Urinary 17-Hydroxycorticosteroid Determination with p-Hydrazinobenzenesulfonic
Acid-Phosphoric Acid
A J I T SANGHVI, P H . D . , CARL W I G H T , M.T.,
BHARAT PARIKH,
M.S.,
AND HASMUKH DESAI, B . S C .
Department of Pathology, Division of Clinical Chemistry, University of Pittsburgh, 201 DeSoto Street,
Pittsburgh, Pennsylvania 15213
ABSTRACT
Sanghvi, Ajit, Wight, Carl, Parikh, Bharat, and Desai, Hasmukh: Urinary
17-hydroxycorticosteroid determination with p-hydrazinobenzenesulfonic
acid-phosphoric acid. Am. J. Clin. Pathol. 60: 684-690, 1973. A new
reagent system with p-hydrazinobenzenesulfonic acid in phosphoric acid for
the determination of urinary 17-hydroxycorticosteroids is described. Use
of this reagent results in increased sensitivity of detection of compounds
possessing a dihydroxyacetone side chain while it retains the same specificity
as obtained with the Porter-Silber reagent. The reaction conditions employed
with the new reagent allow the assay to be performed within 4 to 8 hours
after hydrolysis of the samples of urine.
DETERMINATION
of 17-hydroxycorticosteroids* in biologic fluids provides a
critical evaluation of adrenal cortical activity under various physiologic and pathologic conditions. This task was immensely
simplified with the publication by Porter
and Silber 5 in 1950 of a reagent system
consisting of
phenylhydrazine-sulfuric
acid-absolute ethanol which reacted specifically with compounds possessing a diReceived February 9,1973; accepted for publication
March 20, 1973.
* Systematic names of the steroids referred to in the
text by their trivial names only are: Cortisol (compound
F), 11/3, 17a, 21-trihydroxy-pregn-4-ene-3,20-dione;
cortisone (compound E), 17a, 21-dihydroxy-pregn-4ene-3, 11,20-tnone; 11-desoxycortisol (compound S),
17a, 21-dihydroxy-pregn-4-ene-3,20-dione; tetrahydrocortisol (THF), 3a, 11/3, 17a, 21-tetrahydroxy5/3-pregnan-20-one; tetrahydrocortisone (THE), 3a,
17/3, 21-trihydroxy-5/3-pregnan-l 1,20-dione; tetrahydro-11-desoxycortisol (THS), 3a, 17a, 21-trihydroxy5/3-pregnan-20-one; corticosterone, 11/3, 21-dihydroxy-pregn-4-ene-3,20-dione; 11-dehydrocorticosterone, 21 -hydroxy-pregn-4-ene-3,11,20-trione;
11-desoxycorticosterone,
21-hydroxy-pregn-4-ene3,20-dione; testosterone 17/3-hydroxy-androst-4-ene3-one.
684
hydroxyacetone moiety in their structure.
Thus, all the steroids that contain a 17,
21-dihydroxy-20-keto group at C-17 react
with the Porter-Silber reagent to form a
yellow chromogen having maximum absorption at 410 nm. This chromogen is
characterized as a 17-deoxy-20-ketosteroid-21-phenylhydrazone. 34 Several investigators 2,6 have proposed modifications
of the original Porter-Silber procedure
to suit their individual needs, without
altering the constitution of the reagent.
Recently, we 7 have described optimal
conditions for the use of p-hydrazinobenzenesulfonic acid (HBS) in phosphoric acid
for the analysis of 17-hydroxycorticosteroids. Briefly, steroids with a dihydroxyacetone side chain at C-17 when heated
at 100 C. for 60 minutes with 1.0 mg.
