PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN IN VITRO

Transcription

1 GASTROENTEROLOGY Cpyright 1970 by The Williams & Wilkins C. Vl. 58, N.1 Printed in U.S.A PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN IN VITRO J. DONALD OSTROW, M.D., AND ROGER V. BRANHAM Department f Medicine, Case Western Reserve University, and University Hspitals, Cleveland, Ohi Decmpsitin f bilirubin and biliverdin under intense flurescent light was studied in vitr at ph levels ranging frm 7.4 t Phtdecay f bilirubin was mre rapid at a higher ph if albumin was absent, but mre rapid at a lwer ph if albumin was present, whereas biliverdin was mre stable at a lwer ph, with r withut albumin. During illuminatin at lwer ph levels, albumin stabilized biliverdin but accelerated decay f bilirubin; these effects were nt altered by cmpetitive uncupling f bilirubin frm albumin r by chelatin f cntaminant divalent catins with ethylenediaminetetraacetic acid. Studies with a variety f flurescent lamps demnstrated that wavelengths between 400 and 600 mjl were mst effective in destrying bilirubin, ultravilet light was less effective, and red light (>600 mjl) was ineffective. Phtdecay f bth bilirubin and biliverdin was xidative, but exclusin f xygen did nt arrest pht decay f bilirubin if albumin was present als. Presumably, albumin itself r the vinyl side chain f bilirubin substituted fr xygen as a hydrgen acceptr. By radichrmatgraphy and by reilluminatin f phtderivatives f 14e-bilirubin, it was shwn that the prcess f phtdecay prceeded frm bilirubin t biliverdin t weakly diaz-reactive derivatives t highly plar, diaz-negative prducts. Expsure f infants t flurescent lights cmmnly is utilized t treat nenatal jaundice,2 but the mechanisms invlved and the prducts f bilirubin phtdecay Received June 18, Accepted August 5, A preliminary reprt f this wrk was presented at the Annual Meeting f the Federatin f American Scieties fr Experimental Bilgy n April 15, 1969, at Atlantic City, New Jersey, and has been published in abstract frm.' Address requests fr reprints t: Dr. J. Dnald Ostrw, Department f Medicine, Uniyersity Hspitals, Cleveland, Ohi The authrs are indebted t Mr. Carl Allen, Large Lamp Divisin, General Electric Cmpany, Nela Park, Cleveland, Ohi, fr pryiding the light canpy and flurescent bulbs and fr the data in table 1 cncerning the emissin spectra f the bulbs. They are grateful als fr the advice and suggestins f Dr. Edwin W. Abrahamsn, Department f Chemistry, Case Western Reserve University. 15 have nt been identified. Many investigatrs have apprached this prblem by examinatin f the phtdecmpsitin f bilirubin in vitr,3-10 but the cnditins emplyed ften have been neither physilgical nr standardized, and the factrs affecting the kinetics f bilirubin phtdecay have nt been evaluated systematically. The present in vitr studies were perfrmed t assess the effects f ph, albumin, xygen, and the wavelengths f the incident light upn the phtdecmpsitin f bilirubin and t determine whether biliverdin is an intermediate in the prcess. Materials and Methds Materials. Crystalline un cnjugated bilirubin, btained frm the Pfahnstiehl Cmpany, Waukegan, Ill., was used withut further purificatin. Biliverdin dihydrchlride, purchased frm Sigma Chemical Cmpany, St. Luis, M., was recrystallized frm ht methan!." On paper chrmatgraphy," the prduct

2 16 OSTROW AND BRANHAM Vl. 58, N.1 migrated as a single spt, free frm bilirubin, and exhibited characteristic absrptin spectra in several slvents." Human serum albumin, in the frm f Chn fractin V, free frm preservatives and cntaining less than 0.02% w /w heavy metals, was purchased frm Nutritinal Bichemicals Crpratin, Cleveland, Ohi (lt 6942). Human serum was btained frm a fasting nrmal vlunteer, frzen in aliquts at -10 C fr n mre than 7 days, and thawed and centrifuged just prir t use. All ther chemicals and slvents were reagent grade. Slutins. Bilirubin r biliverdin HCl, 1800 ± 20,ug, was disslved fr 1 min in 1.2 ml f 0.1 N sdium hydrxide and then centrifuged rapidly. Mst f the biliverdin did nt disslve. In a lo-ml Kimax beaker, 2.4 cm in diameter and 3.0 cm tall, 0.5 ml f the supernatant fluid was mixed with 4.5 ml f: (a) 0.1 M phsphate buffer, ph 7.25'3 ; (b) 0.1 M brate buffer, ph 8.30'3 ; (c) 0.1 M bicarbnate-carbnate buffer, ph 9.80'3 ; r (d) 0.1 N sdium hydrxide. Final ph levels f these slutins were 7.4, 8.5, 9.9, and 13.0, respectively. In sme experiments, 