Gastroenterologia Japonica Copyright 9 1977 by The Japanese Society of Gastroenterology
Vol. 12, No. 6 Printed in Japan
--Origlna Article
S U L F O B R O M O P H T H A L E I N A N D I N D O C Y A N I N E GREEN B I N D I N G BY THE HEPATIC C Y T O P L A S M I C P R O T E I N S Toshihiko N A M I H I S A , M.D., Masaji NAMBU, M.D. and Yuji Y A M A S H I R O , M.D.
Department of Gastroenterology, Internal Medicine, Juntendo University, School of Medicine, 1-1, Hongo, 2-chome, Bunkyo-ku, Tokyo, Japan Summary The elution patterns of human and rat liver supernate mixed with BSP or ICG have been demonstrated using G-100 Sephadex gel filtration. The elution pattern in the rat liver remained the same as that shown by Levi et al. In the human liver BSP localized to Y protein could hardly be demonstrated, whereas ICG was detected. This pattern was quite similar in Rotor's hyperbilirubinemm and in Crigler-Najiar syndrome Type II. These observations suggest that the imparired hepatic transportation of organic anions in congenital hyperbilirubinemias may not be related to cytoplasmic organic anion-binding proteins.
Key Words: sulfobromophthalein (BSP), indocyanine green (ICG), hepatic cytoplasmic proteins, congenital hyper-
bilirubinemia. Two hepatic cytoplasmic proteins, Y(ligandin) and Z, which bound various organic anions in vitro and in vivo, including bilirubin, sulfobromophthalein (BSP) and indocyanine green (ICG), were separated by G-75 Sephadex gel filtration and appeared to be important in the transfer of organic anions from the plasma into the liver 1~. Although these proteins have been identified in the liver of various vertebratesl, 2~, the elution p a t t e r n of h u m a n liver supernate mixed with BSP or I C G has not been demonstrated. We observed the patterns of h u m a n liver in normal volunteers, Rotor's hyperbilirubinemia and Crigler-Najjar syndrome T y p e I I , and the normal pattern was compared with the result of the rat liver.
Materials and Methods The h u m a n liver tissues, including normal volunteers, one of Rotor's hyperbilirubinemia
and Crigler-Najjar syndrome Type II, were obtained by biopsy under the laparoscopy or the laparotomy. Wistar rat livers were used in the control study. The procedure for preparation was performed using about 0.2 g of the h u m a n liver. The liver specimens obtained by the biopsy were immediately washed in ice-cold isotonic saline and perfused through the needle pricked into the specimens until all traces of blood were washed out. A 25% homogenate was prepared in 0.25 M sucrose-0.01 M phosphate buffer, p H 7.4, using Teflonglass, motor driven homogenizer. The homogenate was centrifuged at 2~ in automatic preparative ultracentrifuge ( H I T A C H I 65 P) at 100,000 g for 120 min.. T h e separated supernatant fraction was mixed with 6 • .3 moles of BSP or 2 • 10 -4 moles of I C G (final concentration) and placed on a Sephadex G-100 with 0.01 M phosphate buffer, p H 7.4, for using downward flow system. The column
December 1977
BSP and ICG Binding by Hepatic Cytoplasmic Proteins
in size of 1.2 • 140 cm was used, and the flow rate was 1.5 ml/tube/20 min.. Protein concentration was estimated by absorption at 2 3 0 n m and 2 8 0 n m , I C G at 8 0 5 n m and BSP at 580 nm after alkalization with NaoH, using H I T A C H I 100-20 spectrophotometer. T h e procedure for preparation and gel chromatography of the rat liver supernate were essentially the same as that of Levi et al.]>
467
Elution patterns of h u m a n liver in patients with Rotor's hyperbilirubinemia and CriglerNajjar syndrome T y p e I I remained the same as normal h u m a n liver (Fig. 3, 4).
O0
0.7.
1
Protein
- ....... . . . .
