1. Absorption maxima of hydrochloric biliverdins derived
from the natural indirect bilirubin existed at 680 m&#956; and 375 m&#956;, but the maxima of biliverdins purified on the column of silica gel existed at 640 m&#956; and 390 m&#956;. 2. The natural salt-form bilirubin was oxidized by hydrochloric acid to biliverdin, of which absorption maxima existed at 685 m&#956; and 370 m&#956; in a methanolic solution as well as in 5% hydrochloric methanol, but the purified biliverdin in chloroform solution showed the maxima at 640 m&#956; and 390 m&#956;. 3. The natural ester-form bilirubin could be transformed into biliverdin by oxidation of its alcoholic solution in the presence of hydrochloric acid. The crude biliverdin had absorption
maxima at 645 to 655 m&#956;, 600 m&#956; and 320 m&#956;, and the crude hydrochloric biliverdin had the maxima at 665 to 675 m&#956;, 620 m&#956; and in the near ultra-violet range, while the purified biliverdin in chloroform solution had the maxima at 640 m&#956; and 380 m&#956;. 4. The biliverdins derived from the indirect, salt-form and ester-form bilirubin had quite similar absorption maxima after purifications by adsorption chromatography.</p

Sakamoto, Takeshi

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Studies on Bile Pigments III. Biliverdins Derived From Natural Bilirubins

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 10, Issue 1 1956 Article 5JANUARY 1956Studies on Bile Pigments III. BiliverdinsDerived From Natural BilirubinsTakeshi Sakamoto∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on Bile Pigments III. BiliverdinsDerived From Natural Bilirubins∗Takeshi SakamotoAbstract1. Absorption maxima of hydrochloric biliverdins derived from the natural indirect bilirubinexisted at 680 mµ and 375 mµ, but the maxima of biliverdins purified on the column of silica gelexisted at 640 mµ and 390 mµ. 2. The natural salt-form bilirubin was oxidized by hydrochloricacid to biliverdin, of which absorption maxima existed at 685 mµ and 370 mµ in a methanolicsolution as well as in 5% hydrochloric methanol, but the purified biliverdin in chloroform solutionshowed the maxima at 640 mµ and 390 mµ. 3. The natural ester-form bilirubin could be trans-formed into biliverdin by oxidation of its alcoholic solution in the presence of hydrochloric acid.The crude biliverdin had absorption maxima at 645 to 655 mµ, 600 mµ and 320 mµ, and the crudehydrochloric biliverdin had the maxima at 665 to 675 mµ, 620 mµ and in the near ultra-violetrange, while the purified biliverdin in chloroform solution had the maxima at 640 mµ and 380 mµ.4. The biliverdins derived from the indirect, salt-form and ester-form bilirubin had quite similarabsorption maxima after purifications by adsorption chromatography.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLSTUDIES ON BILE PIGMENTSIll. BILIVERDINS DERIVED FROM NATURALBILIRUBINSByTakeshi SakamotoDepartment of Internal Medicine, Okayama UniversityMedical School(Director: Prof. Dr. K. Yamaoka)Received for publication on 1 November 1955.IntroductionThree fractions of the natural bilirubin, the so-called in-direct, salt-form and ester-form bilirubin, were well separablechromatographically on the adsorption column as described inthe preceding reports. Though their a ttitudes towards chloro-form transferability, saponification and hydrolysis were quitedifferent, such a colour test peculiar to the bilirubin as thediazo, Gmelin and Fouchet reaction or a formation of hydro-chloric azobilirubin agreed well among them, but their spectro-chemical properties were not so. Bilirubin, on the other hand,is apt to transform into biliverdin though poorly in an alkalinesolution, which was proved to owe to autoxidation by Lembergl) ,while the colour of the isolated salt-form and ester-form directbilirubins would easily turn into greenish yellow in alcoholic orneutralized aqueous solutions.The main purpose of this report existed in an attempt tocompare the oxidized products among these three isolated natu-ral bilirubins.Experimental1) Materials.Three fractions of the natural bilirubin were isolated andpulverized after separation on the adsorption column from thedog's gallbladder bile as described in the preceding reports.These powders