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Radioimmunoassay for 3,3′,5′-triiodothyronine (reverse T3, R-T3) in unextracted human serum
497
Clinico Chimica Acta, 69 (1976) 497-504 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7845
RADIOIMMUNOASSAY FOR 3,3’,5’-TRIIODOTHYRONINE T3, R-T3) IN UNEXTRACTED HUMAN SERUM
M. HCFNER
and M. GRUSSENDORF
Medizinische Poliklinik, Universitiit Heidelberg, (Received
(REVERSE
January
Hospitalstrasse 3, 69 Heidelberg
(G.F.R.)
14, 1976)
Summary
Highly specific antibodies against 3,3’,5’-triiodothyronine (reverse T3, R-T3) have been produced in rabbits. The crossreaction with T4 is about 0.05%. A radioimmunoassay for R-T3 in unextracted serum was developed. ANS is used for blocking the binding of tracer and endogenous R-T3 to TBG. The sensitivity of the assay is 0.06 ng/ml plasma. The mean normal R-T3 concentration is 0.20 ng/ml. Thyrotoxic patients show elevated levels; in most hypothyroid patients R-T3 concentrations are below the detection limit.
Introduction
The peripheral metabolism of thyroxine (T4), especially the monodeiodination of T4 to triiodothyronine (T3) has drawn increasing interest in recent years. Chopra first showed that 3,3’,5’-triiodothyronine (R-T,) is a normal constituent of human serum, using a specific radioimmunoassay [l]. Further studies by this author revealed a dissociation of T3 and R-T3 serum concentrations under certain physiological and pathophysiological conditions [ 1,2]. These findings question the theory of a random monodeiodination of T4 in the periphery [2]. Because of the great theoretical interest of this metabolite and its possible clinical significance we have developed a simple radioimmunoassay for R-T3 in unextracted human serum. Materials and methods
A carbodiimide conjugate of R-T3 to bovine serum albumin (R-T,-BSAconjugate), 3,3’-diiodothyronine (T,) and R-T3 standard was a gift of the Henning GmbH (Berlin). 1251 for radioiodination and 3,5-diiodotyrosine were obtained from Fa. Hoechst (Frankfurt), L-T3 and L-T~ from Henning GmbH (Berlin). Double antibody solid phase (DASP) was purchased from Organon,
498
%anilino-1-naphthalene-sulfonic acid (ANS) and merthiolate from Serva, Freund’s adjuvant from Difco Laboratories (Detroit), Sephadex G-25 from Pharmacia Fine Chemicals (Uppsala). Human serum albumin (HSA) and bovine serum albumin (BSA) came from Behring Werke (Marburg). Production
of labelled R-T3
1251-labelled R-T3 was produced by radioiodination of 3,3’-T2 using the chloramine-T method of Hunter and Greenwood. 10 1.113,3’-T2 (0.5 pg), 20 ~1 0.5 M PO4 buffer pH 7.5, 10 (~1chloramine-T (10 pg) and 10 ,ul “‘1 (1.0 mCi) were incubated for 30 s. The reaction was stopped with 50 ~1 metabisulfate (250 pg). The incubation mixture was then put on a Sephadex G-25 column (1.0 cm X 18 cm) and eluted with 0.05 M phosphate, pH 12.0 [4]. Fig. 1 shows the elution pattern obtained. There are three peaks corresponding to unreacted 12’1, labelled 3,3’-T2 and labelled R-T3. Under these conditions the 5-position is not activated (Meinhold, H., personal communication), therefore labelled L-T4 which would elute very close to R-T3 is not generated. The labelled R-T3 was diluted and stored at -20°C. It could be used for at least 2 months without considerable loss of binding.
50
10000 ,125
i;
3ocao.~ \ , I
2 c
2 ”
20000,
10000.
; \
. c
1
2
ii_.-, 3
I
ii 5
6
7
8
9
10 ,I
12 13
IL 15
16 17
I8 19 20
l 22
23 2L
25 26 27
28
29 30
fraction
31
32
33
no.
Fig. 1. Elution of the incubation mixture afier iodination on Sephadex G-26 superfine with 0.05 M phosphate. pH 12.0; fraction volume 4.5 ml. The 3 peaks represent free 1251, labelled 3,3’-T 2 and labelled R-TJ, respectively.
