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Enzymatic determination of estradiol and estrone in plasma and urine
1
CZinico Chimica Acta, 92 (1979) l-9 @ Elsevier/North-Holland Biomedical Press
CCA 9739
ENZYMATIC DETERMINATION PLASMA AND URINE
OF ESTRADIOL
AND ESTRONE
IN
JEAN-CLAUDE NICOLAS *, ANNE-MARIE BOUSSIOUX, BERNARD DESCOMPS and ANDRl? CRASTES DE PAULET I.N.S.E.R.M.
Unitk 58, 60, rue de Nauacelies, 34100 Montpellier (France)
(Received May 26th, 1978)
Summary Plasma and urine estradiol and estrone can be determined using the transhydrogenase function of the estradiol dehydrogenase of human placenta: in the conditions described the transhydrogenase activity which is directly related to the estrogen concentration is measured by spectrophotometry. Estrone and estradiol can be determined together or separately if estrone is previously reacted with hydrazine with a limit of sensitivity of 10 picograms in the sample. On urine samples the assay is performed directly without extraction, a simplification which makes the method highly suitable for numerous routine determinations.
Introduction Nowadays, radioimmunoassay is a favoured method for the determination of steroid hormones in biological fluids. However, the use of this method is restricted by the necessity for expensive instrumentation and by protection against isotope contamination. Thus enzymes were proposed as label instead of radioelements, but for estrogen determination in non-pregnant women’s plasma, enzyme immunoassays are not sensitive enough [l-3]. An enzymatic method using an amplifying system was recently described [4] but this method is relatively tedious, it needs very good technical assistance and is not convenient for routine clinical determinations. We propose an easy enzymatic method which possesses the same sensitivity as radioimmunoassay, and can be used for routine clinical determination either on plasma extract or directly on urine without extraction.
GLUCOSE
6P
GLUCONOL4CTONE 6P
# : Glucose-6-phosphate l : Estradiol
dchydrogenose
transhydrogenase
Fig. 1. Estrogen tmnshydrogenase
reaction.
Estrone and (or) estradiol concentrations are measured by using the transhydrogenase function of the estradiol 17/3-dehydrogenase of human placenta [5--81. In appropriate conditions, there is a linear relationship between estrogen (estradiol or e&one) concentrations and the transhydrogenase activity which is measured by the appearance rate of NADH in the enzymatic system shown in Fig. 1. The hydrogen of NADPH continuously formed by a regenerating system is transferred to NAD through the rapid cyclic enzymatic interconversion of estrone and estradiol. This reaction occurs in a few seconds giving at each cycle an amount of NADH proportional to estrone plus estradiol. Several hours later large amounts of NADH can be accumulated through numerous cycles leading to NADH concentrations in the range of 10e5 to 10e4 M, which is easy to determine by spectrophotometry. In these conditions, there is no appreciable interference of the small amounts of NADPH present in the medium. The main difficulties met (contamination of the enzyme by endogenous estrogens and alteration of the enzyme activity by the sample) were overcome. We have thus developed a very sensitive and easy method for the determination of e&radio1 plus e&one in biological fluids. Estradiol alone can also be determined: the estrone contained in the sample is reacted with hydrazine, leading to the formation of the hydrazone which is not a substrate for the transhydrogenase reaction. Material NAD, NADP, glucose 6-phosphate, glucoseS-phosphate dehydrogenase were purchased from Boehringer, trishydroxyaminomethane and HCl from Merck. Bovine serum albumine was obtained from Sigma, ethanol, ethyl ether, benzene, hexane and sodium from Prolabo. Enzyme preparation 500 g of placenta pH 7.2, 2 M glycerol
were homogenized in 500 ml 0.03 M phosphate buffer, (buffer A) and centrifuged at 50 000 X g for 30 min. The
3
