Radioimmunoassay of total and free corticosterone in rat plasma: Measurement of the effect of different doses of corticosterone

RADIOIMMUNOASSAY OF TOTAL AND FREE CORTICOSTERONE IN RAT PLASMA: MEASUREMENT OF THE EFFECT OF DIFFERENT DOSES OF CORTICOSTERONE Eeva-Liisa Sainio,a Timo Lehtola,b and Pirjo Roininew Department of Pharmacology and Toxicology, University of K~opio,~ P.O. Box 6, SF-70211 Kuopio, Finland; National Board of Health,b P.O. Box 221, SF-00531 Helsinki,

Finland

Corresponding author: Eeva-Liisa Sainio Received April 1, 1987 Revised July 1, 1988

ABSTRACT A radioimmunological method was developed for determining total and free corticosterone in rat plasma. This method was used to determine the dose-response curve of corticosterone and to measure the elimination and study the half-lives of total and free corticosterone in rat plasma with a dose of 5 m&kg. The elimination with a dose of 5 mg/kg, when drawn on the half-logarithmic scale, formed a straight Line. The half-lives for total and free corticosterone were 2.5 and L5 min, respectively.

INTRODUCTION Many drugs are known to be bound mainly to plasma proteins, and the free fraction of the drug is thought to be the pharmaeologically active one. Some steroids also participate

in similar

interactions with binding proteins in blood and are thus known to circulate

in

partially

bound,

partially

free

forms, which

are

usually in a state of equilibrium with each other. The most abundant glucocorticoid in man is hydrocortisone and in the rat, corticosterone bound

to

(1). Over 90% of these hormones are

plasma proteins (transcortin and albumin) (1). The appar-

ent distribution volume of corticosterone depends on transcortin

STEROIDS

51/5-6

May-J une1988(609-622)

609

610

Sainio

et al

concentration (2), and the bound form of corticosterone

is bio-

logically inactive (3). For some reason, however, the total concentration of hormones,

including

corticosterone,

in

plasma is

measured most often. Recently, the concept of determining the free fraction of a hormone has been strongly advocated, based on the view that it is the concentration of free hormone that determines hormone delivery to the target tissue (4). Various methods have been used to measure

plasma levels of

corticosterone in animals and in man. The methods used

include

fluorometric (5-6), protein-binding assay (7-lo), and radioimmunoassay (RIA) methods (11-19) for different animal species. Because the free fraction of corticosterone in plasma forms only about 5% of the totai concentration, a very sensitive method is needed for the free fraction determination. For this reason, the RIA assay is the method of choice. We developed the radioimmunological method for determining

total and free corticosterone

concentrations

in

rat plasma. For separation of total and free fractions in plasma, an ultrafiltration technique was chosen because it is rapid and reliable. The other methods that could be used for this purpose would be separation of different fractions by equilibrium dialysis techniques (20) or dextran-coated charcoal (21). In order

to get

information about the binding capacity of

corticosterone to plasma transcortin and albumin, the present RIA method was used to determine a dose-response curve for cortico-

STEROIDS

51/5-6

May-J une 1988

RIA OF CORTICOSTERONE

IN RAT PLASMA

sterone. The elimination curves for the total and free fractions provided information about the half-lives, and characterized the type of elimination.

MATERIALS AND METHODS

Chemicals Corticosterone for standard solutions and the compounds used in cross-reaction tests were obtained from Sigma Chemical Company (St. Louis, USA). [1,2,6,7-3H] Corticosterone (specific activity 84 Cifmmol) and the ACS-scintillation fluid were obtained from bovine Amersham, England. Cort~costerone-3-~-carboxymethyloxime serum albumin (corticosterone-3-CMO-BSA) (lip Jl-dihydroxypregn4-ene-3,20-dione-3-O-carboxymethyloxime bovine serum albumin) was obtained from Steraloids Ltd (Craydon, England). All other chemicals were purchased from E. Merck (Darmstadt, Federal Republic of Germany). Biological iamples Male Wistar rats (weight 230 - 300 g) were bred from the Af/Han/Mol/Kuo strain. The animals were maintained under regularly alternating periods of light (14 h) and dark (10 h) and were without food for 24 h before the experiment. Corticosterone was given intraperitoneally, and the rats were killed by decapitation. The trunk blood was collected into chilled glass tubes, which had been rinsed with a solution containing 1.5X EDTA in 0.9% sodium chloride. After 15 min, the samples were centrifuged and the plasma was separated. For determination of corticosterone in the unbound fraction, the samples were ultrafiltered. Ultrafiltration of plasma For the ultrafiltration of plasma, 1 mL of fresh plasma was placed into a dialysis sac (Union Carbide Corporation, lZ,OOU12,000-14,000, Cutoff) (Fig. 1) and centrifuged for 1 h (3600 g) at room temperature. This procedure produced 100 uL of ultrafiltered plasma in the bottom of the test tube. The absence of protein in the ultrafiltrate was controlled with Albust& (Ames Company, test-strips (sensitivity range 0.0%0.2g/L) Brigend, England). After the ultrafiltrate had been obtained, the radioimmunoassay (RIA) procedure was started immediately.

