Document R4754Q06Bw62EbqoKj2gqbOv

-. 3624 D.I-. P-4RKE -4SD R. T.WILLLUIS "1 6 IrjjI REFERENCES Bate-Smith. E. C. (ltS9). Biochcnc. SW. Sytnp. na. 3.67. Bray. 8.G.. Tho?. W.V. & white. C. (1950). Eiochcna.J. ET4-8,.971R...\., r Par. W.A. 9 Eonns W.c. (1949). Suture, tond., 164. 674. G m n , G. A. 9 T i i m s . R. T.(1949). Biochem. J . 45,138. Kiilz. E. (1S90) 2. B i d s,%l- Porteous. .1. \V. b \VilYk:m. R T. (1949-z). Biochw. .I. a, 46. Porteous,J. W.8 WiiiLrns,R.T.(19496). Bioclrcm.J.44,jri. Schmjedeberg,0. (18891). Arch. a p . Path. Phannuk. 14.2Yy. Smith, 3. X., Spncer. 13.6Williams, H.T. (1950). Bidurn. J . 47.2% Spencer, B. &. Williams. R. T.(1951). f3ioehcm.J. 48.537. Studies in Detoxication 35. THE SLETAE30LISX O F BESZESE. (a) THE DETERJLISATIOS OF PHESYLMERChPTUC ACID IX URTSE. (b) MERCXPl'URIC ACID EXCRETIOB BY RABBITS RECERIXC BESZESE BY D. \T. P - - f c EXSD R. T. \.\;LLUUlS Department of BiochemisiTy, St Xary'a Ho;rpitd .Medic& Scfwol.London. 1V. 2 (Receieed 26 Octo6et 1950) <:We have shownthat some 60 of a dose of benzene with the mercaptide as e dark-brown spongy solid. To the administered to xabbits can be accounted for by oxidation to phenols ( P o ~ ~ ~do:uwsilliams.1949~.b) mdby elimination in the unchaaged state (Parke& Williams. 1950). Benzene, however, isalso escmted ae phenylmercapturic acid and & Young (1943) have prored this by the isolation of the mercapturic acid in a masbum yield of 0-3i 9'0 of the dose from the u r b of rats dosed with benzene. AIthough Witter (1945) fded to isolate phenyl- mercapturic acid from the urine of rabbits receil-ing benzene, Drummond & Finar (1937) have susested that these a h & also excrete this conjugate. In the present paper we s h d show that rabbits do, in fact, excrete phenylmercapturic acid and that. determined by the quantitative methods developed here. the amount excretedisabout 1 yoof the dose. solution of cuprous mercaptide cooled to 0". there was added a solution of d i a z o t i d aniline (8 g.1 in Sx-H$O, (120 ml.). d brisk evoiution of X*o c c d with the formationof a buff precipitate. After 30mia at 0.. the solutionwan heated at 6Q-iOo for 30 min., 6Ihredand estmcted with ether (30mL) to remove phenol. The Faulting greensolutionwas saturated with Hg3 to remove Cu and, after filtration of the mppcr sulphide, the solutio~yt as boded in cwuo to remoce exr-5 Ha.The solution w a ~then neutr;rliztd with conc. S H , klution followed by glacial acetic acid ( 5 d )and after keeping at 0' ovembjht, the precipitated phenyl-L-cysteine WLW collected by tiltration. At this stage, the compound was hea~ycontaminatedwithcyrrtinewhichwaalargelyremoved b r repeated dissolution in r-NaOH (charcod) and pre. cipitation with 2Sacetic acid. The material wna finally recrystallized from 30 yonqueoua ethanol in which cptine is rerpsperinglyaolub1e(Clarke & Inouye, 1931).Theproduct (2.2 9.) wnaobtainedaawhiteglisteningphtea,lep. 180-181"; L-Phenybner ethanol) waa p nccording t o Z from 1g. of p! acid (mp. 1% mercapturic zu pfipared bios with chlorube s p e c k -of t h apectrophotoi and Table 1. [a]y+lOo (c, 1 ixiO-l~-XaOH). (Zbamky atYoung(lW3) EXPERIXEXTAL give lap. 170-17Pand[lr]~+ 11' h0.lX-NaOH.) Anample prepared by debromination auddeacetylationof biosynthetic - Prepadion of + y L w & ~ cyetsine hrrs been by deriditm. Phenyl-L- & Young (1943). p-bromophenyhercapturic wid (Zbaraky &, Young, 1943) hsd mp. 18O-18Ioand [a]$ +10"(e, 1 in0.1*-NaOH). but we were unable to r e p d u r n their conditions exactly. p-Bromophenfl-bcyatehe WUJ similarly prepared from me prcaent method bu pmduced rather better yields. cyetine (5 9.) and diauotizedpbromo.niline (7.5 g.) (yield, Ta .....