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Radioimmonoelectrophoretic analysis of thyroxinebinding proteins in normal human sera
CLINICA
CHIhfICA
341
ACTA
RADIOIMMONOELECTROPHORETIC BINDING
KlY0SHI
PROTEINS
MIYAl,
KISHIO
I’. ITOH,
7‘1~ First Department ofMedicine Osaka Cniversit_y Medical School, ( IZcceivetl
May
30,
ANALYSIS
IN NORMAL
HUMAN
fllllOSH1
ABE
OF THYROXINE-
SERA
.~ED YUICHI
and the Central Laboratovv fovClinical Fukushima-ku, Osaka (Japan)
KLMAHARA Investigation.
1968)
SUMMARY
A freshly prepared serum was mixed with purified [13rIjthyroxine to give a low concentration and was analyzed by radioimmunoelectrophoresis. When antiwhole human serum was used, five distinct radioactive arcs were demonstrated in both Japanese and occidental normal sera. Evidences were obtained to support that these radioactive arcs were not artifacts but showed thyroxine-binding components. By the radioimmunoelectrophoretic patterns with specific antisera, staining for lipoproteins, and using serum to which diphenylhydantoin had been added or a thyroxine-binding globulin-deficient serum, these five thyroxine-binding components were identified binding globulin
as thyroxine-binding prealbumin (TBPA), (TBG), and a,- and /% (or a,-)lipoproteins.
albumin,
thyroxine-
INTRODUCTION
It is a well known fact that thyroxine is bound to special serum proteins in the blood, called thyroxine-binding proteins. It seems generally accepted, at this time, that at least three kinds of thyroxine-binding proteins, thyroxine-binding prealbumin (TBPA), thyroxine-binding globulin (TBG) and albumin exist in normal human sera. These three proteins can be demonstrated by adding radiothyroxine to the sera and analyzed by various techniques including paper*-4, co1umn4p5 and starch ge12y6 electrophoresis. Several investigators, however, have reported that radiothyroxine can be found in four serum components33497!8, while others have suggested that the fourth component may be an artifact or free thyroxineg+lO. Even though radioimnmnoelectrophoretic analysis, which is one of the most sensitive methods, was used, conflicting reports have appeared regarding the number and identification of the thyroxine-binding proteins 11-13. The present study was designed to demonstrate and identify these thyroxine-binding proteins by utilizing various radioimmunoelectrophoretic techniques.
MIYAI
342
et al.
MA’I’ERIALS AND METHODS
!131I]Thyroxine
obtained
from Abbott
Laboratories
was purified
before
USC.
The 11311]thyroxine dissolved in 50% propylene glycol was applied to filter paper (Toyoroshi No. 50) and ascending chromatography was carried out in a n-butanoldioxan-ammonia system. The area containing the radiothyroxine was determined by autoradiography and cut out, and the radiothyroxine was eluted with 50 y0 propylene glycol. Blood was withdrawn from ZS healthy Japanese and 12 healthy occidental people. A serum from a patient with an idiopathic decrease in TBG was kindly given us by Drs. Hideo Yoshida,
lwao Fukui,
Yoshiki
Takeshita,
Masayuki
Ide and their
collaborators of the First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kvoto, Japan. The clinical data of the patient were reported elsewhere’,‘. Each serum was freshly prepared and mixed with the purified 1lslI ,thyroxine to give a final concentration of 0.05 big/m 1 of :serum, (the radioactivity being 1.1-1.7 L!C/ml of serum), except when a larger amount of ~311]thyrosine was used to investigate was incubated
its effect
at room
to immunoelectrophoresis.
on the radioimnmnoelectrophoretic
temperature
pattern.
for I h, left at 4O overnight
A slight modification
was used for immunoelectrophoresis.