4.0% albumin was disslved in the buffer prir t the additin f the pigment slutin. In ther experiments, disdium ethylenediaminetetraacetic acid (EDTA), sdium salicylate, r sulfisxazle (Gantrisin, Hffmann La Rche, Cmpany, Nutley, N. J.) was added. Fr studies with serum, 0.5 ml f pigment slutin was added t 0.5 ml f 0.1 M phsphate buffer, ph 5.80,'3 plus 4.0 ml f serum. Initial pigment cncentratins averaged 15.0 mg per 100 ml (0.26 mm) bilirubin r 3.0 mg per 100 ml biliverdin; albumin cncentratin was 3.6 g per 100 ml (0.52 mm). All slutins were prepared n ice under dim light. Kinetic studies in air. Beakers cntaining the bilirubin slutins were centered n a sheet f white paper under an aluminum reflecting canpy which cntained six 15-watt flurescent bulbs arranged in a semicircle 10 cm frm the surface f the slutin. Characteristics f the bulbs are given in table 1. The ballast and igniter fr each bulb, plus a built-in cnvectin fan (RE-148 Whisper-fan, Retrn Manufacturing Cmpany, Wdstck, N. Y.), were fastened t the uter surface f the canpy t minimize the heating f the slutins, which remained at 27 t 28 C. After expsure t light, OJ-ml aliquts were withdrawn at specified intervals fr 6 t 8 hr. Each aliqut was diluted with 0.9 ml f 0.1 M phsphate buffer, ph 6.50,'3 with 0.4% albumin added if the irradiated slutin cntained n albumin, and then stred n ice in the dark until analysis. Bilirubin and biliverdin were cmpletely stable under these cnditins. On each sample, bilirubin cncentratin was determined by a micrmdificatin f the methd f Michaelssn," direct spectral absrbance was determined at 315,380,465,575, and 650 mjl3; and, where indicated, absrptin spectra were btained frm TABLE 1. Characteristics f flurescent lampsa Clr f lamp..... Red Pink Gld Cl white Daylite Blue Black General Electric Cmpany cde.. R PK G cw D B BLB Per six lamps IIluminatin b (ft candles) Radiant energyc (mw/cm) m).' m).' m).' m).' m).' m).' a General Electric 15-watt flurescent lamps, catalgue F -15-T-8 series. b Measured with General Electric ft-candle meter, type DO-91. c Radiant energy was measured fr individual lamp n Spectrradimeter and values were adjusted t gemetry f six such lamps in reflecting canpy accrding t ft-candle illuminatin readings.

3 January 1970 PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN t 700 mil against apprpriate blanks. Values were nt crrected fr lsses due t evapratin, which averaged 0.25 ml per hr per 5.0 ml initial vlume. Studies j xygen effects. Flasks and buffer slutins were dexygenated by three cycles f alternate evacuatin t 10-6 mm Hg and reequilibratin with cmmercial nitrgen, fllwed by 30 min f bubbling with nitrgen rendered fully xygen-free by passage ver a red-ht cpper baffle.15 Slutins then were prepared in duplicate as abve in rubber sealed lo-ml Kimax Erlenmeyer flasks, but slutins were transferred and samples were withdrawn with air-tight syringes and needles inserted thrugh the seals. One f the duplicate flasks then was rexygenated by expsure t air fr 2 min. Bth recapped flasks were then placed under the light canpy, and samples were withdrawn and analyzed as described abve. Chrmatgraphic studies. After 70 t 80% f the initial pigment had undergne pht decay, a 3.0-ml aliqut f the illuminated slutin was diluted with 12.0 ml f ph 6.5 phsphate buffer which cntained albumin if necessary. After flcculatin f prteins with 4 vlumes f ethanl-acetne (3: 2, v Iv), rganic slvents were remved by vacuum distillatin f the clear supernatant fluid. The remaining aqueus slutin was adjusted t ph 6.0, and residual bilirubin and biliverdin were extracted with chlrfrm: The remaining water-sluble pigments were cncentrated by the methd f Nir et al." and chrmatgraphed by the ascending technique fr 6 hr n Sand S 598 paper (Carl Schleicher and Schuell, Keene, N. H.) in n-butanl-ethanl-water (6:2:3, v/v)." The chlrfrm extract was reduced t a small vlume under a stream f air and chrma tgraphed simultaneusly. After drying, spts were identified by flurescence under an ultravilet lamp. All f the abve prcedures were perfrmed under dim light r in the dark. Studies j reactin sequences. One milligram f 14C-bilirubin'6 (1154 dpm per ilg) disslved in 3.5 ml f 0.1 N NaOH was placed 18 cm frm an unfiltered mercury spt lamp: At 30, 60, 100, 150, and 210 min, 0.5-ml aliquts were remved, neutralized with 2.5 ml f 0.1 M phsphate buffer, ph 6.0,'3 and the phtderivatives were