Document gb3k6g931aDZraMJeJYoZ8KOe

L-h". '**- ..mi' Titc J-u'Kv.a or T'.bm.ouu.wi, Cjil\*>tmi Xc\. :*31, N'o. 1?. IV.- T-.^r 4/1 Metabolism of Biphenyl and 4-Clilorobiphenyl. in ihe Rabbit* AYai.tku D. Bj.ock and IIehukut II. Couxi.sh From the Department of Dermatology, Medical School, The University oj Michigan, Ann Arbor, Michigan (Received for publication, August 21, 1959) The ability of the rabbit-to metabolize naphthalene a rid chlorin ated naphthalene was markedly influenced l>y the degree of halogenation of the compound according to Cornish and block (2). Naphthalene is related structurally to biphenyl, in which a coaxial linkage connects two phenyl molecules. Whether the presence of a chlorine atom in biphenyl also alters the metabolism of this compound as it does in the case of naphthalene has not been reported. Stroud (3) and West, el at. (1) have studied the metabolism of iinsubstiluted biphenyl respectively in rabbits and in rats. The present investigation is a comparative metabolic study of the urinary end-products of biphenyl and 4-clilorobiphenyl in the rabbit. FArKniUUNTAI. Tbc compounds biphenyl1 and 4-ehlorobiphenyl' were recrystailizcd from alcohol until the melting points were constant, 09-iCP and 7(5-77, respectively. Six male albino rabbits (2 kg in weight), fed a diet of oats and cabbage, were used for each compound studied. Twenty-fourhour urine samples were collected for a -1-day period both before and after administration of cither 1 g of biphenyl or 1 g of 4-clilorohiphcnyl in torn oil given by stomach tube. Each animal served as his own control. The urine samples were analyzed for their content of the fol lowing metabolites: glucosidiiror.ie acids, phenolic compounds, ethereal sulfates, aral mercuptunc acids (neutral sulfur). Creati nine content was also determined. Ghicosuluronic acids were as.-ayed by the procedure of 1 lanson el at. (5). Phenolic compounds, wa-re determined with the J'olinCiucalteu reagent (tl'l, wifii -1-it\dniwbipl.inyl as a standard. Sulhir partitions were doc.e by the barium sulfate precipitation procedure of Ed!in (7.1. ('reatinbie foment was determined by a photometric mudil:, -ation of Fchn's picric acid method (s). Tie- amount ol the mclabnluc e.\eietod be each rabbi! during ihr 4 days win u lieitlu i biphenyl nor -t-rbl.uuliiphenyI was fed, was :;ubt re.otcd from the amount. c\erctod during the 4 days after the ciimpound was fed. '1 hr increment, which was presumed to lie cau-'-i d hy tei diiig i.f l!ie hyc::i-carbon, was calculated in terms of milligrams of either hiph' uyl i.r 4-chlori.biphcnvl. For studies in which sec era! ui the excretory products derived from biphenyl ami 4-i I.lure!/:],la n\ 1 wen.- isolated from urine, 1 g of pound was fed ('i rabbit- fed each compound] and the * 1 hi : work was .-copor: ,-.l in part hv Foiled Slates. Puhlie llcslih mavire l.ranl ttf <-1. A piebiuiaaiy repori has heen ' -d ,11. 1 1J..j-hi'ti\ 1 was otilam, d fii-.ii l.a-ancm Organic Chemicals. Hi.-iiI ton lV.ilocl~; tachi-'.M.s. Ian !h--i. r, New V'-r];. -1 C Ido"' -i, i 1 -1:011 \ 1 c,;e e!il aim .| 11: sm; :'.h lie eoillle.-c of Monsanto <`l., :.,i. a! Comj'.anv, Si. I.o-ii-g Ati-ouri. urine collected for 4$ hours. Urine collected from animals fed the same compound was pooled and frozen until analyzed. An aliiiuot of the urine was adjusted to pH 7.2 to 7.3 with 10% Ne.OlI and continuously extracted with ether for 2-1 horn's. The ether extract was evaporated to dryness and Hie residue taken up in hot absolute alcohol and water added until the solution became, turbid. The mixture was placed in the rold (15r) for 24 hours and a crystalline phenolic compound obtained. After several reerystallizations this compound gave a constant melting point. The ether-extracted urine was concentrated to one-half its original volume under