The influence of plasma protein binding on the extra-adrenal metabolism of cortisol in normal, hyperthyroid and hypothyroid subjects

The Influence of Plasma Protein Binding on the Extra-Adrenal Metabolism of Cortisol in Normal, Hyperthyroid and Hypothyroid Subjects By WILLIAM R. BEISEL, VINCENT C. DIRAIMONDO, PING Yu CHAO, JORGE M. ROSNER AND PETER H. FORSHAM 334 minutes. Unbound cortisol always disappeared from plasma more rapidly than bound. The per cent of total plasma cortisol bound to protein was essentially the same at any given concentration of total plasma cortisol throughout the range (7 to 2OO~gJlOO ml.) studied, irrespective of the metabolic state. This implied that the association constants and binding capacities of corticosteroidbinding proteins were unchanged in hyper- or hypothyroidism. Therefore, differences in the disappearance of cortisol observed between normal, hyperthyroid, and hypothyroid subjects did not appear to be a function of protein binding, but could be explained solely by alterations in the rate of hepatic and peripheral cortisol metabolism.

The role of plasma protein binding in the disappearance of cortisol from plasma in vivo has not been previously studied in normal individuals and those with altered thyroid function. Using an ultracentrifugation technic described herein to measure protein binding, the half-disappearance time of the unbound and protein-bound fractions of plasma cortisol was determined. In 6 normal subjects the mean half-disappearance time of total cortisol from plasma was 134 minutes, while that of its unbound fraction was shorter (81 min.) and that of the bound fraction was longer (163 min.). In hyperthyroid subjects these times were only 53, 40 and 71 minutes, respectively, while in hypothyroidism they were all slowed to 288, 160 and

T

HE ROLE OF PLASMA PROTEIN BINDING has not been investigated directly in previous studies of the disappearance of cortisol from plasma. The accelerated rate of disappearance in hyperthyroidism and the slowed rate in hypothyroidism have been attributed to alterations in the hepatic metabolism of cortisol,l-” but in these studies the rate of disappearance of the proteinbound and unboutid fractions of plasma cortisol were not differentiated. The delayed disappearance of cortisol from plasma in late pregnancy has been attributed to increased protein binding, -I-‘; but this has not been measured directly. The rate of disappearance of cortisol from plasma is also related to the amount administered,‘,” being shortest with radioactively tagged tracer doses and longest when large loads are given. Thus it seemed that measurement of disappearance rates of protein-bound and unbound cu>rtisol in normal subjects and those with altered thyroid function might provide a better understanding of the influence of plasma protein’ binding on the peripheral metabolism of cortisol. From the Metabolic Research Unit and the Department California School of Medicine, Sun Francisco, California.

of Blecficinc, University

of

This work was supported by a grant-in-aid from the lrpjohn Company and Grant MY 3951 of The National Institute for Mental and Newological Diseases, Il. S. Public Health SerGce. Received for publication Apr. 1, 1964. 942 h~ETABOLISM,

VOL.

13,

No.

10 ( OXOHHH),

1964

INFLUENCE

Utilizing a centrifugation disappearance unconjugated subject.

reasonably simple, high precision technic employing ultrato measure cortisol binding we have determined the halftimes ( tl/&) of the proteiti-bound and unbound fractions of cortisol in plasma of normal, hyperthyroid, and hypothyroid

MATERIALS Studies primary

were

made

in 6 normal

hypothyroidism.

in alcohol*

dissolved

pharmacologic binding

Each in 560

subject

solution

jects,

all blood samples were heparinized; the

apparent

distribution

volume

of cortisol

within

obtained beginning 1 hour after corticosteroids were quantitated

the method

et al .I The per cent of cortisol

period.

