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(From the Department of Biochemistry, Southwestern Medical College of the
University of Texas, Dallas)
(Received for publication, July l&1949)

Calorimetric methods for the quantitative determination of many com-

pounds have been developed, based on their specific chemical or physi-
cochemical properties. Such tests include the Zimmermann (1) reaction
for androgenic assay and the Kober (2) reaction for estrogenic assay.

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Modifications of each of these have been reported by Callow et al. (3),
Cohen and Bates (4), Szego and Samuels (5), and Holtorff and Koch (6).
Many of these modifications are for the determination of estrogens and
androgens obtained from the extract of urine, feces, and body tissues.
The value of these variations lies in the fact that certain groups inherent
in the perhydro-1, 2-cyclopentanophenanthrene nucleus or its derivatives
are involved. The color development in the Zimmermann reaction (Zim-
mermann (1) and others) depends upon a carbonyl group adjacent to a
methylene carbon (-CH -CO-). For this reason many compounds not
related to 17-ketosteroids will give positive results. Callow (3) has implied
that the group -CH2--CO-CHX--- found in cyclopentanone is necessa-
rily the one found in perhydro-1, 2-cyclopentanophenanthrene compounds
giving a positive Zimmermann. If this were true, only estrone-16 would
give a positive reaction. These data do not confirm this.
Marrian (7) has postulated that 16-ketoestrone is the basis for the red
color developed in the Kober reaction, and that only or-diketones could give
a positive Kober test. This was before 16-ketoestrone was synthesized
and tested. Marlow (8) showed that little or no color is developed when
16-ketoestrone is treated with the Kober or Zimmermann reagent as com-
pared to other estrogens.


The compounds used in these experiments are pure crystalline com-

pounds of proved structure. The quantity treated with the Zimmermann
reagent has an arbitrary concentration of 72 y, while that treated with
the Kober reagents has 30 y. Some of these compounds (estrone-16, 16-
ketoestrone, etc.) have not been reported as subjected to the Kober and
Zimmermann tests, since they have only been synthesized and their struc-
tures established very recently. These compounds are critical ones struc-

turally for the determination of groups involved in the color development

of the Kober and Zimmermann reagents. The other compounds have been
reported but are included for comparative effects.
The methods involved necessitated only minor modifications in those
cited in references above. For clarification they are briefly outlined below.
Zimmermann4.2 ml. of solutions (0.2 ml. of redistilled 95 per cent al-
cohol as blank) containing 72 y was treated with 0.2 ml. of 5.5 N KOH
and 0.2 ml. of 2 per cent m-dinitrobenzene in alcohol. The tubes were
stoppered and allowed to stand for 45 minutes. They were then diluted
with 10 ml. of 70 per cent alcohol solution.
Kober-1 .O ml. of an alcohol solution (1 ml. of redistilled alcohol as blank)

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containing 30 y of the substance to be examined was evaporated to dryness
in a hot water bath. 0.6 ml. of a fresh, modified Kober reagent (0.625
gm. of fi-naphthol dissolved in 25 ml. of concentrated HzSOh) was added.
The tubes were heated for 2 minutes (shaking at 45 and 90 second inter-
vals) in a boiling water bath and then cooled in ice water for a few seconds.
0.6 ml. of acidulated water was added with agitation while the tubes were
in the ice water. They were then reheated in boiling water for l+ min-
utes, being shaken well at 45 seconds. The tubes were transferred to an
ice water bath for 10 seconds. Then 1.8 ml. of 65 per cent HzS04 (1 :l)
were added. The solutions were mixed well and then transmission was
determined in the Cenco-Sheard spectrophotelometer.
Spectrophotelometer-The instrument was set to give 100 per cent trans-
mission at 508 rnp. (This is the peak exhibited by most of the compounds
studied.) When the sliding exit slit was 5 mm., the entrance slit for
light was generally set at about 0.8 mm. The transmission generally was
measured from 400 to 550 rnp to establish the transmission curve over the
significant part of the visible spectrum. Since several samples were read
successively (a special sliding mechanism was employed which will be de-
scribed in a later paper), the transmissions of the blanks were reset at the
original figures to correct for any drift in the machine due to line voltage
variations. In this way comparative successive readings could be made.
The K value recorded was evaluated from the equation K = log I,/I,
which is numerically equivalent to the optical density calculated from D =
2 - log10 T, or D = 2 - log I,/I. All derivatives were equilibrated to be
equivalent to a basic compound.

