CLIN.CHEM.23/6, 982-985 (1977)
Effect of SodiumSulfateon the Hydrolysisof
17-Hydroxycorticosteroidand p-Nitrophenyl-glucuronides
with fl-GlucuronidasePreparationsfrom BovineLiver
Etsuko Furuya
Previous papers dealt with the discovery that hydrolysis
of 17-hydroxycorticosteroid
glucuronides
in urine with
fi-glucuronidase preparations from bovine liver is increased by adding sodium sulfate to the incubation medium. Here, we conclude that sodium sulfate not only increases the activity of bovine liver /9-glucuronidase on
17-hydroxycorticosteroid
glucuronides and p-nitrophenyl
glucuronides, but also removes the inhibitory activity of
substances of high molecular weight in urine and, moreover, inhibits urine putrefaction during the hydrolysis. The
net effect is an increased yield of urinary 17-hydroxycorticosteroids. In the incubation with sodium sulfate (Na2SO4,
final concentration
80 9/liter, 500 Fishman units of 3glucuronidase per milliliter of urine, pH 5.0, 48 #{176}C,
18 h)
the analytical recovery of 17-hydroxycorticosteroid glucuronides added to 12 urine samples proved to be 98 ±
1.8% (95-100).
The main difficulty in all known methods for determining urinary steroids is the hydrolysis of the steroid
conjugates (1, 2).
Our previous papers (3-5) reported the increased
hydrolysis of urinary 17-hydroxycorticosteroid
glucuronides by fi-glucuronidase
(EC 3.2.1.31) preparations
from bovine liver when sodium sulfate was added to the
incubation medium. The purpose of the work described
in this paper was to determine whether sodium sulfate
(a) increases the enzyme activity for both 17-hydroxycorticosteroid
glucuronides
and nonsteroid
glucuronides, (b) removes the inhibitory activity on the enzyme
of high-molecular-weight
substances in the urine, or (c)
if other reactions occurring in the presence of sodium
sulfate cause the increase of the hydrolysis, or if possibly
the effect is explained by some combination
of these
three factors.
Department
of Biochemistry,
School of Dentistry, Hokkaido
University, North 13 West 7 Kits-ku, Sapporo 060, Japan.
Received Jan. 25, 1977; accepted Feb. 11, 1977.
982
CLINICALCHEMISTRY.Vol. 23, No. 6, 1977
Procedures
Separation of 17-Hydroxycorticosteroid
Glucuronides
One liter of urine was dialyzed at 5 #{176}C
for one day
through a cellophane membrane
against 10 liters of
distilled water. The dialysate was applied to a 5 X 20cm
column containing Amberlite XAD-2. The column was
washed with 500 ml of water. The steroid glucuronides
were eluted from the resin with 500 ml of methanol.
After removing the methanol under reduced pressure,
we dissolved the residue in 10 ml of water, washed the
solution three times with 30 ml of methylene chloride,
and applied it to a 4 X 50 cm column containing Sephadex G-25. The steroid glucuronides were eluted with
water, 20-ml fractions being collected. Fractions 18 to
20 contained the 17-hydroxycorticosteroid
glucuronides
(solvolyzable
17-hydroxycorticosteroids,
the steroid
sulfate, made up less than 5% of these fractions).
Separation
of Inhibitors of /3-Glucuronidase
Urine was centrifuged at 3000 rpm (1000 X g) for 20
mm, and the supernatant
fluid was applied to a Amberlite XAD-2 column as mentioned above. The unadsorbed fraction was dialyzed against water. After
heating at 60#{176}C
for 30 mm to inactivate urinary fi-glucuronidase the high-molecular-weight
(nondialyzable)
fraction was used as fi-glucuronidase
inhibitor. In another experiment
the supernatant
fluid was directly
dialyzed against water.
Assay of Enzyme Activity with
17-Hydroxycorticosteroid
Glucuron ides as
Substrate
The reaction mixture, consisting of 4 ml of water, 0.5
ml of acetate buffer (0.67 mol/liter, pH in the range of
4 to 6), 0.5 ml of 17-hydroxycorticosteroid
glucuronide
solution (0.6 mmol/liter,
as cortisol) and 0.1 ml of di-
luted 9-glucuronidase solution (300 Fishman units), was
incubated at 37 #{176}C
for 3 h in the presence of sodium
sulfate (final concentration, 80 g/liter). After incubation,
0.5 g of sodium sulfate was added to the hydrolysate,
followed by extraction with 30 ml of methylene chloride.