Phosphate
of the Grahar buffer
(pH
The mixturcx
and then subjected
and Williams 7.4, ionic
technique
strength
0.05)
in the experiment to investigate the effect of buffer on immunoelectrophoresis. Following electrophoresis on 1 qb buffered agar plate (3.0-3.5 mm thick) at a constant was usually
voltage
used
(2.5-3.5
and borate
V/cm)
buffer
for 2.0-28
(pH
8.6, ionic
11, about
strength
0.05)
0.36 ml of antiserum
was used
was added
only
to eaclr
formation of precipitin arcs, each plate was washed twice with saline and water and dried. Autoradiography was performed by exposing the plates to x-ray film for a period of z- 4 weeks. Amidoblack IO B was used for staining the proteins, and oil red 0 or Sudan Black 1: for staining the lipoproteins. Horse anti-human sera were obtained from the Kesearch Institute for Microbial
trough.
After
diffusion
for 24-48
11 in order to permit
Diseases, Osaka University, Osaka, Japan (Biken), (the antiserum was prepared against Japanese sera), Hyland Laboratories, Los Angeles, Calif. I-.S.A. (Hyland) and Behringwerke A.G., Marburg-Lahn, West Germany (Behringwerke). Rabbit antisera against prealbumin, albumin, q-lipoprotein and p- (or a,-)lipoprotein were obtained from Behringwerke A.G. In some experiments the globulin fraction was separated from the horse anti-human serum by the sodium sulfate precipitation method and used. Diphenylhydantoin sodium (DPH) was dissolved in a solvent (propylene glycol, 40 o/0v/v and ethanol, 10.5 o/0 v/v in H,O, pH 12) and added to the serum to give a final concentration
of 3.6 x
IO-~ M and 1.4 x IO-I M.
RESCLTS
Radioimmunoelectro$ho~etic demo&ration of thyroxine-biding components in normal swa by anti-humagt serum Fig. I shows a typical pattern of the immunoelectrophoresis and its corresponding autoradiogram of normal serum. When Biken’s anti-human serum was used, at
THYROXINE-BINDING
least 5 distinct in both Japanese
PROTEINS
radioactive
IN
SERUM
343
arcs could be observed
and occidental
sera,
although
on the autoradiogranl arc V was usually
(I-V
very thin.
incl.) The
precipitin arcs could be observed by the protein stain corresponding to the radioactive arcs I, II, III and V, even though some were very thin. However, it was difficult
to find a precipitin
arc corresponding
to the thick
radioactive
arc IV. On
Fig. 1. Xaclioimmunoelectrophoresis of normal human serum with [1311]thvroxine added. a: immunoelectrophoretic pattern, b: autoradiogram, c: schematic representation of autoradiogram; ES\&‘: Behringwcrkc’s anti-human serum, Bik: Biken’s anti-human serum, Hy : Hpland’s antihuman serum.
the other hand, under the same conditions employed by us, 3 or 4 radioactive arcs were shown by Hyland’s or Behringwerke’s antisera. Under the following conditions, similarly five radioactive arcs could be demonstrated by Biken’s anti-human serum, although some differences were observed in each pattern, such as, when [l”II]thyroxine was added to the troughs after forming the precipitin arcs, the larger amount of [1311]thyroxine was added to the serum (about 6 PC, 0.3 pg/ml of serum), the y-globulin fraction of the antiserum was added to the troughs, or the borate buffer (pH 5.6) was used instead of phosphate buffer. CEin. Chim. Acta, 22 (1968) 341-347
344
MIYAI
at.
When PI]thyroxine dissolved in 50~; propy lene glycol was added to the serum, no changes were observed in the protein patterns of immunoelectrophoresis. By this method, the free / 1311]thyroxine is eluted bv washing; in fact, neither precipitin arcs nor radioactive areas were shown when only /1311]thyroxine to which serum had not been added, was identically treated. The chemical fog on the X-ray fihn produced by the dried immunoelectrophoretic plate using serum to which rnlI]thyroxine had not been added, could be neglected.
Fig. 2. Radioimmunoelectrophorcsis of [IS1I lthyroxine-added normal human serum using specific antisera. P4: anti-prealbumin serum, ,x-Lip: anti-a,-lipoprotein strum. .Alb: anti-albumin serum, ,5-Lip : anti-jT(or a,-)lipoprotein serum, HS : Riken’s anti-human serum. a : illl111~~11~)electropl~~r~ti(. pattern, b: autoradiogram, c: schematic representation of autoradiogram.