were stored in a dessicator up to use.A) Oxidation of the indirect Bilirubin.a) HydrochloricAcid Method..A. chloroform solution of the pulverized indirect bilirubin1Sakamoto: Studies on Bile Pigments III. Biliverdins Derived From NaturalProduced by The Berkeley Electronic Press, 195648 Takeshi Sakamoto:was added to two volumes of methanol, then concentrated hy-drochloric acid was introduced dropwise in a ratio of 5 to 10vol. % to the mixture, and it was left standing in a dark placeat room temperature (lO° C), during about 12 to 18 hours ayellow colour of the bilirubin turned into blue to greenish blue,then the chloroform phase was separated by shaking after addi-tion of a large amount of water. The crude biliverdin was obta-ined in a chloroform solution after washing the chloroformphase with water several times. As the solution might containpretty amounts of other intermediate substances, it was puri-fied on the adsorption column of silica gel.b) .A Modification of Lemberg and Legge's Method2l •An equal volume of ethanol containing 1 % hydrochloric acidwas added to the chloroform solution of the indirect bilirubin,then hydrogen peroxide was added dropwise in 1 % with shak-ing and the mixture was left standing at room temperature (20 0to 25 ° C), and then a yellow colour turned into blue during 15to 20 min. Further separation and purification of biliverdinfollowed the above.B) Oxidation of the Salt-form and Ester-form Bilirubins.Alcoholic solution of the pulverized salt-form or ester-formbilirubin was added to half a volume of chloroform, then con-centrated hydrochloric acid was introduced dropwise in a ratioof 5 to 10 vol. %. The mixture was left standing in a darkplace at room temperature (lO° C) for about 12 to 18 hours,then a yellow colour of the bilirubin turned into blue to greenishblue. Further procedures were the same as the above.11) Adsorbents and Ch1'omatography.Slicia gel was availed after activating as described in thepreceding reports. Adsorption columns were formed in a usualmethod.Chloroform was chiefly a vailed as a developing solvent,and the rate of flow was also measured in necessity.111) Measurements of the Extinction Coefficients.Electrophotometric Spectrophotometer, Type QB-50 (Shimn-dzu) was used to measure the extinction coefficient, and a tun-gsten bulb was used in the visible range.2Acta Medica Okayama, Vol. 10 [1956], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss1/5Studies on Bile Pigments 49Results and Discussion1) Biliverdin Derived from the Indirect Bilirubin.A) Biliverdin Oxidized by Hydrochloric Acid Method.When coneentrated hydrochloric acid was further intro-duced into a mixture of chloroform-methanolic (l: 2) solutionof the isolated indirect bilirubin, a light brownish yellow solu-tion grew suddenly into light reddish brown. While this solu-tion was left standing in a room for about half a day or more,the colour turned into blue and did not show any yellow tone.Chloroform phase was then separated by adding a large amountof water, and the most of the blue pigments were transferredinto chloroform. After washing with water several times thechloroform solution was filtrated, and the filtrate was driedin vacuo. The dried blue pigment was then dissolved into 5 %hydrochloric methanol and the absorption curve was calibrated.The absorption maxima were recognized at 680 mu and 375 mp.,and the minimum at 490 to 500 m/u (Fig. 1, 1.).The absorption curve was clearly the same as the oneof hydrochloric biliverdin and agreed well with hydrochloricbiliverdin derived from the so-called bilirubin, the crystallinebilirubin.When a chloroform solution of the crude biliverdin wasadsorbed on the column of silica gel and developed with chloro-form alone, a pink zone flowed out first of all, and then a bluezone was clearly separable and was followed by a fairly diffuseviolet zone leaving a brown zone behind on the surface of thecolumn (Fig. 2.). The blue pigment in the effluent was introdu-ced into a chloroform solution after evaporating the solvent.When absorption curves were calibrated on the solution, themaxima existed at 640 mp. and 390 mp. and the minimum at500 mp- (Fig. 1, 11.).B) Biliverdin Oxidized by a Modification of Lemberg andLegge's Method.When 1 % hydrochloric ethanol was added to the chloro-form solution of the indirect bilirubin in an equal volume andhydrogen peroxide was further added, the yellow colour turnedgradually into blue within 15 to 20 min. The blue pigment wastransferred into chloroform by addition of water. The chloro-3Sakamoto: Studies on Bile Pigments III. Biliverdins Derived From NaturalProduced by The Berkeley Electronic Press, 195650 Takeshi Sakamoto:Fig. 1. Absorption Curves of the Biliverdin Derived from the IndirectBilirubin.I ...... 