Docsity
Docsity

Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity


Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan


Guidelines and tips
Guidelines and tips

Steroid Formation by Adrenal Tissue from Hypertensives: A Comparative Study, Lecture notes of Medicine

The results of a study investigating steroid formation by adrenal tissue from normotensive and hypertensive patients. The researchers used adrenal tissue from cancer patients as a baseline to evaluate hypertensive alterations. The study found that steroid formation decreases with increasing diastolic blood pressure and that adrenal weight increases with increasing diastolic blood pressure, partially compensating for the reduced rate of steroid formation. ACTH stimulation did not significantly alter the differences between steroid formation of normotensive and hypertensive groups or within the hypertensive series.

What you will learn

  • What are the implications of these findings for the understanding of essential hypertension?
  • What is the relationship between steroid formation and diastolic blood pressure in hypertensive patients?
  • How does ACTH stimulation affect steroid formation in normotensive and hypertensive patients?
  • How does steroid formation by adrenal incubates differ between normotensive and hypertensive patients?
  • What is the role of adrenal cortical hormones in the pathogenesis of hypertension?

Typology: Lecture notes

2021/2022

Uploaded on 09/12/2022

prouline
prouline 🇬🇧

4.6

(7)

221 documents

1 / 13

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
STEROID
FORMATION
BY
ADRENAL
TISSUE
FROM
HYPERTENSIVES
1
By
DAVID
Y.
COOPER,2
JOSEPH
C.
TOUCHSTONE,
JOHN
M.
ROBERTS,
WILLIAM
S.
BLAKEMORE,
AND
OTTO
ROSENTHAL
WITH
THE
TECNICAL
ASSISTANCE
OF
MARIA
KASPAROW
(From
the
Harrison
Department
of
Surgical
Research,
Schools
of
Medicine,
University
of
Pennsylvania,
and
the
Surgical
Clinic
of
the
Hospital
of
the
University
of
Pennsylvania,
Philadelphia,
Pa.)
(Submitted
for
publication
March
15,
1958;
accepted
July
3,
1958)
The
adrenal
cortical
hormones
have
been
thought
to
play
a
role
in
the
pathogenesis
of
hy-
pertension
for
the
following
reasons:
1)
Hyper-
tension
is
a
characteristic
finding
in
Cushing's
disease;
2)
large
doses
of
desoxycorticosterone
(DCA)
and
salt
administered
to
Addisonian
pa-
tients
produce
hypertension
(1);
3)
hypertensive
patients
who
developed
Addison's
disease
had
blood
pressure
falls
to
normal
limits
which
were
restored
to
previous
levels
with
DCA
(2)
;
and
4)
80
per
cent
of
the
patients
with
severe
hypertension
have
had
significant
improvement
for
three
to
seven
years
following
90
to
100
per
cent
adrenalec-
tomy
and
limited
sympathectomy
(3).
Moreover,
animal
experiments
(4,
5)
have
shown
that
hy-
pertension
following
renal
artery
occlusion
can-
not
be
produced
in
the
absence
of
adrenal
cortical
steroids.
Despite
these
observations,
altered
adrenal
function
in
hypertension
has
not
been
clearly
demonstrated.
While
mean
levels
of
cortical
ster-
oids
in
blood
and
urine
(6-9)
are
not
elevated
in
hypertensive
patients,
disturbances
in
salt
and
wa-
ter
metabolism
observed
in
early
hypertension
prior
to
the
development
of
renal
damage
by
Green,
Johnson,
Bridges
and
Lehmann
(10)
and
Braun-
Menendez
(11)
indirectly
support
the
idea
that
the
pattern
of
adrenal
corticoids
is
altered.
Brady
(12)
found
that
slices
of
canine
adrenal
tissue
produce
large
quantities
of
steroids
when
incubated
in
vitro
in
autologous
plasma
and
pro-
posed
the
measurement
of
steroid
formation
in
vitro
as
a
direct
assessment
of
the
functional
ca-
1
This
work
has
been
supported
by
Public
Health
Grant
CY-3644,
Heart
Association
of
Southeastern
Pennsylvania,
The
Pennsylvania
Heart
Association
and
the
American
Cancer
Society.
2Charles
0.
Finley
Fellow
of
American
College
of
Surgeons.
pacity
of
adrenal
tissue
(13).
The
use
of
adrenal-
ectomy
for
the
palliative
management
of
advanced
cancer
(14,
15)
and
the
treatment
of
severe
hy-
pertension
(16)
enabled
us
to
apply
this
method
to
human
adrenal
tissue
and
to
demonstrate
that
the
amount
of
steroids
formed
by
the
human
adrenal
incubates
is
sufficiently
large
for
semi-
quantitative
estimation
and
identification.
In
the
present
report
we
are
presenting
steroid
analyses
of
adrenal
tissue
incubates
from
3
nor-
motensive
cancer-free
patients,
8
normotensive
pa-
tients
with
carcinoma
of
the
breast
or
prostate
and
23
patients
with
severe
hypertension.
The
dif-
ferences
in
the
steroid
formation
by
the
incubates
from
normotensive
and
hypertensive
subjects
and
the
relationship
between
clinical
picture
and
ster-
oid
formation
within
the
hypertensive
group
will
be
discussed.
In
addition,
the
steroid
pattern
in
the
incubates
from
hypertensive
patients
will
be
compared
with
that
in
the
adrenal
vein
blood
ob-
tained
at
operation.
Studies
concerning
the
iden-
tification
of
the
steroids
will
be
reported
elsewhere.
EXPERIMENTAL
1.
Patients.
The
clinical
data
on
the
normotensive
patients
are
listed
in
Table
I.
In
two
of
the
three
can-
cer-free
patients
only
a
portion
of
one
adrenal
gland
(200
to
600
mg.)
was
excised,
while
in
the
third
a
uni-
lateral
adrenalectomy
was
performed
because
of
a
hemor-
rhage
occurring
in
the
gland
during
the
operation.
No
abnormalities
were
found
by
histological
examina-
tion.
The
eight
normotensive
cancer
patients
were
am-
bulatory
and
not
nutritionally
depleted
at
the
time
of
adrenalectomy
as
evidenced
by
the
satisfactory
levels
of
serum
proteins
(Table
I),
although
all
had
far
advanced
carcinoma
of
the
breast
or
prostate.
The
adrenals
from
these
patients
were
carefully
examined
grossly
and
mi-
croscopically
for
evidence
of
metastatic
tumor.
None
was
found.
Table
II
furnishes
the
clinical
data
of
14
of
the
23
hypertensive
patients
studied.
In
these
patients
a
com-
1524
pf3
pf4
pf5
pf8
pf9
pfa
pfd

