THE INTRAHEPATIC CONJUGATIONOF SULFOBROMOPH-THALEIN AND GLUTATHIONEIN THE DOG*

By NORMANB. JAVITT,t HENRY0. WHEELER,t KATHARINE J. BAKER,OSWALDOL. RAMOS§ AND STANLEYE. BRADLEY

(From the Departmnent of Medicine, Columbia University College of Physicians antd Surgeons,and the Presbyterian Hospital, New York, N. Y.)

(Submitted for publication April 1, 1960; accepted June 19, 1960)

In 1950 Brauer, Krebs and Pessotti (1) re-ported that S35-labeled sodium phenoltetrabro-mophthalein disulfonate (more commonly knownas sulfobromophthalein, Bromsulfalein or BSP)may be recovered from the bile of the dog, duringconstant intravenous infusion, in four fractionswhich are separable by column chromatography.Similar fractions, accounting for the bulk of BSPexcreted and having absorption spectra like thoseof standard BSP, have also been detected in thebile of sheep, rat, cat and chicken and in bile pro-duced by the isolated perfused liver of the rat (2).More recently, Meltzer, Wheeler and Cranston(3) have found that BSP in the blood and bile ofthe dog and man may also be fractionated by paperchromatography and that several of the fractionsyield a positive ninhydrin reaction suggesting thepresence of amino acid. Glutamic acid and gly-cine (but not cysteine) were identified by two-di-mensional chromatography following 6 N hydro-chloric acid hydrolysis of an eluate of the fractions.Hence they concluded that BSP is conjugated withamino acid, presumably in the liver, since the con-jugates were found in the bile prior to their ap-pearance in the blood. Subsequent work by Javitt,Wheeler, Baker and Ramos (4) has shown thatcysteine is also present in the conjugates. Similarfindings have been made independently by otherworkers (5-7) during the past year. Glycine andglutamic acid have been demonstrated in acid hy-drolysates by Combes (5) and cysteine has beenfound in hydrobromic acid hydrolysates of BSPconjugates by Grodsky, Carbone and Fanska (6,7). As both Combes and Grodsky have alsonoted, the appearance of glycine, glutamic acid,

* Supported by a grant from the New York HeartAssociation.

t American Heart Association Fellow.t Markle Scholar in Medical Science.§ Rockefeller Traveling Fellow in Medicine.

and cysteine together would strongly suggest thatglutathione might be conjugated with BSP. Thisinference has been substantiated in the course ofthe studies to be presented below.

METHODS

The biliary excretion of BSP was measured in 5 trainedunanesthetized fasting dogs before and after the initra-venous administration either of single doses ranging from200 to 300 mg or of BSP infusions over a prolonged pe-riod, in most instances with the establishment of highplasma BSP concentrations. Bile was collected con-tinuously by means of an indwelling catheter placed inthe common bile duct under direct visioni tlhrough aThomas duodenal cannula (8, 9). Cholecystectomyl andsplenectomy were performed when the duodenal fistulawas prepared and the cannula inserted, several monithsprior to study. All bile samples were refrigerated orfrozen as soon as possible after collection.

Aliquots of whole bile were applied directly to \NVhat-man no. 1, 3 or 3 MMfilter paper for one-dimensionalchromatography or electrophoresis. Preliminary re-moval of proteins and bile salts was necessary in thosespecimens to be hydrolyzed for amino acid analysis.For this purpose 2 ml aliquots of bile were dialyzed inVisking cellulose tubing for 24 hours against equal vol-umes of distilled water. The dialysates were lyophilizedto dryness, extracted three times with absolute ethanol(10) and the residue reconstituted in distilled water.

Covered glass chambers were used for one-dimensionalascending and descending chromatography with butanol:acetic acid: water (4: 1: 2) as the solvent mixture.For paper electrophoresis (11), a Spinco series B Dur-rum type apparatus was used with a constant voltagepower supply. Electrophoresis was carried out at 400 vfor 4 hours or 120 v for 18 hours using 5 N acetic acid(pH 1.9) as the conducting medium. Two-dimensionalascending chromatography according to Dent (12) wascarried out with phenol: water (4: 1) followed by 2,4-collidine: 2,4,6-lutidine: water (1: 1: 1) as the solventsystems. Following electrophoresis or chromatographythe papers were air-dried at room temperature.

