373
Clinica Chimica Acta, 87 (1978) 373-381 0 Elsevier/North-Holland Biomedical Press
CCA 9484
CORRELATION OF FREE THYROXINE INDEX AND THYROXINE : THYROXINE-BINDING GLOBULIN RATIO WITH THE FREE THYROXINE CONCENTRATION AS MEASURED BY THE THYROXINE AND THYROXINE-BINDING GLOBULIN RADIOIMMUNOASSAYS
MICHEL LECUREUIL a**, GtiRARD and CHRISTIAN CHOFFEL a
CROUZAT-REYNES
a, JEAN-CLAUDE
BESNARD
b
a Laboratoire de Physique et Biophysique pharmaceutique, Faculte’ des Sciences Pharmaceutiques, Pbis boulevard TonnellC, 37032 Tours Chdex (France) and b Laboratoire de Biophysique mkdicale, Facultk de Mbdecine, 2bis boulevard Tonne114 3 7032 Tours Ce’dex (France) (Received
February
8th, 1978)
Summary The concentration of thyroxine-binding globulin in the serum can now be measured by a simple and specific radioimmunoassay. Triiodothyronine uptake and measurement of total thyroxine have been combined to yield a free thyroxine index which has been found to correlate with the clinical state of the patients. An estimate of the free thyroxine concentration, as measured by the thyroxine and thyroxine-binding globulin radioimmunoassays, provided a good correlation with the free thyroxine index and the thyroxine : thyroxine: thyroxine-binding globulin binding globulin ratio. However, the thyroxine ratio is inaccurate when thyroxine-binding globulin concentrations are high or low.
Introduction The major fraction of circulating thyroid hormones, thyroxine (T4) and triiodothyronine (T3) are bound to plasma proteins. The most important binding protein is thyroxine-binding globulin (TBG). Most of the remaining T4 and T3 is transported by thyroxine-binding prealbumin (TBPA) and albumin so that approximately 0.05% of the T4 is not protein-bound but free; measurement of this free thyroxine concentration is unsuitable for routine use. However, the
* To whom all correspondence should be addressed.
374
concentration of circulating TBG is known to fluctuate under the influence of hormones, drugs, diseases and genetic factors [ 121. Some estimate of TBG concentration is therefore necessary for the correct interpretation of serum T4 values; this has led to the development of indirect assays of TBG such as the triiodothyronine uptake tests. These tests assess the number of unoccupied T4-binding sites in a sample by partitioning radiolabelled T3 between the serum sample and a solid phase. T3 uptake tests have been combined with measurements of total T4 to yield a free thyroxine index (FTI calculated as T4 X T3 uptake) which has been found to correlate with the clinical state of the patients. Direct assays of TBG concentration, such as radioimmunoassays, have been described and commercial kits are now available. We recently described a highly specific and precise radioimmunoassay for TBG [ 31. In this study we calculated the concentration of free thyroxine from T4 and TBG radioimmunoassay values and examined the relation between total T4 and TBG concentrations in healthy euthyroid individuals, women taking oral contraceptives and patients with myxoedema and thyrotoxicosis; in addition we calculated the relation between FTI, T4 : TBG ratio and free thyroxine concentration. Lastly we compared the ability of FTI, T4 : TBG ratio and free thyroxine concentration to differentiate between thyrotoxic, myxoedematous and euthyroid states. Patients and methods Total serum thyroxine and TBG were measured in 335 patients. The following groups were studied: 195 healthy euthyroid adults, 24 women taking oral contraceptives, 66 patients with clinically and biochemically proven thyrotoxicosis and 50 patients with myxoedema. Serum TBG was measured by a radioimmunoassay method. TBG purified by a method described elsewhere [4] was used for immunization, radioiodination and standardization. The simple antibody technique was used in the assay. The separation of bound and unbound antigen was carried out in polystyrene test tubes whose walls were coated with antibody [3] or with antibody bound to activated cellulose (Commercial kit-TBGK-CIS). Serum thyroxine was measured by a radioimmunoassay method (Tetra-Tab RIA, Nuclear Medical Laboratories) and T3 uptake by a commercial kit method (T&Tab, Nuclear Medical Laboratories). FTI, T4 : TBG ratio and free thyroxine concentration F were calculated for each sample. The free thyroxine concentration F was calculated from equation 1 derived from the law of mass action [ 51: Fk,(l =0
+ .pzk,Cpz + rz3k3Cp3)+ F(nlklCpl + nzkzCpz + n3k&3
+ 1 - k,TJ) - T4 (1)
nl, n2, n3 are the number of binding sites with association constants kl, k2, k3 on the three proteins, TBG, TBPA and albumin. CpI, Cp2, Cp3 are the respective molar concentrations of these proteins.