BSP
0.6.
Results
T h e elution patterns of rat liver supernate with BSP or I C G were shown in Fig. 1. Three peaks, designated as X, Y and Z protein, were prominent and preferential localizations of BSP and I C G binding to these proteins were demonstrated. In elution patterns of normal h u m a n liver the second peak of BSP binding to the protein was obscure and the second peak of I C G was demonstrated (Fig. 2). The pattern remained the same when the double amount of liver supernate was used.
o.5 ~
0.4'
0.$" 0.2" I
/--~
o.i-
20
'
50
40
Protein
No
Protein . . . . . .
........
Tube
Fig. 2. T h e elution p a t t e r n of n o r m a l h u m a n liver supernate mixed with BSP or ICG.
OD
OD
0.7"
tCG
BSP
0.7'
ICG
8SP
0.6'
0.6"
0.5" 0.50.4" 0.4"
0.3 0.3
0.2 0.2
0.1'
20
3"o
4'o
4o v . b . No
Fig. 1. T h e elution p a t t e r n of rat liver supernate mixed with BSP or I C G .
io
io
V.bo No
Fig. 3. T h e elution p a t t e r n of the liver supernate mixed with BSP in a patient with Rotor's hyperbilirubinemia.
468
T. NAM1HISA E l AL.
OD
Protein ........ 0.7-
. . . . . . .
ICG BSP
0.6-
0.5-
0.4"
0.$ b
~
0.2
0.I
~
/ "~
', .... 20
S0
40
Tub, No
Fig. 4. The elution pattern of the liver supernate mixed with BSP or ICG in a patient with CriglerNaJ~ar syndrome Type II.
Vol. 12, No. 6
conditions. It is suggested that there is a difference of nature in Y protein between rats and humans. In patients with Rotor's hyperbilirubinemia and Crigler-Najjar syndrome Type II the elution patterns of liver supernate bound BSP and I C G were quite similar as that in normal human liver. Fleischner et al. 3) reported that the ligandin concentration revealed normal value in the liver of Gilbert's syndrome, Crigler-Najjar syndrome Type I and Type II, and Dubin-Johnson syndrome using methods by immunodiffusion and radioimmunoassay. These observations suggest that the impaired hepatic transportation of organic anions dyes in congenital hyperbilirubinemias 4,5) may not be related solely to cytoplasmic organic anionbinding proteins. References
Discussion
Levi et al. t) demonstrated two proteins which bound certain organic anions in vivo and in vitro. These proteins were described as being present in the liver of rats and other vertebrates, including humans 2). Although in the rat liver the elution pattern obtained by us remained the same as that shown by Levi et al., in human liver the elution pattern was very different from that. The BSP localized to Y protein could hardly be detected, whereas I C G localized to Y protein was demonstrated. This result was confirmed by performing repeated studies under defined
1) Levi AJ, et al: Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J Clin Invest 48: 2156, 1969 2) Levine RI, et al: Phylogenetic study of organic anion transfer from plasma into the liver. Nature New Biology 231: 277, 1971 3) Fleischner G, et al: Human ligandin : Characterization and quantitation. Gastroenterology 69: 821, 1975 4) Namihisa T, et al: The constitutional conjugated hyperbilirubinemia (Dubin-Johnson syndrome and Rotor's type of hyperbilirubinemia). New definitions based on studies of transport with indocyanine green and bromosulfophthalein. Gastroenterologia Japonica 8: 217, 1973 5) Namihisa T, et al: Hepatic dye transport in constitutional hyperbilirubinemia and constitutional ICG excretory defect. Acta Hepatologica Japonica 16: 25, 1975
Received October 6, 1977. Accepted November 14, 1977. Addres~ requestsfor reprints to: Dr. Toshihiko Namihisa, M.D., Department of Gastroenterology, Internal Medicine, Juntendo University, School of Medicine, 1-1 Hongo 2-chome, Bunkyo-ku, Tokyo, 113 Japan.