extracted, cncentrated, and chrmatgraphed as abve." Fr radiassay, aliquts f the chlrfrm r petrleum ether extracts were blended with 15 ml f tluenebased scintillatr.'s Aliquts f ther phases were blended with 10 ml f dixane-based scintillatr.l7 Flurescent spts were cut frm the dried chrmatgrams and eluted int 1.0 ml f 50% ethanl in a 20-ml liquid scintillatin via!,'6 and 10 ml f Bray's scintillatr'7 were added. Each vial was cunted fr 40 min r 10,000 cunts in a Nuclear-Chicag mdel 720B liquid scintillatin spectrmeter, and then quench-crrected accrding t efficiencies determined with added HC-tluene (New England Nuclear Crpratin, Bstn, Mass.) as internal standard. Cunting efficiency ranged frm 42 t 57%, and ttal recvery f radiactivity frm all phases and chrmatgrams exceeded 95%. Pure phtderivatives were prepared by chrmatgraphy f large quantities f extracted pigments n multiple sheets f paper. After drying, individual bands were cut, pled, and eluted with 50% ethanl, and the alchl was remved by vacuum distillatin. Half f each aqueus cncentrate was re-expsed t light fr 90 min and then rechrmatgraphed as abve alngside f the unexpsed aliqut. In this fashin, the purity f the spts and the phtderivatives frmed frm each culd be determined. Results Effect f ph and albumin. In the absence f albumin (fig. 1) at ph 13.0, bilirubin was unstable even in the dark, O. D. DIAZO "'..'-. ph 13.0 '''l,-. NO LIGHT '"' " " ,--,..--,----,r-..,--, DURATIOr..; OF EXPOSURE - HOURS FIG 1. Phtdecay f bilirubin in the absence f albumin. Bilirubin, 026 mm in 0.1 M buffers at tw ph levels, was expsed t 1700 ftcandles f illuminatin frm six daylight flurescent lamps in a reflecting canpy. Serial samples were neutralized with 9 parts 0.1 M phsphate buffer, ph 6.5, and residual bilirubin cncentratin was measured by the diaz methd f Michaelssn."

4 18 OSTROW AND BRANHAM Vl. 58, N.1 with a first rder decay f diaz reactivity at a rate f 26.5% per hr. At this high ph, expsure t daylight lamps apprximately dubled the initial decay f pigment and led t the appearance after 2 hr f a secnd,. D. DIAZO 0.50.!! ,...-.,..---.,---, r-,..--, I DURATION OF EXPOSURE - HOURS FIG. 2. Phtdecay f bilirubin at varius ph levels in the presence f 0.52 mm human serum albumin. Cnditins and methds therwise as fr figure 1. TABLE 2. Effect f human serum albumin n phtdecay f bilirubin a Slutin Brate buffer, ph Bicarbnate-carbnate buffer, ph 9.9. Decay rate Withutl With albumin albumin albumin %/hr I D i e f e a Bilirubin, 0.26 mm; albumin, 0.52 mm, expsed t daylight flurescent bulbs. mre rapid cmpnent which was first rder als. Tw cmpnents were bserved als at ph 9.9 and 8.5, but the initial phase was slwer at these lwer ph levels. With albumin present, bilirubin was cmpletely stable in the dark at ph 7.4 r 8.5. Under illuminatin, hwever (fig. 2), decay f bilirubin ccurred at all ph levels with tw first rder cmpnents. 8 Unlike the findings withut albumin, the first cmpnent was mre rapid, and decay was faster at lwer ph levels. Similar kinetics were bserved in serum, althugh at slwer rates. 8 At ph 8.5 and 9.9, additin f albumin accelerated the initial decay cmpnent (table 2), but the effect was much less at ph 9.9. Hwever, when the secnd decay cmpnent supervened, bilirubin was ultimately stabilized by albumin wing t the slwing f the reactin in albumin while the reactin accelerated in simple buffer slutin. In a 4% albumin slutin, additin f 12.5 mm salicylate did nt alter pht decay f bilirubin,18 whereas additin f 12.5 mm sulfisxazle accelerated pigment decay (table 3). These cncentratins f salicylate 18 and sulfisxazle 18, 19 are knwn t displace bilirubin frm binding t albumin. In the absence f albumin, salicylate accelerated bilirubin decay, whereas sulfisxazle had n effect. T assess the rle f heavy metal catins, bilirubin phtdecay was studied in the presence f EDTA (table 4). Withut albumin, 1.0 mm EDT A arrested the de- TABLE 3. Effect f uncupling frm albumin n phtdecay f bilirubin a Slutin Albumin a in phsphate buffer, ph 7.4 Brate buffer, ph 8.5 Anin added mm Salicylate, 12.5 Sulfisxazle, 12.5 Salicylate, 12.5 Sulfisxazle, 12.5 Decayb %/hr 62.1 ± 3.3 (SD) 64.8, < 20.4 ± 2.9 (SD) 31.3< 18.8 a Bilirubin cncentratin, 0.26 mm; albumin cncentratin, 0.52 mm. Slutins were expsed t blue flurescent lamps. b Calculated frm initial expnential cmpnent (first 75 min) f the decay curve. < Significantly different frm cntrl, P < 0.01.

