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Documenti di Professioni
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(Submitted for publication May 13, 1963; accepted July 22, 1963)
L.S., L.M., G.S., R.S., J.S., C.Z., and M.G. were healthy
young adult volunteers C.S. was a 27-year-old white
woman in complete remission after treatment for metastatic choriocarcinoma. Regular menses had occurred
for the 6 months before study. E.H. was a 27-year-o'd
Negro woman with normal menstrual function admitted
for treatment of local recurrence of carcinoma of the
breast.
Absolute ethanol 1 was redistilled by the method of
Peterson and his associates (6). Water was glass-distilled after the addition of a few crystals of KMnO4.
n-Hexane, ether, chloroform, and methanol were prepared
as previously described (7, 8). Ligroin 2 was prepared
exactly like n-hexane; on redistillation, the fraction
boiling from 1030 C to 1060 C was collected. Benzene,
ethyl acetate, and acetic anhydride were redistilled.
Pyridine was allowed to stand overnight over calcium
hydride and then redistilled under anhydrous conditions.
Sulfuric acid, reagent grade, was used as supplied.
1 U. S. Industrial Chemicals, Inc., Baltimore, Md.
2 Eastman Kodak #P-1628, Eastman Kodak Co.,
Rochester, N. Y.
3 Fisher Scientific Corp., Boston, Mass.
1753
4
5
1754
System
Composition
Hexane
CHC13
ml
ml
2% CHCl3
98% Hexane
15% CHCl3
85% Hexane
392
30% CHC13
70% Hexane
60% CHCl3
40% Hexane
80% CHC13
20% Hexane
C
D
E
340
8
60
H20
ml
ml
50
50
50
50
224
96
40
40
160
240
50
50
40
160
25
25
Eluate
Volume
collected
Typical component
ml
16-androstene-3a-ol
A2
Bi
100
80
200
C1902-17-ketosteroids
B2
300
C1902-diols
C,903-17-KS, pregnanetriol, 5-preg-
Cl
200
260*
250
150t
250
Al
C2
Dl
D2
E
Testosterone, epitestosterone,
nenetriol
Tetrahydro S
Tetrahydro E
Tetrahydro F, cortolone
Cortol
More polar ketols
* After collecting 200 ml of C2, the column head is allowed to run down for a further 60 ml retaining a 15-ml head.
t Eluates D2 and E contain overlapping components and are therefore combined.
sample.
Reliability of results. The following studies were performed to demonstrate the reproducibility, accuracy, and
sensitivity of the method.
Equal samples of the same final testosterone fraction
were measured in 12 successive assays (Table II).
According to the criterion of Grubbs (11), the disparate
value .154 may be discarded, giving a SE of the method
of 0.0066, or 6% at that level.
Samples of final testosterone preparations containing
10
1 755
TABLE II
Ag
.114
.128
.116
.133
.119
.127
Ag
.130
.126
.154*
Mean .1235
SD .0066
Ag
.128
.114
.124
L.S.
L.S.
L.M.
G.S.
G.S.
R.S.
Period
1
2
1
1
2
jig
jig
.20
.20
.20
.20
.20
.20
.22
.20
.22
.21
.21
.19
RESULTS
In 4 women, 2 receiving corticosteroids, testosterone production rates ranged from 0.94 to 2.8
mg daily (Table VII). There was an increase
after FSH administration and a doubling of baseline values when HCG was added. These increases were greater than the experimental error
of the method (p <.05).
Plains, N. J.
12
1 756
Trivial name
4-Androstene-1 7,-ol-3-one
4-Androstene- 1 7a-ol-3-one
1 -Androstene-3,17-dione
173-Acetoxy-4-androstene-3-one
1 -Androstene-1 7,-ol-3-one
4-Androstene-3, 17-dione
Relative % fluorescence
(testosterone = 100%)
Testosterone
Epitestosterone
100
100
67
60
50
33
30
20
20
15
10
2
Testosterone acetate
Androstenedione
4-Androstene-3#, 1705-diol
4-Pregnene-1ljl,1 7a,21-triol-3,20-dione
4-Pregnene-l11,21-diol-3,20-dione
4-Pregnene-2 1 -ol-3,20-dione
5-Androstene-3#1,1 71-diol
4-Androstene-6,f-ol-3,17-dione
Cortisol
Corticosterone
Desoxycorticosterone
4-Pregnene-1 7a-ol-3,20-dione
4-Pregnene-1 7a,21-diol-3,11,20-trione
5a-Androstane-3a-ol- 17-one
5j1-Androstane-3a-ol- 17-one
5a-Androstane-3t3-ol- 17-one
5-Androstene-3,3-ol- 17-one
5a-Androstane-3a,1 7#3-diol
17a-Hydroxyprogesterone
Cortisone
Androsterone
16-Androstene-3,1-ol
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Etiocholanolone
Epiandrosterone
Dehydroepiandrosterone
Androstanediol
Etiocholanediol
Pregnanediol
Pregnanetriol
1 1,-Hydroxyandrostenedione
5j3-Androstane-3a,1 7#-diol
Pregnane-3a,20a-diol
Pregnane-3a, 1 7a,20a-triol
4-Androstene-1 1,-ol-3,17-dione
1,4-Androstadiene- 1 7,-ol-3-one
5a-Androstane-1 7fl-ol-3-one
5,3-Androstane-1 7,6-ol-3-one
* 0.2 and 0.5 jug of steroid were assayed by the method described in the text. The fluorescence of testosterone is set
at= 100%.
DISCUSSION
TABLE V
Period
L.S.
2
2
1
2
3
IL.M.
G.S.
G.S.
