THYROXINEDISPLACEMENTFROMSERUMPROTEINS ANDDEPRESSIONOF SERUMPROTEIN-BOUNDIODINE BY

CERTAIN DRUGS

BY J. WOLFF, M. E. STANDAERTAND J. E. RALL

(From the Clinical Endocrinology Branch, National Institute of Arthritis and MetabolicDiseases, National Institutes of Health, Bethesda, Md.)

(Submitted for publication February 6, 1961; accepted April 6, 1961)

A number of compounds such as 2,4-dinitro-phenol,' salicylate, tetrachlorothyronine, and di-phenylhydantoin have been shown to lower theprotein-bound iodine level of the blood (1-5). Inthe case of DNPand salicylate, it was concludedthat they acted in part via an inhibition of the pitu-tary TSH mechanism and partly by an acceleratedloss of T4 from the circulation (2, 6). A likelysite of action was, therefore, expected to involvebinding of T4 to serum proteins.

The ability of various analogs of thyroxine tobind to proteins of serum has been extensivelystudied with electrophoretic techniques. At leastthree serum proteins and perhaps four have beendescribed which bind thyroxine (7). Less directmethods of studying this binding of the thyroidhormones consist of measurement of the "uptake"of triiodothyronine by erythrocytes (8) and therate of dialysis of T4 through a cellophane mem-brane (9). By these techniques several com-pounds have been found that appear to alter thebinding of T4 to serum proteins when studied bythese techniques but if whole serum is used, theeffect cannot be localized to specific proteins.Thus it has been suggested from dialysis studiesthat salicylates and DNP act by inhibiting T4binding to serum proteins. Electrophoretic stud-ies in barbital buffer of sera containing salicylatefailed to show any influence on the distributionof T4 between TBG and albumin, nor was thereany change in the T4 binding capacity measuredunder these conditions (2). Since then it hasbeen shown that barbital buffer inhibits T4 bind-ing to prealbumin (10) and Ingbar has stated that

1 The following abbreviations have been used: DNP=2,4-dinitrophenol; PBI = protein-bound iodine, T4 = thy-roxine; TBG= thyroxine-binding globulin of the inter-alpha zone in paper electrophoresis; TBPA= thyroxine-binding prealbumin; TCT=DL-tetrachlorothyronine; TSH=thyroid-stimulating hormone.

salicylate could interfere with T4 binding in elec-trophoretic systems in buffers other than barbital(11). It was therefore desired to examine T4binding in the presence of these four agents in asystem where prealbumin binding of T4 could beobserved. Furthermore, the possible correlationbetween the effect of inhibition of thyroxine bind-ing to TBPA and to TBGby various drugs withthe in vivo effect of these drugs on the PBI wasstudied.

METHODS

A single batch of pooled normal human serum obtainedfrom the Baxter Laboratories was used in all the studies.Butanol solutions of thyroxine were evaporated to dry-ness in vacuo prior to the addition of serum. The mix-tures were incubated at 4° C for at least 24 hours beforeuse and were kept in the cold until used.

Electrophoresis in 0.12 Mammonium carbonate buffer,pH 8.4, was performed at room temperature on 3.75 X49 cm strips of Whatman no. 3 MMfilter paper. Theedges of the strips were coated with a thin border ofparaffin. The strips were saturated with buffer and sus-pended horizontally between two plastic clamps in aclosed system. The strips were equilibrated for at least1 hour before the serum was applied. The anodal cham-ber received an excess of buffer solution just before thecurrent was turned on. Thirty ,ul of serum was addedapproximately 8 cm from the anode just before the vol-tage was applied. In nearly all cases the reverse flowtechnique was employed (12) with a constant voltage of80 v.

Two types of studies were carried out. In the first,the inhibitor was added to the serum which was thenrun in reverse flow electrophoresis. In the second, wefollowed the suggestion of Ingbar (11) that addition ofsalicylate to the buffer permitted the demonstration ofinterference in protein-binding of T4. Conventional elec-trophoresis was used in some cases to rule out the pres-ence of unbound T4 in the albumin (origin) area in reverseflow electrophoresis.

