The effect of a rabbit ACTH preparation on adrenal steroid biosynthesis

605

THE EFFECT OF A RABBIT ACTH PREPARATION

ON

ADRENAL STEROID BIOSYNTHESIS

Howard B. Drummond and H. Richard Fevold Department of Chemistry University of Montana Missoula, Montana 59801 Received

12/2/71 ABSTRACT

Adrenocorticotropic hormone (ACTH) fractions were prepared from both rabbit and steer pituitaries. The preparations assayed at 48 and 25 Units per mg, respectively, by the in vitro rat adrenal slice assay procedure using an Armour ACTHar standard. Each preparation showed one major band on acrylamide gel electrophoresis. The mobilities of these major bands at pH 9.3, when compared with a highly purified commercial pig-ACTH preparation, were in the relative order of rabbit>steer>pig. Comparison of the rabbit-ACTH preparation and pig-ACTH with regard to their ability to stimulate cortisol production by rabbit adrenal tissue homogenates after 2-days injection (26-40 Units, IM, twice daily) showed that the rabbit-ACTH preparation markedly increased cortisol production, but only to the extent of 10% t h a t observed in tissue stimulated with pig-ACTH. In separate experiments the steer-ACTH preparation appeared to stimulate cortisol production to approximately the same degree as observed for the pig-ACTH. These results suggest that although rabbitand steer-ACTH have similar electrophoretic mobilities, both of which are significantly greater than pig-ACTH at pH 9.3, the ability of the rabbitACTH preparation to stimulate 17~-hydroxylation is much less than either the steer- or pig-ACTH preparations. INTRODUCTION Daily injection of rabbits with pig adrenocorticotropin day periods has been demonstrated

to result in a decrease

of corticosterone

in the amount of cortisol

and an increase

the adrenal vein blood of the stimulated are of interest since cortisol by rabbit adrenals,

rabbits

is normally produced

but is the predominant

pig, the source of the adrenocorticotropin. of ACTH from a predominantly

(1,2,3).

corticosterone

for 21-28

in the amount secreted in These results

only in trace amounts

corticosteroid produced by the Determination secreting

of the ability

species

to

S T E R O ID S

606 stimulate

17~-hydroxylation

and, hence,

19:5

cortisol production,

adrenal tissue might indicate whether a species ACTH molecule significance. is identical shown

Synthetic

8

1-24

-corticotropin,

to the amino-terminal

from similarly stimulated

a primarily

of the preparation species.

whose amino acid sequence

24 residues of porcine ACTH, has been synthesis by adrenal tissue

animals.

In the present studies,

producing

exists in the

and whether this action of ACTH may be of physiological

(4) to cause an increased cortisol

rabbits,

specificity

by rabbit

ACTH was isolated from pituitaries

corticosterone-producing

procedure,

from the steer,

A comparison

species,

of

and for a control

a primarily

cortisol-

of in vitro corticosteroid

biosynthesis

by adrenal tissue from rabbits stimulated by rabbit, pig, and steer adrenocorticotropin

is presented.

MATERIALS AND METHODS ISOLATION OF ACTH. ACTH fractions were prepared by published procedures (5,6,7). After ion exchange chromatography on carboxymethyl cellulose (CMC) (7), the resultant fractions from the steer pituitaries were assayed using quartered rat adrenals (8,9). The porcine ACTH and the CMC-fractions from the rabbit pituitaries were also assayed using quartered rat adrenals by the method of Rerup (i0) as modified by Camiener and Tree (ii). In the latter assays, each test material including an Armour ACTHar standard was evaluated at five different dose levels extending over a 100-fold concentration range, and a log doseresponse curve was drawn for the standard. The optical densities of the extracts of the test samples were converted into IU and these test values (averages of duplicates) were plotted on log-log paper against the respective concentrations of the test material used. The activity of the test material in milliunits per ~g was determined at the point where the straight line of best fit intercepted a line corresponding to a concentration of 1 ~g per flask. ELECTROPHORESIS OF ACTH. Disc electrophoresis using 7.5% polyacrylamide gel was carried out on the pig-, steer-, and rabbit-ACTH preparations using a temperature-regulated Buchler Polyanalyst apparatus and the method of Ornstein (12) and Davis (13). Samples were layered on top of the large-pore gel in sucrose solution. Runs were carried out at pH 9.3 with 2.5 ma per tube. Gels were stained one hour with Amido Schwarz in 7% acetic acid. After destaining, the gels were scanned at 660 nm by means of a Gilford Model 2410 Linear Transport at a speed of