Radioinwnu~zoelectrophoresis by speci$c antisera against various human semna jwoteim As shown in Fig. 2, only one radioactive precipitin arc was demonstrated hq anti-prealbumin serum. From the electrophoretic mobility and the confluent pattern, it can be seen that this arc corresponds to arc I which is demonstrated by antihuman serum. It is shown similarly that arc II corresponds to a,-lipoprotein, arc III to albumin and arc V to p- (or cr,-)lipoprotein.
THYROXINE-BINDIh-G
PROTEINS
IN
SERUM
Staining for lipoproteins The precipitin arcs corresponding 0 or Sudan Black
345
to arc II and V were stained
with Oil red
B.
Radioilnmunoelectrophoretic patterns of a TBG-deficient Fig. 3 shows the radioimmunoelectrophoretic
serwm and DPH-added semm pattern of a TBG-deficient
serum. Arc IV was not detectable in the serum while the other arcs were demonstrated. Arc IV was also thin or mostly absent when DPH-added sera were similarly analyzed
(Fig. 4).
d
b
1 J
liig.
3.
: control, of autoradiogram.
L: 3.6 x IO-~ .M, 3: 1.4 x lo- 1 M. b: autoradiogram, Bik: Biken’s anti-human serum.
c: schematic
representation
DISCUSSION
In the present thyroxine-added
study the five radioactive
serum
was analyzed
arcs were demonstrated
when r1311]-
by radioimmunoelectrophoresis.
_ ‘1311]thyroxine
was added to serum to give a low concenClin.
Chim.
Acfa, 22 (1968)
341-347
MIYAI
346
et al.
tration of 0.05 ,q/ml and phosphate buffer (pH 7.4) was used for immunoelectrophoresis in our experiment; these conditions were used to simulate physiological ‘13rI]thyroxine was bound to serum proteins under conditions except that the L conditions i~z vitro forming the precipitin arcs of antigen-antibody complex. It is unlikelythat theradioimmunoelectrophoreticpatterns were modified by the thyroxinebinding proteins contained in the horse antiserum, because a similar pattern was observed
even when the y-globulin
fraction
of antiserum
was used. If other thyroxine-
binding components with low affinity or capacity of binding to thyroxine are present in human serum, they can be demonstrated by adding larger amounts of 1311Ithyroxine to the serum, but such a pattern could not be shown in our experiment. Different patterns of radioimmunoelectrophoresis were observed when antisera from various sources were used, but no difference in pattern was observed between Japanese and occidental sera when the same antiserum was used. Variation in antibody titer of each antiserum may result in different patterns. These observations support the fact that the five radioactive arcs demonstrated not artifacts but thyroxine-binding components. II,
III
From the radioimmunoelectrophoretic patterns and V were identified as TBPA, a,-lipoprotein,
protein respectively. Since react with cr,-lipoprotein, has been examined by us. arc II and V contained the
in our experiment
arc
using specific antisera, arc 1, albumin and p- (or z,-)lipo-
the anti-p-lipoprotein serum of Behringwerke may crossthe differentiation between the two is difficult, as far as Staining with Oil red 0 or Sudan Black B revealed that lipid.