499
Results 1. Production of antibodies against R-T3 R-T,-BSA-conjugate was dissolved in distilled water and emulsified in an equal volume of complete Freund’s adjuvant. Three rabbits were injected of 1 mg of conjugate in 3-4 weeks intervals. The injections were performed intraand subcutaneously at multiple sites. Fig. 2 shows titration curves of the 3 antisera obtained after the third immunisation. In all 3 animals antibodies could be detected. H 981 showed the highest titer and also, as demonstrated later, the best specifity. Therefore all further investigations were performed with this antiserum. 2. Characterisation of the antibody The most important requirement for an R-T3 antibody used for measure-. ment of serum samples is a very low crossreaction with L-T+ because this compound is present in more than lOO-fold higher concentration. Fig. 3 shows displacement curves of labelled R-T3 with unlabelled R-T3 and L-T~, respectively. The two curves are completely parallel in all sections. A crossreaction of the antibody with L-T~ of 0.048% is obtained in this experiment. In several assays the range was 0.04 to 0.07%. Because a contamination of the T4 by R-T3 in the same order of magnitude cannot be excluded (Dr. Scheiffele, E. (Berlin), personal communication), it remains open to question if the apparent crossreaction is really a property of the antibody or represents a contamination with R-T3.
qF
.
3.O$
I::
Ii
981
x
H 982
0
Ii 963
:
‘:‘;;&l$300
1:5600
I:11 200
1:22400
l:L4600 Serumdilution
Fig. 2. Antibody titration curves of 3 antisera obtained from rabbits after the third immunisation.
500
. 10
20
50
100
pg R-T3
L
20
50
100
Fig. 3. Displacement curves of labelled R-T3 by unlabelled R-T3 and L-T4, respectively. of crossreaction of L-T& is indicated at different points of the standard curve.
20OngTL The percentage
Therefore we did not correct serum measurements for this apparent crossreaction. The crossreaction with L-T~,3,3’,-T2 and diiodotyrosine is less than 0.05%, as shown in Table I. These values are not of practical significance. 3. Serum measurements of R-T3 The measurement of thyroid hormone in serum is complicated by its binding to serum proteins, especially TBG [5]. In recent years two main compounds have been used to block this so-called nonspecific binding, ANS [6] and merthiolate [7]. Fig. 4 demonstrates that this binding to serum proteins does also occur with R-T3, though to a lesser degree than with L-T~ and L-T~. ANS (150 pg/tube) as well as merthiolate (1 : 1000, w/v) are equally effective in blocking this binding. The following studies were performed with ANS. The incubation procedure is summarized in Table II. The standards were diluted in thyroid-hormone-free serum prepared with charcoal according to Mitsuma et al. [6]. The sensitivity of the assay was 3 pg/tube or 60 pg per ml of plasma.
TABLE
I
RELATIVE
BINDING
OF DIFFERENT
Compound
Relative
Reverse Tg L-T4 L-T3 3,3’-Diiodothyronine Diiodotyrosine
100.0 0.05 0.02 co.01
R-T3
is set as 100%.
IODOAMINOACIDS
binding to the antibody
TO R-TyANTIBODY
(%)
501 l
- buffer
O- buffer
+ ANS
x-buffer
+ merthiolote
I
* 10
20
30
40
50
60
70
60
90
100 JII plasma
Fig. 4. Increasing to the incubation
binding of labelled mixture. Inhibition
R-T3 to serum proteins by addition of increasing amounts of plasma of this binding by AN.3 (150 fig/tube) or merthiolate (1 : 1000, w/v).