C.S.
Radioactivity
administered
Day 1
dpm
dpm
9.5
9.5
1.1
1.0
2.3
X 106
X 105
X 106
X 106
X 106
Day 2
Radioactivity recovered
Total
On day 3
dpm
dpm
8.2 X 104
4.8 X 104
3.3 X 105
6.7 X 103
4.3 X 103
3.0 X 104
3.1 X 104
4.5 X 104
75
66
80
78
74
.9
.7
3.4
4.0
2.6
7.1 X 10'
6.2 X 105
7.7 X 105
7.0 X 105
1.3 X 106
Day 3
1757
Patient
Age
_---
Daily dose
d sys
lug
dp)n per
Prednisone, 15 mg
Prednisone, 5 mg
HCG, 1,000 U
Prednisone, 15 mg
17
8
6
Prednisone, 5 mg
HCG, 1,000 U
Cortisone, 15 mg
17
8
5
Cortisone, 15 mg
HCG, 1,000 U
12
6
38
26
60
36
22
11
6
3
29
26
39
22
13
6
29
18
56
48
49
45
82
80
47
40
31
31
24
22
120
125
89
90
years
L.S.
L.M.
21
26
1
2
G.S.
21
2
J.S.
26
R.S.
23
The production-rate
Testosterone
IDuration* recoveredt
Period
assay was
Radioactivity
given
SA
Ag
9.5 X 105
6.6 X 105
9.5 X 105
1.1 X 106
X
ratet
nzg
dpm
6.6 X 105
1.0
Testosterone
production
106
2.3 X 106
2.3 X 106
per day
5.9
6.9
9.7
10.5
4.0
4.1
10.1
11.9
11.8
11.8
13.9
15.2
6.4
6.1
8.6
8.5
t Duplicate values represent estimates after repetition of the acetylation and saponification procedure used to isolate
testosterone.
a pro-
1758
Patient
--
Period
Age
Treatment
Daily dose
Testosterone
Duration*
years
E.H.
27
C.S.
27
M.G.
18
C.Z.
18
ratet
pg
dpm per pg
dOm
890
800
545
2.5 X 106
346
390
2.5 X 106
0.94
1.0
1.5
1.8
2.4
2.1
1.6
.18
3.0
.20
1.7
.027
705
684
545
505:
470
2.3 X 106
9.7
8.5
3.5
1.9
1.2
296
278
348
510
342
2.3 X 106
Cortisone, 15 mg
Human FSH, 0.4 ml
Cortisone, 15 mg
Human FSH, 0.4 ml
HCG, 2,000 U
Cortisone, 15 mg
11
5
15
9
4
5
Cortisone, 15 mg
Human FSH, 0.4 ml
Cortisone, 15 mg
Human, FSH 0.4 ml
HCG, 2,000 U
11
5
15
9
4
Testosterone
production
given
4.1
2.7
5.6
.12
5.6
4.5
Radioactivity
Cortisone, 15 mg
SA
days
recoveredt
445$
400
2.4 X 106
2.4 X 106
2.3 X 106
2.3 X 106
1.1
1.1
1.4
1.6
1.7
1.9
2.6
2.8
2.211
1.5
2.2
* The production-rate assay was performed during the last 3 days of each medication period.
t Duplicate values represent estimates after repetition of the acetylation and saponification procedure used to isolate
testosterone.
1: SE of counting, 7% or less.
SE of counting, 20%.
1I This assay was repeated twice because of the poor initial agreement after duplication.
Theoretical considerations. The isotope-dilution method depends upon the dilution of the labeled testosterone by testosterone from all sources.
When all the testosterone is secreted by the glands,
then a secretion rate is obtained. If, however, a
portion of the testosterone is derived from other
steroids such as androstenedione as a result of
peripheral metabolism, then the isotope-dilution
DEHYDROEPIANDROSTERONE
TESTIS
OTHER GLANDS
ANDROSTENEDIONE
7i TESTOS~trOE r
ark
SYTEMFOTROTHE
FIG.O1.NADMODEL ax
,
,
TESTOSTERONEI
|TESTOSTERONE
eT~~~~HER
,,PROUCTIONOANDE
META~~~BOLITE.S
1759
REFERENCES
1. Finkelstein, M., E. Forchielli, and R. I. Dorfman.
Estimation of testosterone in human plasma. J.
clin. Endocr. 1961, 21, 98.
2. Schubert, K., and K. Wehrberger. Isolierung von
Testosteron aus Normalharn.
Naturwissenschaften 1960, 47, 281.
3. Camacho, A. M., and C. J. Migeon. Isolation, identification and quantitation of testosterone in the
urine of normal adults and in patients with endocrine disorders. J. clin. Endocr. 1963, 23, 301.
4. Hudson, B., J. Coghlan, A. Dulmanis, and I. Ekkel.
Measurement of testosterone secretion. Proc.
Endocr. Soc. 44th meeting, Chicago, 1962, p. 16.
5. Wilson, H. Absorption spectra of A5-3#-hydroxy
steroids in several sulfuric acid reagents. Analyt.
Biochem. 1960, 1, 402.
6. Peterson, R. E., J. B. Wyngaarden, S. L. Guerra,
B. B. Brodie, and J. J. Bunim. The physiological
disposition and metabolic fate of hydrocortisone in
man. J. clin. Invest. 1955, 34, 1779.
7. Wilson, H., J. J. Borris, and M. M. Garrison.
Chromatographic procedure for the determination
of urinary corticosteroids and C1, steroids. J. clin.
Endocr. 1958, 18, 643.
1760
20.
21.
22.
23.
24.
25.
26.
27.
28.