For purposes of comparison, the chromatographic andelectrophoretic migration of tetrabromphenolphthalein(Eastman Organic Chemical), tetrabromphenolphthaleindisulfonate (BSP, Hynson, Westcott and Dunning), so-

1570

SULFOBROMOPHTHALEINCONJUGATION

dium taurocholate, and various pure amino acids (Nu-tritional Biochemicals Corp.), glutathione (Schwarz Lab-oratories), taurine (Eastman Organic Chemicals), cys-teic acid, and purified BSP conjugates-dissolved ineither distilled water or canine bile-were determinedseparately or in various combinations. The proteinspresent in bile were dyed on the paper with bromphenolblue (11). Amino acids, taurine, cysteic acid, and glu-tathione were detected by spraying with 0.1 per cent nin-hydrin in water-saturated butanol or by dipping the paperinto 0.1 per cent ninhydrin in acetone and heating at 1000C for 5 minutes. Bile salts were detected by Kritchevskyand Kirk's reagent (13) and tetrabromphenolphthalein,BSP, and BSP conjugates, by spraying with 15 per centsodium carbonate or exposure to ammonia vapor. TheBSP conjugates, as reported previously (3), were alsofound to develop color with ninhydrin. It was noted thatBSP conjugates could be detected more readily at lowerconcentrations by the purple color appearing on alkalini-zation than by ninhydrin treatment. Hence, small butdetectable amounts of the conjugates might be errone-ously considered ninhydrin-negative.

Hydrolysis was carried out on the lyophilized dialyzedbile samples or on solutions of the various BSP conju-gates prepared therefrom in the following manner. Pre-liminary electrophoresis was used to separate BSP andits conjugates as anions from the amino acids, pigmentsand other cations. The zone of the electrophoretic sheetbearing BSP conjugates (ninhydrin-positive) was cutout and eluted with water (adjusted to pH 6.0 to 6.5).The eluate was concentrated by lyophilization and ap-plied to ascending or descending chromatograms fromwhich the individual conjugates were cut out and eluted.Acid hydrolysis was performed in 6 N HCl at 100° C for14 to 24 hours in sealed glass ampules. The hydrolysateswere filtered, evaporated to dryness with gentle heatingand air stream, and the last trace of HCl was removedin vacuo over KOH. Aliquots of the dried residue weredissolved in distilled water for two-dimensional chroma-tography. Alkaline hydrolysis was carried out in cov-ered test tubes in 0.3 N NaOHat 600 C for 2 hours in awater bath. These hydrolysates were "desalted" electro-lytically (Research Specialties Co., model 1930 A), andconcentrated by evaporation prior to chromatography.

Reactive sulfhydryl groups before and after alkalinehydrolysis were detected by the sodium azide-iodine reac-tion described by Feigl (14). Neutralization of thealkalinized samples was necessary to prevent interferenceowing to formation of sodium iodide. Inorganic bro-mide was isolated from the synthetic reaction mixturesby paper chromatographic separation, described by Pol-lard and McOmie (15), using acetone: water (4:1) asa solvent. Bromide was precipitated on the paper byimmersion in an ammoniacal (0.7 M NH4OH) ethanolic(95 per cent) solution of silver nitrate (0.1 M) and "de-veloped" by exposure to blue fluorescent light understandard conditions. The concentration could be de-termined quantitatively, by comparison with appropriatestandard solutions of NaBr on the same chromatogramsby using a Spinco analytrol. Over the range employed

(0.015 to 0.03 M, 10 ,ul aliquots), concentration anddensitometric readings showed a satisfactory linearcorrelation.

Glutathione in the conjugates isolated by electropho-resis and chromatography was determined by the methodof Sachs and Brand (16) as glutamic acid. Since thismethod involves the formation of hydroxamic acid froma lactone ring, BSP itself might be expected to interfere.However, under the actual conditions employed (pHclose to 7.0) it was found that BSP developed only aminimal amount of color compared with an equimolarconcentration of glutathione. BSP could be recoveredcompletely from the chromatograms by elution with aphosphate buffer solution (0.1 M, pH 10.4) and colori-metric analysis. Since glutathione did not interfere andsince no change in optical density occurred in mixtures ofglutathione and BSP during or after conjugation, con-jugated BSP could be evaluated by reference to a stand-ard BSP calibration curve. Glutathione labeled with S35(specific activity 16.6 /Ac per mg) was obtained fromSchwarz Laboratories. Radioautograms were obtainedby placing Eastman Kodak No-screen X-ray film (infilm holders) against the dried chromatogram or elec-trophoretogram and leaving it for 6 weeks prior todevelopment.