To R-T3 free serum, 250, 500 and 1000 pg/ml R-T3 were added and samples were tested in 12 consecutive assays. The recovery and interassay variance is summarized in Table III. At all 3 concentrations the coefficient of variation was about 0.13. In view of the low concentration of this compound this reproducibility seems acceptable. Furthermore, to 4 normal sera 250, 500 and 1000 pg/ml R-T3 were added and subsequently tested. Table IV demonstrates the result of this experiment. At all 3 concentrations the recovery is satisfactory. Using the method described we have measured the R-T3 concentrations in
TABLE
II
ASSAY
PROCEDURE
FOR R-T3
Incubation mixtura Buffer (0.06 M phosphate + 0.9% NaCl + 0.1% R-T; (6000 cpm) Antibody (final dilution: 1 : 25 000) Standard or sample
I-ISA + 150 pg ANS
50 111 50 /ll
Total volume 1. Incubation: 20 h Temperature: 22’C Separation of bound and free: 2. Incubation: 1 h
500 /lI 50 pl
650 !.d
500 ~1 DASP(1
: 10)
502
TABLE
III
INTERASSAY
VARIANCE
To R-Ts-free serum different amounts of R-T3 were added. These sera were tested in 12 successive for determination of interassay variance and recovery. Added R-T3 (pgfml) Recovery (%) Var. Coeff. (%)
250 105 13.9
assays
1000 113 13.8
500 111 12.7
60 normal subjects, 34 thyrotoxic and 14 hypothyroid patients (Fig. 5). The mean normal concentration is 0.204 ng/ml f 0.091 with a range of 0.064.45 ng/ml. Of the 60 normal subjects only one shows an R-T3 concentration below the detection limit of 0.06 ng/ml. In thyrotoxic patients generally elevated R-T3 levels are found with some overlap with the normal range. Most hypothyroid patients show R-T3 levels below the detection limit of 0.06 ng/ml.
R-T3 nglml
A 20
.
.
I.8 .
1.6 ..
1.c . .
1.2 ..
. .** .
1.0
l* ,=-• . . l-.-.% l
ll* l
l?* . ..
.**
..1.*.**. Euthyr Fig. 5. R-T3 is indicated.
levels in euthyroid,
Hyperlhyr thyrotoxic
l
4
Hypothyr. and hypothyroid
patients.
The mean and standard
deviation
503
TABLE IV RECOVERY To 4 different normal sera different amounts of R-T3 were added and subsequently tested for recovery. ______ R-T3 found (pg/ml) Added R-T3
-
.___
0
250
500
1000
Patient H. U. s.s
220 160
G.N. LA.
140 270
+240 +200 +210 i270
+420 +440 +440 +520
+ 920 + 860 +1060 +1000
+230
+450
+ 960
92
90
(pglml):
Mean (pglml) Recovery (o/o)
96
Discussion Chopra [l] first described specific antibodies against R-T3 which he used for a radioimmunoassay for R-T3 in ethanol extracts of human serum. However, the titer of the antibodies of 1 : 250 was very low compared to the 1 : 25 000 reported here; similar potent antibodies have been obtained by Meinhold et al. [8]. The crossreactivity with L-T~ is similar in all three reports. However, it is not yet clear if the apparent crossreaction represents a contamination of the L-T4 by R-T3 or is a property of the antibody itself. ANS has been used extensively for blocking the TBG binding of thyroid hormones. As shown here it is also very useful for the reliable measurement of R-T3 in unextracted serum. Through changes of R-T3 levels in different physiological and pathophysiological conditions described by Chopra [1,2] have been confirmed by other laboratories, the absolute levels and especially the normal range is not yet established. Chopra found a mean normal range of 0.4 ng/ml. This is about double the value reported here and by Meinhold et al. [8]. One difference to Chopra’s method is the fact that he used a racemic mixture of D,L-R-T3 for immunisation whereas here pure L-R-T, was used. Other factors probably involved are the specifity of the antibodies and the purity of iodoaminoacids used. By this simple method it will be possible to make R-T3 measurements in large numbers and to get more information about the significance of this thyroid hormone metabolite. Acknowledgement This study was supported by the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Miss M. Kniipfle is gratefully ciated.
appre-
504
References 1 2 3 4 5 6
I.J. Chopra, J. Clin. Invest., 54 (1974). 583 I.J. Chopra. B.F. Crandall. N. EngI. J. Med. 293 (1975). 740 M.I. Surks. A.R. SchaIdow. J.M. Stock and J.H. Oppenheimer, J. Clin. Invest. 52 (1973). 805 K. Horn, G. HaIIhuber. T. Ruhl and P.C. Scriba, Acta Endocrinologica (Kbh.) SUPPI. 152 (1971). 85 M. Hiifner, H.D. Hesch. CUn. Chhn. Acta 44 (1973). 101 T. Mitsuma, J. Colucci. L. Shenkman and C.S. HoIIander. Biochem. Biophys. Res. Commun. 46 No. 8 (1972), 2107 7 M. Hiifner. H.D. Hesch, Acta endocrinol. 72 (1973). 464 8 H. MeInhold, P. Schiimbrand. K.W. Wenzel. Acta Endocrinol. Suppl. 199 (1975). 343
Clinico Chimica Acta, 69 (1976) 497-504 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7845
RADIOIMMUNOASSAY FOR 3,3’,5’-TRIIODOTHYRONINE T3, R-T3) IN UNEXTRACTED HUMAN SERUM
M. HCFNER
and M. GRUSSENDORF
Medizinische Poliklinik, Universitiit Heidelberg, (Received
(REVERSE
January
Hospitalstrasse 3, 69 Heidelberg
(G.F.R.)
