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Rapid transesterification of bilirubin glucuronides in methanol
Life Sciences Vol . 15, pp " 2069-2078 Printed is the II .S .A .
Pergamon Press
BAPm TBANSBSTSBIFICATZ(li OF BII.IRUBIIQ GIÜCUI:QTID&S IN MSTBANOL* Ma~el Salmon and Catherine Feneelau Department of Pharmacology and Bzperimeatal Therapearice Julio 0. Colder and Gerard B. Odell Department of Pediatrics The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 (Received is final form 12 November 1974) SDilfABY The current studies present aridence that bilirubin taajugates derived frost rat bile undergo rapid treneesterification, n i n vitro, in solnrions containing methanol . The conjugates of bilis~sbin sad the methyl esters formed from them by ezposura to methanol orate isolated by thin layer chromatography . The isolates were chemically quaaritated for their bilirubin and glncuronic acid coe~oeition. Characterization of the bilisvbin methyl esters was performed by mass spectrometric analysis of the trimethylsilyl sad pheaylazo derivatives . A large body of literature eziste on the heparic ezcrerion of bilirubin is many animal species (1,2) .
Two major products of bilisubin in normal
mamataliaa bile have bean identified ae the mono and diglucurori.des is which the glucuroaic acid is esterified with bilirubin at its propionic acid aide chains (3,4,5) .
Smaller fratrions of the bilirubin is bile have been des-
cribed as acylglycosidas of glucose and nylons, ae well as disaccharides of hw~+++~^^+~
acids that vary is differnat species (6,7) . Currently uncertainty
exists concerning the quantitative importance of the glucuronide conjugates, particularly ori.th reference to the relative amounts of bilirubin that are ,t
~
This work was supported by grants from the United Staten Public Health Service, N>B GM-16492, AO-4-fd~i-70417 and HD-02268 and in part by wade from the Andrew W. Mellon Foundation . Oa sabbatical leave from the Inetituto da Quimica de la Universidad National Antonama da Mexico, Mexico, 20, D.F . 2069
2070
Transesterifioation of Silirubin Glucuronides
e=~reted ae the moao and diglucuronide esters
Vol. 15, No . 12
(8,9) .
The current report preseate data that demonstrate rapid transaeterification o£ the ccgjugatea of bilirubin when exposed to methanol
(Schema 1) .
,H SCHEME I
These observations have sigaificant implications on the uaderatandiug of the hepatic excretion of bilirubin.
Solvent partitioning of polar conjugates of
bilirubin in bile have frequently employed methanol and chloroform (S) .
Be-
cause of the is-vitro capacity of the glucuronidee of bilirubia to undergo transeeterification is methanol, the resultant methyl esters would be preferentially recovered in the ap~iar chloroform layer.
Subsequent chemical
analysis by the diazo reaction would lead to both a qualitatively and quantitatively arroaeoue interpretation of the excretion of unconjugated bilirubin in bile, because the methyl esters of bilirubin have the same reaction requirements as unconjugatad bilirubia with diazotizad aulfanilic acid, ethyl anthranilate and phe~l diazonium chloride .
Such tranaeaterification may
have contributed to the differences observed by various woiicers in the amounts of free, mono and diglucuronides of bilirubin recovered is bile . nized this reaction offers as alternative to traasamination (10)
Dace reeogfor the
characterization of conjugates of bilirubia. METHODS A~ MATSR7N.5 The conjugates of bilirubin were obtained by canaalation of the cosmos
Vol.
15, No . 12
Tranaeaterification of Bilirubin Glucuro~nidea
bile duct of Wiatar rats receiving, bilirubin (11) .
2071
intravenously, an aqueous infusion of
Ia moat eaperimeate the bile was sequentially washed with
heaane and chloroform folloaPed by lyophilizatioa, and then analyzed by thin layer chromatography (see Figure 1) .
In same cease an aliquot of whole bile
was directly reacted with diazotized aulfanilic acid and the polar azodipyrroles were isolated by thin layer chromatography an cellulose 1ßt300 plates of 250p, thickaees aiaclcherey, Nagel, Co,) using ethyl methyl ketane :propioaic acid :water (75:25:35) . Quantitative measurements were made on the major pigments derived by chromatographic separation . with methanol .
These ware scraped from the plates sad eluted
The concentration of bilirubin from each spot was determined
colormetrically by com~ersion to aulfanily] .azo derivatives using standards prepared directly free crystallise bilirubin (12) .