RESULTS

Excretion of BSP and its conjugates in the bileof the dog. Regardless of the method of adminis-tration, biliary BSP always proved to be separableinto several fractions by chromatographic analysis(Figure 1). All reacted with ninhydrin, with theexception of the fastest moving component (frac-tion I) which assumed the same chromatographicposition as the commercially available or "stand-ard" BSP. The ninhydrin-positive fractions va-ried considerably in number and concentration.As a rule, three major bands were distinguishablebut, with improved chromatography, other com-ponents were detected. Fractions II and IV ap-peared to be homogeneous, whereas fraction IIIcould be resolved into two additional components(a and b, Figure 1). On electrophoresis, also,biliary BSP could be separated into a number offractions having differing mobilities (Figure 2).All moved toward the anode and the most rapidlymoving component appeared again to be identicalwith standard BSP. When individual BSP con-jugate fractions were eluted from the chromato-grams and subjected to electrophoresis it wasfound that fractions II, IIIa and IV all migrated tothe position represented by the darkest band inFigure 2. Fraction IIIb migrated in part to thisposition, but a major portion also appeared in the

1571

JAVITT, WHEELER,BAKER, RAMOSAND BRADLEY

ever, in association with a small amount of aninhydrin-positive material which behav-ed chro-

E matographically like cysteine. Conversioni. of therelatively unstable cysteine to cysteic acid by theaddition of hydrogen peroxide made it possible to

NAlI demonstrate that cysteine was regularly l)resent inalkaline hydrolysates in significant amotunt (Fig-ure 3A). The azide-iodine test for reactiv-e sulf-hydryl groups, which was negative wNhen per-formed on unhydrolyzed conjugates. becamestrongly positive after alkaline hydrolysis. Theseobservations suggested that fraction IV is a com-plex of BSP and glutathione (glutamyl-cysteinvl-glycine) and that the linkage involves the sulf-hydryl group of cv-steine. The BSP-reactingninhydrin-positive miioiety observed after acid hy-

8 S Pstandard

ALA

BILE

ALK N/A+

FIG. 1. CHROMATOGRAMOF SULFOBROMOPHTHALEIN

(BSP) AND BSP CONJUGATESIN BILE OF THE DOG. Ali-quots of solution of BSP (5 pg), BSP standard, and bilewere applied at the points indicated by the broken lines.After alkalinization (Alk.) of the strips, standard BSPappeared as a single component whereas BSP appears

in at least four additional fractions in bile-here labeledI, II, IIIa, IlIb, and IV for ease of reference. FractionI corresponds to standard BSP, and does not give a posi-tive ninhydrin reaction as do the remaining fractions(NIN). (These figures, and those that follow, havebeen prepared from tracings shaded in black to showrelative intensities of colored bands more precisely thanis possible with photographic reproduction.)

region marked by the upper ninhydrin band inFigure 2. The absence of significant hydrolysisunder the conditions employed during electroplho-resis was indicated by the fact that free amiiino acidscould not be detected when purified conjugateswere subjected to this procedure.

Analysis of the BSP conjugates. Acid hydroly-sis of fraction IV yielded approximately equialamounts of glycine and glutamic acid. Althoughfree BSP appeared in the reaction mixture, a

large moiety of the BSP continued to migrate on

chromatography as a ninhydrin-positive fraction.With alkaline hydrolysis also, large amounts ofglycine and glutamic acid appeared, usually, how-

lP l

Glutomic

ocid

FIG. 2. ELECTROPHORETICPATTERNSOF BSP AND BSPCONJUGATESIN BILE OF THE DOG. Stanidard BSP anid itsconj ugates in the bile migrate toward the anode (+),whereas free amino acid (glutamic acid) that appears

in the bile during the excretion of BSP moves towardthe cathode (-) from the point of applicatioin (hrokenline).

BSPstandard

AL A'

B I L

A L K.

1I ,:

mc

Ilb:1'

iz

1572

A'. ....::-%:.,r..:,.%%%, ":'..V

SULFOBROMOPHTHALEINCONJUGATION

drolysis of fraction IV might be a complex ofBSP and cysteine.