The extract was successively washed with 3 ml of 0.1
mol/liter NaOH in 100 g/liter Na2SO4 and 3 ml of 100
g/liter Na2SO4. Ten milliliters of the methylene chloride
extract was added to 1.0 ml of ethanol, followed by
shaking with 1.5 ml of Porter-Silber
reagent (1 mg of
phenylhydrazine.HC1
per milliliter of diluted sulfuric
acid (concentrated
H2S041H20 64/36, by vol).1 For the
sample blank, instead of Porter-Silber
reagent, 1.5 ml
of the diluted sulfuric acid was used. After removing the
methylene
chloride layer, we incubated
the reagent
layer at 60 #{176}C
for 30 mm, the surface of the layer being
kept 1 to 2 cm lower than that of the water bath. During
the incubation
the remaining
methylene
chloride
evaporated. The absorbance of the sample vs. the blank
was measured at 410 nm in 10 mm cuvettes (value A).
Reagent blank, with water instead of 17-hydroxycorticosteroid glucuronide solution, was carried through the
same procedure
without 13-glucuronidase
(value B).
Value B was subtracted from value A to obtain the true
absorbance of the sample. Cortisol was used as a standard: 5.0 ml of the working standard solution (10 mg of
cortisol per 1000 ml of water) plus 0.5 ml of acetate
buffer without the enzyme was carried through the
procedure, starting with methylene chloride extraction,
in the presence of 1 g of sodium sulfate. This procedure
is reported in detail in previous papers (3, 4).
We then conducted the same experiment,
using a
starting mixture without sodium sulfate, and added 1
g of Na2SO4 to the medium after incubation. The rest
of the procedure was conducted as mentioned above.
In other assays with 9-glucuronidase
inhibitor, 4 ml
of the inhibitor solution instead of water were used for
incubation.
Assay
of Enzyme
Glucuronide
Activity
with p-Nitrophenyl
as Substrate
The enzyme activity was measured according to a
modification
of Nobenaga’s
method
(6). Reaction
mixtures consisted of 0.1 ml of 40 mmol/liter p -nitrophenyl glucuronide,
0.3 ml of 0.67 mol/liter acetate
buffer (pH in the range of 4 to 6), 0.5 ml of water or 0.5
ml of inhibitor solution, and 2.2 ml of water, and were
incubated with 0.1 ml of diluted -glucuronidase
at 37
#{176}C
for 1.5 h in the absence or in the presence of sodium
sulfate (final concentration, 80 g/liter). After incubation,
1 ml of 0.2 mol/liter NaOH was added to stop the reaction and the p -nitrophenol
liberated was measured at
400 nm in a photometer.
In another assay, we used 0.1 ml of 10 mmol/liter
phenolphthalein
glucuronide
instead of 0.1 ml of 40
mmol/liter p -nitrophenyl
glucuronide and, after incubation (pH 5.2, 37 #{176}C,
1 h), 1.5 ml of glycine buffer (pH
1 Diluted sulfuric acid: 64% (by vol) H2S04 (“super special” grade),
or 66% (by vol) H2S04 (concentrated, analytical grade).
IIl/Iiter
#{149}
NOCI
20
02
40
OI
P
10
20
20
40
50
50
00
20
20
020
00
70
FroCtIOs0r
Fig. 1. DEAE-cellulose chromatographic
pattern of a /3-glucu-
ronidase preparation from bovine liver
10.7, 3 mol/liter)
NaOH.
Determination
Urine
instead
of 1 ml of 0.2 mol/liter
of 17-Hydroxycorticosteroids
in
Mixtures containing 5 ml of urine adjusted to pH 5.0,
0.5 ml of acetate buffer (2 mol/liter, pH 5.0), and the
stated number of Fishman units of /3-glucuronidase were
incubated at the stated temperature
(see Results) for
the indicated time in the presence or absence of sodium
sulfate. After incubation and subsequent addition of 1
g of Na2SO4 to the reaction mixture to which it had not
been added initially, the hydrolysate was extracted with
30 ml of methylene chloride. The rest of the procedure
was again conducted as mentioned above.