supernatant was applied on a DEAE cellulose column (5 X 20 cm) and after washing with one liter of buffer A, the enzyme was eluted by one liter of 0.1 M phosphate buffer, pH 7.2,2 M glycerol (buffer B). The fractions containing the estradiol 17/3-dehydrogenase activity were pooled and the enzyme was precipitated by adding one volume of ammonium sulfate saturated solution. The precipitate was collected by. centrifugation (30 min at 50 000 X g) and dissolved in one liter of buffer A. The solution was applied on a second DEAE cellulose column (2 X 10 cm), the column was washed by 300 ml of buffer A containing 4 M ethanol, the enzyme was eluted by a linear gradient formed by adding 400 ml buffer B to 100 ml of buffer A. The fractions containing the estradiol dehydrogenase were pooled: the concentration of glycerol was raised to 5 M, then 1O-4 M of diethylstilbestrol was added to the buffer. This solution was left at 65°C for 4 h and dialysed twice against two liters of buffer A. The enzyme solution was applied on a third DEAE cellulose column (2 X 5 cm). After washing by 300 ml of buffer A containing 4 M ethanol, the enzyme was eluted directly by 200 ml of buffer B. The enzyme was stored in separated tubes at 4°C. The enzyme activity was determined as described in Ref. 8. Sample preparation (a) Plasma samples: 3 ml of plasma were extracted twice by 4 ml ethyl ether. The organic phase was washed with 2 ml of sodium bicarbonate 0.1 M: the ether was evaporated on a nitrogen stream. The residue was dissolved in 1 ml NaOH 2 M and washed by 2 ml of benzene/heptane (2 : 1, v/v). After neutralisation by pure phosphoric acid the aqueous phase was extracted twice by 2 ml of ethyl ether, the ether was washed by 2 ml of water and evaporated in tubes (10 X 50 mm). The residue was dissolved in 50 ~1 of ethanol and then in 1.5 ml of 0.1 M Tris buffer, pH 7.4, 2 M glycerol, 1 g/l bovine serum albumine. (b) Urine samples: The pH of urine samples (4 ml) was adjusted to 6.5 by adding 50 ~1 of 2 M acetate buffer, pH 6.5. Estrogen conjugates were hydrolyzed by glucuronidase Pasteur (one drop) for 30 min. Salts were precipitated by adding one ml of cold ethanol to one ml of hydrolyzed urine maintained at 4” C for 30 min. After centrifugation, 50 ~1 of supernatant were added to 500 ~1 of 0.1 M Tris buffer pH 7.4,2 M glycerol, 1 g/l serum albumin. Estradiol and estrone determination Three different tubes were necessary for the enzymatic determination: blank, assay and standard. Each tube contained 500 ,ul of sample prepared as previously described, 500 ~1 of 0.1 M Tris-HCl buffer, pH 7.4 containing 10e3 M NAD, 10m6 M NADP, 3 X 10-j M glucose 6 phosphate and 0.1 I.U. of glucose-g-phosphate dehydrogenase, 2 M glycerol, 1 g/l bovine serum albumin and: in the blank tube: 100 ~1 of water in the assay tube: 100 ~1 of estradiol dehydrogenase (0.025 I.U.) in the standard tube: 100 ~1 of estradiol dehydrogenase (0.025 I.U.) + 800 pg of estradiol as an internal standard. The tubes were incubated at room temperature for 4 or 16 h according to
4
the precision required Zeiss Spectrophotometer.
and the absorbance
The results were calculated
according
A(assay) -A(blank) - A(assay)
wlm
= A(standard)
at 340 nm was determined
with a
to the following. formula:
x S V
C: concentration (pg/l) of estrone + estradiol in the biological fluid. A (assay), A (blank) and A (standard) are respectively absorbances of assay, blank and standard tubes. S: internal standard expressed as pg of estradiol (usually 800 pg). V: volume of the sample, expressed as ~1 (usually 1000 for plasma and 5 to 25 ~1 for urine). Specific estradiol determination The estrone of the sample was reacted with hydrazine 1 M at pH 7.2 for 1 h at room temperature. For plasma estradiol determinations the treatment was made on the ethyl ether residue dissolved in ethanol. For urine estradiol determination the treatment was made in urine after enzymatic hydrolysis of the conjugates. Results In the assay system NADH appearance rate was a linear function of estradiol concentrations between 0 and 2000 pg (Fig. 2). This linear relationship was obA 340 nm
I
0
1
Fig. 2. Effect incubation.