5 January 1970 PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN 19 cay f bilirubin 4 but, with albumin present, EDTA had n effect even at 5.0 mm cncentratin. Effectiveness f different flurescent lamps. Since similar kinetics were bserved under all flurescent lamps studied, the fractinal decay cnstant, calculated by the methd f least squares 20 frm the initial cmpnent f the phtdecay curve, culd be utilized t assess the relative effectiveness f each type f lamp. In figure 3, these phtdecay rates in albumin (ph 7.4), serum (ph 7.4), and 0.1 N NaOH (ph 13.0) are pltted against the radiant energy incident upn the surface f the slutin frm each set f six lamps in the canpy. Fr each set f lamps, the decay in alkali was crrected fr the 26.5% per hr lss f bilirubin which ccurred in this medium in the dark and was therefre nt light-related. In all three slutins, bilirubin was as stable under red lamps (R), which emit nly at wavelengths abve 600 mft, as in the dark. Since these red wavelengths did nt affect bilirubin decay, nly shrter wavelengths, between 300 and 600 mft, were cnsidered in calculating the effective radiant energy f the ther six types f lamps. The relative effectiveness f different lamps was similar in all three slutins, with phtdecay in albumin generally much mre rapid,8 and in alkali smewhat less rapid, than in serum. Except fr the black bulbs (.), the rate f bilirubin phtdecay in each medium was rughly prprtinal t the ttal radiant energy emitted between 300 and 600 mft, even thugh the relative cntributins frm varius prtins f the spectrum varied widely amng different clred lamps (table 1). This is best illustrated by cmparisn f the blue (B), daylight (D), and cl white (W) lamps, which have similar ttal radiant energies and caused similar rates f bilirubin decay despite marked differences in the prprtin f energy emitted in the vilet and blue (400 t 500 mft) as cmpared with the green and yellw (500 t 600 mft) regins f the spectrum. Hwever, the gld lamps (G), which emit exclusively abve 500 mft, TABLE 4. Effect f EDT A n phtdecay f bilirubin a Slutin EDTA Decayb mm %/hr Brate buffer, ph (SD) < Albumin a in phs (SD) phate buffer, ph a Bilirubin cncentratin, 0.26 mm; albumin cncentratin, 0.52 mm. Slutins were expsed t blue flurescent lamps. b Calculated frm initial expnential cmpnent (first 75 min) f the decay curve. c Significantly different frm cntrl, P < % PER HOUR ALBUMIN ph r&l SERUM ph [QJ lid x NOH O.IM, ph-13.0!wi B 20 0 w RADIANT ENERGY OF LAMPS ( TlJI) m W/cm 2 FIG. 3. Phtdecay f bilirubin in three different slutins under seven different flurescent lamps. Fr details, see text. were nly half as effective as the pink lamps (P), which emit less energy in this green-yellw regin but give ff, in additin, several intense spectral lines at 400 and 440 mft. The black lights, which emit almst exclusively in the ultravilet, were nly half as effective as the ther five lamps which emit principally in the visible spectrum. Rle f biliverdin in bilirubin phtdecay. Under blue r daylight lamps, serial spectral changes f bilirubin in 0.1 N NaOH (fig. 4) resembled thse btained in prir studies with an intense mercury lamp,3 but prceeded abut ne-fifth as rapidly. First rder decay f the bilirubin peak at 465 mft was accmpanied by increased absrbance f the maximum at 315 mft, and waxing and then waning f

6 20 OSTROW AND BRANHAM Vl. 58, N.1 in O.lN NOH in 4% ALBUMIN ph 7.4 :I: <!) Z w 0.50.J w L-A.--_. 380l1\li > mp u u w a. (f) 0.01 ci L ci DURATION OF EXPOSURE - HOURS FIG 4. Serial changes during illuminatin in spectral absrbance f a slutin f bilirubin in 0.1 N NaOH (left) r albumin slutin (right). Cnditins as in figures 1 and 2. Each curve shws the change with time f absrbance at the specified wavelength, determined frm the ptical density f serial samples taken frm the illuminated slutins and diluted 10-fld fr spectrscpic analysis. ALBU Mlli r AT mp the biliverdin peaks at 380 and 650 mp' and f the absrbance "shulder" near 575 mp.. In cncrd with this pattern, the initially range slutin became temprarily greenish brwn, but later develped a glden brwn clr which prgressively faded thereafter. By cntrast, in neutral albumin slutin, the biliverdin peaks and t he 575 mp' shulder prgressively increased as the initially yellw