375
All the calculations for which the albumin and TBPA concentrations have constant values (6.4 X 10d4 M for albumin, 3.57 X 10m6 M for TBPA) were performed by taking the number of binding sites and the association constants, respectively, equal to n, = 1, k, = 0.4 X 10” M-l, n2 = 1, k2 = 1.7 X 10’ M-l, n3 = 2, k3 = 0.375 X lo6 M-l for TBG, TBPA and albumin. Results Fig. 1 shows the relation between serum thyroxine concentration and TBG in euthyroid, thyrotoxic and myxoedematous subjects. In the euthryoid subjects a nearly linear correlation (r = 0.61) was obtained between the two values; no correlation was observed in thyrotoxic and myxoedematous patients. Fig. 2 illustrates the relation between FTI and F with respect to all the subjects studied. This relation was not linear, but a good correlation was found between the two values. A curve was found to fit the data better than a straight
l
Euthyroid
0 Contraceptive
user5
D Myxoedema o Thyrotoxlc
10
20 TBG
30 Concentration
Img/l
40
)
Fig. 1. Correlation between T4 and TBG concentrations in 24 women taking oral contraceptives, 165 healthy euthyroid adults, 50 Patients with myxoedema and 66 thyrotoxic subjects. The straight fie i&icates the correlation in euthyroid subjects.
Fig. 2. Correlation between FTI and F with respect to all the subjects studied. The curve indicates the line best fit to the observed data in the myxoedematous and euthyroid individuals and oral contraceptive users.
4 0
0. 0
0.
.P
5 0.
iz I-
2
0
/
1
2
6
4 Free
T4
concentrat,on
8 , x 10”
M
10
1
Fig. 3. Correlation between T4 : TBG ratio and F. The straight line indicates the relation between these two values in the myxoedematous. euthyroid and thyrotoxic individuals. Euthyroid women taking oral contraceptives are indicated by open circles.
line; however if one takes into account myxoedematous individuals, euthyroid individuals and euthyroid oral contraceptive users, a straight line shows a relatively good correlation between FTI and F since the equation for the curve over this range is: FTI = 0.48 X (10” F)‘.06 (r = 0.98) It should be noted that the values were in good agreement with those Fig. 3 illustrates the significant With reference to the hyperthyroid, a good linear correlation (r = 0.97)
of FTI in euthyroid oral contraceptive users of F. correlation between T4 : TBG ratio and F. euthyroid and the hypothyroid individuals, is obtained, the equation for this line being:
= 0.067 X 10” F
&
It is important,
in retrospect,
to take note of the fact that all the results for the
+ I.““...
BX3tt: I...“... 6
.
,
I.
6
. .
tY 4
.. .. tt’ 2
i :. &:*.
‘is”.. ,p* ‘”
-
Fig. 4. FTI values in healthy euthyroid persons and patients with thyroid disorders.
