Document v16oVwdGRRGj9BdMrMo1aeoJb

f PLAINTIFF'S gf EXHIBIT /&'Ob i! .I- - 1. -idam, N.IL (1923). J . chem Sw. n.io. . k h i b d d , R. >L(1913). J . b i d Chem. 159,(193. &chibald, R.M. (1946). J . biol. C h . 165,443. lf.L k IIarl;ios. W. D. (1M). J . &m. Phya. 14, 640. corrin. JLL. & Harkins, \V. D. (1945). J . .imer. chem. Soc. Krebs, H.A. (1948). Biochcm. J . 43.51. I Lerene, P.A. &Taylor,F. A (1951). J . biol. Chon. jg. :ab; Mci3aain,3. JV. (1944). Colbid C h m . 5,102. Putman, F. W.(1948). Adwne. Prolcin C h .4.79. Piahton, A. W.(1946). Ann. N.Y. dcad. Sci. 48.3.51. Scott, F. B. &Tartar,R I-.(1943). J . dmcr. chem. .+. s. 69, GS3. Gale, E.F. (1947). Bt2tm1.J. 43,Pii H d e y , G. S. (19B). Tram.F A YSoc. 30,444. Hughes. D.E.(1948). J . bid C h n . 176.15i3. Hrrghea. D. E. (1949). B i d e m . J . 45,313. &mm, 0.k Xamel. C.S. (1920). J. AM. chan. Soc. 42, 299. 692. Vsllee. 15. L. & Gibson, J. G. (1948). J . b i d . Chm. 433. Vickery, H.P.. Pucher, G. W.k Clark, E E. (1935). J . bid. Chem. 109.39. Vickery, E P., hcher. 0.W.,Clark. E E.. Chibnall. L FFestall, R. G.j1933). Biodccm. J . 29,2710. QdO \ 3QC+ fiicoi 7 ' IC1 so 4236-243 6 Studies in Detoxication 30. THE 3lETABOLIS31 O F BESZEXE. (a)TEE DETERlIINATIOX O F BENZESE. (a) THE ELINEXTIOX O F b%-CILASGED BEXZEXE BY RABBITS - _r - 2i - ''1 --a BY D. V. PARKE R. T. WILLIA3lS Deyartnient of Biochemistry, St Mary's Hospital .Medical School, h n d o n , IF. 2 (Received 14 September 1949) - -- In general. the metabolismofbenzeneproceedsalong That orally administered benzene might be esfour paths, namely, (1) oxidation to phenols, c r e w unchanged through the lungs was first (2) elimination unchanged, (3) formation of phenyl- gested by Schultzen & Naunyn (1867), but Uunk mercapturic acid. and (4) ring *ion to muconic (1876) doubted this possibility. In 1883, Xencti dr acid and other producta. The first of these w 1 ~ 9 Sieber. after experiments on men, dogs snd rabbits. quantitatively studied by Porteous & Williams expressed the view that approximately one-third of (19491, who concluded that in the rabbit an ever~lge benzene wna excreted 88 phenol conjugatea, one of 21 yo or, at most, about 30% of an oral dose of third as catecholand quinol and the rewining third benzene at the level of 500 mg.lkg. waa metabolized must come out unchanged. The firat quantitative to phenols. At least 70% of the admitered ben- experimente on the elinination of unchanged baa- mne muat takeother paths. The prwent work desle zene -re carried out by Ishmenn, Stohr,KIeinerL with the second path, the elimination unchenged. Gundermenn (1910). h h e o t o m i z e d rabbi& 'Rub It h found that at 8 Ievel of 500mg./kg. S h u t 40% dowed to inhale benzene-eir&urea, and from M -red benzsae h eliminated up- d p h of the +ired and erpired8U. the changed through the lungs. Thna the first two paths of benzene dxmrbed WBB ascertained During 1hr. ! CBP account or 86 least 60% or, taking the higher after benzene ceased to be inhaled, 25% of the valuee of some experimente, up to 8Cr-WY' Pf the abaorbed bemzenewas exhaledunchanged. When t b . -period waa increased to 4 hr. then 34% was exhaled. &e benzene. p p i g s inhaling lungs in 3 min g was not kl observation on the kidney ;i;tcdthatfieebenz mug2gabbiS only a h a the only r -ation of free + $ (1910). but d c only lasted 4 @tion waa st th whr. Ourexpert of benzene w *!rb-enzeaewssad pvlner by the oral 9- L.