Comparison of plasma corticosterone- and progesterone-binding activity in rat and human

355

2669

COMPARISON OF PLASMA CORTICOSTERONEACTIVITY Steve

E.

IN RAT AND HUMAN

Calvano

and

Department University Santa Barbara, L.

AND PROGESTERONE-BINDING

Robert

W. Reynolds

of Psychology of California California

Donald

93106

Keith

Division of Endocrinology of Oregon Health Science Portland, Oregon 97201

University

Center

Received-Z-80 ABSTRACT Corticosterone-and progesterone-binding activity were measured by with dextran-charcoal separation, in plasma obsaturation analysis, tained from male and female rats, and a normal male and female human. In plasma from normal, male and female rats, progesterone was much less H-corticosterone from effective than corticosterone in displacing plasma protein binding sites although the parallelism of the displacement curves indicated competition for the same binding sites. In and progest rone were plasma from the normal male huma?, corticosterone H-corticosterone. However , 4Hequally effective in displacing progesterone showed no apparent binding to either rat or human plasma proteins, suggesting that dextran-charcoal effectively removed progesterone from transcortin binding sites at 4°C. This observjtion was cjonfirmed by multiple equilibrium dialysis. In dialysis, H-corticostIerone and Hgprogesterone were bound equally by human plasma, but rgt plasma bound H-corticosterone to a much greater extent than it did Hprogesterone. These data indicate that, in contrast to human plasma, r’at plasma has much greater affinity for corticosterone than for progesterone. Plasma

corticosteroids

are

glycoproteins

(cortisosteroid-binding

hlumans,

rats,

and

is

bound

to

also

ation for

of

other CBG

progesterone

saturation

Vohme 36, Number

(6,7).

3

of

by high

(l-5).

The

In

limited

human CBG has serum

S

affinity,

globulin,

mammals

with

analysis

bound

or

plasma

TEBROXDD

low

CBG,

transcortin)

human plasma,

capacity, provided

capacity

high

in

progesterone affinity

the

essential

progesterone

(8,g).

associelement

September, 1980

It the

is

clear

Using

rat.

meters

less

for

rat

the

and

capacities,

corticosterone

were

considerably

corticosterone

two

male

rats,

functional

rat.

binding

progesterone

progesterone

is

to to

same binding

of

see site

high

the

some moiety with

displace if,

in with

studies

low

high

relatively

whether protein

other In

from

equal

that

in than

addition,

these

of

dis-

of

obtained a known

sex. progesterone the

rat,

the

steroids

and

and

CBG capable

human CBG was

of

a high

As a further

absence this

and

for

plasma

to

both

than

utilized

determine

corti-

however,

capable

in

plasma

(8))

sites.

and

para-

progesterone

progesterone

affinity.

species,

not

binding (10,ll)

corticosterone this

was

in

constants

reported

concentration

rat

and

progesterone

same binding in

serum

(11)

affinity

binding

Murphy

for al.

the

bound

association

that

affinity

plasma

for

the

same protein.

attempted

compete

there

order

the

in

et

similarly

progesterone

progesterone

above

effort

whether

of

the

significance present

the

Keller

from of

both

concluded

plasma

despite

corticosterone

in

by

is

determined

similarities

bound

the

(10)

CBG with

Westphal

corticosterone

The

the

concentration

complication, from

of

lower in

physiological placing

of

progesterone

Westphal

interaction

Because

found

plasma

sera,

costerone. binding

that

of

ability

of

determined occupy

the

affinity.

METHODS Human blood was obtained by finger prick from a healthy male, age 25, and a healthy female, age 28. Rats were of the Sprague-Dawley strain. Blood was collected by tail tip incision (12), centrifuged, and the plasma removed and stored at -15’6 unti 1 determinations were made. Removal of endogenous steroids was accomplished by adsorption with dextran-coated charcoal (13). Briefly, an aliquot of plasma (50 ul for male rats and 25 1-11 for human and female rats) was incubated with 5 ml dextran-coated charcoal suspension (0.025% dextran T-70, 0.25% Norit A in phosphate-gelatin buffer, 0.1 M phosphate, 0.14 M NaCl, 0.1% sodium azide, pH 7.4) for 1 h. at 37°C. Suspensions were centrifuged (30009