5 of estradiol on hydrogen transfer from NADPH
10 to NAD.
. "9 E2 Abkbince
is read ifter 6 h
5
served for incubation times in the range O-40 h. After an overnight incubation (16 h) one nanogram of estradiol led to an increase in absorbance at 340 nm of about 0.5 unit. The transhydrogenase system is very sensitive to salt and solvent concentrations; thus the addition to the assay system of biological extract resulted in the decrease of enzyme activity. This inhibition changed between 20 to 50% depending upon the sample, but whatever the sample may be, the linear relationship between NADH appearance rate and estradiol concentration was maintained (Fig. 3). Thus, the degree of inhibition introduced by the biological extract can be accurately estimated by the value obtained for the internal standard (800 pg of estradiol).
Sensitivity When pure estradiol or urine are analysed, the limit of detection is about 8 pg owing to the low absorbance of the blank tube. With plasma samples the variation of the blank raises the sensitivity limit to 15 pg. _: <:
Accuracy and precision The recoveries of estradiol and estrone added to a pool of plasma (Table I) are 91 +- 5%. For urine, as there is no extraction process, the recovery is 100%. With both plasma and urine, there is no interference between estradiol and estrone determination for different estrogen concentration. The precision was calculated from 8 separate analyses on pooled plasma or urine and was 8.5% and 4.5%, respectively.
Fig. 3. NADH production versus estradiol concentration after 16 h incubation in: -, assay SYstem o-----o, assay system + 10 ctl urine; a--a, assay system + ether extract of 1 ml of plasma.
alone:
6 TABLE
I
Estrogens added to 0.5 ml plasma (PP)
Estrogens
E2
E2
El
E2
El
33 t 3 II 126 245 127 128 120 118
42 + 4 41 46 40 38 131 274 510
93 95 87 95 96 90 89
97 109 95 90 78 94 94
El
0 50 100 250 100 100 100 100
0
0
0 0
0 125 250 500
measured
% Recovery
Specificity The interference of various steroids in our enzymatic estrogen determination was resolved. The results are shown in Table II. Amongst the compounds tested, equilenine, estrone 3-methyl ether and 7-ketoestradiol were the only compounds which theoretically could interfere in the assay. Actually, plasma or urinary concentration of these steroids is too low to interfere in the assay, as shown by the following results comparing enzyme assay and radioimmunoassay : The e&radio1 plus estrone concentration of a pooled plasma estimated by our enzymatic method was 115 + 10 pg, the radioimmunoassay methods gave 110 + 10 pg. The plasma samples of twenty pregnant women were analyzed with both radioimmunologic and enzymatic methods. The results are shown in Fig. 4. Regression analysis gave the formula: y = 1.1 x - 2 where y was the result of the radioimmunoassay and x the result of the enzyme assay. The correlation coefficient was 0.95. TABLE
II
INTERFERENCE TION
INDEX
FOR
VARIOUS
STEROIDS
IN ENZYMATIC
ESTROGEN
DETERMINA-
Values indicate the percentage contribution for each steroid if it is present at equal concentration under standard conditions for estrogen determination. Experiments are done with various steroids concentration in the range of 50 to 2000 ng per liter. Estradiol-17@ Estrone Estrone a-methyl ether Esuilenine Estradiol S-sulfate Estradiol B-glucuronide 2-Hydroxyestradiol a-methyl 7-Ketoestradiol 11 &Hydroxyestradiol Androstenedione Dehydroepiandrosterone Cortisol Estdol
ether
100 100 130 110 3 0.5 2.5 15 1 0 0 0 0
ia
,
I
I
10
w Enzymatic pwtttre
1
10
30
Fig. 4. Cormlation between estrone + estradiol concentration of 20 samples of pregnancy plasma as measured by radioimmunoassay and enzymatic method. A
pg/I LCI-
3C I-
21D-
Et+E2
IIo-
I
0
.
