DE2020506A1 - Means and method for determining a dehydrogenase activity - Google Patents

Description

See / GKL M 2TO3

Anerican Hospital Supply Corporation, Evanston, 111. / USA Means and method for the determination of a dehydrogenase

activity

There has long been a need for a. Fast,. accurate and reproducible method for determining eonsentration of the six diagnostically important dehydrogenases in blood serum or in other body fluids, with one such a method should be carried out routinely, without that «doing an expensive UV system and a well-trained one β personnel are required.

The aim of the invention is to create a color reagent aur fast, precise and re-modifiable determination of a

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Dehydrogenase activity and thus a dehydrogenase concentration, where these two terms are directly related to each other Are connected, also falls within the scope of the invention the creation of methods for determining the activity of the diagnostically important dehydrogenases in blood serum and in other body fluids.

Another important feature of the invention is its creation of an earbreagense also see »that for! carrying out Methods can be used in which known amounts of dehydrogenase are used to remove unknown amounts of Blood sugar, serum glucose and blood alcohol concentrations To determine this, this is done by combining well-known Amounts of Ölucose Dehydrogenaee (in the case of a blood sugar test) » Olyoerin debrogenase (in the course of a serum glyceride barrier) and alcohol behydrogenase (in the case of: • in blood alcohol determination) in the reaction systems, and «was in the glucose is blood, the glycerine, which by saponification from serum triglyoerides "and the alcohol, which the substrates XOr the glucose dehydrogenase, glycerine dehydrogenaee bsw, forms alcohol dehydrogenaee.

The present invention therefore relates to a Parb reagent and a method of determining the activity of Enssyaen mir Transfer of hydrogen (dehydrogenase), with all of these Need Bneyme Ooensyn I or HAD as a hydrogen absorptor, namely sram purpose of qualitative measurement of an unknown in a body fluid »whereby this unknown can be one of the already described sehydrogenases or a substrate in a sample of a body fluid with which a dehydrogenaee reacts. In every case there is the tarbreagene from likotinamidademine dinucleotide (IAD) ml Hydrogen acceptor, phenasin methoeulfate as electron carrier

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and a color-forming Xetrazoliumsaiz, which with the reduced phenazismethOBulfate forming a distinctly colored formazane reacts. You depth the color of the generated Formazane is proportional to the activity of dehydrogenase, from which either the concentration of the dehydrogenase can be calculated, or from which the (Contains serum triglycerides, blood glucose or blood alcohol can be determined, depending on the respondents as well as depending according to the test carried out in each case.

The embodiment of the invention, which with the reagents and the method for determining the concentration of the diagnostically important dehydrogena in small samples contained in blood serum or other body fluids is described first. The other embodiment of the invention, which essentially uses the same method and color reagent for the purpose of determination the concentration of serum triglycerides, blood sugar and blood alcohol is explained below.

All six diagnostically important dehydrogenases, which have been described above, are to be brought to the common denominator that in the reaction sintering, when the dehydrogenase acts on the substrate, the oxidized form of coenzyme I (HAD) acts as a hydrogen acceptor, which then the electron to an electron carrier, namely phenazine methosulfate. Phenazine methosulfate in turn makes the electron available to a xetrazolium dye. The xetrazolium dye is quantitatively reduced to the formazan, which has a different color from the dye, the color of the formazane produced being proportional to the activity and concentration.

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- 4 tration of dehydrogenase in the body fluid.

The reactions that take place can be summarized as follows:

(Dehydrogenase

in the body »fluid test)

(1) Substrate + NAD <r> Oxidized substrate + RADH ₂

(2) NADHg + phenazine methosulfate (IMS) -> NAD + BiSH ₂ (5) EMSH ₂ + tetrazolium sala> PMS + formazan

The nicotinamide demine dinucleotide are most useful (NAD), the phenazine methosulfate (PMS) and the tetrazolium salt each component of a single comprehensive staining reagent. The amount of each of the three components of the comprehensive color reagent is not critical, provided that that in each case there is a noticeable excess in order to prevent the reaction with the dehydrogenase in the sample to bring the testing body fluid to an end.