changes blood

This

in cortisol

samples

were

in hyperthyroid promptly,

had usually

wero based on samples Plasma unconjugated of Peterson

a 30 minute

was separated

concentrations

and 3 with

of 100 mg. cortisol

of possible

or for 5 hours

the plasma

As plasma

infusion

After the infusion

to 2 hours for 7 hours,

C. until studied.

hyperthyroidism,

during

investigstion

over a wide range of plasma concentrations. of one-half

with

an intravenous

saline

to permit

at intervals

with

3 patients

received

ml. normal

load was employed

AND METHODS

adults,

taken

stored at -10

943

OF BINDING ON CORTISOL DISAPPEARANCE

1 hour,

sub-

frozen,

reached

and

equilibrium

determination

of t%,

completion of the infusion. as Porter-Silber chromogens bound

to protein

by

was determined

in duplicate by an ultracentrifugation technic modified from one used to determine the protein binding of calcium.3 This measured the total protein binding of cortisol due to both albumin

and corticosteroid-binding

globulin

( CBG,

ULTRACENTRIFUGATION Plasma 4-C’4f cortisol and

diluted

with an equal volume

in trace amounts

added to 4 ml. of plasma

had

an activity

of normal

was ultracentrifuged

of 8,000

increased

transcortin).

METHODS saline solution

at a relative the initial

cortisol

disintegrations/minute.

which

contained

force of 115,006 concentration

The

diluted

pipetted into 6.5 ml. cellulose centrifuge tubes which permit by adsorption.‘0 After 16 hours of equilibration at 4 C.,

plasma

cortisol-

G.9 The radioby 2 pg./ml. mixture

was

no significant loss of cortisol ultracentrifugation was per-

formed for 6 hours at 37 C. in a Spinco Model L-HT machine at 40,000 rpm using a 40.3 type fixed angle rotor which permitted a maximum of 18 tubes to be spun simultaneously. The

37 C. temperature

was selected

for ultracentrifugation

in an attempt

to maintain

in

viva conditions, as low temperatures are known to increase the affinity of CBG for cortisol.l’-1s After ultracentrifugation in this manner, 2.2 ml. of the uppermost portion was aspirated with care to prevent disturbance of the layering or inclusion of superficial lipids. This portion was free of protein portion

were

extracted

with

as determined

by Biuret.

15 ml. of methylene

Duplicate

dichloride,

1.0 ml. aliquots

dried

under

of this

vacuum

in a

counting vial, and redissolved in 5 ml. scintillation fluid.$ Radioactivity of each duplicate was determined during a 20 minute period by a Packard Tricarb liquid scintillation spectrometer

using tap 3, attenuator

1, with a high voltage

setting

of 10 to 50 volts.

It was assumed that the radioactivity of the upper portion of the spun sample represented the unbound cortisol concentration in plasma water. As water normally comprised 93 per cent *We

of the pIasma

we indebted

volume 9 the water

to Dr. H. C. Peltier

of The

content Upjohn

of plasma

diluted

Company,

with

Kalamazoo,

an equal Michigan,

for the supply of intravenous Cortefcn) in 50 p&r cent ethyl alcohol. fobtained from the Endocrine Study Section of The Nation11 Institutes of Health, Public Health Service, and purified further by paper chromatography in successive systems to achieve a constant specific activity. IReagent grade toluene p-bis (2-( 5-phenyloxazolyl)

containing 0.4 per cent 2,5 diphenyloxazole ) l-benzene.

and 0.004

U. S. Bush

per cent

BEISELET AL.

944 volume

of saline solution

was considered

to equal

96.5

per cent of the volume.

The total

radioactivity in plasma water was calculated and compared to the radioactivity added to the specimen to determine the per cent of bound and unbound cortisol. It was found

that the protein

In a preliminary radioactive

binding

of cortisol

was reduced

study using a single large pool of normal

cortisol

to give 5,300

counts/minute/ml.

by diluting

plasma

initially

the plasma.