IO
20
30 jours
estrone + estradiol: l-, Fig. 5. Edradiol and estrone levels in urine of normal woman. o----O, e&radio1 alone; O- - - - - -0, estradiol + estrone of menopausal woman’s urine.
8
Urinary estradiol during normal menstrual cycle
Fig. 5 shows urinary estradiol levels during a normal menstrual cycle and in a menopausal woman. Discussion The method described is very useful for the determination of urine e&radio1 and e&one concentrations in both men and women: it does not need extraction and more than 40 duplicate determinations can be performed in one day, even without automatisation. However, the method can be easily adapted to automatic spectrophotometers since there are only three steps: addition of sample and standard to the transhydrogenase buffer, incubation and absorbance measurement. For the determination of estrogens in the plasma of nonpregnant women, the method needs only extraction and a partition step to remove steroid binding protein and neutral lipids which can inhibit completely the transhydrogenase activity; as for the plasma of pregnant women, only a single step of extraction is necessary. The sensitivity of this method is in the range of the radioimmunoassay, and better than enzyme immunoassay [l-3]. As the absorbance is a linear function of the concentration over a wide range, a standard curve is not ‘necessary and only an internal standard is added to the sample to determine the rate of the transhydrogenase activity as a function of e&radio1 concentration. The cyclic interconversion of estradiol and estrone occurs at about lob9 M, so the reaction needs a large excess of enzyme, more than one thousand times, the molar equivalent. Thus this enzyme should be completely free of endogenous estrogen, a condition which is satisfied by the ethanol treatment proposed; and certainly the presence of any estrogen contaminants in the enzyme should be carefully checked before assay as follows: the difference of absorbance at 340 nm between transhydrogenase buffer plus enzyme and transhydrogenase buffer alone should not be greater than 10 milliunits of absorbance after an overnight incubation at 25’C. The specificity of the present method is based on the substrate selectivity of the estradiol dehydrogenase of human placenta which is highly specific for Cl8 steroids [g-11]. In these conditions steroids of the Cl9 and CZl series do not interfere with estrogen determination and the only estrogen derivatives which could be substrate of the enzyme are not present in sufficient concentration to interfere with the assay in humans (cf. Results, .,Table II). Moreover, the specificity for transhydrogenase is higher than the specificity for dehydrogenase since e&radio1 3aulfate or 3_glucuronide, which are good substrates for dehydrogenase, do not work as substrate for transhydrokenase JTable II). So, the values found with the enzymatic method are in agreement with the results of radioimmunoassay as shown by the good correlation obtained Fig. 4. It can be observed that the enzymatic method gave higher values ‘than the ritdioimmunoassay. This fact was also reported by authors using an enzyme assay [4] or gas chromatography coupled with mass fragmentograpfiy [ 121 and it appeared that the difference could not be attributed to a lack of specificity in these methods.
References 1 2 3 4 6 6 7 8 9 10 11
Van Veemen. B.K. and Schuurs, A.H.W.M. (1972) FEBS Lett. 24.77-81 Van Veemen, B.K. and Schuurs. A.H.W.M. (1975) Immunochemistry 12.667-670 Numazawa, M., Haryu. A., Kurosaka, K. and Nambara. T. (1977) FEBS Lett. 79.396-398 Harkonen. M.. Adlercreutz. H. and Croman, E.V. (1974) J. Steroid Biochem. 5. 717-726 TaIaIay, P. and William Ashman, H.G. (1968) Proc. NatI. Acad. Sci. U.S.A. 44.15-21 ViIIee, CA. and Hagerman. D.D. (1958) J. Biol. Chem. 233,42-54 Langer, L.J. and Engel. L.L. (1968) J. Biol. Chem. 233.583-592 Jerabak, J., Adams, J.A., WiBii-Ashman, H.G. and TaIaIay. P. (1962) J. Biol. Chem. 237.345-357 Adams, J.A.. Jsrabak. J. and TaMlay, P. (1962) J. Biol. Chcm. 237.3069-3073 Descomps, B. and Crastes de PauIet. A. (1969) Bull. Sot. Chim. Biol. 51.1591-1611 Langer, L.J.. Alexander, J.A. and Engel. L.L. (1969) J. Biol. Chem. 234,2609-2614
CZinico Chimica Acta, 92 (1979) l-9 @ Elsevier/North-Holland Biomedical Press
CCA 9739
ENZYMATIC DETERMINATION PLASMA AND URINE
OF ESTRADIOL
AND ESTRONE
IN
JEAN-CLAUDE NICOLAS *, ANNE-MARIE BOUSSIOUX, BERNARD DESCOMPS and ANDRl? CRASTES DE PAULET I.N.S.E.R.M.