The glucuronic acid con-
tent corresponding to the bilirubin spots was quantitated by the naphthoresorciaol assay (13) .
The standard glucuroaic acid aolutioae ware made up to
contain the same concentration of bilirubia to eliminate color interference in the spectrophotometric readings of the naphthoresorcinol assay for glucuroaic acid, Phenvlazo derivatives were obtained from bilixnbin and its esters by treatment with a chilled solution of phenyldiazonium chloride (14) .
The
colored products of this reaction were chramatographed on thin layers of 3102 GF, using chloroform :matheaol (99:1, 99 .5 :0 .5 or 5 :95) . Trimathvlsilvl derivatives ware prepared by reaction with N,0-bis(trimethyleilyl)trifluoroacetamide containing
lx
trichloramethylsilane,
l+tass
spectra were measured immediately after eaceas reagent was removed in vacuo . Authentic bilirubia dimethyl diester was prepared from ca~ercially available bilirubin (Sigma Chemicals) .
Treatment with diazomethaae provided
the authentic dimethyl diester of bilirubin, which was separated from smaller amounts of trimathyl and tetramathyl derivatives by this layer chromatography and recrystallized,
This compound was characterized by thin layer chromsto-
Transesterification of Silirubin Glucuronidea
2072
Vol. 15, No . 12
graphy with chloroferm :methaaolavater (64:30:6), mana spectrometry and ultraviolet spectroscopy . Mass Svactra were measured on a CEC 21-110 double focusing mess spectrometer using the direct probe inlet system .
Source temperatures between 200
and 250° C ware used for the various compounds . RESiII.TS and DISCOSSION Charactarizatios of the major bile pigments . After the rat bile had bees washed with hexane and chloroform and lyophilized the residue was found to separate on this layer chromatography initially into two major components figure la),
Tasted with anisidine phthalate (18), these two components gave
positive taste for uronic acids, but negative taste for hexosea or pentoeea . lheae compamde were recovered from the plates and assayed for bilirubin and glucuronic acid .
~e results of thane assays confirm that glucuronic acid and
bilirubin era present in the faster moving spot in a molar ratio of .% /1, and is the slower moving major spot is a 2,01/1 ratio (Table 1) . When the slow TA~S 1 Concentrations of Conjugate Components Spot rf
Glucuronic acid
Bilirubin
Malar ration
0.13
30 .25 4ig/ml
45 6~g/ml
2.01/1
0.25
10 .8 Fig/ml
34 4~g/ml
0 .%/1
running spot was converted to eulfanilylazo derivatives, one class of derivatives was observed by TLC oa cellulose and silica gel plates .
This suggests
that the too halves of the bilixvbin molecule are identically conjugated .
The
faster spot was converted to two classes of sulfaailylazo derivatives separable by TLC, which suggests, along with the molar ratio, that only one half of the tetrapyrrole ie conjugated . When the washed bile residua was converted to sulfaailylazo derivatives without prior separation of the two major campoa" ante, two kinds of azo derivatives nould be separated by TLC .
The faster had
the same retention time as the sulfaailylazo derivative formed from unconju-
Vol . 15, No . 12 gated bilisnbin.
2073
Transesterification of Bilirubin Glucuronidee
The ratio of glucuronic acid to azoviaylneasanthobilirubinic
acid was found to be 0/1 .
11îe slower sulfanilylazo derivative was found to
contain glucuronic acid and azovinylaeazaathobilirubinic acid in the ratio of 1.14/1.
Based on these analyses, the two major components of the washed bile
are ideatifiad as the mono and diglucuronidn conjugates of bili:sbia.
7hese
results were consistently found is 4 bile samples derived from 3 rats after iafueion with bilirubia. Characterisation of the trsnsesterification vroducta .
Thin layer chroma-
tograms era shown in Figure 1 of (a) the miztura of bilirubin mono and diglu-
SOLVENT FRORT
r ___ â _____b ____ç____
d
___ ORIGIN
FIGORE 1 Thin layer chromatograms of di.asopoeitive solid material recovered from a methanol solution of bilirubia glucuronidas sampled at (a) 0 minutas, (b) 20 minutes, and (c) 120 miautae . Ia chromatogram (d) authentic bilirubia stays at the origin sad bilirubin dimethyl diestar moves sear the aolveat front. All four chromatograms wale stm is the same system, using chloroform :mathaaol: avatar (64:30 :6) . curonide freshly isolated from rat bile, (b) this sample after 20 minutes in methanol solution at room temperature is the dark, sad (c) the sample after two hours in methanol solution at roam temperature is the dark .