HBS per ml. of a 0.6:1 (v/v) solution of
H3PO4-H2O were shown to manifest a
characteristic absorption maximum at 355
nm. There was a linear rise in absorbance
with the HBS reagent over a 1 to 100
November 1973
685
17-HYDROXYCORTICOSTEROIDS
ixg. concentration range of Cortisol. A
similar linear increase in absorbance with
increasing concentrations of Cortisol was
also obtained with the Porter-Silber (P-S)
reagent, but the absorption intensity was
an average of 68% greater with the HBS
reagent per unit weight of steroid. When
tested with a large number of steroids
of known structure, the HBS reagent
showed the same degree of specificity for
the so-called Porter-Silber chromogens as
that obtained with the Porter-Silber
reagent.
This report concerns the application
of the HBS reaction to routine analyses
of urinary 17-hydroxycorticosteroids in
clinical specimens received in our
laboratory.
Materials and Methods
Apparatus. Spectra for the routine specimen analyses were obtained using a PerkinElmer model 124 double-beam grating
spectrophotometer with a wavelength accuracy of ± 5 X 10"1 nm. Spectra for the
recovery studies of tetrahydrocortisol
(THF), tetrahydrocortisone (THE), tetrahydro-11-desoxycortisol (THS), and Cortisol (F) from urine samples were recorded
on a Beckman Acta V double-beam recording spectrophotometer. All spectra
were recorded at ambient temperature
using 1.0 cm. optical pathlength matched
fused silica cells.
Reagents. All chemicals used were of
analytic reagent grade. Water was distilled
or deionized. Steroids were obtained from
Steraloids, Inc. HBS was purchased from
Eastman Organic Chemical and was recrystallized prior to use to a constant
melting point of 283 to 283.5 C , as described before. 7 Ketodase was from Warner-Chilcot. The complete reagent contained 1.0 mg. HBS per ml. of 37.5%
H3PO4 solution (37.5 ml. 85% H3PO4
+ 62.5 ml. water). Incomplete reagent was
37.5% H3PO4 only.
Procedure. The pH of a urine specimen
is adjusted to 5.0 and a 5.0-ml. amount
is transferred into a 50 ml. heavy-wall
conical centrifuge tube. After overnight
incubation (16 to 18 hours) at 42 C. with
1.0 ml. ketodase (1,000 units of j3-glucuronidase per ml. urine), the samples
are extracted with 25 ml. of chloroform,
centrifuged at 2,000 r.p.m. for about 10
minutes, and the aqueous phase is discarded. The chloroform extract is washed
successively with 2 ml. of 0.1 N NaOH and
2 ml. of water, then placed in two 10ml. aliquots into 15 ml. centrifuge tubes
and evaporated to dryness in a 40 C.
water bath under a stream of nitrogen.
T o one of the tubes is added 4 ml. of
the complete reagent, while to the other
tube is added 4 ml. of incomplete reagent
which contains 37.5% H3PO4 only. The
tubes are covered with aluminum foil and
incubated at 100 C. for 60 minutes. The
absorbance of the yellow chromogen
formed with each urine sample is scanned
between 400 to 300 nm. against its own
similarly treated blank. T h e peak absorbance of the sample at 355 nm. is corrected
for nonspecific contributions by the threepoint correction procedure described by
Allen. 1 Absorbances at 350 nm. for 10,
20, and 30 /ug. Cortisol standards treated
with the HBS reagent as described are
also measured with each run of urine
samples and absorbance per /ng. of Cortisol
is calculated. T h e concentration of 17hydroxycorticosteroids in each sample of
urine is then estimated by comparison with
the absorbance of the cordsol standard.
Results
Reproducibility of the HBS Method. Reproducibility of results obtained using the
HBS procedure was determined first.
Replicate analyses at five different concentrations of 17-hydroxycorticosteroids
(17-OHCS) in urine specimens were performed over a period of one month. The
concentration range of 17-OHCS employed in these studies was 2.39 to 37.27
mg. per 24 hours. A total of 48 analyses
was carried out. The mean and standard
686
A.J.CP.—Vol.
SANGHVI ET AL.
60
deviation for the five concentrations used
were: 3.20 ± 0.59 (n = 10); 6.36 ± 0.52
(n = 10); 13.01 ± 1.21 (n = 9); 25.54
^g- per 5 ml. 1Urine
±
1.36 (n = 9); and 32.58 ± 2.25 (n = 10).
