Test system and procedure for the determination of nad(p)h

A TEST SYSTEM AND PROCEDURE FOR THE DETERMINATION OF NAD(P)H

Abstract

The invention relates to a test system having an extended range of measurement and an appropriate procedure 5 for the determination of NAD(P)H or of substrates or enzymes which react to form or consume NAD(P)H in fluids. The test system contains, at one and the same time, several substances acting independently of one another as electron acceptors with respect to NAD(P)H and having different 10 electrochemical potentials. Addition of the test system to the sample solution gives rise to different end-products which can be analytically differentiated and which are evaluated by a technique of measurement or visually.

A Test System and Procedure for the Determination of NAD(P)H

The invention relates to a test system and a pro- cedure having an extended range of measurement for the determination of NAD(P)H or of substrates or enzymes which react with the formation or consumption of NAD(P)H.

In the majority of cases, the determination of clinical parameters is carried out using highly specific dehydrogenase reactions during the course of which NAD(P)H is indirectly or directly formed or consumed. The amount, absolute or per time unit, of NAD<P>H reacted thereby is a measure of the concentration of the substance to be investigated in a fluid. The dehydrogenase reaction has proved to be especially advantageous in respect of both stoichiometry and low susceptibility to interference. On the other hand, it is a disadvantage that, due to the absorption properties of the coenzyme molecule, evaluation of the reaction for measurement is only possible using photo- meters with a UV measurement range; purely visual evaluation cannot be carried out. The latter is only made possible by coupling the actual reaction which forms NAD (P)H with a colour reaction. A large number of procedures which achieve this by direct transfer, or transfer using electron transfer agents, such as phenazine methosulphate or diaphorase, of the redox equivalents to a variety of redox indicators have been described. The latter substances include, for example, cytochromes, complexed or chelated iron ions, di ch lorophenoL-indophenoI, tetrazolium salts and the Like.

Coupling the reaction for measurement with a reaction sequence is also known (German Aus Leges chrift 1 ,598,263, European Patent Specification 54,146), the formation of the dyestuff taking place via a substance formed as an intermediate. A sequential course of reactions, in which each product from the first part reaction is the starting substance for the second and so on, is common to all these systems.

A procedure is described in Patent Application

P 32 11 167 in which a substance to be determined is reacted to give a variety of products which can be differentiated so that, compared with the conventional tests, a wider range of measurement with the same accuracy of measurement is obtained. This is achieved by using several enzyme systerns which, independently of one another, react with the same substance to be determined, one system being a NADdependent dehydrogenase and another being a NAD-independent dehydrogenase. The only disadvantage of this procedure is that the NAD-independent enzymes necessary for a major proportion of the substances to be investigated in clinical diagnostics are not commercially available, in contrast to the corresponding NAD-dependent enzymes.

The invention has the object of developing a test system for the determination of NAD(P)H, which has a much larger range of measurement compared with the conventional systems and which can be carried out with enzymes which can be obtained commercially at any time.

Surprisingly, it has been found that, under certain conditions, independent reaction of NAD(P)H with a variety of redox indicators is possible. In this context, independent denotes that, for example, the reaction of the second redox indicator (having the lower electrochemical potential) only takes place after the reaction of the first (having the higher electrochemical potential) is Largely complete, so that separate evaluation is possible. This was all the more surprising since it was to be expected that, when the redox indicators are present at one and the same time, interference will occur due to the interaction between the individual substances and due to outside effects (oxygen, test substances).

Accordingly, the invention relates to a test system having an extended range of measurement for the determination of NAD(P)H, or of substrates or enzymes which react with the formation or consumption of NAD(P)H, which is characterised in that it contains, at one and the same time, several substances which act, independently of one another, as electron acceptors with respect to NAD(P)H and which have different electrochemical potentials.

The invention also relates to procedures for the determination of NAD(P)H, or of substrates or enzymes which react with the formation or consumption of NAD(P)H, in aqueous solution, which are characterised in that the sample solution is treated, at one and the same time, with several substances which act, independently of one another, as electron-acceptors with respect to NAD(P)H and which have different electrochemical potentials, these giving rise to different end products which can be analytically differentiated and which are evaluated by a technique of measurement or visually.

Depending on the type of redox indicator, the transfer of the electrons can take place directly or using a so-called electron transfer agent. The test system preferably contains electron transfer agents which exert a catalytic effect on the redox indicators. Examples of suitable electron transfer־agents are phenazine methosulphate (PMS), phenazine ethosulphate (PES), methoxyphenazine methosulphate (MPMS), Meldola's blue, diaphorase and the like, and mixtures of these substances.

In principle, suitable redox indicators are all substances which have a higher electrochemical potential than NAD(P)H/NAD(P), and the oxidised and reduced forms of which can unambiguously be differentiated visually, by photometry or using electrochemical procedures. Examples of suitable redox indicators for the test system according to the invention are oxazine and thiazine dyestuffs, tetrazolium salts and the like (German Patent Specification 2,834,743, Proc. Soc. Exp. Biol. 104, 407 (1960)), preferably di chlorophenol-indophenoL (DIP), 3-(4-iodophenyL)-2(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) and

3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The use of these redox indicators in dehydrogenase test systems has hitherto been restricted to systems having only one redox indicator. Surprisingly, it has now been possible to show that test systems having an extended range of measurement can be made available when several redox indicators having different standard potentials are present. The standard potentials of the redox indicators should be between -0.3 and +0.5, in particular between -0.3 and +0.3. As a rule, the determination is carried out in buffered aqueous solutions, it being necessary for the pH to be between 4.5 and 8.0, preferably between 5.0 and 7.0. Examples of buffers suitable for setting up these pH values are phosphate buffers, citrate buffers, 2-Nmorpho I ו no)ethanesuLphonic acid sodium salt, bis (2-hydroxyethyl)ami notris(hydroxymethyl)methane, pi peraz וne-N,N bis(2-ethanesuLphonic acid) dipotassium salt and the Like, preferably phosphate buffers and citrate buffers.