May 1972

ST ER O ID S

607

1 cm per min and an aperture width of 0.05 mm. TISSUE PREPARATION. Male New Zealand white rabbits weighing 2.3 4.2 kg were injected twice daily for two days with approximately 26 Units purified porcine-ACTH (Upjohn, Lot No. 7275) or the equivalent of 26 U steer- or 40 U rabbit-ACTH in 0.2 ml of 5% beeswax in peanut oil containing 0.5% phenol. Control animals were injected with beeswax - peanut oil vehicle. Animals under light pentabarbital anesthesia were exsanguinated and the adrenals were removed, trimmed, and weighed. Adrenal glands from each of four similarly treated rabbits were pooled and homogenized at 0 ° in sufficient Krebs-Ringer phosphate buffer (pH 7.35) to give a tissue concentration of i00 mg/ml. The buffer also contained i00 mg each of E-D-glucose and glucose-6-phosphate per i00 ml, 40 mM nicotinamide, 0.i mM sodium fumarate, and 0.4 mM each of NAD, NADP, and ATP. INCUBATION PROCEDURE. Pregnenolone-4-14C, 55.7 mCi/mM (NuclearChicago), was chromatographically purified prior to use. The specific activity was adjusted to 1.8 ~Ci/vmole with nonradioactive pregnenolone (Sigma), and 0.165 ~mole of this material was added to 25-mi incubation flasks in 0.2 ml of ethanol-propylene glycol (I:i, v/v). The ethanol was evaporated at 40 ° under nitrogen. Approximately two units of glucose-6phosphate dehydrogenase (Sigma Type VI) were included in each flask with 2 ml of adrenal homogenate. The flasks were gassed with 95% 02-5% CO2, stoppered, and placed in a constant temperature, shaking water bath at 37.5 ° All incubations were performed either in triplicate or duplicate, as indicated in the results, and terminated after three hours by the addition of 5 ml dichloromethane-ethyl acetate (i:i, v/v). The contents of each flask were mixed thoroughly and kept at -20 ° until analyzed. EXTRACTION AND PRODUCT ISOLATION. Approximately 0.05 ~Ci each of chromatographically purified 3H-labeled cortisol, 82.8 ~Ci/~g (NuclearChicago), corticosterone, 158 ~Ci/~g (Tracer Lab.), and ll-deoxycorticosterone, Ii0 ~Ci/~g (Tracer Lab.), were added to each flask to permit the estimation of percent recoveries of these compounds. The incubation media were extracted four times with double volumes of dichloromethane-ethyl acetate (i:i, v/v). The combined extracts were evaporated and the residue was initially separated into four major fractions by paper chromatography in the heptane-formamide system (14), developed for 1 hour after the mobile phase had reached the end of the strip, followed by redevelopment in the benzene-formamide system. Each of the three fractions from these initial chromatograms was recovered by elution with 15.0 ml of ethanol. The recovered fractions were further purified and separated into their components as follows. All chromatographic separations were performed on Whatman No. 1 filter paper. Fraction I (cortisol) was acetylated and chromatographed in chloroform-formamide. The area corresponding to cortisol-21-acetate was eluted, and the recovered material was used for mass and radioactivity determinations. Fraction II (cortisone) was acetylated and chromatographed in benzene-formamide for a period of four hours after the mobile phase had reached the end of the strip. The area corresponding to cortisone-21acetate was eluted, and the recovered material was used for mass and radioactivity determinations. Fraction III (corticosterone, ll-deoxy-