A freeze-drying of the comprehensive color reagent conserved ' fourth, its activity for longer periods of time, although it should be noted that it is freeze-drying not essential to the practice of the present invention is. If a freeze-dried reagent is used, then such a color reagent must be used to carry out the Tests can be brought back to its original state. To preserve the color reagent, a stabilizer, such as gamma globulin, can be incorporated into the reagent formulation. The amount of this way added gamma globulin can be used within a wide range

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rich waver. It is to be pointed out that the exhaustion the stabilization is related to the amount of stabilizer in the formulation, but with excess Singeing, although they do not produce a noticeable increase in stabilization, does not detrimentally Influence heaktion. Satisfactory results could obtained with amounts of gamma globulin between 20 and 60 # with a preferred range between 40 and 50 j £, based on weight, is

When carrying out the method according to the invention, the freeze-dried reagent is returned to its original state with a measured amount of the corresponding buffered substrate. The enzyme activity in the sample of the body fluid with the dehydrogenase to be examined is determined in such a way that a measured amount of the body fluid is added to the comprehensive color reagent, which has been converted into its original state again with the corresponding substrate and to a temperature has been heated between about 25 and about 40 ^{β C.} The preferred temperature is 37 ° C. In any case, it is important that the selected temperature is maintained during the incubation time, the incubation time must be precisely determined * Depending on the dehydrogenase to be tested, the incubation time can generally be between 5 and 60 Minutes. If lower temperatures are observed, longer times are required, while higher temperatures cause a faster reaction.

As can be seen from the equations given above, take at the reaction, which catalyzes by the dehydrogenaee

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are, substrates tell, which are capable of being oxidized to become when using nicotinamide adademine dinucleotide (NAD) implemented in the presence of the corresponding dehydrogenase will. In a test to determine lactic acid dehydrogenase activity the substrate consists of an aqueous solution of a salt of lactic acid, while at the Performing a malic acid dehydrogenase Xest an aqueous Solution of a salt of malic acid is used. When performing a glutamic acid jtohydrogenaae test an aqueous solution of a salt of glutamic acid is used, etc. Each of the substrates to test the activity of the six diagnostically important dehydrogenases should be associated with one suitable alkaline buffer to a pH between about 7 and 9, the respective pH at a given test depends on the particular enzyme. Iris (hydroxymetbyl) aminomethane is preferred, but other alkaline buffers, such as sodium barbital, Trisodium oephate or the like can be used.

The color change in the Heaktionesyetem takes place when the tetrasolium dye is reduced to its Pormasan. Any suitable Xetrasolius dye included in the Is able to produce with reduced phenazine metbosulfate of foraazan to react with a color different from that of the dye can be used will. For example, blue p-nitrotetrazolium chloride, Iodonitrotetrazolium violet, thiaeolyl blue, blue m-nitrotetrazolium chloride or the like. Blue p-nitrotetraolium chloride has been found to be special proven effective

The method according to the invention is suitable for bedding

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the dehydrogenases in any body fluids, such as for example blood serum, spinal fluid, serosal Effusions or the like. The rate of reaction between the substrates and NAD caused by each of the six diagnostically important dehydrogenases are catalyzed, which are present in a sample of such a body fluid, can be determined in such a way that the depth of the color. which is determined by the reduction of the tetrazolium dyes is generated, in comparison to the depth of color achieved by reducing the same colorant if a standard is examined in parallel that has a known concentration of the dehydrogenase in question contains. Units of the dehydrogenase examined can be read from a constructed curve or from a Basic formazan standard test can be calculated, with yourself the unknown can be determined according to the following formula:

QD of sample X International

OD of the basic units = international single standards (Standard) units in the sample

Note: In the above formula is the unit value

a function of the respective dehydrogenase testo.

