DE2908957A1 - PROCESS FOR THE ENZYMATIC DETERMINATION OF OESTROGENS AND READY-TO-USE COMBINATION FOR PERFORMING THE SAME - Google Patents

Description

DR. GERHARD RATZEL

PATENT Attorney file

eecc Mannheim ι, f, ^ 1 ™ Sedtenheimer sir. 36a, Tel. (0621) 406315

Potitcheckkonto: Frankfurt / M No. 82S3-603 Bank: Deutsche Bank Mannheim No. 72/00066 Te Ieο r. -C οde: Gerptt Tele »463570 Pin D

INSTITUT NATIONAL DE LA SANTE ET DE LA HECHERCHE MEDICALE - OKi] TOM 101 rue de Tolbiac 756 ^ 5! ¹ ARIS CEDEX 13 / France

Process for the enzymatic determination of (JstroS ⁶¹¹⁶¹¹ as well as a ready-to-use combination for carrying out the same.

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The present invention relates to a method for enzymatic Determination of estrogens, especially estrone and estradiol, as well as a ready-to-use combination for implementation this provision.

Accurate knowledge of the amount of steroid hormones, particularly the amount of estrone and estradiol, that are present in the urine and plasma is of growing importance for the clinician, especially the gynecologist, in determining a safe Diagnosis and the creation of an effective therapy, especially in the case of ovarian isufficiency, in the case of disorders of the i: stro ^ en biosynthesis, in monitoring problematic pregnancies eic, However, such knowledge requires that one has techniques of estrogen determination that are specifically, are accurate and sensitive, reliable, reproducible and relatively inexpensive and can be carried out, without the technician performing the determination must require special skills and abilities.

Several methods of micro- Proposed determination of estrogens to meet the demands of clinicians. The most commonly used so far Methods of micro-determination are:

micro-determination by fluorescence, radioimmunological determination.

The former, i.e. determination by fluorescence, determines the

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total urinary phenols, ^M stradio1, oestrone and oestriol and comprises four stages: a stage of hydrolysis of the conjugated compounds; an extraction stage which is an expensive and lengthy phase of determination and requires the use of large, narrow, very pure solvents; a stage in the development of the chromophore; and a level of reading. In any case, the value of this provision is limited, since it does not have great specificity and can therefore only be used by medical professionals with the utmost caution.

As for the second type of provision proposed so far, namely the radioimmunological determination of whether it is much more accurate than the determination by fluorescence. and allows it the separate determination of estradiol, estrone and estriol im Plasma, however, has the disadvantage that it is expensive and requires the use of radioactive isotopes, thereby their use is limited to laboratories which have suitable, very expensive equipment and due to are entitled to do so under a special agreement of the Ministry of Health.

By the way, methods for the enzymatic determination of estrogens are described in the literature (M.H \ RKONEN, H.ADLERKREUZ, E.V. GROMAN, Journal of Steroid Biochemistry 197 ^ 4 Vol. 5, pp 717 - 7 ^ 5), but because of their difficulty you cannot used clinically. These methods describe the determination of estradiol and estrone using 17 P - '' ^ Dehydrogenase from the placenta, which with NADP (phospho-

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Nicotinamide adenine dinucleotide, coenzyme of dehydrogenase) or. NADΠI, the estradiol being purified from the other reactive steroids present in the plasma by chromatography over an alumina column. The enzymatic dismutation between NADP and NADPH% is carried out according to the method described in the above publication, using glucose-6-phosphate dehydrogenase (g6PDH) and Güirfcamas dehydrogenase, a gain of 10,000 being achieved; the enzymatic circuit is necessary in order to measure the NADPH formed during the reaction, because in the ⁿ 1as ma of the non-pregnant woman there is only a very low concentration of stradiol (4-1 nmol / l). The 6-phosphogluconate formed is determined by fluorometry using 6-phosphogluconate dehydrogenase and NADP. It is stated in this publication that the method described permits a detection sensitivity of 25 JFMoI steroid and an accuracy of determination of ik ';i> and that its specificity is comparable to radioimmunological determination. However, the authors of the publication point out the difficulty of the method, especially with the enzymatic cycle, which prevents the application of their method in routine analyzes in the laboratory.

