394

D. Reinwein, H.A. Duner, H. Wiermann, J. Löhnert and A. von zur Mühlen

extra-pancreatic effects of the drug. Lower dosages than those used in this experiment have been shown to reduce pancreatic IRI levels to near 0 (Junod et al. 1967), so it is doubtful that the 2 injections induced varying levels of hypoinsulinism. Indeed, hyperglyc-

emia, the prirnary criterion for diabetes mellitus, was similar for the 2 diabetic groups (Fig. 1). There is no apparent explanation for the slow onset of the muscle effects at this time.

Referenees

Requests for reprints should be addressed to: Dr. R.B. Armstrong, Biology Department, 2 Cummington Street, Boston University, Boston, Massaehusetts 02215 (USA)

Horm. Metab. Res. 8 (1976) 394-399

© Georg Thieme Verlag Stuttgart

The Thyroidal T 4fT 3 Ratio and its Regulation in Non-taxie Goitre D. Reinwein, H.A. Durrer, H. Wiermann, J. Löhnert and A. von zur Mühlen Abteilung für klinische Endokrinologie, Medizinische Klinik und Chirurgische Klinik der Universität Essen (GSH), Abteilung für klinische Endokrinologie, Dept. Innere Medizin, Medizinische Hochschule Hannover

Summary In 35 patients with non-toxie goitre, surgieally reseeted thyroid tissue was hydrolyzed with Pro nase under anaerobie eonditions in the presence of methylmereaptoimidazole. Total iodine, PBI, L-thyroxine (T4-RIA and T4 (D» as weil as trüodothyronine (T 3-RIA) were determined in the tissue hydrolysates. The data obtained were eompared with T4fT 3 ratios in the serum, TRH test and thyroidal 131 I uptake before operation. The total iodine, arnounted to 112.6 ± 14.6 ugfg tissue. The T 4fT 3 ratio was 11.37 ± 1.80. Using this procedure of tissue hydro lysis lind RIA assays for iodothyronines, the yield for T4 and T 3 is eonsiderably higher than the one obtained with previous methods. As in animal experiments, evidenee was given for human goitre that T 3 is predominantly produced fllst in the ease of deereased T 4 produetion. Second in the case of iodine defieiency, and third in eases of inereased TSH response after TRH. Patients with a large arnount of iodine and r 4 in their thyroids have a mueh smaller inerease in serum TSH after TRH than do subjeets with a mueh lower thyroidal T4 eoneentration. This oeeurs even though the thyroid hormone levels in serum in these groups are not different. No eorrelation between thyroidal T 4fT 3 ratio and serum T4fT 3 ratio eould be demonstrated even under extreme eonditions. This finding favors the assumption of apredominant extrathyroidal regulation for T 4fT 3 ratio in serum.

Received: 22 Jan. 1976

Aceepted: 30 Apr. 1976

Key-Words: Non·toxie Goitre - Intrathyroidal lodothyro· nines - T4 /T 3 Ratio - lodine Defieieney

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of streptozotoein-diabetic rats to exereise training. BioArmstrong, R.B., P.D. Gollniek, C.D. lanuzzo: Histoehemical chem.Biophys.Res.Comm. 58: 107-111 (1974) properties of skeletal muscle fibers in streptozotocin-diaJunod, A., A.E. Lambert, L. Orei, R. Pietet, A.E. Gonet, betie rats. Cell Tiss.Res. 162: 387-394 (1975) A.E. Renold: Studies of the diabetogenie action of strepCooperstein, S.J., A. Lazarow, N.J. Kurfess: A mierospeetrotozotoein. P.S.E.B.M. 126: 201-205 (967) photometrie method for the determination of suecinic Peter, J.B., R.J. Barnard, V.R. Edgerton, C.A. Gillespie, K.E. dehydrogenase. J.BioI.Chem. 186: 129-139 (1950) Stempel: Metabolie profiles of three fiber types of skeletal Gollniek, P.D.,C.D. lanuzzo: Hormonal deficiencies and the muscle in guinea pigs and rabbits. Biochemistry 11: 2627metabolie adaptations of rats to training. Amer.J.Physiol. 2633 (972) 223: 278-282 (1972) lanuzzo, C.D., M. Lesser, F. Battista: Metabolie adaptations

Abbreviations used: FT 41 for free L-thyroxine index; MMI for methylmereaptoimidazole; PBI for protein bound iodine; TI for total iodine; T 3 for L-triiodothyronine; T 4 for Lthyroxine; TRH for thyrotropin re leasing hormone; TSH for thyrotropin.

