United States Patent (19) 11 Patent Number: 4,791,057 Misaki Et Al

Home , Hydroxysteroid, Ketosteroid

United States Patent (19) 11 Patent Number: 4,791,057 Misaki et al. (45) Date of Patent: Dec. 13, 1988 (54) HIGHLY SENSITIVE ENZYMATIC ASSAY 57 ABSTRACT METHOD A highly sensitive quantitative assay method for a com 75 Inventors: Hideo Misaki; Shigeru Ueda, both of ponent which is 3,3-hydroxysteroid or 3-ketosteroid, in Shizuoka, Japan a specimen to be assayed, comprising causing this com ponent in the specimen to take part in the cycling reac 73) Assignee: Toyo Jozo Kabushiki Kaisha, Shizuoka, Japan tion (21) Appl. No.: 757,953 36-hydroxysteroid oxidase 22 Filed: Jul. 23, 1985 (30) Foreign Application Priority Data O2 H2O2 Jul. 23, 1984 (JP) Japan ...... 59-151438 3,3-hydroxy 3-ketosteroid 51 Int. Cl." ...... C12Q 1/26; C12O 1/32; steroid C12Q 1/28 NAD(P) reduced NAD(P) 52 U.S. Cl...... 435/26; 435/25; 435/28; 436/817 3,3-hydroxysteroid 58) Field of Search ...... 435/25, 26, 28, 192; dehydrogenase 436/817 and measuring a detectable change in the reaction sys (56) References Cited tem. There is thus provided a 36-hydroxysteroid - 3 U.S. PATENT DOCUMENTS ketosteroid cycling reaction using 3,3-hydroxysteroid oxidase, which consumes O2 and generates H2O2 and 4,230,797 10/1980 Boguslaski et al...... 435/7 3-ketosteroid, with a substrate of 36-hydroxysteroid, or OTHER PUBLICATIONS 3,3-hydroxysteroid dehydrogenase, which consumes reduced NAD(P) and generate NAD(P) and 36 Hurlock et al. (1956) Society of Experimental Biology hydroxysteroid, with a substrate of 3-ketosteroid. Ex and Medicine Proceedings, vol. 93, pp. 560-564. ample of specimens are specimens which contain 3 Yamagouchi et al. (1981) Analytical Letters, vol. 14, hydroxysteroid or 3-ketosteroid, or which liberate or No. B6, pp. 433-438. generate such a component. By proceeding at a rate of Dolin, 1957 Science, vol. 225, pp. 557-573. more than ten cycles per minute and measuring the Blaedel et al. (1978), Analytical Chemistry, vol. 50, No. amount of a detectable change in the reaction, the com 8, pp. 1026-1032. ponent in a specimen can easily and sensitively be de Primary Examiner-Robert J. Warden tected with good accuracy. Assistant Examiner-Jack Spiegel Attorney, Agent, or Firm-Young & Thompson 8 Claims, 3 Drawing Sheets U.S. Patent Dec. 13, 1988 Sheet 1 of 3 4,791,057

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l O O O2Z - O 5 CD Z, 3. O (?) to (i. m O O O S d NW O / WN Ot/9 LW S39)NWHO NOLOS 9W 4,791,057 1. 2 by heating under alkaline conditions, and the said alkali HIGHLY SENSTIVE ENZYMATIC ASSAY then be neutralized, are required. METHOD The concentration of steroid hormones in vital speci mens is generally low and so direct assay by spectro This invention relates to a quantitative assay method 5 photometry has been difficult. In the prior art of assay for a component in a specimen to be assayed, which methods, hydroxysteroid is treated with 3-hydroxys component can be 3,3-hydroxysteroid or 3-ketosteroid. teroid dehydrogenase and NAD to generate reduced More particularly the present invention relates to a NAD which is colorimetrically measured at 350 mm. highly sensitive enzymatic assay method which com But this method is of low sensitivity and hence trace prises reacting a component in the specimen with the 10 steroid horomones in serum, except cholesterol, cannot components of the cycling reaction be measured. We have now discovered a 3.3-hydroxysteroid-3- 3,3-hydroxysteroid I ketosteroid cycling reaction using 3.3-hydroxysteroid oxidase oxidase, which consumes O2 and generates H2O2 and O2 H2O2 15 3-ketosteroid, with a substrate of 3,3-hydroxysteroid, and furthermore 3,6-hydroxysteroid dehydrogenase, 3.3-hydroxy- 3-ketosteroid which consumes reduced NAD(P) and generates steroid NAD(P) and 36-hydroxysteroid, with a substrate of NAD(P) reduced NAD(P) 3-ketosteroid. 