Results and Discussion
Direct
Effect of Sodium Sulfate on /3-Giucuronidase
To determine if sodium sulfate increases the enzyme
activity for 17-hydroxycorticosteroidand p-nitrophenyl-glucuronides,
we fractionated
a commercial
/3-glucuronidase preparation from bovine liver (Sigma)
on diethylaminoethyl(DEAE)-cellulose.
The enzyme
preparation
was applied to a DEAE-cellulose
column
(3 X 8 cm), previously equilibrated
with tris(hydroxymethyl)aminomethane.HC1
(5 mmol/liter, pH 8.0), and
then was successively eluted with this same buffer and
this buffer with 0.1 and 0.2 mol/liter of added NaC1.
Fractions of 10 ml were collected, and 0.1 ml of each
fraction was tested for 9-glucuronidase
activity at pH
5.2 in the absence of sodium sulfate with the abovementioned method and with p-nitrophenyl
glucuronide
as substrate. As seen in Figure 1, the elution diagram
showed three fractions with /3-glucuronidase
activity:
Fraction I (fraction No. 8), Fraction II (No. 54), and the
starting preparation
were used for determining
the effect of sodium sulfate on the enzyme activity in the pH
range from 4.0 to 6.0.
Figure 2 shows the pH curves with p-nitrophenyl
glucuronide. Without sodium sulfate the activities of
Fraction I and II show no pH optimum with increasing
pH and are always lower than those with sodium sulfate
in the pH range from 4.4 to 6.0, which have an optimum
CLINICALCHEMISTRY,Vol. 23, No. 6, 1977 983
Fraction I
Fraction I
SCOoting Enzyw
PrepOootlon
lii
30
a’
I:
20
ii
U
0
0
00
200
z
00
200
300
50
55
60 40
Urn.
ft-GL
2010
300
4010
Anny
Fig. 4. Effect of dialysis on urinary /3-glucuronidase activity
50 55 60 40 4.55.065 60
45
000
400
Ogn.I
1.0 4.5
7
Substrate: p.nitrophenyl giucuronide
514
Fig. 2. Effect of sodium sulfate on /3-glucuronidase activity for
p-nitrophenyl glucuronide
-o
Na2SO4 5, inhibitor 50--
A-A
#{149}‘
2#{176}4
e, inhibitor S(S
=
added; 0
FrOctuOlI
Na2SO4 0,
-
- -
to that without sodium sulfate, for the enzyme preparations
(Sigma, USA; Hokuiken
Sapporo,
Japan;
Tokyo-zoki, Tokyo, Japan; latron, Tokyo, Japan) the
figures were 1.31, 1.30, 1.54, and 1.66 for p-nitrophenyl
glucuronide;
1.35, 1.37, 1.38, and 1.44 for phenolphthalein glucuronide; and 1.64, 1.80, 1.86, and 1.82 for
17 -hydroxycorticosteroid
glucuronides, respectively.
absent)
=
FrOCt30AI
Stortir#{231}
Enzyac
Preparation
10
Effect of Sodium Sulfate on Inhibition of /3Glucuronidase by Urinary High-Molecular-Weight
Substances
40
-
40
IS
50 55 tO
40
45
II
5,5 5040
1.
(5
50
55
60
pH
Fig. 3. Effect of sodium sulfate on /3-glucuronidase activity for
17-hydroxycortlcosteroid glucuronides
#{149}-#{149}
Na2SO4 ,
Na2SO4,
A-A
Na2SO4 ,
inhibitor 50--
-o Na2SO4 0,
-
- -
e, inhibitor S
between 4.4 and 4.8. The activity of the starting enzyme
preparation
without sodium sulfate has an optimum
between pH 4.4 and 5.0; when sodium sulfate is added
this optimum shifts to pH 5.0 to 5.4 and a considerably
greater activity is observed.
Figure 3 shows the pH curves with 17-hydroxycorticosteroid glucuronide solution. Without sodium sulfate
the activities of Fraction I, II, and the starting preparation have a pH optimum between 4.0 to 4.4, between
4.4 and 4.8, and between 4.2 and 4.6, respectively. With
sodium sulfate present the activities have a pH optimum between 4.4 and 4.8, between 4.6 and 5.0, and between 4.8 and 5.2, and considerably greater activity is
observed. We conclude that sodium sulfate increases the
activity of /3-glucuronidase
from bovine liver on both
substrates, p -nitrophenyland 17-hydroxycorticosteroid glucoronides.