Vol. 12, No. 6 Printed in Japan
--Origlna Article
S U L F O B R O M O P H T H A L E I N A N D I N D O C Y A N I N E GREEN B I N D I N G BY THE HEPATIC C Y T O P L A S M I C P R O T E I N S Toshihiko N A M I H I S A , M.D., Masaji NAMBU, M.D. and Yuji Y A M A S H I R O , M.D.
Department of Gastroenterology, Internal Medicine, Juntendo University, School of Medicine, 1-1, Hongo, 2-chome, Bunkyo-ku, Tokyo, Japan Summary The elution patterns of human and rat liver supernate mixed with BSP or ICG have been demonstrated using G-100 Sephadex gel filtration. The elution pattern in the rat liver remained the same as that shown by Levi et al. In the human liver BSP localized to Y protein could hardly be demonstrated, whereas ICG was detected. This pattern was quite similar in Rotor's hyperbilirubinemm and in Crigler-Najiar syndrome Type II. These observations suggest that the imparired hepatic transportation of organic anions in congenital hyperbilirubinemias may not be related to cytoplasmic organic anion-binding proteins.
Key Words: sulfobromophthalein (BSP), indocyanine green (ICG), hepatic cytoplasmic proteins, congenital hyper-
bilirubinemia. Two hepatic cytoplasmic proteins, Y(ligandin) and Z, which bound various organic anions in vitro and in vivo, including bilirubin, sulfobromophthalein (BSP) and indocyanine green (ICG), were separated by G-75 Sephadex gel filtration and appeared to be important in the transfer of organic anions from the plasma into the liver 1~. Although these proteins have been identified in the liver of various vertebratesl, 2~, the elution p a t t e r n of h u m a n liver supernate mixed with BSP or I C G has not been demonstrated. We observed the patterns of h u m a n liver in normal volunteers, Rotor's hyperbilirubinemia and Crigler-Najjar syndrome T y p e I I , and the normal pattern was compared with the result of the rat liver.
Materials and Methods The h u m a n liver tissues, including normal volunteers, one of Rotor's hyperbilirubinemia
and Crigler-Najjar syndrome Type II, were obtained by biopsy under the laparoscopy or the laparotomy. Wistar rat livers were used in the control study. The procedure for preparation was performed using about 0.2 g of the h u m a n liver. The liver specimens obtained by the biopsy were immediately washed in ice-cold isotonic saline and perfused through the needle pricked into the specimens until all traces of blood were washed out. A 25% homogenate was prepared in 0.25 M sucrose-0.01 M phosphate buffer, p H 7.4, using Teflonglass, motor driven homogenizer. The homogenate was centrifuged at 2~ in automatic preparative ultracentrifuge ( H I T A C H I 65 P) at 100,000 g for 120 min.. T h e separated supernatant fraction was mixed with 6 • .3 moles of BSP or 2 • 10 -4 moles of I C G (final concentration) and placed on a Sephadex G-100 with 0.01 M phosphate buffer, p H 7.4, for using downward flow system. The column
December 1977
BSP and ICG Binding by Hepatic Cytoplasmic Proteins
in size of 1.2 • 140 cm was used, and the flow rate was 1.5 ml/tube/20 min.. Protein concentration was estimated by absorption at 2 3 0 n m and 2 8 0 n m , I C G at 8 0 5 n m and BSP at 580 nm after alkalization with NaoH, using H I T A C H I 100-20 spectrophotometer. T h e procedure for preparation and gel chromatography of the rat liver supernate were essentially the same as that of Levi et al.]>
467
Elution patterns of h u m a n liver in patients with Rotor's hyperbilirubinemia and CriglerNajjar syndrome T y p e I I remained the same as normal h u m a n liver (Fig. 3, 4).
O0
0.7.
1
Protein
- ....... . . . .
BSP
0.6.
