209

Clinica

Chimica

0 Elsevier

Acta,

Scientific

59 (1975)

Publishing

209-213 Company,

Amsterdam

-- Printed

in The Netherlands

CCA 6877

CONVERSION OF THYROXINE (T4 ) AND TRIIODOTHYRONINE (T3 ) AND THE SUBCELLULAR LOCALISATION OF THE CONVERTING ENZYME

R.D. HESCH*, Department

(Received

G. BRUNNER

of Medicine,

October

and H.D. SOLING

University

of Gijttingen,

(G.F.R.)

9, 1974)

Introduction The majority of biochemical investigations on the effects of thyroid hormones have ignored the possibility that thyroxine (T4) and triiodothyronine (Tj ) may have different actions at the subcellular level [l].It has been known for some time that in vivo T, is converted to T, and recent reports have further clarified this process [2,3]. In addition it has been demonstrated that T4 and T3 have different subcellular binding sites and that nuclear receptors have a higher affinity for T3 than for T, [4,5]. It is still controversial as to whether or not T, acts only as a prohormone for T3 [6]. Whereas earlier investigations used radiolabelled hormones, and subsequent chromatography to demonstrate the conversion of T4 to T3, this communication describes the use of direct radioimmunoassay for the measurement of T3. The results demonstrate that T4 is converted to T3 by the intact liver and by isolated liver cells. The conversion takes place in the microsomal fraction of homogenized liver.

Materials

and Methods

Rut livers (male Wistar rats, weight about 240 g, liver weight 6-7 g) were perfused in a recirculating system after a recovery phase of 15 minutes as previously described [7]. The flow rate was 6 ml/g/min. Initially a 30 minute perfusion with T4 -free medium was carried out, followed by a further 105 minute perfusion with T4 -containing medium (32” C). Aliquots of the medium were measured in the radioimmunoassay without prior extraction. Isolated liver cells were prepared by a modification of the method of Berry and Friend [8], (Soling et al., 1974, in preparation). * The laboratory is supported by a grant of the Deutsche Forschungsgemeinschaft

He 593.

210

100 and 200 1-11 aliquots of liver cells, equivalent to 15-25 mg dry weight, were incubated at 37°C in 2 ml medium, with or without T4 for the times indicated. After incubation the cells were sedimented by centrifugation at 500 X g. The cell pellet was sonicated, rapidly frozen at -80” C and lyophilized. T3 was measured in the supernatant and the resuspended pellets after extraction with absolute ethanol. Aliquots of 50 1.11 of the extract were assayed directly in the radioimmunoassay [9] . As a control, liver cells were immediately frozen at -80°C after addition to the medium. Subcellular fractions of the liver were prepared by differential centrifugation by the method of Schneider [lo], in a medium containing 0.25 M Sucrose, 10 mM triethanolamine buffer and 1 mM EDTA (PH 7.3). The supernatant from the first mitochondrial sedimentation step was centrifuged at 17 000 X g for 20 min to remove fragmented mitochondria and then at 144 000 X g for 60 min. The pellet obtained at this point is referred to as the “microsome” fraction. 10 mg of the respective fractions were incubated in 2.0 ml incubation medium for different time periods and with varying concentrations of T, ,at 37°C. The reaction was stopped by rapid freezing at --80°C. Control tubes were immediately frozen after addition to the medium. After thawing at 2 to 4”C, 100 ,ul of the medium were extracted with 200 ~1 of absolute ethanol and 50 ~1 of the extract were assayed as previously described [9]. The incubation medium for all experiments (perfusion experiments, liver cell incubation, cell fractions) was Krebs-Ringer bicarbonate buffer containing 0.25% (w/v) human serum albumin (Behring-Werke, Marburg). T4 was a highly purified preparation (containing 0.3% T3 ) from Dr Henning, Berlin-Tempelhof. For liver perfusion the T4 concentration was 10 pg/lOO ml. Liver cells and subcellular fractions were incubated with 1000 ng T4 /2.0 ml of incubation medium. For kinetic experiments increasing amounts of T4 (25-10 000 ng) were used. Unless otherwise stated the incubation time was 60 minutes. T3 was either measured directly in the medium (100 ~1 per tube), or ethanol extracts (50 ~1) were added to the radioimmunoassay system as described elsewhere [9]. Each experiment was carried out in duplicate and each incubation sample was measured in triplicate in the assay. The control value (chilled at zerotime) was subtracted from the test value after incubation. An out-layer-screening in the on-line-computer calculation rejects measurements with a deviation greater than ? 5% (S.D.). Results In Fig. 1 is shown a typical curve of T, generated during liver perfusion with T4. Conversion starts after about 10 minutes and exhibits a curvilinear behaviour for another 20 minutes. The conversion is then linear over a further period of 75 minutes. During a 30 minute period of perfusion 35 pg of T3 were generated per 100 ~1 of medium; the total amount of T3 generated by the whole liver was therefore approximately 450 ng or 2.5 ng per g liver per minute. Without T, in the medium T, could not be detected in the effluents. After incubation of T4 with liver cells, T3 could only be detected after disruption of the cells by sonication. There was no detectable T3 in the super-

211 13 (Pgl gmemted

from

1‘

m 100,ut of the medrum assayed L IVERPERFUSION

100

60

LO

20

80. :/!I/ 10 Fig.