- EA A. T l r e i fie+ methob. benzene the mer 935) isverysensit to this. FPeltzer (1933) dc gdhtrOb0nZene .ooncentrated*l F Y a n t (1936) de determinetion of thismethod benze .misture of conce -idthe m - h i t r o fiom the neurral ketone. A purple 4ueous 407; (w' (1943). Alekseeva described modifx. Themethodof -$pg.ofbenzenew %3long time (1.5 h eolour,which &e haw now inv c3 . - / , * . - ; I /,! L / ' c- ,. --;{-It;' L OF BGVZELUE 237 condense with the e n d f o m of ketones to form coloured quinonoid structures. (1) The choice of &one. In order to ertrsct m-dinitro- benzene from the neuMitprl uitrating mixture, whilst avoiding such aolventa aa ether. L ketone incompletely miscible with water WM necesssrp. With aliphatic ketone3 it wan foundthat the intensity of the colourdiminished with incrensing number of C atoms. and we were immediaalp limited to methyl ethyl ketone and &hesarione. Sin the coIour developed in cyd4hUsnone waa much more sensitive to alkali concentration than that in methyl ethyl ketone (Fig.1). the latter ketone becnme the obviouschoice. i .. a more controlled EXPERDE"IXL A. 2% determination of benzene - mveral methods available for the determination ne the method of Schrenk, Pearce & Yant verysensitive, andwedirected our attention (1933) described colour reactions between benzene and ketones in the presence of t (1936) developed a micromethod for the ream of air into 0 / 1OU)u)4050~ lima (a4 Fig. 1. The inauencsof allrrliconcentration on the stability ofthe colourformedbetaanmdinitrobenzeneandmethyl ethyl kctone or cyclohexanone. The data for this figure wm obtained by taking2 mL of p-dinitrobenzene solution (40pg./ml.) in methyl ethyl ketone or cqrchhelanoneand adding0.1m l of ethanolia NaOH (concentration0-4,0-5, 0-75 and 1% (wlv)) and adring up to 10 mi. with the ketone. Reading wem taken at internah with the SpeWrer absorptiometer. Final alk& concentrations are given on the figure. Precipitation oc~nrredin some of the cydohexanonesolutiona Full linea, methyl ethyl ketone; broken lines, cycbhesanone. colour development. discussed 1. ie that, in the p"en0e of (2) The d u r e ond conunlmlwn of the alkali. The strong organio basea piperidine and benzylamine gave no colour with m-dinitrubenzene in ray of the ketonea tested. Xa ethoxide in dry ethanol geve an unstable violet solution which rapidly turned bmwa owing to reaction between the ketone end ethorida In order to amid obtaining L twop b system which mulb h m the uua of aqueom alkali we tried ethanolie NaOE snd KOH. In general, it ~*sd fonnd that the c o h r waa mors atable with NaOH than with KOH. In methyl ethyl ketma thebolour ia completely deatmyed in 1- thnn 6 min. if tha aLali concentration ir greater than 0.5%. Betaesa 0.05 rad 0.5%. there may be pdpitation of alkali. Below o.OoZ% alkali the full colonr ia not damloped. Some of our mdta are ahom in Fig. 2 whioh again shorn the differencebet- cyclohexanoneand methyl ethyl ketona The concazltmtion of rlkli W y sdopted waa O.ooS% NIOE (3) The effed of CLAOrd "he rhbility of the mlour ir a h aEffibdby ethanol, 10- of ahioh had to be d k r r a e the k t alkaliW t foundmue t h r a o l o NaOH and uome ethanol waa necmssuytolruintrio L homogeasoar 8pt6m. The effect ofethano1concentration ia uhomin Fe.3. It m a found that if the ethanol concentsstion in much below A% -. _-_. - pmcjpiiJ&n of ti;,: r&Ji ~ N I I X - I . ..