Zimmermann Test For Ketosteroids

It will be seen in Table I that there is some change in the transmission
of light, or shift of the peak, when an OH group, or other groups (as meth-
oxy, benzyloxy), is at position 3. For instance, 3-methyoxyestrone, 3-
benzyloxyestrone, and estrone gave K values in that descending order.
H. W. MARLOW 169

Androsterone acetate is about as efficacious as those mentioned above,

indicating neither interference of the acetate radical nor need of unsaturation
in Ring A for highest color development. In all these cases the carbonyl
group is at position 17, and a methylene carbon is at position 16. Estrone-

Stru,clural ConJiguration of Steroids and Zimmermann and Kober Reagwh
Compound Position Group at immermann Kober reaction*
occupied position reaction’

Estrone (1,3)t.. 17 Carbonyl 0.278 1.025

Benzyloxyestrone (1, 3 i 17 “ 0.303 0.871

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Methoxyestrone (1,3) 17 “ 0.382 1.115
Androsterone acetate (1 9
3) . 17 0.326 No peak
Equilenin (1, 3). 17 0.349 0.807 (490)
Equilin (1, 3). 17 0.304 0.494 (470)
E&one-16 (2, 4). . . 16 No peak No peak
Methoxyestrone-16 (1!,
4) . 16 “ “ “ “ “
Benzyloxyeatrone-16 (24
4) . . . 16 “ “ “ “ “
Estriol (1, 2). 16, 17 OH “ “ 0.479 (464); 0.498
Isoestriol-A (1, 2). 16, 17 “ “ ‘I 0.695
a-Estradiol (1, 3). . 17 1‘ “ “ 0.532 (436); 0.497
&E&radio1 benzoate (I ‘1
3) . 17 17 OH “ “ 0.497
estradiol (1, 2). 16, 17 16 0, 17 OH “ “ 1.056
16-Ketoestrone (1, 2) 16, 17 Carbonyl 1‘ “ No peak
trone (1, 2). . 16, 17 “ ‘I “ “ “
Testosterone (1, 3). 17 17 OH “ “ 0.334 (490)
Cholesterol (1, 3). 17 It “ I‘ No peak
Doisynolic acid.. Ring D out 1.‘ “ “ ‘I
Marrianolic acid. “ ‘I “ “ “ “ “
* Peak at 508 nn.~ unless otherwise indicated in parentheses.
t Meaning of figures in parentheses in this column as follows: (1) 13 terl-carbon;
(2) 15 CH2; (3) 16 CH,; (4) 17 CHs.

16 differs in that a carbonyl group is now at position 16, with methylene

carbons at both positions 15 and 17. No color is obtained with this com-
pound and the Zimmermann reagents. 16-Ketoestrone, with carbonyl
groups at both positions 16 and 17 and an adjacent methylene carbon in
position 15, develops no color. When an OH or an R group is at position
17 and methylene carbons at both the 15 and 16 positions, no color de-

velops. If Ring D has been broken, as in doisynolic acid and marrianolic

acid, no color develops.

Kober Reaction
The above modified Kober reaction was employed with p-naphthol in
concentrated HzS04. Table I shows the peak values at 508 rnp as in the
Zimmermann reaction. Here again, when the carbonyl group is at posi-
tion 17 and the methylene carbon is at position 16, the greatest amount of
color is produced. However, 3-methoxy-lG-keto-a-estradiol is an excep-
tion to this. When carbonyl groups are at positions 16 and 17 and a
methylene carbon is at position 15 (conditions under which the Zimmer-

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mann reaction gives no color), from 10 to 20 per cent absorption of light is
noted, but no definite peak is observed.

