223

Clinica Chimica Acta, 67 (1976) 223-230 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7491

TOSHIHIDE

YAMAMOTO

*, KEI DOI, KIYOSHI

MIYAI and YUICHI

KUMAHARA

Endocrine Laboratory, Center for Adult Diseases, Osaka and Central Laboratory Investigation, Osaka University Hospital, Osaka (Japan) (Received

August

for Clinical

8, 1975)

cI._______ -_-_ WJmlnary

By means of measurement of the serum free triiodothyronine fraction (%FT3) by equilibrium dialysis, the influence of varying serum concentrations of thyroxine (T4) on %FT3 was studied in conditions associated with lack or excess of serum T4 and in serum enrichment with T4 in vitro. The enrichment of pooled serum with T4 caused significant increases of %FT, when T4 was added to elevate its T4 content between 10 and 25 E.cg/dl. In 4 subjects with primary hypothyroidism treated with T3 (25 pg once daily for two weeks, twice daily thereafter), progressive increases of serum T3 were observed while their Tq, free T4 and T3 fractions remained unchanged. Consequently, the serum free T3 and thyrotropin concentrations became normal after the serum T3 concentrations became hypernormal. When comparisons of the serum thyroid hormone levels were made between 6 subjects with T3 toxicosis and 8 with a usual variety of hyperthyroidism matched for serum T3 concentration, the former sub-group of hyperthyroidism showed significantly lower serum free T3 concentration (0.51 + 0.22 ng/dl versus 0.79 + 0.21 ng/dl, p less than 0.05). The amount of T4 in serum is considered to affect its free T3 concentration by virtue of sharing serum-binding proteins. Introduction It has been known that thyroxine (T4) and triiodothyronine (T3) circulate bound to specific binding proteins in a firm and reversible way [ 1,2] . Most of the circulating T3 is bound to thyroxine-binding globulin (TBG), less intensely than T4 and a lesser amount of T3 to thyroxine-binding prealbumin (TBPA) has been demonstrated [4,5] . As T3 shares with T, its plasma-binding proteins, the increase or decrease of plasma T4 concentration conceivably affects its free T3 * Address

correspondence to: Dr. T. Yamamoto, Higashinari-ku, Osaka. Japan.

Center for Adult Diseases, 3-3, Nakamichi 1-chome,

~(~I~~~elltratiorl. Robbins and Rail 16 1 calculated serum free ‘I’I t,t.,ncet?trat,iotls in the presence of varying amounts of serum T.$: which, however, remain to be confirmed. In the present communication, the problem of how lack or excess of TJ in vivo influences serum free TJ concentration ( FT3) was studied. ‘I’he effects of added T, (or T,) to pooled serum on its free TJ and T, fractions (‘AFT,. ‘/rFTA) were studied as well.