It has now been found that the difficulty of this method is not the only reason for its use for enzymatic purposes To prevent routine determination in the laboratory.

As can also be seen from the prior art, it is

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knows that 17 ft -estradiol dehydrogenase (the enzyme is designated as EC1.1.1.6? according to the international classification) not only has a dehydrogenase effect (NAD ⁺ - * NADH; NAD ⁺ = nicotinamide-adenine-dinueleotide), but also has a transhydrogenase effect: in the presence of NADI ¹ H (in small amounts), a transfer and formation of NADH takes place (whereby the estrogen in oxidized or reduced form takes part in this transhydrogenation reaction).

The cycle of formation of NADP .—> NADPH in the presence of an enzyme such as glucose-6-P-dehydrogenase is also known. It is sufficient therefore, when the following cycle is formed (which is shown in scheme 1), under such conditions of concentration of gofactors, that NADH is produced in measurable amounts that are proportional to the concentration of estradiol and estrone:

NADH

(estron)

NADP

NADI ¹ H

Enzyme:

Estradiol dehydrogenation

, Glucose-6-phosphate (G6P)

Gluconolactone

Enzyme: glucose-6P dehydrogenase

This method of enzymatic determination of the total estrogen oestrone (E ₁ ) + oestradiol (Ep) is much simpler than that

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determination rivet testicles actually used and theoretically very much tempting; unfortunately, however, it is not satisfactory in practice, particularly with regard to the accuracy of the ones obtained Result: the reaction rate, which is determined by the kinetics of the occurrence of NADH, is namely a puncture the concentration of estrogens E and E.

Now the 17 ß -f'stradiol dehydrogenase from placenta is afflicted with two types of impurities, which considerably influence the accuracy of the determination}

it contains steroids, especially estradiol, in non-negligible amounts, the presence of which falsifies the results to a greater extent as it is necessary to use the 17 fi -estradiol dehydrogenation in relatively large amounts, which results in an even greater amount in the environment of impurities is introduced;

it also contains other non-estradiol-dependent contaminants -Transhydrogena sen, whose presence is another

Represents source of error.

The aim of the present invention is therefore to create a new process ZiU? enzymatic determination of estrogens E and E _n, which better meets the requirements of practice than the previously known methods and especially specific, very accurate, reliable, a little expensive, relatively simple, feasible and in V _{er gy} I said in the literature described method for enzymatic determination not only the

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non-estradiol-dependent-Transhydrogennsen eliminated which the I7P estradiol dehydrogenase accompany from Piacentn, which is used for the reduction of NAD to NADH and a total elimination of the SS with the 17-estradiol dehydrogenase from placenta entrained estradiol ensured, that the enzymatic determination takes place under conditions as precise and reliable as could not be achieved in the prior art.

The subject of the present invention is a method for the enzymatic determination of the estrogens E and E ₀ in biological media "in particular in plasma and urine, characterized in that one uses a 17 ß -estradiol dehydrogenase from placenta, which is not estrogenic from steroids and parasitic" dependent transhydrogenases is freed, and that one works in the presence of NAD or an NAD analogue, which is recognized by the enzyme, of NADP or one of its analogues, as well as of a specific enzyme NADP dehydrogenase and a substrate forming NADP «

Under the conditions of the method according to the invention - in contrast In addition to the classic enzymatic determinations - the substance to be determined disappears, in the present case the estrogens E .. and E ", not; it is not the limiting factor in the speed of reaction and the amount of NADH formed is directly proportional to the estrogen concentration.

According to a preferred embodiment of the process according to the invention select the specific enzyme NADP dehydrogenase

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of the following group: tetrahydrofolate dehydrogenase (international classification 1-5-1-13 »1-5-1-15), isocitrate dehydrogenase (international classification 1-1-1- ^ 2), phosphogluconate deh} '- drugase (international classification 1-1-1-kk), glucose-6-phosphate-Befrydrogenase (international classification 1-1-1-1) 9), lactaldehyde dehydrogenase (international classification 1-1-1-55), benzaldehyde- Dehydrogenase (international classification 1-2-1-7), glyceraldehyde phosphate dehydrogenase (international classification 1 -? - 1-9 £ "I -2-1 -I 3_J), L-aspartate-semi-aldehyde dehydrogenase enase (international classification 1-P-1-11), glyoxy-1ate dehydrogenase (international classification 1τ2-1-17).