The major
now, partially resolved, spots in chromatogramw b and c were lass polar sad imoediately diazopositive.
The increase is iatansity of the sew spots was as-
sociated with a decrease in the iatensitiea of the more polar glucuroaidas . The ratio of glucuronic acid to bilirubin in this sew material vas found to ba
2074
Transeaterification of Hilirubin Glucuronides
Vol . 15, No . 12
0/1 . The two new campounde were isolated together by preparative thin layer
396 371
M` i 378
20 I~~ I~ I~II ~ ~~lul~ll ~~L~ 360 380 400 420 440
460
480
M/E
100
F
371
386 TRIMETHYL$ILYL OERhgTNES DF T}~E 7RaNSesr~icnTari PRODUCTS
80-
Z
W F
w o:
60-
40M 378
20~
360
380
429
M` . 407
400
420
M/E
443
440
460
480
FIGURE 2 Partial mesa spectra of authentic bis(trimethylsilyl)bilirubia dimethyl dieeter, and the trimethyleilyl derivatives of traasesterification products . chromatography . ty .
Part of the sample was analyzed directly by mere epectramet-
A second portion was trimethyleilylated (TM5) and aaalyaed by mass epec
tramatry .
A third portion was converted to pheaylazo derivatives of viayl-
aeoxaathobili=nbinic acid .
These two classes of derivatives were made because
Vol. 15, No . 12
Transeaterification of Bilirubin Glucuronides
2075
they had been found to produce the most satisfactory mesa spectra in as earlier study (15) . The mass spectrum of the silylated tetrapyrrole mutate contained molecular ion peeks at m/e 756 sad 814, accampaaiad by M-15 peaks at 741 and 799. Doubly charged molecular ions vets detected at 378 and 407 (Figure 2) .
These
molecular weights suggest that the silylated sample is a mutate of dimethyl di(trimethylsilyl)bilirubin and monomethyl tri(trimethylsilyl)bilirubin, and that the tvo products detected after solution is methanol for 2 hours and isolated by thin layer chromatography are dimathyl and mommethyl bilirubins . The middle mass range from the spectrum of the silylated transesterification products is shown in Figure 2, along with a portion of the spectrum of authentic bis(trimethyleilyl)bilirubin dimethyl duster,
The intense peaks
at m/e 371 and 385 is the reference spectrum are formed by cleavage at the methyleae bridge, ae i~icated for & ~ CH3 is Schema 2 .
OTMS
rMSo
a p " c~y., a" aas, b"an R " nws,
o "~u, b"aa
SCHEME
2
Ions of mass 371 and 385 in the spectrum of the methanol reaction products are presumed to arise by analogous cleavage of the mathylene bridge bonds in bie(trimethylailyl)bilirubin dimethyl theater . In the case of the tris(trimethyleilyl) derivative of the monaa~ethyl reaction product cleavage around the methylane bridge will lead to fwr ions prominent is the spectrum,
Their mesa values are indicated in fragmentation
2076
Traaseaterification of Hilirubin Gluauronidea
Vol . 15, No . 12
Schema 2 for R ~ TL+~ . Additional structural evidence was obtained by examination of the pheaylaso dnrivativee of vi~+l- and isoviaylneaxsnthobilirubini.c acids derived from the traaseeterification products .
Tao classes of phe~leao campounde were
identified and purified by thin layer chromatography . tors were 0.19 and 0 .09 in chloroform :methanol (99:1) .
Their retention facThe mass spectrum of
the less polar a~ more abundant pheaylaso compound is shown in Figure 3.
~n en
eo
eo
do
iJo
ào
roo
zôo
zpo
x+o w~
zeo
xeo
aoo
~,
aso
s+o
~eo
aeo
~ao
FIGIIRS 3 Mass spectrum of the phenylazo derivativee of vinyl- and ieoviaylaeoaaathobilixvbinic acid methyl ester obtained from th~ tranaesterificatioa products, The molecular weight, 404 atomic mesa units, ie consistent with a methylated ccwpou~, and the praeeace of a methyl ester is confirmed by cleavage in the propionate side chain,
specifically formation of mesa 317 (M-87) and 331
~I-73) ions ae shown in Figure 3 .