After a run of 17 hours the strips were air-dried,scanned on a continuous recording strip counter, andradioautographed on No-Screen X-ray film. Strips wererealigned on the film and the radioactive fractions cut

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out, using the radioautograph as a guide to their exactlocation.. The filter paper segments were counted in awell scintillation counter. A duplicate strip was stainedwith bromphenol blue to show the protein location. Allresults are averages of duplicate or quadruplicate de-terminations and are reported as fractions of the totalthyroxine on each protein fraction.

It is felt that with the reverse flow technique directcounting offered greater accuracy than quantitation ofthe strip counter recordings. This is especially true in in-stances where the various agents have displaced all buttrace amounts of thyroxine from a binding site. Suchquantities are difficult to measure accurately on thestrip counter.

For calculations, serum PBI measured by a modifica-tion of the Zak procedure (performed by the BostonMedical Laboratories) was assumed to consist entirelyof thyroxine. Concentrations of T4 listed in Table Ihave been corrected for the endogenous T4 of the serumused. Labeled thyroxine was obtained from Abbott Lab-oratories and chromatographed before use; when neces-sary it was purified until better than 90 per cent pure(12). DNP and sodium salicylate were reagent gradeand TCT was a generous gift of Dr. Jacob Lerman.When drugs were added directly to the ammonium car-bonate buffer, the pH was adjusted after addition of thedrug.

Male guinea pigs of the NIH strain were used. Inexperiment III (Table II) thyroidectomy was checkedby thorough examination of the tracheal region, and allanimals suspected of incomplete thyroidectomy werediscarded. They were then divided into three groups: anuninj ected control group, a thyroxine-inj ected controlgroup, and a group receiving both thyroxine and TCT.The thyroxine treatment was started 8 days after thy-roidectomy and was continued for 12 days. During thelast 5 days the third group of guinea pigs received, inaddition, subcutaneous injections of TCT. All animalswere exsanguinated 3 to 4 hours after the last injection.

RESULTS

Initial experiments, in which salicylate or DNPwas added to the serum before electrophoresis,showed that both these agents tended to displaceT4 from TBPA onto TBG. However, experi-ments in which these agents were added to the buf-fer gave more reproducible results, and most of thestudies presented here will deal with the lattertechnique.

FIG. 1. THE EFFECT OF 2,4-DINITROPHENOL ON THEELECTROPHORETICDISTRIBUTION OF RADIOACTIVE THYROXINE(0.28 AG PER ML OR 0.36 /LM) IN HUMANSERUM. Inthis and succeeding figures a record of the radioactivityis above the stained paper strip and a radioautograph ofthe paper strip is below it. A) Control serum; B) 1 X10'M dinitrophenol in the buffer; C) 1 X 104M dini-trophenol added to serum.

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It can be seen from Figures 1 and 2 that the ad-dition of 1 x 10 4M DNPor 3 X 10 3 MNa sa-licylate to human serum containing 3.6 x 10-7 MT4 caused significant displacement of T4 from theprealbumin peak to TBG. This effect was moremarked when DNPor salicylate had been addedto the (NH4),2CO3 buffers (Figures 1B and 2Bas compared with Figures 1C and 2C). At 2.7 x10-7 M T4 (Figure 3C) there was practically noradioactivity left in the prealbumin area. At theselow T4 concentrations most of the T4 displacedfrom TBPAwas bound by TBG. In the presenceof higher amounts of T4, the TBG became satu-rated and the displaced T4 was bound to albumin(see Table I). That there was no displacementfrom albumin was ascertained in studies withstandard paper electrophoresis in which albuminis permitted to migrate from the origin. Thyrox-ine stayed with the albumin and no radioactivitywas found at the origin. Displacement of T4 fromprealbumin as a function of DNP or salicylateconcentration is also demonstrated in Table I.Displacement by salicylate could still be demon-strated at 3 X 10 4 1\1, a level easily attained dur-ing salicylate therapy (4.1 mg per 100 ml), andwas marked at 1 to 3 x 10-3 M levels which havebeen shown to be accompanied by lowering of thePBI (2).

