Endocrinol. Japon.

An

1979, 26 (1), 1-7

Attempt to Analyze Various Thyroid Stimulators the Receptor Assay using hTSH Radioimmunoassay

by

YuKIo OCHI1, TAKASHIHACHIYA2, TADAYOSHIMIYAZAKI2 YOSHIHIROKAJITA AND MANABUYOSHIMURA2 1Second

Department of Internal Medicine, Shiga University of Medical Science, Otsu, Shiga520and 2Second Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, 602, Japan

Synopsis In

an

attempt

procedure petition. by

The

other

thyroid unlabelled

bovine

receptor,

samples

level

activity

like

This

of TSH

is

in

On

negative

show with

serum

showed

receptor

increased

TSH

method

hormone

value other

is

or

over

hand,

the to

in

animal

serum

can

useful

for

can

be be

also

determined

be

due

sensitivity

difficult

this

by

RIA

Evidence has been accumulated that polypeptide hormones bind to specific highaffinity receptors located on the cell surface of each target organ. There are several reports of radio-receptor assays of TSH and thyroid stimulating immunoglobulin (TSI) of Graves' disease (Amir et al., 1973; Manley et al., 1974; Smith and Hall, 1974; Verrier et al., 1974; Mehdi and Nussey, 1975; Mukhtar et al., 1975; Schleussner et al., 1975; Teng et al., 1975; Endo et al., 1976). In these assays labeled TSH binds to the specific receptor preparation in a Received

January21,

1978.

has

the

bound

or

(0.1ml)

or

free

high

thyroid

more

assay

of

activity

from

the

and42sera did

activity

of

not LATS

method.

because by

as

stimulating

derived

binding

clinically

that

such

than5ƒÊU/ml

hTSH LATS

lower

serum

range,

the

the

human

hTSH.

with

the

in

useful

hyperthyroidism

this

previously

serum

untreated

determined

examining an

to of

assay be

with

with

comdisplaced

with

hTSH

the

that the

by

hTSH which

than in

serum

sera

patients

lower use

of

estimated

might

hTSH

human

eighteen

from This

the

of

test

assay

RIA. other

toxin

displacement for

an

receptor

bound of

by

Cholera

RIA

were

stimulators

TSH.

is

may that

rat

developed with

preparation

addition

measured

of

we

by

hTSH

by

was

serum

combination

receptor of

thyroid

the

activity

hTSH

it

ox

applicable

displacement.

thyroidal

of0.5-4.0ƒÊU

also

assay

in

determination

displaced

the

not

any

a

(0.1ml)

TSH

the

is the

hTSH

activity

LATS

Although

method

determine

the

serum.

with

this

then

bovine

assay because

test

this

in

(RIA)

from

and

can

stimulators

Practically4ƒÊU

displacement

mU/ml

TSH,

of

hTSH.

assay

the

thyroid

radioimmunoassay

stimulators

dissolved

This

analyze

hTSH principle

bound

has

to

using

this

receptor

of assay. binding

its

low The of

sensitivity, principle other

of peptide

method.

membrane or solubilized receptor preparation and is displaced by unlabeled TSH or TSI. However, the sensitivity of the assay was reported to be limited due to the fact that the extent of binding of the labeled hormone to the membranes was affected by the presence of serum proteins or of salt or bivalent cations in the incubation medium. The authors attempted to analyze animal TSH or other thyroid stimulators by radioimmunoassay (RIA) of unlabeled hTSH which was displaced from thyroid membranes by the thyroid stimulators to be assayed. This was expected to avoid the

OCHI

2

loss of biological activity of the hormone by the labeling procedure and the nonspecific influence of proteins in the conventional receptor assay. We would like to describe the experimental results by this receptor assay method.

Endocrinol. Japon. February1979

et al. consisted 2%

of0.5ml

normal

antiserum

for

dilution) was

and

up

and

bation

was

LATS

bioassay

Bioassay of

the

of

hTSH

was

performed

Mckenzie

by

method

a

(Ochi

minor

et

modification

al.,

fractions

from

human

and

et

bovine

volume

thyroid Thyroid

glands

with

Graves'

were

homogenized

10mm

The

the

fraction.