STEROfDS

51/5-6

May-June 1988

611

Sainio et al

612

Antiserum immunogen The

eorticosterone3-CIAO-BSA, used for the production of corticosterone antiserum, was emulsified in Freund's complete adjuvant (22). The rabbits were immunized in multisite subcutaneous injections at four-week intervals for six months. Two were weeks later the rabbits bled. The antiserum used for RIA was lyophifized before storage.

CORK

-DIALYSIS ;($W&ING

!u

SAC

3cm

POLYETHENE TUBE

Radioimmunoassay First, 100 pL of a standard solution, an unknown solution, or a phosphate-buffered saline solution (PBS) (ph 7.2) of a compound used in the cross-reaction tests was added to the test tubes. Then, 100 IJL of a 1X solution of human gamma-globulin in PBS, 50 nL tritiated corticosterone, and 50 1.11 of the antiserum diluted in PBS was added to the tubes, using antiserum dilution (1:9000) with which 50X binding of labeled antigen was obtained. The tubes were shaken in a Vortex mixer and incubated overnight at 4°C. Bound and free antigen were separated by polyethyleneglycol (PEG) precipitation (23) using 500 pL of a '25X solution of PEG 6000 in PBS. After centrifugation (2000 g, 20 min), the supernatant solution was decanted, mixed with 5 mL of scintillation solution, and counted in a liquid scintillation counter (LKB-Wallac 1216 Rackbeta). Figure 1. Separation of total and free fractions of plasma corticosterone by ultrafiltration. See detailed information in Materials and Methods.

RESULTS Tne sensitivity, defined equivalent value

to twice

of the

RIA,

as

the

detectable

the standard deviation of was

found

concentration

the zero-binding

to be 5 pg in the

100 nL

plasma

samples. The measuring range of the assay extends from 10 pg (0.1 ng/mL) to 2 ng (20 ng/mL) of corticosterone. The total number of mol/L)

and

the

antibody binding sites (A, = 5.1 x low6

corresponding

value

for

the

maximum affinity

constant (K, = 1.2 x l.Og L/mol) were determined (24).

STEROIDS

51/5-6

May-June 1988

RIA OF CORTICOSTERONE

the

cross-reactivity of

The

613

IN RAT PLASMA

corticosterone

antiserum

in

plasma samples is presented in Table 1.

Table 1. Cross-reactivitya of the corticosterone antiserum

Name

Cross-reaction X

of compound

100

Corticosterone Aldosterone Cholesterol Cortisol (Hydrocortisone) Estradiol 18-Hydrocorticosterone 17a-Hydroxypregnenolone 5-Pregnen-3P-ol-ZO-one Spironolactone Testosterone

.o

14.1 0 2.7 0 6.0 0 0 0 6.3

a Calculated at SO% inhibition level.

The cross-reactivities of aldosterone, cortisol, lg-hydroxycorticosterone, and testosterone were 14.1X, 2.7%, 6.0% and 6.3%, respectively. No interference was observed for the other compounds tested. Recovery of corticosterone added to the total and ultrafiltered plasma pools before ultrafiltration was 96.05% f 6.86 (n=12) for

total

plasma.

plasma

and

97.48

f

7.38%

(n=12)

for

ultrafiltered

Intra- and inter-assay variations for the total plasma

were 5.85% (n=30) and 7.44% (n=9), respectively. The corresponding values for the ultrafiltered plasma were 6.37% (n=9). Linearity was

investigated by the sample dilution method

(25).

STEROIDS

51/5-6

(n=30) and 8.69%

May-June 1988

614

Sainio

et al

Dose-resoonse curve and the elimination of corticosterone With

different

doses of corticosterone

(0.1 - 10.0 mg/kg)

the maximum binding percentage was about 20% for both the 5 mg/kg and 10 mg/kg dose. With doses lower than Smg/kg the dose response curve for free corticosterone concentration was steeper than that for total corticosterone (Fig. 2).