TCT also could displace T4 from serum pro-teins, in this case from TBG(Figures 3A and 3B).Some displacement of T4 from TBG can be ob-served at TCT levels as low as 10-7 M. The dis-placement can also be demonstrated when the com-pound is added directly to serum. Comparison ofFigures 3B and 3C reveals the striking differencebetween TCT and salicylate in the electrophoreticdistribution of T4. In contrast to salicylate andDNP, T4 displacement is from TBG. At lowlevels of TCT, the T4 that is not bound to TBG isfound to be associated with TBPA. At very highlevels of TCT there is also inhibition of bindingof T4 to TBPA, and the thyroxine thus displacedappears on albumin (Table I).

In view of the potent T4 displacing action ofTCT, it was important to establish that this com-

FIG. 2. THE EFFECT OF SALICYLATE ON THE ELECTRO-PHORETIC DISTRIBUTION OF RADIOACTIVE THYROXINE (0.28,UG PER ML OR 0.36 AM) IN HUMANSERUM. A) Controlserum; B) 3 X 103M salicylate in the buffer; C) 3 X 10-3Msalicylate added to serum.

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pound could, in fact, lower the PBI when adminis-tered to the intact animal since previous observa-tions were not unequivocal (3, 4). Although wewere able to demonstrate such an effect in rats,the results with guinea pigs were more dramaticand are listed in Table II. A 30 to 35 per centdepression in the PBI could be demonstrated, al-beit at rather large doses of TCT. Since this T4analog does have very weak thyromimetic activity,it seemed important to rule out an indirect effectof TCT, e.g., via the pituitary TSH mechanism.For this reason thyroidectomized guinea pigs onconstant doses of T4 were utilized. It can be seenthat under these conditions TCT lowered thePBI by nearly 50 per cent despite the very highinitial PBI level. A similar PBI depression inathyreotic subjects receiving replacement therapyhas been reported after DNP treatment in man(13) and after salicylate treatment in rats (14).

Oppenheimer, Fisher, Nelson and Jailer haverecently shown that patients chronically treatedwith diphenylhydantoin show depressed valuesof PBI and have suggested that this might haveresulted from T4 displacement from one of thethyroxine-binding proteins (5). This proved tobe the case (see Table I), and at drug concentra-tions that obtain clinically (15) there was markeddisplacement of T4 from TBG to prealbumin. Itmay also be noted that in the presence of a con-stant amount of diphenylhydantoin, increasing con-centrations of thyroxine result in increasingamounts of T4 bound to TBG. With 10-4 M di-phenylhydantoin, the amount of T4 bound to TBGincreased from 0.03 Mtg per ml at T4 concentrationof 0.07 Fkg per ml to 0.065 Mg per ml at a T4 con-centration of 0.28 MAg per ml, and at the highest T4concentration to 0.15 /,g per ml. This would beexpected when two compounds compete for thesame site. Similar considerations apply to theother compounds studied. Even at the highestlevel of diphenylhydantoin (we were limited bythe solubility at this pH) there was no displace-ment from prealbumin, in marked contrast to theresults obtained with TCT (Table I). This may

FIG. 3. COMPARISONOF THE DISPLACEMENTOF RADIO-ACTIVE THYROXINE (0.15 /AG PER ML OR 0.27 AtM) FROMHUMANSERUMPROTEINS PRODUCEDBY DL-TETRACHLORO-THYRONINEAND SALICYLATE. A) Control serum; B) 3 X10'M tetrachlorothyronine in the buffer; C) 3 X 10-'Msalicylate in the buffer.

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DRUGDISPLACEMENTOF THYROXINEFROMSERUMPROTEINS 1377

TABLE I

The distribution (in per cent) of thyroxine on serum protein fractions as a function of theconcentration of various displacing agents

0.15 jzg T4/'ml1 (0.19 MM) 0.28 lg T4/ml (0.36 MM) 1.54 Mg T4/ml (2.0 MM) 2.08 Mg T4/ml (2.7 MM)Agent (molarity-

in buffer) TBG A PA[12]t TBG A PAENl] TBG A PA[5] TBG A PALS]

Control 38 20 43 38 25 37 13 32 55 15 37 48±SD 4 2.4 i 1.2 i 1.1 i 4.6 ± 2.2 + 5.7 ± 1.0 ± 1.3 ± 1.7 4t 1.2 ± 8.6 ± 9.4