According to a preferred embodiment of the method according to the invention if the specific enzyme is MDJ'-dehydrogenase GöPDll is, the HIDP-forming substrate is G6P.

According to a further embodiment of the method according to the invention, the determination takes place in a medium which consists of a buffer with a pH value in the vicinity of 7. k , the ionic strength being equal to or less than that of a 0.1 molar buffer .

According to a further preferred embodiment of the method according to the invention, the buffer also contains 0.5 per mil to ifo protein without any significant affinity for estrogens.

According to a special embodiment of this variant, the protein added to the milieu is serum albumin, especially cattle albumin. _ ■ 909839/0735

■ Ab-

Serum albumin.

According to the invention, the determination in the presence of FAD or carried out one of its analogs, such as acetyl-NAD or thio-NAD.

According to a further preferred embodiment of the process according to the invention, the NAD and the NADP-forming substrate are present in excess.

According to a further embodiment of the process according to the invention

-3- k rens, the amount of NAD in the reaction medium is 10 to 10 mol, that of NADP is 10 to 10 mol and the concentration ratio of NAD to NADP is about 10.

It has been found that the absolute and relative proportions of the NAD or NADP introduced into the medium contribute considerably to the accuracy of the determination in that the amount of NadP present must not exceed the proportions given above, otherwise the activity of the enzyme 17/3 ^f istradiol dehydrogenase is hindered and the results are therefore falsified.

According to a further preferred embodiment of the invention Procedure will eliminate the parasitic

non-estrogen-dependent transhydrogenase enzymes by heating the solution of 17 ^ -estradiol dehydrogenase, which is a polyol contains, causes, namely 100 to 150 minutes at one temperature

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from 60 to 7o ⁰ C in the presence of 4o to 60% of this polyol, and purified by chromatography on DEAE - cellulose.

According to a special embodiment of this variant there is the polyol made from glycerine.

According to a further preferred embodiment, the solution containing the polyol contains 17/5 'oestradiol dehydrogenase also a small amount of diethylstilbestrol (10 ~ M).

According to a further embodiment of the method according to the invention, the steroids are removed from the 17 $ * estradiol dehydrogenase by washing the enzyme, which was previously adsorbed on BEAE cellulose or something similar, with a phosphate buffer, the pH of which is close to about 7- »2 and which contains about 15 to 25 % of a polar solvent.

According to a special embodiment of this variant, the phosphate buffer contains about 20 % ethanol.

According to a further preferred embodiment of the erfindungsgeinäßen process, the reaction mixture containing the ReaktionskoiEponenten and the substances to be determined is, prior to reading the optical density of 2 to 12 hours, preferably 3 to 9 hours, at a temperature of from 2o to 4o ⁰ C, preferably 25 to 38 ⁰ C incubated.

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"The driving wheel" for the total dosage consists namely in the conversions of E ^ and E ₂ and then from E ₂ to E ^ (as indicated above in Scheme 1). These conversions lead to the formation of a stoichiometric amount of NADH in each cycle. These conversions take place during the incubation period. Thus, at the end of χ cycles, which correspond to a certain incubation period, an amount of NADH is created that corresponds to x times the stoichiometric amount of the substrate (E ^ and / or E ₂ ) in the environment, which amount of NADH can then be easily determined .

According to a preferred embodiment of the method according to the invention, the samples of the biological media which _{contain the estrogens E 1} and E ₂ to be determined are subjected to a preparation, extraction and / or purification process before the determination process.

According to a preferred embodiment of this variant,

If the biological medium containing the estrogens to be determined consists of plasma, this is previously subjected to a known extraction process with, for example, ether, after which it is washed several times with the appropriate solvents, evaporated and the last evaporation residue in a mixture of a polar solvent and a Buffer the ionic strength of which is practically the same as that of a 0.1 M buffer and which dissolves a small amount of a protein with no significant affinity for estrogens and 15 to 25 %

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of a polyol.