LOG

DOSE

6.0 10.0 ld00RTICOSTERONE mg/kg

E

0

0.1

1.0 LOG

DOSE

6.0 CORTICOSTERONE

10.0 mg/kg

Figure 2. Dose-response curves for total (A) and free fractions (B) of corticosterone. Figure C is drawn on the basis of figures A and B. The rats were injected intraperitoneally (0.5 mL/lOO g rat body weight) with a suspension of corticosterone in 0.9% saline, diluted from a basic suspension (the base suspension was 2 mg/ qL, corresponding to a dose of 10 q/kg). Magnetic stirrer was used. The rats were killed by decapitation 15 min after the injection. These values are the means f S.E.M for each group of five rats.

STEROIDS

51/5-6

May-J une 1988

RIA OF CORTICOSTERONE

IN RAT

PLASMA

615

2o.c

I

/ ’ 0%

TIME

30

45

60

90

30

TIME &)

45

-xc--

60

Figure. 3. Elimination of total (A) and free (B) corticosterone. Corticosterone (5 mgfkg) was injected intraperitoneally. The rats were killed and blood was collected 30, 60, and 90 min after the injection. These values are the means for each group of five rats.

Elimination

of corticosterone

(dose 5 mg/kg) was

Because the first phase on the half-logarithmic

scale

biphasic. formed a

straight line this type av elimination can be considered of

the

min

for

first order

(Figs. 3A and 3B). The half-lives were 25

total corticosterone and 15 min for free corticosterone.

DISCUSSION Although mining

the literature

corticosterone,

sterone in rat plasma which

the

describes

many

only a few exist

methods

for measuring

(18). To our knowledge

free fraction has

for deter-

been measured

cortico-

there is none

in

with an RIA method.

However the percentage of free corticosterone determined

in our

study

humans

agrees

STEROIDS

with

51/5-6

earlier

results

May-J une 1988

(with

rats

[25] and

616

Sainio et al

[Z&21])

measured with methods other than RIA. D'Agostino and eo-

workers (X6) obtained a somewhat higher percentage of free fraction corticosterone than found here. This may be because that they used the competitive protein binding method (10) to measure total serum corticosterone, and dextran-coated charcoal (21) followed by counting

the supernatant to measure

free corticosterone.

In our

method the corticosterone is measured in both fractions with the same RIA method.

As far as

reliability

of

RIA

methods

is

concerned,

the

essential question is the of cross-reactivity with certain chemically related compounds. In the present study. the specificity of antiserum was good. Aldosterone showed the highest percentage of cross-reactivity, but in the methodological context very

important

because

aldosterone

concentration

that is not is

only

one

thousandth of corticosterone concentration in rat plasma. Cholesterol, present

in plasma in quite large amounts (about SOug/dL),

did not show significant cross-reactivity in our method. We assumed that technical error

here would easily swamp the method-

ological error, since small volumes (1OOuL) of samples were being handled. We considered there was no reason to use prior extraction of plasma samples.

The dose-response and elimination of corticosterone The dose-response curves for total and free corticosterone in rat plasma followed the typical dose-response patterns

STEROIDS

51/5-6

for drugs

May-June 1988

RIA OF CORTICOSTERONE

IN RAT

PLASMA

(27). The maximum percentage of free corticosterone was 20% (with doses

of 5

and

Smg/kg the dose

lOmg/kg

corticosterone). curve

response

With

doses

lower

for free corticosterone

than

concen-

tration was steeper than that for total corticosterone. This indicated that there was a greater

increase in

free than in

total.

corticosterone concentration at the Smg/kg dose and that an equilibrium, or saturation of binding sites, was achieved at doses of Smg/kg or higher. The study time used here was 90 min maximum. The corticosteroid-binding globulin in plasma has been shown to exhibit daily fluctuations (27) and thus the profile obtained may fluctuate with changes in plasma proteins. The half-life of total corticosterone obtained (25min) agrees with earlier studies (28-30). Unfortunately, we have not succeeded in locating other studies of half-life determination of free corticosterone. Our results showed that the half-life of free corticosterone sterone.

in

These

plasma results

was are

shorter

than that of

consistent

with

the

total corticodose-response

curves obtained. One of the possible explanations concerning different halflives is that transcortin-bound corticosterone is perhaps not released at the same speed that the target organs take up free corticosterone from plasma. There is never a complete balance between all pharmacodynamic factors. The finding that the half-life for free corticosterone

in plasma is shorter than for total cortico-

sterone is interesting and needs to be studied further.