Partial preview of the text

Download Steroid Formation by Adrenal Tissue from Hypertensives: A Comparative Study and more Lecture notes Medicine in PDF only on Docsity!

STEROID FORMATION BY^ ADRENAL^ TISSUE^ FROM

HYPERTENSIVES 1

By DAVID Y. COOPER,2 JOSEPH C. TOUCHSTONE, JOHN M. ROBERTS, WILLIAM S. BLAKEMORE, AND OTTO ROSENTHAL WITH THE TECNICAL ASSISTANCE OF MARIA KASPAROW (From the Harrison Department of Surgical Research, Schools of Medicine, University of

Pennsylvania, and the Surgical Clinic of the Hospital of the University of

Pennsylvania, Philadelphia, Pa.)

(Submitted for publication March 15, 1958; accepted July 3, 1958)

The adrenal cortical hormones have been

thought to play a role in the pathogenesis of hy-

pertension for the following reasons: 1) Hyper-

tension is a characteristic finding in Cushing's

disease; 2) large doses of desoxycorticosterone

(DCA) and salt administered to Addisonian pa-

tients produce hypertension (1); 3) hypertensive

patients who developed Addison's disease had

blood pressure falls to normal limits which were

restored to previous levels with DCA (2) ; and 4)

80 per cent of the patients with severe hypertension

have had significant improvement for three to

seven years following 90 to 100 per cent adrenalec-

tomy and limited sympathectomy (3). Moreover,

animal experiments (4, 5) have shown that hy-

pertension following renal artery occlusion can-

not be produced in the absence of adrenal cortical

steroids.

Despite these observations, altered adrenal

function in hypertension has not been clearly

demonstrated. While mean levels of cortical ster-

oids in blood and urine (6-9) are not elevated in

hypertensive patients, disturbances in salt and wa-

ter metabolism observed in early hypertension

prior to the development of renal damage by Green,

Johnson, Bridges and^ Lehmann^ (10)^ and^ Braun-

Menendez (11) indirectly support the idea that

the pattern of adrenal corticoids is altered.

Brady (12) found that slices of canine adrenal

tissue produce large quantities of steroids when

incubated in vitro in autologous plasma and pro-

posed the measurement of steroid formation in

vitro as^ a^ direct assessment of^ the^ functional^ ca-

(^1) This (^) work has been supported by Public Health Grant CY-3644, Heart Association of Southeastern^ Pennsylvania, The Pennsylvania Heart^ Association^ and^ the^ American Cancer Society. 2Charles 0. Finley Fellow of American College of Surgeons.

pacity of adrenal tissue (13). The use of adrenal-

ectomy for^ the^ palliative^ management^ of^ advanced

cancer (14, 15) and^ the treatment^ of^ severe^ hy-

pertension (16) enabled us to apply this^ method

to human adrenal tissue and to demonstrate that

the amount of steroids formed by the human adrenal incubates is sufficiently large for semi-

quantitative estimation and identification.

In the present report we are presenting steroid

analyses of adrenal^ tissue^ incubates^ from^3 nor-

motensive cancer-free patients, 8 normotensive^ pa-

tients with carcinoma of the breast^ or^ prostate and

23 patients with severe hypertension. The^ dif-

ferences in the steroid formation by the incubates

from normotensive and hypertensive subjects and

the relationship between^ clinical^ picture and^ ster-

oid formation within^ the^ hypertensive group will

be discussed. In^ addition, the^ steroid^ pattern in

the incubates from hypertensive patients will^ be

compared with that in the adrenal vein blood ob-

tained at operation. Studies concerning the iden-

tification of^ the^ steroids^ will^ be^ reported elsewhere.

EXPERIMENTAL

  1. Patients.^ The^ clinical^ data^ on^ the^ normotensive patients are^ listed^ in^ Table^ I.^ In^ two^ of^ the^ three^ can-

cer-free patients only a^ portion of^ one^ adrenal^ gland

(200 to 600 mg.) was^ excised, while^ in^ the^ third^ a^ uni- lateral adrenalectomy was^ performed because^ of^ a^ hemor-

rhage occurring in the gland during the^ operation.

No abnormalities were found by histological examina- tion. The eight normotensive cancer patients were^ am- bulatory and^ not^ nutritionally depleted at^ the^ time^ of adrenalectomy as^ evidenced^ by the^ satisfactory^ levels^ of

serum proteins (Table I),^ although^ all^ had^ far^ advanced

carcinoma of the^ breast^ or^ prostate. The^ adrenals^ from these patients were^ carefully examined^ grossly and^ mi- croscopically for evidence of^ metastatic^ tumor.^ None was found. Table II furnishes the clinical data^ of^14 of^ the^23 hypertensive patients studied. In these patients a^ com-

STEROID FORMATION BY^ ADRENAL^ TISSUE^ FROM HYPERTENSIVES

TABLE I Clinical data on normotensive patients

Blood Duration Blood Height Weight pressure of Serum urea Urine Adrenal- No. Patient^ Sex^ Age in.^ Ibs.^ mm.^ Hg Diagnosis* disease^ protein^ nitrogen albumin^ ectomy