Per Cent
Recovery of Steroids. Data from recovery
Added
Found
Recovery
Recovered
studies are presented in Tables 1 to 4.
0
140
Recoveries of tetrahydro steroids from
20*
20
100
160
urine were estimated as, under normal
50
63
127
203
249
109
100*
109
circumstances, these compounds constitute
191
331
95.5
200
the bulk of the 17-OHCS in the urine.
400
560
420
105
As indicated in the Tables, various amounts
734
874
91.7
800
of Cortisol, T H F , T H E , and T H S in
MEAN±S.E.
104.7 ± 5 . 1 3
dioxane (10 fig. per fil. dioxane solution)
* Average of three determinations. All other recoveries are averages were added to 5 ml. of urine. After adof four determinations.
justing the pH to 5 they were incubated
for 18 hours at 42 C. with ketodase and
17-OHCS were measured as described earTable 2. Recovery of Tetrahydrocortisol lier. T h e mean recoveries from urine of
Cortisol and its tetrahydro derivative T H F
(THF) Added to Urine and Analyzed
were 104.7 and 84.5%, respectively. Simiby the HBS Method*
larly, T H S and T H E showed mean reA*g- per 5 ml. Urine
coveries
of 95.9 and 94.7%, respectively.
Per Cent
Table 1. Recovery of Cortisol (F) Added to
Urine and Analyzed by the HBS Method
Added
Found
Recovered
Recovery
0
20
50
100
250
500
1000
25.0
45.2
66.2
97.9
221.5
440.4
916.9
20.2
41.2
72.9
196.5
415.4
891.9
101.0
82.4
72.9
78.6
83.1
89.2
MEAN ±
84.5 ± 3 . 9 6
S.E.
* All recoveries averages of four determinations.
Table 3. Recovery of Tetrahydro-11desoxycortisol (THS) Added to
Urine and Analyzed by
the HBS Method
Comparison of HBS Results with P-S Results. Figure 1 compares 17-OHCS values
obtained using the P-S and the HBS procedures. T h e equation for the regression
line is Y = 0.9455 X - 0.1501. T h e intercept of the regression line is not significantly different from zero by F test, although the slope of the regression line
is significantly different from 1. T h e coefficient of correlation between the two
methods is 0.994.
Precision of the HBS Method. T h e withinTable 4. Recovery of Tetrahydrocortisone
(THE) Added to Urine and Analyzed
by the HBS Method
Mg- per 5 ml. Urine
Mg- per ml. Urine
Added
Found
Recovered
Per Cent
Recovery
0
19.85
49.62
99.25*
248.12
496.25
992.50
22.4
42.3
71.4
112.8
245.5
495.5
1014.4
19.9
49.0
90.4
223.1
473.1
991.9
100.2
98.8
91.4
89.9
95.3
99.8
MEAN ±
S.E.
95.9± 1.81
* Average of three determinations. All other recoveries are averages
of four determinations.
Added
0
22*
55*
110
275
550*
1100
MEAN±
S.E.
Found
Recovered
Per Cent
Recovery
19.6
40.4
74.4
117.2
268.7
550.8
1101.0
20.8
54.8
97.6
249.1
531.2
1081.4
94.5
99.6
88.7
90.5
96.6
98.3
—
94.7 ± 1.77
* Average of three determinations. All other recoveries are averages
of four determinations.
November 1973
17-HYDROXYCORTICOSTEROIDS
687
FIG. 1. Comparison of 17-OHCS
values obtained using the HBS and
P-S procedures. T h e dotted line represents all values when Y = X. T h e
solid line is the regression line.
MG/TOTAL VOLUME
run precision of the HBS procedure was
compared with the precision of the P-S
procedure in the following manner. Duplicate analyses for 17-OHCS were performed on random 24-hour urine specimens as received in our laboratory with
both the P-S and the HBS methods. Estimates of S.D. were calculated from the
formula
/2n (d = difference between
the duplicate values, n = 48), which gave
0.51 for the HBS method and 0.44 for
the P-S method. These values are not
significantly different from each other by
F test.
