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Isolation of oestriol-16 (17?)-glucosiduronate by means of an ion exchange resin
SHORT
3%
COMMUNICATIONS
2 R. S. Ya~ow AND S. A. BERSON, J. Clin. Iwest., 39 (1960) ~157. 3 A. S. MCFARLANE, Nature, 182 (1958) 53. 1 N. VEALL AND H. VETTER, Radioisotope techniques in clixical vewnvch and diagnosis, Butterworth, London, 1958. 5 A. S. MCFARLANE, EIiochenz. J., 62 (1956) 135. B S. r\. PERSON, R. S. YALOW,~~.BAUMAN,M. A. ROTHSCHILD ANU K. XEWERLY, J.Cli+l. Iwest., 35 (1956) 170. 7 S. A. BERSON AND R. S. YALOW, J. Clin. Iwvest., 40 (1961) 1803. 190 (1961) 1211. 8 E. SAMOLS AND H. S. WILLIAMS, .vaturP, 9 J. D. PEARSON, Unmet, i (1959) 967. l” K. \T:ISEMAN AND B. E. BALTZ, Endncrixolog~,68 (1961) 354. I1 P. J. KESTENS, \V. G. AUSTENAND IV. V. MCDERMOTT JR., SurgicalForum, IO (195~) 225. 1% 1’. J, KESTENS, J, A. FARELL~ AND \V. V. MCDERMOTT JR,,J. Suvg. Research, I (1961) 53. I5 I’. J. KESTENS AND W. V. MCDERMOTT JR., Swgery, 50 (1961) rgb. I1 P. J. KESTENS, J. J, HASHE, L. LALIBOTTE AND C. LAMBOTTE, Rev. Intern. Hepntol., 11 (1902)
Received,
August
zgth,
1962
Isolation of oestriol-16 (17?)-glucosiduronate by means of an ion exchange resin In a previous
paperr
a method
for the quantitative
separation
of oestrogens
from pregnancy urine by means of ion exchange chromatography was described. Because unhydrolysed urine was used for the separation procedure it was claimed that the oestrogenic material in the column eluate was present in the form of conjugates. Further investigations on this point have now been made and the results are presented in this paper. For the separation of the oestrogen conjugates from urine column chromatography on De-acidite FF was used as described beforer. The eluate containing the oestrogens was acidified with hydrochloric acid to pH 2-3 and saturated with sodium chloride. This solution was extracted three times with half volumes of n-butanol. The extract was washed twice with one tenth of its volume of saturated sodium chloride solution, pH approximately 2, and dried with sodium sulfate. The sodium sulfate was filtered off (glass wool) and the butanol extract evaporated to dryness in vacua. The dry residue was dissolved in methanol. This solution of oestrogen conjugates in methanol is referred to as “conjugate solution”. Identification
of oestriol-lh(I7?)-glzscosid~vonate
in the conjugate
solution
(a) Paper chromatography. Paper chromatography of the “conjugate solution” was performed in three different solvent systems. According to the solvent mixture used 3 to 7 distinct spots could be found on the chromatograms using the FolinCiocalteu reagent, the most intense of which behaved like authentic oestriol-r6(r7 ?)glucosiduronate (Table I). Paper electrophoresis in two buffer systems of different (b) Paper electrophovesis. pH values gave further evidence that oestriol-rb(r7 ?)-glucosiduronate is present in the “conjugate solution” besides other substances (Table II).
300
SHORT COMMUNICATIONS TABLE I ICp V*LuE op D~~TR~L-I~(I 7 ?)-GLUC~SIDURONATE
St. LOUiS, 1..S..4.)
(sigma,
THE
IN
“CONJUGATE
COMPARISON SOLUTION”
WITH FROM i?F
Soloelrt sJste,n
AT,,
I 1
3
_
ELECTROPHORESIS WITH
“CONJUGATE
SPOT
OF
?dtlt!