STEROIDS

51/5-6

May-J me 1988

617

618

Sainio et al

Biphasic elimination of both total and free corticosterone in rat plasma can be explained by the fact that there are several binding sites on transcortin, which operate in the order determined by their physico-chemical properties. The ultrafiltration

technique seemed to serve our purposes

very well, being faster than equilibrium dialysis. We have found 15

min

centrifugation

sufficient

to

obtain

the

sample

volume

(1OOul) needed for RIA free corticosterone determination. Although both

equilibrium

cepted methods

dialysis and ultrafiltration

for separating free fractions

are generally acfrom protein bound

compounds, both methods have their merits and drawbacks. With the ultrafiltration technique the duration of centrifugation and the temperature used are variables commonly used. On the other hand, it

is

possible

that

some

conformational

changes

occur

in

the

proteins during 16 hours of dialysis at 37°C (31). Also, there can be an increase in microbe counts. Comparison of these methods has been discussed

by Goldie

and coworkers

(1969) (32). They *found

similar concentrations of cortisol in both dialysate and

ultra-

filtrate. Thus choice

the

here

ultrafiltration

because

method

it is not only

should rapid,

be but

the also

method

of

accurate,

simple, reproducible and inexpensive (31). The binding capacity of corticosterone

binding proteins may

change in different physiological (33) or pathological conditions

STEROIDS

5-l/5-6

May-J une 1988

RIA OF CORTICOSTERONE

(34) and thus the total and free corticosterone

IN RAT PLASMA

measurements do

not always correlate with the free corticosterone value in plasma. Our method surmounts this problem and offers a highly

promising

new approach to studying the effects of glucocorticoid hormones.

We are greatly indebted to Jaakko-Juhani Himberg M.D.for his help with the interpretation of pharmacokinetics and critical reading of this manuscript. This study was supported by Kymenlaakso Cultural Foundation and by personal grants to Timo Lehtola from the Emil Aaltonen Foundation and the Heikki and Hilma Honkanen Foundation (Finland).

REFERENCES 1.

McDonald LE. Hormones influencing metabolism. In: Veterinary Pharmacology and Therapeutics (Jones LM, Booth NH and McDonald LE, eds.), The Iowa State University Press, Ames, USA (1977).

2.

Acs Z, and Stark E (1973). The role of transcortin in the distribution of corticosterone in the rat. HORM METAB RES 5: 27Y-282.

3.

Slaunwhite, WR, Jr., Lockie GN, Back N, and Sandberg AA (1962). Inactivity in vivo of transcortin-bound cortisol. SCIENCE 135:1062-1063.

4.

Ekins R. Free hormones in blood: Their physiological significance and measurement. In: Practical immunoassay (Butt B, ed), THE STATE OF ART (1984), pp. 217-251.

5.

Sweat ML (1954). Sulfuric acid-induced flwrexence ticosteroids. ANAL CHEM 2~773-776.

6.

Solem JH, and Brinck-Johnsen T (1965). An evaluation of a method for determination of free corticosteroids in minute quantities of mouse plasma. SCAND J CLIN LAB INVEST 17 (Suppl 80):1-14.

STEROIDS

51/5-6

May-June 1988

of cor-

619

620

Sainio et al

7.

Newsome HH, Clements AS, and Borum EH (1972). The simultaneous assay of cortisol, corticosterone, IL-deoxycortisol, and corticone in human plasma. .J CLIN ENDOCRINOL METAB -34: 473-483.

8.

Chattopadhyay S, Johnson D, and Jaques LB (1971). Rat serum corticosterone assay: use of radiosteroid assay method. STEROIDS -18:411-419.

9.

Cameron EH, and Scarisbrick JJ (1973). Determination of corticosterone in rat plasma by competitive protein binding assay and its use in assessing the effects of C-B154 and perphenazine on adrenal function. J ENDOCRINOL -58:27-28.

10.

Henning SJ measurement 673-683.

11.

Gross HA, Ruder HJ, Brown KS, and Lipsett MB (1972). A radioimmunoassay for plasma corticosterone. STEROIDS -20:681-695.

12.

Nabors CJ, West CD, Mahajan DK, and Tyler FH (1974). Radioimmunoassay of human plasma corticosterone: method, measurement of episodic secretion and adrenal suppression and stimulation. STEROIDS -23:363-378.

13.

Etches RJ (1976). A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry. STEROIDS -28~763-773.

14.

Schoneshofer M (1977). Simultaneous radioimmunoassay for corticosterone and deoxycortisol in human serum: Sex differences in the mean serum concentrations. J STEROID BIOCHEM -8:9951OOY.