A. Cancer-free patients 1 E. O. M^58 69 176 140/80 Sigmoid 10 yrs. 9 0 9/15/ diverticulitis (^2) G. B.t F (^58 69 126) 110/70 Congenital 6 mos. 12 trace (^) 3/29/ cystic dis. of kidney 3 R. H. M (^49 67 167) 130/80 Hematuria (^1) yr. 11 trace (^) 2/ 7/ undiagnosed

B. Cancer patientsl

1 A. E. F 51 66 150 128/80 Breast (^2) yrs. 9 0 2/17/ 2 D. B. F 48 61.5 139 120/80 Breast 6 yrs. 7.2 (^13 0) 12/20/ 3 L. F. F 48 63 140 110/80 Breast 2 yrs. 6.0 12 trace 11/30/ 4 W. S. M 62 61 135 130/60 Prostate 1-2 (^) yrs. 6.1 (^13 0) 4/ 8/ 5 J. B.§ M 57 64 125 132/80 Prostate 2 yrs. 5.4 16 0 1/22/ 6 G. M.§ M 58 67 173 114/68 Prostate (^3) yrs. 5.4 (^16) 1+ 1/19/ 7 J. H.§ M 71 71 160 130/70 Prostate 3 yrs. 5.9 13 0 3/31/ 8 J. G.§ M 67 67 128 130/80 Prostate 4 yrs. 6.4 21 0 4/12/

  • (^) Primary site of carcinoma in cancer cases. t (^) Subsequently found to have (^) papilloma of bladder, six months hematuria (^) prior to (^) operation. All patients previously castrated^ (except those^ indicated^ by §). Castrated at time of adrenalectomy.

plete chromatographic steroid analysis of the adrenal in- cubates was obtained. Blood (^) pressure and adrenal (^) weight records of the (^) remaining eight patients in whom (^) only the hydrocortisone values^ of^ the^ incubates^ are^ available^ are included with the^ latter^ values^ in^ Table^ V.^ The^ patients in Tables II and V are^ arranged according to^ ascending diastolic blood pressure. Those^ in^ Table^ II^ are^ divided for statistical analysis into^ three^ groups according to their clinical picture and diastolic blood pressure. All patients were severe hypertensives who^ had^ been followed by the staff of the Hypertension Section of^ the Edward B. Robinette Foundation of the Hospital of the University of Pennsylvania. At the time (^) adrenalectomy and a modified Adson type of (^) sympathectomy were rec- ommended (^) they had failed to (^) respond to several (^) types of medical (^) therapy and were (^) showing progressive symp- toms of the disease.^ All^ antihypertensive therapy had been (^) stopped two weeks (^) prior to (^) operation. Twenty-one of the patients were on the usual hospital house^ diet without any restrictions. Two of the patients were in congestive heart failure as indicated in^ Table II^ and were on a salt-poor diet.

  1. Preparation and extraction of the adrenal incubates. All adrenal tissue used in this study was obtained at the first stage of a bilateral adrenalectomy. No previous surgery had been done within one (^) year in all cases studied. Adrenal (^) glands were (^) dropped into an ice cooled beaker immediately after removal (^) and transferred to a cold room '(4° (^) C.) where (^) the periadrenal fat and capsule were removed. The glands were then weighed, a por- tion (^) removed for (^) histologic examination and the re- mainder sliced by means of a Stadie Riggs tissue slicer.

Approximately 1.0 Gm. batches of slices were transferred to chilled 125 ml. glass-stoppered Erlenmeyer flasks equipped with gas inlet and outlet tubes in the stoppers. Ten ml. of the patient's heparinized plasma with 50, units of penicillin and 0.1 Gm. of streptomycin were added to each flask. (^) The flasks were agitated in a water bath at (^) 37.5° C. for (^24) hours while a slow current of a gas containing 95 per cent oxygen and 5 per cent carbon dioxide was continuously passing through the gas space of (^) the flasks. The shaking rate was 50 to 60 oscillations per minute through an amplitude of 4 to 5 cm. The time interval between removal of the gland and the be- ginning of incubation was 45 minutes or less. Five units of adrenocorticotropin (Armour) was added to selected flasks. After incubation, the tissue and medium were quanti- tatively transferred to and homogenized in a Potter Elvehjem all-glass homogenizer. Five volumes of acetone was added to precipitate the protein which was separated by filtration and washed several times with additional acetone. (^) The acetone was removed in vacuo in a water bath at a temperature not exceeding 450 C. The aque- ous phase remaining was extracted twice with 50 ml. of ethyl acetate and twice with 50 ml. of chloroform. The combined organic extracts were washed twice with 25 ml. portions of 0.1 N sodium hydroxide and twice with 50 ml. portions of distilled water. The organic phase was dried over sodium sulfate and evaporated to dry- ness in vacuo. Adrenal (^) vein blood was treated in a similar (^) manner.