\--'ne~:irf ~it rtsc.c.r~is 2% the maximum development of colour is impaid. Methanol can be.aubetitutedfor ethanol. (4).fled o/ tmkr. Since, in the determinationof benzene, the m.diaitrobenzene formed by nitration luu eo be es- tracted with methyl&hyl ketone f h m an aqueous solution, the iduence of the concentrstioa of water on the deveiopm a t of the colour had to be in9eSti@d. The ruulta are w, A' I '& I Qoos I 00075 bnctncrarion of alkali (tr) Fig. 2. Effect of XaOH or XOH concentration on the intenrrity ofthe colourfrommdinitrobenzene andmethyl ethyl ketone (brokenline) or &hexanone (full lines). Solutions prepared as for FB. 1 and mad 5 m h after mling. ketone. The colour WM developed with 1 mL of I)+$.; ethanolic &OH. volume made up to 10mL ai& & ketone and readings taken after 5 mh. I I II I I I I 0 102030105060 Tim (mln.) pig. 3. Effect of ethanol concentretion. !Fhe carvej w m obtained by adding to1 mL of kdipitrobenzenein methyl ethyl ketone (loo /zg./d). 0.1 mL of 1%. 0.2 mL of @6%, 0.6 mL of0.2% rad1d.Of 0.1yo~thrnolioNaOH rupectively andthenmrLingupto 10 mLwiththe ketone. The colour ma then rcad in the abaorptiometsr at in- taroak Ethanol ~ a n c a n t r s tai ~m~givcm OII the figrus -.. The alkaliconcantmtiw waa conatant throughout. * ed in Prig. 4.Water in c(I, onmtmtitl up to 0.6% hu noin0uurceonthe dour. Above this conccmtrrtion,the d o u r in dowur to derdop and thaFe in (I, l a r of intensity. - .- Binoe the solubility of nrhin methylethyl *ne u 10%. theoriginal ketone extract mnmt be diluted at l s y t 1 : with ketone prior ta caloar development ta thrtthe concartrationofwater ia ha b 0.6%. (a) Theinj?umuo/o-cudpdbdro6cnrcnc h thepresmt - . wwrk the nihticm of baarsne w u crnied oat at raw tempmature (18-269 with am& H,SO, and fumiag FfNO,, 10 mL with the ketone. The ooloarr a~ ~ .. 1. Thcrccovayof t -4. Benzene slowly ?. Benzene dded bg-) 435 413 309 303 466 902 832 d o t i of the atundai 239 le 1. The recowry of bsnzerre added directly to a nitding mixture Benzene recorered am deaaibedin the test, p. 239. the ketone and midng. the mbur wu mad, after 5 min. standing, in the Spekkar aimorptiometez wing the colour filter Ilfordno. 605mdheat aterno. E503 and a 1cm. cell. A standard blank wm prepared by miriag 1 mL 'dkali reagent' with 9 mL methyl ethylketone.The curveobtained is linear from 0 t o .cO pg.110 mL and gppmsimptel~linear ion of rhc stundard cuwe f o r mdinilroben- fill Recovery of mdinitrobenzens from the nitrating mizlwe Varioas amounts of pnm mdinitrobenzene were added to 20mL of the nitrating mixture and left for 2 hr. The mirtum was diIuted with an equal volume of wafer and Table 2. Rccaccry of added mdinikobaztns fmanikcrtingmiaws driedorsrdy- f on of b.p. 80-810 Added to 20 mL of Reaorared I th the 40% qu60ru N d H . the tsmpemturs being kept below W under thetap. Afmdropof thek L i were then addedinex- Themixture, which hd(L volume , WM e m t a d psi& 2 ~ 1 5 d a. d A n i d . Chinchill0rabbits, 23kg. in weight, WM ased The animal chamber (Fig. 7) con3isted of a Pe-s tank of dimemiom 60.6 x 336 x 22.5 cm.aith an internalvolume of45-6 L The lid of the tankwan removableand was secnred by twelve wing-nub to the tank w a b 4th a rnbber diaphrsgminbetweentoensansirtightness.ThisdiaphrsPm -- tlie'draught nt the air inlet and produce a current of air acm= the tank, airwas admittedthrough a3 mm. g b tube fitted into the g h tube dingonally oppmite the copper outlet tube. The other copperair tube w m d e d Th.e-tion kain (Fig. 7). Thin wnsbted of two 50 d I)nsehelgao bottlea in &ea with pdg h hesds each containing20 mlof I! mm. gIaasbeada and20 mLof nitrating miannr A third bottle, oimilarly sed, wag connected in pMuel and WM used to check the in-going air for buwna Table 3. Recovny of benzenefrom the reapirdwn drambcr - Benzene in Rataof h t i o n o f -tion ' suation With s M y killed rabbit in the chamber (mtext, above). of the animal and re.: . . ... Table 4. The eliminatwn of unchanged betrrenc inthe expired uir of rabbits receiving benzcne orully Duration of experiment (hr.) Percentage of dose recovemi (coorncted for 0- and p ditrobenzenc) Benzene catabolized Injected. 