lo46. 1936. 6. Ka.rrer, t>f . !Ielv. ch im. acta. 20: 369, 1937. 7,, Meiklejohn, A.1?. :Bi()-()hem. J. 31t 1441, 1937. 8. Ochoa. s. 9. Schop:f'e:r, W.R. e.nd. A. Jung. Ve Cong. Int. Tooh. Chim. d. Indust ..~rio. Scheveningue 1937. p.22. Biochem. J. 33r l262t 1939-t 10. Sinclair, H. M. 11. Tauber, H. Qu~,rt. J. Hod . 7: 591, 1938. Enzymo1ogia 2: 171, 1937. 12, Wail-Mtlherbe, H. Dioch.em. J. 33: 1997, 1939. 14. Westanbrink , H.G. K. P....7.tl J. GoudsmH. Rec. trav. ch im. 56;803, 1937. 15. 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