378
euthyroid individuals taking oral contraceptives were situated below the above mentioned straight line. Fig. 4 shows the FTI values in oral contraceptive users, and thyrotoxic, myxoedematous and euthyroid subjects. The T4 : TBG ratio (Fig. 5) differentiated relatively well between thyrotoxic, myxoedematous and euthyroid subjects with normal TBG concentration; although 23 of the women taking oral contraceptives were within 2 SD of the normal mean the T4 : TBG ratio gave results in the “myxoedematous-euthyroid” range and the mean value was significantly lower than normal. The free thyroxine concentration F (Fig. 6) appeared to have made a very good distinction between euthyroid subjects with normal TBG concentration and those with thyrotoxicosis and myxoedema. In euthyroid patients with raised TBG concentration F gave values in the euthyroid range with the same mean value.
Fig. 5. ~~ : TBG ratio in healthy euthyroid persons and patients with thyroid disorders.
379
R
Fig. 6. Free thyroxine concentration
F in healthy euthyroid persons and patients with thyroid disorders.
Discussion Direct measurement of serum TBG will probably soon be available as a routine diagnostic aid. The radioimmunoassays we used are simple, precise and specific and may be applied to large numbers of samples. The normal values for serum TBG we found are comparable with those reported by some investigators [6-g], although higher or lower values have been suggested by others [ 10-161. Direct comparisons are difficult owing to the lack of an international standard for TBG. A respectable linear correlation was found in euthyroid subjects between total T4 and TBG concentrations, but no correlation was obtained in thyrotoxic and myxoedematous individuals. This fact can be explained by the fundamental relationship between total T4 and TBG concentrations obtained by considering the contribution of the three binding proteins. Equation 1 may be rewritten as:
T4 = CPI
____ 1 + k,F
F + (n2k2Cp2 + n3k3CP3 + 1) F
380
The expressions (n,k,/l + klF)F and (nzkzCpz + n3k3Cp3 + l)F remain relatively steady in euthyroid subjects because free T4 values have a slight variation, so that a straight line could be found to accurately fit the data. This approach is no longer applicable in thyrotoxic and myxoedematous subjects because the free T4 range is much more important than in euthyroid subjects. This relationship also suggests that serum TBG is not the only major determinant of total T4 concentration in euthyroid subjects and that the contribution of other binding proteins must be considered. The linear correlation between T 4 : TBG ratio and F in all subjects studied can be explained by the relationship between total T4 and TBG concentrations (Equation 2) rewritten as: T4 TBG
nlkl ( 1 +k,F
TBG
-F
1
The sum of the two expressions niki/(l + klF) and (n2k2Cp2 + n3k3Cp3 + l)/ TBG remained nearly constant because the first one decreased as the other increased when myxoedematous, euthyroid and thyrotoxic subjects were successively studied. This was due to the fact that the increase of TBG values in myxoedema balanced the decrease of free T4 concentration and that the decrease of TBG values in thyrotoxicosis balanced the increase of free T4 concentration. The free thyroxine concentration in euthyroid oral contraceptive users was found to be in the euthyroid range, this being in good agreement with most reports. In this case, because the TBG concentration has clearly increased, the preceding relationship shows that the T4 : TBG ratio has decreased, which theoretically justifies the results obtained in Fig. 3. This relationship shows that in euthyroid patients with elevated TBG concentrations the T 4 : TBG ratio gave values in the “myxoedematous-euthyroid” or myxoedematous range because the free T4 concentration was in the euthyroid range. It also shows that in clinically euthyroid patients with reduced TBG concentrations, the T4 .* TBG ratio was increased and situated in the thyrotoxic range. Lastly, the use of T4 : TBG ratio is no longer valid in those individuals possessing either no or insufficiently measurable quantities of TBG. All of these data demonstrate that the free thyroxine concentration is one of the best indices for differentiating between euthyroid subjects with normal, high or low TBG concentrations and those with thyrotoxicosis and myxoedema. References 1 Inada. M. and Sterling, K. (1967) J. Clin. Invest. 46,1442-1450 2 Marshall. J.S., Levy. R.P. and Steinberg, A.G. (1966) New Eng. J. Med. 274, 1469-1473 3 Crouzat-Reynes, G.. Lecureuil, M.. Besnard, J.C. and Cboffel. C. (1977) C. R. Acad. Sci. 285,11751178 4 Crouzat-Reynes, G., Lecureuil. M.. Babillot, P. and Lecureuil, N. (1977) Brevet Francais No. 77 36668 5 Lecureuil, M., Crouzat-Reynes. G., Besnard. J.C. and Choffel. C. (1978) C. R. Acad. Sci. 286, 81-83 6 Rudorff, V.K.H., Herrmann, J., Kroll. H.J. and Kruskemper. H.L. (1976) Clin. Chem. Clin. Biochem. 14.3136
381 7 Cavalieri.