31.0 64.3 60-9 69.0 3;i.i suspended in S o d . water by a stomach tube made of `Pcrtex' p o l m y 1 plastic which doea not absorb benzene - __--..-- . ..-.. . ..... . . -. ._ .,' --, gij a. .. * wem a n d y r d for dinitrobenzene. The second bottle rarely contained more than O2yo of the dose of benzene. The mults ore summarized in Table 4 and in Figs. Y and 9, the r c s u l t ~for rabbit no. IS are espressed graphidy. It is to be noted that the elimination of unchanged benzene takes long& when it is injected than when fed by mouth. E-Wn of unchanged benzene in the urine catabolizes benzene at about 350 m?.22. two-thirds is elinhated benzen2 at 500 mg./kg. is oxidized glucuronides, 9.5 ?/o as ethereal & Williams. 1949) and 39-1O First we required to know whether benzeneadded to urine could be recovered by aeration. Accordingly, 165 mg. of benzene were added to 100 mL fmshlp voided rabbit urine in a Dceschel bottle and thoroughly mired. A current of air WM then padeed for 6 hr. at 20 l./hr. through the urine into the absorption circuit and the mdinitrobenzeneestimatd as befort. The benzene recovered wm 145 mg. or 94O6 (corrected d u e ) . It was noticed on some occasions that ahen a rabbit in the t.pL voided urine there WUI I a jump in the benzene elimination. This suggested that some unchangd benzene appem in tho urine. Pabbits rere therefore given 5oC) mg.1 kp. of benzene omllr; placed in metabolism age3 and the urine collectedin M e l bottlesplrrced beneath the funnels rupportingthe csgs &.a soonas urinewaa passed its benzene contentWUJ estimsted by emti011. It vaa found that at this dow level no unchanged benzene appeared in the urine. DISCCSSIOX rabbits, and, if all the estirnatio bolites could be carried Out s h u l t The problem of getting IL balance sheet ;;;r bolism of benzene is. however, one d -?I. owing- to the scatrer of rhe results: :LY -x. merage d u e s obtained 50 far OW iut;j.r~m: As glucuronides As etherenl sulphates Aa phenol Unchanged benzene Po) .&.2-'12.; 5.9-1B.J 8-3-174 %-O-XO The eliminationof unchanged benzene by the rabbit at a dose level of 500 mg./kg. appears to be entirely via.the lungs, no detectable benzene appearing inthe urine. The elimination at different dose levels is Other kspects of the netablism of as the formation of muconic acid mercapturic acid, ace being actively p summarizedin Table 4. Since the anima?3were kept in RP enclosed chamber i t is possible t h a t our estimates of the unchanged benzene are slightly ! m ~ , 1. Tile m&thodfor the de:ermh;.x: :: f because the animal will rebreathe and absorb some of the benzene it has eshaled. At the dose level of ment of a colo 500 mg./kg. the average concentratiou of benzene in benzene and e the chamber is aboiit 4mg.D. at t h e period of masimum excretion. Honever, basing our calcu- lations OR the experimental findings of Lelimann et al. (1910), we have assessed the amount of benzene reabsorbed during an experiment as being not more than5-10 mg.,despitethefactthatduringmaximum excretion the total amount of benzene breathed in would be 120-140 mg.jhr. Our resulta on thin basis would not be more than 0.6 yolow. The striking featam of Table 4 ia that at the high dose level of 1g./kg. a much greater percentage of the dose ia eliminated unchanged than at the lower levela. This indicates that there is a limit to the amount of benzene which the rabbit cau metabolize. This shows more clearly in the laat column of Table 4. The rsbbit therefore deals with benzene by el;minatincl it unchanged and by catabolizing it, 