14, 1976)
Summary
Highly specific antibodies against 3,3’,5’-triiodothyronine (reverse T3, R-T3) have been produced in rabbits. The crossreaction with T4 is about 0.05%. A radioimmunoassay for R-T3 in unextracted serum was developed. ANS is used for blocking the binding of tracer and endogenous R-T3 to TBG. The sensitivity of the assay is 0.06 ng/ml plasma. The mean normal R-T3 concentration is 0.20 ng/ml. Thyrotoxic patients show elevated levels; in most hypothyroid patients R-T3 concentrations are below the detection limit.
Introduction
The peripheral metabolism of thyroxine (T4), especially the monodeiodination of T4 to triiodothyronine (T3) has drawn increasing interest in recent years. Chopra first showed that 3,3’,5’-triiodothyronine (R-T,) is a normal constituent of human serum, using a specific radioimmunoassay [l]. Further studies by this author revealed a dissociation of T3 and R-T3 serum concentrations under certain physiological and pathophysiological conditions [ 1,2]. These findings question the theory of a random monodeiodination of T4 in the periphery [2]. Because of the great theoretical interest of this metabolite and its possible clinical significance we have developed a simple radioimmunoassay for R-T3 in unextracted human serum. Materials and methods
A carbodiimide conjugate of R-T3 to bovine serum albumin (R-T,-BSAconjugate), 3,3’-diiodothyronine (T,) and R-T3 standard was a gift of the Henning GmbH (Berlin). 1251 for radioiodination and 3,5-diiodotyrosine were obtained from Fa. Hoechst (Frankfurt), L-T3 and L-T~ from Henning GmbH (Berlin). Double antibody solid phase (DASP) was purchased from Organon,
498
%anilino-1-naphthalene-sulfonic acid (ANS) and merthiolate from Serva, Freund’s adjuvant from Difco Laboratories (Detroit), Sephadex G-25 from Pharmacia Fine Chemicals (Uppsala). Human serum albumin (HSA) and bovine serum albumin (BSA) came from Behring Werke (Marburg). Production
of labelled R-T3
1251-labelled R-T3 was produced by radioiodination of 3,3’-T2 using the chloramine-T method of Hunter and Greenwood. 10 1.113,3’-T2 (0.5 pg), 20 ~1 0.5 M PO4 buffer pH 7.5, 10 (~1chloramine-T (10 pg) and 10 ,ul “‘1 (1.0 mCi) were incubated for 30 s. The reaction was stopped with 50 ~1 metabisulfate (250 pg). The incubation mixture was then put on a Sephadex G-25 column (1.0 cm X 18 cm) and eluted with 0.05 M phosphate, pH 12.0 [4]. Fig. 1 shows the elution pattern obtained. There are three peaks corresponding to unreacted 12’1, labelled 3,3’-T2 and labelled R-T3. Under these conditions the 5-position is not activated (Meinhold, H., personal communication), therefore labelled L-T4 which would elute very close to R-T3 is not generated. The labelled R-T3 was diluted and stored at -20°C. It could be used for at least 2 months without considerable loss of binding.
50
10000 ,125
i;
3ocao.~ \ , I
2 c
2 ”
20000,
10000.
; \
. c
1
2
ii_.-, 3
I
ii 5
6
7
8
9
10 ,I
12 13
IL 15
16 17
I8 19 20
l 22
23 2L
25 26 27
28
29 30
fraction
31
32
33
no.
Fig. 1. Elution of the incubation mixture afier iodination on Sephadex G-26 superfine with 0.05 M phosphate. pH 12.0; fraction volume 4.5 ml. The 3 peaks represent free 1251, labelled 3,3’-T 2 and labelled R-TJ, respectively.