Arc IV was not detectable by radioimmunoelectrophoresis in a TRG-deficient serum. On the other hand, the decreased thyroxine-binding capacity of TBG in the serum was estimated by acetate membrane electrophoresisl”. The discrepancy in the results may still be explained by the lower separability on acetate membrane electrophoresis or the lower sensitivity of radioimmunoelectrophoresis. DPH which has been reported to displace thyroxine from TBG to other thyroxine-binding proteins6T13 also decreased the arc IV. Thus it is reasonable to assume that arc 11: represents the so-called “TBG”. Previous studies have shown that three or four thyroxine-binding components were demonstrated by radioimmunoelectrophoresis. Tllyroxine-binding to prealbumin, albumin and xc,- (or a,-)lipoprotein was found by Hollander et d.12; binding to albumin, R~- and a,-lipoproteins by Clausen and Munkner”, and to prealbumin, albumin, a,-lipoprotein and another cl-globulin by- Lightfoot and Christianl3. However, Sterling et al.16 postulated that thyroxine-binding ~-globulin was not a lipoprotein of the major lipoprotein classes, on basis of their cluantitativ,c studies using ultracentrifugation and precipitation with specific antisera. Our studies using radioimmunoelectrophoresis have shown that thyroxine was bound to al- and /3- (or cc,-)lipoproteins in addition to the three major thyroxine-binding proteins, namely prealbumin, albumin and the so-called TBG. The specificity and the physiological significance of thyroxine-binding to serum lipoproteins are unknown. Relationship between these lipoproteins and additional thyroxine-binding components which were detected by other methods *,3,~ is also still unclarified, and further studies are necessary
Clin.
Chim.
Acta,
to solve these problems.
2~ (1968)
341 -.j+i
THYROXINE-BINDING
PROTEINS
IS
347
SERUM
ACKNOWLEDGMENTS
W’e wish to express We are greatly English.
I L 3 4 5 6 7 8 9 ICI IL
indebted
our thanks
to Dr. S. Tanabe
for his valuable
to Dr. C. Ito for his help in preparing
suggestions.
this manuscript
S. H. Ixcam, ~ndocrinologv, 63 (1958) 256. 31. 11’. HAMOLSKY, D. B. FISHER AND A. S. FREEDBERG, Ewdocvinology, 66 (1960) B. S. BLUMBERG AND J. ROBBINS, Endocvimlogy, 67 (1960) 368. J, Ii. TATA, C. C. WIDNELL AND W. B. GRATZER, Clin.Chim. Acta, 6 (1961) 597. TV. E. BALFOURMVD H. E.TUNNICLIFFE,J. Physiol., 153 (1960) 179. I<. SQUEF, M.MARTINEZ.~ND J. H. OPPENHEIMER, Proc. Soc.Exptl.Bml.Med., 113 B. S. BLUMBERG, L. FBRER, J. F. RALL AND J. ROBBINS, Endocvinology, 68 (1961) S. C. THORSON, W. N. TAUXE AND H. F. TASWELL, J. Clin. Endocrinol. Metab., 26 L. S. FARER, J. ROBBINS AND B. S. BLUMBERG, E+zdocYinology, 70 (1962) 679. J. S. MARSHALL, R. 1'.LEVY, J. Clin. Endocrinol. Metab., 26 (1966) 87. J, CL.AUSEN .~XD T. MUNKNER, Proc. Sot. Exptl. Biol. Med., 104 (1960) 40.
in
780.
(1963) rj. (1966)
837. 181.
IL C. S. HOLLAXDER, B. H. LATIMER, T. E. PROUT, D. H. LOCKWOOD XXII S. P. &PER, Mctabolisw, IL (1963) 45. 13 Ii.M'. LIGHTFOOT, Jr. AND C. L. CHRISTIAN, J. Clin. Endocrinol. Metab., 26 (1966) 305. 14 H. WOSHIDA, &I.IDE, I. FUKUI, Y. TAKESHITA, T. YMAZAKI, R. ~OSHIMATSU, H. ~A~MM~T~, N. HIROT~, E. SAWASHIGE, S. YOSHIKAWA, N. MAEDA AND N. HAMANAKA, Folia Endocrinologica Japonica,
42 (1967)
1200.
15 I'.GRAB.~R AND C. A4.V:ILLIAMS, Biochim. Biophys. Acta, IO (1953) 193. 16 Ii.STERLING, h1. A. BRENNER AND H. G. ROSE, Endocrinology, 77 (1965) 398. C&L. Chim.
Addendum
Acta,
21 (1968) 341-347
to page 344, line 7
Paper chromatographic analysis of ethanol-acetone extract oftheimmunoelectrophoretic plate indicated that only about IO % of inorganic r/II was liberated from the [ *JrI]thyroxine, and no distinct radioactive areas were demonstrated when equivalent amount of [‘3r]sodium was added to the serum and analyzed by radioimmunoelectrophoresis.