The following examples illustrate the invention, specifically with a view to determining the enzyme activity of a dehydrogenase in unknown samples of body fluids.

example 1

A comprehensive color reagent for the determination of dehydrogenases can using the following ingredients per test unit be prepared in the manner described below;

0 0 9 8 iU / U? 8th

Weight in ψψ:

Blue p-nitrotetrassolium

chloride 3.00

Nicotinamide adademine dinucleo-

Ud (MSl) 2.00

Phenassin methosulfate 0.03

Gamma Globulin (Bovine) 4.00

g of a crystalline bovine (gamma globulin) dissolved in water with stirring. This solution is filtered through a filter pad with suction. Then this will be Solution 1.6 g of the tetrazolium dye (preferably blue p-nitrotetrazolium) ssugesetzt, whereupon after stirring the ! solution is filtered again. 11g nicotinamide adademine dinucleotide are added. The solution, which is now made up to 1000 ml, is kept in a bottle wrapped in aluminum foil to protect it from exposure to light shield. Immediately before filling, 160 mg of phenazine methosulfate are added, whereupon the mixture is stirred. The solution is filled into amber vials at a rate of 2.2 ml per vial. Direct after filling the vials, they are placed in dry ice placed to freeze them before freeze drying.

The comprehensive reagent is then used for preservation freeze-dried of its activity for prolonged periods of time. Prepared in this way, the freeze-dried color reagent is stable at 4 - 8 ° C during Periods of more than 1 year, as can be determined by stability tests.

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Example 2

Buffered substrates for the determination of the enzyme activity of a any of the six diagnostically important dehydrogenases can can be produced as follows:

(a) Lactic acid DetLvdrogenaee (U) H)

Assembly of the buffered

Substrates,

for each test

Weight in mg

Idthiumlac did 6,000

Magnesium lactate 0.500

Triehydroiyaethylaminomethane,

3 times crystallized

(adjusted to a pH of 8.2) 6 * 0 ^ 0

(to) q-Hydroxybtttterflänre-ltehTdrogenaae (HHD)

Assembly of the buffered

Substrate,

for every seat

weight in mg

a-hydroxyl maternal lure 20,000

Triehydroxjeethylaminomethane,

3-faoh crystallized

(adjusted to a pH of 8.2) 6.050

(o) AT apple acid dehydrogenase (MD)

Teaming up the buffered

Substrates,

for everyone! Seat

Ordained in day Natriuaaalat 718? 0 Triehjdroaysaethylaiiinooethan,

3-faoh krietalllfliert

(• divided into tin · »pH τοη S ₉ 4} 6.050

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(d) glutaiaiiiBäur-Dehydrogenaee (SPH)

Composition dea buffered

Substrates,

for each test

Sodium glutamate
Adenosine triphosphate epridoxy! Phosphate

Xrishydroxy methylaminomethane, 3-faoh crystallized (adjusted to a pH of 7.3 and freeze-dried for reconversion to the original Condition with 1 ml water)

Weight in mg

9,400
1,400
0.002

6.050

(e) 8orpitol-Psh3rdrogenasa (SPH)

Composition of the buffered

Substrates,

feast for everyone

d ~ sorbitol

Partial hydroxymethylaminoethane, 3-faoh crystallized (adjusted to a pH of 8.0) weight in 1,000

6.050

(f) Alcohol-Petodrogenaa »(APH)

Pairing of the buffered

Substrate,

for everyone

Ethyl alcohol

Triflhydroaymethylaminoiaethane, 5-faoh crystallized (adjusted to a pH of 8.8) Qewioht in ag 100,000

6.050

£ 09884/142 *

Example 3

You quantitative examination of six diagnostically important Dehydrogenases can be broken down by the following method perform, using the comprehensive color reagent and one of the buffered substrates specified in Examples 1 and 2 be used. All substrates of Example 2, with the exception of the substrate for glutamic acid dehydrogenase, are located is in a liquid state ready for immediate use. The substrate for grlutamic acid dehydrogenase requires conversion to the original state using a predetermined amount (1 ml) water,