Unitk 58, 60, rue de Nauacelies, 34100 Montpellier (France)
(Received May 26th, 1978)
Summary Plasma and urine estradiol and estrone can be determined using the transhydrogenase function of the estradiol dehydrogenase of human placenta: in the conditions described the transhydrogenase activity which is directly related to the estrogen concentration is measured by spectrophotometry. Estrone and estradiol can be determined together or separately if estrone is previously reacted with hydrazine with a limit of sensitivity of 10 picograms in the sample. On urine samples the assay is performed directly without extraction, a simplification which makes the method highly suitable for numerous routine determinations.
Introduction Nowadays, radioimmunoassay is a favoured method for the determination of steroid hormones in biological fluids. However, the use of this method is restricted by the necessity for expensive instrumentation and by protection against isotope contamination. Thus enzymes were proposed as label instead of radioelements, but for estrogen determination in non-pregnant women’s plasma, enzyme immunoassays are not sensitive enough [l-3]. An enzymatic method using an amplifying system was recently described [4] but this method is relatively tedious, it needs very good technical assistance and is not convenient for routine clinical determinations. We propose an easy enzymatic method which possesses the same sensitivity as radioimmunoassay, and can be used for routine clinical determination either on plasma extract or directly on urine without extraction.
GLUCOSE
6P
GLUCONOL4CTONE 6P
# : Glucose-6-phosphate l : Estradiol
dchydrogenose
transhydrogenase
Fig. 1. Estrogen tmnshydrogenase
reaction.
Estrone and (or) estradiol concentrations are measured by using the transhydrogenase function of the estradiol 17/3-dehydrogenase of human placenta [5--81. In appropriate conditions, there is a linear relationship between estrogen (estradiol or e&one) concentrations and the transhydrogenase activity which is measured by the appearance rate of NADH in the enzymatic system shown in Fig. 1. The hydrogen of NADPH continuously formed by a regenerating system is transferred to NAD through the rapid cyclic enzymatic interconversion of estrone and estradiol. This reaction occurs in a few seconds giving at each cycle an amount of NADH proportional to estrone plus estradiol. Several hours later large amounts of NADH can be accumulated through numerous cycles leading to NADH concentrations in the range of 10e5 to 10e4 M, which is easy to determine by spectrophotometry. In these conditions, there is no appreciable interference of the small amounts of NADPH present in the medium. The main difficulties met (contamination of the enzyme by endogenous estrogens and alteration of the enzyme activity by the sample) were overcome. We have thus developed a very sensitive and easy method for the determination of e&radio1 plus e&one in biological fluids. Estradiol alone can also be determined: the estrone contained in the sample is reacted with hydrazine, leading to the formation of the hydrazone which is not a substrate for the transhydrogenase reaction. Material NAD, NADP, glucose 6-phosphate, glucoseS-phosphate dehydrogenase were purchased from Boehringer, trishydroxyaminomethane and HCl from Merck. Bovine serum albumine was obtained from Sigma, ethanol, ethyl ether, benzene, hexane and sodium from Prolabo. Enzyme preparation 500 g of placenta pH 7.2, 2 M glycerol
were homogenized in 500 ml 0.03 M phosphate buffer, (buffer A) and centrifuged at 50 000 X g for 30 min. The
3