608

ST ER O I D S

19:5

corticosterone, ll-dehydrocorticosterone, progesterone, and pregnenolone) was chromatographed in chloroform:benzene (i:i) - formamide. The most polar area of Fraction III, corresponding to corticosterone, was eluted, and the resulting material acetylated and chromatographed in heptane: benzene (i:I) - formamide for twenty-two hours after the mobile phase had reached the end of the strip. The areas corresponding to corticosterone21-acetate and ll-deoxycortisol-21-acetate were eluted and the resulting materials analyzed for mass and radioactivity. The center area of Fraction III, corresponding to ll-deoxycorticosterone and ll-dehydrocorticosterone, was acetylated and chromatographed in the benzene-formamide system. The areas corresponding to ll-deoxycorticosterone-21-acetate and ll-dehydrocorticosterone-21-acetate were eluted and analyzed for mass and radioactivity. The third area of Fraction III, corresponding to progesterone and pregnenolone, was eluted, acetylated, and chromatographed in the heptane:benzene (i:i) - formamide system. The areas corresponding to progesterone and pregnenolone-3~-acetate were eluted and analyzed for radioactivity. ANALYSIS. All final paper chromatograms were run on 2-cm strips of filter paper that had been previously washed for 5 days with deionized water and for 5 days with freshly redistiiled absolute methanol. Chromatographic fractions were recovered by elution with 15 ml of redistilled absolute ethanol, and one tenth of each eluted sample was taken in duplicate for the determinations of 14C- and 3H- content by dual-channel liquid scintillation spectrometry. The mass of the isolated metabolites was determined spectrophotometrically on a second set of duplicate aliquots by the reaction of Porter and Silber (15) for cortisol-21-acetate, and by the blue tetrazolium reaction (16) for corticosterone-21-acetate, ll-deoxycorticosterone-21-acetate, and ll-dehydrocorticosterone-21-acetate. The analytical values for cortisol, corticostercne and ll-deoxycorticosterone were corrected for the percent recovery as determined by the amount of the appropriate 3H-labeled steroid recovered from the final chromatogram. All values were corrected for zero-time controls in which the organic solvent had been added to the flask immediately after homogenate addition. Samples were counted a sufficient time to achieve less than 2% error at a 95% confidence level. Calculation of 3}{_ and 14C- content was done by programming the standard equations for dual-labeled samples (Nuclear-Chicago, Liquid Scintillation Manual) for an IBM 1620 data processing system (17). Pooled samples of the cortisol-21-acetate, and corticosterone-21acetate, cortisone-21-acetate, and ll-deoxycorticosterone-21-acetate were repeatedly crystallized to obtain constant specific activity after the addition of 25 mg of authentic compound. Crystals were weighed on a Cahn Electrobalance in aluminum foil cups, and radioactivity was determined by liquid scintillation spectrometry. When performed, acetylation reactions were carried out in 0.2 ml of pyridine-acetic anhydride (4:1, v/v) and oxidations were done in glacial acetic acid saturated with CrO 3.

May 1972

ST ER O ID S

609

RESULTS

Figure extract,

1 represents

the CMC elution pattern

which w~s similar to the elution pattern obtained w i t h the

steer pituitary extract and to that reported and Li

of the rabbit pituitary

(7).

The shaded area

the corresponding

188-200)

represented

65 mg and

peak for the steer preparation

(not shown)

represented

llO mg dry weight.

(fractions

for sheep extracts by Birk

The activity of the porcine-ACTH

d e , at±on was found to be 62 ± 2 IU/mg in two assays, 48 ± 7 IU/mg for the rabbit-ACTH,

assayed

with the standard as compared with

four times.

A representative

graph of the results from an assay of these two preparations Figure 2.