CHIhfICA
341
ACTA
RADIOIMMONOELECTROPHORETIC BINDING
KlY0SHI
PROTEINS
MIYAl,
KISHIO
I’. ITOH,
7‘1~ First Department ofMedicine Osaka Cniversit_y Medical School, ( IZcceivetl
May
30,
ANALYSIS
IN NORMAL
HUMAN
fllllOSH1
ABE
OF THYROXINE-
SERA
.~ED YUICHI
and the Central Laboratovv fovClinical Fukushima-ku, Osaka (Japan)
KLMAHARA Investigation.
1968)
SUMMARY
A freshly prepared serum was mixed with purified [13rIjthyroxine to give a low concentration and was analyzed by radioimmunoelectrophoresis. When antiwhole human serum was used, five distinct radioactive arcs were demonstrated in both Japanese and occidental normal sera. Evidences were obtained to support that these radioactive arcs were not artifacts but showed thyroxine-binding components. By the radioimmunoelectrophoretic patterns with specific antisera, staining for lipoproteins, and using serum to which diphenylhydantoin had been added or a thyroxine-binding globulin-deficient serum, these five thyroxine-binding components were identified binding globulin
as thyroxine-binding prealbumin (TBPA), (TBG), and a,- and /% (or a,-)lipoproteins.
albumin,
thyroxine-
INTRODUCTION
It is a well known fact that thyroxine is bound to special serum proteins in the blood, called thyroxine-binding proteins. It seems generally accepted, at this time, that at least three kinds of thyroxine-binding proteins, thyroxine-binding prealbumin (TBPA), thyroxine-binding globulin (TBG) and albumin exist in normal human sera. These three proteins can be demonstrated by adding radiothyroxine to the sera and analyzed by various techniques including paper*-4, co1umn4p5 and starch ge12y6 electrophoresis. Several investigators, however, have reported that radiothyroxine can be found in four serum components33497!8, while others have suggested that the fourth component may be an artifact or free thyroxineg+lO. Even though radioimnmnoelectrophoretic analysis, which is one of the most sensitive methods, was used, conflicting reports have appeared regarding the number and identification of the thyroxine-binding proteins 11-13. The present study was designed to demonstrate and identify these thyroxine-binding proteins by utilizing various radioimmunoelectrophoretic techniques.
MIYAI
342
et al.
MA’I’ERIALS AND METHODS
!131I]Thyroxine
obtained
from Abbott
Laboratories
was purified
before
USC.
The 11311]thyroxine dissolved in 50% propylene glycol was applied to filter paper (Toyoroshi No. 50) and ascending chromatography was carried out in a n-butanoldioxan-ammonia system. The area containing the radiothyroxine was determined by autoradiography and cut out, and the radiothyroxine was eluted with 50 y0 propylene glycol. Blood was withdrawn from ZS healthy Japanese and 12 healthy occidental people. A serum from a patient with an idiopathic decrease in TBG was kindly given us by Drs. Hideo Yoshida,
lwao Fukui,
Yoshiki
Takeshita,
Masayuki
Ide and their
collaborators of the First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kvoto, Japan. The clinical data of the patient were reported elsewhere’,‘. Each serum was freshly prepared and mixed with the purified 1lslI ,thyroxine to give a final concentration of 0.05 big/m 1 of :serum, (the radioactivity being 1.1-1.7 L!C/ml of serum), except when a larger amount of ~311]thyrosine was used to investigate was incubated
its effect
at room
to immunoelectrophoresis.
on the radioimnmnoelectrophoretic
temperature
pattern.
for I h, left at 4O overnight
A slight modification
was used for immunoelectrophoresis.