Glycine and cysteic acid could be found follow-ing similar treatment of the chromatographicallyfaster moving component of fraction III (Figure3B). Glutamic acid appeared in trace amounts

r -

A

I II II II I

Cysteicacid

lycine

a. ocid

PHENOL H21

1 81~~~~~~~yti

acid

Glycine

cw

za

-J

wza-j-_0

0

J:0

I

III:

-:

II

0

z

-J

0

30

wz0-J-J43

,a

PHENOL-H20

FIG. 3. CHROMATOGRAMS(TWO-DIMENSIONAL) OF AL-

KALINE HYDROLYSATESOF ISOLATED BSP CONJUGATES.A. Hydrolysate of fraction IV showing presence of gly-cine, glutamic acid, and cysteic acid following treatmentwith peroxide. B. Hydrolysate of fraction IlIa in whichglycine and cysteic acid, but not glutamic acid, are

detectable.

mGmb3JI

BILE

A I. K.

T -i:.:-.

.A

... ... !:..

V"

SYN. MIXBSP £GSH

ALK

ASP-K.

{3SP-GSH

BSP-(GSH)2

FIG. 4. CHROMATOGRAM1SOF BSP AND BSP CONJU-GATES IN BILE AND IN A MIXTURE OF BSP AND GLUTA-THIONE. BSP and glutathione combined spontaneouslyin aqueous solution at 37° C to yield two detectable nin-hydrin-positive conjugates. One having the chromato-graphic and electrophoretic properties of fraction IV ap-peared to be an equimolecular combination of BSP andreduced glutathione (GSH). The other, occasionallydetectable in minute quantities in the bile, contained twomolecules of glutathione for each molecule of BSP[BSP- (GSH) 2].

in early preparations but disappeared altogetheras the separative procedure was improved. Themore slowly moving moiety of fraction III whichcould be separated into two components by elec-trophoresis was not obtained in sufficient amountfor complete analysis.

Fraction II behaved as a single substance bothon chromatography and electrophoresis. Like thechromatographically faster moving portion of frac-tion III, it yielded glycine and cysteic acid with

1573

I

I

IIIIIIIII GIIIIIL - -

JAVITT, WHEELER,BAKER, RAMOSAND BRADLEY

appropriate treatment. Glutamic acid did not ap-

pear in the hydrolysates.Protein-free (dialyzed) bile obtained from four

normal fasting dogs not receiving BSP containedbarely detectable quantities of free amino acidswhich were not significantly increased by hydroly-sis. Eluates of the paper employed in these stud-ies yielded on hydrolysis insignificant amounts ofninhydrin-positive material. An abundance of

free glutamic acid did appear in the bile duringexcretion of BSP but, since the amino acid mi-grated toward the cathode, it could be easily re-

moved by electrophoresis. Thus, amino acids,peptides, or other amino acid conjugates present

in the bile, reagents or paper could not account

for the abundance of glycine and glutamic acidwhich was observed in the hydrolysates of conju-gates separated from the bile of dogs receivingBSP.

Synthesis of the BSP conjugates. BSP andglutathione were found to combine spontaneouslyin vitro in slightly alkaline aqueous solution at

370 C to yield two ninhydrin-positive conjugates(Figure 4). At the outset the mixture was

brought to a pH of 7.3 by the addition of 0.1 NNaOH. As the reaction proceeded, the mixturebecame more acid and the color of the BSP faded.The pH was therefore maintained by dropwise ad-mixture of the alkaline reagent over a 24-hourperiod. With exhaustion of glutathione by com-

bination with BSP or by oxidation, the reactioncame to a halt. The reaction was resumed if more

glutathione was added. On both chromatographyand electrophoresis two easily separable ninhy-drin-positive BSP-reacting bands were demon-strable in addition to ninhydrin-negative standardBSP. Neither glycine nor glutamic acid was de-tectable. The major component appeared to beidentical with fraction IV, chromatographicallyvelectrophoretically and analytically. The secondfraction seemed also to have its counterpart in a

conjugate occasionally appearing in trace am--ounts

in the bile of both the dog and rat.