By a similar assay method,

estimated

the steer-ACTH preparation was

to contain 25 IU/mg.

Figure

3 compares

the electrophoretic

steer- and rabbit-ACTH preparations phoresed

is shown in

at the same time.

was more homogeneous accounting,

pattern

at pH 9.3 of the

with that of pig-ACTH electro-

The porcine preparation

(Figure 3a and 3c)

than either the rabbit or steer preparations,

at least in part,

for its greater activity.

The mobil±ties

of the steer- and rabbit-ACTH

appear to differ slightly

from each other,

but both are significantly used as a standard.

greater than the mobility of the pig-ACTH

The greater anodal mobility of the steer-ACTH

(Figure 3b) compared with the pig-ACTH from the previously

(Figure 3a) is to be expected

reported structures

of these molecules

Table I shows the values for cortisol production

by rabbit adrenal homogenates

stimulation with pig- or rabbit-ACTH. increase

in the formation of cortisol

corticosterone

formation

(18).

and corticosterone

after two days in vivo

Pig-ACTH

caused a large

and a concomitant

decrease in

compared with that of the non-stimulated

STEROIDS

61o

0.01 M

O.IOM

l~4,e

.6

19:5

TO O. IOM

im 4,6

_t

O~IOM

9~ 6 . 7

pH 6 . T

TO 0 . 2 0 M pH 8 . ?

.5

m-

~.4 tLI 0 Z m .3, 0 m In .2

20

401

•

80

IO0

120

! 40

;60

J80

" 188

200

FRACTIONS ( 4 M L )

Figure

i

E l u t i o n p a t t e r n of a r a b b i t - A C T H p r e p a r a t i o n f r o m a 60 x 1 cm C a r b o x y m e t h y l C e l l u l o s e Column. Shaded area r e p r e s e n t s m o s t active fraction. F r a c t i o n size - 4.0 ml Abscissa: Fraction number Ordinate: A b s o r b a n c e , 278 n m A m o u n t of m a t e r i a l p l a c e d on column:

2 g

220

May 1972

ST ER O ID S

611

1000

100 e~

o x v

<

m

10

>.-

1.0 0.t

I

I

1.0

10

CONCENTRATION,

,ug/ASSAY FLASK

Figure 2 Typical in vitro ACTH assay results using an ACTHar standard. Activity in IU is plotted against the quantity assayed. Specific activity in mU/~g is read on the ordinate at the point where the line of best fit intersects the 1.0 ~g concentration line. o

Pig-ACTH

•

Rabbit-ACTH

i 100

6t2

ST ER O l D S

19:5

PeG

PIG

.1.0"

o.Ts.

|

~

STrEet

I~SSJT

m*

D*

I

I

I

I

3

4

5

•

i Bm

I !

~

3

•

S

•

cM

Figure

3

Disc e l e c t r o p h o r e t i c patterns o f A C T H preparations. 75-200 Vg of each p r e p a r a t i o n were run on 0.5 x 7.5 cm 7.5% p o l y a c r y l amide gel columms in Tris b u f f e r pH 9.3, w i t h a current of 2.5 ma per column for a duration of 90 rain. The p i g - A C T H was run as a standard w i t h both the steer- and rabbit-ACTH. The gels were stained with A m i d o - B l a c k and s c a n n e d at 660 nm. E l e c t r o p h o r e t i c patterns A and B, and C and D were o b t a i n e d from gels run at the same time. S h a d e d areas indicate the major p r o t e i n bands. A b s o r b a n c e at b e g i n n i n g and end of gels is due to the i r r e g u l a r i t i e s of the gels at these points, and not stained material.

May 1972

ST ER OID S

613

Table 1---Amount of Cortisol and Corticosterone Isolated and Percent Conversion of Pregnenolone-4-14C per I00 m g of Homogenate of Adrenal Tissue from Control and ACTH-Stimulated Rabbits. Values are corrected for percent recovery and zero-time incubations and are averages of triplicate incubations.