Salicylate3 X10-4 52 24 24 18 35 471 X10-3 61 27 12 21 59 203X10-3 61 30 9 71 25 5

2 ,4-Dinitrophenol1 X10-5 42 17 423 X10-5 39 25 36 13 39 481 X10-4 55 32 13 56 28 16 11 54 353X10-4 63 32 5 15 74 11

Tetrachlorothyronine1 X10-7 32 27 413 X10-7 20 36 44 18 41 411 X10-6 11 48 42 14 45 413 X10-6 6* 47* 48* 5 57 381X10- 4 69 26 4 77 19

5,5-Diphenvlhydantoin3 X10-s 29 30 411 X10-4 21 30 49 24 29 47 10 31 593 X10-4 14 27 59 15 30 556 X10-4 11 29 60 10 33 54

* T4 concentration for this experiment of 0.19 jg per ml (0.24 MAM).t Number of determinations at each level in brackets.

imply a difference at the prealbumin binding site, tion of thyroxine are functions of the concentra-although higher concentrations of the drug might tion of unbound thyroxine (7). Any factor whichhave shown displacement here also. In any case, decreases protein binding of T4 will, if the totalthe present finding, that diphenylhydantoin com- concentration of thyroxine is unchanged, increasepetes with thyroxine for sites on TBG is a reas- the concentration of free thyroxine. Consequently,onable explanation for the depression of PBI. It an important factor in the control of the PBI isis surprising, however, that a compound so unlike the serum concentration of available binding sitesthe iodothyronines can compete for what appears for the T4. Given a constant output of T4 fromto be a highly specific binding site on TBG. the thyroid, loss of these sites by competition with

a variety of drugs leads inevitably to a depressionDISCUSSION in the PBI level. This cause for the reduction

There is available a body of evidence which sug- of the PBI concentration in the circulation must begests that both the effect and the rate of degrada- clearly differentiated from that resulting from a

TABLE II

The effect of tetrachlorothyronine on the PBI of guinea pigs

No. ofguinea

Experiment Treatment pigs PBI P*

I Control 5 3.0 i 0.34TCT 0.6 mg/100 g/day for 5 dayst 6 2.0 ± 0.33 <0.001

II Control 6 2.6 + 0.28TCT 0.4 mg/100 g/day for 1.5 dayst 6 1.7 i 0.16 <0.001

III L-T4 6 ,g/100 g/dayT 6 14.7 :1: 1.0(All guinea pigs L-T4 6 g/I100 g/dayt 5 7.6 ± 0.86 <<0.001thyroidectomized) TCT 1.0 mg/100 g/dayT

Control 4 1.1 + 0.29

* p Values are with respect to the control groups in Experiments I and II and with respect to the T4-injected groupin Experiment III.

t In two divided doses per day.t The injection schedule is listed in Methods.

J. WOLFF, M. E. STANDAERTAND J. E. RALL

decrease or absence in the thyroxine-binding pro-teins, as in the case of nephrosis (16), congenitalabsence of a binding protein (17), or after treat-ment with methyl testosterone (18). Presumablythe homeostatic mechanisms operating to reducethe PBI are similar in all these circumstances.

Inhibition of thyroxine binding to prealbuminhas been clearly shown with both salicylate anddinitrophenol. Since there is no evidence that, inthe concentrations used, either drug denatures se-rum proteins, and since there is a good correla-tion between concentration of drug and degree ofinhibition of thyroxine binding, it may be as-sumed that these drugs compete with thyroxinefor binding sites on TBPA. It would appear fromthe quantities of drug required that the associationconstant of both of these drugs for TBPA is sev-eral orders of magnitude lower than that of thy-roxine. Neither of these drugs affects T4 bindingto TBG.

It is probable that addition of the drug to thebuffer is the better method. If the associationconstant of drug for TBPA is not high it seemsquite likely that if drug is added only to serum,there will be, during electrophoresis, a continualremoval of unbound drug. If drug is added to thebuffer, a reservoir is provided so that the concen-tration of drug in the buffer will approach the con-centration of free drug. As a result of the un-certainty in the concentration of drug bound toprotein, it is not possible to calculate accurately aratio of binding constants of drug/T4 for TBPA.Doubt has recently been expressed regarding thephysiological importance of TBPA as a bindingprotein for T4 at pH 7.6 (19). The present datawith salicylate and DNP are nevertheless com-patible with a significant role for TBPA.