15.

Giordino ND, Koletsky RJ, and Koletsky S (1976). Simultaneous determination of plasma aldosterone, corticosterone, ll-deoxycorticosterone, 18-hydroxydeoxycorticosterone, cortisol, and ll-deoxycortisol. AM J CLIN PATHOL -66:588-597.

16.

Kley HK, Elsasser W, Dehnen H, and Kruskemper HL (1978). Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free Lldeoxycorticosterone. STEROIDS -32:223-232.

17.

Sippel WG, Bidlingmaier F, Becker H, Brunig T, Ddrr H, Hahn H, Golder W, Hollmann G, and Knorr D (1978). Simultaneous radioimmunoassay of plasma aldosterone, corticosterone,

(1980). A sensitive and convenient method of corticosterone in rat serum. STEROIDS

STEROIDS

51/5-6

for -35:

May-June 1988

RIA OF CORTICOSTERONE

IN RAT

PLASMA

ll-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, ll-deoxycortisol, cortisol and cortisone. STEROID BIOCHEM 9:63-74. 18.

Al-Dujaili EAS, Williams BC, and Edwards CRW (1981). The development and application of adirect radioimmunoassay for corticosterone. STEROIDS -37:157-175.

19.

Mathew JK, Marsaw B, Curtis JC, and Mrotek JJ (1981). An improved method for the extraction of corticosterone from cell homogenate and subcellular fractions of the rat adrenal cortex. STEROIDS 37:497-509.

20.

Cohen A (1982). Rgpartition des glucocorticosteroides entre fraction libre et fraction 1iGe dans le plasma maternel foetal et neonatal du rat, au tours de la periode perinatale. C R SOC BIOL 176: 781-786.

21.

Martin CE, Cake MH, Hartmann PE, and Cook IF (1977). Relationship between foetal corticosteroids, maternal progesterone and parturition in the rat. ACTA ENDOCRINOL -84:167176.

22.

Freund J (1951). The effect of paraffin oil and mycobacteria on antibody formation and sensitization. AM J CLIN PATHOL 21: 645-656.

23.

Shaw W, Schulman L, and Spierto FW (1975). The carrier effect of gamma globulin when a polyethylene glycol separation technique is used in digoxin radioimmunoassay. CLIN CHIM ACTA 60:385-389. -

24.

Scatchard G (1949). The attraction of proteins molecules and ions. ANN NY ACAD SC1 -51:660-672.

25.

Henning SJ (1978). Plasma concenrations of total and free corticosterone during development in the rat. AM J PHYSIOL 235:E451-E456.

26.

D'Agostino J, Vaeth GF, and Henning SJ (1982). Diurnal rhythm of total and free concentrations of serum corticosterone in the rat. ACTA ENDOCRINOL 100:85-90.

27.

Curry SH, Drug disposition and pharmacokinetics, Scientific Publications, Oxford (1977) pp. 99-102.

28.

Glenister DW, and Yaters FE (1961). Sex difference in the rate of disapperance of corticoscerone-4-C14 from plasma of

STEROIDS

51/5-6

May-June 1988

for

small

Blackwell

621

622

Sainio et al

intact rats: Further evidence for the influence of hepatic activity on cortical hydrogenase adrenal *4-steroid function. ENDOCRINOLOGY -68:747-758. cortical

29.

in adrenal Kitay JI (1961). Sex differences secretion in the rat. ENDOCRINOLOGY -68:818-824.

30.

Schapiro S, Percin CJ, and Kotichas FJ (1971). Half-life of plasma corticosterone during development. ENDOCRINOLOGY -89:284-286.

31.

Slaunwhite WR Jr., and Rosenthal H. Quantitation of Specific Binding Proteins. In: Hormones in human blood, Detection and Assay (Antoniades HN, ed), Harvard University Press, Cambridge, England (1976), pp. 777-794.

32.

Goldie DJ, Hasham N, Keane PM, Pearson J, characteristics binding Cortisol (1969). dialysis. J ENDOCRINOL -43:127-128.

33.

Schiilke B, and Tegeler G und rungsproteinqualitXt TIERERNtiRUNG -25~453-462.

34.

Abasheva GK, and Mikhneva LM (1983). Plasma content of free and protein-bound corticosterone in white rats exposed to hypoxia and ACTH administration. KOSM BIOL AVIAKOSM MED 17: 47-50.

and Walker measured

WH by

(1975) Beziehungen zwischen NahNebennierenrindenfunktion. ARCH

STEROIDS

51/5-6

May-J une 1988