From a study of the groups affecting the intensity of color development
in the Zimmermann and Kober reactions, the following results are re-
Zimmermann Reuction-(1) Maximum color is developed when the com-
pound contains a carbonyl group at position 17 and a methylene carbon ad-
jacent at position 16 in the perhydro-1, 2-cyclopentanophenanthrene com-
pounds. (2) When a carbonyl group is at position 16 and the two adjacent
carbons are methylenic, or when there are carbonyl groups at both po-
sitions 16 and 17 and amethylenic carbon at position 15, no color is produced.
(3) When an OH or R group is at position 17 with an adjacent methylenic
carbon at position 16, no color development is observed. (4) If position
3 has a methoxy or benzyloxy group on it, there is a significant effect on
the transmission of light. (5) Unsaturation of Ring A or Rings A and
B does not significantly affect the color development.
Modified Kober Reaction-(l) Maximum color is developed when the
compound contains a carbonyl group at position 17 and a methylenic
carbon at the 16th position of all the estrogens tested. (2) If an OH
group is at position 17, or at both positions 1G and 17, together with
adjacent methylenic carbons, the K value is roughly 50 per cent of that
of compounds with a carbonyl group at the 17t.h position. (3) Meth-
oxyestrone gives more color but the benzyloxy derivative gives less color
than estrone in the Kober test. (4) Carbonyl groups at position 16 or at
both the 16th and 17th positions gave no color and hence showed no peak
value. (5) A hydroxyl group at position 17 and a carbonyl group at po-
sition 16 show as much transmission activity as a carbonyl at position
17 with an adjacent methylenic carbon.
H. W. MARLOW 171


1. In the Zimmermann reaction, maximum color development depends

on a carbonyl group being at position 17 and a methylene carbon at 16.
No other group on or modification of Ring D of the perhydro-1, 2-
cyclopentanophenanthrene nucleus gives a positive value.
2. In the modified Kober reaction, maximum color development is noted
as in the Zimmermann reaction above. Other groups in other positions
give definite color development. Especially noteworthy is 3-methoxy-
16-keto-cu-estradiol which replaces the methylenic carbon at position 16
with a carbonyl group. Equilenin, which has an aromatic Ring B, gives
a very high peak. Equilin, which is less unsaturated than equilenin, has

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a K value of approximately one-half that of equilenin.

The author wishes to acknowledge his indebtedness to the following for

their contributions to t.his paper by generously furnishing pure samples for
the tests: Research Division, the Schering Corporation, Bloomfield, New
Jersey, for cy-estradiol, p-estradiol benzoate, testosterone, and androsterone
acetate; Research Laboratories, Parke, Davis and Company, Detroit,
Michigan, for equilenin, theelin, and theelol; Dr. Ernest J. Umberger,
Pharmacologist, Food and Drug Administration, Washington 25, D. C.,
for equilin; and Dr. Max N. Huffman and coworkers for benzyloxy- and
methoxyestrone, 16-ketoestrone, 3-methoxy-16-keto-a-estradiol, estrone-
16, isoestriol-A, doisynolic a.nd marrianolic acids. The author also wishes
to express his gratitude for the help of his technicians, Mrs. Tom Morgan
and Mrs. John Little, for their contribution to the paper.


1. Zimmermann, W., Z. physiol. Chem., 233, 257 (1935).

2. Kober, S., Biochem. J., 32, 357 (1938).
3. Callow, N., Callow, R., and Emmens, C. W., Biochem. J., 32, 1312 (1938).
4. Cohen, H., and Bates, R. W., J. Clin. Endocrinol., ‘7, 701 (1947).
5. &ego, C. M., and Samuels, L. T., J. Biol. Chem., 161, 587 (1943).
6. Holtorff, A. F., and Koch, F. C., J. Biol. Chem., 136,377 (1940).
7. Marrian, G. F., Harvey Lectures, 34, 37 (193839).
8. Marlow, H. W., Endocrinology, 42, 479 (1948).
9. Umberger, E. J., and Curtis, J. M., J. Biol. Chem., 178,275 (1949).