Phosphate
of the Grahar buffer
(pH
The mixturcx
and then subjected
and Williams 7.4, ionic
technique
strength
0.05)
in the experiment to investigate the effect of buffer on immunoelectrophoresis. Following electrophoresis on 1 qb buffered agar plate (3.0-3.5 mm thick) at a constant was usually
voltage
used
(2.5-3.5
and borate
V/cm)
buffer
for 2.0-28
(pH
8.6, ionic
11, about
strength
0.05)
0.36 ml of antiserum
was used
was added
only
to eaclr
formation of precipitin arcs, each plate was washed twice with saline and water and dried. Autoradiography was performed by exposing the plates to x-ray film for a period of z- 4 weeks. Amidoblack IO B was used for staining the proteins, and oil red 0 or Sudan Black 1: for staining the lipoproteins. Horse anti-human sera were obtained from the Kesearch Institute for Microbial
trough.
After
diffusion
for 24-48
11 in order to permit
Diseases, Osaka University, Osaka, Japan (Biken), (the antiserum was prepared against Japanese sera), Hyland Laboratories, Los Angeles, Calif. I-.S.A. (Hyland) and Behringwerke A.G., Marburg-Lahn, West Germany (Behringwerke). Rabbit antisera against prealbumin, albumin, q-lipoprotein and p- (or a,-)lipoprotein were obtained from Behringwerke A.G. In some experiments the globulin fraction was separated from the horse anti-human serum by the sodium sulfate precipitation method and used. Diphenylhydantoin sodium (DPH) was dissolved in a solvent (propylene glycol, 40 o/0v/v and ethanol, 10.5 o/0 v/v in H,O, pH 12) and added to the serum to give a final concentration
of 3.6 x
IO-~ M and 1.4 x IO-I M.
RESCLTS
Radioimmunoelectro$ho~etic demo&ration of thyroxine-biding components in normal swa by anti-humagt serum Fig. I shows a typical pattern of the immunoelectrophoresis and its corresponding autoradiogram of normal serum. When Biken’s anti-human serum was used, at
THYROXINE-BINDING
least 5 distinct in both Japanese
PROTEINS
radioactive
IN
SERUM
343
arcs could be observed
and occidental
sera,
although
on the autoradiogranl arc V was usually
(I-V
very thin.
incl.) The
precipitin arcs could be observed by the protein stain corresponding to the radioactive arcs I, II, III and V, even though some were very thin. However, it was difficult
to find a precipitin
arc corresponding
to the thick
radioactive
arc IV. On
Fig. 1. Xaclioimmunoelectrophoresis of normal human serum with [1311]thvroxine added. a: immunoelectrophoretic pattern, b: autoradiogram, c: schematic representation of autoradiogram; ES\&‘: Behringwcrkc’s anti-human serum, Bik: Biken’s anti-human serum, Hy : Hpland’s antihuman serum.
the other hand, under the same conditions employed by us, 3 or 4 radioactive arcs were shown by Hyland’s or Behringwerke’s antisera. Under the following conditions, similarly five radioactive arcs could be demonstrated by Biken’s anti-human serum, although some differences were observed in each pattern, such as, when [l”II]thyroxine was added to the troughs after forming the precipitin arcs, the larger amount of [1311]thyroxine was added to the serum (about 6 PC, 0.3 pg/ml of serum), the y-globulin fraction of the antiserum was added to the troughs, or the borate buffer (pH 5.6) was used instead of phosphate buffer. CEin. Chim. Acta, 22 (1968) 341-347
344
MIYAI
at.
When PI]thyroxine dissolved in 50~; propy lene glycol was added to the serum, no changes were observed in the protein patterns of immunoelectrophoresis. By this method, the free / 1311]thyroxine is eluted bv washing; in fact, neither precipitin arcs nor radioactive areas were shown when only /1311]thyroxine to which serum had not been added, was identically treated. The chemical fog on the X-ray fihn produced by the dried immunoelectrophoretic plate using serum to which rnlI]thyroxine had not been added, could be neglected.
Fig. 2. Radioimmunoelectrophorcsis of [IS1I lthyroxine-added normal human serum using specific antisera. P4: anti-prealbumin serum, ,x-Lip: anti-a,-lipoprotein strum. .Alb: anti-albumin serum, ,5-Lip : anti-jT(or a,-)lipoprotein serum, HS : Riken’s anti-human serum. a : illl111~~11~)electropl~~r~ti(. pattern, b: autoradiogram, c: schematic representation of autoradiogram.