0.01
0.26
OF ONE
OESTRIOL-Ih(l7 OF
SOLUTION” (IOO
Verona1 B Phosphate B
INTENSE URINE
0.0”
~~ ~~~
COMPARISON
MOST
oestriol-I6(17?)ghcosiduronate
)z-butyl acetateu-butanol-IO:, formic acid, 8 : 2 : IO (ref. 3) 0.~91 SH,OHPethylacetate-u-butanol, 200: I75 : 25 (ref. 4) Toluene-Al-butanolSH,OH cont.-water, 50: I j0: 10: 180 (ref. 5)
PAPER
THE
PREGNANCY
8.6 6.8
THE
i)-
CONJUGATES FROM
GLUCOSIDURONATE PRESENT
PREGNANCY
IN
IN THE
URINE
V, 5 mA, 16 h)
37 II
(c) Hydrolysis with enzymes. When the “conjugate solution” hydrolysis with a ,B-glucuronidase preparation (Ketodase, Warner hydrolysate chromatographed in solvent system I (see Table I), oestriol-I6(r7 ?)-glucosiduronate position disappeared. On the other with sulfatase of the “conjugate solution” did not influence the
36 TL
was subjected to Chilcott) and the the spot in the hand, incubation paper chromato-
graphic behaviour of the spot concerned. (d) Identijcation of the oestrogen @art qf the conjugate. After elution of the spot suspected to be oestriol-I6(I7 ?)-glucosiduronate from a paper chromatogram in solvent system I (Table I) the material was subjected to an oestrogen determination according to BROWNS (hydrolysis, solvent distribution, methylation, column chromatography on aluminium oxide, Kober reaction). The material behaved in all respects like oestriol. Furthermore, some oestriol-I6(r7 ?)-glucosiduronate like material from the chromatogram was hydrolysed with hydrochloric acid and the oestrogen part characterized by paper chromatography in benzene-methanol-water (IO : 8 : 2). A single spot with the &-value of oestriol (0.16) was found on the chromatogram. The data presented provide sufficient evidence for the presence of oestriol-I6 Authentic oestriol-r6(r7 ?)(17 ?)-glucosiduronate in the “conjugate solution”. glucosiduronate and the main substance in the “conjugate solution” behaved identically in paper chromatography in three different solvent systems, paper electrophoresis at two different PH values, hydrolysis with @-glucuronidase and BROWN’S
307
SHORT COMMUNICATIONS
method. As in late pregnancy urine the oestriol amounts to about 90% of the total oestrogens and as it is further known that the predominant oestriol conjugate is oestriol-r6(r7
?)-glucosiduronate,
its identification
in the “conjugate
solution”
proves
that with the procedure described in our previous paper not only quantitative recovery of the oestrogenic material of pregnancy urine is possible, but that the main portion of the isolated material is present in its native state, i.e. as conjugate. The author oestrone,
would like to thank
oestradiol-rT,!l,
oestriol,
Schering
oestrone
A.G.,
sulfate,
Berlin,
for a generous
QzceefzElizabeth II Coronation Gift Fund Trust arzd Department of Biochemistry, Royal Perth Hospital, Perth (Australia)
1 R. HKHNEL, Clin. Chim. Acta, 7 (1962) 768. 2 J, B. BROWN, in Adva?lces i?z Clilzical Ckemistr)‘, Vol. s 4 5 6
P. M. J. E. E.
gift of
and steroid sulfatase.
III,
ROLAND
ilcademic
l’ress, New
HAHNEL
York,
1960,
‘57. L. L~WBART AND J. J. SCHNEIDER, Nature, 176 (1955) 1175. J. SCHNEIDER AND M. L. LEWBART, J. Biol. Chem., 222 (1956) 787. MENINI AND E. DICZFALUSY, Endocrinology, 68 (1961) 492. MENINI AND E. DICZFALUSY, E)zdocri)zoZogv, 67 (1960) 500.