5% HCI methanolic solution of the hydrochloric biliverdin, derivedfrom the chloroform-methanolic (1: 2) solution of the isolated indirectbilirubin in the presence of HCl.Il ...... Chloroform solution of the chromatographically separated biliverdin.The biliverdin was derived from the natural indirect bilirubin in thesame way as the above. The crude biliverdin was developed withchloroform on the adsorption column of silica gel, and then purifiedblue biliverdin was got in powders after drying in vacuo. Chloro-form solution of this purified dried biliverdin was subjected to cal-ibration.III ......Chloroform solution of the chromatographically separated biliverdin.The biliverdin was derived from the natural indirect bilirubin by amodification of Lemberg ani Legge's method2 ). The crude biliverdin-was purified chromatographically like the above, and the purifieddried biliverdin was calibrated in a chloroform solution.Absorption Maxima. Absorption Minima.I 680 mjl. 375 mjl. 490 to 500 mjl.Il 640 ~~ 390" 500 "III 390 ~/ 390 ~/ 500 //form solution was washed furthermore several times withwater, and subjected to further analysis.The chromatogram of this chloroform solution was identi-cal with the former, and a blue zone flowed out lately follow-ing a slight pink zone. Ab30rption maxima of the chloroformsolution of the blue pigment after evaporating the solvent ofthe effluent were recognized at the same place as the former(Fig. 1, 111.).11) Biliverdins D3rived from the Salt-form Bilirubin.A yellow colour of the n-propanolic solution of the salt-4Acta Medica Okayama, Vol. 10 [1956], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss1/5Studies on Bile Pigments 51I_ •• DARK bROWNIf!''"'!'~':~~;''' YELLOWC .1 ~ la.. ........,....T r~.:.f'D'~ VIOLE Tb t BLUEFig. 2. Chromatogram of the Oxidized Pro-ducts of the Natural Bilirubin.Material· .. ··· Chloroform solution of the oxi-dized products of the naturalindirect bilirubin.Column Chloroform packed silica gelcolumn.Developing solvent Chloroform alone.The pigments contained in the b-zone wasidentified with the biliverdin, showing the ab-sorption maxima at 640 m,u and 390 m,u in chloro-form solution, and the chloroform solution ofeach oxidized products of three fractions of thenatural bilirubin formed the same chromatogram.R (b) = 0.30, R (c) = 0.28.form bilirubin turned remarkably into blue in half a day ormore. The blue pigment was almost completely transferred intoa diethyl ether phase when a mixture of the blue n-propanolicsolution was shaken hard with some quantities of ether and alarge amount of water. After washing the ether phase withwater several times, it was dried in vacuo to obtain the bluepigments. The pigment was easily and completely soluble inmethanol. The absorption maxima of the methanolic solutionexisted at 685 mp. and in the near ultra-violet range, and theminimum at 500 mp. (Fig. 3, 1.). The absorption curve in 5 %hydrochloric methanol was very similar to the above (Fig. 3, 11.).After leaving a 5 % hydrochloric methanolic solution of it stan-ding in a room, it was filtrated and calibrated the absorptioncurve on the filtrate, and the maxima existed at 685 mp.. and370 mp.(Fig. 3, 111.). A yellow colour of a mixture of a metha-nolic solution of the salt-form bilirubin, chloroform and hydro-chloric acid turned into blue in half a day or more. The chloro-form phase was then separated, and chloroform was evaporatedto obtain the blue pigments. The absorption maxima of thedried blue pigments exsited at 685 mp. and 370 mp. when theywere dissolved into methanol (Fig. 3, IV.) or 5 % hydrochloricmethanol (Fig. 3, V.).The dried blue pigment was dissolved into chloroform andwas subjected to chromatographic analysis. The chromatogramobtained on the adsorption column of silica gel by development5Sakamoto: Studies on Bile Pigments III. Biliverdins Derived From NaturalProduced by The Berkeley Electronic Press, 195652 Takeshi Sakamoto:Absorption Curves of the Biliverdin Derived from the Salt-formAbsorption Minima.490-480 mp.500-490 ~r480 ~r480 ~r500 ~r500 rrFig. 