Corticosteroid

ACTH

Formed (~g/100 mg Tissue ± Is)*

Conversion of Substrate (Percent ± is) *

Cortisol

Pig Rabbit Control

32.8±4.5 3.5±0.6 0.6±0.1

16.3±0.3 4.9±1.8 1.2±0.3

Corticosterone

Pig Rabbi t Control

26.4±1.5 30.0±3.1 51.8±6.8

18.6±0.9 24.1±0.9 31.1±1.9

Corticosteroid EXPERIMENT I

EXPERIMENT

II

Cortisol

Pig Steer

10.0±1.6 7.3±1.6

Corticosterone

Pig Steer

2.1±0.1 5.4±0.1

* S = standard deviation

614

ST ER O I D S

control tissue. production steroids

The rabbit-ACTH

and a decrease

elicited

19:5

a smaller increase

in corticosterone

production.

in cortisol

The other cortico-

for which analyses were performed were present in amounts equal

to or less than 2 ~g/100 mg tissue, which approached analytical

methods.

tissue previously

In a separate

stimulated with steer-ACTH

version of the pregnenolone approached

experiment

substrate

(Table i), incubations also showed significant

to cortisol

the amount converted by pig-ACTH

the limits of the

(7.3 ± 1.6%)

stimulated

tissue

of con-

and closely (i0.0 ± 1.6%)

incubated at the same time. The corticosteroids ic mobilities acetylation In addition,

of the parent compounds

according

and their derivatives

to the scheme presented

the identities

cortisol-21-acetate, acetate,

were identified by partition paper chromatograph-

of the isolated

above for their separation.

14C-labeled metabolites,

ll-deoxycortisol-21-acetate,

ll-deoxycorticosterone-21-acetate,

terone and remaining pregnenolone-38-acetate

obtained by

corticosterone-21-

ll-dehydrocortisol,

proges-

were confirmed by crystalliza-

tion to constant specific activity after the addition of approximately 25 mg of the authentic,

non-labeled

isolated 14C-cortisol-21-acetate tissue incubations

steroid in question.

from the rabbit- and pig-ACTH

was also oxidized with CrO3,

acetate isolated and crystallized addition of 25 mg of authentic tiated the formation

to constant specific

cortisone-21-acetate.

stimulated

stimulated

the 14C-cortisone-21activity after These data substan-

of cortisol from the pregnenolone-4-

by both pig- and rabbit~ACTH

A portion of the

14

C substrate

adrenal tissue.

DISCUSSION The primary purpose of these studies was to compare

the effects of

May 1972

ST ER O ID S

stimulating

rabbits with ACTH from a corticosterone-producing

those already established experiment

species with

for ACTH from a cortisol-producing

dealing with steer-ACTH

species.

The

stimulation was carried out to deter-

mine whether an ACTH fraction prepared producing

615

in our laboratory

species would show a similar stimulation

from a cortisol-

of cortisol production

as seen with the commercial pig-ACTH preparation. Comparison

of the electrophoretic

rabbit-ACTH preparations minor components, heterogeneous.

indicated that the pig-ACTH

whereas

Since homogeneous

the biological

electrophoretic indicate

of 100-120 IU/mg,

component.

Thus, the pig-ACTH the rabbit-ACTH

and the steer-ACTH

tion of the stainable material.

dicates

mobilities

Assuming

of steer-

bilities,

to the major

activity of 48 IU/mg a purity

for this large a propor-

that the major e l e c t r o p h o r e t i c

rabbit-ACTH

(Figures 3a and 3c).

3 in-

(Figure From the

(18) it is expected that their

From these differences

appears

Figure

(Figure 3b) and rabbit-ACTH

of pig- and steer-ACTH

should differ.

to

activity of 62 IU/mg would

accounts

3d) are six to seven times that of pig-ACTH known structures

it is reasonable

the active ACTH fraction of each preparation,

that the mobilities

cortico-

activity of 25 IU/mg a purity of 20-25%.