reduced pressure, acidified to pi I 2.0 with 10% 1I.C1, and placed in the cold (15) for 24 hours. A yellow precipitate high in glueosiduronie acid content was se cured. This material was recryslaUized from hot absolute alco hol until a constant melting point was obtained. UESULTS 9 Metabolic Studies Table I shows average 4-day exeretion values of various uri nary metabolites both before and after ingestion of biphenyl and of 4-chlorobiplienyl. Because normal daily excretion of these metabolites is variable, changes in exeretion of less than 20% are not considered meaningful. Amounts of ingested bi phenyl and 4-chlorohiplienyl accounted for by the four urinary metabolites determined were essentially the same, 039 mg and G17 mg. Approximately 20% or 202 mg of the biphenyl was excreted as the glueosiduronie acid derivative, but.. 50'% or 505 mg of the ingestcd 4 ehltirnbipheHylwas excreted in this manner. Free phenolic compounds in tbc urine accounted for 243 mg of the ingested biphenyl as contrasted to .30 mg of the 4-chlorobipheiiyl. Ethereal sulfate excretion accounted for 1-3 and 11 %, respectively, of the ingested biphenyl and 4-ehlorobiphonyl. Xciilier biphenyl nor 4-eblorobipheny] seemed to be exi ieled as men aptuire acid derivatives. Isolation Studies Hiphenyl Mt tnbdiles-- 4-1 lydroxybipln nyl (m.p. 105) was iso lated from the ether-soluble fraction of urine excreted by rabbits fed biphen\l. Tiie acetate and benzoate derivatives of this rompoiind were prepared and found to ha ve melting points of SS and 151 h respectively, which arc consistent with values reported in the literature (9). Hiplc iiylghico.-illumine acid (m.p. 1 S3-- IN I' ) v.-c isolated from the ctlu T-inSoluble fraction of the urine. 11 vi holy sis of 11 io (i-l a poii in I w nil acid or)> ghtcnionidase \ icldrd -1 -hvilro ,ylliph'-ayl (m.p. lEV). Addition of Known ! hydrnxyhipl'e-nyl did not d'-;iri-`s the iiualuc; point. Acetal.' and I <-.)zoa to deiio.il j\< ; of the oluled -I hvuVo'.ybiplit nr 1 bad m.-lung points ol S7-Nn and ] III", rc-a..lively. DSW 192857 STLCOPCB4052025 02 Mrlahnlixm of HiplmpiI cid i Cltliniil'iphi nill Yd. No. 12 Tuu.i; 1 I'1'" ! of 1`ngciion of biphenyl and f-tfdinnbiJ'f.tn yl on csiretion of urinary is. !id..,litr\ by the i\d>bil KOk-umI M<r .in* solute i tu;i ,n:il l OIucomM- I Tree j utonio j'Snolo aci.l ' y --y' Toial i inrrc.nciit r? . r. ' es-r! as (>m y a 'puml fcil E? 3 t z- 3 ?.ti 3 J u, n I if c I3 Control.. Biphenyl. Contiol......... I CMorobi- 1'ilifiiyl....... MS "IS* *4 37 71 65 134 75 1i 41 IIS' 1 1 5S 112 891 * tf`4 519 311 Sol 202 3S1 2(3 G39' G1 512i 291 1)82 505 .300 017 05 * The values represent the total -l-day excretion. Each value is the average. of results obtained on six different animals. Hydrolysis by (jghieuroiiidasc of the plucosiduroiihles i-nlated from tie' urines of animals fed either compound indicated a beta linkage v'ith the ghieosuluronic acid moiety. Although lii|iheoyl is related structurally to naphthalene ir> that both are composed of two phenyl-groups but with differ. i,` linkages, the pathways for metabolism, by the rabbit, of the two compounds are dissimilar. Cornish and Block (2; reported 19', of the ingested naphthalene was excreted as mereapturie ;ni! derivatives, anil h",<, as free phenolic compounds. In the pre.vur study, no increment in mereapturie acid excretion caused hv feeding biphenyl could he detected, and, as mentioned hefme, free phenolic derivatives were an important excretory product. Cornish and Block (2) did find that more. /-chloronaphth.dcna and dichloronaphlhalcne than naphthalene was excreted as the glucosiduromde. The present study woidd coe.f'rm the. finding that glucosiduromde formation lwoomes more important in metabolism of the ingested hydrocarbon by the vahl.it when the compound is halogcnatcd, and that apparently the rabbit lacks a mechanism for (lie. deludogcnution in vivo of such compounds (2). 