When performing each test, the freeze-dried color reagent is first returned to its original state with the appropriate amount of the buffered substrate solution. The color reagent and the buffer are mixed by shaking the container and then left to stand for 5 minutes with stirring. A measured amount of the Sarbreagenses and the buffered substrate solution (0,5 - 2,0 ml) is anschlieesend each χ in two 16 125 mm test tubes-pipetted with "unknown" and "blank ⁿ are marked Both test tubes are in a water bath. preheated at a temperature of 37 ° C for a period of 5 minutes, using a "To Contain" pipette 0.01-0.50 ml of the body fluid to be tested is added to the test tube marked "unknown" while 30 seconds later from 6.01 to 0.05 ml of distilled water to the test tube, the contents are supplied, which is referred to as "blank" of each tube are anschlieesend in the water bath at a temperature of 57 ⁰ C for a given period incubators.

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~ 12 -

beer, "for a period of time" necessary for each of the various dehydrogenases develops its activity. The incubation times for each of the major dehydrogenases are as follows: laotat dehydrogenase (IlDH) 5-20 minutes, α-hydroxybutyric acid dehydrogenase (HHD) 10 - 30 minutes, malic acid dehydrogenase (MDH) 10 to 30 minutes, glutamic acid dehydrogenase (GDH) 10-30 Minutes »Sorbitol Dehydrogenase (SDH) 10-30 minutes and Alcohol dehydrogenase (ADH) 10-30 minutes.

Subsequent to such an incubation are under Compliance with the above-described 30 second inter-. vallzeitplanes 2 - 5 ml of a 0, in-hydrochloric acid solution added to each tube, first the tube marked "unknown" and then the one marked "blank sample" labeled tube, whereupon the contents are mixed to stop the ensym reaction. 30 minutes after using the addition of the hydrochloric acid a 'spectrophotometer at a wavelength of 500 to 560 nm read the unknown sample against the blank sample, namely with a 100 ^ igen permeability or at an optical density of 0. The units of the examined dehydrogenase are then constructed by a Curve read off or from a dehydrogenase basic standard test calculated with the unknown. Such a basic standard consists of a stable freeze-dried one Mixture of a xetrazolium paraffin, Phenazine methosulfate and a stabilizing agent (such as e.g. gamma globulin). After its transfer to its original state, this standard represents a Solution with a known concentration of tetrazolium and, with appropriate treatment, provides a coloring

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intensity, which is a known level of dehydrogenase activity is proportional.

As mentioned before, another embodiment of the invention relates to a similar reaction system in which the Substrate contains the substance of unknown value, therefore the substrate consists of the body fluid sample to be tested, each such sample containing an unknown amount of glucose, serum triglyoers, blood alcohol, or the like.

The determination of the blood glucose according to the method of this embodiment consists in converting the glucose and nicotinamide amine dinucleotide (SAD) to form the reduced coenzyme I (NADH ₂ ) and d-gluconolactone in the presence of glucose dehydrogenase as a catalyst. The nicotinamide amine dinucleotide is a rope of a comprehensive color reagent which, as already mentioned, also contains phenazine methosulfate and a xetrazolium salt, which is able to react with reduced phenazine methosulfate to produce a formazane with a different color. The color reagent also contains a stabilizing agent, such as gamma globulin, and is preferably freeze-dried to enable extended storage.

To determine the glucose content of a blood sample, the comprehensive color reagent is first returned to its original state, preferably using a solution that contains a known amount of glucose dehydrogenate and an alkaline buffer.This solution is then mixed with a blood sample that contains an unknown amount of gluose . The reactants are at a temperature zwisohen 25 and 4O ⁰ C

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(preferably 37 ⁰ C) incubated for a period of 5-30 minutes. The glucose in the blood sample is influenced by the enzyme »which has the consequence that the coenzyme I (SAD) is reduced, whereby the phenazine methosulfate is reduced» which in turn reduces the tetrazolium salt to its 3? Omnazane. The intensity of the color reaction is proportional to the concentration of glucose in the blood sample. If the reading is carried out in parallel to a standard with a known glucose concentration, the concentration of glucose in the test sample can be calculated.