  1. Separation of the steroids. The residue (^) of the ex- tracts of (^) the adrenal incubates was subjected to paper chromatography by means of a modification of the method

STEROID FORMATION BY ADRENAL TISSUE FROM (^) HYPERTENSIVES

of Burton, Zaffaroni and Keutmann (17). Three main fractions resulted. Fraction X consisted of material found on the paper in^ a toluene-propylene glycol system after 13 ml. of effluent had been collected per 0.5 inch width of strip. The effluent from fraction X was evapo- rated and rechromatographed until 3 ml. of effluent per half inch strip was collected to give fraction Y. The effluent from fraction Y was (^) evaporated to (^) dryness and re- chromatographed in^ a methylcyclohexane-propylene gly- col system until 17 ml. of effluent per 0.5 inch strip was collected to give fraction Z. In all instances aliquots of the extract corresponding to approximately 0.15 Gm. of adrenal tissue were placed on each half inch width of chromatographic strip. The strips, after drying in air, were scanned at (^245) mju in a Beckman model D.U. spectrophotometer (^) equipped with an (^) adapter as described (^) by Tennent, Whitla and (^) Florey (18). The instrument was set to 100 per cent transmit- tance against an area of the paper strip one inch above the starting line. Representative strips were sprayed with blue tetrazolium reagent and read at 600 mjt for detection of a ketolic substances. Absorption curves for all strips were obtained by plot- ting the optical (^) density of each (^) point against the distance from the starting line. Quantitative determinations of the amount of steroid^ were^ made by comparison of^ the area under the peak of absorption at 245 m~u with the area under the curve produced by known amounts of cortisol, chromatographed under identical conditions as fraction X. In nine determinations of hydrocortisone by this (^) method, agreement with the Porter-Silber values on (^) aliquots from the same zone was better than (^95) per cent in^ all^ cases. Results of the incubation experiments are expressed as (^) ptg. steroid formed per 24 hours per Gm. of tissue. The standard error of measurement computed from 20 duplicate determinations of adrenal incubates was (^) plus or minus (^20) /Ag. for the F position and plus or minus 40

/Ag. for^ the^ steroids^ of^ the^ Y^ and^ Z^ positions.^ This^ cor-

responds (^) approximately to a (^) reproducibility of (^) plus or minus 5 per cent for^ the^ F^ position and^ plus or^ minus 8

per cent for the B-region. Recoveries of added cortisol were 95 to 105 per cent (13).

RESULTS

1. Chromatographic pattern in adrenal incubates

and adrenal vein blood

The chromatograms shown in Figure 1 repre-

sent the absorption curves at (^245) m1A from the ex- tracts of the adrenal (^) incubate and the adrenal vein

blood of a patient. The pattern is characteristic

for the entire group of patients studied. Five defi-

nite areas of absorption on the chromatograms

from the adrenal incubates have been found. The

first area, corresponding to material more polar

than (^) compound F, has been labeled the "before F"

region. It contains several steroids not as yet

identified. The second peak has been labeled as

hydrocortisone on the basis of the following char-

acteristics: The color reaction with blue tetrazolium

gave a quantititative value agreeing with the de-

gree of absorption of ultraviolet light at (^245) mpu

and the results of Porter-Silber determinations on

selected zones. The mobility of the eluted zone in

toluene-propylene-glycol and chloroform-forma-

mide paper chromatograms as the free alcohol and

as the acetate was the same as that of authentic hy-

drocortisone. The spectrum in sulfuric acid from

220 to (^600) m/u was the same as that of the refer-

ence steroid under equivalent conditions. The

characteristic green fluorescence was also shown

by the eluted zones. The third peak has a mobility

comparable to compound E in this system. Since

several unidentified substances are present in this

region, the area has been labeled the E-region.

§1 0- A m 15 BF

.M Adrenali1 In, S~w 1 5

10-IBFf\

. (^5) F I- Distance 10 20 Fraction (^) X

Ul -oni

icubate -j B..is 11>>3-Ot

10 20 10 20 cm. Y z FIG. 1. CHROMATOGRAMS OF (^) EXTRACTS OF ADRENAL VENOUS (^) BLOOD AND ADRENAL INCUBATES

COOPER, TOUCHSTONE, ROBERTS, BLAKEMORE, AND^ ROSENTHAL

The fourth peak may be in either the Y or Z frac-

tion and corresponds to^ the^ area^ in^ which^ com-

pound B would separate. This area, designated

the B-region, has recently been further separated

by use of the dimethyl (^) formamide-methylcyclo-

hexane paper chromatography system and shown

to consist of about 70 per cent corticosterone and

10 per cent Substance S^ [17a-hydroxydesoxy- corticosterone ( 19) (^) ]. The rema ing^20 per cent

of this region has not been identified. The fifth

area represents (^) A'-androstene-11,8-ol-3,17-dione as

has been previously reported by us (20).

The five areas representing the different steroids

on the chromatograms from^ the^ adrenal^ vein^ blood

are similar to those from the adrenal incubates.

It is possible, however, that there are differences

in the individual compounds which form the five

areas since all of the substances have not been

identified.

2. Steroid formation by adrenal incubates without

added ACTH

a. Normotensive Group. Comparison of the

values (Table III) for^ steroid^ formation^ by

adrenal incubates from the three^ "normal"^ and

eight carcinoma patients suggests that^ the^ latter

do not differ greatly from those of the former, al-

though the number of patients in the "normal"

group is too small for statistical evaluation. While

no comparative data on in vitro steroid formation

of normal^ human^ adrenal^ tissue^ are^ available^ in

the literature, Sweat (21) has^ obtained^ ratios^ of

2.8 to 3.0 for hydrocortisone to^ corticosterone^ in

peripheral blood of normal subjects-a value^ simi-

lar to the averages of 2.62 for carcinoma and^ 3.

for "normal" adrenal incubates in our series.

b) Hypertensive group. From Table IV it is

seen that the pattern of steroid formation in the

hypertensive group did not differ markedly from

that in^ the normotensive^ series.^ In^9 of^ the^14

cases of Table IV, however, the^ rate^ of^ steroid

formation was considerably higher than^ in^ adrenal incubates from the normotensive patients.

Steroid formation and diastolic blood^ pressure

To explore whether the differences in^ rate^ of

steroid formation within the hypertensive groups were related to the severity of the disease, hydro- cortisone formation^ by the^ incubates^ from^ the^23

patients of Tables IV and V was plotted against

diastolic blood pressure. The results are illus-

trated in Figure 2. A significant negative corre-

lation (r =-0.64; p^ <^ 0.01)^ was^ obtained.