Analysis of the eluted synthetic conjugates fromiglutathione and BSP indicates that the comiiponent

apparently identical with fraction IV consists ofan equimolecular combination of the two sub-stances, whereas the second component (whichis fluorescent in the ultraviolet) contains two

molecules of glutathione for each molecule ofBSP. During the reaction, hydrobromic acid isformed, accounting for the progressive acidificationof the reaction mixture. Since the bromide re-

leased was found to be proportional to the ambuntof conjugate formed and since the conjugate con-

tained no reactive sulfhydryl groups, BSP andreduced glutathione (GSH) probably com-ibine bythe formation of a thio-ether with replacemiient ofbromine on the phthalic acid ring (Table I).

Cysteinylglycine prepared from pure GSH bypartial hydrolysis with dilute phosphoric acid (17)also formed a conjugate with BSP in vitro whenheld at pH 7.4 and 37° C for 24 hours. Owingto the difficulty imposed by the high phosphate

TABLE I

Release of bromide during conjugation of sulfobromophthalein (BSP) and glutathione in vitro *

AmountNaOHadded

Composition of Distribution of BSP in mixture during re- Computed Observedsynthetic mixture after reaction action to hydrogeni bromide

Exper. keep pH ion ionno. BSP GSH Free BSP-GSH BSP- (GSH) 2 constant released released

inmole % inmole

I 0.30 0.30 52.1 43.4 4.5 0.13 0.16 0.1511 0.42 0.42 44.4 51.4 4.2 0.21 0.25 0.24

III 0.14 0.25 22.7 68.0 9.3 0.10 0.12 0.12IV 0.10 0.29 10.1 72.5 17.4 0.10 0.11 0.12

* In each experiment (except IV) aqueous solutions of BSP and reduced glutathione (GSH) containing the totalamounts indicated under "Composition," were mixed and allowed to react at 370 C. The pH was raised to 7.3 and closelymaintained by continuous titration with NaOH(0.1 N) in the total amount indicated in column 7. At equilibrium, 22.7to 52.1% of the BSP remained unconjugated in Experiments I to III, 43.4 to 68.0% was conjugated as BSP-GSH and4.2 to 9.3% as BSP-(GSH)2. The amount of hydrogen ion which should have been released, computed as the sum ofBSP-GSHand 2 X BSP-(GSH)2, did not differ significantly from the amount of alkali actually added or the quantity ofbromide ion released. In Experiment IV, 0.2 mmole of GSHwas added to an aliquot of equilibrium mixture from Experi-ment II containing 0.1 mmole free BSP and 0.12 mmole conjugated BSP having the distribution shown for II. Underthese circumstances the reaction came closer to completion and yielded a much larger qtiantity of BSP- (GSH)2. Againthe calculated value did not differ materially from observation.

1574

SULFOBROMOPHTHALEINCONJUGATION

BILE

ALK.

SYN. mIX

BSP Xi CySH-gly( Isol. comp.)

ALIK.

I[o

Xb

*33.

FIG. 5. CHROMATOGRAMSOF CONJUGATI

AQUEOUSMIXTURES OF BSP AND CYSTEI

CYSTEINE. Cysteinylglycine prepared fr4reacted with BSP to yield at least one cc

behaved like fraction II when isolated fro]mixture (Syn. Mix, center). At leastappeared in a mixture of BSP and cysteinresembled fraction IIIb (Syn. Mix, right)

concentration of this mixture, a

chromatographic separation was ne

which only the fastest moving portlated. This material behaved electrchromatographically (Figure 5) an

(two-dimensional chromatography a

sis) like fraction II. Moreover, mildsis of fraction IV (or glutathione-Bin the production of glutamic acidII. Thus, fraction II appears to

glycine-BSP synthetically as well aw

Analysis of the faster component ofindicates that it, too, is a cysteinylCysteine and BSP have been found 1

vitro to yield two conjugates of whiseems to be identical chromatographislower moiety of fraction III (Figelectrophoresis this conjugate movedtion marked by the upper ninhydrinure 2.

Incorporation of glutathione sulfur in conjugatesin vivo. Additional evidence that cysteine, de-

SYN. MIX rived from glutathione, appeared in the conjugateswas adduced from the behavior of radioactive

BSP(ACySH sulfur incorporated in glutathione during the ex-

cretion of BSP; 5 to 6 mg of glutathione labeled

ALAI. with S35 (50 to 100 ,c) was injected intravenously2 hours prior to administration of 300 mg of BSP.Bile samples were collected in the usual way andthe radioactivity, in counts per minute per milliliterand BSP concentrations were determined in suc-cessive samples collected at 10- to 22-minute in-tervals. The biliary radioactivity and BSP con-centrations rose, reached a peak, plateaued, andthen fell simultaneously (Figure 6).