L-T4 and L-TJ used for enrichment were obtained from Sigma Chemical Co. (St. Louis, MO., U.S.A.). The preparations of T, and T3 were estimated to contain less than 1% of T3 and TJ, respectively, by Sephadex LI-I column chromatography [7] . Serum T4 concentration was measured by competitive proteinbinding assay after extraction with ethanol using a commercial test kit (Tetrasorb “, Dainabot Laboratory, Tokyo, Japan). Serum T3 concentration was measured by radioimmunoassay (MA) using specific anti-T,s antibody raised in rabbits according to Chopra et al. [S] . Serum thyrotropin (TSH) concentration was measured by double-antibody RIA [9] employing a commercial test kit (Daiichi Radioisotope Laboratory, Tokyo, Japan) with Research Standard 68/38 as a reference standard. Serum %FTJ and %FT3 were measured simultaneously by equilibrium dialysis in a modification 1101 of the method described by Sterling and Brenner [ll] . To 1.2 ml of serum to be assayed, [ ‘3111T, with specific activity of approx. 50 &X/pg (Daiichi Radioisotope Laboratory) and [ ‘25X]T3 (spec. act., 300 @Zi/pg, Dainabot Laboratories) were added to concentrations of 2 pg/dl and 25 ng/dl, respectively. Both 1i3’I]Tj and ]‘*‘I] T3 were preliminarily purified by dialysis according to Schussler and Plager [12]. 0.5 ml of serum with tracers added was placed in Visking dialysis tubing S/32 inch X 8 inch) by means of a 500 /.A disposable tip of an Eppendorf ~,,i-~~.+;~ IP;nottn rJnrl n“..I. i -1 ix,,ac ;m ‘l.U”“IIIULIIcI lr.Jr;UU.z Ullcl 111,I\f “I thn UIIC.-.llm O.z71Ulll ““Lli)cc~.mrl DLI”CXA 111n u ,rr\,,nt;rrm ~“u,,lilllg t,,hfi LIcIvr;nrrnL”IItaining 2.9 ml of phosphate buffer (total, i.e. dialysable and non-dialysable, fraction). The dialysis tubing was bent into a “V” shape, placed in a 25 ml Ehrenmeyer flask containing 9 ml of phosphate buffer (ionic strength 0.15, pH 7.4 without inclusion of antiseptics) and immersed therein. Both ends of the dialysis tubing were suspended by a rubber stopper and dialysis was carried out at 37°C for 18 h in a shaking water bath. After the completion of dialysis, the tubing was discarded unopened and the dialysate was mixed with 1 ml of pooled serum. The mixture was left at room temperature for 10 min and a scoopful of anion-exchange resin, Rexyn 201 Cl-SO, (Fisher Scientific Co., New York, N.Y., U.S.A.) was added to the serum/dialysate mixture. The resin and serum/diaiysate mixture was shaken vigorousiy for 2 min in the shaking water bath to allow contaminating inorganic iodide (in the form of 13’1 and 12’1) to be adsorbed onto the resin. Three ml was pipetted from the resintreated dialysate and placed in a counting tube (dialysable fraction). The radioactivity of the dialysable and total fractions were counted for 13iI and “‘1 simul~neously in an automatic well-type scintillation counter employing a dual channel spe~trophotometer (Model 4233, Nucle~~hi~ago Corp., Des Plaines, Ill., U.S.A.). The net count of 12’1 was corrected for background of 1311 in ‘251 region using a counting ratio of ‘2sIf’3’I obtained with a sample

225

containing only 13’I. The %FT4 (and %FT3) was calculated as a ratio of the count of the dialysable fraction to that of the total fraction (multiplied by 30). Serum free T4 (FT,) and FT3 concentrations were calculated as the product of total hormone concentration and the respective percent free value. 1. In vitro study A series of 1.2 ml aliquots of pooled serum was prepared, to which 10 ,ul _l? r\ -Im I___.:._-- ____ -_ aiuuf~w~ _,L..-_:._ 111 r._ 110mial ._---_-1 siamt: __,I._^ COiitZiiii~g VSjGig izrrlOiiIltS 01 u.170 uuvme serum of L-T~ were added to make final concentrations of 0,0.5,1,5,10,25 and 100 pg/dl elevated above the endogenous T4 concentration, Another series of serum aliquots was similarly enriched with L-T~ to give T3 concentrations 0, 0.5, 1, 5, 10, 25 and 100 pg/dl above the endogenous concentration. %FT4 and %FTI1 levels of these series of sera were determined in duplicate. 2. Clinical study No. 1 Four subjects with untreated primary hypothyroidism were treated solely with T3. They were given 25 pg T3 once in the morning for the first 2 weeks, _..E.:-1, : _^_^^^^ ,l cr. ..C c...:,, sa3,:,.. +1,,,,..Pc,.. I:, ,,A ,.%,A WlllCl‘ ._.^^ was ,11txeilwzu b” or LJ yg CWILX?uauy ciltxtxlictx \“’ CL, bllt: -,,,:,... 1I1”111111& callil c’vcning). Blood samples were obtained periodically as shown in Table I before and after the replacement with T3 and analysed subsequently for serum TSH, T4 and T3 concentrations as well as %FT4 and %FT3. The samples were drawn at approximately one pm., at the time of clinic visits. 3. Clinical study No. 2 Six subjects with T3 toxicosis [13] and 8 with the usual variety of hyperthyroidism were arbitrarily selected from patients attending our endocrine clinic. Their average serum T3 concentrations were comparable. Serum T4 and V “8.“.,-.,-.,..+rn+;~~nozT;lV GzVW A,-.+,-:,,A 13 L”IIL~IIuacI”II3) /or rq, 101‘ 13, FT, and FT3 were u~~cT~.Ll,llcx4 of 32iz from these subjects and comparisons were made of these values between two subgroups of hyperthyroidism. An unpaired t-test was used to determine statistical significance between two sub-groups. Results 1. In vitro study (Fig. 1) When pooled serum con~ning 8.7 pg/dl of T4 and 165 ng/dl of T3 was enriched with T4 or T3, %FT4 remained unchanged by the presence of added Tq or T3 less than 5 pg/dl and it showed progressive increases with higher concentrations of added T4 or T3. Similarly, %FT3 remained unchanged below 10 pg/dl of added Tq or T, and increased above this concentration. 2. Cli~icul study No. 1 (bugle I, Fig. 2) When 4 subjects with untreated primary hypothyroidism were given 25 pg of T3 orally daily initially, then 50 pg in two equally divided doses every twelve hours subsequently, progressive increases in serum T3 concentration were observed while T4 concentration, %FT4 and %FT3 remained unchanged. In the absence of increases of %FT3, FT3 appeared to increase solely depending upon

added

T4or T3

( rUd#)