Side chain cleavage hoe been used is other
studies (16) to confirm the nature of carbaxylate modification . Reference material was prepared by reaction of authentic bilirubia dimathyl dineter with phenyldiasoaium chloride .
Standard and unlmown compounds
showed the same retention factor on thin layer chromatography and similar mass spectra . the second, more polar and less abundant phmylazo compound was identical to that of a standard prepared from camtaercial bilinibia (r .f . 0 .09 is chloroform :methaaol (99:1) and r.f . 0 .23 in chloroform :methanol (5 :95)] .
Its bis-
Vol. 15, No . 12
Transesterification of Bilirubin Glucuronides
2077
(trimethyleilyl) derivative was prepared and analyzed by mass spectrometry . Tha molecular ion weighed 534 atomic mass unite, consistent with formation of a bis(trimethylsilyl) derivative and the presence is the campauad of a free carboxylic acid .
When a portion of this acidic phe~lazo compou~ was treated
with ethereal diazostathane, the product had the same chromatographic mobility as the less polar phenylezo methyl ester, Additional confirmation of the assignment of the dimathyl diester st :vctore to the major product of transesterification by methanol was obtained by comparison to the authentic dimethyl diester of bilirubin.
The ultraviolet
absorption curves were identical, with maximum absorption at 400 nm is chloroform, and corresponded to that value reported is the literature for bilirubin dimethyl diester (17) .
Sta~ard and ualozown compounds showed the same retea-
tioa factors in this layer chromatography, .95 in chloroform :methanol :water (64:30:6) and .26 is chlorofornt . Bilirubin itself remained at the origin in these systems (Figure ld), 11îe mass spectre era also similar with very small molecular ion peaks at m/e 6l2 and major peaks at m/a 227, 241, 300 and 313.
The latter pair of ions
are formed by cleavage aroa~ the mathylena bridge (and transfer of a hydrogen atom in one case) analogous to that shown in Scheme 2 . ßSFSRSNC85 1.
G . H. IATHE, Sasavs in Bi.ochem . 8 107-148 (1972) .
2.
J . FSVSßY, B. VAN DAt4~, ß. MICHIBi.B, J. De Gß00TS, a~ H . P. M. HSIHiiSGH, J . Clin . Irrrast. 51 2482-2492 (1972),
3.
ß. SCH~, J . Biol . G1zem. 229 881-888 (1957) .
4.
B . H. BILLING, P, G. COl.S, and G . H. IATHS, Biochem. J. 65 774-784 (1957) .
5 . D. SCBAGR7ilt, Sciexe ~ 507-508 (1957) . 6.
J. FSVS$Y, P. LSßOY, and R. P. M. HSIItiIS(~, Biochem . J. 129 619-633 (1972) .
7. C. C . R~LS, Biochem. J, 119 411-435 (1970) . 8 . J. D . 08Tß0ä and N . H, MQRPHY, Biochem. J . 120 371-327 (1970) .
2078
9.
Tranaeaterification of Hilirubin Glucuronidea
Vol . 15, No . 12
P . L . M . JANSSN, Clin . Chew . Acta 49 233-240 (1973) .
10 .
P . L . M . JANSSN aad B . H . BILLIIiG, Biochem . J . 125 917-919 (1971) .
11 .
J . C . NATZSCH&A and G . B . ODELL, Pediatrics 37 51-61 (1966) .
12 .
H . T . MALiAY and R . A . $VSLYN, J . Biol . Chem . 119 481-490 (1937) .
13 .
G . J . DUTTON, Glucuronic Acid . Free and Combined, p . 116 Academic Prass, London (1966) .
14 .
C . C . RIISI~lLS, Biochem . J . 119 387-394 (1970) .
15 .
M. SAiliON and C . F~i.SSI1~II, Biomed . Mass Svectrom .
16 .
F . COMPSRNOaJ~E, F . H . JANSSN, and R . P . M . HEIRWBGH, Hiochem . J . 120
1
in press .
891-894 (1970) . 17 .
M . JIItSA, J . P . DICKINSON, and G . H . IATHB, Nature 220 1322-1324 (1968) .
18 .