In Tables II and IV the hypertensive patients

have been divided into three equal sub-groups ac-

cording to clinical criteria of the severity of the

disease. Groups I and III comprise the least and

most severe cases,^ respectively,^ at^ the^ time^ of^ op-

eration while the intermediary cases make up

Group II as recorded in Table II. In general

this classification agrees well with the diastolic

blood pressure arrangement, the mean diastolic

pressures being 109, 134 and 156 mm. Hg for the

three groups. All patients of Group III with the

exception of P. B. have subsequently^ died.^ The

grouping of our patients may^ require^ slight^ re-

vision when a greater number of cases becomes

available.

The mean hydrocortisone formation (372 pg.)

by the incubates of Group I adrenal tissue was sig-

nificantly greater than that (184 (^) pug.) of the nor-

motensive cancer patients (Tables III and VI),

whereas the rate of hydrocortisone formation (

pg.) by the Group III adrenal incubates was on

the average not higher than that of the normo-

tensive control group (the lower curve in Figure

3 and Table VI). The rate of hydrocortisone

formation per unit weight of^ adrenal tissue thus

decreased with increasing severity of hypertension.

,Ug./gml2./Z4hr 500-i

300 ' 4 *4^ I $0 200--I

100 -

r= - -

0 P< -oi

0 0 0 0 0

  • s

(^0 )

  • 0

-Diastolic 1.P.

1 m (^160) mm.Hy

FIG. 2. RELATIONSHIP BETWEEN CORTISOL FORMATION

AND DIASTOLIc BLOOD PRESSURE

COOPER, TOUCHSTONE,^ ROBERTS,^ BLAKEMORE,^ AND^ ROSENTHAL

  • ) r-01-o

0 .In. co Cl0- 0'C

C_eCC_Cl

@0o~ oo t--4 tif)@. 0 U) (^) if) Cl if Cl Cl 0-. (^) co tr

V W') Cl^ mU)fk a'0" 4-

Cl+Cl

Cl@0'0.0C-

oo (^) co - -

00 W40C C - C C- C- '

"N 4

(^0) _N0'0 0 NoV

mmm.0r

co Vo0'o NO )if i if)

~U) soo4- eOos oo _0 -H C'4. U.0m4H

r-^ U).No^ oCW) 0*orl@ IU)v

0* 0.

-, -^ -H co t>V> _+qpW

U C! U> ooest V

o 44-^ - 4

@0 cl

  • -H

Cli t- if)

0"-H

I'!

. N

C-C

O'-H

_i

"4 Cl 0%

@00'@.

'Or0'

tc O0 0%

C- (^) 0' - U)

  • (^) Cl

00 @0 Cl

(^10) '0"40 U)^ C- ONNO 0-~

C " eCti) i Cl

QC'4i) 1U)

Womwo (^) lq~bIN (^) CO ON-"NoN

a'Clifapi U (0-@

_ie vle W Gort- m

' UW) _-

'0, 00- -H

-4_ Clm --H

t~if In 0%

Cl-H

0'N

0 W

U@0+

(4-H

(^10) o 'CV ou.

-H

C4I 0 'OU (^) 00e eq 0 @-O. 11) cmcm (^) m-H

W)

  • -H

vsv-m0 04-

__ 4 oto0 _o

__0-0^ *.^.^.^ CO

C-0toIf ) C-@

U)'o 'o v o

o'0UU

U) (^) -'O4 -4 i)

C--o@0-c0 Cl - U) if)

  • C-0C-0C I^ m -,n -4l)ve^ - ON

0 0-Clf P*Qa

O-:tSj¢+

_____^4 M

m 9T

I

: 0

14

m

04

9

4i

0

Exl

m 0

14

4

m

rX

mm

I'

04 e-

Q 04

0 0*0co

@0- 0i

at-H

-H

U)

_ I-

0-H_R

0 _

O-H

n

-H

U)

10:

(^10) 'O m

0 I

4)0^ oo^ c

  • bo (4^00 a 0

t: 8 o I I

.

4310 oS4-

I r-

i-A : R (^13) L

i .r

  1. : 0
  1. c A4-i P.^ ct

: cI c

STEROID FORMATION BY ADRENAL TISSUE FROM HYPERTENSIVES

There was also a slight though less significant diminution (from 216 to (^163) jpg.) in the synthesis of steroids of the B region from Subgroups I to III. In all the three hypertensive groups, how- ever, the rate of formation of B-region steroid was significantly higher than in the cancer group (cf. Figure 4 and Table VI). The increasing pre- ponderance of formation of B-region steroids with increasing diastolic pressure can be most readily seen by inspecting the ratio of synthesis of hydrocortisone to synthesis of B-region steroids in Tables III and IV. This ratio declined from a mean of 2.6 in the group of normotensive pa- tients (^) with cancer to 1.6 in Group I of the hyper- tensive patients and, finally, to 1.1 in the patients with the most severe hypertension.

Steroid formation and adrenal weight Inspection of the data in Tables IV and V re- veals that the rate of hydrocortisone formation by incubates from glands weighing more than 6 Gm.

was in 7 out of 9 instances below 200 jug. per Gm.