A one-dimensional ascending chromatogramwas made from each sample and used for thepreparation of a radioautograph by contact withX-ray film over a period of 6 weeks. The carbo-nate-sprayed chromatogram of two successive pe-riods selected at the concentration peak, 40 min-utes after the administration of BSP, and the ra-

ES PRODUCEDIN dioautograph of the same patterns are illustrated:NYLGLYCINE OR in Figure 7. In every instance radioactive ma-om glutathione

)njgat, wichterial was observed in the regions correspondingnm the synthetic to BSP conjugates but not in the position oc-two conjugates cupied by unconjugated BSP. Since free glu-e, one of which tathione was never detected in bile during any of

the present studies, it is apparent that the S35 inthese patterns must have been bound in the BSP

preliminary conjugates.cessary from It may be concluded, therefore, that sulfur de-tion was iso- rived from glutathione is a constant constituent*ophoretically, of the BSP conjugates. It seems not unlikely thatd analytically all of the conjugates have their origin in the com-fter hydroly- bination of glutathione and BSP, fractionation re-acid hydroly- sulting from the formation of isomers and from'SP) resulted the cleavage of glutamic acid and glycine. Theand fraction relatively small quantity of radioactive material de-

be cysteinyl- tectable in the bile prior to administration of BSPs analytically. was probably attributable to the excretion off fraction III breakdown products, since GSH did not appearlglycine-BSP. in the bile in the absence of BSP.to combine inich the major DISCUSSIONcally with the The results of this study indicate that BSP,ure 5). On and glutathione combine both in vitro and in vivoI to the posi- in the liver of the dog, presumably with the forma-band in Fig- tion of a thio-ether linkage and release of bromine.

In support of this view is the observation that a

1575

1576 I.T \W1111\LFR BA\IKER RhAMOSAND PRADLEY

250 300 mg

,%. 200 j

It 50

100

Iq 50

U)I)

1000

800

e600

400

200

o 80

MINUTES

180

F1tc(. 6. BII, IX' xN ETiI ')N.\ ()I- I I). AND) RAI)TOAT(''I'V\'; t1I'1. ['1 1.N \' (1F I( I\V 1EN ISP \\ \ACIVE N 2 110t115 A FTER JNTEAVINItS AIM)IN STISTRATION IF S'-L.\1IIELII:1 I 'TAT II II N F:. The bilia-voutput ot radioactive itiaterial inicrease(d markedly above the relatively lowv control valuie aISB S P appeared in tile ile. Bililary ratlioactivitv anid( BSP conceentrationi rose atli(I fell to(g-etller.

na<jor B S j) CIljtl,;Iigte inl (oog b ile ( fracti (lI V inlFiglre 1) a inlihlyrini-p)ositiV e C"111pI)( Itill w hi'hvields ogive ilne, CV-steille, an(l Iti t IlliCCJ1tiai' -1oli I-drolysis, caii lIe synthic itidzed b spoItaVllc mits (' ll-

linationI of BSP w\\ith litre o-Itithtll"IFl( 111Icr - CIIl-dlitions that p)reCll(lude hydrol y t(s STo . Sincean equnliniolar Iuanitity of hydroogen 1 Ironti(le evIolivesdtitrinog Sy'nthesi.s anlld slint ttle cnlij tigyatc pI)S(sc-e's.si11 free st1lfhvdrvl orotups it may be inferred thatConillnatilln inyllOives linKace lbetXcX s'tilfnr and

a carbon of tile plhthalic acid ring. A similar re-actioni lhas been (lescrilbled ( 18) a a llmealls of lIe-patic (letoxifhcatioll Iof h'alogeiatedl nitr Ilweinzeneandi related colpl)oidn(ls (inring tict fI rlllatim(111 Itfnmercal)turic aCiEl.