(with

sufficient

added

and a concentration

of 20.5

pg. of

17-OHCS/lOO ml. of plasma) the per cent of bound cortisol had an inverse relationship with the logarithm of the dilution (see fig. 1). At dilutions of 1 in 20, binding was reduced to 35 per cent then chosen difficulties samples

and was barely

for routine

studies

in accurately

adding

and pooled plasma

ied to determine 2 dilution

detectable because

a minute

containing

and used to correct propriate undiluted pendix.

plasma.

dilution.

of tracer

concentrations

between

The

less plasma

amount

varying

the exact relationship

and in undiluted

at a greater it required

1 in 2 dilution

and eliminated

to each

sample.

Individual

of carrier

cortisol

were stud-

the per cent of unbound

By this method,

was

technical

an average

cortisol

at a 1 in

ratio of 0.68’7 was derived

the per cent of unbound cortisol found in diluted plasma to an apvalue. Sample calculations with this method are shown in the Ap-

At pH values below 5 there is apparent instability of CBG’s but as shown by Chen, Mills and Bartterl2 the degree of steroid binding of cortisol was not highly dependent on pH between higher

the range

pH values.

that an increase As pH within

of 7.0 to 8.2,

Our preliminary

although studies

of pH from 6.7 to 8.1 caused

the sealed ultracentrifuge

ultracentrifugation,a

no initial

binding

using

affinity was slightly

pooled

plasma

this,

less than a 2.5 per cent increase

tube is not measurably

pH adjustments

greater

confirmed

changed

were made in preparing

at the showing

in binding.

by standing plasma

or by

samples

for

variations

in

the procedure. To gain information cortisol

binding

on the usefulness

at different

plasma

of this ultracentrifugtion

concentrations

were studied

method, in normal

subjects:

blood

was obtained between 7 and 10 a.m. from 40 fasting individuals, between 4 and 6 p.m. in 9 subjects to obtain low plasma concentrations, and during or after cortisol infusion in 10 normal

subjects

to study high levels

produced

in vivo. The

cortisol at varying levels of plasma cortisol in these normal 2. These slopes are virtually identical with those obtained cortisol

concentration

C. to obtain

Milks (a

constant,

subjects

The CBG

capacity

subjects

by Peterson

of unbound

are shown in figure a smaller range ot

et al.5 who ultrafiltered

plasma

at 37

their data.

The association our normal

in normal

concentrations

individuals throughout

binding

of 24.3

K, for CBC

calculated

after correction

was 4.7 x 107 L./ mole as compared capacity

pg./100

at 37 C. was 4.3 x 10-T

ml. was calculated

for albumin

binding8

to that of 5.2 s 107 reported moles/L.,

if no correction

in by

or 15.4 yg./lOOml.

was made

for albumin

binding). No differences in binding could be detected centrifugation method, duplicate determinations

because of sex or race. With throughout the entire range

concentrations showed excellent agreement, with the coefficient of variation deviation of differences x lOO/mean) being 1.7 per cent. Binding of cortisol

this ultraof plasma (standard was also

determined in more than 60 of these same samples by an equilibrium dialysis method” but these showed a poorer coefficient of variability (8.9 per cent) between duplicates and a less exact correlation between unbound and total cortisol concentrations.

RESULTS The average rate of disappearance (half-disappearance time, t?&) of cortisol from plasma in our 6 normal subjects was 134 minutes, within the over-all range of normal values (113 to 146 min.) reported by various 3*5-6~8 after a pharmacologic load of cortisol. The rate of disappearance grOUPS

