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
- 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.
- 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.
- 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
(^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
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
Cli t- if)
0"-H
I'!
. N
C-C
O'-H
_i
"4 Cl 0%
@00'@.
'Or0'
tc O0 0%
C- (^) 0' - U)
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)
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
t: 8 o I I
.
4310 oS4-
I r-
i-A : R (^13) L
i .r
- : 0
- 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
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.
- 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,
- Goldblatt, H. Studies on experimental hypertension. V. The pathogenesis of experimental hypertension due to renal ischemia. Ann. intern. Med. (^) 1937, 11, 69.
- (^) Blalock, A., and Levy, (^) S. E. Studies on the etiology of renal hypertension. Ann. Surg. 1937, 106, 826.
- Selye, F. L. Biochemical changes in hypertension. Canad. med. Ass. J. 1947, 57, 325.
- Tobian, L., Jr., and Joseph, H. Adrenal cortex ac- tivity in essential hypertension. J. clin. Endocr. 1949, 9, 677.
- Hetzel, B. (^) S., and Hine, D. C. Adrenal cortical func- tion in (^) hypertension. J. clin. Endocr. 1952, i2, 296.
- (^) 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.
- 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.
- 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.
- Brady, R. 0. A rapid method for qualitative and quantitative estimation of the (^) physiological ac- tivity of the adrenal cortex. Endocrinology (^) 1953, 52, 49.
- Cooper, D. Y., Roberts, J. M., and Touchstone, J. C. Steroid production by incubated human adrenal tissue. Surg. Forum 1955, 5, 583.
- 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.
- Huggins, C., and Bergenstal, D. (^) M. Inhibition of human (^) mammary and prostatic cancer by adrenal- ectomy. Cancer Res. 1952, 12, 134.
- 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.
- 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.
- Tennent, D. M., Whitla, J. B., and Florey, K. Two techniques in paper chromatography; (^) application to ketosteroids. Analyt. (^) Chem. 1951, 23, 1748.
- Touchstone, J. C. The (^) identification of compound S and other steroids in human adrenal venous blood. Fed. Proc. 1958, 17, 323.
- 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.
- Sweat, M. L. Adrenocorticosteroids in peripheral and adrenal venous blood of man. J. clin. Endocr. 1955, 15, 1043.
- (^) 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.
- Sarason, E. L. Adrenal cortex in systemic disease: Morphologic (^) study. Arch. intern. Med. 1943, 71,
- Page, I. H., and Corcoran, A. C. Arterial Hyper- tension: Its Diagnosis and Treatment. Chicago, The Year Book Publishers, Inc., 1949, p. 132.
- 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.
- 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.
- 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.
- 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.
- Selye, H. Interactions between gluco-corticoid and mineralo-corticoid hormones (abstract). J. clin. Invest. 1950, (^) 29, 844.
- 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.
- 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.
- 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.