A similar test can be used to determine the concentration of Xriglyoerids can be performed in a serum sample. the Triglycerides are first saponified in a known manner, after which the liberated glycerin in glyceraldehyde in the presence of Glyoerin-Dehydrogenas® and the described comprehensive Color reagents is converted. The setrazolium salt of the comprehensive Color reagents are related to their formazan in the reaction reduced, which then takes place when the glycerol dehydrogenase to catalyze the conversion of glycerin to glyoeraldehyde is used.

The comprehensive color reagent is first returned to its original state, preferably with a solution containing glycerine dehydrogenase and an alkaline buffer. Such a solution is then mixed with a substrate containing glycerine, which has been produced by saponification of triglycerides in a sample of the serum to be tested.The reaction catalyzed by glycerine dehydrogenase is carried out according to a precise schedule, with the temperature constant is kept at a value between 25 and 40 ^ö 0. If the test is carried out in parallel with a standard with a known concentration of glycerin, then the concentrations can

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tration of the produced by the saponification of the serum triglycerides Glycerine and consequently the concentration of glycerides can be calculated in the original serum sample.

The comprehensive PaCbreagenß is used to determine blood alcohol first restored to its original state, and preferably with a solution called alcohol dehydrogenase and contains an alkaline buffer. Sine such Solution is mixed with a substrate, usually a serum, that contains alcohol. The reaction caused by the Alcohol dehydrogenase is catalyzed after one carried out on an exact schedule, maintaining the temperature at a constant level within the range of 25-4O ° G. If you run the test in parallel by using a standard with a known alcohol concentration, then the concentration of alcohol in the serum can be determined be calculated.

The following examples illustrate the application of the method as well as the comprehensive color reagent with a view to the determination of blood glucose, serum triglyceride and blood alcohol content:

Example 4

To carry out every best, the freeze-dried Color reagent first with a buffer solution (preferably a solution containing glucose dehydrogenase) in transferred to its original state. The mix will ditched. After a suitable interval, a measured amount of the color reagent as well as the buffer solution is added (0.5 - 2.0 ml) each in two 16 χ 125 mm test tubes pipetted marked with "unknown" or "blank sample"

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are. Both test tubes are preheated in a water bath (22 - 37 ° 0) for a period of 5 minutes. Using an "Io Contain" pipette, 0.01-0.50 ml of the body fluid to be tested is added to the test tube marked "unknown", whereupon 30 seconds later O _f O1 -0.50 ml of distilled water is added to the test tube marked "Blank." The contents of the test tubes are then incubated in the water bath (22-37 ° C) for a certain period of time, namely for a period of time that is necessary for the glucose dehydrogenase to completely remove the glucose in the body fluid oxidized.

Following the incubation, at the same 30 second interval schedule, 2-5 ml of 0, hydrochloric acid are added to each test tube, first to the test tube marked "unknown" and then to the test tube marked "Blank". Fifty minutes after the addition of the acid, the unknown relative to the blank sample is read using a spectrophotometer at a wavelength of 500-60 µm, with a transmittance of 100 f * or an optical density of 0. The amount of glucose in the body fluid is then read from a constructed curve or calculated from a glucose basic standard test with the unknown.