TABLE V Steroid formation by adrenal tissue from (^) patients with hypertension-Cases with hydrocortisone determination only

Adrenal Blood Patient weight pressure F no. Gm. mm. Hg pg./24 hrs.fGm. 15 3.31 200/100 312 16 5.30 198/105 405 17 4.00 200/130 440 18 3.67 245/130 210 19 5.85 270/130 263 20 5.76 210/140 314 21 10.49 220/140 112 22 7.14 240/140 127 23 6.52 240/140 156

per 24 hours, whereas only 2 out of 14 incubates from lighter glands displayed similarly low rates. In Figure 5 the regression of rate of hydrocorti- sone formation on weight of gland is^ depicted. The correlation proved to be significant statistically

(r =-0.65; p < 0.01). Since the adrenal

weights include both the weight of cortex and of medulla, it cannot be decided at present which ana-

ILE VI The differences in steroid formation and adrenal weight between hypertensive groups and cancer group Adrenal Before differences*^ Group^ weight(Gm.) F^ F^ E^ B^ 11-0-OH^ Total Wag.) (jg.)^ Wug.) (Wg.)^ (jig.)^ (Wg.) Without ACTH Mean differences :1^ standard errort I-Cancer 0.12 41.6 187.6 109.8^ 134.4^ 33.3^ 506. :1:0.557 ±10.45 (^) 4:40.68 ±30.31 4140.21 4:21.91 ±90. p <.01 p^ <.01 p^ <.01 p^ <.01 p^ <. I (^) I-Cancer 0.80 20.1 104.4 84.5 132.5 42.2 384. 4:0.681 41:16.29 :4:44.42 425.85 ±32.29 427.13 ±110. p <.05 p <.01 p <. III-Cancer 1.94 - 0.2 -11.2 7.5 81.0 26.9 128. 40.629 411.92 ±35.78 418.68 ±18.94 ±19.99 ±73. p .02^ p^ <. With ACTH I-Cancer 61.2 344.6 135.1 77.2 28.1 646. 438.51 ±71.90 ±37.7 444.3 436.61 ± 166. p <.01 p <.01 p <. II-Cancer -10.1 205.3 172.0 178.4 5.9 551. 433.91 466.06 ±41.77 ±55.78 ±45.50 ±161. p <.02 p <.01 p <.02 p^ <. III-Cancer -43.2 -29.0 4.5 123.8 -25.1 32. 427.33 468.85 ±20.69 448.71 ±40.99 ± 108. p <.

* Number of experiments are 5, 4 and 5 in hypertensive groups I, II and III, respectively, 8 in cancer group without

ACTH and 7 with ACTH. t Standard^ error^ =^4 2+ - (2 -,)^ (1^ +1^.^ Unless^ p^ value^ is^ recorded,^ difference^ is^ not significantly different from zero statistically.

1531

STEROID FORMATION BY ADRENAL TISSUE FROM HYPERTENSIVES

Group III was due to steroids of the B-region

as compared with 17 to 19 per cent in both Group I hypertensives and the normotensive cancer group after ACTH stimulation.

DISCUSSION

This study aimed^ at^ assessing^ the^ functional^ ca-

pability of normotensive and hypertensive adrenals

by estimating in vitro steroid formation of tissue

slices incubated in the autologous plasma. The

close resemblance of the steroid patterns in

adrenal vein blood^ and^ adrenal^ incubates^ supports

the soundness of this method. The^ use^ of^ adrenals

from the normotensive cancer patients as baseline

for evaluating hypertensive alterations appeared

justifiable as the data from the incubation of

adrenal tissue from three normotensive cancer-free

patients were in close agreement with the average

of those from the cancer group.

The experiments yielded three findings of pos-

sible interpretative significance: 1)^ a^ negative

correlation between steroid formation per Gm. of

tissue and diastolic blood pressure in the hyper-

tensive group associated with a fall of the ratio of

F/B steroids due to the fact that formation of

B-region steroids^ declined^ significantly^ less^ with

increasing diastolic blood^ pressure than^ hydrocor-

tisone formation; 2) a^ doubling of^ the^ rate^ of^ for-

mation of all steroids, with the exception of^1 1f-

hydroxyandrostene-3,17-dione, in the less^ severe

hypertensive groups when compared with the

,u g./gm./24 Ir 500_

400-ai-

300- F.-J 1 200-

I Inn

r= -^ - 0 p<^ * 0 0

  • (^) a 0 0 0 0

0

0

*.

I&

w --II 1 II I - 2 4 6 10 gm. -AdrenaZ (^) weight FIG. 5. RELATIONSHIP BETWEEN CORTISOL FORMATION AND ADRENAL WEIGHT

Total Adrenal Steroicd Output 'A9. 4800- (^) GP. 1 4 3600 -GP.

-400 -

1200 -

_. (^).... *.

I- a 80 IIb 100 i 120 I~ .~ 140 I^. 160 o

-Diast. B.P, mm. Hg FIG. 6. TOTAL CAPACITY OF FORMATION OF CORTISOL AND B-REGION STEROIDS WITHOUT ACTH Representation as in Figure 3. For details consult the text

normotensive group; and 3) a positive correlation

between adrenal weight^ and^ diastolic^ blood^ pres-

sure compensating, in^ part,^ for^ the^ reduced^ rate^ of

steroid formation per Gm. of tissue in advanced

hypertension. In evaluating the significance of the quantitative

differences in steroid formation outlined above,

consideration must^ be^ given to^ the^ fact^ that^ our

assay system includes in^ the^ tissue^ slice^ and

plasma two potentially independent^ variables.

The question thus arises to what extent differences

in the supply of substrates and ACTH by the

individual plasmas contributed to the differences

in rate of steroid synthesis.

If the difference in rate of steroid formation

between the adrenal^ incubates^ from^ normotensive

and hypertensive patients were^ due^ only to^ dif-

ferent amounts of corticotropin in the autologous

plasmas used for the incubates, supplementation of

the plasma medium with an excess of corticotropin

should eliminate these differences. Exploratory

experiments (13) have^ shown^ that^ the^ addition

of 5 units of^ ACTH to^ the^ assay system produced

maximum stimulation of steroid^ formation^ by both

normotensive and hypertensive tissue. While hy-

pertensive adrenal tissue was slightly though not

significantly less stimulated than normotensive

tissue by ACTH, the differences between the val-

ues from the hypertensive and normotensive

groups as^ well^ as^ among^ the^ hypertensive^ groups

1533

F

COOPER, TOUCHSTONE, ROBERTS,

remained essentially unaltered when^ the^ plasma

was supplemented with 5 units of ACTH.