According to a further preferred embodiment of this variant, if the biological medium containing the estrogens to be determined consists of urine, this is previously subjected to a preparatory process with the aid of an enzyme which hydrolyzes the conjugated compounds, such as "β- glucuronidase" Pasteur.

The invention also relates to a ready-to-use determination combination (determination kit), which is used in particular for determining the estrogens E ^, and Ep in urine are useful, thereby characterized in that it essentially contains the following previously determined and lyophilized ingredients:

the enzyme 17 β ^ estradiol dehydrogenase, which is freed from steroids and parasitic non-estrogen-dependent OJranshydrogenasen and is preserved in a suitable polyol, in particular glycerol;

an internal standard (containing a known excess of estrogens);

- a control (blanc);

- an enzyme for the hydrolysis of the conjugated compounds contained in the urine, such as ,, P> -glucuronidase ^M Pasteur, which was previously determined as a function of the amount of the urine sample to be treated:

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NAD: 1o " ² - 1o ~ ⁴ Μ

Glucose-6-PDH: ο, 1 IU / ml of the sample to be determined

NADP: 1o ~ ⁶ - 1o ~ ^{8 sts}

Glucose-6-P: 1o ~ ² - Ίο Μ

Buffer SBA (bovine serum albumin): 0.5 per thousand to 1 Buffer Tris-HCL 0.05 M, pH about 7.5

According to the invention, the control (blanc) consists of a mixture of buffer Tris-HCL + 15 to 25 % polyol + 0.5 to 1 % SBA (buffer A) and about 0.5 ml of buffer A + 2 10 "r M NAD , 2 1o ~ ⁶ M NADP, 2 χ 1o "" ⁵ M glucose-6-phosphate, 0.2 IU glucose-δ-phosphate dehydrogenase / ml (buffer B).

According to a preferred embodiment of the invention, the internal standard consists of a mixture of the above buffers B and of buffer A, which contains 16oo picograms of estradiol / ml and 0.02 I.U. 17 /? - contains estradiol dehydrogenase.

In addition to the above-mentioned forms of listing, the invention relates to also other embodiments that emerge from the following description emerge.

The process according to the invention is explained in more detail in the following examples, without being restricted thereto target.

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example 1 Determination of estrogens in plasma

A - Bas enzyme

The 17β estradiol hydrogenase from human placenta is determined by chromatography over DEAE cellulose and by affinity chromatography according to the method described by JCNICOLAS, M. PONS, B. DESCOMPS and A.CSASTES de PADLET, Febs Letters YoI. 23 n ° June 2, 1972, pp 175-179. The enzyme solution / ice thus obtained V is heated for 2 hours at 65 ⁰ C in the presence of 5o% glycerol (and 1o ~ M diethylstilbestrol) to destroy all parasitic other enzymes. Is then removed by washing containing the steroids of the enzyme, which was previously adsorbed on DEAE-cellulose, with 3oo ml phosphate buffer (o.o3 M, pH = 7.2) 2o fo ethanol.

B - Extraction and purification of the estrogens of the plasma

100 aiI sodium pyrophosphate buffer are added to 3 »5 ml of plasma (0.1 M, pH 9.). The pH value of the plasma sample is then 8.9 »

Then it is extracted twice with 3 ml of ether and the enriched extract is then evaporated under nitrogen

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and concentrated to 2 ml, then washed twice with 1 ml of sodium carbonate (1 per thousand). The ether is then evaporated to dryness and the dry residue is taken up in 1 ml of 2 M sodium hydroxide solution. The aqueous phase is then washed twice with 2 ml of benzene / heptane (2: 1 V / V), then neutralized with 2oo All 10 MH ^ P (X and extracted twice with 3 ml of ether. After evaporation, the residue is washed with "looyul ethanol and 25o / ul buffer-Tris-HCl (0.1 M, pH 7.4), which contains 1 per mill bovine serum albumin and 20 % glycerol.

The estrogen determination is carried out with 100 μl of this extract.

C - Determination of estrogens

The reaction medium consists of 1 ml of buffer Tris-HCl (0.05 M, pH 714)> 5 per thousand serum albumin, 5 · 1o ^ M glucose-6-P, 5.1o " ⁴ M NAD, 5.1o ~ ⁷ M NADP , o, 1 IU glucose-6-P dehydrogenase and o, o3 IU 17 ^ oestradiol dehydrogenase The estrogen concentration of the extract is o, o2 to 2 pmol.