Example 5

To carry out the test, the triglycerides are in one Aliquot of a serum first saponified in a known manner. A Rope of the saponified serum and a rope of the nioht-saponified Serums are processed as follows:

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To carry out each seat, the freeze-dried ParbreagenB is first converted to its original state with a buffer solution, a solution which contains glycerol dehydrogenaee preferably being used. The mixture is left to stand. After a suitable interval, a measured amount of the Sarbreagent and the buffer solution (0.5-2.0 ml) is pipetted into each of three 16 χ 125 mm test tubes labeled "unknown test", "unknown blank ^R and" reagent blank "are marked All test tubes are in a water bath.. - preheated (22 37 ⁰ C) for a period of 5 minutes using a" to Contain "pipette 0.01 - 0.50 ml of the saponified serum tested the is to be added to the test tube marked with "unknown test". 30 seconds later, 0.01-0.50 ml of the untreated serum is added to the test tube that is marked with "unknown blank", while 30 seconds later 0, 01 - Add 0.50 ml of distilled water to the test tube labeled "Reagent Blank". The contents of the test tubes are then incubated in the water bath (22 - Y ( ⁰ O) for a certain period of time, namely for a period of time that is necessary for the glycerin dehydrogenase to completely oxidize the glycerol in the body fluid.

Following such an incubation, 2 - 5 ml of 0.1 »hydrochloric acid are added to each reagent glass (first to the tube labeled" unknown test ", then to the tube labeled" unknown blank sample ¹¹ "and then to the tube labeled" unknown blank sample "11) in compliance with the same 30 second interval schedule aneohlieeeend the reagent tube marked "reagent blank"), whereupon the contents are mixed

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after adding the acid is made using a »spectrophotometer at a wavelength of 500 - 560 nm the unknown read against the blank at 100% transmittance or at an optical density of 0. The amount of triglyceride glycerin in the body fluid is then read from a constructed curve or from a ßlyoerin basic standard experiment with the unknown calculated.

Example 6

At the office of each festival, the freeze-dried color reagent is first converted to its original state using a buffer solution, a solution containing alcohol sehydrogenase being preferably used. The mixture is left to stand. At an appropriate time interval, a measured amount of the color reagent and buffer solution (0.5-2.0 ml) is pipetted into each of two 16 × 125 mm test tubes marked "unknown" and "blank". Both test tubes are preheated in a water bath (22-57 ^° C.) for a period of 5 minutes. Using a "So Contain" pipette, 0.01-0.50 ml of the body fluid to be tested is added to the test tube marked "unknown", while 30 seconds later 0.01-0.50 ml of distilled water is added to the test tube Test tube labeled "Blank" must be given. The contents of the test tubes are then incubated in the water bath (22 - 37 ⁰ O) for a certain period of time, namely during a period of time that is necessary for the alcohol hydrogenase to completely oxidize the alcohol in the body fluid.

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Subsequent to this incubation are observing the 30 second interval schedule described above 2 - 5 ml of an O, In-hydrochloric acid in each Beagensglas added, and «was first designated as" unknown " Test tube and then the test tube marked with "blank sample", whereupon the contents are mixed. 30 minutes after the addition of the acid, the unknown is compared using a spectrophotometer at 500-560 nm read the blank sample at a transmittance of 100 56 or an optical density of 0. The The amount of alcohol in the body fluid is then read from a constructed curve or from an alcohol standard test calculated with the unknown.

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Claims (20)