Perfusion

1.

minutes

and

TABLE

I

Incubation

20

the liver

is linear

between

of isolated

Tg formed

by

LO

of

liver

the sonicated

60 with

80

30 and

cells cells

Total

100 mm

Tq-containing

(15

105

and

is given

T3 formed

medium

(10

fig/100

ml).

Generation

of T3

25

mg)

in pg/mg

with

T4-containing

medium

T3 formed

(1000

ngj2.0

(pg)/mg

60

100

/.d cells

860

990

9.2

10.6

200

/.d cells

1362

1062

14.6

11.4

INCUBA T/ON OF MICROSOMES

Fig. ble

2.

Microsomes

after

by further

6 minutes. metabolism

(10

mg)

were

It reaches of T3.

ml).

The

total

min

WI TH r,

PERIODS

incubated

a maximum

10

protein

30 min

60 min

TIME

after

cell protein.

(pg)

30 min

FOR DIFFERENT

starts

min.

in Tqcontaining after

60

minutes

medium.

Conversion

and

decreases.

then

of T4 This

to T3

could

is detecta-

be explained

212 TABLE

II

Incubation homogenate

of subcellular fractions of liver (10 mg protein) and the microsomal fraction convert Tq to T3.

with

Tq-containing

medium.

Only

the

_____ Incubation buffer Homogenate Mitochondria Mitochondria (sonicated) Microsomes Cytosol

100% 185% 111% 108% 600% 95.5%

natant. The amount of T3 generated by the cells is given in Table I. Lower levels of T3 were observed after incubation for 60 minutes with 25 mg of liver cells. At the subcellular level, significant conversion of T, to T3 could only be detected with the homgenate and the microsomal fraction (Table II). The microsomal conversion was measurable after 6 minutes and reached a maximum after 60 minutes (Fig. 2). After 120 minutes a decrease in measurable T3 was observed, similar in character to the results obtained with isolated liver cells. In the absence of microsomes there was no spontaneous degradation of T4 to T, during the incubation period. The amount of T3 generated from increasing amounts of T, is shown in Table III. Even at a concentration of 10 r.lgl2.0 ml the enzymatic system in the microsomal fraction is not saturated. From the decreasing percentage of T, converted to T3 it can be assumed that saturation can be expected at higher concentrations of T, . Discussion Recent results have indicated that a greater understanding of the conversion of T, to T3 is necessary in order to elucidate the biochemical effects of these hormones on the metabolic status of disturbed thyroid function [ 111. With perfused rat livers the conversion of T4 to T3 is linear with time after a delay of 30 minutes. The exact amount of T3 cannot be accurately

TABLE

III

Microsomes (10 mg) were incubated with increasing percentage of T4 converted to Tj decreases, a saturation

T4 (ng)

T3 (pg) generated per mg microsomal protein

% of T4 converted to T3

10000 5 000 1000 500 250 25

1248 804 756 324 288 60

0.012 0.016 0.075 0.064 0.115 0.240

amounts (25-10000 ng) of of the system is ndt observed.

T4.

Although

the

213

calculated for this experiment since an open perfusion system is necessary for this purpose. In addition corrections should be made for the further deiodination of T3 by the liver [12] . However, the high capacity of the liver for T4 conversion is immediately evident. Isolated liver cells also convert T4 to T3 although the T3 formed is measurable only in the disrupted cells and not in the incubation medium. This is in agreement with the results of Sterling et al. [13]. Increasing the amount of liver cells and the incubation time does not lead to a proportional increase in T3, presumably as a result of conversion to other iodothyronines. The enzyme converting system is located in the microsomal fraction of homogenized liver. This, to our knowledge, is the first evidence that the converting system is confined to a particular subcellular fraction. The amount of T3 formed increases with time, reaching a maximum after 60 minutes. The subsequent decrease may be due to a number of reasons, the most likely of which is that further metabolism of T3 occurs at a greater rate than the T4 to T3 conversion. Preliminary kinetic experiments reveal that the in vitro system is only saturable appear that the at unphysiologically high levels of Tq. It would therefore converting system is never saturated in vivo. The converting activity of isolated microsomes is 75-fold higher, on a protein basis, than that of intact liver cells. Our results confirm reports on the in vivo and in vitro conversion of T, [2,3,13] and, in addition, a quantitative estimate of the system has been made by direct radioimmunoassay. It is not possible to conclude whether or not T3 is the only active hormone [6] but the velocity of the conversion process in the microsomal fraction would suggest that prior to any known biochemical effect a considerable amount of T, is metabolized to T3. The latter may then of T, , bind with high affinity to other subcellular fractions [4,5]. The inhibition of the converting system will show whether or not T, alone has any biochemical effects. References 1

J. Robbins

2

R.

3

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I.E.

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