supernatant was applied on a DEAE cellulose column (5 X 20 cm) and after washing with one liter of buffer A, the enzyme was eluted by one liter of 0.1 M phosphate buffer, pH 7.2,2 M glycerol (buffer B). The fractions containing the estradiol 17/3-dehydrogenase activity were pooled and the enzyme was precipitated by adding one volume of ammonium sulfate saturated solution. The precipitate was collected by. centrifugation (30 min at 50 000 X g) and dissolved in one liter of buffer A. The solution was applied on a second DEAE cellulose column (2 X 10 cm), the column was washed by 300 ml of buffer A containing 4 M ethanol, the enzyme was eluted by a linear gradient formed by adding 400 ml buffer B to 100 ml of buffer A. The fractions containing the estradiol dehydrogenase were pooled: the concentration of glycerol was raised to 5 M, then 1O-4 M of diethylstilbestrol was added to the buffer. This solution was left at 65°C for 4 h and dialysed twice against two liters of buffer A. The enzyme solution was applied on a third DEAE cellulose column (2 X 5 cm). After washing by 300 ml of buffer A containing 4 M ethanol, the enzyme was eluted directly by 200 ml of buffer B. The enzyme was stored in separated tubes at 4°C. The enzyme activity was determined as described in Ref. 8. Sample preparation (a) Plasma samples: 3 ml of plasma were extracted twice by 4 ml ethyl ether. The organic phase was washed with 2 ml of sodium bicarbonate 0.1 M: the ether was evaporated on a nitrogen stream. The residue was dissolved in 1 ml NaOH 2 M and washed by 2 ml of benzene/heptane (2 : 1, v/v). After neutralisation by pure phosphoric acid the aqueous phase was extracted twice by 2 ml of ethyl ether, the ether was washed by 2 ml of water and evaporated in tubes (10 X 50 mm). The residue was dissolved in 50 ~1 of ethanol and then in 1.5 ml of 0.1 M Tris buffer, pH 7.4, 2 M glycerol, 1 g/l bovine serum albumine. (b) Urine samples: The pH of urine samples (4 ml) was adjusted to 6.5 by adding 50 ~1 of 2 M acetate buffer, pH 6.5. Estrogen conjugates were hydrolyzed by glucuronidase Pasteur (one drop) for 30 min. Salts were precipitated by adding one ml of cold ethanol to one ml of hydrolyzed urine maintained at 4” C for 30 min. After centrifugation, 50 ~1 of supernatant were added to 500 ~1 of 0.1 M Tris buffer pH 7.4,2 M glycerol, 1 g/l serum albumin. Estradiol and estrone determination Three different tubes were necessary for the enzymatic determination: blank, assay and standard. Each tube contained 500 ,ul of sample prepared as previously described, 500 ~1 of 0.1 M Tris-HCl buffer, pH 7.4 containing 10e3 M NAD, 10m6 M NADP, 3 X 10-j M glucose 6 phosphate and 0.1 I.U. of glucose-g-phosphate dehydrogenase, 2 M glycerol, 1 g/l bovine serum albumin and: in the blank tube: 100 ~1 of water in the assay tube: 100 ~1 of estradiol dehydrogenase (0.025 I.U.) in the standard tube: 100 ~1 of estradiol dehydrogenase (0.025 I.U.) + 800 pg of estradiol as an internal standard. The tubes were incubated at room temperature for 4 or 16 h according to
4
the precision required Zeiss Spectrophotometer.
and the absorbance
The results were calculated
according
A(assay) -A(blank) - A(assay)
wlm
= A(standard)
at 340 nm was determined
with a
to the following. formula:
x S V
C: concentration (pg/l) of estrone + estradiol in the biological fluid. A (assay), A (blank) and A (standard) are respectively absorbances of assay, blank and standard tubes. S: internal standard expressed as pg of estradiol (usually 800 pg). V: volume of the sample, expressed as ~1 (usually 1000 for plasma and 5 to 25 ~1 for urine). Specific estradiol determination The estrone of the sample was reacted with hydrazine 1 M at pH 7.2 for 1 h at room temperature. For plasma estradiol determinations the treatment was made on the ethyl ether residue dissolved in ethanol. For urine estradiol determination the treatment was made in urine after enzymatic hydrolysis of the conjugates. Results In the assay system NADH appearance rate was a linear function of estradiol concentrations between 0 and 2000 pg (Fig. 2). This linear relationship was obA 340 nm
I
0
1
Fig. 2. Effect incubation.