slutin became and then remained deep green in clr (fig. 4). These results suggested that biliverdin was an early intermediate in the phtdecay f bilirubin and that biliverdin was stabilized in neutral albumin slutin. This was cnfirmed by study f the phtdecay f biliverdin under varius cnditins (fig. 5). With albumin present, biliverdin was cmpletely stable at ph 7.4 r 8.5, but decayed slightly at ph 9.9 and rapidly at ph Thus, unlike bilirubin, biliverdin in albumin slutin was mre stable at lwer ph levels. On the ther hand, withut albumin, biliverdin, like bilirubin, decmpsed mre rapidly at higher ph levels. Cmparisn f the results with and withut albumin at ph 8.5 and R f SPOT FLUOR Orange BI-Gr r + BI-Gr I , , , DURATION OF EXPOSURE - HOURS FIG. 5. Phtdecay f biliverdin at varius ph levels with r withut added albumin. Biliverdin cncentratin in each sample was assessed frm ptical density at 650 mp' after dilutin with 9 parts phsphate buffer, ph 6.5. Initial cncentratins: 0.05 mm biliverdin and 0.52 mm albumin. Lighting cnditins as in figure Yellw Yellw Red Blue FIG. 6. Ascending paper chrmatgram f phtderivatives f bilirubin r biliverdin, shwing fr each derivative, P-l thrugh P-7, its flurescence clr under ultravilet light. Chrmatgram n Sand S 598 paper, develped fr 6 hr with n-butanl-ethanl-water (300: 100: 150). Shading represents relative flurescence intensities f derivatives btained during illuminatin f bilirubin in 0.1 N NaOH.

7 January 1970 PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN 21 tives detected when bilirubin in 0.1 N N aoh was allwed t decay in the dark. Reactin sequence. Radiassay f labeled pht derivatives extracted at different intervals after illuminatin f 14C-bilirubin (fig. 7) revealed that the chlrfrmsluble pigments, bilirubin and biliverdin, and the rapidly migrating derivatives, P-5, P-6, and P-7, cntained mst f the label during the early phases f phtdecay. Of the residual diaz reactivity at 30 and 60 min, 25 and 56%, respectively, was nt extracted int chlrfrm and represented the diaz-psitive derivatives, P-5 and P-6. As the reactin prgressed, these diaz-reactive prducts waned, and increasing prprtins f radiactivity appeared in the slwer mving derivatives P-3 and P-4 and eventually in P-2 and in the clrless, highly water-sluble prducts which did nt extract int butanl. When the separated pht derivatives were eluted, individually re-expsed t light, I- > u cr cr II::..J cr I l- I&. z 2 l II:: IN CHCI, PHASE IN COLORLESS AQUEOUS PHASE 9.9 indicated that albumin itself retarded biliverdin decay at these ph levels. Paper chrmatgraphy f the water-sluble prducts frmed during phtdecay f biliverdin yielded the same pattern as the phtderivatives f bilirubin (fig. 6). Bth bile pigments yielded the same derivatives at all ph levels, with all types f lamps, and whether r nt albumin was present, but the relative prprtins f the seven spts, designated P-l thrugh P-7, varied depending upn the cndiins emplyed. Thus (a) P-4 was detected nly when illuminatin was perfrmed with a strng ultravilet emitter such as black flurescent bulbs r the mercury spt lamp.3 (b) The slwly migrating spts with red (P-2) and yellw (P-3) flurescence were mst prminent when bilirubin was irradiated in the presence f albumin. (c) In the absence f albumin, the rapidly migrating spts, P-5, P-6, and P-7 were mst prminent, and were the nly deriva- 40'1. IN CHROMATO- GRAPHIC 30'1. SPOTS P-2 - P-7 20'1. 10'1. 22"1. 6% 10% 3"1. 5% 3% 10% 17% 35% 51% II:: G MINUTES OF ILLUMINATION FIG. 7. Prprtins f radiactivity fund in varius derivatives at intervals during phtdecmpsitin f 14e-bilirubin, 30 mg per 100 ml, in 0.1 N NaOH, under unfiltered mercury spt lamp. Pigments were extracted frm samples taken at intervals and then chrmatgraphed as in figure 6. The varius extracts and the spts P-1 thrugh P-7 cut frm dried chrmatgram were assayed fr radiactive cntent and expressed as percentage f ttal initial radiactivity in the sample. CHCI. phase, radiactivity extracted int chlrfrm, principally bilirubin and biliverdin. Clrless aqueus phase, radiactivity in clrless derivatives which remained in aqueus phase during butanl extractin. P-2 thrugh P-7 refer t spts frm chrmatgram (see figure 6). Spt P-1 never cntained mre than 0.2% f ttal radiactivity.