INFLUENCE

OF BINDING ON CORTISOL DISAPPEARANCE

945

lOOr

60-

40-

DILUTION

OF

PLASMA

Fig. L-The effect of dilution on cortisol binding. Protein-bound cortisol was determined in multiple dilutions of a single pool of human plasma with added cortisol-4-C14. of cortisol from plasma was accelerated iti hyperthyroidism and slowed in hypothyroidism. Deviation of tYszd of cortisol from normal values in each patient was roughly proportional to the clinical and laboratory assessment of the metabolic abnormality present (see table 1). In all subjects the rate of disappearance of unbound cortisol was more rapid than that calculated from the total plasma cortisol concentrations while the disappearance of the bound fraction was slower. In normal subjects the mean tl/zd for the unbound cortisol was 81 minutes (see fig. 3) while that of the bound component was 163 minutes. Disappearance of both unbound and bound components of cortisol were more rapid in hyperthyroidism (see fig. 4). Again the average tlhd of the unbound fraction was faster (46 min.) and that of bound cortisol slower (71 min.) than the t$& calculated from the total (53 min.) In hypothyroidism the half-life disappearance time of total plasma cortisol (mean, 288 min.) as well as that of the bound and unbound fractions was longer than normal. Again, the tl/zd of unbound cortisol was shorter (mean, 160 min.) and that of the bound fraction was longer (mean, 394 min.) than that of the total. The binding of cortisol by plasma proteins did not appear to be influenced by either hyper- or hypothyroidism. The correlation between total plasma cortisol and its unbound fraction in hyper- or hypothyroid plasma showed no

946

BEISEL ET AL.

SO-

2.0 -

1.0 G

6-/O 0 4-6

l

8 5; a

I

16

AM PM

I

I

20

rOTAL PLASMA CORTISOL C AS I7-OHCS) pyboo

2s

ml

Fig. 2.-Relationship of unbound to total plasma cortisol (17-OHCS) in normal subjects. The relationship of total plasma cortiso1 (measured as I7-OHCS) to its unbound fraction is shown. In A (above) points represent variations of endogerwus cortisol in normal subjects. The least squares line pictures was calculated from morning specimens only. B (below) shows this relationship at elevated concentrations in normal subjects produced by cortisol infusion. The slope of the least squares line pictures was steeper at elevated levels because of the weaker binding affinity of plasma albumin when compared to corticosteroid-binding globulin.

INFLUENCE OF BINDING ON CORTISOL DISAPPEARANCE

Table I.-Cortisol

Clinical Impression

PBl (Mr./ 100 ml.)

Age and Sex

947

Disposal Rates

AA1 Uptake (?‘c 24 hr.)

Ta-RBC uptake (‘ic)

Cholestel.01 (IIlK./ 100 ml.)

NCX-?d Average

-_____ tYz, Total (min.)

ts.i Unbound (min.)

t%, Bound (min.)

134

81

163

32 48 78

30 38 63

53 71 E4

63

40

71

432 210 222

225 144 110

664 246 372

288

160 __~-

394

Hyperthyroid 2. K.--severe P. K.-moderate R. P.-mild

31 F 23 M 67 F

17.8 9.8 11.0

82 74 63

29 36

169 130 154

Average Hypothydd M. Y.--severe

68 M

2.1

1.0

C. F.--moderate R. S.--moderate

68 F 41 F

4.1 2.7

0.3 3.0

Average __-___-~

significant other.

-

---

differences

11 13 16

from normal

(as depicted

361 629 340

in figure. 2)

or from each

DISCUSSION

The observation that unbound cortisol disappeared from plasma about twice as rapidly as its protein-bound fraction added support to several concepts regarding the physiologic role of protein-bound cortisol. Protein binding appeared to block the physiologic activity of cortisol’4 and also served to prevent excessive urinary loss of cortisol. l5 Although subjects with increased CBG were not studied herein, these findings in normal subjects provided additional support for the postulated role of heightened protein binding in pregnancy in slowing the tl& of cortisol. The experimental design of these studies did not yield information concerning rates of protein binding in vivo, as a prolonged time elapsed during determinations of binding which permitted cortisol to equilibrate with its binding proteins. However, no differences were detected in binding if cortisol were added to plasma in vitro or in vivo (either during or following an intravenous infusion). The binding of cortisol in normal subjects was dependent upon the concentration of cortisol in plasma as governed by the physical chemical factors of the binding affinities and capacities of plasma components for cortisol. This implied a reversible binding, independent of metabolic “work” in the liver or elsewhere, compatible with the concept that bound cortisol served as a circulating reserve of temporarily inactivated cortisol in a state of equilibrium with unbound, physiologically active hormone. Although the half-disappearance time of cortisol was markedly influenced by the level of thyroid function, the per cent of unbound cortisol increased in its unsual normal linear relationship with total plasma cortisol at elevated concentrations, irrespective of the metabolic state. Neither decreased protein binding of cortisol in hyperthyroidism nor increased binding in hypothyroid-