Results
T h e elution patterns of rat liver supernate with BSP or I C G were shown in Fig. 1. Three peaks, designated as X, Y and Z protein, were prominent and preferential localizations of BSP and I C G binding to these proteins were demonstrated. In elution patterns of normal h u m a n liver the second peak of BSP binding to the protein was obscure and the second peak of I C G was demonstrated (Fig. 2). The pattern remained the same when the double amount of liver supernate was used.
o.5 ~
0.4'
0.$" 0.2" I
/--~
o.i-
20
'
50
40
Protein
No
Protein . . . . . .
........
Tube
Fig. 2. T h e elution p a t t e r n of n o r m a l h u m a n liver supernate mixed with BSP or ICG.
OD
OD
0.7"
tCG
BSP
0.7'
ICG
8SP
0.6'
0.6"
0.5" 0.50.4" 0.4"
0.3 0.3
0.2 0.2
0.1'
20
3"o
4'o
4o v . b . No
Fig. 1. T h e elution p a t t e r n of rat liver supernate mixed with BSP or I C G .
io
io
V.bo No
Fig. 3. T h e elution p a t t e r n of the liver supernate mixed with BSP in a patient with Rotor's hyperbilirubinemia.
468
T. NAM1HISA E l AL.
OD
Protein ........ 0.7-
. . . . . . .
ICG BSP
0.6-
0.5-
0.4"
0.$ b
~
0.2
0.I
~
/ "~
', .... 20
S0
40
Tub, No
Fig. 4. The elution pattern of the liver supernate mixed with BSP or ICG in a patient with CriglerNaJ~ar syndrome Type II.
Vol. 12, No. 6
conditions. It is suggested that there is a difference of nature in Y protein between rats and humans. In patients with Rotor's hyperbilirubinemia and Crigler-Najjar syndrome Type II the elution patterns of liver supernate bound BSP and I C G were quite similar as that in normal human liver. Fleischner et al. 3) reported that the ligandin concentration revealed normal value in the liver of Gilbert's syndrome, Crigler-Najjar syndrome Type I and Type II, and Dubin-Johnson syndrome using methods by immunodiffusion and radioimmunoassay. These observations suggest that the impaired hepatic transportation of organic anions dyes in congenital hyperbilirubinemias 4,5) may not be related solely to cytoplasmic organic anionbinding proteins. References
Discussion
Levi et al. t) demonstrated two proteins which bound certain organic anions in vivo and in vitro. These proteins were described as being present in the liver of rats and other vertebrates, including humans 2). Although in the rat liver the elution pattern obtained by us remained the same as that shown by Levi et al., in human liver the elution pattern was very different from that. The BSP localized to Y protein could hardly be detected, whereas I C G localized to Y protein was demonstrated. This result was confirmed by performing repeated studies under defined
1) Levi AJ, et al: Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J Clin Invest 48: 2156, 1969 2) Levine RI, et al: Phylogenetic study of organic anion transfer from plasma into the liver. Nature New Biology 231: 277, 1971 3) Fleischner G, et al: Human ligandin : Characterization and quantitation. Gastroenterology 69: 821, 1975 4) Namihisa T, et al: The constitutional conjugated hyperbilirubinemia (Dubin-Johnson syndrome and Rotor's type of hyperbilirubinemia). New definitions based on studies of transport with indocyanine green and bromosulfophthalein. Gastroenterologia Japonica 8: 217, 1973 5) Namihisa T, et al: Hepatic dye transport in constitutional hyperbilirubinemia and constitutional ICG excretory defect. Acta Hepatologica Japonica 16: 25, 1975
Received October 6, 1977. Accepted November 14, 1977. Addres~ requestsfor reprints to: Dr. Toshihiko Namihisa, M.D., Department of Gastroenterology, Internal Medicine, Juntendo University, School of Medicine, 1-1 Hongo 2-chome, Bunkyo-ku, Tokyo, 113 Japan.