Fig. 1. Effects of added Tq and T3 on %FTq and %FT3. A, effect of added T3 on “oFT4; B, effect Tq on “/oFTa; C, effect of added T3 on %FT3: D, effect of added T4 on %FT3.

of added

increases of serum T3 concentration. Consequently, in 3 subjects, FT3 and TSH concentration became normal after serum T3 concentration became hypernormal. In one subject (I.H.) given 50 pg of T3 daily, FT3 became within normal range but TSH concentration still remained in the hypothyroid range. 3. Clinical study No. 2 (Table II) When comparisons were made of serum thyroid hormone levels between subjects with T3 toxicosis and those with usual hyperthyroidism, %FT3 and FT3 of the former sub-group (0.187 * 0.033%, 0.51 k 0.22 ng/dl, respectively) were significantly lower (p < 0.01, p < 0.05) than those of the latter sub-group (0.263 f 0.051%, 0.79 * 0.21 ng/dl). Meanwhile %FT4 levels, 0.32 + 0.008% for the former and 0.033 f 0.003% for the latter, were not significantly different.

I I

0

ZSPB

day

a&

trllodothyrO”lM 3

I

1

1-Z

1-2

wks

3-4wks

5-6wks

Fig. 2. Changes of serum FTq, FT3 and TSH of subjects with primary hypothyroidism treated with triiodothyronine. Full lines, TSH; dotted lines. FT3: and fine lines. FTq. Shaded areas on ordinate are normai ranges.

TABLE

I

AND

kg

kg5

54 kg

I.H.

47

ST.

42

S.N.

detectable.

y weight

§Bd o

8

8

8

* Not

Normal

4.

3.

2.

55kg

1. K.O.

Subject

(6th

50 /a

before

replacement

0.05

i S.E.

less then

Range

Mean

therapy.

pUlm1.

7.4-12.4

0.018

* 0.005 -0.036 1.53

2.5

0.024

10.3

+ 1.5

0.09

0.023

0.4

(4th

50 fig T3

0.13

(2nd

0.025

0.08

0.026

0.5

0.07

0.022

0.03 0.06

0.020

0.22

0.025

0.020

0.20

0.24

0.33

0.27

0.22

0.19

0.028

0.024

0.024

0.039

0.031

0.027

0.19

“.A”

nrr.

n2n

n

0.026

V.““”

FT4

WITH

0.004

50 pg T3

week)

PATIENTS

%FT4

IN

0.08

TSH

0.020

0.2

0.6

week)

(Ccg/dI)

AND

0.014

0.3

(1st

week)

week)

0.3

25 /.% T3

(3rd

50 pg T3

week)

Control

(1st

50 /.a T3

0.2

0.3

0.9

0.9

25 /.a T3

week)

week)

week)

Control

(2nd

(4th

50 /x

T3

(2nd

50 fig T3

1.4

1.0

week)

0.7

0.7

0.7

0.7

0.5

25 /.a T3

week)

week)

week)

week)

T4

HORMONE

Control

(1st

(3rd

50 j&S T3

T3

(1st

T3

50 /.a T3

25 /a

(1st

THYROID

Treatment

FREE

SUPPLEMENTATION

TOTAL

-

3.42

+ 0.5

(ne/dI)

PRIMARY

113

147

235

170

165

45

255

260

120

46

310

340

170

75

205

245

220

190

30

T3

-

*

185

19

(m/dI)

0.138

0.175

0.094

0.086

0.111

0.094

0.107

0.097

0.086

0.078

0.105

0.089

0.019

0.109

0.170

0.130

f -

0.026 0.239

0.17

0.25

0.22

0.15

0.18

0.04

0.27

0.25

0.10

0.04

0.33

0.30

0.15

0.08

0.34

0.33

0.29

0.21

0.130

0.03

FT?

AND

0.114

_

BEFORE

0.112

%FT3

HYPOTHYROIDlSM

-

0.33

? 0.05

(w/dI)

AFTER

4.5