R.R..
McMahon.
F.A. and Castle, J.N. (1975)
J. Clin. Invest. 56, 79-87
8 Gershengom, M.C., Larsen. P.R. and Robbins, J. (1976) J. Clin. Endocrinol. Metab. 9 Horn, K., Kubiczek, Th. and Pickardt, C.R. (1977) Acta Endocrinol. 84, 111-112 10
Levy, R.P.. Marshall, J.S. and Velayo,
N.L. (1971)
J. Clin. Endocrinol.
Metab.
42, 907-911
32, 372-381
11 Bastomsky. C.H.. Kalloo, H. and Frenkel-Leith. D.B. (1977) Clin. Chim. Acta 74. 5167 12 Hesch, R.D., Gatz, J., McIntosh, C.H.S. and Janzen. J. (1976) Clin. Chim. Acta 70, 3342 13 Burr. W.A.. Ramsden, D.B., Evans, S.E., Hogan, T. and Hoffenberg, R. (1977) Br. Med. J. 1, 485488 14 Bradwell. A.R., Burnett, D., Ramsden, D.B., Burr, W.A.. Prince, H.P. and Hoffenberg, R. (1976) Clin. Chim. Acta 71, 501-510 15 Chopra, I.J., Solomon, D.H. and Ho, R.S. (1972) J. Clin. Endocrinol. Metab. 35, 565-573 16 Lecureuil, M.. Lecureuil. N. and Crouzat-Reynes, G. (1977) Lyon Pharmaceutique 28, 223-237
Clinica Chimica Acta, 87 (1978) 373-381 0 Elsevier/North-Holland Biomedical Press
CCA 9484
CORRELATION OF FREE THYROXINE INDEX AND THYROXINE : THYROXINE-BINDING GLOBULIN RATIO WITH THE FREE THYROXINE CONCENTRATION AS MEASURED BY THE THYROXINE AND THYROXINE-BINDING GLOBULIN RADIOIMMUNOASSAYS
MICHEL LECUREUIL a**, GtiRARD and CHRISTIAN CHOFFEL a
CROUZAT-REYNES
a, JEAN-CLAUDE
BESNARD
b
a Laboratoire de Physique et Biophysique pharmaceutique, Faculte’ des Sciences Pharmaceutiques, Pbis boulevard TonnellC, 37032 Tours Chdex (France) and b Laboratoire de Biophysique mkdicale, Facultk de Mbdecine, 2bis boulevard Tonne114 3 7032 Tours Ce’dex (France) (Received
February
8th, 1978)
Summary The concentration of thyroxine-binding globulin in the serum can now be measured by a simple and specific radioimmunoassay. Triiodothyronine uptake and measurement of total thyroxine have been combined to yield a free thyroxine index which has been found to correlate with the clinical state of the patients. An estimate of the free thyroxine concentration, as measured by the thyroxine and thyroxine-binding globulin radioimmunoassays, provided a good correlation with the free thyroxine index and the thyroxine : thyroxine: thyroxine-binding globulin binding globulin ratio. However, the thyroxine ratio is inaccurate when thyroxine-binding globulin concentrations are high or low.
Introduction The major fraction of circulating thyroid hormones, thyroxine (T4) and triiodothyronine (T3) are bound to plasma proteins. The most important binding protein is thyroxine-binding globulin (TBG). Most of the remaining T4 and T3 is transported by thyroxine-binding prealbumin (TBPA) and albumin so that approximately 0.05% of the T4 is not protein-bound but free; measurement of this free thyroxine concentration is unsuitable for routine use. However, the
* To whom all correspondence should be addressed.