Received July myth, 1962 (revised manuscript November
sth, 1962) CliK ChiJ,l. ‘-I&, 8
Zur Standardisierung
(1963) 305-307
esterspaltender Enzyme
Bei der Durchftihrung der Bestimmung esterspaltender Enzyme des Serums (Phosphatasen, Lipase, Cholinesterase, Glukuronidase, Sulfatase) werden eine Vielzahl von Methoden angewandt, in denen fur das gleiche Enzym verschiedene Substrate, Pufferlijsungen und Inkubationszeiten benutzt werden. Die Vielfalt der Methoden bedingt, dass die Ergebnisse der Aktivitatsbestimmungen nicht ohne weiteres miteinander verglichen werden kiinnen. Es wurden deshalb Standardmethoden vorgeschlagenl, bei denen die Aktivitatsbestimmung aller genannten Enzyme auf einem gemeinsamen Prinzip beruht : Spaltung eines fur jedes Enzym spezifischen Phenolesters als Substrat und Messung der freigesetzten Menge Phenol durch die Reaktion mit dem Reagenz nach Folin und Ciocalteu. Dieses Vorgehen hat den Vorteil, dass mit einem Reagenz und einer Eichbeziehung mindestens 6 verschiedene Enzyme bestimmt werden konnen. Ausserdem wird nur ein einfaches Wasserbad und ein normales Fotometer beniitigt. Als Enzymeinheit wird in Ubereinstimmung mit dem Vorschlag der Iiommission fur klinische Chemie der Internationalen Union fur reine und angewandte Chemie definiert: I int. E = pmol (Mikromol) freigesetztes Phenol/Min./~ooo ml Untersuchungsgut. KIND
Reagenzes
UND
verwandten und Ciocalteu
KINGLY 3
von Folin
bei der Phosphatasebestimmung anstelle des cc-Aminoantipyrin. Dies hat den Vorteil einer Clin. Chin.
dctn,
8 (x963)
307-308
3%
COMMUNICATIONS
2 R. S. Ya~ow AND S. A. BERSON, J. Clin. Iwest., 39 (1960) ~157. 3 A. S. MCFARLANE, Nature, 182 (1958) 53. 1 N. VEALL AND H. VETTER, Radioisotope techniques in clixical vewnvch and diagnosis, Butterworth, London, 1958. 5 A. S. MCFARLANE, EIiochenz. J., 62 (1956) 135. B S. r\. PERSON, R. S. YALOW,~~.BAUMAN,M. A. ROTHSCHILD ANU K. XEWERLY, J.Cli+l. Iwest., 35 (1956) 170. 7 S. A. BERSON AND R. S. YALOW, J. Clin. Iwvest., 40 (1961) 1803. 190 (1961) 1211. 8 E. SAMOLS AND H. S. WILLIAMS, .vaturP, 9 J. D. PEARSON, Unmet, i (1959) 967. l” K. \T:ISEMAN AND B. E. BALTZ, Endncrixolog~,68 (1961) 354. I1 P. J. KESTENS, \V. G. AUSTENAND IV. V. MCDERMOTT JR., SurgicalForum, IO (195~) 225. 1% 1’. J, KESTENS, J, A. FARELL~ AND \V. V. MCDERMOTT JR,,J. Suvg. Research, I (1961) 53. I5 I’. J. KESTENS AND W. V. MCDERMOTT JR., Swgery, 50 (1961) rgb. I1 P. J. KESTENS, J. J, HASHE, L. LALIBOTTE AND C. LAMBOTTE, Rev. Intern. Hepntol., 11 (1902)
Received,
August
zgth,
1962
Isolation of oestriol-16 (17?)-glucosiduronate by means of an ion exchange resin In a previous
paperr
a method
for the quantitative
separation
of oestrogens