No other component in these preparations

band represents

were quite

and synthetic

activity of the present preparations

a purity of 50-60%,

of 40-50%,

ACTH preparations

and

contained very few

the rabbit and steer preparations

tropins have specific activities ascribe

results of the pig-, steer-,

in electrophoretic

to be at least as different

mo-

from pig-ACTH as

is steer-ACTH. It is evident from the incubations imately the same ability as pig-ACTH of the rabbit adrenal cortex.

results that steer-ACTH has approx-

to activate

the 17~-hydroxylase

This is not surprising

system

since it has been

616

ST ER O I D S

previously

demonstrated

which is identical

increase

(4) that the synthetic

to the NH2-terminal

steer- and pig-ACTH,

24 amino acid residues

by the rabbit adrenal cortex,

a much lesser extent than the pig-ACTH. of stimulation

ed that proteolytic

^1-24 5 -corticotropin

has the ability under similar conditions

in cortisol production

the magnitude

19:5

enzymes

analog,

of both to cause an although

to

The reason for this difference

has not been established.

in

Landon has suggest-

attack the smaller molecule more readily

(19).

Of greater interest is the fact that ACTH from the rabbit, whose adrenals normally produce primarily of cortisol,

stimulates

rabbit adrenal cortex.

the production

this difference

in stimulating

cortisol production.

may reflect structural

differences

abilities

system in the rabbit adrenal

ACTH stimulates it appears

of 17~-hydroxylated

the synthesis of cortisol

that in the rabbit-ACTH

the total production

preparation

was

On the other hand,

in the two molecules

to induce or activate cortex.

clear.

Although

adrenal tissue incubations

was reduced approximately

stimulated

a 17~-

The mechanism by which

is not completely

stimulated

of corticosterone

same extent as in the pig-ACTH

products by the

(ii), which could account for the differences

which could affect their relative hydroxylating

with only trace amounts

It was noted that the rabbit-ACTH

less soluble than the pig-ACTH in their activities

corticosterone

to the

adrenal tissue incubations

(Table i), cortisol production was not stimulated to the same extent. This would suggest that inhibition rable from the stimulation (Fevold, unpublished and pig-ACTH

of cortisol production.

observations)

stimulated

mixtures

production

is sepa-

In other experiments

of homogenates

of unstimulated

rabbit adrenal tissue showed no evidence of either

an inhibitor of cortisol production tor of corticosterone

of corticosterone

production

in unstimulated

tissue,

in the ACTH-stimulated

or an inhibi-

tissue.

The

May 1972

STEROIDS

reason for the lack of an increase the decrease rabbit-ACTH

in corticosterone stimulated

in cortisol production

production

a corticosterone-producing

cortisol production activity appears producing

However,

to with

the results

that ACTH from pituitaries

of the

species, has the ability to stimulate

by rabbit adrenal glands,

although the potency of this

to be less than that of ACTH from pituitaries

of cortisol-

species.

Whether or not the action of ACTH to stimulate in rabbit adrenal tissue is of physiological producing

species experiencing

question.

Kass a n d c o - w o r k e r s

situations

formation.

antibody production prolonged

of prolonged

stress is open to

in rabbits.

Such a response to infections

the sudden die-off seen during peak population

that

in 17~-hydroxylation

High doses of ACTH were also shown

cycles.

in corticosterone-

(20) published a report suggesting

stress more susceptible

marked population

cortisol production

significance

rabbits might respond to stress with an increase cortisol

equivalent

in the present experiments

adrenal tissue is unknown.

of these studies clearly demonstrate rabbit,

617

and

(21) to decrease

could make animals under and thus,

contribute

years of species

Secretory rates of both cortisol

to

exhibiting

and cortico-

sterone in organ culture have been shown to be higher from adrenals of small mammals such as voles and lemmings stress,

than in summer,

densities

(22).

physiological dominantly natural

in winter,

and to also correlate

a period of high

directly with population

While these types of data suggest the possibility

role of the stimulation

corticosterone-producing

animal populations

quired for a definitive

of cortisol production

species,

of a

by a pre-

more direct evidence

from

under severe stress conditions will be re-

answer.