948

BEISEL ET AL.

1234567

HOURS

Fig. 3.-Disappearance of plasma 17-Hydroxycorticosteroids after 100 mg. intravenous cortisol in normal subjects: comparison of protein-bound and unbound fractions. The disappearance of the protein-bound and unbound fractions of plasma cortisol is shown in normal subjects as the mean * SE of the group.

ism, inferred indirectly from determinations of cortisol uptake by red blood cells,r6 was confirmed by our direct measurement of plasma protein binding at either normal or elevated concentrations of cortisol. Accordingly, the acceleration of the t%,, of total, bound and unbound cortisol above normal in hyperthyroidism, or its slowing in hypothyroidism, could not be explained by alterations in plasma protein binding. Metabolic degradation of cortisol, controlled chiefly within the liver by the rate of initial saturation of .the A-ring to form dihydrocortisol, was speeded in hyperthyroidism and slowed in h.ypothyroidism *i-‘!’ because of increased or decreased activity of the rate-limiting enzyme, delta-4-hydrogenase. The assumption that only unbound cortisol could be metabolized”’ was compatible with the findings of this study. Slower disappearance of cortisol from plasma after large doses as compared to trace quantities could be explained also if the increased concentration of unbound cortisol after large loads exceeded the capacity of the rate-limiting enzyme to remove it. Slowed degradation would also result if the equilibration of cortisol and its binding proteins were less than instantaneous and amounts were “trapped” in a bound form.

INFLUENCE

OF BINDING ON CORTISOL DISAPPEARANCE

949

1SOr

150

nYPAW..YRO/D

100 L

K_

60

s___ i

20

,.:,:.:.:.:.:.:.:.:.:.:.:: :...;.;.: ..: ........ ...~.......-.....~.....-.-...~.~.-.-,-.-. .;;;;;:::. -:;::..:“,,-;:-~~~~:;. :. ,..,...;..,.. .:::::.

-

. ,,min

‘.‘.‘.:.;.:.::::::; ..::::::::::::::::::::::::::::::::::: ~.~.~.,.,,, __ ”

‘.‘.~.:.;. ...... ,,,,_ UNDOUND .. ..... .......................;.: ,,,,, ‘~.‘.‘.:.:~~~~:,:,:,:,~.:.:.:.:.:.:.:.:.:.:.:.:.~ :.:.; ,....,

‘.‘~~~.:.:~~~~~~.~,.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.~.:.:.:.:.~ ,.,.,,, .........,: .........i ,.,.,.,.; ,.,.,.,.,,.,.,...,...... .,.,.......,., .:. ..i, .,..c .............................,.,.....,.......,.::::;, ,..........:,.,.... c

6-

6

min

.,.,,~~.~~~~~,~.:,t,t~ * 16o

10 -

4-

~.~.~.~.~.~.~.~.__. .,.(,.,.,.,.,.,.,.,.,.,: :... .:;;::::. .:,..., -..,~.~,~,~,~.~.~,~,~.~.~.~.‘. ‘.‘,~~~r~;;;;~;;;;:‘;.‘~;;~..~.-.~. ..........: ........:.,...... ......:.. ......,.::. .,.~.,.,.,.,.,.,.,.,.,...,.,.,.,...,.,.,. .....-::: ‘.‘.‘.:.:.:.:.:‘.:.:.:.:.:.:.::.:.:.:.:.~~~ .........._._._._ .... ..:_ .::,. _ ._~~~_._., __., .,.,.,.,.,.,.,.,.,.,.,.,.,.,...,.,.,.,.,.,.,., ,.,.,., .; ,.,..., .::.