Introduction

Precise direct determinations of L-triiodothyronine (T 3) in thyroid tissue were not possible before radioimmunoassays were applicable to systemic investigations. Previous data based on tracer studies and chromatographie procedures are subject as weil to substantial errors du ring the hydrolysis of the homogenate (Inoue and Taurog 1967, Ermans, Kimhaert, Delcroix and Collard 1968). This fact and the recent findings of iodine deficiency in our area (Wiermann, Du"er, Mühlen. Ruppert. Schmidt and Reinwein 1975) prompted us to reinvestigate the compösition of iodothyronines in non-toxic goitrous tissue. The question we attempted to answer in patients with sporadic non-toxic goitre were: 1: Which factor might control the T 4 IT 3 ratio? and 2. Are there any correlations with extrathyroidal changes of iodine metabolism and TSH?

The Thyroidal T4/T 3 Ratio and its Regulation in Non-toxie Goitre

33 patients with non-toxie goitrc were investigated. The patients were 4 males and 29 females, aged 21 to 69, with an average of 45.2 years. There were 5 patients with a reeurrenee of goitre after operation. Solitary nodules were found in 18, multinodular goitre in 8, and diffuse goitre in 7 cases. Except for 4 patients, who had been treated with thyroid hormones within 3 and 12 months, no patient received thyroid drug treatment until 3 months before operation.

Recollery: Recovery was studied with 125I-T4 and 125I-T3 added to tissue homogenates. The yield of traeer in the centrifuged hydrolysate depended strongly on the consistence of the tissue. The average yield in the supernatant was 81.0 ± 2.0% ror T 4 and 75.0 ± 3.0% for T 3. When cold T4 was added in a concentration of 0.7 lJg per 7.0 ml homogenate 83.8% could be recovered in the supernatant by T4 -RIA. All data given in the results are corrected for losses in recovery.