In an experiment in which we used several /3-glucuronidase preparations
from the same source (bovine
liver), for the enzyme preparations with p-nitrophenyl-,
phenolphthalein-,
and 17-hydroxycorticosteroid
glucuronides (incubation pH: 5.2,5.2, and 5.0, respectively),
we found the same effect of sodium sulfate; if the effect
is expressed as the ratio of the value with sodium sulfate
984 CLINICALCHEMISTRY,Vol. 23, No. 6, 1977
We used the above starting /3-glucuronidase
preparation as enzyme and p-nitrophenyland 17-hydroxycorticosteroid glucuronides as substrate. The inhibitor,
the Amberlite XAD-2 treated urinary high-molecularweight fraction, was prepared as described above.
As seen in Figures 2 and 3, in the absence of sodium
sulfate the rate of hydrolysis with the /3-glucuronidase
preparation
in the case of both substrates clearly decreased at each pH when the urinary inhibitor was
added to the reaction system, but this inhibition was
remarkably suppressed by addition of sodium sulfate,
the rate of hydrolysis being very nearly the same as if
no inhibitor were present.
When 15 urine samples that had only been dialyzed
against water were used as inhibitor, the comparative
rates of hydrolysis for 17-hydroxycorticosteroid
glucuronides were as follows (incubation:
300 Fishman
units of /3-glucuronidase,
pH 5.0, 37 #{176}C,
3 h): with no
sodium sulfate present, the values without and with the
inhibitor were respectively 66 ± 3.6% (mean ± standard
deviation) and 40 ± 7.4% of the value found without the
inhibitor but with sodium sulfate present. The value
with inhibitor present was 97 ± 2.1% when sodium sulfate was also present.
Figure 4 shows the effect of sodium sulfate and of the
dialysis on the activities of urinary j3-glucoronidase
(substrate: p-nitrophenyl
glucuronide, pH 4.6). Without
sodium sulfate the values for urinary f3-glucuronidase
in the dialyzed fractions (designated as /3-Glap) are lower
than those in the corresponding
original urines, while
with sodium sulfate both values-those
of dialyzed
fractions (designated as /3-Gl) and those of the original
urines-are
almost the same. At the same time the
values with sodium sulfate present are always higher
.
.
#{149}#{149}.if,
#{149}#{149}
#{149}
S.
00’
#{149}
#{149}.#{149}
S.
.
#{149}
.#{149}.
.
#{149}
30
0
#{149}
S
We conclude
putrefaction.
that sodium sulfate does inhibit urine
Effect of Sodium Sulfate and Temperature
Glucuronidase Activity
on /3-
S
S
In the experiment concerned with the effect of sodium
sulfate (final concentration,
80 g/liter) and of tem10’
#{149}
I
perature
on
the
enzymatic
hydrolysis
(500 Fishman
#{149}. #{149}#{149}
30
Se
..
units/ml
of
urine)
of
urinary
17-hydroxycorticosteroid
-J
.
glucuronides:
(a) at the incubation temperature
of 37 #{176}C
(18 h), the
value (n = 11) without sodium sulfate was 88 ± 2%
I 7 -OHCS
(84-92) of that with sodium sulfate;
Fig. 5. Correlation between /3-glucuronidase inhibitor and 17(B) in the presence of sodium sulfate the value (n =
hydroxycorticosteroids In urine
15) on incubation at 37 #{176}C
(18 h) was 92 ± 696(78-104)
-Gi Inhibitor: -Gl0 minus $-GI, (jig of p-nitrophenol released per 100 ml per
of
that
at
48
#{176}C;
hour). 17-Hydroxycorticosteroid
concn in mg/liter, expressed as cortisol
(c) at the incubation temperature
at 48#{176}C
(18 h), the
value (n = 46) without sodium sulfate was 94 ± 3%
than the values when it is absent. We conclude that the
(90-101) of that with sodium sulfate;
urinary high-molecular-weight
fraction plays a major
and (d) on incubating for 4.5, 12, and 18 h with sodipart in the inhibition of urinary /3-glucuronidase
and
um sulfate at 48#{176}C,
the values for the first two were 97
that such inhibition is removed by sodium sulfate.