K . ßAND~A.TH, Thin Layer Chromatoptrauhy, p . 200 Academic Preae, New York (1964) .
Pergamon Press
BAPm TBANSBSTSBIFICATZ(li OF BII.IRUBIIQ GIÜCUI:QTID&S IN MSTBANOL* Ma~el Salmon and Catherine Feneelau Department of Pharmacology and Bzperimeatal Therapearice Julio 0. Colder and Gerard B. Odell Department of Pediatrics The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 (Received is final form 12 November 1974) SDilfABY The current studies present aridence that bilirubin taajugates derived frost rat bile undergo rapid treneesterification, n i n vitro, in solnrions containing methanol . The conjugates of bilis~sbin sad the methyl esters formed from them by ezposura to methanol orate isolated by thin layer chromatography . The isolates were chemically quaaritated for their bilirubin and glncuronic acid coe~oeition. Characterization of the bilisvbin methyl esters was performed by mass spectrometric analysis of the trimethylsilyl sad pheaylazo derivatives . A large body of literature eziste on the heparic ezcrerion of bilirubin is many animal species (1,2) .
Two major products of bilisubin in normal
mamataliaa bile have bean identified ae the mono and diglucurori.des is which the glucuroaic acid is esterified with bilirubin at its propionic acid aide chains (3,4,5) .
Smaller fratrions of the bilirubin is bile have been des-
cribed as acylglycosidas of glucose and nylons, ae well as disaccharides of hw~+++~^^+~
acids that vary is differnat species (6,7) . Currently uncertainty
exists concerning the quantitative importance of the glucuronide conjugates, particularly ori.th reference to the relative amounts of bilirubin that are ,t
~
This work was supported by grants from the United Staten Public Health Service, N>B GM-16492, AO-4-fd~i-70417 and HD-02268 and in part by wade from the Andrew W. Mellon Foundation . Oa sabbatical leave from the Inetituto da Quimica de la Universidad National Antonama da Mexico, Mexico, 20, D.F . 2069
2070
Transesterifioation of Silirubin Glucuronides
e=~reted ae the moao and diglucuronide esters
Vol. 15, No . 12
(8,9) .
The current report preseate data that demonstrate rapid transaeterification o£ the ccgjugatea of bilirubin when exposed to methanol
(Schema 1) .
,H SCHEME I
These observations have sigaificant implications on the uaderatandiug of the hepatic excretion of bilirubin.
Solvent partitioning of polar conjugates of
bilirubin in bile have frequently employed methanol and chloroform (S) .
Be-
cause of the is-vitro capacity of the glucuronidee of bilirubia to undergo transeeterification is methanol, the resultant methyl esters would be preferentially recovered in the ap~iar chloroform layer.
Subsequent chemical
analysis by the diazo reaction would lead to both a qualitatively and quantitatively arroaeoue interpretation of the excretion of unconjugated bilirubin in bile, because the methyl esters of bilirubin have the same reaction requirements as unconjugatad bilirubia with diazotizad aulfanilic acid, ethyl anthranilate and phe~l diazonium chloride .
Such tranaeaterification may
have contributed to the differences observed by various woiicers in the amounts of free, mono and diglucuronides of bilirubin recovered is bile . nized this reaction offers as alternative to traasamination (10)
Dace reeogfor the
characterization of conjugates of bilirubia. METHODS A~ MATSR7N.5 The conjugates of bilirubin were obtained by canaalation of the cosmos
Vol.
15, No . 12
Tranaeaterification of Bilirubin Glucuro~nidea
bile duct of Wiatar rats receiving, bilirubin (11) .
2071
intravenously, an aqueous infusion of
Ia moat eaperimeate the bile was sequentially washed with
heaane and chloroform folloaPed by lyophilizatioa, and then analyzed by thin layer chromatography (see Figure 1) .
In same cease an aliquot of whole bile
was directly reacted with diazotized aulfanilic acid and the polar azodipyrroles were isolated by thin layer chromatography an cellulose 1ßt300 plates of 250p, thickaees aiaclcherey, Nagel, Co,) using ethyl methyl ketane :propioaic acid :water (75:25:35) . Quantitative measurements were made on the major pigments derived by chromatographic separation . with methanol .
These ware scraped from the plates sad eluted
The concentration of bilirubin from each spot was determined
colormetrically by com~ersion to aulfanily] .azo derivatives using standards prepared directly free crystallise bilirubin (12) .