Specificity of the HBS Reagent and Absorption Characteristics. T h e specificity of the
HBS procedure described here resides
in part in the favorably selective extraction
of steroids from an aqueous milieu into
the chloroform phase, and in part in the
ability of steroids to form hydrazone derivatives with p-hydrazinobenzenesulfonic
acid. These characteristics are central to
our method, as they are to the method
of Porter and Silber. Figure 2 shows absorption curves for a number of steroids
of known structure after reaction with
HBS-H3PO4 acid. The absorption data at
different wavelengths are presented in
terms of absorbance per mM of steroid
per liter, for the comparisons are then
made on a mol-for-mol basis. As can be
gleaned from these data, the HBS reagent
is highly specific for steroids possessing
a I7,21-dihydroxy-20-keto group at C-17.
Absorbance responses with increasing
concentrations of Cortisol, cortisone, 11desoxycortisol, and their tetrahydro derivatives were determined with HBS and
P-S reagents. Figure 3 represents the data
from these experiments. T h e concentrations tested for each compound were 5,
10, 20, and 50 fig., except for Cortisol,
for which a concentration range from
5 to 100 £ig. was used. In all cases the
responses were linear with both reagents,
but the absorption intensities were considerably greater with the HBS reagent
for all the compounds examined than
with the P-S reagent. T h e bar diagram
depicts these data in terms of molar
absorptivity ± S.E. of the mean.
688
AJ.C.P.—Vol.
SANGHVI ET AL.
60
v 20
E
E
y
15
10
325
350
WAVELENGTH
375
325
350
WAVELENGTH
375
300
325
350
WAVELENGTH
375
nm
FIG. 2. Absorption characteristics of the steroid-HBS reaction. A (left), o
o, cortisone; o
D,
tetrahydrocortisone; o
o, 11-dehydrocorticosterone; x
x, testosterone. B (center), o
o, Cortisol;
o
o, tetrahydrocortisol; x
x, corticosterone. C (right), o
o, 11-desoxycortisol; o
o,
tetrahydro-11-desoxycortisol; x
x, 11-desoxycorticosterone.
Discussion
Two of the important parameters one
considers when instituting a new methodology in the laboratory are the reproducibility of the results obtained with a new
method, and the recovery of the test material from biologic fluids. Our results show
the HBS procedure to be very satisfying
regarding both these parameters. The reproducibility studies performed between
the concentrations which ranged from 2.39
to 37.27 mg. 17-OHCS per 24 hours represent extremes of concentrations and
should encompass values encountered in
most pathologic states.
T h e regression analysis shows the 17OHCS values obtained using the proposed
HBS method to be highly correlated
(r = 0.994) with values obtained using the
P-S method. T h e fact that statistically,
by the F test, the intercept of the regression line is indistinguishable from zero
implies an identity of material measured
by two different procedures. On the other
hand, by the same criterion, the slope of
the regression line is significantly different
from one, showing that on an average
the HBS procedure yields 17-OHCS values
which are about 5% lower than those
obtained by the P-S method. We cannot
explain this difference on the basis of
data we have at hand. In practical terms,
however, this difference does not critically
alter the clinical utility of the results obtained by the HBS method. Of particular
importance is the fact that we have not
included five pieces of paired data in the
regression analysis, since these specimens
developed interfering pink colors when
carried through the P-S procedure, which
consequently resulted in artifactually elevated 17-OHCS values. This interfering
color development was not observed with
the HBS method.
Precision of the HBS method compares
well with that of the P-S method. This
mode of obtaining an estimate of precision
is suggested by Snedecor 9 to be particularly
valuable, since it gives information concerning the precision of a method under
the conditions encountered during routine
daily use.