S

TIIROIDS

One hundred microliter x 10 min) and the supernatants retained. aliquots (containing ca. 1 ~1 steroid-free plasma for male rat samples and 0.5 ~1 steroid-free plasma for male humTn and female rat samples were incubated with 15,000 cpm of (1,2,6,7H)-corticosterone (ca. 75 pg, S.A. 82 Ci/mmol, New England Nuclear) plus varying massesof unlabeled progesterone or unlabeled corticosterone (0-, O.l-, 0.4-, l-, 1000 ng, Sigma) for 1 h at 22’C agd 12 h at lo-, 40-, loo-, 400-, 4-s Identical aliquots were also incubated with (l,2,6,7H)-proges4°C ,, New England Nuclear) under similar terone (ca. 75 pg, S.A. 98 Ci/mmol, High-affinity conditio= but without addition of unlabeled steroid. bound steroid was separated by incubation with 1 ml dextran-coated charcoal suspension for 15 min at 4°C followed by centrifugation (30009 x 10 min). The supernatant was decanted into scintillation vials (Blovials, Beckman) containing 2 ml of scintillation solution (0.2% 2,5-diphenyloxazole in toluene) and counted to 3% error in a Beckman LS-150 scintillation counter. Multiple equilibrium dialysis was performed by dialyzing steroidfree male and female human, an9 male and female fat3plasma, at a diluH-corticosterone or H-progesterone at tion of 1:200, against either a concentration of 125 pg/ml (ca. 25,000 cpm/ml). Dialysis was carried out at 4°C and terminated afteF72 h. Aliquots of the inside and outside solutions were removed, and the radioactivity determined as described above. RESULTS In

plasma

effective

than

affinity

(11,

sites

indicated

Furthermore,

male

binding

14),

female

rat

but

little

was

evident. In

the

compete

by

the

of

increasing

to

human plasma

saturated

of

male

to

with

plasma

in

the for

affinity

either

which

high

3H-corticosterone.

of

the

greater

the

curves unlabeled affinity

been

previously

binding

capacity in

and

(Fig.

steroid

corticosterone

same binding 2.)

sites

generated

progesterone

or

lack

of

binding

high

1).

was

no evireported

than

male

binding

appeared as evidenced by addition

corticosterone

corticosteroid-binding The

less

from

(Fig.

there

difference

progesterone

much

displacements

sites

separation,

sex

for

was

H-corticosterone

same binding

As has

showed

plasma,

3

parallelism

the

qualitative

displacement of

progesterone

displacing

3H-progesterone.

equal

mass

rats,

dextran-charcoal

human

overlapping

with

in

although

the

of

plasma,

female

competition

using

rat

and

corticosterone

binding

curves

dence

from

sites of

were

3H-progesterone

S

358

TSX1EIROXD6

60

50 -

0

$ao-

P ;r 30 ai

-

I

,I 20 -

0

0.1

0.4

I

4

IO

MASS OF STEROtD

40

100

400

1000

(ngf

Displacement OF 3H-corticosterone (3H-B) from corticosteroidFig. 1. binding proteins by either corticosterone (8) or progesterone (P) in pfasma obtained from Top: male rat5 or #ottom: female rats, @ . + - - @ to plasma from two different rats. Varyjng masses and X . . . . X refer of 6 or P were added to steroid-free plasma preincubated with H-B. Following equilibration, free steroid was yeparated from prgtein-bound M-progesterone ( H-P) was steroid by addition of dextran-charcoal. aIs. checked for binding to steroid-free plasma.

359

MASS

OF

STEROID

(ng)

Figl. 2. Displacement of 3H-corticosterone (3H-B) from corticosteroidbinding proteins by either corticosterone (B) or progesterone (P) in huma plasma obtaiged from a norma13male Conditions were as described 3. in Fig. I. H-prggesterone ( H-P) and H-P purified by celite column chromatography (p H-P) were also checked for binding to seroid-free plasma.

in

male

human plasma

were

equally

there

was

amount

effective

in

no apparent

of

the

female

that

a sex

tion

(15)

progesterone failed

was

determination showed to

that

bind

in

3 the

not of

progesterone

to

male as bind

responsible. the

human CBG.

In

human plasma much as

corticosterone

even

also

3H-progesterone

activity were

addition, when

seven-fold.

of not

the

the Plasma

indicating

Chromatographic

specific

factors

and

H-corticosterone.

increased

failed

these to

since

displacing

was

human also

difference and

puzzling

binding

3H-progesterone

from

steroid

was

purificathe

3H-

reason

this

S

360

multiple

However,

equilibrium

progesterone

binding

steroid-free

human plasma

to

extent,

a similar

displacement from its

The sis that

to

fact

that

but

not

the

sites

bind

results

present

the

able 1. Ability of H-Corticosterone or Equi 1 ibrium Dialysis

‘I

CBG (Table

results

(Table

and

binding the

removed

separation,

the

was

3

a large

3

to

present

Rat

competitive unbound

H-corticosterone,

in

experiments.