The results with TCT are more easily inter-preted because of the apparent high affinity con-stant of this compound for TBG; although boundless strongly, its binding affinity for TBG prob-ably differs from that of T4 by less than an orderof magnitude. Since equilibrium is not attainedin electrophoresis, a more exact statement of therelative affinities is not possible. At high concen-trations TCT also inhibits T4 binding to TBPA.Diphenylhydantoin appears not to have this sec-ond action. None of the compounds tested ap-peared to inhibit T4 binding to albumin. This maybe a quantitative phenomenon, since albumin is

present in serum at a concentration of - 5 X 10-4M [with four sites for T4 (20)] and extremelyhigh concentration of the drugs would be required.Furthermore, it is unlikely that T4 binding to al-bumin normally plays a significant role in thyrox-ine metabolism, since the PBI of analbuminemicpatients is normal (21). It is highly probablethat other drugs will be found that can alter thePBI by this mechanism. It would behoove in-vestigators studying thyroid function during ther-apy with presumably unrelated drugs to take cog-nizance of this fact.

It may be concluded, therefore, that a decreasein the number of available binding sites either inTBGor in prealbumin, produced by TCT and di-phenylhydantoin or by DNP and salicylate, will(by mass law relations) increase the free T4 atthe expense of the fraction bound. This will inturn accelerate T4 disappearance from the circu-lation and ultimately lower the PBI to a newsteady state value at which the free T4 concentra-tion may be perfectly normal. The in vivo ex-periments with TCT, which produced a consistentfall in the PBI both in normal guinea pigs and inthyroidectomized, thyroxine-treated animals, of-fer only indirect support for this hypothesis, sinceno electrophoretic results on guinea pig sera arereported. Studies of guinea pig sera by theabove techniques showed T4 binding only in thealbumin area, and T4 displacement from one pro-tein to another within this area would not bedetectable.

The above considerations may also be applicablein certain short-term assays of thyromimetic (andantithyroxine) materials. Are such effects, asfor example, goiter prevention, due to inherentthyroxine-like activity or do they merely repre-sent an elevation of free T4 as a result of thyroxinedisplacement from one of the serum proteins?

Can decreased T4 binding explain the otherphenomena produced by DNPand salicylate? Ofthe diverse effects of salicylate on thyroid func-tion, loss of binding sites can probably explain theaccelerated disappearance of radioactive T4 fromthe circulation (2, 22, 23). Whether or not theother effects can be similarly explained is not cer-tain at present. The whole problem must be re-considered from the point of view of intracellularbinding proteins and the effect of the drugs uponthese.

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DRUGDISPLACEMENTOF THYROXINEFROMSERUMPROTEINS

SUMMARY

The binding of thyroxine to human serum pro-teins has been studied by paper electrophoresis in(NH4)2CO3 buffer at pH 8.4.

1. Salicylate and 2,4-dinitrophenol inhibit thy-roxine binding to prealbumin, the displaced thy-roxine appearing in the thyroxine binding a-glob-ulin area.

2. Diplhenylhydantoin inhibits thyroxine bindingto the thyroxine-binding a-globulin, displacing itonto prealbumin.

3. DL-tetrachlorothyronine displaces thyroxinefrom the thyroxine-binding a-globulin, and at highdrug concentrations also from prealbumin. Tetra-chlorothyronine given in vivo lowers the level ofserum protein-bound iodine in intact and in thy-roidectomized, thyroxine-treated guinea pigs.

4. It is concluded that loss of binding sites oneither of the major thyroxine-binding proteinscan explain the lowering of the serum protein-bound iodine produced by the four drugs studiedwhen they are given in vivo.

REFERENCES

1. \Volff, J., Rubin, L., and Chaikoff, I. L. The influ-ence of 2,4-dinitrophenol on plasma protein-boundiodine. J. Pharmacol. exp. Ther. 1950, 98, 45.