from pregnancy urine by means of ion exchange chromatography was described. Because unhydrolysed urine was used for the separation procedure it was claimed that the oestrogenic material in the column eluate was present in the form of conjugates. Further investigations on this point have now been made and the results are presented in this paper. For the separation of the oestrogen conjugates from urine column chromatography on De-acidite FF was used as described beforer. The eluate containing the oestrogens was acidified with hydrochloric acid to pH 2-3 and saturated with sodium chloride. This solution was extracted three times with half volumes of n-butanol. The extract was washed twice with one tenth of its volume of saturated sodium chloride solution, pH approximately 2, and dried with sodium sulfate. The sodium sulfate was filtered off (glass wool) and the butanol extract evaporated to dryness in vacua. The dry residue was dissolved in methanol. This solution of oestrogen conjugates in methanol is referred to as “conjugate solution”. Identification
of oestriol-lh(I7?)-glzscosid~vonate
in the conjugate
solution
(a) Paper chromatography. Paper chromatography of the “conjugate solution” was performed in three different solvent systems. According to the solvent mixture used 3 to 7 distinct spots could be found on the chromatograms using the FolinCiocalteu reagent, the most intense of which behaved like authentic oestriol-r6(r7 ?)glucosiduronate (Table I). Paper electrophoresis in two buffer systems of different (b) Paper electrophovesis. pH values gave further evidence that oestriol-rb(r7 ?)-glucosiduronate is present in the “conjugate solution” besides other substances (Table II).
300
SHORT COMMUNICATIONS TABLE I ICp V*LuE op D~~TR~L-I~(I 7 ?)-GLUC~SIDURONATE
St. LOUiS, 1..S..4.)
(sigma,
THE
IN
“CONJUGATE
COMPARISON SOLUTION”
WITH FROM i?F
Soloelrt sJste,n
AT,,
I 1
3
_
ELECTROPHORESIS WITH
“CONJUGATE
SPOT
OF
?dtlt!
0.01
0.26
OF ONE
OESTRIOL-Ih(l7 OF
SOLUTION” (IOO
Verona1 B Phosphate B
INTENSE URINE
0.0”
~~ ~~~
COMPARISON
MOST
oestriol-I6(17?)ghcosiduronate
)z-butyl acetateu-butanol-IO:, formic acid, 8 : 2 : IO (ref. 3) 0.~91 SH,OHPethylacetate-u-butanol, 200: I75 : 25 (ref. 4) Toluene-Al-butanolSH,OH cont.-water, 50: I j0: 10: 180 (ref. 5)
PAPER
THE
PREGNANCY
8.6 6.8
THE
i)-
CONJUGATES FROM
GLUCOSIDURONATE PRESENT
PREGNANCY
IN
IN THE
URINE
V, 5 mA, 16 h)
37 II
(c) Hydrolysis with enzymes. When the “conjugate solution” hydrolysis with a ,B-glucuronidase preparation (Ketodase, Warner hydrolysate chromatographed in solvent system I (see Table I), oestriol-I6(r7 ?)-glucosiduronate position disappeared. On the other with sulfatase of the “conjugate solution” did not influence the
36 TL
was subjected to Chilcott) and the the spot in the hand, incubation paper chromato-
graphic behaviour of the spot concerned. (d) Identijcation of the oestrogen @art qf the conjugate. After elution of the spot suspected to be oestriol-I6(I7 ?)-glucosiduronate from a paper chromatogram in solvent system I (Table I) the material was subjected to an oestrogen determination according to BROWNS (hydrolysis, solvent distribution, methylation, column chromatography on aluminium oxide, Kober reaction). The material behaved in all respects like oestriol. Furthermore, some oestriol-I6(r7 ?)-glucosiduronate like material from the chromatogram was hydrolysed with hydrochloric acid and the oestrogen part characterized by paper chromatography in benzene-methanol-water (IO : 8 : 2). A single spot with the &-value of oestriol (0.16) was found on the chromatogram. The data presented provide sufficient evidence for the presence of oestriol-I6 Authentic oestriol-r6(r7 ?)(17 ?)-glucosiduronate in the “conjugate solution”. glucosiduronate and the main substance in the “conjugate solution” behaved identically in paper chromatography in three different solvent systems, paper electrophoresis at two different PH values, hydrolysis with @-glucuronidase and BROWN’S