Radioinwnu~zoelectrophoresis by speci$c antisera against various human semna jwoteim As shown in Fig. 2, only one radioactive precipitin arc was demonstrated hq anti-prealbumin serum. From the electrophoretic mobility and the confluent pattern, it can be seen that this arc corresponds to arc I which is demonstrated by antihuman serum. It is shown similarly that arc II corresponds to a,-lipoprotein, arc III to albumin and arc V to p- (or cr,-)lipoprotein.
THYROXINE-BINDIh-G
PROTEINS
IN
SERUM
Staining for lipoproteins The precipitin arcs corresponding 0 or Sudan Black
345
to arc II and V were stained
with Oil red
B.
Radioilnmunoelectrophoretic patterns of a TBG-deficient Fig. 3 shows the radioimmunoelectrophoretic
serwm and DPH-added semm pattern of a TBG-deficient
serum. Arc IV was not detectable in the serum while the other arcs were demonstrated. Arc IV was also thin or mostly absent when DPH-added sera were similarly analyzed
(Fig. 4).
d
b
1 J
liig.
3.
: control, of autoradiogram.
L: 3.6 x IO-~ .M, 3: 1.4 x lo- 1 M. b: autoradiogram, Bik: Biken’s anti-human serum.
c: schematic
representation
DISCUSSION
In the present thyroxine-added
study the five radioactive
serum
was analyzed
arcs were demonstrated
when r1311]-
by radioimmunoelectrophoresis.
_ ‘1311]thyroxine
was added to serum to give a low concenClin.
Chim.
Acfa, 22 (1968)
341-347
MIYAI
346
et al.
tration of 0.05 ,q/ml and phosphate buffer (pH 7.4) was used for immunoelectrophoresis in our experiment; these conditions were used to simulate physiological ‘13rI]thyroxine was bound to serum proteins under conditions except that the L conditions i~z vitro forming the precipitin arcs of antigen-antibody complex. It is unlikelythat theradioimmunoelectrophoreticpatterns were modified by the thyroxinebinding proteins contained in the horse antiserum, because a similar pattern was observed
even when the y-globulin
fraction
of antiserum
was used. If other thyroxine-
binding components with low affinity or capacity of binding to thyroxine are present in human serum, they can be demonstrated by adding larger amounts of 1311Ithyroxine to the serum, but such a pattern could not be shown in our experiment. Different patterns of radioimmunoelectrophoresis were observed when antisera from various sources were used, but no difference in pattern was observed between Japanese and occidental sera when the same antiserum was used. Variation in antibody titer of each antiserum may result in different patterns. These observations support the fact that the five radioactive arcs demonstrated not artifacts but thyroxine-binding components. II,
III
From the radioimmunoelectrophoretic patterns and V were identified as TBPA, a,-lipoprotein,
protein respectively. Since react with cr,-lipoprotein, has been examined by us. arc II and V contained the
in our experiment
arc
using specific antisera, arc 1, albumin and p- (or z,-)lipo-
the anti-p-lipoprotein serum of Behringwerke may crossthe differentiation between the two is difficult, as far as Staining with Oil red 0 or Sudan Black B revealed that lipid.
Arc IV was not detectable by radioimmunoelectrophoresis in a TRG-deficient serum. On the other hand, the decreased thyroxine-binding capacity of TBG in the serum was estimated by acetate membrane electrophoresisl”. The discrepancy in the results may still be explained by the lower separability on acetate membrane electrophoresis or the lower sensitivity of radioimmunoelectrophoresis. DPH which has been reported to displace thyroxine from TBG to other thyroxine-binding proteins6T13 also decreased the arc IV. Thus it is reasonable to assume that arc 11: represents the so-called “TBG”. Previous studies have shown that three or four thyroxine-binding components were demonstrated by radioimmunoelectrophoresis. Tllyroxine-binding to prealbumin, albumin and xc,- (or a,-)lipoprotein was found by Hollander et d.12; binding to albumin, R~- and a,-lipoproteins by Clausen and Munkner”, and to prealbumin, albumin, a,-lipoprotein and another cl-globulin by- Lightfoot and Christianl3. However, Sterling et al.16 postulated that thyroxine-binding ~-globulin was not a lipoprotein of the major lipoprotein classes, on basis of their cluantitativ,c studies using ultracentrifugation and precipitation with specific antisera. Our studies using radioimmunoelectrophoresis have shown that thyroxine was bound to al- and /3- (or cc,-)lipoproteins in addition to the three major thyroxine-binding proteins, namely prealbumin, albumin and the so-called TBG. The specificity and the physiological significance of thyroxine-binding to serum lipoproteins are unknown. Relationship between these lipoproteins and additional thyroxine-binding components which were detected by other methods *,3,~ is also still unclarified, and further studies are necessary
Clin.