8 22 OSTROW AND BRANHAM Vl. 58, N.1 TABLE 5. Reilluminatin f purified phtderivafives a Pure derivative Qualitative flurescence intensity f derivatives detected after re-expsure t light P-7 P-6 P-5 P-4 P-3 P-2 P-l P-7 trb H P-6 tr H 2+ tr tr P-5 tr 2+ H P-4 tr 2+ tr P-3 tr H P-2 tr 2+ a Each pure derivative was illuminated fr 90 min in neutral aqueus slutin under intense mercury fld lamp, unfiltered. Slutin was then chrmatgraphed as in figure 6, and spts were identified by flurescence under ultravilet lamp. b Trace. - 2 x---x NO BILIVERDIN in 0.1 N NaOH BILIRUBIN in 4% ALBUMIN ph , -, DURATION OF EXPOSURE-mins. FIG. 8. Phtdecay f bile pigments in air (02) and under dexygenated nitrgen (n 02). Cnditins and methds as in figures 1 and 5, but pigment cncentratins are expressed as percentage f initial cncentratin befre illuminatin. Graph f decay f bilirubin in albumin, ph 7.4 (lwer right), represents mean f three almst identical experiments. and rechrmatgraphed, each derivative yielded nly spts with slwer R f values, althugh nt all f the slwer mving spts were btained frm any given derivative (table 5). The general sequence f phtdecay was, therefre, bilirubin, biliverdin, rapidly migrating less plar derivatives with weak diaz reactivity, and finally, slwer migrating mre plar derivatives which were diaz-negative. Effect f dexygenatin (fig. 8). In 0.1 N NaOH, phtdecay f bilirubin and biliverdin was virtually arrested in the dexygenated system. By cntrast, in three experiments in neutral albumin slutin, anxia slwed but did nt arrest bilirubin phtdecay (mean ± BE: with xygen, 69.8 ± 0.8% per hr; withut xygen, 59.9 ± 0.7% per hr; t = 10.22; P < 0.001). Similar slwing withut arrest f phtdecay was bserved with bilirubin in human serum, with 42.5% decay under xygen and 16.3% under nitrgen. Prlnged dexygenatin did nt alter these results. Discussin These studies demnstrate that, during phtdecmpsitin f bilirubin in vitr, changing cnditins f ph, lighting, and albumin cncentratin mdified the relative rates f the sequential reactins and the relative stability f the varius intermediates withut altering the nature f the derivatives frmed. Indeed, the same prducts were btained in the absence f light during decmpsitin f bilirubin in 0.1 N NaOH. Irrespective f cnditins, a green clr and a peak at 650 mp' appeared early during the phtdecay f bilirubin and suggested, as thers have nted,4-6, 21 frmatin f biliverdin. Hwever, the present demnstratin that illuminatin f biliverdin yielded the same water-sluble derivatives as illuminatin f bilirubin cnstitutes the first clear prf that biliverdin is an early intermediate in the phtdecay f bilirubin. Further steps in the reactin sequence were clarified by extractin and chrmatgraphy f labeled phtderivatives (fig. 7) and reilluminatin f the purified phtpigments (table 5). The results f bth f these studies were in agreement and indicated that the faster migrating, diazreactive derivatives were frmed earlier in the sequence and were the precursrs f the slwer migrating, mre plar, diaznegative cmpunds. If it may be assumed that the spts detected were the

9 January 1970 PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN 23 nly prducts frmed frm illuminatin f a given derivative, the data in table 5 suggested three degradative pathways: (a) biliverdin...,. P-7...,. P-2...,. P-l; (b) biliverdin...,. P-6...,. P-5...,. P-3...,. P-2...,. P-l; and (c) biliverdin...,. P-4...,. P-3...,. P-2""'" P-l. The last f these pathways apparently ccurred nly under lamps with significant ultravilet emissin, and the cnversin f P-5 t P-3 apparently required the presence f light. This schema can be cnfirmed nly by islatin and identificatin f the prducts, but it ffers a tenable explanatin fr the alteratins in the kinetics f the tw cmpnents f bilirubin decay in different media. With bilirubin in alkali in the dark, nly the first cmpnent was bserved and nly the mre rapidly migrating phtderivatives were frmed. Since P-5 and P-6 are diaz-psitive, they presumably cntributed t the diaz reactivity f the slutin 9 and thus resulted in false verestimatin f the cncentratin f bilirubin which