374
concentration of circulating TBG is known to fluctuate under the influence of hormones, drugs, diseases and genetic factors [ 121. Some estimate of TBG concentration is therefore necessary for the correct interpretation of serum T4 values; this has led to the development of indirect assays of TBG such as the triiodothyronine uptake tests. These tests assess the number of unoccupied T4-binding sites in a sample by partitioning radiolabelled T3 between the serum sample and a solid phase. T3 uptake tests have been combined with measurements of total T4 to yield a free thyroxine index (FTI calculated as T4 X T3 uptake) which has been found to correlate with the clinical state of the patients. Direct assays of TBG concentration, such as radioimmunoassays, have been described and commercial kits are now available. We recently described a highly specific and precise radioimmunoassay for TBG [ 31. In this study we calculated the concentration of free thyroxine from T4 and TBG radioimmunoassay values and examined the relation between total T4 and TBG concentrations in healthy euthyroid individuals, women taking oral contraceptives and patients with myxoedema and thyrotoxicosis; in addition we calculated the relation between FTI, T4 : TBG ratio and free thyroxine concentration. Lastly we compared the ability of FTI, T4 : TBG ratio and free thyroxine concentration to differentiate between thyrotoxic, myxoedematous and euthyroid states. Patients and methods Total serum thyroxine and TBG were measured in 335 patients. The following groups were studied: 195 healthy euthyroid adults, 24 women taking oral contraceptives, 66 patients with clinically and biochemically proven thyrotoxicosis and 50 patients with myxoedema. Serum TBG was measured by a radioimmunoassay method. TBG purified by a method described elsewhere [4] was used for immunization, radioiodination and standardization. The simple antibody technique was used in the assay. The separation of bound and unbound antigen was carried out in polystyrene test tubes whose walls were coated with antibody [3] or with antibody bound to activated cellulose (Commercial kit-TBGK-CIS). Serum thyroxine was measured by a radioimmunoassay method (Tetra-Tab RIA, Nuclear Medical Laboratories) and T3 uptake by a commercial kit method (T&Tab, Nuclear Medical Laboratories). FTI, T4 : TBG ratio and free thyroxine concentration F were calculated for each sample. The free thyroxine concentration F was calculated from equation 1 derived from the law of mass action [ 51: Fk,(l =0
+ .pzk,Cpz + rz3k3Cp3)+ F(nlklCpl + nzkzCpz + n3k&3
+ 1 - k,TJ) - T4 (1)
nl, n2, n3 are the number of binding sites with association constants kl, k2, k3 on the three proteins, TBG, TBPA and albumin. CpI, Cp2, Cp3 are the respective molar concentrations of these proteins.
375
All the calculations for which the albumin and TBPA concentrations have constant values (6.4 X 10d4 M for albumin, 3.57 X 10m6 M for TBPA) were performed by taking the number of binding sites and the association constants, respectively, equal to n, = 1, k, = 0.4 X 10” M-l, n2 = 1, k2 = 1.7 X 10’ M-l, n3 = 2, k3 = 0.375 X lo6 M-l for TBG, TBPA and albumin. Results Fig. 1 shows the relation between serum thyroxine concentration and TBG in euthyroid, thyrotoxic and myxoedematous subjects. In the euthryoid subjects a nearly linear correlation (r = 0.61) was obtained between the two values; no correlation was observed in thyrotoxic and myxoedematous patients. Fig. 2 illustrates the relation between FTI and F with respect to all the subjects studied. This relation was not linear, but a good correlation was found between the two values. A curve was found to fit the data better than a straight
l
Euthyroid
0 Contraceptive
user5
D Myxoedema o Thyrotoxlc
10
20 TBG
30 Concentration
Img/l
40
)
Fig. 1. Correlation between T4 and TBG concentrations in 24 women taking oral contraceptives, 165 healthy euthyroid adults, 50 Patients with myxoedema and 66 thyrotoxic subjects. The straight fie i&icates the correlation in euthyroid subjects.