- 20 claims / Iy Comprehensive color reagent for the quantitative determination of a Enzyme activity evidences a selected dehydrogenase and a selected substrate which is produced by the dehydrogenase in In the presence of nicotlnasddademine dinucleotide can, characterized by a mixture of ifikotinamidademine dinucleotide, Phenazlnmethosulfat and a color-forming tetrazolium salt, the one with reduced phena κ .tn metho sulfate with extraction of a formazane is able to react with a color different from that of the salt. 2. Color reagent according to claim 1, characterized in that it also contains gamma globules as a stabilizing agent. 3. Color reagent according to claim 1, characterized in that the mixture is freeze-dried. 4. Color reagent according to claim 1, characterized in that it indicates the activity of a dehydrogenase which is derived from milk » acid dehydrogenase, a-hydroxybutyric acid dehydrogenase, apple Acid Uehydrogenaee, Glutamine Acid Dehydrogonaee, Sorbitol * - There is dehydrogenase or alcohol dehydrogenase. 5. Color reagent according to claim 1, characterized in that it is able to indicate the enzyme activity between a substrate of known composition and a dehydrogenase of unknown amount in a sample of a body fluid, the dehydrogenase being able to oxidize the selected substrate in the presence of nicotinamidademine dinucleotide . 00988WU28 ■ - 21 - 6. Color reagent after. Claim 5, characterized in »that the coloring tetrazolium salt of blue p-Nltrotetrazolium-. Chloride, iodonitrotetrazolium violet, thiazolyl blue or blue m-ffitrotetrazoliumchlorld consists. 7. larb reagent according to claim 6, characterized in that the color-forming tetrazolium salt of blue p-ffitrotetrazolium chloride consists· 8. Color reagent according to claim. 5, characterized _t that it is able to display the activity of a dehydrogenase with a buffered aqueous substrate solution, the subsolution being stepped from a sodium lactate, a-hydroxybutyric acid, ffodium oalate, There is sodium glutamate, d-sorbitol or ethyl alcohol solution. 9. Parbreagens according to claim t, characterized in that there is the enzymatic activity between the aforementioned dehydrogenase and able to indicate to a subseat that from a sample a body fluid that contains an unknown amount of a substance made up of a serum triglyceride, glucose or an alcohol. 10. A method for the quantitative measurement of the reaction between a selected dehydrogenase and a selected substrate which is capable of being oxidized by the dehydrogenase in the presence of hikotinamidademine dinucleotide, characterized in that the dehydrogenase and an aqueous solution of the substrate with a color reagent are mixed, which consists of a mixture of Hikotlnamidademin-Dinulcleotld, Phenaainnethosulfat and a color forming Xetrazollumsalz "· which refer to the lag is to react a formazan with the reduced Phenazinmethoeulfat to produce, in the 003884714 its color is different from that of the salt, and the mixture is kept at a uniform temperature between about 25 and 40 ⁹ C! for a period of about 10-60 minutes to develop a visible color with a depth proportional to the intensity of the reaction, is incubated. 11. The method according to claim 10, characterized in that also the depth of the color to determine the intensity the leaktlon is measured. 12. The method according to claim 10, characterized in that the dehydrogenase used consists of lactic acid dehydrogenase, while the substrate used is a buffered aqueous solution of an alkali or alkali metal salt of lactic acid. 13. The method according to claim 10, characterized in that the dehydrogenase used from α-hydroxybutanoic acid sehydrogenase and the substrate used consists of α-Ifydroxybtttteräure consists. 14. The method according to claim 10, characterized in that the dehydrogenase used is malic acid dehydrogenaae, while I am adding the substrate used from an aqueous solution of Batriunoalat. 15. The method according to claim 10, characterized in that the dehydrogenase used from glutamic acid dehydrogenae « consists, while the substrate used consists of an aqueous solution of sodium glutaoate susasnensetst « §09884 / 1428 16. Procedure after. Claim 10, characterized in that the dehydrogenase used is sorbitol dehydrogenase while the substrate used is composed of an aqueous solution of d-sorbitol. 17. The method according to claim 10, characterized in that the dehydrogenase used is an alcohol dehydrogenase, while the substrate is ethyl alcohol. 18. The method according to claim 10, characterized in that the dehydrogenase used consists of crlucose behydrogenase, while the substrate used is a sample of a body fluid that contains an unknown concentration of d-Glucoae. 19 · Method according to claim 10, characterized in that the dehydrogenase used consists of lycerol dehydrogenase, while the substrate is a sample of a liquid that contains an unknown concentration of glycerin, which has accumulated during the saponification of serum triglyoerides. 20. The method according to claim 10, characterized in that the dehydrogenase used is an alcohol dehydrogenase, while the substrate is a sample of a liquid that contains an unknown concentration of alcohol. 009884 / U28