-. -- &+e (5 9.) vu d ~ c byd boiling for 2 hr. in 100 m~ 2.66.). It formed cobde8~.W n h g p k a , m.p. 193' X.H$O, with 3 g. of 2% daat. a few pi- (total, 19.) of (notdeprssredb~*..mp14~P1. Q 4 a ~ p ~ b ~ d ~ t ~ l ~ g n n d a t d Zn ( A X ) beingadded periodically. The solution tion of biosynthetia p b r o m o p h e n y l m e n m ~ oacid a~. - - -- ateredhot .04in order to fscilitate the formation of the ~ ~ p r ornmemrgtids. 1mL of mna HCl w a a~dded (cf. cotding to Baumuui ck (c, 1in0 1N-N~OH). 1881). and [aJir+20" MC,193,1). The hot solution WM now trssbd with h h l y pChlon+uyZ-~'nm. Thir eompound h u not bean, . -* p k p i h t e d -0 dirolved on a (3.6g. in 2 0 d water) which alowiy previounly deacribad dthongh the DL-fOm (m.p. 2020) hM m to @= a grrao 001utioa When the been e y n t h e s i d by Bshringat k F ~ k l o r(lW9).It WM ddition of the ~ridien n e u completionthe mercaptide may p m p d from cy.tine (6 9.) .nd cliuntized pehlorrrnibe . .- .-tendto cow oat Ofsalution (ua ahita pracipitatc, but this (5.5 g.) aa dcseribadabove for phenylcyatoina The yield WM can d y ba redimolved by adding a little Z N - ~ O , . 2g. dter r s c r y s ~ u l i o nfmm 6 0 % e h L It formed - Exasn-0 ahouldbeavooided,.inceittsndrrtoco-pncipitnte colourlean gliatsningc k m g a u p h b q m.p. 190". [aJy+18. (c I in 0-la-SaOH). (Found: C. 46-9; H. 4-7; CZ 15.1. a.C,H,,O,SSC1 requiresC. 16.7; H.44; 15.3 ?&.) A sample scu also prepared by hearing bios>mtheticp-chlomphenyl. mercapturic ofid (0.1 8.) under r e d u with 5 ml. -L-u-HSO, for 45 min. After cooling, the dalution wan neutralizedwith >%, solution. and after keeping overnightthe precipitateof p-chknophcnyi-Lcy&inc var, vrshed with water and R- a y s t a l l t e d from 50 ye aqueous ethanol (yield. 50 mg.) (m.p. 189-190; [ZIT+19' (e, 1 in 0-14-%OH)). (Found: CL 14-9 O&.) Fig. 1. Lltnriolet absorption spectra of phen?l-t-cpteine (d).p-chlomphenyl-L-cpteine( B )and p-bmmophenyi-t. - cysteine (C) in 0-lx-NaOH (-) and O-~X-ECI - -{- -1. Sea de0Table 1. L-Phenybercapturic acid (m.p. 1*2'20; ( z ] 7 -!!lo in ethanol) .was p r e p a d by acet;rlation of phenpl-Lcpsteine according to ZbarsLT h Young (1943). The geld wsa 0.6 g. from 1 g. of phenylcysteine. tp-chloropbybempmercspturic -acid (m.p. 15T; [XID W in ethanol) and p-bromuphcnyl- -mercapturic acid ( u p . 1j.Y and (.ID 13O in ethanol) were prepared biosynchetidy from the Urine of rabbits dosed D.V. P,utKE h i ! R. T.WIT.,LLUIS qjx cpteines require up to Jx-dkali for complete hydrolpk The aubtit8ted phenylmercapturic acids were mom readily b y d r o l y d than the ansubstituted denvativea. WM heated on a sand bath (105') for 40 min. After cwiinq under the tap, the solution WM seidifini with 17mL of 2-9-H,SO,. Ethanol (23 mL) and ntmch solution (10 werethensddedandthemiaarsti~talinanicebathaith 0-Olu-I,. A blank titrationWM perfolmed on 40mi. of the clear solution without hydrolysis with XaOH. The turbidimefric method The small amounts of phen~lmcrcnpturicacid expcrtel in benzene urines r e n d 4 the iodometric method uncerrnin o m n g t o thevariabilityofthenormdblurluandthepreaenrm