Fig. 2. Correlation between FTI and F with respect to all the subjects studied. The curve indicates the line best fit to the observed data in the myxoedematous and euthyroid individuals and oral contraceptive users.
4 0
0. 0
0.
.P
5 0.
iz I-
2
0
/
1
2
6
4 Free
T4
concentrat,on
8 , x 10”
M
10
1
Fig. 3. Correlation between T4 : TBG ratio and F. The straight line indicates the relation between these two values in the myxoedematous. euthyroid and thyrotoxic individuals. Euthyroid women taking oral contraceptives are indicated by open circles.
line; however if one takes into account myxoedematous individuals, euthyroid individuals and euthyroid oral contraceptive users, a straight line shows a relatively good correlation between FTI and F since the equation for the curve over this range is: FTI = 0.48 X (10” F)‘.06 (r = 0.98) It should be noted that the values were in good agreement with those Fig. 3 illustrates the significant With reference to the hyperthyroid, a good linear correlation (r = 0.97)
of FTI in euthyroid oral contraceptive users of F. correlation between T4 : TBG ratio and F. euthyroid and the hypothyroid individuals, is obtained, the equation for this line being:
= 0.067 X 10” F
&
It is important,
in retrospect,
to take note of the fact that all the results for the
+ I.““...
BX3tt: I...“... 6
.
,
I.
6
. .
tY 4
.. .. tt’ 2
i :. &:*.
‘is”.. ,p* ‘”
-
Fig. 4. FTI values in healthy euthyroid persons and patients with thyroid disorders.
378
euthyroid individuals taking oral contraceptives were situated below the above mentioned straight line. Fig. 4 shows the FTI values in oral contraceptive users, and thyrotoxic, myxoedematous and euthyroid subjects. The T4 : TBG ratio (Fig. 5) differentiated relatively well between thyrotoxic, myxoedematous and euthyroid subjects with normal TBG concentration; although 23 of the women taking oral contraceptives were within 2 SD of the normal mean the T4 : TBG ratio gave results in the “myxoedematous-euthyroid” range and the mean value was significantly lower than normal. The free thyroxine concentration F (Fig. 6) appeared to have made a very good distinction between euthyroid subjects with normal TBG concentration and those with thyrotoxicosis and myxoedema. In euthyroid patients with raised TBG concentration F gave values in the euthyroid range with the same mean value.
Fig. 5. ~~ : TBG ratio in healthy euthyroid persons and patients with thyroid disorders.
379
R
Fig. 6. Free thyroxine concentration
F in healthy euthyroid persons and patients with thyroid disorders.
Discussion Direct measurement of serum TBG will probably soon be available as a routine diagnostic aid. The radioimmunoassays we used are simple, precise and specific and may be applied to large numbers of samples. The normal values for serum TBG we found are comparable with those reported by some investigators [6-g], although higher or lower values have been suggested by others [ 10-161. Direct comparisons are difficult owing to the lack of an international standard for TBG. A respectable linear correlation was found in euthyroid subjects between total T4 and TBG concentrations, but no correlation was obtained in thyrotoxic and myxoedematous individuals. This fact can be explained by the fundamental relationship between total T4 and TBG concentrations obtained by considering the contribution of the three binding proteins. Equation 1 may be rewritten as:
T4 = CPI
____ 1 + k,F
F + (n2k2Cp2 + n3k3CP3 + 1) F
380