Receptor

obtained

by

to

Nagai's

600•~g

method

layer

min,

After the

sucrose

were

Assay

two

band were

stored

procedure.

of0.15M,

pH7.8phosphate

the

mixed

with0.2ml

TSH

(hTSH,

incubated ml

of

buffer

test

serum by

continued 2,

for

hTSH

divided RIA

duplicate

In

the

at5•Ž, and as

the each0.5ml

temperature.

were

blood

injection

was of

test

of positive

After

min, Mckenzie

and

LATS

the

cen-

supernatant

bioassay injected obtained

(Ochi ip

in

a

at9and

sample.

Results

A) Preliminary experiment Components in assay system; A typical standard curve of the RIA for determination of hTSH, using0.1ml of test serum is shown in Fig.1. This figure represents

frac-

according

receptor

Lab.,

assay, (PB)

Tokyo),

and several

fraction

and

(NHS

the

incuba-

hours

centrifugation supernatant

was human

serum

added

con-

Then0.2

human

following

After

of

the

purified

absorb4ƒÊU

stimulator in

temperature.

the

to

samples

PM of

two

receptor

of

was

by

the

for60

containing8ƒÊU

normal

by and8mg

the

between

protein)

RIA)

3and6hrs).

for30min

was

or

TSH

then after

of0.32M

(0-100mg

room

and

performed

used.

Daiichi-radioisotope at

to

buffer

fraction

for60min

1ƒÊU/ml tion

performed

receptor

was

applied

These

thyroid

following

between

solutions

until

the

plasma according

interface

collected.

for were

was

at81,500•~g

at-20•Ž

procedure

taining

was

the

assays

receptor

thyroid

fraction

sucrose

at

crude

for12.5min

fraction

All

0.6ml

g

The

incu-

of

obtained

centrifugation

milky layers

tions

the

containing

and1.2M.

to

(1977).

first

at5•Ž, was

for30

Assay

The

Purified

was

of

The

Kobe)

water.

bovine

method.

curve

at10,000•~g as

from

room

assayed

the

SF.

with0.1ml

at

of0.5ml after

the

enough

at15,000•~g was

standard

of

PM

thyroid

1977).

A

containing0.1ml

activity

crude

for15min.

used

and20,000•~g

Then,

upper

ice

centrifuged

fraction)

for10min

obtained.

in

was

similar

(receptor

24hrs

patients

with5volumes

was

pellet fraction

a

Clinic,

at800•~g

fraction and

from

Thyroid

pH7.6,

centrifuged

supernatant

for30min,

operation

gently

buffer, was

membrane

at

(Kuma very

tris-HCl

homogenate

1,

obtained

disease

LATS

for30min

al.,

rabbit 20,000

PB

for3days

incubated

trifugation fraction

Receptor

human

of

NIAMDD)(1:

antibody.

of

were

sera

1977).

mg

containing

of

(24hr)

of

Thirty PM

instead

second

PB, 0.1ml

125I-hTSH.

performed

Absorption

Methods

by

incubation the

of

(supplied of

hTSH

0.2ml (NRS),

using0.5ml

second

adding

Materials

hTSH

SF,

serum

and0.1ml set

NHS

a

of

rabbit

at fraction was

(0.5,

15,000•~ (SF) used

for

follows.

hTSH-RIA The standard curve of RIA for hTSH using0.1ml of test serum was set up according to the design of the NIH TSH kit. The incubation medium was

Fig.1.•@ serum

Standard (ml)

curve by

double

of

RIA

antibody

for

hTSH

method.

in

test

Vol.26, the

RECEPTOR

No.1

average

assay

in3experiments.

coefficient

6assays 2,

for

5,

15,

of

10,

14,

measured

in

were10,

8,

curve

8,

was

respectively.

up

gradually. of of

hTSH

was

determined

fraction

after

of

4

receptor

the

the

varying and

serum

of

than0.3ƒÊU.

it

is

of

less

serum

subtraction. hTSH

in

that

possible

were to

having

receptor

test

the

(NHS)

Thus

displac-

by

by

stimulator

activity the

hTSH

displaced

serum

by

the

than

4ƒÊU

by0.1ml

measure displace of

test

the less

hTSH

serum.

frac-

supernatant

fraction

calculated

thyroid

of

receptor

were

control

ment

absorb

receptor the

test

activity

amounts

thyroid

amounts

normal

the

suitable to

of

containing

the

(i.e.,

the

from

of

displacement

The the

the

from

centrifugation

medium

from

of

examined. by

serum;

ed

and then flat-

fraction

were hTSH

of

(0.1ml)

to40ƒÊU/ml

receptor

test

The

Therefore,

the

in-

samples

through8assays

amounts

ties

above

duplicate

steep

Absorbed

The

the

7and8%,

tended

ed

of

of test serum)

μU/0.1ml

4ƒÊU

each

displacement activity examined at30min.