The incubation period is 6 to 16 hours at 25 ° C.

The reaction rate is at 34-0 mm with the help of a ZEISS spectrophotometer MD 1 or a colorimeter LKB. The absorption of the various estrogen concentrations

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solutions containing ions at 34-O mm will be at the end of the incubation period ' had read.

D - results

The optical density at 34-0 mm becomes after 6 to 16 hours Reaction time determined. The attached figure shows curve I for the standard obtained with estradiol levels between o, o2o and 2 picomoles, that is, 5 to 500 pg after 6 Hours of reaction time; curve II shows the optical density that is obtained with the same amounts of estradiol after 16 hours Incubation is maintained.

The calibration curve is linear over a very large range: In any case, it covers the physiological range over a wide range Concentrations, including the immature Child and man. Usually, the determinations are made by the fluorometric methods that are currently in use be carried out in the prior art, the sensitivity is limited by the size of the basic fluorescence, which is contributed by the biological extract. According to the determination method according to the invention, it is because of the large "amplification is not necessary, the readings by fluorescence or in a very high sensitivity range. This is why it is contributed by the biological extract Absorption negligible compared to the absorptions

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the products of the enzymatic reaction.

Example 2 Determination of estradiol and estrone in urine

A - Preparation of urine samples

The collection is carried out with urine taken before standing up. 1 ml urine is adjusted to a pH of 6.5 to 7 by adding 50 μl (0.1 M) citrate buffer. This solution is treated with .einem drop "ß-glucuronidase" -Pasteur Jo minutes at 37 ° C. The tubes are then cooled and treated with 1 ml of ethanol, the mixture was held for about 3o minutes at 24 ⁰ C and then centrifuged at 4ooo g Half of the supernatant is diluted with buffer Tris-HCl (pH 7.4), 1 per mill bovine serum albumin, 20 % glycerol, and the dosage is carried out with 100 μl of this extract.

B - provision

The following buffers are used: Buffer A: Tris HCl 0.1 M, pH 7.4, 20 % glycerol, 1 per mill SBA; Buffer B: Buffer A + 2.1o "" ⁵ M NAD, 2.1o "" ⁶ M NADP, 2.1o " ⁵ M glucose-6-phosphate, 0.2 units per ml glucose

6-phosphate dehydrogenase; 909839/0735

A -

Buffer C i buffer A + 160 pg estradiol per ml.

For each sample, 3 tubes are used, which are 100 ml Urine dilution and the following amount of reaction components contain:

Tube 1 ϊ control = 0.5 ml buffer A + 0.5 ml buffer B Tube 2 ϊ determination = 0.5 ml buffer A + 0.5 ml buffer B

+ o, o25 IU / 3-estradiol dehydrogenase Tube J: excess = 0.5 ml buffer B + 0.5 ml buffer C.

+ o, 25 IE i? 4-estradiol dehydrogenase.

The tubes are incubated for 4 to 16 hours at 7 ° C. The optical density is determined at 34-Ο mm after this incubation period.

C - calculation of estrogen concentrations

The concentration of the estrogens contained in the samples is calculated in comparison with the excess absorption value using the following formula

( j control tube C xier / l = absorption of the determination tube - absorption of the J

Absorption of the excess tube - absorption of the Determination tube

multiplied by 800

X

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C: Estrogen concentration in urine or plasma (jug / liter) 800: corresponds to the estradiol excess in the tube X: Volume of the biological medium removed in microliters. In the present example it is loooyul in the case of plasma and 25 / oil in the case of urine.

Example 3 Specific determination of estradiol

The specific determination of estradiol can be carried out provided that the estrone has been obtained beforehand of the milieu reacts with hydrazine.

For this purpose, 0.5 ml of the biological medium (plasma or urine hydrolyzed with glucuronidase) is left with 100 nl an aqueous hydrazine solution (5 M, with HCl to pH 7 to brought) react 30 minutes at 37 ° C. The estradiol is determined according to the method previously described in Examples 1 and 2.