The question whether the substrate concentra-

tion of the plasma was rate-limiting cannot be de-

cided directly from the available experimental evi-

dence. Whenever tested, steroid formation by the incubates appeared to proceed almost linearly

during the first 12 hours and to decline but gradu-

ally during the second 12 hour period. Routine

determinations of the time course which might

have revealed instances of lack of substrate were

not deemed advisable because of the limited amount

of tissue available and the need for comparatively

large quantities of steroids for separation and

identification. The fact^ that^ in^ three^ exploratory

experiments substitution of plasma from a normal

donor for the hypertensive patient's plasma failed

to alter significantly the rate of steroid formation

by the incubates indicates a lack of substantial

differences in substrate concentrations between the

plasmas of the various groups. The fall of the

F/B ratio with^ increasing^ severity^ of^ hyperten-

sion appears to be a significant observation. Since

the steroids of these two regions arise from the

same precursors, the shift of the ratio indicates

changes in the functional capability of the adrenal

cortical cells rather than differences in the supply

of substrates.

Since corticosterone constitutes 70 per cent of

the B-region steroids elaborated by the incubates,

the fall of the F/B ratio suggests that there de-

velops a deficiency of the 17-hydroxylase with

progressing hypertension. To substantiate this

interpretation it^ would^ be^ desirable^ to^ get quan-

titative information^ on^ the^ rate^ of^ synthesis^ of^ 17-

hydroxycorticosterone (Substance^ S) and^ 18-

aldocorticosterone (aldosterone), the formation of

which should be affected by a deficiency in 17-

hydroxylase in a similar manner as compounds

F and B.

Our findings of^ increased^ adrenal^ weights^ in

advanced hypertension, occasionally associated

with nodularity of the cortex, corroborates the ob-

servations of earlier investigators [Rinehart, Wil-

liams and Cappeller (22); Sarason (23); Page

and Corcoran (24) ]. According to observations

of Page and Corcoran which we can confirm, hu-

man adrenals in advanced hypertension frequently

show arteriolar^ sclerosis.^ Reports in^ the^ litera-

ture (25) indicate that repeated trauma and

chronic illness may cause hypertrophy of^ the

adrenal cortex, increased weight of^ the^ glands and

abnormally high rises in the plasma levels of corti-

costeroids following administration of ACTH.

Whether incubates^ of^ adrenal^ slices^ from^ such

glands would^ show^ alterations^ in^ the^ rate^ of^ steroid

formation similar to those in^ the^ adrenal^ incubates

from the enlarged glands of^ hypertensive patients

remains an open question.

The decrease of the F/B ratio in^ the^ adrenal

incubates from the patients with far advanced hy-

pertension is^ of^ clinical^ interest^ since,^ besides^ al-

dosterone, corticosterone^ is^ the^ only^ salt-retaining

steroid which can be detected^ in^ human^ adrenals

(26). Perera and Pines (27), Woodbury, Rosen-

berg and Sayers (28), and Selye (29) suggested

that decreased formation of glucocorticoids rather than over-production of mineralocorticoids might

be the essential feature of hypertension, since in

the dog the pathological changes produced by ad-

ministration of the mineralocorticoid desoxycorti-

costerone could^ be^ prevented by simultaneous

treatment with cortisone (28). There^ are^ several

clinical reports of increased mineralocorticoid

activity in essential hypertension. Genest and^ as-

sociates (30, 31) found increased urinary aldos-

terone excretion in 50 per cent of patients with ad-

vanced essential^ hypertension. It^ is^ of^ interest^ that

aldosterone is^ metabolically related^ to^ corticos-

terone in^ not requiring 17-hydroxylation. The

disturbances of the salt and^ water^ metabolism^ de-

scribed by Green, Johnson, Bridges and^ Lehmann

(10) and by Braun-Menendez (11) also support

the concept of increased activity of mineralocorti-

coids. It^ should^ be^ noted,^ however,^ that^ the^ latter

disturbances were^ manifest^ in^ the^ early^ hyperten-

sives, whereas in^ the^ incubates^ the^ F/B ratio^ was

markedly reduced only in^ the^ most^ severe^ cases.

Precise information on the F/B ratio^ in^ the^ periph-

eral blood of hypertensive patients is^ as^ yet not available. This question is under investigation in

our laboratory.

The increased^ rate^ of^ steroid^ formation^ in^ the

adrenal incubates from the less^ severe^ hypertensive

patients and the decline of the rate^ of^ cortisol^ syn-

thesis with the progress of the disease might be^ the

reason for the wide range of urinary corticoid ex-

cretion in the hypertensive patients, studied by other (^) investigators. Hypercorticoiduria was ob- served by F.^ L.^ Selye (6), using bioassays, in^6

1534 BLAKEMORE, AND ROSENTHAL

COOPER, TOUCHSTONE, ROBERTS, BLAKEMORE, AND ROSENTHAL

J. T. Clinical studies on bilateral complete adrenalectomy in patients with severe hypertensive vascular disease. Ann. intern. Med. 1952, 37, 972.