± 3% (9 1-102) and 99 ± 2% (96-100) that of the last.
Figure 5 shows a correlation between urinary /3-gluSummarizing
these results, the values with sodium
curonidase inhibitory activities and total 17-hydroxysulfate present are always higher, whether the incubacorticosteroid
values in the same urines. In this expertion is at 37 or 48#{176}C,
than without; values on incubation
iment the hydrolysis of 17-hydroxycorticosteroid
gluat 48 #{176}C
are higher than those at 37 #{176}C;
and the value
curonides was conducted
with bovine liver /3-glucuwith sodium sulfate at 48 #{176}C
(18 h) is about 25% higher
ronidase (500 Fishman units/ml urine) in the presence
than that without sodium sulfate at 37 #{176}C
(18 h).
of sodium sulfate (final concentration
80 g/liter, 48 #{176}C,
In our experiment
starting with addition of 17-hy18 h). When assuming that the difference between fl-Gl
droxycorticosteroids
glucuronides to 12 urine samples
and /3-Glap reflects the activity of the high-molecularwith 500 Fishman units of /3-glucuronidase per milliliter
weight inhibitors, we can say that the inhibitor action
of urine, the analytical recovery was 98± 1.8% (95-100)
increases logarithmically
as 17-hydroxycorticosteroid
in the presence of sodium sulfate (48 #{176}C,
18 h).
glucuronides
increase in urine. This can be one of the
main reasons for decrease in the efficiency of the enzymatic hydrolysis in urines containing large amounts of
Prof. Dr. Osaniu Nishikaze provided unfailing guidance throughout
such glucuronides.
the course of this work.
I-
#{149}.
30
S#{149}
#{149}#{149} S
.
#{149}
#{149}S
S
0?
Effect of Sodium Sulfate on Urine Putrefaction
during Incubation
References
We adjusted the pH of 32 urine samples obtained
1. Curtinus, H., and Mueller, M., Gas-liquid chromatography
of
from hospitalized
patients to pH 5.0 with diluted sul17-ketosteroids and progesterone metabolites of urine: Comparison
of different methods of hydrolysis. J. Chromatogr. 30,410 (1967).
furic acid, and then incubated without acetate buffer
2. Van Kamper, E. J., and Anker, A. P., Steroid spectra, pattern
(37 or 48 #{176}C,
18 h). Without sodium sulfate present,
recognition and quantitative
analysis. Clin. Chim. Acta 34, 241
after incubation at 48 #{176}C,
eight of the 32 samples stayed
(1971).
at pH 5.0 and the pH of the rest had shifted to a mean
3. Nishikaze, 0., and Furuya, E., A new method for the determination
pH of 6.4 (range, 5.4 to 8.0). With sodium sulfate present
of 17-OHCS (Porter-Silber
chromogens) in urine. I. The effect of
sodium sulfate on the -g1ucuronidase
catalyzed reaction. Jpn. J. Clin.
(final concentration,
80 g/liter) the pH of 16 samples
Pathol. 17,634 (1969) (in Japanese).
stayed at pH 5.0, the rest shifted to a mean pH of 5.6
4. Furuya, E., A new method for the determination
of 17-hydroxy(range, 5.2 to 6.0). The results after incubation at 37 #{176}Ccorticosteroids in urine. Jpn. J. Clin. Chem. 2, 107 (1973).
were almost the same.
5. Graef, V., Furuya, E., and Nishikaze, 0., Hydrolysis of steroid
glucuronides with fl-glucuronidase
preparations
from bovine liver,
Sodium sulfate thus inhibits the pH shift, presumably
Helix pomatia, and E. coli. Clin. Chem. 23,532 (1977).
by inhibiting the growth of urinary microorganisms that
6. Nobenaga, S., Studies on fl-glucuronidase. II. Clinical application
destroy steroids, and an optimal pH for the enzyme
of the assay method of i9-glucuronidase activity using p-nitrophenyl
glucuronide as the substrate. Fukuoka Acta Med. 52,300(1961).
hydrolysis is better maintained.
CLINICALCHEMISTRY,Vol. 23, No. 6, 1977 985