3.Bilirubin.I······Methanolic solution of the crude biliverdin which was extracted bydiethyl ether from the oxidized products of the natural salt-formbilirubin in n-propanolic solution in the presence of HCI.n ... 5 % HCI methanolic solution of the crude biliverdin derived like theabove.Ill"·5% HCI methanolic solution of the pulverized salt-form bilirubin,after leaving in a room for a day.IV", Methanolic solution of the crude biliverdin which was extracted bychloroform from the oxidized products of the natural salt-form bili-rubin in methanolic solutions in the presence of HCI.V ... 5% HCI methanolic solution of the crude biliverdin derived like theabove (IV).VI .. · Chloroform solution of the chromatographically separated biliverdin.The crude biliverdin was derived like the above (IV). A chloroformsolution of the crude biliverdin was developed with chloroform onthe adsorption column of silica gel and the purified biliverdin wasobtained.Absorption Maxima.1. 685 m/-! near ultra-violetn 685 N near ultra-violetIll. . .. 685 N 370 m/-!IV. 685 ~~ 370 ~rV 685 N 370 NVI. 640 ~r 390 ~rwith chloroform was quite similar to the former and a blue zonewas clearly separated. The absorption maxima of the blue pig-ment in the chloroform solution existed at 640 m/l. and 390 m/l.(Fig. 3, VI.) and were identical with the one derived from theindirec t bilirubin.6Acta Medica Okayama, Vol. 10 [1956], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss1/5Studies on Bile Pigments 53111) Biliverdins Derived from the Ester-form Bilirubin.A light greenish yellow tone of the methanolic solution ofthe ester-form bilirubin, isolated with the column of silica geland further pulverized as described in the preceding report,turned into a clear blue tone ina day when some quantities ofconcentrated hydrochloric acid had been added beforehand.After separating the blue pigment by addition of chloroformand water like the above, the chloroform phase was washed withwater several times and dried in vacuo in a dessicator. Thedried blue pigment was then dissolved into methanol (Fig. 4, 1.)~mI~~~\~--rr."':-:=-I--;;::-.... ......-..-f 20WAVROOTH <-f) ,Fig. 4. Absorption Curves of the Biliverdin Derived from the Ester-formBilirubin.Biliverdin was derived from the ester-form bilirubin isolated on the ad-sorption column of silica gel. Oxidation was carried out on a methanolic or anaqueous solution of the bilirubin with HCI, and the blue oxidized productswere transferred into chloroform by washing the reactants with water, andthen the chloroform solution was dried in vacuo to obtain the green powders.I······Methanolic solution of the green powder from the methanolic oxidi-zed products.Il ... 5% HCI methanolic solution of the green powder from the methano-lic oxidized products.III···Methanolic solution of the g-reen powder from the aqueous oxidizedproducts.IV···Chloroform solution of the purified biliverdin. A chloroform solutionof the green powder from the methanolic oxidized products was deve-loped with chloroform alone on the adsorption column of silica gelto obtain the purified biliverdin of R = 0.30.A bsorption Maxima. Absorption Minima.1······645 to 655 m.u 600m.u 320 m.u 510-500 m.uIl ... 665 to 675 ~~ 620 N near ultra-violet 510 ~~Ill·· ·650 to 665 ~~ 600 N' 320 ~~ 520 ~~IV· .. 