equilibrium

from at

CBG binding

4°C.

or Human Plasma to Bind as Determined by Multiple

Cort i costerone

Progesterone

0.93”

1.51

Fema e Human

1.39

1.04

Male

1.97

0.87

9.40

1.46

Male

Human

Rat

Fema e Rat

“Bound

Steroid/Free

dialyQ suggested

separation

out

dialysis,

discrepancy

competition

H-progesterone carried

the

separate

dextran-charcoal

was

‘Zjteroid-Free H-Progesterone

showed

In

3H-corticosterone in

to

as opposed in

1).

obtained

1)

obtained

following

procedure

rat

dextran-charcoal

plasma

3H-

demonstrated

H-progesterone

the

‘H-progesterone

though

3

bound

3H-progesterone

the

latter

even

human and

using

Rat

supporting

dialysis

confirming

steroid.

ability

again

by both

experiments

bound

CT11R8XRIs

Steroid DISCUSSION

In the

function

proteins. ogical

spite

of of

There significance

considerable high is

affinity, evidence

(16-20).

experimentation, Few capacity that

only

Competitive

the

little

is

steroid-binding unbound

about

plasma

steroid

displacement

known

of

has

physiol-

progesterone

in

S from

CBG by surges

increase

of

from

gonads

the

surges

in

free

in

TRROXD1)

corticosterone

progesterone or

adrenals.

progesterone

could

361

could

provide

a mechanism

independent

of

Conversely,

proestrus

conceivably

secretion

of and

increase

for

progesterone

pregnancy

unbound

corticoste-

rone. The there for

rat

is

data

little

in

in which

than

progesterone.

for

means has in

reducing

been

found

increasing

established In

Use of of

have

the

by multiple

was

to

bound

CBG in

for

of

plasma

specificity

species

progesterone equilibrium

competitive

therefore

binding

be a valuable Horse

progesterone cortisol

extent

protein

by progesterone.

affinity

this

a much greater

might

caused

in

and

confirmed

corticosteroids

low

that

corticosterone

rat

interference

to

suggested

(Zl),

CBG also

and

measurement

its

has

use been

(21,22). contrast

costerone and

was

corticosterone

measurement

for

study

between

This

sites.

dialysis

assays

present

competition

CBG binding

was

the

and

to

rat

plasma,

progesterone

progesterone

were

rone

from

CBG binding

were

also

bound

to

the the

male

human plasma

same extent

equally

effective

sites.

3H-corticosterone

equally

by male

results

were

and

in

female

since

bound

corticosterone

displacing

‘H-corticoste-

and plasma

corti-

3H-progesterone

in

multiple

equili-

b’t-ium dialysis. Discrepant corticosterone the

by progesterone

dextran-charcoal

charcoal moving a general

in

the

separation concentrations

progesterone phenomenon

from

observed and

binding

method employed

CBG binding

associated

between

with

was

of

displacement

of

3H-progesterone

used.

apparently

Thus, is

3Hwhen

dextrancapable

sites.

Furthermore,

binding

protein-progesterone

this

of is

renot

interactions

since

antiserum is

to

progesterone

frequently

ability

of

radioi~unoassay

performed

dextran-charcoal

system

removes

that is

caution

based

absolute

comparisons

across

but

a specific

not (23).

when

The

CBG

from

between

binding of

equilibrium

(24)

dialysis

that at

should have

as

should

such

assays

4°C

a binding

therefore

methods

Westphal

antiserum

such

separation two

pig

sites

techniques

methods

the

and

guinea

observation

evaluation

an equilibrium

different

from

The

laboratory.

from

separation

As Gala

this

separation

as

be

dialysis.

not

observed, are

of

even question

value. In

contrast

affinity,

estradiol also

in

separation

concordance

a specific

dextran-charcoal

reported

be used

using

progesterone

progesterone

with

be expected.

high

been

Disruptive

necessarily

able

strip (PBG),

should

by comparison

However,

identical

on non-equilibrium

dextran-charcoal. validated

to

has

progesterone

difficulties

globulin

progesterone,

suggests

an

without

dextran-charcoal

progesterone-binding against

and

for

shows

to low

(25). very

human plasma, capacity

rat

binding

The

results

little

high

of

has

been

proteins

for

testosterone

study

suggest

this

affinity

plasma

binding

of

shown

that

rat

to

lack and

plasma

progesterone.

ACKNOWLEDGEMENTS This work was Grant #144,

supported

University

of

California

Faculty

Research

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