Cross reactions: The T 4 -antibody cross reaction with T 3 was Studies prior to operation. Besides thyroidal 13lI-uptake, less than 0.1 %. In order to evaluate possible cross reactions 13 PB 1J, thyroid scan, serum T4 (D), RT 3 U (Thyopac-3, Buch- with thyroglobulin rsp. hydrolysed material, the following ler, Amersham), PBI and T 3-RIA, a TRH-test with 200 J..I8 experiments were performed: 1. T 3-RIA was measured in a TRH (Hoechst) was performed 24 ho urs before operation. thyroid tissue hydrolysate at three different dilutions before TSH was determined before and 30 min after TRH. and after the addition of 9 ng T 4 /m!. The hydrolysate eonStudies after operation. The resected goitrous tissue was tained between 0.03 and 0.2 ng T 31m!. There was no difference in the experiments with and without the addition of stored deep-frozen until used. The clinical diagnosis in each T 4 • 2. Thyroid hydrolysate was used at various dilutions as patient was confirmed by histological examination of a portion of the gland analyzed. 2 to 4 pieees of tissue, 400 to donor for T 3 in a T 3-R1A standard curve. The curve obtained with hydrolysate was eongruent with the usual T 3 standard 700 mg taken from different parts of the reseeted goitrous curve. These experiments exclude interference for T 3 meatissue, were analyzed in exactly the same way. The tissues surements under the conditions studied here. The T 3-antiwere dissected, rinsed with saline, homogenized for 2 min in a Potter-Elvehjem glass homogenizer with Pronase under body cross reaction with T 4 was less than 0.1 %. anaerobic conditions according to Chopra. Fisher, Solomon Chemieals: L-thyroxine and L-T3 labelIed with 125 I in the and Beall (197 3~. The tubes were charged with nitrogen and prime positions were purchased from Abbott. L-T 4 purissiineubated at 37 C for 16 hours in a Dubnoff metabolie mum was obtained from Henning, Berlin; it contained less shaking incubator. The hydrolysate was centrifuged, and tothan 0.1% T 3 . Pronase E 70000 PUK/g came from Merck, tal iodine, PBI, T 3-RIA and T 4 -RIA measured in the superDarmstadt. Methylmercaptoimidazole was obtained from natant. T 4 (D) was determined in butanol extraets. Kali-Chemie, Hannover. Assays Table 1. Yields of thyroidal T4 and T 3 during the preparaT 4 -RIA was measured in a test kit from Henning, Berlin. tion with and without MMI. The standard curve of the RIA was linear between 200 and 1000 g, and that of T 4(D) between 10.0 and 90 ng. For the The concentrations of T 4 and T 3 obtained with MMI are expressed as percentage of the values without MMI. T 4(D) determination butanol extracts from the hydrolysate (1 ml hydrolysate + 2 ml acid n-butanol) were prepared for MMI the T4(D) determination. After shaki'lg for 1 min, the butanol extract was centrifuged, and the butanol phase collected. 0.01 M 0.05 M The extracts were evaporated to dryness, since butanol af239 ± 12 T 4 (n = 5) 176 ± 12 fected the determination unspecifically. Thc dried extract was dissolved in 1.0 ml aqua bidest. Control experiments 303 ± 20 T 3 (n=5) 170 ± 13 exc\uded interfering effects of non-specific protein binding. The properly diluted extract was used for T 4(D) determination. Compared to T 4 -RIA, T 4 (D) values matched well in Results tissues comprising of more than 20.0 ug T 4/g tissue. Below Table 2 shows the average values of thyroid para20.0 Jlg ,T 4/g, T 4(D) values were 29.7% ± 10.8% lower. meters of 33 patients with non-toxie goitre before Measurement of T 3-R1A was performed in a test kit from operation. Group B received estrogen-containing drugs, Byk Mallinekrodt. When T 4 or T 3-values were outside the range of the standard, appropriate dilutions were used. All while group A did not. PBI, T 4(0), RT 3 U and T 3determinations were run in duplieate at 3 different dilutions. RIA were significantly higher in group B than in Serum-TSH was measured by a radioimmunoassay (Mühlen, group A. Group A differed significantly from average Emrich. Hesch and Köbberling 1971). PBI and total iodine values of our controls with respect to T 4 (0), T 3 -RIA were determined by Technicon autoanalyzer. Two different and FT 41. The TRH-response was normal (ll TSH less dilutions were used. Additions of monitrol (DADE) control serum to the sampies were taken for recovery studies. In the than 15 J.LU/ml) in 17 patients, subnormal oe negative case of total iodine the Isobeads Resin was omitted before in 9 patients (ll TSH less than 1.2 J.LU/ml), and exagthe reaction took plaee. gerated (ll TSH more than 15 J.LU/ml) in 6 patients. All data given are expressed as IJg/g tissue (wet weight) and The mean values of intrathyroidal iodine compounds represent means ± standard deviations. Student's t-test was employed for tests of signifieance. were as follows: total iodine (TI) 112 ± 14.6 J.Lg/g tissue, PBI 95.2 ± 13.3 J.Lg/g, PBI in % of TI 79.4 ± Control experiments 2.9%, T 4 -RIA 18.59 ± 5.0 J.Lg/g, T 3 -RIA 1.5 ± 0.2 Deiodination: Deiodination of T 4 and T 3 during the prepaJ.Lg/g and T 4 /T 3 ratio 11.37 ± 1.8. Maximal T 4 /T 3 ration was studied in experiments with and without the adratios up to 48.6 were seen in iodine-rich tissues, dition of methylmercaptoimidazole (MMI). Table 1 demonwhereas minimal ratios of 0.5 were found in iodinestrates the yields of T 4 and T 3 in the presence of 0.01 and poor tissues only. In one goitre, whieh showed scin0.05 M MMI. Whereas the thyroid hormones increase by a faetor of 2 to 3, the T4/T 3 ratio did not change significant- tigraphically cold areas and a low 13lI_uptake of 11%, Iy. For all experiments the concentration of 0.05 M MMI only T 3 but no T 4 could be detected. The T 3 conwas used.

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Materials and Methods

395

396

D. Reinwein, H.A. Durrer, H. Wiermann, J. Löhnert and A. von zur Mühlen

Table 2. Thyroid function parameter of 33 patients with non-toxic goitre before operation. Group A patients without medication of estrogens. Group B patients under medication of estrogens. Normal values are for PBI 6.39 ± 0.23 pg/IOO ml; for T 4 (D) 7.8 ± 0.4 pg/l00 ml; for RT 3 U is 103 ± 2; for FT4 I is 7.05 ± 0.56; for T 3 RIA 153.1 ± 8.5 pg/l00 ml. TRH-test Maximal T4 (D) PBI RT 3 U T 3 -RIA FT 41 Patients RI-uptake TSH in uU/ml 1Jg/100 ml pg/l00 ml pg/l00 ml 0' % of dosage 30' m ± SEM m ± SEM m ± SEM m ± SEM m ± SEM m ± SEM m ± SEM m ± SEM Group A 5.70 ± 0.25 7.22 ± 0.40 108.8 ± 1.7 184.8 ± 9.5 6.7 ± 0.38 2.03 ± 0.82 7.9 ± 0.46 55.2 ± 2.5 n = 27 7.98 ± 0.62 10.47 ± 1.0