As shown in Figure 2 absorbance per
mM of steroid is greater in each instance
when the steroid contains a A 4 -3-keto
group in addition to the dihydroxyacetone
structure at C-17. It is postulated that
Cortisol gives rise to a 3,21-dihydrazone
when treated with phenylhydrazine. 8 Our
earlier results 7 on the kinetics of color
development when Cortisol is reacted with
November 1973
689
17-HYDROXYCORTICOSTEROIDS
the HBS reagent have shown a shift in
the absorption maximum from 338 nm.
to 350 nm. during the initial 30 minutes
of reaction and an absence of such a shift
in the absence of a A 4 -3-keto group. Together with the results presented here,
these observations support the notion
that in these instances a 3,21-dihydrazone
may also be formed. We are currently
investigating further the constitution of the
final product(s). Frequently encountered
androgens, estrogens, and metabolites of
progesterone have been shown 7 not to react
with the HBS reagent.
Compounds F, S, and E all exhibit molar
absorptivities which are somewhat greater
in comparison with those for their ring
A-saturated analogs T H F , T H S , and T H E ,
respectively (Fig. 3). The greater molar
absorptivities for compounds F, S, and E
may be ascribed to the presence of an
a,/3-unsaturated 3-ketone in ring A of their
structures and consequent formation of
a 3,21-dihydrazone, as alluded to above.
Further, cortisone and tetrahydrocortisone
both show molar absorptivities which are
an order of magnitude greater than those
obtained for Cortisol, tetrahydrocortisol,
11-desoxycortisol,
and
tetrahydro-11desoxycortisol. In this instance, it appears
that the presence of an 11-keto group
in cortisone and tetrahydrocortisone is
responsible for their increased molar absorptivities. No such correlation, however,
can be made with respect to P-S reagent,
and this perhaps is due to the fact that
the absorption maxima of interest in the
P-S reaction occurs at 410 nm., whereas
3-hydrazones of A 4 -3-keto steroids absorb
light in the vicinity of 340 to 360 nm., 8
and thus do not contribute significantly
to absorption at 410 nm.
Urine specimens received for analysis of
17-OHCS are set up for overnight hydrolysis at 42 C. with ketodase at the end
of a working day. The time required to
carry the specimens through the HBS
procedure after hydrolysis and to complete
the analysis, including calculations for 17OHCS, is estimated to be about 4 hours
10
A
- I %
|i£|
P-S METHOD
^
HBS METHOD
J
i J lI lj i
m•a
%
-
4.
111
FIG. 3. Comparison of molar absorptivities of steroids
with P-S and HBS reagents.
for ten specimens run in duplicate together with appropriate standards. The
results can thus be reported the day after
receipt of the samples. Similar treatment
of specimens with the P-S procedure after
initial enzyme hydrolysis, on the other
hand, takes a much longer time, since
the extracted steroids need to be incubated
overnight (—18 hours in darkness) with
the phenylhydrazine reagent. While it is
true that time of analysis with the P-S
procedure can be abbreviated by heating
the extracted steroids at 60 C. for about
30 minutes with phenylhydrazine-sulfuric
acid, in our hands the blanks with this
approach have tended to be appreciably
higher and the results less reliable. Thus,
with the HBS reagent the time of analysis
can be reduced by a full day. We have
investigated the possibility of further reducing the time needed to carry out the
HBS procedure by increasing the enzyme
concentration from 1000 units of ketodase
to 1500, 2000, and 2500 units of ketodase
per ml. of urine. T h e samples were incubated at 42 C. and removed at 1-hour
intervals to assay for 17-OHCS. In each
instance, with increased enzyme concentration, the amount of 17-OHCS liberated
after 4 hours was the same as the maximum
amount liberated after 18 hours of hydrolysis with 1,000 units of ketodase per
ml. of urine. Thus, if necessary, it is possible
to accelerate the time of analysis by increasing the enzyme concentration.
690
SANGHVI ET AL.