499
Results 1. Production of antibodies against R-T3 R-T,-BSA-conjugate was dissolved in distilled water and emulsified in an equal volume of complete Freund’s adjuvant. Three rabbits were injected of 1 mg of conjugate in 3-4 weeks intervals. The injections were performed intraand subcutaneously at multiple sites. Fig. 2 shows titration curves of the 3 antisera obtained after the third immunisation. In all 3 animals antibodies could be detected. H 981 showed the highest titer and also, as demonstrated later, the best specifity. Therefore all further investigations were performed with this antiserum. 2. Characterisation of the antibody The most important requirement for an R-T3 antibody used for measure-. ment of serum samples is a very low crossreaction with L-T+ because this compound is present in more than lOO-fold higher concentration. Fig. 3 shows displacement curves of labelled R-T3 with unlabelled R-T3 and L-T~, respectively. The two curves are completely parallel in all sections. A crossreaction of the antibody with L-T~ of 0.048% is obtained in this experiment. In several assays the range was 0.04 to 0.07%. Because a contamination of the T4 by R-T3 in the same order of magnitude cannot be excluded (Dr. Scheiffele, E. (Berlin), personal communication), it remains open to question if the apparent crossreaction is really a property of the antibody or represents a contamination with R-T3.
qF
.
3.O$
I::
Ii
981
x
H 982
0
Ii 963
:
‘:‘;;&l$300
1:5600
I:11 200
1:22400
l:L4600 Serumdilution
Fig. 2. Antibody titration curves of 3 antisera obtained from rabbits after the third immunisation.
500
. 10
20
50
100
pg R-T3
L
20
50
100
Fig. 3. Displacement curves of labelled R-T3 by unlabelled R-T3 and L-T4, respectively. of crossreaction of L-T& is indicated at different points of the standard curve.
20OngTL The percentage
Therefore we did not correct serum measurements for this apparent crossreaction. The crossreaction with L-T~,3,3’,-T2 and diiodotyrosine is less than 0.05%, as shown in Table I. These values are not of practical significance. 3. Serum measurements of R-T3 The measurement of thyroid hormone in serum is complicated by its binding to serum proteins, especially TBG [5]. In recent years two main compounds have been used to block this so-called nonspecific binding, ANS [6] and merthiolate [7]. Fig. 4 demonstrates that this binding to serum proteins does also occur with R-T3, though to a lesser degree than with L-T~ and L-T~. ANS (150 pg/tube) as well as merthiolate (1 : 1000, w/v) are equally effective in blocking this binding. The following studies were performed with ANS. The incubation procedure is summarized in Table II. The standards were diluted in thyroid-hormone-free serum prepared with charcoal according to Mitsuma et al. [6]. The sensitivity of the assay was 3 pg/tube or 60 pg per ml of plasma.
TABLE
I
RELATIVE
BINDING
OF DIFFERENT
Compound
Relative
Reverse Tg L-T4 L-T3 3,3’-Diiodothyronine Diiodotyrosine
100.0 0.05 0.02 co.01
R-T3
is set as 100%.
IODOAMINOACIDS
binding to the antibody
TO R-TyANTIBODY
(%)
501 l
- buffer
O- buffer
+ ANS
x-buffer
+ merthiolote
I
* 10
20
30
40
50
60
70
60
90
100 JII plasma
Fig. 4. Increasing to the incubation
binding of labelled mixture. Inhibition
R-T3 to serum proteins by addition of increasing amounts of plasma of this binding by AN.3 (150 fig/tube) or merthiolate (1 : 1000, w/v).