Calculation

was18,

respectively. for

3

hTSH-RIA

to

TSH/ml

12and17%, CV's

from

corresponding

USING

the was

inter-

(CV's)

sample

20and40ƒÊU

traassay

tion

The

variations

each

ASSAY

incubatquanti4ƒÊU

of

B) Application Animal TSH and cholera toxin; Various amounts of bovine TSH (bTSH, Thytropar, Armour Comp., Chicago) in0.1ml NHS

hTSH. As was

shown

in

augmented

receptor

fraction

tionship of

Fig.2, by and

was

the

absorbed

increasing a

More

receptor

than20mg obtained

from

bovine

plete had

absorbing activity. Human less absorbing activity.

purified

showed

receptor

used,

8mg

of

of

necessary

to

rela-

fraction

thyroid

almost

human

the

of

dose-response

obtained.

crude

hTSH amounts

receptor

absorb4ƒÊU

comthyroid

When

the

thyroid

was

fraction

was

of

hTSH

com-

pletely. Conditions cation

for of

ed

with

mg

8mg

assay;

or

the

hTSH. on

ceptor

was

RIA)

displacement

was

room

temperature.

5mU

of

showed

the

fraction

effect

of0.1ml from

When was

human of

absorbing

hTSH

used,

observed

containing

strong

of20 of

4ƒÊU test the

NHS no

re-

(TSH, Fig.2.•@

significant

within6hr

However,

bTSH a

for

observed. by

fraction

receptor

thyroid

displacing

1ƒÊU/ml

perform-

or30mg

purified

Then

serum

appli-

was

receptor

thyroid

human

practical

assay

crude

bovine

of

The

receptor

the

of

thyroid,

of

the

at

more NHS

displacement

than

receptor

change

Displacing

Upper:

within6hr

incubation.

did Therefore,

hTSH

from

by

Crude

of

not

the

bovine

PM

crude

thyroid

from

Graves'thyroid

(0.1ml) at30min

activity

of

fraction

or

purified

or

Graves'

thyroid.

Lower:

incubation.

Absorption

All

used

thyroid for

or

absorption

hTSH.

Purified used

bovine was

PM for

values

from

Graves'thyroid

absorption are

of expressed

was

hTSH. as

mean•}SD.

4

OCHI

were by

assayed.

The

hTSH-RIA.

In

more

than

placed

5mU

hTSH

human

of

thyroid.

eptor,

the

was

shown

bTSH

N.

Y.)

in

or

by

toxin

In from

bTSH.

cholera

et

the rats

al.,

of

ob-

cholera

activity

receptor.

not

dose-

was

of

However,

show

any

cross

re-

hTSH.

When treated

The hTSH

than1ƒÊg

the

experireceptor

displacement

did

with

other

(Schwartz-Mann,

of

from

toxin

action

bTSH (2.2-0.3)

(Fig.3).

the

hTSH

re-

human

toxin

More

showed of

bovine was0.3ƒÊU.

the

displacement

served

dis-

of1mU

displaced

examined

of

bTSH

hTSH

Table1.

cholera

was

dependent

4ƒÊU

from (NHS)

as1.9ƒÊU

hTSH

by

of

activity

dis-

receptor

mU

hTSH

displaced

calculated

ment,

completely crude

control

detected

experiment,

bTSH the

of the

not

typical

One

while

Thus

was

the

from

placed2.2ƒÊU

as

bTSH

Endocrinol. Japon. February1979

et al.

serum

of

containing

1974)

with

normal

also

produced

was rabbit

methyl-thiouracil

increased

TSH

assayed

after

serum

(NRS),

complete

(Ochi dilution

rat

Fig.3.•@

displacement

hTSH

bTSH

TSH

or

Upper:

of

displaced cholera Displacement receptor

hTSH

(Table1).