We 7 have reported previously the use
of ethyl acetate as the extraction solvent
for 17-OHCS. With the use of this solvent,
however, we have sporadically observed
(1) high nonspecific background absorption, (2) a shift in the absorption maximum from 355 nm. to 360 nm., and
(3) a skewed absorption curve. We have
now replaced ethyl acetate with chloroform
and find the results to be much more
satisfactory. One explanation for the occasionally spurious results obtained with
ethyl acetate may be its greater polarity
relative to chloroform, which might have
affected the extraction of other compounds
from some urine specimens which contributed to the nonspecific background
absorption.
One of the concerns in analyzing for
constituents in biologic fluids obtained
from patients is the possible interference
by either various drugs or their metabolites,
or both. Both P-S and HBS procedures
are subject to these interferences, although,
at least in some instances, the HBS procedure appears to be less susceptible. This
problem, however, is easily overcome by
simply extracting the urine after enzyme
hydrolysis first with a nonpolar solvent
such as 30 to 60 degree petroleum ether
and then proceeding with the regular
method. In most cases our experience
with this approach has been satisfactory.
T h e presence of sugars, for example,
glucose, may present a potential problem,
since they are known to form hydrazones
and osazones with phenylhydrazine under
appropriate conditions. This is observed
with the P-S reagent, and the HBS reagent
is no exception. However, the selective
extraction of urine or plasma with organic
solvents excludes most compounds, including sugars. In our experience with
diabetic urines we have not found sugar
content to be a problem in the measurement of 17-OHCS with either the P-S
or the HBS procedure (for details see
ref. 7). Moreover, even when glucose (or
fructose) is reacted directly with the HBS
A,J.C.P.—Vol.
60
reagent at 100 C , its absorption maximum
at 425 nm. (443 nm. for fructose) is quite
remote from that observed for 17-OHCS
and thus it poses no great problem.
We have also determined the concentration of unconjugated 17-OHCS in urine
using the HBS method. The specimen
is first extracted with chloroform without
enzyme hydrolysis and the chloroform
extract is then subjected to the rest of the
procedure. Determined in this manner,
the unconjugated or "free" 17-OHCS in
urine constitutes between 5 and 6% of the
total 17-OHCS in urine, and this figure
compares with that published earlier by
Silber and Porter. 8
We have successfully used the HBS procedure in the clinical laboratory for four
years and have found the method to be
reliable, stable, and well accepted by technical and clinical staff.
Acknowledgment. Dr. Joseph S. Amenta aided with
the statistical analysis of the data.
References
1. Allen WM: A simple method for analyzing complicated absorption curves, of use in the
colorimetric determination of urinary steroids.
J Clin Endocrinol 10:71-83, 1950
2. Glenn EM, Nelson DH: Chemical method for the
determination of 17-hydroxycorticosteroids
and 17-ketosteroids in urine following hydrolysis with /3-glucuronidase. J Clin Endocrinol Metab 13:911-921, 1953
3. Lewbart ML, Mattox VR: T h e mechanism of the
Porter-Silber reaction. I. Rearrangement of
the dihydroxyacetone group of steroids. J
O r g C h e m 29:513-521, 1963
4. Lewbart ML, Mattox VR: The mechanism of the
Porter-Silber reaction. II. Formation of 17deoxysteroidal 21-phenylhydrozones. J Org
Chem 29:521-527, 1963
5. Porter CC, Silber RH: A quantitative color reaction
for cortisone and related 17,21-dihydroxy-20ketosteroids. J Biol Chem 185:201-207, 1950
6. Reddy WL, Jenkins D, Thorn GW: Estimation
of 17-hydroxycorticosteroids in urine. Metabolism 1:511-527, 1952
7. Sanghvi A, Taddeini L, Wight C: Determination
of 17-hydroxycorticosteroids with p-hydrazinobenzenesulfonic acid-phosphoric acid.
Anal Chem 45:207-210, 1973
8. Silber RH, Porter CC: T h e determination of
17,21-dihydroxy-20-ketosteroids in urine and
plasma. J Biol Chem 210:923-932, 1954
9. Snedecor GW: Query No. 92. Biometrics 8:8586, 1952