To R-T3 free serum, 250, 500 and 1000 pg/ml R-T3 were added and samples were tested in 12 consecutive assays. The recovery and interassay variance is summarized in Table III. At all 3 concentrations the coefficient of variation was about 0.13. In view of the low concentration of this compound this reproducibility seems acceptable. Furthermore, to 4 normal sera 250, 500 and 1000 pg/ml R-T3 were added and subsequently tested. Table IV demonstrates the result of this experiment. At all 3 concentrations the recovery is satisfactory. Using the method described we have measured the R-T3 concentrations in
TABLE
II
ASSAY
PROCEDURE
FOR R-T3
Incubation mixtura Buffer (0.06 M phosphate + 0.9% NaCl + 0.1% R-T; (6000 cpm) Antibody (final dilution: 1 : 25 000) Standard or sample
I-ISA + 150 pg ANS
50 111 50 /ll
Total volume 1. Incubation: 20 h Temperature: 22’C Separation of bound and free: 2. Incubation: 1 h
500 /lI 50 pl
650 !.d
500 ~1 DASP(1
: 10)
502
TABLE
III
INTERASSAY
VARIANCE
To R-Ts-free serum different amounts of R-T3 were added. These sera were tested in 12 successive for determination of interassay variance and recovery. Added R-T3 (pgfml) Recovery (%) Var. Coeff. (%)
250 105 13.9
assays
1000 113 13.8
500 111 12.7
60 normal subjects, 34 thyrotoxic and 14 hypothyroid patients (Fig. 5). The mean normal concentration is 0.204 ng/ml f 0.091 with a range of 0.064.45 ng/ml. Of the 60 normal subjects only one shows an R-T3 concentration below the detection limit of 0.06 ng/ml. In thyrotoxic patients generally elevated R-T3 levels are found with some overlap with the normal range. Most hypothyroid patients show R-T3 levels below the detection limit of 0.06 ng/ml.
R-T3 nglml
A 20
.
.
I.8 .
1.6 ..
1.c . .
1.2 ..
. .** .
1.0
l* ,=-• . . l-.-.% l
ll* l
l?* . ..
.**
..1.*.**. Euthyr Fig. 5. R-T3 is indicated.
levels in euthyroid,
Hyperlhyr thyrotoxic
l
4
Hypothyr. and hypothyroid
patients.
The mean and standard
deviation
503
TABLE IV RECOVERY To 4 different normal sera different amounts of R-T3 were added and subsequently tested for recovery. ______ R-T3 found (pg/ml) Added R-T3
-
.___
0
250
500
1000
Patient H. U. s.s
220 160
G.N. LA.
140 270
+240 +200 +210 i270
+420 +440 +440 +520
+ 920 + 860 +1060 +1000
+230
+450
+ 960
92
90
(pglml):
Mean (pglml) Recovery (o/o)
96
Discussion Chopra [l] first described specific antibodies against R-T3 which he used for a radioimmunoassay for R-T3 in ethanol extracts of human serum. However, the titer of the antibodies of 1 : 250 was very low compared to the 1 : 25 000 reported here; similar potent antibodies have been obtained by Meinhold et al. [8]. The crossreactivity with L-T~ is similar in all three reports. However, it is not yet clear if the apparent crossreaction represents a contamination of the L-T4 by R-T3 or is a property of the antibody itself. ANS has been used extensively for blocking the TBG binding of thyroid hormones. As shown here it is also very useful for the reliable measurement of R-T3 in unextracted serum. Through changes of R-T3 levels in different physiological and pathophysiological conditions described by Chopra [1,2] have been confirmed by other laboratories, the absolute levels and especially the normal range is not yet established. Chopra found a mean normal range of 0.4 ng/ml. This is about double the value reported here and by Meinhold et al. [8]. One difference to Chopra’s method is the fact that he used a racemic mixture of D,L-R-T3 for immunisation whereas here pure L-R-T, was used. Other factors probably involved are the specifity of the antibodies and the purity of iodoaminoacids used. By this simple method it will be possible to make R-T3 measurements in large numbers and to get more information about the significance of this thyroid hormone metabolite. Acknowledgement This study was supported by the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Miss M. Kniipfle is gratefully ciated.
appre-
504
References 1 2 3 4 5 6
I.J. Chopra, J. Clin. Invest., 54 (1974). 583 I.J. Chopra. B.F. Crandall. N. EngI. J. Med. 293 (1975). 740 M.I. Surks. A.R. SchaIdow. J.M. Stock and J.H. Oppenheimer, J. Clin. Invest. 52 (1973). 805 K. Horn, G. HaIIhuber. T. Ruhl and P.C. Scriba, Acta Endocrinologica (Kbh.) SUPPI. 152 (1971). 85 M. Hiifner, H.D. Hesch. CUn. Chhn. Acta 44 (1973). 101 T. Mitsuma, J. Colucci. L. Shenkman and C.S. HoIIander. Biochem. Biophys. Res. Commun. 46 No. 8 (1972), 2107 7 M. Hiifner. H.D. Hesch, Acta endocrinol. 72 (1973). 464 8 H. MeInhold, P. Schiimbrand. K.W. Wenzel. Acta Endocrinol. Suppl. 199 (1975). 343