Chim.
Acta,
to solve these problems.
2~ (1968)
341 -.j+i
THYROXINE-BINDING
PROTEINS
IS
347
SERUM
ACKNOWLEDGMENTS
W’e wish to express We are greatly English.
I L 3 4 5 6 7 8 9 ICI IL
indebted
our thanks
to Dr. S. Tanabe
for his valuable
to Dr. C. Ito for his help in preparing
suggestions.
this manuscript
S. H. Ixcam, ~ndocrinologv, 63 (1958) 256. 31. 11’. HAMOLSKY, D. B. FISHER AND A. S. FREEDBERG, Ewdocvinology, 66 (1960) B. S. BLUMBERG AND J. ROBBINS, Endocvimlogy, 67 (1960) 368. J, Ii. TATA, C. C. WIDNELL AND W. B. GRATZER, Clin.Chim. Acta, 6 (1961) 597. TV. E. BALFOURMVD H. E.TUNNICLIFFE,J. Physiol., 153 (1960) 179. I<. SQUEF, M.MARTINEZ.~ND J. H. OPPENHEIMER, Proc. Soc.Exptl.Bml.Med., 113 B. S. BLUMBERG, L. FBRER, J. F. RALL AND J. ROBBINS, Endocvinology, 68 (1961) S. C. THORSON, W. N. TAUXE AND H. F. TASWELL, J. Clin. Endocrinol. Metab., 26 L. S. FARER, J. ROBBINS AND B. S. BLUMBERG, E+zdocYinology, 70 (1962) 679. J. S. MARSHALL, R. 1'.LEVY, J. Clin. Endocrinol. Metab., 26 (1966) 87. J, CL.AUSEN .~XD T. MUNKNER, Proc. Sot. Exptl. Biol. Med., 104 (1960) 40.
in
780.
(1963) rj. (1966)
837. 181.
IL C. S. HOLLAXDER, B. H. LATIMER, T. E. PROUT, D. H. LOCKWOOD XXII S. P. &PER, Mctabolisw, IL (1963) 45. 13 Ii.M'. LIGHTFOOT, Jr. AND C. L. CHRISTIAN, J. Clin. Endocrinol. Metab., 26 (1966) 305. 14 H. WOSHIDA, &I.IDE, I. FUKUI, Y. TAKESHITA, T. YMAZAKI, R. ~OSHIMATSU, H. ~A~MM~T~, N. HIROT~, E. SAWASHIGE, S. YOSHIKAWA, N. MAEDA AND N. HAMANAKA, Folia Endocrinologica Japonica,
42 (1967)
1200.
15 I'.GRAB.~R AND C. A4.V:ILLIAMS, Biochim. Biophys. Acta, IO (1953) 193. 16 Ii.STERLING, h1. A. BRENNER AND H. G. ROSE, Endocrinology, 77 (1965) 398. C&L. Chim.
Addendum
Acta,
21 (1968) 341-347
to page 344, line 7
Paper chromatographic analysis of ethanol-acetone extract oftheimmunoelectrophoretic plate indicated that only about IO % of inorganic r/II was liberated from the [ *JrI]thyroxine, and no distinct radioactive areas were demonstrated when equivalent amount of [‘3r]sodium was added to the serum and analyzed by radioimmunoelectrophoresis.