3.0. by oxidation. The second way is, however, quantitatively limited,and i t appears from Table 4 that it doeenot exceed about 300-350 mg. benzene/ kg+f rabbit in about 24 hr. Thus up to 600 mg./kg. about two-mhaofthe benzene adminietared cornea 2. The elimination o rabbits receivingbenzene has been investigated. subsequently determiningthe m-dinitro dud 3. The benzene was eliminated dUrin about 40 % being recovered uncb4& levale of 0.25 aud 0.6 g.fig. and Myo at 4. The resulta have been d i s c d in out unchanged and three-fifths i s catabolized. At 31. THE I CYLY OF Deua are also The expensesofthis work were in partdetrayed br P grsot REFERESCES X. V. (1940). C h .Z. 1.1712. Pearco. S. J., Schnk, H. E & rant,W. P. (1936). h p . ,H. D.(1943). Id&. Ewng C h .(i5nal. d). US.Bur. Xin. no. 3302. Peltzer, J. (1933). C k 2.57,162. . w.FDdsLrry~bb. 11. Wi. Pernunet, 11. (19%). J . PAarm. Chim, Paris. 21.303. (1938). Bkui~emJ. . 92 79. Portcow, J. & W i m s , R T. (1949). Bioehm J. 44. IJ. & b m v , J. 3.(1931). 46. SchrraL. E K. P ~ a r aS. . J. & rant,"7. P. (1933). &p. Kleiner, R. & Gundermann. C.S. Bur. Vin.. no. 3337. . Schultzen, 0. S Saanyn, B. (1887). Arch. -it&. Physiot. .. i. J .3 f .-? -. _. Studies in Detoxication d 31. THE ISOLXMOZT OF m- Ah73 p-CY-AXOPHEXOLS AS XETABOLITES OF CYANOBEXZESE (BENZONTl3,ILE) AND THE PROBLEX OF THE n+- ORIENTATION OF HYDROXYL GROUPS FORJIED IN VIVO -.-- * -I L iLh uf .Dsa BY 3. X. SXXTH AND R. T. TVTLLL-LB Department of Bioehtmkiy,St Xary'a Eio4piLal :Medical School, tondon, IY. 2 (Received 16 September 1949) * m,onosubstituted benzenes c a q i n g the stituted phenols. or both, ifiespective ofthe nature &-pm-direothg groups (0.q. ofthesubstituent. ,03)arefed to animals,it isfoundthat the Now Smith b;\VilIb,u (1948) pointedout that the conjugatesof the correspondingortho- orientation of hydror$ation of aoet@dide-atad tituted pheaols. or both, e.g. acetanilide aniline in the rabbit vas similar to that foundduring ,ISM),aniline (Smith&Williams, nitration, i.e. pcrm in acatadide and ortho :arton &williams, 1949)and chloro- and p m in aniline. This suggested to US that one 5 (speocer & Williams, unpublished ~beer- possibleinterptetetionoftbeonentationofbiologic s).Little definite hfonnation, however, is hydroqlation could be dong the k e a used to bb about the position of biological hydroxy- explain aromatic substitution in purs organio esi-'tutad benzcmse cemying the chemistry, i.e. that substitution ia either ionic or meta-dhctiug groups (0.g. NO,, CN, dhg to Meyer (1906) 89ullIamocmte hsueb-srtiatudtiiodnimnenHsteuyre&(fworeckikaw, m19*o3n7,1o9ff4r8e;ew-r8ated4ircsl -hen01 owur in the urine of rabbita re- 1948; Dewar, 1949). whilet other inkpretationn of pOg~benz8w,butthiswaenotamequivody the orientatio~o~f biologicd orid&ion (M eleo humanu (1863) isolated m n d amounta of possible, we studied the f&e of ~yenobsllEsnaa9d benzoio acid with the purely o h e m i d theory of (Mmstic mbstitution in mind Working on these asmm3ptions we thought,tbat if the orimtation of ~~meybethefirstproductoftheoridetionhydroxylation in oioo wera known for both orsho-, ~ ~ d d b ~ o e r h i n b s d s r i ~ ( ~ e l e o pmS r -r ,and mcb-d'usating group, then wd aould && Evans, 1949). The idonnetion available decide whether biologicd hydroxylation had the that monoeu&tituted tummme, iforidized characteristics of ionioBubstitutiDnor of free-rsdical &b in vivo, give riee to ortho- or pam-aub- aubstitutioa,lwmudnpj, of anuae. the& the hydroxy- 3 *E4 --?2 4& '? -- 1 -..-----., 2 _---.- ------f.s -:*bV? km ---.- --. . .*f_ -43y -... I - -.- -1 i.-g- :Id .----. -* <;-?A