Group A+B 6.16 ± 0.28 n = 33

7.81

±

0.43

117.3±4.1

217.5 ± 8.64

8.9 ± 0.65

5.1 ± 3.42

25.5± 11.4

61.8 ± 5.0

110.3 ± 1.7

190.8 ± 8.2

7.1 ± 0.4

2.2 ± 0.79

10.5 ± 2.34

56.3 ± 2.3

,Yg Tig tissue

T4/T3 ratio

T4 1T3 ratio

40

SE'rum

40 •

T4

0 VTJratio

30

30

20

20

10

10

< 50 (n= 7)

50-120

) 120

(n=IO)

(n=15 )

"'9TI/gtissu~

Fig. 1. Dependence of the intrathyroidal T4 and T 4/T 3 ratio on total iodine (TI).

GO

40

. ..... . ~ . .......

20

10

20

30

40

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Group B n =6

T4 /T3 ratio tissuE' cent ration was 1.62 p.g/g tissue. Recovery experiments of added T4 in order to exclude inhibitory effects Fig. 2. Comparison of serum T4/T 3 ratio with tissue T4/ T 3 ratio. have not been performed because of shortage of tissue. Microscopically, Iymphocytic inflltration was found. goitres are subdivided into two groups according to The iodine content of different pieces of resected its T 4 content. Again the correlation between high tissue varied considerably. In some cases they difT4 with a high T4 /T 3 ratio is seen in the iodine-rich fered by a factor of 3, although goitrous tissue aptissue being significantly different from the iodinepeared scintigraphically and macroscopically uniform. poor tissue. In the serum of tbis group, the TSH inWe therefore chose only mean valucs of each goitre. creases after TRH is somewhat smaller and the T4 /T 3 ratio in the serum is lower than in goitres with a Figure 1 demonstrates the dependence of intrathyroidal hormones on TI in the t,issues. lf one separates very low T 4 content. These differences, however, are the tissue according to its total iodine into three not significant. groups, less than 50, 50-120 and more than 120 p.g Correlations between TSH response after TRH and TI/g tissue, one finds a significantly higher T 4 and intrathyroidal and serum T 4, T 3 are shown in Table 4. T 4 /T 3 ratio in iodine-rich glands than in iodine-poor The patients are subdivided according to their TSH glands. The PBI values not shown here reflect the response into three groups. Patients with a low same dependence. ß TSH are characterized by a relatively high thyroidal

lf one compares the T 4 /T 3 ratio in the thyroid gland with the T 4 /T 3 ratio in serum, one obtains the results given in Figure 2. There is no correlation between the two ratios. The slope of the line is insignificant.

T 4 and T 4 /T 3 ratio, being significantly different from the data of patients with aß TSH of more than 15 p.U/mI. The total iodine reflects the pattern already shown before, but in this particular case a significant difference could not be ascertained. The correspondWe looked therefore for special conditions where cor- ing serum T 4 values and T 4 /T 3 ratios, on the other relations between serum and intrathyroidal data might hand, do not change. show up. One is demonstrated in Table 3 where Another extreme condition was looked for with respec

The Thyroidal T4fT 3 Ratio and its Regulation in Non-toxie Goitre Table 3. Dependence of intrathyroidal T 4fT 3 ratio, TI and serum T 4fT 3 ratio, Mean values and deviation as in Table 1.

D. TSH on intrathyroidal T 4 .

Thyroid Tissue

Serum

D. TSH tlUfml

T4 pgfg

n

T 4fT 3 ratio

TI w,fg

T 4fT 3 ratio

>20.0 m = 51.2

7

21. 7 ± 5.8"

227.9 ± 35.8

42.4 ± 5.0

5.0 ± 2.3

15

6.9 ± 1.0*

40.6 ± 10.4

48.5 ± 4.3

13.3 ± 5.6

Table 4. Correlations of intrathyroidal T 4, T 4fT 3 ratio, TI and serum T4fT 3 ratio to

D. TSH tlUfml

n

2.0

D. TSH

Thyroid Tissue

Serum

T4 w,fg

T 4fT 3 ratio

TI w,fg

9

45.0 ± 17.6*

18.5 ± 4.9*

174.2±37.7

6.81 ± 0.6 (6.45)**

40.84 ± 3.0 (40.21)**

< 15.0

17

12.6 ± 2.4

8.3 ± 1.3

105.9 ± 15.8

7.88 ± 1.0 (7.50)**

46.73 ± 3.9 (47.62)**

> 15.6

6

7.7 ± 1.0*

92.0 ± 28.2

8.37 ± 1.0 (6.53)**

46.0 ± 5.3 (40.7)**


40

5

84.3 ± 10.2

10.2 ± 2.8

Group 2