of other iodine-conaaming compounds in benzene urine (* later). Fnrthumore, the more specificgmimetric metlmi uas not adaptable to small amounr~.However. an obwci. tion that when mercuric chloride was added to hydrolywl benzene &ea atable mqensi~nr ere fonmd suggested a turbidimetric method. II I11 0 1234s. Concentration of NaOH (N) Fig.3. Estent of hydrolysis to the thiophenols of phenyl. Xdcntiry of fhemcrclyiGmereupti&. Onalkaline hydmlysis phenylmercapturic acid gives thiophenol which rea& in wid ethanolic solution with HgcZ to give phenylmercuric mercaptide, (C,E,S).Hg. On standing, according to Lecher (1920). an equilibrium. 2C,H$HgClt(C,H,S),HR +HgCL. rnerclrpturic acids (A; ansubstituted, *- * p-chloro- is attained. The mercaptide. which fonna colourleaa plate --and pbromo deriratives, ----) and phenylcysteines (decompoaeaat MY),prepared h m phengknereapturicacid (B; UMubStitoted. ,p-chloro and p-bromo den- waa found to be identicalwith that prepand from authentic vatives, ----) by vafious coocentrationa of NaOH in thiophenol and in a g r a v i m e ~ cexperiment, 49.1 mg. pun 30 mia phenylmercapturic acid yielded 43.5 mg. of the mercaptidr (calc. for CI2H,&Hg. 439 mg.). The deternrination of phenylmercapturic acid Turbidimetric eatiwtion of pknylmacapluric acid. (u) I n pure d u f i o n . The principle of the method is that JTethoda for the determination of pbromophenylmer- phenylmercapturic acid ir hydrolysed to thiophenol by apturic acid have ken deoised by Stekol (1936) and alkali. then neutralized by acid. The solutionis then trelrtcl B d e y (1949). I n Stekol'r method, the mercapturic acid with ethanol to keep thiophenol in solution. and gelatin to waa hydmlyned by 0.35x-NaOH to pbromothiophenol stabilize the turbidity developed on addingHgCZ.Turbidity which wan then estimstcdiodometndy. or gravimetrically is then measured in the Spekler absorptiometer. M p b m m o p h e n ~ l r n e d cmercaptide. Binlrley's method, Careful studies, which need not be detailed here, were which only indirectly involves the mercuric mercaptide. is made of the effects of the conantnuion of gelatin, electro- colorimetric and usea Folin'r reagent. Jiethoda involving lyte.~,HgCI,and ethanol andof the effectof pH and tempern- . the formation of a phenylmercdo mercaptide are mors tareontheatabilityofthe tarbidityproduced bytheformation apecific than the iodometrio method, but Stekol's gravi- of the phenylmercaptidc. Bridy, 0003-0-02 yogelatin gave metric method cannot bsam&tJy ueedona dd e . complete stabilization. with no precipibtion ofthe mercuric It is a h dear h m the preceding &ion that whcnsa mercaptidein 1hr. Outaidethismngeprrcipitationocenrml. 035r-NaOHcompletely hydrolysedpchloro- and p-bromo. The electrolyte mixturea, E,So,-Na,SO,, HCI-NaQ and phenyimemsptnrio acids, mora conwntmted alkali U HOAo-NaOAo wem testedfor theiieffect: the bst prodaced necumu-y for phenylmmqtmio mid, whilst for the unstable suepensionq the matad canned alow development phenylcpteines S N - N ~ mH y be n-. -- rTha e#.. of d i d on V n m &idby & iodomchic - - mwihod. Whom mercapturia aoid. are srtirn.ted iodo. _-nretzicallyinurine,it unsoay todotarminea braa vdne of opacity, whilst the lint wam the moat mitable at L Sat oonamhtionofabout04% A 0011ce.ntdion of 0.1yoHgCI, gavethe k t smpeumion~and MOtWhLamcantrationthem wanrlowdevelopment . n d prwipitstia A Eoncentrationof in fsrms of the average titre of a day'r oatput of 16% ethanolor 1- did not gratly&ect the atability ofthe - . ......