From the preceding description it follows that a method can be used regardless of the types of execution. enzymatic determination of estrogens E ₁ and E _{2 is} obtained, which has significant advantages compared to the previously known methods of determination, some of which have already been achieved

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mentioned, in particular:

- the process is very inexpensive in terms of components and materials, using only one Photometer in the near U.V. needed.

- You don't need radioisotopes

- The process can be easily automated and is carried out directly at im Commercially available automatic systems can be used (EECHNICON or LKB)

- Another advantage is the great stability of the reaction

CjUa non) components and enzymes (condition sine ^^ a commercialization on a large scale-.

- Bas procedure can be used in a very broad biological range can be applied

- There is no parasitic reaction

- The method has a very high specificity

- No enzymes are destroyed

- The process is simple, reliable and completely reproducible and very sensitive

- The method can be used in a large number of biological environments

- The method can be used in biological environments, such as urine, without first extracting the estrogens su must or in which one is relatively simple and uses rapid extraction or purification methods.

As can be seen from the above, the invention is not

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limited to the embodiments described in detail; Rather, it encompasses all variants that occur to the person skilled in the art on the basis of the description, without departing from the scope of FIG Invention to submit.

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Claims (1)

2908357 a tent claims . ! Procedure for enzytnati see determination of the estrogenic E and K _? , in biological media, in particular in plasma and in urine, characterized in that that one uses a 17 β -estradiol dehydrogenase from placenta that is freed from steroid and parasitic non-estrogen-dependent transhydrogenases, and that in the presence of NAD or an NAD analog which is recognized by the enzyme, of NADP or one of its analogs, as well as a specific enzyme NADP dehydrogenase and an NADP-forming substrate works. Method according to claim 1,
characterized, that the specific enzyme NADP dehydrogenase is selected from the following group: tetrahydrofolate dehydrogenase (international classification 1-5-1-13, 1-5-1-15), isocitrate dehydrogenase (international classification 1-1-1- ^ 2), Phosphoglucona t-dehydrogenase (international classification 1-1-1- hk} _} glucose-6 - ^T> phosphate-dehydrogenase (international classification 1-1-1-itO) »Lacta ldehyde hydrogenase (international classification 1-1-1-55) »Benzaldehyde dehydrogenase (international classification 1-2-1-?), Glyceraldehyde- ^ phosphate- ^ ehydrogenase (international classification 1-2-1-9 £ i -? -1-1 3 }) I.-Aspartate-semi-aldehyde-dehydrogenase ν internati onale Klassi fikafion 1 - "- 1-1i), Glyoxylat- ^ ehydrogenase \ international 909839/0736 BATH ORIGINAL - Tf- 2908357 Classification 1-2-1-17). 3. The method according to claims 1 and 2, characterized in that the UADP-forming substrate is G6P, if the specific Enzyme NADP dehydrogenase is G6PDH. 4. Process according to claims 1 to 3, characterized in that reaction that the determination takes place in a medium consisting of a buffer with a pH value close to 7.4, the ionic strength being equal to or less than that of a 0.1 molar buffer. 5. The method according to claim 4, characterized in that the buffer also 0.5 parts per thousand to 1 $ protein without essential Contains affinity for estrogens. 6. The method according to claim 5 »characterized in that the protein added to the medium is serum albumin, in particular Bovine serum albumin is. 7. The method according to claims 1 to 6, characterized in that the determination in the presence of NAD or one of its 909839/0735 - Ύ- It is carried out analogously, such as acetyl-NAD or thio-NAD. 8. The method according to claims 1 to 7, characterized in that that the NAD and the NADP-forming substrate are present in excess are. 9. The method according to claims 1 to 8, characterized in that that the amount of NAD in the reaction medium 1O "- ⁵ - up to 10" is -molar, 6-7 that of the NADP 1o - and the concentration ratio is up to 10 NAD NADP -molar to about 10 ^'5. 10. The method according to claim 1, characterized in that the elimination of the parasitic, non-estrogen-dependent transhydrogenase enzymes by heating the solution of Λ? Β -estradiol dehydrogenase, which contains a polyol, is effected, namely 100 to 150 minutes to a temperature of 6o to 7o ⁰ G in the presence of 4o to 6o % of this polyol, and by chromatography on DEAE cellulose. 11. The method according to claim 10, characterized in that the polyol consists of glycerol. 12. The method according to claims 10 and 11, characterized, 909839/0736 that the solution of 17/3 estradiol dehydrogenase containing the polyol also contains a small amount of diethylstilbestrol 13 method according to claim 1,