3,3-hydroxysteroid 20 These reagents can be obtained at low cost and good dehydrogenase quality, and moreover an effective cycling reaction can and measuring the amount of detectable change in the be achieved in spite of coexisting oxidatively active reaction system. H2O2 and reductively active reduced NAD. Further Enzymatic cycling assay methods such as NAD cy- 25 more, we have found that in the cycling reaction, 343 cling, NADP cycling or CoA cycling are known. For hydroxysteroid or 3-ketosteroid in a specimen is reacted example, alcohol dehydrogenase has been subjected to with the remainder of the other components in the cy reaction with ethanol as a substrate in the presence of cling reaction system consisting of 3,3-hydroxysteroid, NAD to form reduced NAD which is oxidized to NAD 3-ketosteroid, 36-hydroxysteroid oxidase, 3,3-hydrox by the action of malic dehydrogenase on a substrate of 30 ysteroid dehydrogenase, NAD(P), reduced NAD(P), oxalacetate to constitute an NAD-reduced NAD cy O2 or H2O2, and that, by proceeding at a rate of more cling reaction. Jap. Biochem. Soc. Ed. "Experimental than ten cycles per minute and measuring the amount of Methods in Biochemistry”, Vol. 5, "Research Methods in a detectable change in the reaction, the target compo Enzymology', p. 121-135, Tokyo Kagaku Dojin Pub nent in a specimen can easily and sensitively be detected lishing Co., August 1975, Mori, A. Ed. "Manual of 35 with good accuracy. Assay Methods in Neuro-transmittance', p. 165-172, An object of the present invention is to provide a Ishiyaku Publishing Co., November 1979. simple and highly sensitive enzymatic assay method, Another method is known as follows: without necessitating complicated pre-treatment as in In the above reaction, malic dehydrogenase is re known assay methods for 33-hydroxysteroid or 3-ketos placed by reduced NAD oxidase to constitute a cycling teroid in a specimen, by the use of a coenzyme cycling reaction wherein oxygen and reduced NAD are con reaction. sumed and H2O and NAD are generated Institute for These objects are achieved, according to the present Phys. and Chem. Sci. Ed.: “Present and Future of Life invention, by the method which we have discovered, Science', p. 30-32, K. K. Creative Life Sci. Res. Assn., which comprises reacting a component in the specimen March 1981). Hydroxysteroid is treated with hydroxys 45 with the components of the cycling reaction teroid dehydrogenase to reduce NAD to reduced NAD which is converted to NAD with the formation of for 3p3-hydroxysteroid I mazane by the action of a transferase such as diaphorase oxidase in the presence of a tetrazolium salt to constitute the O2 H2O2 cycling reaction Jap. Pat. Unexam. Publ. No. 50 56-144096). Glutathione and dehydroascorbate are treated with 3p3-hydroxy- 3-ketosteroid glutathione dehydroascorbate oxido-reductase, thereby steroid converting dehydroascorbate to ascorbate, which con NAD(P) reduced NAD(P) sumes oxygen and generates H2O and dehydroascor- 55 3,3-hydroxysteroid bate by the action of ascorbate oxidase to constitute a dehydrogenase cycling reaction. Jap. Pat. Unexam. Publ. No. 56-151498. NAD cycling wherein oxygen is consumed and measuring the amount of a detectable change in the and hydrogen peroxide is generated by using reduced reaction system. NAD oxidase is also known Jap. Pat. Unexam. Publ. Examples of specimens are specimens which contain No. 56-78599). 3,3-hydroxysteroid or 3-ketosteroid, or which liberate As mentioned hereinabove, several kinds of cycling or generate such a component. In the latter case, meta reaction systems have been reported, however, the one bolic systems and biosynthetic systems of various ste most frequently used, in which reduced NAD oxidase roid hormones, which are classified into, for example, generates peroxide, suffers from the difficulty of purifi- 65 androgens in male animals, estrogen or gestagen in cation, so that an accurate assay cannot be expected. female animals, and corticoid in both sexes, can be men Furthermore, in NAD cycling methods, complicated tioned. An enzymatic activity which characterizes these processes wherein excess NAD has to be decomposed systems, or the amount of 3,3-hydroxysteroid or 3 4,791,057 3 4. ketosteroid, can be determined. Examples thereof are as follows: 3,3-hydroxysteroid I 3,3-hydroxysteroid: oxidase (1) Androgen: O2 H2O2 pregnenolone, 17a-hydroxypregnenolone, dehydro- 5 epiandrosterone, androstene-3,3,1713-diol, androstane 3,3-hydroxy 3-ketosteroid 36,176-diol, A-androsterone-3p3,17a-diol, androstane steroid 3,3,116-diol-17-one. (2) Estrogen: ,36-hydroxysteroid reduced NAD(P) estradiol, estrone, estriol, equilin, equilenin, 10 dehydrogenase ethynylestradiol. 3-ketosteroid: In the reaction I), one mole of 3,3-hydroxysteroid is (1) Androgen: converted with the consumption of one mole of O2, progestreone, 17a-hydroxyprogesterone, testoster generally dissolved oxygen, to one mole of 3-ketos one, androstenedione. 15 teroid with the generation of one mole of H2O2 by the (2) Corticoid: action of 3,3-hydroxysteroid oxidase to constitute the corticosterone, cortisol, cortisone. 3p3-hydroxysteroid oxidase reaction system. Then one In an assay, 33-hydroxysteroid oxidase, 3,3-hydrox mole of 3-ketosteroid is further converted by the action ysteroid dehydrogenase and reduced NAD(P) are of 3,3-hydroxysteroid dehydrogenase in the presence of added to a specimen which contains any one of the one mole of reduced NAD(P) to one mole of 36 above components to be asssayed. hydroxysteroid with the generation of one mole of The concentration of the substrate, i.e. 33-hydroxys NAD(P), to constitute the 3,3-hydroxysteroid dehydro teroid or 3-ketosteroid, can optionally be adjusted to be genase reaction system. Furthermore, the thus 5 nM-30 M/5-20 ul. The reaction temperature can be generated 3,3-hydroxysteroid is converted to 3-ketos a temperature at which a smooth cycling reaction pro 25 teroid to constitute the 36-hydroxysteroid oxidase reac ceeds, and is preferably 37° C. The reaction time will tion system, thereby constituting the 3,3-hydroxys vary depending on the substrate concentration, the teroid-3-ketosteroid cycling reaction. The said cycling enzyme concentration and the reaction temperature, reaction I proceeds in the presence of the six compo and is usually more than one minute, preferably 2-10 nents, 36-hydroxysteroid, 3-ketosteroid, 36-hydroxys minutes. 30 teroid oxidase, 3,3-hydroxysteroid dehydrogenase, re An example of 36-hydroxysteroid dehydrogenease is duced NAD(P) and O2; however, 3,3-hydroxysteroid a commercially available product obtained from Pseu and 3-ketosteroid can be formed from each other by a domonas testeroni (Sigma Chem. Co., U.S.A.) The cycling reaction, and therefore either one thereof can be amount to be used is more than 0.05 unit/ml in one used as desired. reaction, preferably 2-4 units/ml. 35 Furthermore, in the cycling reaction I), H2O2 and An example of 33-hydroxysteroid oxidase is a com reduced NAD can be linked to form NAD, wherein one mercially available enzyme, generally known as choles mole of H2O2 and one mole of reduced NAD are con terol oxidase (EC 1.1.3.6), which is produced from a sumed to form two mols of H2O and one mole of NAD microorganism Streptococcus, Brevibacterium, Schizo by the action of NAD peroxidase (EC 1.11.1.1) J. Biol. phylum, Corynebacterium, Cellulomonas, Arthrobac Chem..., 225; 557, 1957 as shown by the following reac ter, Nocardia or Mycobacterium. The amount of the tion:

3p3-hydroxysteroid (II) oxidase NAD(P)peroxidase O2 H2O2 N / 3,3-hydroxy- 3-keto steroid / steroid f NAD(P) reduced NAD(P) N2H2O + NAD(P) 3,3-hydroxysteroid dehydrogenase

55 In cycling reaction II, H2O2 generated in cycling enzyme used is more than 0.2 unit/ml, preferably 10-30 reaction I) is consumed with reduced NAD(P) to form unit/ml. NAD(P), and so two moles of reduced NAD(P) are The amount of reduced NAD(P) to be added is gen consumed in one cycle of cycling reaction II), which erally a stoichiometric excess as compared with the provides twofold more rapid changes in the amount of substrate to be assayed, and is preferably 0.1-2.0 mM. 60 reduced NAD(P) and a more highly sensitive assay The above concentrations of substrate, enzyme and method. the other necessary component in the reaction system Alternatively, NAD(P) can be converted by another can optionally be varied and are not limited. dehydrogenase (herein called a second dehydrogenase) The cycling reaction I of the present invention can and its substrate, to reduced NAD(P), to constitute an be illustrated in the following reaction schema for 36- 65 NAD(P) cycling reaction consisting of NAD(P), the hydroxysteroid, 3-ketosteroid, 3.3-hydroxysteroid de second dehydrogenase and a substrate thereof. The said hydrogenase, 3,3-hydroxysteroid oxidase, NAD(P), NAD(P) cycling can be linked with cycling reaction I reduced NAD(P), O2 and H2O2. as illustrated by the following reaction: 4,791,057 5 6 excess of the amount of 36-hydroxysteroid, for example 3,3-hydroxysteroid (III) more than 50 times, preferably 100-10,000 times. The oxidase magnitude of the detectable change after the cycling O2 H2O2 reaction is completed can be determined by the amount 5 of consumed O2 or reduced NAD(P) or generated 3,3-hydroxy- 3-keto H2O2. As shown in cycling reaction II hereinbefore, steroid steroid the generated H2O2 and consumed reduced NAD(P) NAD(P) reduced NAD(P) can be linked for highly sensitive assaying. In this linked 36-hydroxy- 10 steroid reaction, NAD(P) peroxidase is used, in general, in an dehydrogenase amount more than 0.5 unit per one test, preferably 1-20 units. The magnitude of the detectable change can pref erably be determined by measuring the consumed re substrate for second oxidized 1 duced NAD(P). second dehydro- substrate 5 (b) Component in specimen; 3-ketosteroid: dehydrogenase genase The schema is illustrated in the following reaction: For example, a component in the specimen (shown as -) and a component which constitutes cycling reaction 20 3,3-hydroxy (Ib) (I) (shown as ) can be illustrated as follows: steroid dehydrogenase (a) Component in specimen; 36-hydroxysteroid: reduced NAD(P NAD(P) Ia 3,3-hydroxy- 25 o steroid -G-3-keto- 3,3-hydroxy oxidase H2O2 steroid steroid -e 3,3-hydroxy- 3-keto 4 3,3-hydroxy- Y.O2 steroid steroid steroid 30 oxidase NAD(P) reduced NAD(P 3,3-hydroxysteroid Any of 3,3-hydroxysteroid oxidase, 3,3-hydroxy dehydrogenase steroid dehydrogenase, O2 and reduced NAD(P) can be 35 the component which completes cycling reaction (Ib). In cycling reaction Ia), if the component in the speci In this reaction, 3-ketosteroid-313-hydroxysteroid cy men is 3,3-hydroxysteroid, then 3,3-hydroxysteroid oxi cling is characterized by consuming one mole of 3 dase, 36-hydroxysteroid dehydrogenase, O2 and re ketosteroid and reduced NAD(P) to generate one mole duced NAD(P) are used for the components which of NAD(P) and 33-hydroxysteroid, then by consuming constitute the cycling reaction. 36-hydroxysteroid oxi- 40 one mole of 36-hydroxysteroid and O2 to generate one dase is the substrate, which consumes one mole of 33 mole of H2O2 and 3-ketosteroid. O2 can be supplied by hydroxysteroid and O2 and generates one mole of H2O2 dissolved oxygen and so 36-hydroxysteroid oxidase, and 3-ketosteroid. Furthermore, 3,3-hydroxysteroid 343-hydroxysteroid dehydrogenase and reduced dehydrogenase acts on a substrate of 3-ketosteroid to 45 NAD(P) are simply added in stoichiometric excess. The consume one mole of 3-ketosteroid and reduced amount of 36-hydroxysteroid dehydrogenase and of NAD(P) and generate one mole of 36-hydroxysteroid 3,3-hydroxysteroid oxidase is generally more than 0.05 and NAD(P), thereby to complete the 36-hydroxys unit/ml for the former, and more than 0.2 unit/ml for teroid-3-ketosteroid cycling reaction. In the reaction, the latter, preferably 10-30 units/ml for 36-hydroxys O2 can be supplied by dissolved oxygen in the system 50 teroid dehydrogenase and 2-4 units/ml for 36-hydrox and so 3,3-hydroxysteroid oxidase, 36-hyroxysteroid ysteroid oxidase. The amount of reduced NAD(P) to be dehydrogenase and reduced NAD(P) should be sup used is determined by the product of the amount of plied in excess amounts as reagents for the assay. The 3-ketosteroid in the specimen times the number of cy amount of 36-hydroxysteroid in the specimen is not 55 cles, and is in general, in stoichiometric excess as com limited but will naturally be diluted if at high concentra pared with the amount of 3-ketosteroid, for example tion. more than 50 times, preferably 100-10,000 times. The The amount of 3,3-hydroxysteroid oxidase and 36 magnitude of the detectable change after completion of hydroxysteroid dehydrogenase depends on the amount the cycling reaction can be determined by consumed of 36-hydroxysteroid in the specimen and is usually 60 oxygen or reduced NAD(P) or generated H2O2. High more than 0.2 unit/ml per one test, preferably 10-30 sensitivity of assay can be achieved by using NAD(P) units/ml for 36-hydroxysteroid oxidase and more than peroxidase as hereinbefore illustrated. 0.05 unit/ml per one test, preferably 2-4 units/ml for Hereinafter, an assay method using a second dehy 3,3-hydroxysteroid dehydrogenase. The amount of re- 65 drogenase and its substrate as illustrated in reaction (III) duced NAD(P) can be more than the product of the hereinbefore will be explained. amount of 36-hydroxysteroid in the specimen times the The cycling reaction is illustrated by the following number of cycles, and is generally in stoichiometric reaction: 4,791,057 7 8

IIIa) reduced oxidized- - NAD(P) H2O2 substrate 36-hydroxy- 3,3-hydroxysteroid dehydrogenase steroid -Ge oxidase dehydrogenase substrate for second dehydrogenase NAD(P) 2 36-hydroxy. steroid

Any one of 3-ketosteroid or 33-hydroxysteroid dehy- -continC ued drogenase, 3,3-hydroxysteroid oxidase, O2, any one of D-galactose a d-D-galactose-y-lactone + reduced NAD NAD(P) or reduced NAD(P), the second dehydro genase and the substrate for the second dehydrogenase 20 in cycling reaction (Id), NAD in the specimen is are used as the components which complete the cycling converted to reduced NAD by the action of the second reaction IIIa. In this reaction, the second dehydro dehydrogenase in the presence of a substrate therefor to genase acts on NAD(P), and one mole of NAD(P) and take part in the NAD cycling reaction, then the thus the substrate for the second dehydrogenase are con generated reduced NAD participates in the same cy sumed to generate one mole of oxidative product and 25 cling reaction as in Ic hereinbefore. reduced NAD(P), then the reduced NAD(P) is used in The amount of reagents used in cycling reaction the 3-ketosteroid-313-hydroxysteroid cycling reaction. IIIa can be in stoichiometric excess of the amount of 3,3-hydroxysteroid dehydrogenase acts on the gener NAD(P) or reduced NAD(P). Furthermore, the said ated reduced NAD(P) and an equimolar ratio of 3 NAD(P) cycling reaction can be linked by the action of ketosteroid to generate one mole of NAD(P) and 343 30 NAD(P) peroxidase as illustrated in cycling reaction hydroxysteroid, and then one mole of 3,3-hydroxys II). teroid and O2 are consumed by the action of 3,3-hydrox Then any one of the components 3,3-hydroxysteroid ysteroid oxidase to form one mole of H2O2 and 3-ketos or 3-ketosteroid in the specimen or a specimen which teroid. In this reaction, O2 can be supplied from dis liberates or generates the said component is measured solved oxygen in the mixture, as also 3,3-hydroxysteroid 35 and the said component or specimen can be assayed oxidase, 36-hydroxysteroid dehydrogenase and based on the above cycling reaction Ia), Ib) or III), or NAD(P) or reduced NAD(P)), and the second dehy IIIa), or II). In the reaction, the number of cycles is drogenase and its substrate should be supplied as rea more than 10, and so the amount of reagents used is gents for assaying in excess amounts. The magnitude of preferably in molar excess of the number of cycles. the detectable change can be determined by measuring 40 Furthermore, trace amounts of specimen or diluted consumed O2 or generatd H2O2. specimen are advantageously used. The second dehydrogenase used in the cycling reac The reaction medium can be a stable pH buffer solu tion IIIa) can be an enzyme which catalyzes a reaction tion, generally weakly acidic to weakly alkaline. For on a substrate for the said dehydrogenase and NAD(P) example, a phosphate buffer of pH 6.5-8.5, a tris-HCl to form oxidative products of the said substrate and 45 buffer, an imidazole-HCl buffer, a dimethylglutarate reduced NAD(P), and is illustrated as follows: ...Y NaOH buffer or a PIPES-NaOH buffer can be used. The reaction proceeds generally at 37 C. for more than (a) Dehydrogenase; D-arabitol dehydrogenase (A Dase) one minute. (EC 1.1.1.11) and its substrate: D-arabitol: Cycling reaction proceeds usually at a rate of more D-arabitol - NAD - a Se D-xylose -- reduced NAD 50 than 50 cycles per minute, although this value varies depending on the amount of enzyme used or its Km (b) Lactate dehydrogenase (LDH) (EC 1.1.1.27) and L-lactate: value, and so the amount of enzyme and reagents can be provided preferably for more than 60 cycles/min. of L-lactate - NAD - Li spyruvate -- reduced NAD reaction. (c) Malate dehydrogenase (decarboxylating) (MDH) 55 Next, the magnitude of a detectable change in the (EC 1.1.1.28) and L-malate: reaction is measured after the reaction proceeds, and the said detectable change is the quantity of component, L-malate -- NAD MD Spyruvate - CO2 - reduced NAD which consumes or generates one molar ratio of the component, in one reaction cycle wherein one mole of (d) Glucose dehydrogenase (GDH) (EC 1.1.1.47) and glucose: 3,3-hydroxysteroid or 3-ketosteroid is consumed or 3-D-glucose - NAD on S generated during cycling reaction I). The preferred D-glucono-6-lactone + reduced NAD component to be measured is consumed O2 or reduced NAD(P), or generated H2O2. (e) Formate dehydrogenase (FDH) (EC 1.2.1.2) and formate: The amount of consumed O2 can usually be deter mined by the value of electro-chemical changes using formate + NAD FESCO2 + reduced NAD 65 an oxygen electrode. The amount of consumed reduced (f) Galactose dehydrogenase (Gal DH) NAD(P) can be determined by subtracting the amount (EC 1.1.1.48) and D-galactose: of reduced NAD(P) remaining at the end of the reac 4,791,057 9 10 tion from the initial amount of reduced NAD(P). Mea FIG. 5 is the quantitative assay curve for pregneno suring these amounts of reduced NAD(P) can be done lone, androstenedione, dehydroisoandrosterone and by various known assay methods. For example, the androstene-3,6,1743-diol; and specific absorbency of reduced NAD(P) and the non FIG. 6 is the quantitative assay curve for pregneno specific absorbency of NAD(P) can be measured. Since 5 lone. NAD(P) has its specific absorption maximum at 260 nm. and reduced NAD(P) at 260 nm and 340 nm, the absor EXAMPLE 1. bency at 320-360 nm, preferably at 340 nm is measured The following reaction mixture is used for assay: for assaying reduced NAD(P). 50 mM phosphate buffer (pH 7.5) Another assay method for reduced NAD(P) is a col 10 3,3-hydroxysteroid oxidase (Toyo Jozo Co., pro orimetric assay using an electron transport chromogen duced from Streptomyces): 20 units/ml having an electron acceptor from reduced NAD(P). 3,3-hydroxysteroid dehydrogenase (Sigma Chem. Examples of electron transport chromogens are tetrazo Co., produced from Pseudomonas teststeroni): 2 lium salts such as 3-(p-iodophenyl)-2-(p-nitrophenyl)-5- units/ml phenyl-2H-tetrazolium chloride, 3-(4,5-dimethyl-2- 1.5 mM reduced NAD (NADH2) thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 3,3'- 0.1% Triton X-100 (4,4'-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium The above reaction mixture (0.5 ml) was introduced chloride), 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis(2- into a small test tube and preincubated at 37 C. Speci (p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride mens (0 ul) containing pregnenolone (0, 5, 10, 15 and 20 (=nitro-tetrazolium: NTB), 3,3'-(3,3'-dimethoxy-4,4'bi 20 uM, respectively) were added thereto, and the mixture phenylene)-bis(2,5-bis(p-nitrophenyl)-2H-tetrazolium was incubated for exactly 10 minutes. The reaction was chloride, 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)- stopped by adding 2% sodium dodecyl sulfate (SDS) bis(2,5-diphenyl-2H-tetrazolium chloride), and 2,6- (2.5 ml). Changes of absorbency of the reaction mixture dichlorophenol-indophenol. A preferred example is a 25 were measured at 340 nm. combination of water-soluble tetrazolium salt and di The results are shown in FIG. 1 (O-o) with good aphorase or phenazinemethosulfate. These electron quantitativity. The cycling ratio in this example was 80 transport chromogens are electron acceptors for re cycles/min. Reduced NAD in the above reaction mix duced NAD(P) to form a colored formazane pigment, ture was replaced by reduced NADP, and the same and the thus formed pigment is colorimetrically mea 30 process as above was followed to obtain the good quan sured at the maximum absorption thereof such as titative results shown in FIG. 1 (A-A). Furthermore, 500-550 nm. NADH2-peroxidase (9 units/ml) was added to the reac A further assay method for reduced NAD(P) is fluo tion mixture being reduced NAD hereinabove, and the rometry wherein reduced NAD(P) is treated with di material was assayed as above. The results are shown in aphorase in the presence of a fluorescentreagent such as 35 FIG. 1 (o-o) with two times greater absorbency. resazulin. For assaying reduced NAD(P) in cycling reaction (I), N2O2 should preferably be decomposed EXAMPLE 2 and removed by catalase. NAD(P) peroxidase (EC Reaction mixture: 1.11.1.1) can be added to cycling reaction I for a dou 50 mM phosphate buffer (pH 8.0) bly sensitive assay as illustrated in reaction system II) 3,3-hydroxysteroid oxidase: 20 units/ml herinbefore. Generated H2O2 can be measured as an 36-hydroxysteroid dehydrogenase: 4 units/mi electro-chemical change by using a hydrogen peroxide 0.2 mM reduced NAD (NADH2) electrode, or as a detectable product by reacting with 0.1% Triton X-100 an indicator and H2O2. Examples of indicators are rea The above reaction mixture (1.0 ml) was introduced gents which can be measured by spectrophotometric 45 into a quartz cell (1.0 ml) and was set in a spectropho means, color indicators, fluorescent reagents or lumi tometer assembled with a constant temperature cell nescent reagents. holder at 37 C. The present invention is thus a novel assay method Specimens (20 ul) containing pregnenolone (0, 5, 10, for 36-hydroxysteroid or 3-ketosteroid in a specimen by 15, 20 and 25 M, respectively) were added thereto and means of a cycling reagent of 3,3-hydroxysteroid-3- 50 each mixture was incubated at 37 C. Changes of absor ketosteroid, substrate for a coupling of 3,3-hydroxys bency each 2 minutes from 2 to 4 minutes after starting teroid dehydrogenase and 3,3-hydroxysteroid oxidase, the reaction were measured at 340 nm. The results are at a rate of more than 10 cycles per minute, wherein a shown in FIG. 2 (o-o), in which displays good linear component, namely 3,3-hydroxysteroid or 3-ketosteroid 55 ity as to the amount of pregnenolone and absorbency. in a specimen such as serum or urine, is measured with The cycling ratio in this example was 65 cycles/min. good accuracy and high sensitivity of 100-1,000 ti Human serum, to which pregnenolone was added to mes/10 minutes as compared with known methods. the same concentration as above, was used for assay in The following examples illustrate the present inven the same way to obtain the results as shown in FIG. 2 tion but are not to be construed as limiting. 60 (O-O) with good quantitativity. The results of the examples are shown in the accom panying drawings, in which: EXAMPLE 3 FIGS. 1 and 2 are the quantitative assay curves for Solutions (10 pul) containing testosterone (0, 5, 10, 15 pregnenolone; and 20 M, respectively) were added to a reaction FIG. 3 is the quantitative assay curve for testoster 65 mixture (0.5 ml) of the same composition as in Example one; 1 and treated the same as in Example 1. The results are FIG. 4 is the quantitative assay curve for pregneno shown in FIG. 3, which shows that good quantitativity lone; was obtained in the case of testosterone. 4,791,057 11 12

EXAMPLE 4 3,3-hydroxysteroid oxidase The reaction mixture (1.0 ml) of Example 2 was intro duced into a reaction vessel assembled with a Galvani 5 O2 H2O2 type oxygen electrode and the mixture was prein- 3,3-hydroxy 3-ketosteroid cubated at 37 C. Solutions (10 ul) containing pregneno- steroid lone (0, 5, 10, 15 and 20 M, respectively) were added O NAD(P) reduced NAD(P) thereto and the mixtures were incubated at 37 C. for 10 3,3-hydroxysteroid minutes. Then oxygen consumption was measured. The dehydrogenase results are shown in FIG. 4 and prove the high sensitiv- and measuring an amount of a compound consumed ity of the present method. 15 or generated in said cycling reaction system, and deriving a quantitation for either 36-hydroxysteroid EXAMPLE 5 or 3-ketosteroid based on said measurement. - 2. An assay method according to claim 1, wherein Specimens (20 Jul) containing pregnenolone, andros- NAD peroxidase, which catalyzes a reaction consum terone, dehydroisoandrosterone or androstenedione- 20 I Oe E. so s of t S5 38,176-dione (0, 5, 10, 15, 20 and 254M, respectively) andis used generates to react two said moles Hoan of H2O said and reduced one moleo NAD(P) iny were added to the reaction mixture of Example 1, and said cycling reaction system and an amount of reduced the changes of absorbencys were measured after treat- 25 NADF)3. An assayconsumed method is measured. according to claimo 1, wherein ment the same as in Example 1. The results are shown in said NAD(P) generated in said cycling reaction system FIG. 5, wherein o-o: pregnenolone, o-o; androster- is subjected to a second cycling reaction contained one, A-A: dehydroisoandrosterone and A-A; andros- within said cycling reaction system, wherein a second s y O dehydrogenase and a substrate for said second dehydro terone-3,6,1713-diol. 30 genase are added to said cycling reaction system, said substrate and said generated NAD(P) reacting to oxi EXAMPLE 6 dize said substrate and reconvert said NAD(P) to re Human serum specimens were prepared by adding saidduced recoversion NAD(P), said of saidsecond generated dehydrogenase NAD(P). catalyzing pregnenolone at concentrations of 0, 5, 10, 15, 20 and 25 35 4. An assay method according to claim 1, wherein the M, respectively. The specimens (20 ul) were added to amount of O2 consumed in said cycling reaction system s is measured. the reaction mixture in Example 1, and treated as in 5. An assay method according to claim 1, wherein the Example 1 to measure pregnenolone. The results are amount of H2O2 generated in said cycling reaction sys 40 ten is measured. shown in FIG. 6 and demonstrate good recovery. 6. An assay method according to claim 1, wherein What is claimed is: said detectable change is the amount of consumed re 1. A quantitative enzymatic assay method for 36- duced NAD(P). 7. An assay method according to claim 1, wherein hydroxysteroid or 3-ketosteroid in a specimen to be 45 said 3.3-hydroxysteroid dehydrogenase is an enzyme assayed, comprising the steps of: produced by Pseudomonas teststeroni. 8. An assay method according to claim 1, wherein causing at least one component selected from the said 36-hydroxysteroid oxidase is an enzyme produced group consisting of 36-hydroxysteroid and 3-ketos- by a microorganism of genus selected from the group teroid, and contained in a specimen to be assayed, 50 consisting of Streptomyces, Brevibacterium, Schizo to take parti i ti foll phylum, Corynebacterium, Cellulomonas, Arthrobac o take part in a cycling reaction system as follows ter, Mycobacterium and Nocardia. sk is s sk