2

1 0.6 0.4 i

Fig. 4.-Disappearance of bound and unbound cortisol in hyper-and hypothyroid subjects. The disappearance of the protein-bound and unbound fractions of plasma cortisol is shown in hyperthyroid and hypothyroid subjects as the range of data in each group. APPENDIX Samde a) b) c) d)

Initial

Calcukztiun of Per Cent Protein Bindiw of Co&sol by the Ck?acentrifugation Method-

dilution:

Total radioactivity in ultracentrifuged sample: Volume of protein-free plasma Hz!0 Radioactivity in 1.0 ml. aliquot of protein-free plasma H,O after ultracentrifugation:

e ) Total radioactivity in proteinfree plasma water:

f ) Per cent unbound cortisol, uncorrected g) h)

~

Per cent unbound cortisol, corrected for dilution Per cent protein-bound cortisoi

4 ml. plasma 4 ml. saline-cortisol-C’4

=

6.5 ml. X 500 cpm/ml.

=

3250 cpm total

6.5 ml. X 96.5%

=

6.27 ml.

=

100 cpm/ml.

=

627 cpm

100 cpm/ml. X 6.27 ml. 627 cpm

x loo

8ml. 500 cpm/ml.

= 19.3%

3,250 cpm 19.3% X 0.687 100% - 13.3%

= =

13.3% 86.7%

950

BEISEL

ET AL.

ACKNOWLEDGMENT We

wish

to thank

centrifugation

Dr.

technique

sistance, and encouragement

Lt.

Hans tar

Col. Harry and guidance

F. Loken

use

for

in this

advice

study,

F. Hurd, M.C., in this work.

Miss

and

assistance

Jeanette

U. S. Army,

in adapting

Shinsako

and

Dr.

the

ultra-

technical

as-

G. Biglieri

for

for

Edward

REFERENCES 1. Peterson, Guerra,

R. E., S. L.,

Bunim, position

Wyngaarden, Brodie, B.

J. B., B., and

J. J.: The physiological disand metabolic fate of hydro-

cortisone in man. 1779, 1955. 2. Samuels,

L.

T.,

J. Clin.

Invest.

34:

Brown,

H.,

Eik-Nes,

Moor, P., and Hinnekens, M.: The disappearance rate of plasma corticoids in patients with suppressed

4. Sandberg, Ft., Jr.:

production, 1961.

A. A., and Transcortin:

J. Clin.

Acta

En-

II. Leve!s the effects

Invest.

38:1290,

1959. 5. Peterson,

R. E., Nokes,

Jr., and Black, adrenocortical Clin. 1960. 6. Mills,

I. H., Schedl,

Jr., and estrogen tabolism

G., Chen,

P. S.,

R. L.: Estrogens and function in man. J.

Endocrinol

& Metab.

20:495,

albumin

H. P., Chen,

P. S.,

Endocrinol.

&

7. Cranny, R. C., and Kelley, V. C.: The interpretation of steroid half-life J. Clin. Endocrinol. 1959.

as demon-

ultracentrifugation

& Metah.

8. Mills, I. H.: Transport and metabolism of steroids. Brit. M. Bull. 18:127, 1962. 9. Loken, H. F., Havel, R. J., Gordan, G. S., and Whittington, S. L.: Ultracentrifugal analysis of protein-bound and free calcium in human serum,

and

Endocrinology

57:

1955.

11. Slaunwhite, W. R., Jr., and Sandberg. A. A.: Transcortin: a corticosteroidbinding

protein

of

plasma.