characterized, that the removal of steroids from the 17 P -estradiol dehydrogenase by washing the enzyme previously adsorbed on DEAE cellulose or the like with a Phosphate buffer is effected, the pH of which is in the vicinity of about 7.2 and that of about 15 to 25% of a polar solvent contains. 14 method according to claim 13,
characterized,
that the phosphate buffer contains about 20 $ ethanol. 15 process according to claims 1 to 14, characterized, that the reaction mixture, which the reaction components en
and which contains substance to be determined * is incubated to 38 ⁰ C at 25 prior to reading the optical density of 2 to 12 hours, preferably 3 to 9 hours, at a temperature of 20 to 40 ⁰ G, preferably. 16. The method according to claims 1 to 15, characterized in that 909839/0735 2308957 that the samples the biological media, which contain the estrogens E and E to be determined, before the determination 1 2 drive a preparation or extraction and / or purification process to be subjected " 17. The method according to claim 16,
characterized, that, if the biological containing the estrogens to be determined The milieu consists of plasma, which has previously been subjected to an extraction process known per se, e.g. with ether, whereupon you wash several times with the appropriate solvents, evaporate and the last evaporation residue in one Mixture of a polar solvent and a buffer, the ionic strength of which is practically the same as that of a 0.1 M buffer is dissolves, and which is a small amount of a protein with no significant affinity for estrogens, as well as $ 15 to $ 25 of a polyol. 18. The method according to claim 16,
characterized, that, if the biological medium containing the estrogens to be determined consists of urine, this is first subjected to a preparatory process with the aid of an enzyme which hydrolyzes the conjugated compounds, such as "P -glucuronidase" Pasteur. 909839 / 073S 19 · Ready-to- use combination for determining the estrogenic Έ * and E ₂ in urine by performing the method according to claims 1 to 18,
characterized, that it was determined and lyophilized essentially as follows Components contains: the enzyme 17 ß -estradiol dehydrogenase, which is freed from steroids and parasitic / non-estrogen-dependent transhydrogenases and preserved in a suitable polyol, in particular glycerol, an internal standard (which contains a known excess of estrogens),
a control (blanc), an enzyme for the hydrolysis of the conjugated compounds contained in the urine (such as "^ -glucuronidase" Pasteur), which was previously determined by puncture to the amount of the urine sample to be treated,
NAD: 10 ~ ² - 1O ~ ⁴ M, Glucose-6-PDH: 0.1 ΙΕ / ml of the sample to be determined, KADP: 10 ~ ⁶ - 10 ~ ⁸ M,
Glucose-6-P: 10 ~ ^2-10 ~ ⁴ M, Buffer SBA (bovine serum albumin): 0.5 parts per thousand to M> buffer! Dris-HCl 0.05 M, pH value about 7.5 20. Combination according to claim 19,
characterized,
that the control (blanc) from a mixture of buffer Tris- 909839/0735 * τ * HOL + 15 Ms 25% polyol + 0.5 per mil to 1j6 SBA (buffer A) and about 0.5 ml buffer A + 2 χ 10 ~ ⁵ M NAD, 2 χ 10 " ⁶ M NADP ', 2 χ 1O ~ ^ M glucose-6-phosphate, 0.2 IU glucose-6-phosphate dehydrogenase / ml (buffer B) ". 21. Combination according to claim 19 and 20, characterized in that that the internal standard from a mixture of the above buffer B and the buffer A, the 1600 picograms of estradiol / ml and o, o2 I.U. contains 17 Ρ -estradiol dehydrogenase. 22. New industrial product for the enzymatic determination of the estrogens E. and E "in biological media, namely that Enzyme 17 ß -estradiol dehydrogenase from placenta, which is produced by Steroids and parasitic non-estrogen-dependent transhydrogenases is exempt by applying the method according to claims 10 to 14. 9Ö9839 / 073S