5 of estradiol on hydrogen transfer from NADPH
10 to NAD.
. "9 E2 Abkbince
is read ifter 6 h
5
served for incubation times in the range O-40 h. After an overnight incubation (16 h) one nanogram of estradiol led to an increase in absorbance at 340 nm of about 0.5 unit. The transhydrogenase system is very sensitive to salt and solvent concentrations; thus the addition to the assay system of biological extract resulted in the decrease of enzyme activity. This inhibition changed between 20 to 50% depending upon the sample, but whatever the sample may be, the linear relationship between NADH appearance rate and estradiol concentration was maintained (Fig. 3). Thus, the degree of inhibition introduced by the biological extract can be accurately estimated by the value obtained for the internal standard (800 pg of estradiol).
Sensitivity When pure estradiol or urine are analysed, the limit of detection is about 8 pg owing to the low absorbance of the blank tube. With plasma samples the variation of the blank raises the sensitivity limit to 15 pg. _: <:
Accuracy and precision The recoveries of estradiol and estrone added to a pool of plasma (Table I) are 91 +- 5%. For urine, as there is no extraction process, the recovery is 100%. With both plasma and urine, there is no interference between estradiol and estrone determination for different estrogen concentration. The precision was calculated from 8 separate analyses on pooled plasma or urine and was 8.5% and 4.5%, respectively.
Fig. 3. NADH production versus estradiol concentration after 16 h incubation in: -, assay SYstem o-----o, assay system + 10 ctl urine; a--a, assay system + ether extract of 1 ml of plasma.
alone:
6 TABLE
I
Estrogens added to 0.5 ml plasma (PP)
Estrogens
E2
E2
El
E2
El
33 t 3 II 126 245 127 128 120 118
42 + 4 41 46 40 38 131 274 510
93 95 87 95 96 90 89
97 109 95 90 78 94 94
El
0 50 100 250 100 100 100 100
0
0
0 0
0 125 250 500
measured
% Recovery
Specificity The interference of various steroids in our enzymatic estrogen determination was resolved. The results are shown in Table II. Amongst the compounds tested, equilenine, estrone 3-methyl ether and 7-ketoestradiol were the only compounds which theoretically could interfere in the assay. Actually, plasma or urinary concentration of these steroids is too low to interfere in the assay, as shown by the following results comparing enzyme assay and radioimmunoassay : The e&radio1 plus estrone concentration of a pooled plasma estimated by our enzymatic method was 115 + 10 pg, the radioimmunoassay methods gave 110 + 10 pg. The plasma samples of twenty pregnant women were analyzed with both radioimmunologic and enzymatic methods. The results are shown in Fig. 4. Regression analysis gave the formula: y = 1.1 x - 2 where y was the result of the radioimmunoassay and x the result of the enzyme assay. The correlation coefficient was 0.95. TABLE
II
INTERFERENCE TION
INDEX
FOR
VARIOUS
STEROIDS
IN ENZYMATIC
ESTROGEN
DETERMINA-
Values indicate the percentage contribution for each steroid if it is present at equal concentration under standard conditions for estrogen determination. Experiments are done with various steroids concentration in the range of 50 to 2000 ng per liter. Estradiol-17@ Estrone Estrone a-methyl ether Esuilenine Estradiol S-sulfate Estradiol B-glucuronide 2-Hydroxyestradiol a-methyl 7-Ketoestradiol 11 &Hydroxyestradiol Androstenedione Dehydroepiandrosterone Cortisol Estdol
ether
100 100 130 110 3 0.5 2.5 15 1 0 0 0 0
ia
,
I
I
10
w Enzymatic pwtttre
1
10
30
Fig. 4. Cormlation between estrone + estradiol concentration of 20 samples of pregnancy plasma as measured by radioimmunoassay and enzymatic method. A
pg/I LCI-
3C I-
21D-
Et+E2
IIo-
I
0
.
IO
20
30 jours
estrone + estradiol: l-, Fig. 5. Edradiol and estrone levels in urine of normal woman. o----O, e&radio1 alone; O- - - - - -0, estradiol + estrone of menopausal woman’s urine.