The expressions (n,k,/l + klF)F and (nzkzCpz + n3k3Cp3 + l)F remain relatively steady in euthyroid subjects because free T4 values have a slight variation, so that a straight line could be found to accurately fit the data. This approach is no longer applicable in thyrotoxic and myxoedematous subjects because the free T4 range is much more important than in euthyroid subjects. This relationship also suggests that serum TBG is not the only major determinant of total T4 concentration in euthyroid subjects and that the contribution of other binding proteins must be considered. The linear correlation between T 4 : TBG ratio and F in all subjects studied can be explained by the relationship between total T4 and TBG concentrations (Equation 2) rewritten as: T4 TBG
nlkl ( 1 +k,F
TBG
-F
1
The sum of the two expressions niki/(l + klF) and (n2k2Cp2 + n3k3Cp3 + l)/ TBG remained nearly constant because the first one decreased as the other increased when myxoedematous, euthyroid and thyrotoxic subjects were successively studied. This was due to the fact that the increase of TBG values in myxoedema balanced the decrease of free T4 concentration and that the decrease of TBG values in thyrotoxicosis balanced the increase of free T4 concentration. The free thyroxine concentration in euthyroid oral contraceptive users was found to be in the euthyroid range, this being in good agreement with most reports. In this case, because the TBG concentration has clearly increased, the preceding relationship shows that the T4 : TBG ratio has decreased, which theoretically justifies the results obtained in Fig. 3. This relationship shows that in euthyroid patients with elevated TBG concentrations the T 4 : TBG ratio gave values in the “myxoedematous-euthyroid” or myxoedematous range because the free T4 concentration was in the euthyroid range. It also shows that in clinically euthyroid patients with reduced TBG concentrations, the T4 .* TBG ratio was increased and situated in the thyrotoxic range. Lastly, the use of T4 : TBG ratio is no longer valid in those individuals possessing either no or insufficiently measurable quantities of TBG. All of these data demonstrate that the free thyroxine concentration is one of the best indices for differentiating between euthyroid subjects with normal, high or low TBG concentrations and those with thyrotoxicosis and myxoedema. References 1 Inada. M. and Sterling, K. (1967) J. Clin. Invest. 46,1442-1450 2 Marshall. J.S., Levy. R.P. and Steinberg, A.G. (1966) New Eng. J. Med. 274, 1469-1473 3 Crouzat-Reynes, G.. Lecureuil, M.. Besnard, J.C. and Cboffel. C. (1977) C. R. Acad. Sci. 285,11751178 4 Crouzat-Reynes, G., Lecureuil. M.. Babillot, P. and Lecureuil, N. (1977) Brevet Francais No. 77 36668 5 Lecureuil, M., Crouzat-Reynes. G., Besnard. J.C. and Choffel. C. (1978) C. R. Acad. Sci. 286, 81-83 6 Rudorff, V.K.H., Herrmann, J., Kroll. H.J. and Kruskemper. H.L. (1976) Clin. Chem. Clin. Biochem. 14.3136
381 7 Cavalieri.
R.R..
McMahon.
F.A. and Castle, J.N. (1975)
J. Clin. Invest. 56, 79-87
8 Gershengom, M.C., Larsen. P.R. and Robbins, J. (1976) J. Clin. Endocrinol. Metab. 9 Horn, K., Kubiczek, Th. and Pickardt, C.R. (1977) Acta Endocrinol. 84, 111-112 10
Levy, R.P.. Marshall, J.S. and Velayo,
N.L. (1971)
J. Clin. Endocrinol.
Metab.
42, 907-911
32, 372-381
11 Bastomsky. C.H.. Kalloo, H. and Frenkel-Leith. D.B. (1977) Clin. Chim. Acta 74. 5167 12 Hesch, R.D., Gatz, J., McIntosh, C.H.S. and Janzen. J. (1976) Clin. Chim. Acta 70, 3342 13 Burr. W.A.. Ramsden, D.B., Evans, S.E., Hogan, T. and Hoffenberg, R. (1977) Br. Med. J. 1, 485488 14 Bradwell. A.R., Burnett, D., Ramsden, D.B., Burr, W.A.. Prince, H.P. and Hoffenberg, R. (1976) Clin. Chim. Acta 71, 501-510 15 Chopra, I.J., Solomon, D.H. and Ho, R.S. (1972) J. Clin. Endocrinol. Metab. 35, 565-573 16 Lecureuil, M.. Lecureuil. N. and Crouzat-Reynes, G. (1977) Lyon Pharmaceutique 28, 223-237