-- -. -- - ~ a r i n s b a f o m t h s c o m ~ t o b e 6 t u d i a d c a n bssuspenrion; below 10 yothedevdopmant of opacityh -The I, titrsforoDnnrlminsi.variable u n l e ~ pmgmmivelydomrandL h 16"/o prsCipitation e t cam ir tasn to nuintakthe animab on a conatant A concentnation of 10 yo ethanol w u adopted. The m e at _- diet. On a daily diet of 75 g. rat c n h (J. Thorley. f i e stable supe.naioru were prodoood st L pH t . lo~f .bo& 2. hLondon). the average 6t.m w u ISmL 0-01N-G; on The beet temperstmw for d e d o p m m t of opacity m 75 g. rat cubas+M) g. crbbagz 33d;on 7s g. of an equd between 10 and W ;above 21p the m-ptids to .__ -- -- ~ 0 f b r q p a n d 0 a b . 7 - 6m l ; . P d ~ ~ ' D i e t l l ' ( h i a b ddholve utd below 8. Na,SO,tmded touyddiza outofthe -- * lao&on~onrMillem).7d. solution. ---. f "he lodomemo method for rll nrinen w u crvrid out u o k Each day's & e r u d e up ta200 ml. with watar. US@ 0-02 % elatis 10%athrad.O.~R-N+SO,,0.1% HgCI,. pH <2 and tamperatam 10-20.. opdv ru foand To 100 ml. them wna added 50 mL IO % (w/v) ZnSO, and to develop rapidly d ta remaina b l e for wed hotus. - 60 d Din-NaOH, the dufitwm mired r a d centrifuged The d d p t i o n of the preparationof the standard o ~ - -- - The dar aolution (40mL) mind with 5r-NaOH (10 mL) ._ which follonr gira the method W y &pbd e ... 07 i*r/- Wx 02 ._.. c &. obirined,-ha 1 :':Voi. 48 PHESPLM.ERCXE?CURIC ACID L\; U R S E - 627 R w e n f a . Phenylmercapturic acid; 5-SaOH; 2s- and sy.&So,; 1 (wlv) aqueous HgCI,; 0.2 &: (wiv) gelatm; - &?a,SO, and ethanol. , Xefhod. Phenylmercapturic acid (100 mg.) WM hydro- by heating (sand bath; 103) in 50 mi. of x-XaOH for :: min. The solution WM cooled and made up to %O: ml. -=-rith x-NaOH. This solution contained the equivalent of -- &5mg./mL phenylmercapturic acid. A fourfold dilution I 6 t h x-XaOH was also prepared. i' Volumea of 1-12 mL of the latter solution were added to series of twelve 25 mL graduated Banks each containing t 1--=2-.o5fym-Si.aOetHha. nTohle, 2.5 ml. 0.2 ?& gelatin and from solutionswere mixedand cooled 11 to 0 mL in ice water i dunng the addition of 3 ml. of SX-H$O, and 2.5 ml. of 1$: HgCI, .Thesolutionawereremoredtomom temperature, L.made up to 25 ml. with viater and mired. After 10 min. the opacities were measured in the Spekktr absorptiometer wln3 1 em. cells and Uford spectrum yellow filter no. 606 modheat filrrr H 503. The blank employed was preparcd by -- diluting to 25 mL e mixture of 3.5 mL ethanol, 2.6 mL i 0." o& gelatin, 2.5 ml. 1 X H a . 12 ml. x-X+SO, snd - - 2 mL 2sS-H,SO,. Jlore concentrated solutionswere set up by adding 5.7-5.10 and 12.5 mL of the 0.5 mg.;mL solution of ;1 hydrolysedphenyimerpturicacid~d~flaelircontaining I_ -r-6. 6, 2.6 and 0 ml. of x-NaOH and ethanol and gelatin 1 solotions rn before. The standord c u r e is given in Fig. 1. 071 ~ 10 p& ZnSO, and u - ~ s - S ~ O H ;.