307
SHORT COMMUNICATIONS
method. As in late pregnancy urine the oestriol amounts to about 90% of the total oestrogens and as it is further known that the predominant oestriol conjugate is oestriol-r6(r7
?)-glucosiduronate,
its identification
in the “conjugate
solution”
proves
that with the procedure described in our previous paper not only quantitative recovery of the oestrogenic material of pregnancy urine is possible, but that the main portion of the isolated material is present in its native state, i.e. as conjugate. The author oestrone,
would like to thank
oestradiol-rT,!l,
oestriol,
Schering
oestrone
A.G.,
sulfate,
Berlin,
for a generous
QzceefzElizabeth II Coronation Gift Fund Trust arzd Department of Biochemistry, Royal Perth Hospital, Perth (Australia)
1 R. HKHNEL, Clin. Chim. Acta, 7 (1962) 768. 2 J, B. BROWN, in Adva?lces i?z Clilzical Ckemistr)‘, Vol. s 4 5 6
P. M. J. E. E.
gift of
and steroid sulfatase.
III,
ROLAND
ilcademic
l’ress, New
HAHNEL
York,
1960,
‘57. L. L~WBART AND J. J. SCHNEIDER, Nature, 176 (1955) 1175. J. SCHNEIDER AND M. L. LEWBART, J. Biol. Chem., 222 (1956) 787. MENINI AND E. DICZFALUSY, Endocrinology, 68 (1961) 492. MENINI AND E. DICZFALUSY, E)zdocri)zoZogv, 67 (1960) 500.
Received July myth, 1962 (revised manuscript November
sth, 1962) CliK ChiJ,l. ‘-I&, 8
Zur Standardisierung
(1963) 305-307
esterspaltender Enzyme
Bei der Durchftihrung der Bestimmung esterspaltender Enzyme des Serums (Phosphatasen, Lipase, Cholinesterase, Glukuronidase, Sulfatase) werden eine Vielzahl von Methoden angewandt, in denen fur das gleiche Enzym verschiedene Substrate, Pufferlijsungen und Inkubationszeiten benutzt werden. Die Vielfalt der Methoden bedingt, dass die Ergebnisse der Aktivitatsbestimmungen nicht ohne weiteres miteinander verglichen werden kiinnen. Es wurden deshalb Standardmethoden vorgeschlagenl, bei denen die Aktivitatsbestimmung aller genannten Enzyme auf einem gemeinsamen Prinzip beruht : Spaltung eines fur jedes Enzym spezifischen Phenolesters als Substrat und Messung der freigesetzten Menge Phenol durch die Reaktion mit dem Reagenz nach Folin und Ciocalteu. Dieses Vorgehen hat den Vorteil, dass mit einem Reagenz und einer Eichbeziehung mindestens 6 verschiedene Enzyme bestimmt werden konnen. Ausserdem wird nur ein einfaches Wasserbad und ein normales Fotometer beniitigt. Als Enzymeinheit wird in Ubereinstimmung mit dem Vorschlag der Iiommission fur klinische Chemie der Internationalen Union fur reine und angewandte Chemie definiert: I int. E = pmol (Mikromol) freigesetztes Phenol/Min./~ooo ml Untersuchungsgut. KIND
Reagenzes
UND
verwandten und Ciocalteu
KINGLY 3
von Folin
bei der Phosphatasebestimmung anstelle des cc-Aminoantipyrin. Dies hat den Vorteil einer Clin. Chin.
dctn,
8 (x963)
307-308