8
Urinary estradiol during normal menstrual cycle
Fig. 5 shows urinary estradiol levels during a normal menstrual cycle and in a menopausal woman. Discussion The method described is very useful for the determination of urine e&radio1 and e&one concentrations in both men and women: it does not need extraction and more than 40 duplicate determinations can be performed in one day, even without automatisation. However, the method can be easily adapted to automatic spectrophotometers since there are only three steps: addition of sample and standard to the transhydrogenase buffer, incubation and absorbance measurement. For the determination of estrogens in the plasma of nonpregnant women, the method needs only extraction and a partition step to remove steroid binding protein and neutral lipids which can inhibit completely the transhydrogenase activity; as for the plasma of pregnant women, only a single step of extraction is necessary. The sensitivity of this method is in the range of the radioimmunoassay, and better than enzyme immunoassay [l-3]. As the absorbance is a linear function of the concentration over a wide range, a standard curve is not ‘necessary and only an internal standard is added to the sample to determine the rate of the transhydrogenase activity as a function of e&radio1 concentration. The cyclic interconversion of estradiol and estrone occurs at about lob9 M, so the reaction needs a large excess of enzyme, more than one thousand times, the molar equivalent. Thus this enzyme should be completely free of endogenous estrogen, a condition which is satisfied by the ethanol treatment proposed; and certainly the presence of any estrogen contaminants in the enzyme should be carefully checked before assay as follows: the difference of absorbance at 340 nm between transhydrogenase buffer plus enzyme and transhydrogenase buffer alone should not be greater than 10 milliunits of absorbance after an overnight incubation at 25’C. The specificity of the present method is based on the substrate selectivity of the estradiol dehydrogenase of human placenta which is highly specific for Cl8 steroids [g-11]. In these conditions steroids of the Cl9 and CZl series do not interfere with estrogen determination and the only estrogen derivatives which could be substrate of the enzyme are not present in sufficient concentration to interfere with the assay in humans (cf. Results, .,Table II). Moreover, the specificity for transhydrogenase is higher than the specificity for dehydrogenase since e&radio1 3aulfate or 3_glucuronide, which are good substrates for dehydrogenase, do not work as substrate for transhydrokenase JTable II). So, the values found with the enzymatic method are in agreement with the results of radioimmunoassay as shown by the good correlation obtained Fig. 4. It can be observed that the enzymatic method gave higher values ‘than the ritdioimmunoassay. This fact was also reported by authors using an enzyme assay [4] or gas chromatography coupled with mass fragmentograpfiy [ 121 and it appeared that the difference could not be attributed to a lack of specificity in these methods.
References 1 2 3 4 6 6 7 8 9 10 11
Van Veemen. B.K. and Schuurs, A.H.W.M. (1972) FEBS Lett. 24.77-81 Van Veemen, B.K. and Schuurs. A.H.W.M. (1975) Immunochemistry 12.667-670 Numazawa, M., Haryu. A., Kurosaka, K. and Nambara. T. (1977) FEBS Lett. 79.396-398 Harkonen. M.. Adlercreutz. H. and Croman, E.V. (1974) J. Steroid Biochem. 5. 717-726 TaIaIay, P. and William Ashman, H.G. (1968) Proc. NatI. Acad. Sci. U.S.A. 44.15-21 ViIIee, CA. and Hagerman. D.D. (1958) J. Biol. Chem. 233,42-54 Langer, L.J. and Engel. L.L. (1968) J. Biol. Chem. 233.583-592 Jerabak, J., Adams, J.A., WiBii-Ashman, H.G. and TaIaIay. P. (1962) J. Biol. Chem. 237.345-357 Adams, J.A.. Jsrabak. J. and TaMlay, P. (1962) J. Biol. Chcm. 237.3069-3073 Descomps, B. and Crastes de PauIet. A. (1969) Bull. Sot. Chim. Biol. 51.1591-1611 Langer, L.J.. Alexander, J.A. and Engel. L.L. (1969) J. Biol. Chem. 234,2609-2614