618

ST E R O I D S

19:5

ACKNOWLEDGMENTS This work supported by U.S. Public Health Service Grant AM08386 and U . S . P . H . S . Career Development Award 9392 (H. Richard Fevold). The authors wish to thank Dr. J. W. Hinman of the Upjohn Company for generously supplying the pig-ACTH, and G. W. Camiener and P. N. Tree, of the Upjohn Company, for assaying the rabbit-ACTH preparation.

REFERENCES i.

Kass, E. H., Hechter, O., Macchi, I. A., and Mou, T. W. Proc. Soc. Exp. Biol. Med. 85, 583-587.

2.

Krum, A. A., and Glenn, R. E. 255-258.

3.

Yudaev, N. A., and Anfinogenova, Gormonoter. 6 (i), 19-25.

4.

Fevold,

5.

Li, C. H., Geschwind, I. I., Dixon, J. S., Levy, A. L., and Harris, J. I. (1955). J. Biol. Chem. 213, 171-192.

6.

Pickering, B. T., Andersen, R. N., Lohmar, P., Birk, Y., and Li, C. H. (1963). Biochim. Biophys. Acta 74, 763-773.

7.

Birk, Y., and Li, C. H.

8.

Saffran,

9.

Saffran, M., and Schally, A. V. (1962). "Methods in Hormone Research" (R. Dorfman, ed.), p. 643. Academic Press, New York.

H. R.

(1968).

(1965).

Steroids 1 2

(1964).

M., and Schally, A. V.

C.

(1958).

S. A.

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118,

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Biochim.

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Biophys. Acta 82, Endocrinology 56,

i0.

Rerup,

ii.

Hinman,

12.

Ornstein,

13.

Davis,

14.

Zaffaroni,

15.

Porter, C. C., and Silber, 201-207.

16.

P~ron, F. G. (1962). In "Methods in Hormone Research, Dorfmann, ed.) pp. 199-264, Academic Press, New York.

J. W., Personal

(1954).

430-432. 523-532.

83-92.

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L.

(1964).

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121

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B. J.

(1964).

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(2), 404-427.

A.

(1953).

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R. H.

(1950).

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I"

185,

(R. I.

May 1972

ST ER O ID S

619

17.

University of Montana Computer Center.

18.

Hofmann, K.

19.

Landon, J., James, V. H. T., Crier, R. J., Wynn, V., and Frankland, A. W. (1964). J. Clin. Endocrinol. Metab. 2_~4 (ii), 1206-1217.

20.

Kass, E. H., Hechter, 0., Mou, T. W., and Lurie, M. B. Trans. Assoc. Amer. Physicians 68, 92-100.

21.

(1962).

Ann. Rev. Biochem.

31, 212-246.

Kass, E. H., Kendrick, M. I., and Finland, M. 102 , 767-774.

(1955b).

(1955a).

J. Exp.

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22.

Andrews, R. V.

(1969).

Fed. Proc. 28, 636.

23.

The trivial and systematic names for the steroids referred to in the text are as follows: ll-dehydrocorticosterone, ll-deoxycorticosterone,

21-hydroxy-4-pregnene-3,11,20-trione 21-hydroxy-4-pregnene-3,20-dione

corticosterone, llS,21-dihydroxy-4-pregnene-3,20-dione cortisol, 118, 17e,21-trihydroxy-4-pregnene-3,20-dione cortisone, 17~,21-dihydroxy-4-pregnene-3,11,20-trione pregnenolone,

3~-hydroxy-5-pregneno-20-one

progesterone,

4-pregnene-3,20-dione