  1. Blakemore, W. S., Zintel, H. A., Jeffers, W. A., Sellers, A. M., Sutnick, A. I., and Lindauer, M. A. A comparison of thoracolumbar (^) sympathectomy and adrenalectomy with Adson sympathectomy in the treatment of severe arterial hypertension: A three- to-seven-year follow-up report. Surgery 1958, 43,
  2. Goldblatt, H. Studies on experimental hypertension. V. The pathogenesis of experimental hypertension due to renal ischemia. Ann. intern. Med. (^) 1937, 11, 69.
  3. (^) Blalock, A., and Levy, (^) S. E. Studies on the etiology of renal hypertension. Ann. Surg. 1937, 106, 826.
  4. Selye, F. L. Biochemical changes in hypertension. Canad. med. Ass. J. 1947, 57, 325.
  5. Tobian, L., Jr., and Joseph, H. Adrenal cortex ac- tivity in essential hypertension. J. clin. Endocr. 1949, 9, 677.
  6. Hetzel, B. (^) S., and Hine, D. C. Adrenal cortical func- tion in (^) hypertension. J. clin. Endocr. 1952, i2, 296.
  7. (^) Corcoran, A. C., Page, I. H., and Dustan, H. P. Urinary formaldehydrogenic corticoids: Normal values and observations in hypertension. J. Lab. clin. Med. 1950, 36, 297.
  8. Green, D. M., Johnson, A. D., Bridges, W. C., and Lehmann, J. H. Stages of salt exchange in (^) es- sential (^) hypertension. Circulation 1954, 9, 416.
  9. Braun-Menendez, E. Water and electrolytes in ex- perimental hypertension in Ciba Foundation (^) Sym- posium on Hypertension. Boston, Little, Brown and Co., 1954, p. 238.
  10. Brady, R. 0. A rapid method for qualitative and quantitative estimation of the (^) physiological ac- tivity of the adrenal cortex. Endocrinology (^) 1953, 52, 49.
  11. Cooper, D. Y., Roberts, J. M., and Touchstone, J. C. Steroid production by incubated human adrenal tissue. Surg. Forum 1955, 5, 583.
  12. Huggins, C., and Scott, W. W. Bilateral adrenalec- tomy in prostatic cancer: Clinical features and urinary excretion of (^) 17-ketosteroids and estrogen. Ann. (^) Surg. 1945, 122, 1031.
  13. Huggins, C., and Bergenstal, D. (^) M. Inhibition of human (^) mammary and prostatic cancer by adrenal- ectomy. Cancer Res. 1952, 12, 134.
  14. Wolferth, C. C., Jeffers, W. A., Lukens, F. D. W., Zintel, H. A., and Hafkenschiel, J. H. Observa- tions on the results of subtotal adrenalectomy in the treatment of severe, otherwise (^) intractable hy- pertension and their (^) bearing on (^) the mechanism by which (^) hypertension is maintained. Ann. intern. Med. (^) 1951, 35, 8.
  15. Burton, R. B., Zaffaroni, A., and (^) Keutmann, E. H. Paper chromatography of steroids: II. Corticoster- oids and related compounds. J. biol. Chem. 1951, 188, 763.
  16. Tennent, D. M., Whitla, J. B., and Florey, K. Two techniques in paper chromatography; (^) application to ketosteroids. Analyt. (^) Chem. 1951, 23, 1748.
  17. Touchstone, J. C. The (^) identification of compound S and other steroids in human adrenal venous blood. Fed. Proc. 1958, 17, 323.
  18. Touchstone, J. C., Glazer, L., Cooper, D. Y., and Roberts, J. M. Isolation of A'-androstene-llfi-ol- 3,17-dione from human adrenal incubates. J. clin. Endocr. (^) 1955, 15, 382.
  19. Sweat, M. L. Adrenocorticosteroids in peripheral and adrenal venous blood of man. J. clin. Endocr. 1955, 15, 1043.
  20. (^) Rinehart, J. F., Williams, 0. O., and Cappeller, W. S. Adenomatous hyperplasia of the adrenal cortex associated with essential hypertension. Arch. Path. (Chicago) 1941, 32, 169.
  21. Sarason, E. L. Adrenal cortex in systemic disease: Morphologic (^) study. Arch. intern. Med. 1943, 71,
  22. Page, I. H., and Corcoran, A. C. Arterial Hyper- tension: Its Diagnosis and Treatment. Chicago, The Year Book Publishers, Inc., 1949, p. 132.
  23. Moore, F. D. Endocrine changes after anesthesia, surgery and^ unanesthetized^ trauma^ in^ man^ in Recent Progr. Hormone^ Res.^ G.^ Pincus, Ed.^ New York, Academic Press Inc., 1957, vol. 13, p. 511.
  24. Thorn, G. W., Jenkins, D., Laidlaw, J. C., Goetz, F. C., Dingman, J. F., Arons, W. L., Streeten, D. H. P., and McCracken, B. H. Medical prog- ress: (^) Pharmacologic aspects of adrenocortical steroids (^) and ACTH in man. New (^) Engl. J. Med. 1953, 248, 232, 284, 323, 369, 414, 588, 632.
  25. Perera, G. A., and Pines, K.^ L., Simultaneous ad- ministration of adrenal cortical extract and des- oxycorticosterone; effects on blood pressure of hypertensive patients. Proc. Soc. exp. Biol. (N. Y.) 1949, 71, 443.
  26. Woodbury, D. M., Rosenberg, C. A., and (^) Sayers, G. Antagonism of (^) adrenocorticotropic hormone (ACTH) and adrenal cortical extract (ACE) to desoxycorticosterone (DCA): Pathological changes. Fed. Proc. 1950, 9, 139.
  27. Selye, H. Interactions between gluco-corticoid and mineralo-corticoid hormones (abstract). J. clin. Invest. 1950, (^) 29, 844.
  28. Genest, J., Lemieux, G., (^) Davignon, A., Koiw, E., Nowaczynski, W., and^ Steyermark, P.^ Human^ ar- terial hypertension: A state of mild chronic hyper- aldosteronism? Science 1956, 123, 503.
  29. Genest, J., Koiw, E., Nowaczynski, W., and Leboeuf, G. Further studies on urinary aldosterone in hu- man arterial hypertension. Proc. Soc. exp. Biol. (N. Y.) 1958, 97, 676.
  30. Cooper, D. Y., Kasparow, M., Blakemore, W. S., and Rosenthal, 0.^ Relationship between steroid out- put in^ adrenal vein blood and steroid formation of adrenal incubates of hypertensive patients. Fed. Proc. 1958, 17, 205.