The glucuronic acid con-
tent corresponding to the bilirubin spots was quantitated by the naphthoresorciaol assay (13) .
The standard glucuroaic acid aolutioae ware made up to
contain the same concentration of bilirubia to eliminate color interference in the spectrophotometric readings of the naphthoresorcinol assay for glucuroaic acid, Phenvlazo derivatives were obtained from bilixnbin and its esters by treatment with a chilled solution of phenyldiazonium chloride (14) .
The
colored products of this reaction were chramatographed on thin layers of 3102 GF, using chloroform :matheaol (99:1, 99 .5 :0 .5 or 5 :95) . Trimathvlsilvl derivatives ware prepared by reaction with N,0-bis(trimethyleilyl)trifluoroacetamide containing
lx
trichloramethylsilane,
l+tass
spectra were measured immediately after eaceas reagent was removed in vacuo . Authentic bilirubia dimethyl diester was prepared from ca~ercially available bilirubin (Sigma Chemicals) .
Treatment with diazomethaae provided
the authentic dimethyl diester of bilirubin, which was separated from smaller amounts of trimathyl and tetramathyl derivatives by this layer chromatography and recrystallized,
This compound was characterized by thin layer chromsto-
Transesterification of Silirubin Glucuronidea
2072
Vol. 15, No . 12
graphy with chloroferm :methaaolavater (64:30:6), mana spectrometry and ultraviolet spectroscopy . Mass Svactra were measured on a CEC 21-110 double focusing mess spectrometer using the direct probe inlet system .
Source temperatures between 200
and 250° C ware used for the various compounds . RESiII.TS and DISCOSSION Charactarizatios of the major bile pigments . After the rat bile had bees washed with hexane and chloroform and lyophilized the residue was found to separate on this layer chromatography initially into two major components figure la),
Tasted with anisidine phthalate (18), these two components gave
positive taste for uronic acids, but negative taste for hexosea or pentoeea . lheae compamde were recovered from the plates and assayed for bilirubin and glucuronic acid .
~e results of thane assays confirm that glucuronic acid and
bilirubin era present in the faster moving spot in a molar ratio of .% /1, and is the slower moving major spot is a 2,01/1 ratio (Table 1) . When the slow TA~S 1 Concentrations of Conjugate Components Spot rf
Glucuronic acid
Bilirubin
Malar ration
0.13
30 .25 4ig/ml
45 6~g/ml
2.01/1
0.25
10 .8 Fig/ml
34 4~g/ml
0 .%/1
running spot was converted to eulfanilylazo derivatives, one class of derivatives was observed by TLC oa cellulose and silica gel plates .
This suggests
that the too halves of the bilixvbin molecule are identically conjugated .
The
faster spot was converted to two classes of sulfaailylazo derivatives separable by TLC, which suggests, along with the molar ratio, that only one half of the tetrapyrrole ie conjugated . When the washed bile residua was converted to sulfaailylazo derivatives without prior separation of the two major campoa" ante, two kinds of azo derivatives nould be separated by TLC .
The faster had
the same retention time as the sulfaailylazo derivative formed from unconju-
Vol . 15, No . 12 gated bilisnbin.
2073
Transesterification of Bilirubin Glucuronidee
The ratio of glucuronic acid to azoviaylneasanthobilirubinic
acid was found to be 0/1 .
11îe slower sulfanilylazo derivative was found to
contain glucuronic acid and azovinylaeazaathobilirubinic acid in the ratio of 1.14/1.
Based on these analyses, the two major components of the washed bile
are ideatifiad as the mono and diglucuronidn conjugates of bili:sbia.
7hese
results were consistently found is 4 bile samples derived from 3 rats after iafueion with bilirubia. Characterisation of the trsnsesterification vroducta .
Thin layer chroma-
tograms era shown in Figure 1 of (a) the miztura of bilirubin mono and diglu-
SOLVENT FRORT
r ___ â _____b ____ç____
d
___ ORIGIN
FIGORE 1 Thin layer chromatograms of di.asopoeitive solid material recovered from a methanol solution of bilirubia glucuronidas sampled at (a) 0 minutas, (b) 20 minutes, and (c) 120 miautae . Ia chromatogram (d) authentic bilirubia stays at the origin sad bilirubin dimethyl diestar moves sear the aolveat front. All four chromatograms wale stm is the same system, using chloroform :mathaaol: avatar (64:30 :6) . curonide freshly isolated from rat bile, (b) this sample after 20 minutes in methanol solution at room temperature is the dark, sad (c) the sample after two hours in methanol solution at roam temperature is the dark .