640 ~~ 380 ~~ 500 ~~7Sakamoto: Studies on Bile Pigments III. Biliverdins Derived From NaturalProduced by The Berkeley Electronic Press, 195654 Takeshi Sakamoto:or 5 % hydroc4loric methanol (Fig. 4,11.). The absorption max-ima of the £9rmer ~xisted at 645 to 655 mp., 600 mp and 320 mll,but of the latter at ~55 to 675 mp, 620 mp. and in the near ultra-viol~t ra.nge, while the absorption minima existed at 510 to 500mp. respeetively. Similarly, after transferring the blue pigmentinto chloroform from a hydrochloric aqueous solution of biliver-din, chloroform was washed with water several times. Afterdrying in vacuo, absorption curves were calibrated in a metha-nolic solution (Fig. 4, 111.), and the maxima were quite similarto the one (Fig. 4, 1.) of the above methanolic solution.According to Hosokawa* the absorption maxima at 600 to620 mp. and in the near ultra-violet range of these curves maybeowe to some further oxidized products. To find a completeexplanation of these possiblities, these blue pigments wereexamined on the column of silica gel by development withchloroform. A pink zone moved down first of all, then a splen-did blue zone appeared moving in a low rate of flow (R = 0.30)accompanied with a violet zone in some distance and left a darkbrown to blackish zone on the upper surface. The absorptionmaxima of the chloroform solution of the blue pigment of R =0.30 existed at 640 mll and 380 m/I. (Fig. 4, IV.).According to Lemberg and Legge3 ) the absorption maxima ofa methanolic solution of the biliverdins existed at 640 m/I. and392 mll, and of 5 % hydrochloric methanol at 680 m/I. and 377mf/.. Though these absorption maxima were almost identicalwith the one derived from the indirect bilirubin as describedabove, the ones derived from the bilirubins did not agree withthese results while they were crude, but the ones purified bychromatography agreed well with those reported by Lembergand Leggea>, Amada-t) and Engel5>. But when the crude hydro-chloric biliverdins derived from the salt-form bilirubin are com-pared with the one derived from the indirect bilirubin, there isa parallel point in their maxima. These facts may well be ex-plained as follows: When hydrochloric acid is added to thesalt-form bilirubin it will be transformed into the dibasic acidbilirubin. Hydrochloric acid will further act secondarily as anoxidizer upon the newly formed indirect bilirubin, and then* Personal communication.8Acta Medica Okayama, Vol. 10 [1956], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss1/5Studies on Bile Pigments 55each hydrochloric biliverdin may resemble closely in their max-ima. But the ester-form bilirubin will not be hydrolyzed byhydrochloric acid, and the crude biliverdin derived from it willnot agree with the one derived from the indirect bilirubin.Though it is not easily concluded whether the biliverdinsderived from the indirect, salt-form and ester-form bilirubinare identical with or different from one another, the purifiedones resembled closely at least.Summary1. Absorption maxima of hydrochloric biliverdins derivedfrom the natural indirect bilirubin existed at 680 mp. and 375mp., but the maxima of ~iliverdins purified on the column ofsilica gel existed at 640 mp. and 390 mp..2. The natural salt-form bilirubin was oxidized by hydro-chloric acid to biliverdin, of which absorption maxima existedat 685 mp. and 370 mp' in a methanolic solution as well as in 5 %hydrochloric methanol, but the purified biliverdin in chloro-form solution showed the maxima at 640 mp. and 390 mp..3. The natural ester-form bilirubin could be transformedinto biliverdin by oxidation of its alcoholic solution in the pre-sence of hydrochloric acid. The crude biliverdin had absorptionmaxima at 645 to 655 m/fl, 600 mp. and 320 mp., and the crudehydrochloric biliverdin had the maxima at 665 to 675 mp., 620mfl and in the near ultra-violet range, while the purified bili-verdin in chloroform solution had the maxima at 640 mfl and380 mll.4. The biliverdins derived from the indirect, salt-formand ester·form bilirubin had quite similar absorption maximaafter purifications by adsorption chromatography.The author wishes to thank Prof. K. Yamaoka for his kind guidance andAssis. Prof. K. Kosaka for his valuable advice throughout this investigation.References) Lemberg, R.: Biochem. J., 28,978,1934. - 2> Lemberg, R., and Legge, J. W.:Aust. J. expo Med. Sci., 18, 95, 1940. - :l) Lemberg, R., and Legge, J. W.:Hematin Compounds and Bile Pigments. Interscience Publ. Inc., New York,p. 117, 1949. - 4) Amada, Y.: J. Biochem., 32, 187, 1940. - 0) Engel, M. :Z. f. physiol. Chem., 266, 135, 1940.9Sakamoto: Studies on Bile Pigments III. Biliverdins Derived From NaturalProduced by The Berkeley Electronic Press, 1956

Studies on Bile Pigments III. Biliverdins Derived From Natural Bilirubins

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