4-Otlorobipln it*/! Metabolites- - l-(/i-Cldoropheiiy])phcnol (imp. l-l(i') was isolated from the ether-soluble fraction of urine ex creted by rabbits fed 4-cliloiobiphcnyl. Acetate and benzoate derivatives of this compound had melting points of 110 and 1S2 ', respectively. Those values are consistent with those reported in the literature (10). 4-Cldorobiphenyl glueosiduronide (m.p. lSi'd; was isolated from the ether-insoluble fraction of the urine. Hydrolysis of the compound with acid or /3-glueun>tuduse yielded 1-(p-ch!orophcuy])phenol (m.p. 1-15a) whose acetate and benzoate derivatives had melting points of 110-111and 181, respectively. The isolation procedures used in this study do not rule out the presence of other metabolites of biphenyl and 4-cldumbipheiiyl in the urine of rabbits fed those compounds. DISCUSSION' The resulls of this study indicate that 4-rhlorobipheny] is metabolized as readily as biphenyl by the rabbit. The presence, of a chlorine atom in the 4-position of biphenyl, however, do-xs alter the di<t rihution of the urinary metabolites of the compound. Essentially twice ns much -1-ehlorohiphenyl as biphenyl was ex creted as the gluensiduronic acid derivative. The isolation stud ies indicated that the glucosidurouie acids of both 1-elilorobiph.-nyl ais.l biphenyl were funned by conju'.*,ati<ni m the 4- or p.ini-po-ition. Oxidation cd the -E position of the 4-chlurobiphet.y! to a phenol gTOiip must, have taken place ill order for lb- c.lm .i.-idiuoiiide to be formed, and 4-(/>-eh,.oniplu-nyllplieii..l "as i'olated lrom the ellier-.-.o!uli!e iraetion of mine eolleete.l fr.an rabbits fed -i-<Til.'r.>1 >110 11 vI. Nevertheless, the animal piektint i:i 1 lx f, irrned the glm o id'iron ale d.'rival i ve for excretion 1 .-a air-1* only a small fraction of the ingested 4-chlnrobiphenvl (>'./! was e\. rete.l as the free ph"iiohe compound. This path way was a major one for the excretion r.f biplu-nvl (2-1' () and "as ripc'dlv as important as the formation of glueosiduronide.s in biphenyl metabolism. 6UM.MAHV Twenty-four-hour mine samples were collected for a 4-day period, both before and after oral administration of either 1 g of biphenyl or l g of 4-chlorobiphenyl to rabbits. 'The urine sam ples were analyzed for their content of gtucosiduronic acids, five phenolic compounds, mereapturie acids, and ethereal sulfates. Tl-se metabolites in the urine accounted for 6 f % of the in gested biphenyl or 4-chlorobiphenyl. 4-Chloiobiphenyl was ex creted mainly as the glueosiduronide (50N); free phenolic com pounds (24c/c) and glueosiduronides (2f.be) in the urine accounted for most of the biphenyl. Thus the presence of a chlorine atom in the biphenyl molecule altered the metabolic pathway in the rabbit. . Isolation studies indicated that the gliicosidunmic acid deriva tives of both compounds were formed by conjugation in the- 4 01 /icini-posilion. KKFEKEXCKS 1. Coumsii, H. II., and Block, W. IT, Federation Proc., 13, Cc. (1959). 2. Coaxtsu, H. II., Axn Block, \V\ D., J. Biol. Chen., 231, .o3 Ot'A-S). 3. StibUi'U, S. W J. ICnilon iuol.t 2, 55 (1910). I. West. 11. D., I, a "sox, J. It., Mu.uin, I. 1!., and Matiich-i. Ct. It., .(reft. Iliarhrni. lliophys., GO, 14 (!9n6). - 5. Han.-ox, S. W. Mills, (I. T., and Williams, H. T., /'>* rhrm. J , 38. 271 (1911). ('.. Toi ls', <)., and Cox al rue, V., J. Bint. C/ion., 73, 027 (192. - 7. Toi.ix, O., A >n. J. Physiol., 13, 45 (1905). N. Toux, O., J Biol, (iem., 17, 109 (1911). 9. Colkoms, N. 11., ano T.n i uuux, J. B., Scnnnirrc yw'-hri.:.< oryanir analysts, 2ml edition, Inte.rseieacv 1'uhUshcis. lc..-, New 3 in li, 19a.. 10. b.ivfiv, C. .M., ami Ahuiinatuv, J. I,., J. An. Chan. For., Cl. 2219 (1912.1. DSW 192858 STLCOPCB4052026