The major
now, partially resolved, spots in chromatogramw b and c were lass polar sad imoediately diazopositive.
The increase is iatansity of the sew spots was as-
sociated with a decrease in the iatensitiea of the more polar glucuroaidas . The ratio of glucuronic acid to bilirubin in this sew material vas found to ba
2074
Transeaterification of Hilirubin Glucuronides
Vol . 15, No . 12
0/1 . The two new campounde were isolated together by preparative thin layer
396 371
M` i 378
20 I~~ I~ I~II ~ ~~lul~ll ~~L~ 360 380 400 420 440
460
480
M/E
100
F
371
386 TRIMETHYL$ILYL OERhgTNES DF T}~E 7RaNSesr~icnTari PRODUCTS
80-
Z
W F
w o:
60-
40M 378
20~
360
380
429
M` . 407
400
420
M/E
443
440
460
480
FIGURE 2 Partial mesa spectra of authentic bis(trimethylsilyl)bilirubia dimethyl dieeter, and the trimethyleilyl derivatives of traasesterification products . chromatography . ty .
Part of the sample was analyzed directly by mere epectramet-
A second portion was trimethyleilylated (TM5) and aaalyaed by mass epec
tramatry .
A third portion was converted to pheaylazo derivatives of viayl-
aeoxaathobili=nbinic acid .
These two classes of derivatives were made because
Vol. 15, No . 12
Transeaterification of Bilirubin Glucuronides
2075
they had been found to produce the most satisfactory mesa spectra in as earlier study (15) . The mass spectrum of the silylated tetrapyrrole mutate contained molecular ion peeks at m/e 756 sad 814, accampaaiad by M-15 peaks at 741 and 799. Doubly charged molecular ions vets detected at 378 and 407 (Figure 2) .
These
molecular weights suggest that the silylated sample is a mutate of dimethyl di(trimethylsilyl)bilirubin and monomethyl tri(trimethylsilyl)bilirubin, and that the tvo products detected after solution is methanol for 2 hours and isolated by thin layer chromatography are dimathyl and mommethyl bilirubins . The middle mass range from the spectrum of the silylated transesterification products is shown in Figure 2, along with a portion of the spectrum of authentic bis(trimethyleilyl)bilirubin dimethyl duster,
The intense peaks
at m/e 371 and 385 is the reference spectrum are formed by cleavage at the methyleae bridge, ae i~icated for & ~ CH3 is Schema 2 .
OTMS
rMSo
a p " c~y., a" aas, b"an R " nws,
o "~u, b"aa
SCHEME
2
Ions of mass 371 and 385 in the spectrum of the methanol reaction products are presumed to arise by analogous cleavage of the mathylene bridge bonds in bie(trimethylailyl)bilirubin dimethyl theater . In the case of the tris(trimethyleilyl) derivative of the monaa~ethyl reaction product cleavage around the methylane bridge will lead to fwr ions prominent is the spectrum,
Their mesa values are indicated in fragmentation
2076
Traaseaterification of Hilirubin Gluauronidea
Vol . 15, No . 12
Schema 2 for R ~ TL+~ . Additional structural evidence was obtained by examination of the pheaylaso dnrivativee of vi~+l- and isoviaylneaxsnthobilirubini.c acids derived from the traaseeterification products .
Tao classes of phe~leao campounde were
identified and purified by thin layer chromatography . tors were 0.19 and 0 .09 in chloroform :methanol (99:1) .
Their retention facThe mass spectrum of
the less polar a~ more abundant pheaylaso compound is shown in Figure 3.
~n en
eo
eo
do
iJo
ào
roo
zôo
zpo
x+o w~
zeo
xeo
aoo
~,
aso
s+o
~eo
aeo
~ao
FIGIIRS 3 Mass spectrum of the phenylazo derivativee of vinyl- and ieoviaylaeoaaathobilixvbinic acid methyl ester obtained from th~ tranaesterificatioa products, The molecular weight, 404 atomic mesa units, ie consistent with a methylated ccwpou~, and the praeeace of a methyl ester is confirmed by cleavage in the propionate side chain,
specifically formation of mesa 317 (M-87) and 331
~I-73) ions ae shown in Figure 3 .