Cleavage p)rodcnts an1(I isomllers If a conjnliogateof BSP aln(n GSHcotili( aCcotiIIt for all 4o-f the uil-

hydrin-positive, BISP-reacting substance> alplpear-ilig in tle blile. The appearance of radioactiVesuilfuir in all the l)iliarv conjtugates after adminis.>-tration of S:5-lal)eled glutathlione clearlv stpl)portsthis conclutsioln. The lpresence of cysteinylglycine-

1Ii(;. 7. RAnIOACTIVE S t'LFUUR IN BILIARY I-I Sl Nt' -GATES EINCRETEI AFTFRIZ AWMINISTRATION ()I >-O.F LAE.l.lGLUTAT1ITONE. Autora(liograiii (film) of tile alkallizezdclrotmiatograi (If l)ile ( Alk.) ? radioactivity is (vidciltin every fIractimll except I (free BSP).

B I L EALK FILM

ii -.

i :

=a _IMb~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~j

*& _Afifmb tl57 6

4L.-A.... .....

-.4p...j., .-- g

SULFOBROMOPHTHALEINCONJUGATION

BSP conjugates, invariably in association withfree glutamic acid, suggests that cleavage of glu-tamic acid may occur to some extent during ex-

cretion. The other possible products of cleavage,cysteinyl-BSP, glutamyl-cysteinyl-BSP and tri-bromo-BSP have not been identified. Isomerismcould account for the chromatographic separationof two forms of cysteinylglycine BSP. Indeed,the appearance of a diglutathione conjugate ofBSP in artificial synthetic mixtures attests to atleast two sites on the BSP molecule at which com-

bination may occur.

Regardless of the role of glutathione in BSPtransport, whether essential or merely incidental,the loss of glutathione during BSP excretion couldlead in principle to depletion of hepatic GSH.Such a depletion has actually been- observed fol-lowing the administration of a variety of sub-stances that are excreted as mercapturic acids,but the dosages required have been much largerthan those in which BSP has been administeredin these or in the usual clinical procedures. Pos-sibly a critical loss could be induced more readilyin the liver, damaged by disease or hepatotoxicagents. These studies suggest that BSP may beused as a tool for the investigation of hepatic glu-tathione metabolism and its relation to hepaticdisease.

SUMMARY

Sulfobromophthalein (BSP) and glutathioneappear to combine intrahepatically in the dog toyield a variety of ninhydrin-positive conjugates.Since bromide and hydrogen ions are releasedstoichiometrically during synthesis of conjugatesin vitro, it may be inferred that conjugation in-volves the formation of a thio-ether with replace-ment of bromine. Radioactive sulfur is found inall of the conjugates detectable in the bile whenS35-labeled glutathione is given with BSP, sug-

gesting that all the ninhydrin-positive fractionsmay be derived from the conjugation of BSP andglutathione. This phenomenon may provide an

approach to the quantitative investigation of he-patic glutathione metabolism in the experimentalaninial.

REFERENCES

1. Brauer, R. W., Krebs, J. S., and Pessotti, R. L.Bromosulfonphthalein as a tool for study of liverphysiology. Fed. Proc. 1950, 9, 259.

2. Krebs, J. S., and Brauer, R. W. Metabolism ofsulfobromophthalein sodium (BSP) in the rat.Amer. J. Physiol. 1958, 194, 37.

3. Meltzer, J. I., Wheeler, H. O., and Cranstoi, \V. I.Metabolism of sulfobromophthalein sodium (BSP)in dog and man. Proc. Soc. exp. Biol. (N. Y.)1959, 100, 174.

4. Javitt, N. B., Wheeler, H. O., Baker, K. J., andRamos, 0. L. Intrahepatic conjugationi of brom-sulphalein and glutathione (abstract). J. clin. In-vest. 1959, 38, 1015.

5. Combes, B. The biliary excretion of sulfobromo-phthalein sodium (BSP) in the rat as a conijugateof glycine and glutamic acid. J. clin. Invest. 1959,38, 1426.

6. Grodsky, G. M., Carbone, J. V., and Fanska, R.Metabolism of sulphobromophthalein. Nature(Lond.) 1959, 183, 469.

7. Grodsky, G. M., Carbone, J. V., and Fanska, R.Identification of metabolites of sulfobromophthaleini.J. clin. Invest. 1959, 38, 1981.

8. Thomas, J. E. An improved cannula for gastric in-testinal fistulas. Proc. Soc. exp. Biol. (N. Y.)1941, 46, 260.

9. Wheeler, H. O., and Ramos, 0. L. Determinianltsof the flow and composition of bile in the unan-esthetized dog during constant infusions of sodiumtaurocholate. J. clin. Invest. 1960, 39, 161.

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