Sera

of

Test

sera

hyperthyroid

(TSH250% at9 hrs in assay response) in LATS bioassay, were examined for displacement activity with the receptor fraction of human thyroid or bovine thyroid. However, no positive

5

hTSH-RIA

displacement cases

was

observed

(Fig.4).

sera

were

ment

by

displace-

the

LATS

PM

absorbed

that the

this

incubated

and8mg

enough

LATS

by

with

were PM

were

hTSH,

LATS

When4sera

crude

than25%

ment

the

(0.1ml)

of

of

less

significant

PM;

activity

with30mg

4ƒÊU

these-

no

of

activity

purified

of

observed.

Determination

high

any

negative

tested,

was

in

When42LATS

of

to

activity

absorb was

absorption

lost

experi-

(Fig.5).

Discussion

Fig.4.•@

Receptor

of

assay

untreated

tivity

of

(open

value

in

LATS

hyperthyroidism. hTSH

from

circle)

or

positive

sera

Displacement

the

crude

bovine

receptor

thyroid

acof

(closed

Several investigators have suggested that two thyroid stimulators, TSH and LATS might interact with the same receptor site on thyroid in the radio-ligand receptor assay (Manley et al., 1974; Mukhtar et al., 1975; Schleussner et al., 1975; Teng et al., 1975). However, there is also reports that TSH and LATS do not bind to the same receptor site (Amir et al., 1973). Mehdi's paper (1975) suggested that the receptor site on the thyroid membrane for TSH and LATS were intimately related, if both were different. The questions are remained open whether LATS and TSH have the same receptor site. The discrepancy in these previous reports may be due to the inaccuracy of the method of receptor assay. Several problems may upset the receptor assay. One is the loss of receptor binding activity during the preparation of the thyroid tissue. An other is the loss of binding ability of the receptor due to loss of biological activity of the hormone by radioiodine labeling. Another is the influence of the protein con centration in the incubation medium on

human

circle)

by

LATS.

Fig.5.•@

Absorption

positive of human

sera

LATS

activity

Each

experiment

All

values

*Test ceptor

of

LATS

PM. was

was

activity

positive performed

expressed

receptor

The

assay

This

means

as

with

increased

assay

was that

TSH

sera.

of values

hTSH-RIA were

to

the

determination (more

PM of

mean•}SD.

compared the

LATS

in4mice

having40•`200ƒÊU/ml assay.

in

Crude or purified used for absorption

in4LATS

are

sera

the

by the thyroid

than5ƒÊU/m/)

of

(diluted

similar value

to by

increased should

properly values

RIA,

a

hTSH not

with

obtained high

correlation

appears be

used

NHS) by

to for

be this

was

RIA.

examined When

was

observed

meaningless receptor

by the

and assay.

the

values

reby

(r=0.93). that

sera

6

OCHI

the

binding

of

labeled

Schleussner false

et

positive

results

concentration was

changed.

TSH It

by

vestigators tion

from

found if

is

substances receptor

isolate

the

test

protein

assay

impossible

the

that

the

receptor

stimulating

directly

(1975)

occurred

in

thyroid

hormone.

al.

system

to

determine

in

the

assay.

serum

Most

in-

gamma-globulin

serum

frac-

(usually1mg)

for

the

assay. In of

this

experiment,

biological

activity

labeling4ƒÊU

of

biological the

ment

of

receptor

by

avoid

oretically, the

displacement

μu

of hTSH did

of

rat

usually.

or100ng placing

fact

of

might

finity

be

of

human the

PM LATS

activity of

the

amount

the

LATS

amounts

was

TSH

finity

to

membrane cyclic determined

is

the

the

less

of

hTSH.

than25%

This

means

enough

absorb

the

to

Acknowledgements

large

absorb

the

completely.

is

known

to

action specific

to

have bind

receptor

and

to

AMP.

increase In

by

on

the

the

the with

the mean

stimulatory

thyroid high

human

af-

thyroid production

while

mouse thyroid in the Mckenzie bioassay. The stimulatory action may be caused by the specific binding of LATS with mouse thyroid membrane. There are several evidences that LATS has less stimulating activity for human thyroid (Adams, 1975; Mckenzie and Zakarija, 1977). Smith's data (1974) that showed no significant correlation between the values obtained by receptor assay and LATS biological activity might be explained by the lack of specificity of LATS for the human thyroid receptor. In this receptor assay it is impossible to draw conclusion whether LATS does compete for the same receptor as TSH. However, it is interesting that the bovine and rat TSH which do not react immunologically with hTSH have the displacement activity of hTSH from the receptor. Cholera toxin is known as the universal stimulator of adenyl cyclase in various organs. Mullin et al. (1976) reported that cholera toxin stimulated adenyl cyclase and also bound with the same receptor of TSH in the thyroid. In the previous paper we demonstrated the thyroid stimulating action of cholera toxin (Ochi et al., 1977) and in the present experiment we confirmed an evidence for the binding of both cholera toxin and TSH to the same receptor. Although this receptor assay is not sensitive enough for the clinical use, the fundamental basis of this method may provide as a tool for the study of hormone receptor.