\ftcr mixing. the solution waa centrifuged a d 'Wml. of the :tupenm.tmt liquid hydrolysed by hesting on a s ~ bdnth (105') *Pith 50 mL 5.u-XaOH for 40 min. Solutions of hydrolysed phenylmerc~pturicacid in urine were prepared by heating phenylmercapturic acid (100 mg.) in a mistme of 40ml. ofthe aupernatant fraction of the urine and 10 mL of 5x-XaOH. The solution waa cooled and made up to 200ml. with hydrolysed urine. This .whtion wnd equivalent to 0.5 mg./ml. phenylmercapturic acid. It waa further diluted 1:4 for estimation. The standardcurve in urine(we Fig. 4)waa then prepared in an analogous manner to that for pure solution but omitting the gelatin, by taking 1-12 mL ofthe solutionof hydrol p d mercaptrricacid(O.l25mg.,!ml.)inurine, and 11-0 d of the hydrolysedurine. Recoverim of phenylmercapturic acid from urine were studied NL foUows. Rabbit urinm (24hr. samples) w e n diluted to 2013mL and \-3nOUS volumes (up to 50 ml.) of a 5 mg./mL aqueous solution of phenylmercaptuic acid added. followed by 1OOmL of 10yoZnSO, and jomL x-XaOH. Each solution was diluted to 400mL with water. mixed and centrifuged. T h e aupematant solutinna (16G d) were then heated on B sand bath with 40mL 5r-XaOH for 40min. made up to 200 ml. with water and the phenyl- mercapturic acid estimated both turbidhetrically and iodometricallyaa above. If the opncities in the turbidimetric method were toogreat,suitable dilutionr were made, prior to the formation of the phenylmercuric mercaptide, with hydrolysed urines The I, titre of the normal diluted rabbit wine containing no added phenylmercapturia acid waa a h determined. In the turbidimetrio method the normal b l a n h were negligible. The recoveria of phenylmercapbc acid from urine were 3!k. yo (iodometric) and 93 yo (turbidimetric) a t levels of 1 W 2 5 0 mg.'% hr. urine and 91 04 (iodometric) and 89 ?& (turhidirnetric) a t 20 mg.l-24 hr. urine. Experimertts witla benzene b n a n c n o n of phcnylmeropuricr a d (trgJml.) 4. Standard c w e n for the turbidimetric eatimation of henylmeccapturio acid. A in p~squmue solution, in hydmlyaedrebbit urine. -_. Xn Urirrc. 1lthe above p d u m bapplied to hydraa b b i t urine diluted 1 in 6 the pheoylmsrcnrio mere ~ p i d l yumgalateu. Stable OQPWS~OM ware resdily h o w m , if the gelatin wvxe omitted. There b, e Ipophilio sdmtanca in rabbit urine whioh im JIercapturic acid urrclbn. Rebbita kept on a diet of 75 g. cubes ( h i a t e dLondon FlourU e n ) were given benzene auspendedin 50 mLwater, by mean5of a stomach tube made of polyvinyl plastic. urine waa collected every 24 hr. and analysed both iodometrically and turbidimetridy. For analyeiaeach day's urinewaa filteredand made up to 200 mi. with water. To 100mL of the diluted uxiae t h u s 11- added 50 ml. each of 10 % %SO, and 0.5r-NaOH. The mlutiom weremixedand centrifuged. Thecrupernstmtliquid (80mL) WM then heated on e mud bath (at 1050) with 20 ml. of 5 n-NaOH for 40min. After cooling the ~ i ~ twaia om d~e up to 100mL with rater and 60 d waa d for iodometno eatimetion and 12-5mL (in duplicate) for turbidimetrio eatimation. (During the tihti~the~d o u r of thiophenol WM dwaym mident.) For blrak artinutionn, 40mL of the unhydrolysed urine, stbr h t m a n t with ZnSO,. WU ared for the iodometric blank and 10 ml. for the kvbidimetric blank. Table 2 d F i g . 6 give eumplerof indiridudmdt.. whilst Table 3 g i v a 8 mmmwy of all d b . . reaulta given by the iodometric method appear to be due to ._ 623 & D.1'. PARICE AXD R. T. WILLIAJIS Rabbit Table 3. Phenylmercapturic acid czcrdiorr by m b b b receiving bcnzcnc * Benzene fed .Phenylmercapturic acid (PA)iodometricdy P.1 excretion estimated turbidimetrically f 5 63 M 1. T h e esc a- mbbits remiv -. 