Invest. 38:384, 1959. 12. Chen, P. S., Jr., Mills, Bartter, F. C.: of steroid-protein

I.

Ultrafiltration binding.

crinol. 23: 129, 1961. 13. Daughaday, W. H., and

J.

Clin.

H.,

and

stiidies J. Endo-

Mariz,

I. K.:

globulin:

properties and quantitation. lism 10:936, 1961. 14. Slaunwhite, Black.

W. R., Jr., Lockie, G. N., and Sandberg, A. Transcortin: a corticosteroid-binding protein of plasma. IV. ViVO of transcortin-hound Science 135:1062, 1962.

13. Beisel,

W.

R.,

Cos.

J. J.,

excretion. J. Clin. Metah. in press. 16. Farese,

R. V.,

in vitro cortisol: binding abnormal

and

red blood

Inactivity

states.

N., A,: in

cortisol. Horton,

R.,

P. H.: cortisol

Endocrinol.

Plager,

J. E.:

cell uptake

studies of of cortisol

its

Metabo-

Chao, P. Y., and Forsham, The physiology of urinary

Bartter, F. C.: The effect of administration on the meand protein binding of hy-

drocortisone. J. Clin. Metab. 20:515, 1960.

values. 19:854,

serum

by

Corticosteroid-binding

Slaunwhite, W. a corticosteroid-

binding protein of plasma. in various conditions and of estrogens.

and

strated 456,

the levels of 17-hydroxycorticosteroids in plasma. CIBA Fdn Colloq Endocrinol. 11:208, 1957.

adrenocortical docrinol. 37:1,

4-Cl4

electrophoresis.

K., Tyler, F. H., and Dominguez, 0. V.: Extra-adrenal factors affecting

3. De

J. Biol. Chem. 235:3654, 1960. 10. Westphal, U.: Interaction between hydrocortisone-4-Cl4 or progesteronr-

& The

of C”-

plasma protein in normal and

J. Clin.

Invest.

41:

53, 1962. W. H.: 17. Levin, M. E., and Daughaday, The influence of the thyroid on adrenocortical function. J. Clin. Endocrinol. & Metab. 15:1499, 1955. H., Englert, E., Jr., and Wal18. Brown, lath, S.: Metabolism of free and conjugated 17-hydroxycorticosteroids m subjects with thyroid diseases. J. Clin. Endocrinol. & Metab. 18:167.

INFLUENCE

19. -,

20.

OF BINDING

951

ON CORTISOL DISAPPEARANCE

1958.

FL, Jr.:

and -: Corticosteroid metabolism in liver disease. Arch. Int. Med. 107:

binding protein of plasma. V. In vitro inhibition of cortisol metabo-

773, 1961.

lism. J. Clin. Invest.

Sandberg,

A. A., and

Slaunwhite,

Transcortin:

a corticosteroid-

42:51,

1963.

W.

Lt. Cot. Willium R. Beisel, M.C., U.S. Army; Research Fellow, Metabolic Research Unit. Present afiliation: Chief, Physical Sciences Division, U.S.A. Medical Unit, Fort Detrick, Md. Vincent C. DiRaimondo, M.D., Associate Professor of Medicine; Associate Director, Metabolic Research Unit. Ping Yu Chao, M.D., Research Fellow of the China Medical Board, New York, N. Y.; Assistant Research Physician, Metabolic Research Unit. Jorge M. Rosner, M.D., Fellow of El Cosejo National de lnvestigaciones Cientificas y Tkcnicas de la Repiblica Argentina; Assistant Research Physician, Metabolic Research Unit. Present address: Facultad de Medic& Universidad de1 Salwdor, Buenos Aires, Argentina. Peter H. For&m, M.D., Professor of Medicine and Pediatrics; Director, Metabolic Research Unit; Chief of Endocrinology and Metabolism, Department of Medicine, University of Cflifornia School of Medicine, San Francisco, Calif. IL

.c: