SE441931B - Process and agent for determination of pyro-grape acid or pyruvates - Google Patents

Abstract

Process and agent for determination of pyro-grape acid or pyruvates which include pyruvate oxidase produced from pyruvate oxidase producing micro-organisms especially Aerococcus viridans IFO-12219 or IFO-12317 and with an optimal pH of 6.3-8.5. The agent includes FAD, in addition to pyruvate oxidase, thiamine pyrophosphate, phosphate and ion-releasing salt, chosen from the group which consists of calcium ions, cobalt ions, magnesium ions and manganese ions. Measurement is carried out on the amount of component used as oxygen or a produced component such as hydrogen peroxide.<IMAGE>

Description

8405647-2 composition of agents for pyruvate analysis and an analytical procedure for pyruvic acid. Furthermore, it has been found that the addition of salt of the kind which releases calcium ions, cobalt ions, magnesium ions or manganese ions to said reaction system results in an improvement of the assay. Addition of any color-producing or fluorescent indicator to the reaction system results in a suitable and excellent assay composition resp. analysis.

The invention furthermore relates to a means for carrying out the new method. The invention is explained in more detail in connection with the accompanying drawings which show the following. Pig. 1 an optimal pH value of pyruvate oxidase. Pig. 2 thermal stability of pyruvate oxidase. Pig. 3 pH stability of pyruvate oxidase. Pig. 4 the result of an analysis of pyruvic acid by means of an oxygen electrode and the use of pyruvate oxidase. Pig, the result of an analysis of serum by means of an oxygen electrode and the use of pyruvate oxidase. Pig. 6 the result of an analysis of pyruvic acid by colometric method using pyruvate oxidase. Pig. 7 results of a quantitative analysis of serum pyruvic acid and the use of pyruvate oxidase. Pig. 8 results of an analysis of ADP and the use of pyruvate oxidase.

Pig. 9 results of an analysis of glycerol, triglyceride and serum triglyceride using pyruvate oxidase. Pig. The result of an assay of GPT and GOT activity using pyruvate oxidase.

Pig. ll a correlate diagram of an analysis of GPT activity using pyruvate oxidase. Pig. 12 is a correlate diagram of an assay of GOT activity using pyruvate oxidase. Pyruvate oxidase according to the invention has a catalytic effect on an oxidizing reaction with pyruvic acid, inorganic phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide, and is preferably produced by culturing pyruvate oxidase-producing microbial species. 0867, Streptococcus sp B-0668, Aerococcus viridans IFO 12219 or IFO 12317.

The above-mentioned species B-0667 and B-0668 have the following taxonomic characteristics.

A. Observations in different media, grown at 30 ° C for 2 days: Strain B-0867 Strain B-0668 Tryptosoja broth tryptosoja agar slice tryptosoja agar plate gelatin slice gelatin slice) ßcP-mj 61k (ln dvznl weak plant, homogeneous green woolly precipitate weak plant, pale yellow-gray, no gloss, no emergence of soluble pigment colony formation; small and smooth plant along chopped line no gelatin melting plant along chopped line no gelatin melting no change weak plant, homogeneously cloudy, later woolly precipitation weak plant, pale yellow-gray no gloss, no emergence of soluble pigment colony formation, small and smooth plant along chopped line plant along chopped line no gelatin melting no change B. Microscopic observation candle Strain B-0667 Strain B-0668 Shape size rërlizhet szorer Éramfirgniog syxifzsf ffrg fn: spherical, egg-like, pairwise, tetral-like or short chains 0.5 - l, Û x 1.5 - 1.0 p - spherical, egg-like, paired, tetral-like or short chains 0.8 - 1.0 X 1,? -1.2 u 9 8405647: 2 C. Physiological properties: Strain B ~ Û667 - Strain B-0658 Plant temperature; us ° c - - a7 ° c ao ° c 2s ° c 1o ° cs ° c halogen-tol- or (+) or (+) I + + + + + rans: NaCl 10% 6.5% 5.0% 1.0 % 0% OF-test fermentative fermentative + +++++ + behavior in oxygen optional anaerobic optional anaerobic _ nitrate reduction - indonformation - - hydrogen sulphate formation - - gelatin hydrolysis - starch hydrolysis - 'food hydrolysis + + acetoin formation - MR-oxidase' - - ureaus (SSR) - urease (Christensen) - utilizing citric acid (Christensen) - - acid formation of §sugar: U ïadonitol - Ü i 1 L (+) - arabinos cellobios dulcitol meso-erythritol fructose fucous galactose glucose glycerol inositol insulin lactose maltose mannitol mannos melisitos melibios raffinos L (+) - ramnos salícin L-sorbos sorbitol starch sucrose trehalose xylose tolerance at 60 ° C for 30 min 8405647-2 .8405647r2 As shown in "Bergey's Manual of Determinative Bacteriology, 8th Edition" "and" ST Cowan and KJ Steel, Manual for the Identification of Medical Bacteria, Cambridge Press, l97H ", are B-0667 and B-0668 - having the abovementioned taxonomic characteristics, in particular Gram-positive cooks, with respect to catalase and oxidase negative, fermentative acid formation of glucose and no sugar gasification (glucose) - Mentioned in this literature as belonging to the genus Pediococcus and Streptococcus.

A comparison of these relatives with the identification manual 'as above shows the following.

In the table, + = means more than 85% positive, and - = more than 85% negative and d = varies among genera and species.

Species species _ genus Pedio- genus Strsp- B-0667 B-0668 _ cooks tococci growth at 4500 “- - + d tolerance at 'ä 60 ° C for 30 min - + _ d glycerol (sy- § rabildning) _ - _ _ d arabinos (syrabiia- fl i fl š) - - + d salt tolerance 7 (Nasa 110%) + _ _ 4. _ In the following, the species B-0667 will thus be referred to as the genus Pediococcus or Streptococcus. As can be seen from the above-mentioned "Manual for the Identification of Medical Bacteria" and "J Gen Microbiol, 26, 185 - 197, 1961", the taxonomic characteristics of species B-0667 are almost identical to those of Pediococcus urina-equí, but they in the properties described in "Bergey's Manual of Detaminative Bacteriology, ed. l97H" are somewhat: equal, s4oss47f2 with them. For this reason, the species B-0667 will be referred to as the genus Pediococcer and will be referred to as Pedíococcus sp B-0667.

The genus B-0668 is more similar to the genus Streptococcus than the genus Pedíococcus. Furthermore, the "Manual for the Identification of Medical Bacteria" states that the species B-0668 is similar to Streptococcus faecium var durans, but no taxonomic characteristics have been described in the "Bergey's Manual", so that a detailed comparison is impossible. However, the species B-0668 is referred to as Streptococcus sp B-0668.

Species B-0667 and B-0668 have been deposited for permanent collection in the "Institute for Microbial Industry and Technology, Agency of Industrial Science and Technology, MITI, Japan" under Deposit Nos. FERM-P No 4H38 and FERM-P No 4439 .

In one embodiment of the invention, the above-mentioned Pediococcus sp B-0567, Streptococcus sp B-0668, the per se known Aerococcus viridans IFO-12210 or Aerococcus viridans IFO ~ l23177 are grown in a customary medium for enzyme production. The cultivation can be carried out by means of ordinary liquid cultivation, whereby underwater aeration is suitable for industrial production.

A conventional medium for microorganisms can be suitably used. As carbon sources can serve assimilable carbon sources such as glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid or the like. Assimilable nitrogen sources such as peptone, meat extract, yeast extract, casein hydrolyzate or the like may be used. Various inorganic salts such as phosphate, carbonate, sulfate, salts of magnesium, calcium, sodium, iron, manganese or zinc can be used.

The culture temperature can be selected in the range of microbial cell growth and pyruvate oxidase production and is suitably 25-3700. The culture time can be varied depending on the conditions and terminated when the production of pyruvate oxidase is essentially complete and is usually between 18 and H8 hours.

Pyruvate oxidase is present in cells of microorganisms.

To separate pyruvate oxidase from the cultured cells, the cultured mass is filtered or centrifuged to collect the cells, and burst by mechanical treatment or enzymatically such as with lysozyme. If necessary, pyruvate oxidase is treated by adding ethylenediaminetetraacetic acid (EDTA) and surface treatment agents such as TRITGN K-IgG ° 'or ASEC TSL 30-LQT' to separate the enzyme. The solution of pyruvate oxidase thus obtained can be concentrated if necessary, after which the enzyme is precipitated by salting out by adding soluble salt such as ammonium sulphate or sodium chloride. Low molecular weight contaminants are removed by dialysis. Additional impurities are removed from the pyruvate oxidase solution suitably by chromatographic absorption, ion exchange chromatography or gel filtration. The enzyme solution thus obtained is treated with vacuum concentration and lyophilization to produce pyruvate oxidase powder. Further purification can be accomplished by conventional protein and enzyme purification procedures such as absorption chromatography, ion exchange chromatography or gel filtration.

Pyruvate oxidase prepared according to the invention has the following physicochemical properties, abbreviations being used as follows.

Pediococcus sp B-0567 - B-0667 Streptococcus sp B-0668 - B-0868 Aerococcus viridans IFO-12219 - IFO-12219 Aerococcus viridans IPO-12317 - IFO-12317 (l) Enzyme activity: The enzyme catalyzes the oxidative reaction by renal tartaric acid, inorganic phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide.

CH 3 COCOOH + HOPO 3 + O 2 -9 CH 3 COOPO 3 + CO 2 + H 2 O 2 (2) Optimal pH - The effect of pH on pyruvate oxidase activity is measured.

Phosphate buffer solutions with pH 6-8 are used for testing. The results are shown in Fig. 1 where the optimum pH is as follows.

B-0667 pH 6.3 - 7.5 B-0668 pH 7.5 - 8.5 IFO-12219 pH 7.0 - 8.0 IFO-12317 pH 6.8 - 7.5 A slight change is observed by phosphate concentration and some kind of metal ions. 9 8405647-2 (3) Thermal stability: Enzymatic thermal stability is determined by incubation in 10 mM phosphate buffer (pH 6.5), containing 10 pH FAD at 0, H0, 50, 60 and 70c for 10 minutes according to the method of the enzyme test. As shown in Fig. 2, the enzyme obtained from B-0667, IFO-12219 and IFO-12317 is activated at UOOC and denatured at a temperature higher than 6000. The enzyme obtained from B-0668 is not activated at ROOC and denatured close; at over 6000. (Ä) Stability of the pH value: To each 3ç enzyme solution (0.1 m1) is added 0.2 M phosphate buffer tube pa 6 - 8 (Q, 9 m1) or 0.2 M: rice-Hcl buffer for pa 7-9 (0.9 ml), each solution containing 10 pM FAD; the solutions are allowed to stand for 10 minutes at HOOC. 20 μl of the enzyme solutions are taken out, and the enzyme activity is determined. As shown in Fig. 3, the enzyme from B-0667, IFO-12219 and IFO-12317 is substantially stable at about pH 7 and the a1 B-O668 is stable at a value for acid reaction. (5) Effect of different substances: I 1) The effect of different substances on the enzyme activity is tested by adding 5 mM of the following substance to the test system instead of MgCl2.

Added relative activity ($), ““ b ”t” “5 B-0667 B-0668 Iro-12219 I Iro-12317 No additive 25, ü 72.0 ü2.0 50.3 EDTA ooo 0 ngclz -1oo 1oo 100 100 c c1 ~ 9 2 69, Å 75.0 83, u 78.7 MHCl 2 129.1 102.7 116.2 111.0 coc12 81.3 81.1 85.0 8h, o Baclz 20.6 58.8 23 .9 23.3 2nCl2 16.0 38.2 1h.9 22.8 - EDIA and activated by Mg *, Ca, Hn ** and Co *. As stated above, the enzyme is: the effect is abolished by + ++ 4 2) The effect of the elimination of the following substance from the test system on the enzyme activity is shown below. 0.1 M dimethylglutarate NaOH buffer is used in phosphate elimination.

Eliminated relative activity ($) * “b $ t °“ s B-0667 B-0668 Iro-12219: Fo-12317 No elimination 100 '100 100 100' thiamine pyrophosphate O 0 OO FAD 33.9 f 100 32 It is apparent from the above that the enzyme requires thiamine pyrophosphate and FAD as cofactor and phosphate as substrate.

Furthermore, oxygen consumption and enzyme reaction have been measured by means of an oxygen electrode, whereby oxygen is consumed according to the enzyme activity (formation of hydrogen peroxide). The results are shown in the following Oxygen consumption reaction product (p mol / min) (p mol / min) H 2 O 2 acetyl phosphate B-0667 0.0h2 0.0h2 I 0.038 B-0668 0.022 0.0213 '0.020: Fo-12219 o, oho . 0.038 0.037: Fo-12317 _- o, c35 0.036 'o, o3h Investigations hn Perform by means of the following aids: 8405647-2 ll Oxygen consumption: Measured instrument for dissolved oxygen (YSI-dissolved oxygen-Meter, Model 531) acetyl phosphate: "F Lipmann et al Biol Chem, 134, 463-u64, 19.19 Hydrogen peroxide: method using N, N-dimethylaniline, 4-aminoantipyrine and horse radish peroxide As explained above, the enzyme obtained from the above four species of bacteria is referred to as pyruvate oxidase and flavin protein.

The test method for pyruvate oxidase according to the present invention is as follows. 0.5 M potassium pyruvate 0.1 ml 0.5 M phosphate buffer (pH 7.0) 0.2 ml 0.2% 4-aminoantipyrine 0.1 ml 0.2% N, N-dimethylaniline 0.2 ml 0 2 M Mgclz so μl 10 mM thiamine pyrophosphate 20 μl peroxidase (45 U / ml) 0.1 ml 1 mM PAD 10 μl distilled water 0.22 ml The above reaction mixture is preincubated at 37 ° C for 3 min.

To this solution is added the enzyme solution (20 μl) and preincubated at 37 ° C for 10 minutes. 0.1 M citrate buffer (ph 6.0, Zml), containing 0.1 M EDTA is added to quench the reaction. The violet color formed is measured by colorimetry at 565 nm.

One unit (1 U) of enzyme activity is defined as the activity that produces 1 pmol of hydrogen peroxide / min.

To activate the pyruvate oxidase reaction system, FAD, thiamine pyrophosphate and phosphate are added. Furthermore, for activating the enzyme, ion-releasing salt is suitably added which releases calcium ions, cobalt ions, magnesium ions or manganese ions in the form of chloride. An indicator such as a color indicator or a fluorescence indicator for hydrogen peroxide can be selected according to suitability.

The amount and ratio of the constituents of the enzyme reaction can be selected for substantial enzyme reaction and can be varied according to the amount of pyruvate, the temperature and the duration of the enzyme reaction.

For example, between 1 and 20 μl of pyruvate oxidase, 0.1 - 20 n moles of FAD, 0.05 - 0.5 μmol of thiamine pyrophosphate, 1 - 10 μmol of inorganic phosphate and 0.05 - 10 .mu.l moles of ion-releasing salt per test can be used. with advantage.

Fyruvate oxidase can be microcapsulated or be in the form of inert forms with organic or inorganic carriers or adsorbed by Teachers. The Mo] ratio of hydrogen peroxide indicators is at least equimolar or an excess amount of hydrogen peroxide generated. In the case of peroxidase, 045 - 20 U per test is suitably used.

These components of the enzymatic reaction mixture are preferably used in solution with suitably adjusted pH of the buffer.

The enzymatic reaction system thus prepared is used for the analysis of pyruvic acid. All such samples containing pyruvate can be analyzed. For example, pyruvic acid itself, pyruvic acid in serum or urine and pyruvic acid which form enzyme reaction systems such as lactic acid and lactate dehydrogenase (LDG), ADP and pyruvate kinase, glycerol glycerol kinase and pyruvate kinase may be mentioned. Further detailed examples of enzymatic reactions which form pyruvic acid and test object are as follows. Examination of lactic acid or LGD activity: LDH 7 lactate -ñÃñ - 7 pyruvate + NADH2 _ (2) Examination of ADP or pyruvate kinase (PK) activity: PK ADP + phosphoenolpyruvate ----- > ATP + pyruvate (3) Examination of glutamate-pyruvate transaminase (GTP) ~ activity or ketoglutarate: alanine + vt-ketoglutarate gg: -1) pyruvate + glutamate (H) Examination of glutamate-oxaloacetate-transaminase (GOT) - activity: GOT aspartate + GQ-ketoglutarate ----- α-oxaloacetate + glutamate Oxaloacetate oxaloacetate decarbooxilase> pyruvate + CO 2 (5) Examination of glycerol or glycerol phosphokinase (GK) activity: glycerol + ATP --Eg - aglycerol-3 ADP ADP + phosphoenolpyruvate --Eg-get pyruvate + ATP (6) Investigation of triglyceride: triglyceride lipase or linonrote1nl1nas; glycerol_3_phosphate + ADP ADP + phosphoenolpyruvate --Eg - e> pyruvate + creatine or ATPin (7) ): creatine -Ešâëïšgàâ -> - creatine creatine + ATP --Egg - 9 creatine phosphate + ADP ADP + phosphoenolpyruvate --few- ~ +> pyruvate + A TP (8) Examination of myokinase: ATP + AMF 2 ADP ADP + phosphoenolpyruvate --25 - <> pyruvate + ATP (9) Examination of fatty acid or thiokinase activity: fatty acid + CoA + ATP -ÉÉ9EiEÉâ:> aoyl-CoA + AMF + PPi AMP + ATP 2 ADP ADP + phosphoenolpyruvate --EE - ¿> pyruvate + ATP 1a 84Û5647 "2 These enzyme reactions are mentioned by way of example only; the pyruvate-forming reactions can be carried out by means of different combinations of enzymes and their substrates, for example biological samples. As exemplified above, the assay can be applied not only to pyruvate assays, but also to enzyme, enzyme activity or substrate assays.

The test is performed by incubating the sample with the reagent mixture. This is suitably a composition of necessary reagents. Measuring the amount of oxygen consumed with the help of a meter for dissolved oxygen is suitable for the examination method. In this case, no hydrogen peroxide indicator is necessary. To examine the component produced, it is convenient to measure the amount of hydrogen peroxide, for example by means of a hydrogen peroxide electrode meter such as "IST" oxidase meter or colorimetric or fluoremetric with a hydrogen peroxide indicator. The test can be conveniently carried out for 10-60 minutes at 20 ° C.

An indicator of hydrogen peroxide is a combination of one or more chromo- or fluorescence generators provided by coupling with the hydrogen peroxide source. An example of such indicators is a combination of tetravalent titanium compounds and xylenol-orange which are coupled with hydrogen peroxide to form a stable red color, or a combination of phenol or N, N-dimethylaniline or homovanilic acid, 4-aminoantipyrine and peroxidase to measure color or fluorescence. 4-Aminoantipyrine can be replaced by 4-aminophenazone.

A combination of 2,6-dichlorophenol, indophenol and peroxidase and of guaiacol and peroxidase may also be used. The indicator can be prepared in advance as a solution. Color or fluorometric tests can be performed by measuring the absorbance at a wavelength such as 565 nm.

The amount of pyruvic acid can be measured by calculating from the corresponding standard curves for spent oxygen or hydrogen peroxide generated.

Phosphate as spent component or acetyl phosphate as generated component can likewise be examined by any suitable method.

As explained above, a composition of reagents can be used for analysis, especially diagnostic analysis, including pyruvate oxidase and its application to various assay methods. More specifically, and as described above: diagnostic analysis As analysis of pyruva: in this reagent or in serum or urine, examination of enzyme activity of LDH, pyruvate kinase, GPT, GOT, glycerol kinase, lipase, lipoprotein lipase, creatinine phosphokinase, myokinase or thiokinase, and analysis of biological components such as lactate, ADP, glycerol, triglyceride, creatinine or fatty acid can be advantageously performed by the composition and method of the invention.

The following examples explain the embodiments of the invention.

Example 1 A medium (each medium 100 ml, pH 7) comprises glucose (low), yeast extract (0.5%) NaCl (0.2%), KHZPOH (0.1%) KZHPOR (0.1%), MgSO (0.05%) and CaCO 3 (0.3%) and sterilized in an Erlenmeyer flask at 120 ° C for 20 minutes. In each medium, bacteria of the genus Pediococcus sp B-0667 FERM-P no HMBB, Streptococcus sp B-0668 FERM-P no 4H39, Aerococcus viridans IPO-12219 or Aerococcus viridans IFO-123 17 are inoculated and grown under protection at 30 °. C below ZH h at 300 rpm. The cultured cells are then collected by centrifugation and washed with 10 mM phosphate buffer (pH 6.5) and centrifuged again to collect the bacterial cells. The cells thus obtained are suspended in the 10 mM buffer (10 ml, pH 7.0), containing 0.02% lysozyme and 0.1% TRITON X-100, and incubated at 37 ° C for 60 minutes. The product obtained by centrifugation is collected. It contains pyruvate oxidase. The existing enzyme activity of the product is shown in the following table. related enzyme activity B-0667 0.60 B-0668 0.38 IFO-12219 0.52 IFO-12317 0, u6 Example 2 A medium (20 l) consisting of the same components as described in Example 1 is sterilized by steam in a 30 L fermentation vessels.

Cultured broth (200 ml) Pediococcus sp B-0667 FERM-P No. 4038, grown in the same manner as in Example 1, is added and the whole is now grown at 30 ° C for 24 hours. Bacterial cells collected by centrifugation (approximately 100 g) suspended in the lysozyme solution (0.2 mg / ml, U 1), in addition add TRITON X-100 (Hg), EDTA (Eg) and 1 M phosphate buffer (pH 6.5, # 0 ml) . The whole is stirred at 37 ° for 60 minutes. To the product then obtained by centrifugation is added ammonium sulphate, and the precipitate is collected by centrifugation at 0.54 - 0.73 degree of saturation. Further, acetone (0.3 vol, cold) is added, and the precipitate, which is collected by centrifugation, is dissolved in 10 mM phosphate buffer (pH 6.5, 70 ml) (4750 U, recovery 79.2%).

To the solution is added ammonium sulfate, and the precipitate is collected at 0.5U - ÛJO saturation by centrifugation.

After dissolving the precipitate in 10 mM phosphate buffer (pH 6.5), the solution is filled into a column of SEPHADEf® (6 x 70 cm), and the fraction showing absorbance at 280 nm is collected. The active fractions are combined and lyophilized to obtain pyruvate oxidase in powder form (39H0 U, 758 mg, recovery 65.7%).

Example 3 After dissolving the precipitate in 10 mM phosphate buffer (pH 6.5), the solution is filled into a column of SEPHADE (6 x 70 cm), and the fraction showing absorbance at 280 nm is collected.

The active fractions are combined and lyophilized to obtain pyruvate oxidase in powder form (3940 U, 758 mg, recovery 65.7%).

Example 3 A composition for pyruvate analysis (using oxygen electrode): pyruvate oxidase prepared according to Example 2 (same as in the following examples) 300 U FAD - 0.5 pmol thiamine pyrophosphate 10 pmol MnCl 2 25 pmol 0.2M phosphate buffet (pH 7, 5) 1.0 ml sucrose 0.5 g 0.2M dimethylgbxane fi rNaOH buffer (pH 7.5) 5 ml The above mixture is lyophilized to prepare the pyruvic acid test composition (for oxygen electrode measurement, 50 tests).

Example 4 A composition for pyruvate analysis (for colorimetric examination): mouse-z Reagent (I) pyruvate oxidase g 200 U FAD '0.5 pmol thiamine pyrophosphate 10 pmol 0.2 M phosphate buffer (pH 7.5) 1.0 ml sucrose 0.5 g 0.2 M dimethylglutarate NaOH buffer (pH 7.5) 0.5 ml peroxidase (100 U9mg, horseradish peroxidase) 2.5 mg 0.3% H-aminoantipyrine 5 ml The above mixture is lyophilized to prepare reagent (I) for the composition for pyruvate analysis (for colorimetry, 50 tests).

An additional reagent (II) consists of 0.2% aqueous N, N-dimethylaniline, containing 25 pmol MnCl 2 (50 ml) and breaking reagent (III) consisting of 0.1 M citrate buffer (pH 6.0, 100 ml), containing 0.1 M EDTA is added.

Example 5 A composition according to Example 3 is dissolved in distilled water (50 ml) and a sample amount of solution (1.0 ml) thereof is introduced into a reaction vessel. To this is added 5.0 mM pyruvate solution (each 0-100 μl), or human serum 50 μl and 5.0 mM pyruvate solution (each and an additional 0-100 μl), and the solution is incubated at 37 ° C, after which the oxygen consumption is measured by a galvanic acid electrode. The results are presented in Fig. 4.

Further, a composition according to Example 3 is dissolved in distilled water (25 ml) and an aliquot of solution (0.5 ml) thereof is introduced into a reaction vessel, after which it is incubated with the addition of aqueous solution (0.5 ml) containing human serum (0-0 , 5 ml) at 37 ° C. Fig. 5 shows the result of the examination of the oxygen consumption, measured by means of galvanic-oxygen electrode.

As shown in Figs. 4 and 5, there are good linear conditions.

Example 6 The lyophilized reagent (I) of Example U is dissolved by adding reagent (II) (50 ml), and each aliquot of solution (1 ml) in small travel tubes is incubated at s1 ° c. Harrili sans s m1 potassium pyruvate solution (each 0-50 μl), and human serum (50 μl) are added with 5 mM potassium pyruvate solution (each C-SC μl) and incubated at 37 ° C for 10 hours. my. Breaking solution (2 ml) is added, after which the absorbance is measured at 555 nm. As shown in Fig. 6, there are good linear conditions, quantitative results in the studies shown above and also when this result coincides with the calibration curve for hydrogen peroxide (2.5 mM HQO2, o-sn P1). Additional human serum samples (0-0.5 ml) are introduced into small test tubes and adjusted to 0.5 ml by adding distilled water. Each solution (1.0 ml) of reagent (I) with the addition of reagent (II) according to Example 4 is added and incubated at 37 ° C for 10 minutes. The reaction is stopped by adding breaking reagent (III) (1.5 ml) and examined colorimetrically at 565 nm. As shown in Fig. 7, a good quantitative result is obtained.

Example gel 7 'Reaction mixture: 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 0.2 ml 10 mM MnCl 2 50 μl 0.2% N, N-dimethylaniline 0.2 ml 0.3% 4-aminoantipyrine , 1 ml peroxidase (45 U / ml) 0.1 ml 10 mM thiamine pyrophosphate 20 μl 0.2 M phosphate buffer (pH 7.5) 25 μl 20 mM phosphoenolpyruvate 0.1 ml pyruvate kinase (4000 U / ml) 5 ml s mm The above reaction mixture is adjusted to 1.0 ml by the addition of distilled water, preincubated at 37 ° C, then the solution of pyruvate oxidase (200 U / ml, 20 μl) is added, and incubated at 37 ° C for 10 minutes. my. After stopping the reaction by adding 0.1 m of citrate buffer (pH 6.0, 2.0 ml), the absorbance is measured at 565 nm. As shown in Fig. 8, good quantitative results are obtained in the ADP test by determining hydrogen peroxide which occurs in the reaction mixture of ADP, pyruvate kinase, phosphoenolpyruvate and others.

According to Fig. 8, there is good linearity if 2.5 mN hydrogen peroxide is used instead of 5 mM ADP.

B-406647r2 Example 8 Triglyceride test composition: Reagent (I): 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 10 ml 0.3% H-aminoantipyrine S ml peroxidase (100 U / mg) 2, 5 mg thiamine pyrophosphate 10, umo1 0.2 M phosphate buffer (pH 7.5) 1.25 ml phosphoenolpyruvate 100 pmol pyruvate kinase (4000 U / ml) 0.1 ml pyruvate oxidase (200 U / ml) 2 ml lipoprotein lipase (3000 U / ml ml) 0.5 ml glycerol phosphokinase (300 U / ml) 1.0 ml ATP 100 pmol The above reagent is lyophilized.

Reagent (II): 25 pmol MnCl 2 in 0.2% dimethylaniline 50 ml Reagent (III) (breaking solution): 0.1 M E1 in 0.1 M citrate buffer (pH 6.0) 100 ml Example 9 Reagent (I) according to Example 8 is dissolved in reagent (II) and each sample amount of solution (1.0 ml) thereof is filled into the test tubes.

Each sample amount (0-50 μl) of human serum, containing 1.02 pmol / nl triglyceride, 5 mM glycerol solution, 4.2 mM triolein in 0.1% TRITON X-10 solution or 2.5 mM hydrogen peroxide solution is added and incubated at 37 ° C for 10 minutes. As can be seen from Fig. 9, there is good linearity.

Example 10 Composition for testing serum transaminase for 100 tests: (1) Composition for GTP test (glutamate-pyruvate transaminase) (for 100 tests): Reagent (I): lyophilized reagent, consisting of the following: pyruvate oxidase 000 U FAD 630 nmol thiaminepyrcphosphate Lšßlurol 19 8405647-2 L-alanine »20 mnç; W-ketogluaraf: sal suhros 1 μH H-aminoantipyrine 150 pmol peroxidase N50 U 0.2 M phosphate buffer (pH 7.5) 2.5 ml 0.2 M targeteryiglurarar-Naon (pH 7.5) 'so m1 Reagent (II ) (100 ml used for reagent (I): H2 pmol MnCl2 in 0.2% dimethylaniline solution 210 ml Reagent (III) (breaking solution) 200 ml for reagent (I), 2.0 ml for 1 test: '0, 1 M EDTA in 0.2 M Citrate Buffer (pH 5.0) H 2 O ml (2) composition for GOT test (glutamate oxaloacetate transaminase) (for 100 tests): Reagent (I) lyophilized reagent, consisting of the following: pyruvate oxidase H00 U FAD 500 nmol thiamine pyrophosphate 150 pmol L-aspartic acid 20 mmol Mechetoglutarate 1 mmol oxaloacetate decarboxylase 200 U sucrose - lg H-aminoantipyrine l50, pmol peroxidase H50 U 0.2 M phosphate buffer (pH 7.5) 2.5 ml 0.2 M dimethyl glutarate NaOH buffer (pH 7.5) 30 ml Reagent (II) and reagent (III): same as above (1).

Example II The reagents (I) according to Example 10, (1) (for GPT activity test) and (2) (for GOT activity test) are each dissolved by adding reagent (II) (100 ml). Each sample (1.0 ml) of the solution is introduced into small test tubes and pre-incubated at 37 ° C for 5 minutes (pre-incubated reagent solution (I): Standard serum solution (20 μl) (MAXITOL, Calbichem Co, containing GPT 700 KU / ml and GOT 1000 KU / ml), diluted in a constant ratio, are added and incubated at 37 ° C for 10 minutes The reaction is stopped by adding breaking reagent (III) (2.0 ml) and the absorbance is measured at 565 nM. ...___...______._...-.._._., _._ ß4o5647f2 N As shown in fig.10, the two enzyme activities show linearity all the way up to the optical density of about 1.0 (enzyme activity: approximately 750 KU / ml).

Example 12 V To each pre-incubated solution of reagent (I), obtained in Example 11, is added 20 μl of human serum (US sample) and incubated at 37 ° C for 20 minutes. Breaking reagent (III) (0.2 ml) is added and the absorbance is measured at 565 nm.

The same sample of human serum is examined by the LKB method (UV absorption method, GOT examination composition and GPT examination composition, manufactured by LKB Corp) and correlated with the GPT and GOT activity.

Fig. 11 shows the correlation coefficient: r = 0.966 and the regression equation: y = 0.00295 x '+ 0.0032 for the GPT study.

Fig. 12 shows the correlation coefficient: r = 0.966 and the regression equation: y = 0.00287 x + 0.0l80.

Claims (3)

8405647-2 21 P A T E N T K R A V Method for analytical determination of pyruvic acid or pyruvate in a sample containing pyruvic acid or pyruvate liberating system, characterized in that the sample is reacted in a reaction system consisting of pyruvate oxidase with an optimum pH of at least 6,3 8.5, FAD, thiamine pyrophosphate, phosphate and ion-releasing salt, selected from the group consisting of calcium ion, cobalt ion, magnesium ion and manganese ion and then the amount of a consumed component such as oxygen or a generated component such as hydrogen oxide is measured. A method according to claim 1, characterized in that the pyruvate releasing system comprises releasing pyruvate by means of an enzyme reaction. 3. A process according to claim 1, characterized in that the amount of hydrogen peroxide is measured with an indicator of hydrogen peroxide, suitably consisting of peroxidase, 4-aminoantipyrine and phenol or N, N-dimethylaniline or homovanilic acid. U. Means for carrying out the process according to claim 1, characterized in that it consists of pyruvate oxidase, prepared by culturing the pyruvate oxidase-producing microorganisms Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317 in a nutrient medium and forming oxidized pyruvate. isolated from crushed, separated cells, flavin adenine dinucleotide (FAD), thiamine pyrophosphate, phosphate and a salt that releases calcium ions, cobalt ions, magnesium ions or manganese ions and preferably an indicator of hydrogen peroxide.