remained. Upn illuminatin, cnversin f P-5, P-6, and P-7 t the slwer migrating, diaz-negative prducts, P-3 and P-2 accelerated the disappearance f diaz reactivity, which accunts fr the appearance f the mre rapid secnd decay cmpnent. The presence f albumin and lwer ph stabilized biliverdin and favred frmatin f the slwer migrating derivatives which are diaz-negative, s that bilirubin decay, measured by diaz reactivity, appeared mre rapid under these cnditins. Biliverdin was stabilized markedly at neutral as ppsed t high ph, especially if albumin was present als. By cntrast, during illuminatin, bilirubin was unstable even at weakly alkaline ph and deterirated mre rapidly in the presence f albumin, especially at mre neutral ph levels. Binding t albumin alters the cnfiguratin f the bilirubin mlecule,21 pssibly rendering it mre susceptible t phtdecmpsitin. Hwever, binding f bilirubin is little altered 22 between ph 8 and 10, yet the accelerating effect f albumin n pigment decay diminished greatly when the ph was raised frm 8.5 t 9.9. Mrever, decay f bilirubin was just as rapid when bilirubin was uncupled frm albumin by cmpetitive binding f salicylate r sulfisxazle. (We have n explanatin fr the differences in the effects f these tw anins, shwn in table 3.) Anther pssibility is that divalent catins, cntaminating the albumin, accelerated bilirubin decay. Others 4,23 have shwn that Zn++ ins accelerate and EDT A arrests decay f bilirubin in albumin-free slutins at ph 10 t 11, and ur data cnfirm this finding at ph 8.5. Hwever, the failure f EDTA t alter the rate f bilirubin phtdecay in albumin slutin at ph 7.4 suggests that cntaminant divalent catins did nt accunt fr the effect f albumin in accelerating bilirubin decay. Biliverdins are frmed during xidatin f bilirubins catalyzed by ferrus ins 24 r during the Gmelin reactin,25 and Barac and Gernay26 have shwn that xygen is cnsumed during bilirubin decay in the dark at ph 10 t 11. The arrest f pigment phtdecay in alkali when xygen was excluded (fig. 8) demnstrated that the phtdecmpsitin f bilirubin and biliverdin were xidative als. Since the same derivatives were frmed in neutral albumin slutin and in alkali, and thers7 have fund an increased redx ptential during phtdecay f bilirubin in serum, the phtdecmpsitin f bilirubin in albumin slutin als must be xidative. It was, therefre, surprising that dexygenatin merely slwed but did nt arrest phtdecay f bilirubin in albumin slutin. The same result was btained with native serum, indicating that cntaminants added during preparatin f the albumin were nt respnsible fr this effect. Apparently albumin, r ne f its ligands, can substitute fr xygen as a hydrgen acceptr during the phtreactin. A likely candidate is the vinyl grup f bilirubin itself, since thers have shwn 4,27 that bilirubin can be cnverted t mesbiliverdin by internal prtnatin frm the methane bridge t the vinyl side chain. Hwever, albumin can underg cupled phtreactins with certain dyes,28 and it is nt imprbable that a similar cupled pht-

10 24 OSTROW AND BRANHAM Vl. 58, N.1 xidatin ccurred between albumin and bilirubin. The cmparative studies f different flurescent lights indicated that, taken as a whle, the blue-vilet (400 t 500 m,u) and yellw-green (500 t 600 m,u) regins f the spectrum mst effectively destryed bilirubin, that red light (>600 m,u) was ineffective, and that ultravilet light (300 t 400 m,u) had intermediate ptency. The lesser effect f ultravilet light may have been due in part t absrptin by the glass walls f the vessel. The data cmparing the pink and gld lamps suggested, hwever, that the spectral line at 440 m,u, clse t the absrptin maximum f bilirubin, may be mst effective f all. Sissn's recent reprt 29 has cnfirmed these bservatins. Cremer and c-wrkers 7 drew similar cnclusins, but their data are limited by failure t adjust fr radiant energy emitted in the different prtins f the spectrum. These findings have tw practical implicatins. (a) In handling bilirubin in the labratry, the pigment can be stabilized in serum r in physilgical albumin slutin by perating under red light and in albumin-free slutins by additin