2. Austen, F. K., Rubini, M. E., Meroney, W. H.,and Wolff, J. Salicylates and thyroid function.I. Depression of thyroid function. J. clin. Invest.1958, 37, 1131.

3. Richards, C. E., Brady, R. O., and Riggs, D. S.Thyroid hormone-like properties of tetrabromo-thyronine and tetrachlorthyronine. J. clin. Endocr.1949, 9, 1107.

4. Lerman, J., and Harington, C. R. The physiologicactivity of tetrabromothyronine and tetrachlorthy-ronine. J. clin. Endocr. 1949, 9, 1099.

5. Oppenheimer, J. H., Fisher, L. V., Nelson, K. M.,and Jailer, J. W. Depression of the serum pro-tein-bound iodine level by diphenylhydantoin. J.clin. Endccr. 1961, 21, 252.

6. Wolff, J., and Austen, F. K. Salicylates and thy-roid function. II. The effect on the thyroid-pituitary interrelation. J. clin. Invest. 1958, 37,1144.

7. Robbins, J., and Rall, J. E. Proteins associatedwith the thyroid hormones. Physiol. Rev. 1960,40, 415.

8. Hamolsky, M. W., Ellison, H. E., and Freedberg,A. S. The thyroid hormone-plasma protein com-

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9. Christensen, L. K. Thyroxine-releasing effect ofsalicylate and of 2,4-dinitrophenol. Nature(Lond.) 1959, 183, 1189.

10. Ingbar, S. H. Pre-albumin: A thyroxine-bindingprotein of human plasma. Endocrinology 1958,63, 256.

11. Ingbar, S. H. Communicated at the Meeting of theNew York Academy of Sciences, New York,Nov. 1959.

12. Robbins, J. Reverse-flow zone electrophoresis. Amethod for determining the thyroxine-binding ca-pacity of serum protein. Arch. Biochem. 1956, 63,461.

13. Castor, C. W., and Beierwaltes, W. Effect of 2,4-dinitrophenol on thyroid function in man. J. clin.Endocr. 1956, 16, 1026.

14. Good, B. F., Hetzel, B. S, and Opit, L. J. Effects ofsalicylate on plasma protein-bound iodine in thy-roxine-maintained thyroidectomized rats. J. En-docr. 1960, 21, 231.

15. Kozelka, F. L., and Hine, C. H. Distribution, rateof disappearance and excretion of Dilantin (sodiumsalt of 5,5-diphenyl-hydantoin) (abstract). J.Fharmacol. exp. Ther. 1940, 69, 292.

16. Robbins, J., Rail, J. E., and Petermann, M. L.Thyroxine-binding by serum and urine proteinsin nephrosis. Qualitative aspects. J. clin. In-vest. 1957, 36, 1333.

17. Tanaka, S., and Starr, P. A euthyroid man withoutthyroxine-binding globulin. J. clin. Endocr. 1959,19, 485.

18. Federman, D. D., Robbins, J., and Rall, J. E. Ef-fects of methyl testosterone on thyroid function,thyroxine metabolism, and thyroxine-binding pro-tein. J. clin. Invest. 1958, 37, 1024.

19. Christensen, L. K., and Litonjua, A. D. Is thyroxinebinding by pre-albumin of physiologic importance?J. clin. Endocr. 1961, 21, 104.

20. Tabachnick, M., and Sterling, K. The binding ofthyroxine and its analogues by human serum al-bumin. Clin. Res. 1960, 8, 379.

21. Beck, G. E., and Dorta, T. Un cas d'analbuminemie.Helv. med. Acta 1959, 26, 764.

22. Goldberg, R. C., Wolff, J., and Greep, R. 0. Themechanism of depression of plasma protein-boundiodine by 2,4-dinitrophenol. Endocrinology 1955,56, 560.

23. Escobar del Rey, F., and Morreale de Escobar, G.Studies on the peripheral disappearance of thy-roid hormone. IV. The effect of 2,4-dinitrophenolon the I"s' distribution in thyroidectomized, L-thy-roxine maintained rats 24 hours after the injectionof 131 labeled L-thyroxine. Acta endocr. (Kbh.)1958, 29, 161.

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