2. The p r d e after . IS 2.0 60 1-2 19 20 1-9 53 14 31 o1..18'1,-.Average0.98 21 1-66) 97. 1.9 38 0.8) Twodaya. hydroryquinol, a known metabolite of benzene (Portscms& \Filliams, 19496).Thisconclusion was reached by testing the OridEed with diluta H,SO,, do not react with 1, or @re a precipitate aith Y&4. Hydroxyquinol, howerer, d w rractaithI,foraekuad,forerarupIe, that 100 mg. hydrog. quinoi in 60mL 10 yoaqueous ethanol acidihd with dilute H,SO, immediatelr reduced 15mL of @01~-&and then absorbed further I, more slowly. Hydrosyquinol does not form a prrcipitats with HgCl,. It should be pointed out. however, that the dXerrnce between the t a o methoda h only appreciable There the mercspturic acid is a minor metabolite and ia probablF insi@cant where memapbrio aci& are formed in large amountsan in the case ofthe halogmobenzenea (ae-e SteLol, 1936; Spencer& Willisma, 1960~a.). DISCUSSION The prssent paper shows that phenylmercapturic acid inametabolita ofbenzenein thersbbit. Eetime- tion by the more epeaific ttubidimetrio method s h o d that h u t 0-8 yo of the bepzens fed et a dose level of 1g./kg. and about 1 yoa t the doee level of 0.6 g./kg. b excreted as phonylmercepturio acid. Ueing Qurw obtained in an eerlier papem (Parke & WiIhns. 1960) it CBP be calculatd that, allowing for the eliiminated unchanged. 2.2 yo of the benzene ostabolizud is ercrrrted .a mercapturic acid a t a does of 1g./kg. and 1.7 yo at@6g./kg. . -. .-- - -- ... . . .. r PHESFE'r4JIERC.4PTURK! ACID IX G R I S E ...,. We can now add one furthor figuw w our balrrnce sheet for the metabolism of benzene at 0.5 g./kg. - -.d, ---.L... ,. ?,. -*# -: Benzene eliminatad unchanged Benzene oxidized (andexcreted as glucuronide and ethereal adphates) Benzene eliminsted an phenylmercapturic acid 01 dO 39 21 1 -3 preliminary results have shown that another I metaboiite of benzenet muconic acid, accounts for St least 8further 1 yoof the dose. St3IJfXRY I- %-- . 1. The excretion of phenylmercapturic acid by rabbits receiving benzene has been studied. shos\;nby clcveiopingcr sprcitic rurlidirndr:v .WLILWI of estimation. based on tho formation of phenylmercuric mercaptide from phenylrnercnpturic acid by alkaline hydrolysis. 3. T h e iodometric method for the determination ofmercapturic acid has been shown to be influenced by diet and by hydmxyquinol, a metabolite of benzene, and may give high reaulta when small amounts of mercapturic acids have to be estimated in urine. 4. By the turbidimetric method about 1 9.b of rke . benzene fed at 8 dose level of 0-5 g./kg. and about 0.8 yoat 1g./kg. was excretedaa phenylmercapturic acid, which mas found in the urine for 2-3 d a &~r dosing. 5. Some arylcysteines and mercapturic acids have been syntheaized d their ultraviolet absorp- tion spectra recorded. --&a- Ihummond, J. C. & F i i . L V. (1937). Biochurr J . S2.59. Stekol. J. A. (1936). J . bid C h 113.279. ? .. BY P. ALEXANDER,' D. C-4RTER, C. EARLAND AND 0. E. FORD Research Depart~nent,JVoZsey Ltd., Leicester ( R e m ' d 18 July 1950) f i d y with the c a r b ~p~u~pls in proteins without modifying any 0th- group. Methanol containing 0*1N-hydmChlOnCscid wus UBBd by Blackburn & tumm group la Fra8nkl3l-conret (1944)found thst 1:2*pOxidea reedily eateri6ed csrboxyl group in prateinn ead ehowed that, aithough epoxidee ale0 combiae with amino gmu* their btnio nakus hnot mppmmed and the maation ie themfore suitable for studying