f EDTA r exclusin f xygen. (b) The emissin spectrum f lamps utilized fr phttherapy f the newbrn shuld be selected within the blue-vilet and yellwgreen prtins f the spectrum t eliminate thse wavelengths which mst prminently generate unwanted side effects.3 REFERENCES 1. Ostrw, J. D Factrs affecting the phtdecmpsitin f bilirubin and biliverdin in vitr (abstr.). Fed. Prc. 28: Lucey, J., M. Ferreir, and J. Hewitt, Preventin f hyperbilirubinemia f prematurity by phttherapy. Pediatrics 41: Ostrw, J. D Pht-xidative derivatives f "C-bilirubin and their excretin by the Gunn rat, p In 1. A. D. Buchier and B. H. Billing [eds.], Bilirubin metablism. Blackwell Scientific Publicatins, Oxfrd. 4. DeEwensn, 1. W., F. A. Gianturc, and P. Gramaccini On the stability f bilirubin. Experientia 22: Gign, A., and M. Nverraz Etude cinetique de la diaztisatin du serum sanguin. Schweiz. Med. Wschr. 69: Blndheim, S. H., D. Lathrp, and J. Zabriskie Effect f light n the absrptin spectrum f jaundiced serum. J. Lab. Clin. Med. 60: Cremer, R. J., P. W. Perryman, and D. H. Richards Influence f light n the hyperbilirubinemia f infants. Lancet 1: Lathe, G. H., P. Lrd, and C. Tthill Bilirubin transprt by plasma prtein, p In P. Desgrez and P. M. DeTraverse [eds.], Transprt functin f plasma prteins, Vl. 5. Elsevier Publishing Cmpany, New Yrk. 9. Blndheim, S. H., and N. A. Kaufmann The effect f light n the direct diaz reactin f uncnjugated bilirubin. J. Lab. Clin. M ed. 65: Brughtn, P. M. G., E. J. R. Rssiter, C. B. M. Warren, G. Gulis, and P. S. Lrd Effect f blue light n hyperbilirubinemia. Arch. Dis. Child. 40: Gray, C. H., A. Kulczyka, D. C. Nichlsn, and Z. Petryka The chemistry f the bile pigments. II. The preparatin and spectral prperties f biliverdin. J. Chern. Sc. 2: Nir, B., E. R. Garay, and M. Ryer Separatin and prperties f cnjugated biliverdin. Bichim. Biphys. Acta 100: Gmri, G Preparatin f buffers fr use in enzyme systems, p In S. P. Clwick and N. O. Kaplan [eds.], Methds in enzymlgy, Vl. 1. Academic Press, Inc., New Yrk. 14. Michaelssn, M Bilirubin determinatin in serum and urine. Studies n diaz methds and a new cpper-az pigment methd. Scand. J. Clin. Lab. Invest. Suppl. 5: Hashimt, S., E. A. Glende, Jr., and R. O. Recknagel Hepatic lipid perxidatin in acute fatal human carbn tetrachlride pisning. New Eng. J. Med. 279: Ostrw, J. D., L. Hammaker, and R. Schmid The preparatin f crystalline bilirubin-c". J. C/in. Invest. 40: Bray, G. A A simple efficient liquid scintillatr fr cunting aqueus slutins

11 January 1970 PHOTODECOMPOSITION OF BILIRUBIN AND BILIVERDIN 25 in a liquid scintillatin cunter. Anal. Bichem. 1: Watsn, D The transprt f bile pigments: the binding f sdium bilirubinate t plasma prtein. Clin. Sci. 22: Jsephsn, B., and P. Furst Sulfnamides cmpeting with bilirubin fr cnjugatin t albumin. Scand. J. Clin. Lab. Invest. 18: Edwards, A. L Statistical methds fr the behaviral sciences, Ed. 1. Hlt, Rinehart and Winstn, New Yrk. 21. Fg, J., and E. Jellum Structure f bilirubin. Nature (Lndn) 198: Wennberg, R. P., and M. L. Cwger Spectral identificatin f albumin-bilirubin cmplexes (abstr). Pediatr. Res. 3: Fg, J., and B. Bugge-Asperheim Stability f bilirubin. Nature (Lndn) 203: Lemberg, R Bile pigments. VI. Bilirubin, uterverdin, and cyan. Bichem. J. 28: Siedel. W., and W. Frwis Uber die Vilettstufe der Gmelinschen Reaktin (Mesbilipurpurine). I. Mitteilung liber den Mechanismus der Gmelinschen Reaktin. Zt. Physil. Chern. 267: Barac, G., and J. M. Gernay Recherches sur la cenapse albumin-bilirubinique. Bull Sc. Chim. Bii. (Paris). 31: Gray, C. H., A. Kluczyka, and D. C. Nichlsn Chemistry f the bile pigments. Part III. Prttrpy f bilirubin t a verdinid pigment. J. Chern. Sc. 2: Odell, G. B Effect f cupled phtxidatin with methylene blue n bilirubinbinding by albumin. In D. Y. Y. Hsia [ed.], Bilirubin metablism in the newbrn. Natinal Fundatin Sympsium, Chicag. 29. Sissn, T. R. C Phtbilgic aspects f hyperbilirubinemia, D. Y. Y. Hsia [ed.], Bilirubin metablism in the newbrn. Natinal Fundatin Sympsium, Chicag. 30. Hsia, D. Y. Y. [ed.], Bilirubin metablism in the newbrn. Natinal Fundatin Sympsium, Chicag.