that

to

that

needed

ab-

dose of

and

are

in LATS

from

not

af-

of

which

PM.

activity, PM

activity

stimulating

binding

absorbed

PM

This

receptor

absorb4ƒÊU

purified of

of

LATS

to

high

positive PM.

TSH

in

dis-

binding

observed

enough

by8mg

with

low

the

experiment was

sera

the

the

The was

potent

any

from

to

bTSH

a

show

hTSH

NHS

displaceof

had

not

with

PM

sorption

than40

than0.3mU

due

LATS

less

serum.

significant

TSH

thyroid.

with

is

any

did

displacement

thehaving

of

Eighteen

activity

RIA.

detected

of test

More

activity.

LATS

The by

be

activity

to

protein.

stimulators

in1ml

ob-

serum

determined

thyroid

show

was

test

of

can

not

displace-

receptor of

was

the

with directly

Then,

the

assay

loss

preparation

effect

hTSH

the radioiodine

incubated

fraction. from

receptor

ment

by

hTSH

nonspecific

displaced

NRS

avoid

adding0.1ml

the

This

to TSH

was

hTSH

served

of

a

activity

with

or

of

Endocrinol. Japon. February1979

et al.

LATS action

of

The author is grateful to Dr. Kuma for the supply of thyroid specimens and wishes to thank Dr. L. J. DeGroot (University of Chicago) for his valuable comments. The author wishes to thank The Hormone Distribution Office (NIAMDD), for supplying the preparation of rabbit antiserum against hTSH, and the materials for TSH-RIA. This work was supported in part by Research Grant (No.287067) from the Ministry of Education,

Vol.26,

No.1

RECEPTOR

Science and Culture of Japan. thanks Miss Akiko Moridera for ance.

ASSAY

The author also secretarial assist-

References Adams, D. D.(1975). N. Z. Med. J. 81, 22. Amir, S. M., T. F. Carraway, Jr., L. D. Kohn and R. J. Winand (1973). J. Biol. Chem. 248, 4092. Endo, K., J. Konishi and T. Mori (1976). Abst. V Intern. Cong. Endoc. p.26. Manley, S. W., J. R. Bourke and R. W. Hawker (1974). J. Endocr. 61, 419. McKenzie, J. M. and M. Zakarija (1977). Pecent Prog. Horm. Res. 33, 29. Mehdi, S. Q. and S. S. Nussey (1975). Biochem. J. 145, 105. Mukhtar, F. D., B. R. Smith, G. A. Pyle, R. Hall and P. Vice (1975). Lancet 1713.

USING

hTSH-RIA

7

Mullin, B. R., S. M. Aloj, P.H. Fishman, G. Lee, L. Kohn and R. O. Brady (1976). Proc. Nat. Acad. Sci. 73, 1679. Nagai, Y. and T. Hosoya (1974). J. Biochem. 75, 1091. Ochi, Y., T. Hachiya, M. Yoshimura, T. Miyazaki, T. Majima, I. Kaimasu and H. Takahashi (1974). Endocrinol. Japon. 21, 459. Ochi, Y., T. Hachiya, T. Miyazaki, Y. Kajita and M. Yoshimura (1977). ibid. 24, 277. Schleussner, H., P. Kotulla, I. Kruck, G. Kruck, G. Geissler and F. Adlkofer, Thyroid Research (edited by J. Robbins and L. W. Braverman). Excerpta Medica, Amsterdam p.414 (1975). Smith, B. R. and R. Hall (1974). Lancet II 427. Teng, C. S., B. R. Smith, J. Anderson and R. Hall (1975). Biochem. Biophys. Res. Common. 66, 836. Verrier, B., G. Fayet and G. Lissitzky (1974). Eur. J. Biochem. 42, 355.