Side chain cleavage hoe been used is other
studies (16) to confirm the nature of carbaxylate modification . Reference material was prepared by reaction of authentic bilirubia dimathyl dineter with phenyldiasoaium chloride .
Standard and unlmown compounds
showed the same retention factor on thin layer chromatography and similar mass spectra . the second, more polar and less abundant phmylazo compound was identical to that of a standard prepared from camtaercial bilinibia (r .f . 0 .09 is chloroform :methaaol (99:1) and r.f . 0 .23 in chloroform :methanol (5 :95)] .
Its bis-
Vol. 15, No . 12
Transesterification of Bilirubin Glucuronides
2077
(trimethyleilyl) derivative was prepared and analyzed by mass spectrometry . Tha molecular ion weighed 534 atomic mass unite, consistent with formation of a bis(trimethylsilyl) derivative and the presence is the campauad of a free carboxylic acid .
When a portion of this acidic phe~lazo compou~ was treated
with ethereal diazostathane, the product had the same chromatographic mobility as the less polar phenylezo methyl ester, Additional confirmation of the assignment of the dimathyl diester st :vctore to the major product of transesterification by methanol was obtained by comparison to the authentic dimethyl diester of bilirubin.
The ultraviolet
absorption curves were identical, with maximum absorption at 400 nm is chloroform, and corresponded to that value reported is the literature for bilirubin dimethyl diester (17) .
Sta~ard and ualozown compounds showed the same retea-
tioa factors in this layer chromatography, .95 in chloroform :methanol :water (64:30:6) and .26 is chlorofornt . Bilirubin itself remained at the origin in these systems (Figure ld), 11îe mass spectre era also similar with very small molecular ion peaks at m/e 6l2 and major peaks at m/a 227, 241, 300 and 313.
The latter pair of ions
are formed by cleavage aroa~ the mathylena bridge (and transfer of a hydrogen atom in one case) analogous to that shown in Scheme 2 . ßSFSRSNC85 1.
G . H. IATHE, Sasavs in Bi.ochem . 8 107-148 (1972) .
2.
J . FSVSßY, B. VAN DAt4~, ß. MICHIBi.B, J. De Gß00TS, a~ H . P. M. HSIHiiSGH, J . Clin . Irrrast. 51 2482-2492 (1972),
3.
ß. SCH~, J . Biol . G1zem. 229 881-888 (1957) .
4.
B . H. BILLING, P, G. COl.S, and G . H. IATHS, Biochem. J. 65 774-784 (1957) .
5 . D. SCBAGR7ilt, Sciexe ~ 507-508 (1957) . 6.
J. FSVS$Y, P. LSßOY, and R. P. M. HSIItiIS(~, Biochem . J. 129 619-633 (1972) .
7. C. C . R~LS, Biochem. J, 119 411-435 (1970) . 8 . J. D . 08Tß0ä and N . H, MQRPHY, Biochem. J . 120 371-327 (1970) .
2078
9.
Tranaeaterification of Hilirubin Glucuronidea
Vol . 15, No . 12
P . L . M . JANSSN, Clin . Chew . Acta 49 233-240 (1973) .
10 .
P . L . M . JANSSN aad B . H . BILLIIiG, Biochem . J . 125 917-919 (1971) .
11 .
J . C . NATZSCH&A and G . B . ODELL, Pediatrics 37 51-61 (1966) .
12 .
H . T . MALiAY and R . A . $VSLYN, J . Biol . Chem . 119 481-490 (1937) .
13 .
G . J . DUTTON, Glucuronic Acid . Free and Combined, p . 116 Academic Prass, London (1966) .
14 .
C . C . RIISI~lLS, Biochem . J . 119 387-394 (1970) .
15 .
M. SAiliON and C . F~i.SSI1~II, Biomed . Mass Svectrom .
16 .
F . COMPSRNOaJ~E, F . H . JANSSN, and R . P . M . HEIRWBGH, Hiochem . J . 120
1
in press .
891-894 (1970) . 17 .
M . JIItSA, J . P . DICKINSON, and G . H . IATHB, Nature 220 1322-1324 (1968) .
18 .
K . ßAND~A.TH, Thin Layer Chromatoptrauhy, p . 200 Academic Preae, New York (1964) .