The selection of the redox indicators used for the test system according to the invention takes place according to the possibility of differentiating between the measurement signals produced on reaction, preferably according to the change in extinction at different wave-lengths (photometric evaluation) or according to the change in colour of the test solution which can be differentiated (visual evaluation). In the latter case, combinations of redox indicators, one form of each of which is colourless or only weakly coloured, are especially suitable. Some preferred redox indicators and their normal potentials at a pH of 7 and the appropriate colour changes are listed below.

<td> Substance</td><td> Potential at pH 7.0</td><td> Colour of the oxidised form</td><td> Colour of the reduced form</td>

<td> NADH</td><td>.32</td><td></td><td></td>

<td> DIP</td><td> +0.23</td><td> blue</td><td> co Lou r les s</td>

<td> MTT</td><td> +0.11</td><td> colourless</td><td> blue</td>

<td> INT</td><td> +0.09</td><td> colourless</td><td> red</td>

<td> Thionine</td><td> +0.06</td><td> violet</td><td> colourless</td>

<td> Methylene</td><td> +0.01</td><td> blue</td><td> co Lour Less</td>

<td> b lue</td><td></td><td></td><td></td>

It is clear from the table that the combination of redox indicators DIP/INT, which has a colour transition from blue to colourless in the first stage and from colourless to red in the second stage, is particularly advantag6 eous. The two substances show marked differences in their absorption spectra and in the visual colour impressions both between the oxidised and reduced forms of each substance and between the substances themselves. Reaction of the two substances mixed with NAD(P)H Leads to independent reaction of DIP and INT and thus to a range of measurement which is considerably extended compared with each system containing only one of these substances. The substance having the higher normal potential (DIP) reacts almost completely before the second substance (INT) reacts. In a determination of NAD(P)H using DIP as the only redox indicat or, the concentration range covered is up to about 0.26 mmoL/L, with INT the range is up to about 0.13 mmol/l, and with a combination of DIP and INT, the range is up to about 0.55 mmol/l, that is to say that, using the test system according to the invention, the range of concentration of NAD(P)H covered is increased by from double to more than 4 times. The photometric evaluation can be carried out, for example, by recording the extinction at two different waveLengths (460 and 650 nm). On a visual evaluation, a further increase in the colour stages obtained can be produced by adding a so-called background dyestuff (for example titani urn ye L Low).

On combination of the redox indicators INT and thionine, as is appropriate for the standard potentials, first INT and then thionine is reduced. Depending on the evaluation procedure selected, a wide variety of combinations of redox indicators can be used for the procedure according to the invention.

The test system according to the invention can, in principle, be used for all determinations of NAD(P)H and for all reactions consuming or forming NAD(P)H. The samples to be investigated are preferably body fluids, such as serum, plasma, urine etc. The procedure according to the invention is carried out in the manner customary for enzymatic methods, in general the reaction converting NAD(P)H being initially allowed to go to completion and then the formed or consumed NAD(P)H being determined. 0bviously, in the case of reactions forming NAD(P)H, direct coupling is also possible. Moreover, enzyme activities can be determined by stopping the reaction at a specified time and determining the NAD(P)H formed or consumed in this time. The redox indicators are preferably added to the 5 sample solution at the same time; addition at different times is also possible.

The >test system according to the invention is also suitable, in the form of absorbent materials impregnated with it, as an indicator for the determination of NAD (P)H.

Example 1

Determination of NAD(P)H

The following three mixtures are prepared:

<td rowspan="3"> 1.5</td><td colspan="2"> Mixture A</td><td colspan="2" rowspan="2"> Mixture B 0.01 mmol/l PMS</td><td colspan="2"> Mixture C</td>

<td rowspan="2"> 0.01 mmol/l 0.25 mmo I / I 0.025mmol/l titanium</td><td rowspan="2"> PMS DIP yellow</td><td rowspan="2"> 0.01 mmol/L 0.2 mmol/L 0.25 mmol/l</td><td rowspan="2"> PMS INT DIP</td>

<td> 0.2 mmol/L 0.025mmol/l titanium</td><td> INT yellow</td>

<td> 20</td><td colspan="2"> 0.15 mol/l citrate</td><td> 0.15 mol/l</td><td> citrate</td><td> 0 025mmoI/L titanium 0.15 mol/L</td><td> yellow citrate</td>

<td></td><td> buffer pH</td><td> 5.8</td><td colspan="2"> buffer pH 5.8</td><td colspan="2"> buffer pH 5.8</td>

Various amounts of NADH (NADPH) are added to 2 ml of each of these mixtures. After 10 minutes in each instance, the extinctions at 460 and 650 nm and the colour impression of each test solution are determined. The following results are obtained for the colour impression depend30 ing on the concentration of NADH or NADPH used in the total test solution:

<td rowspan="2"> NADH (NADPH) (mmol/ I) 35</td><td colspan="3"> Resulting colour impression of the test solution</td>

<td> Mixture A</td><td> Mixture B</td><td> Mixture C</td>

<td> 0</td><td> deep blue</td><td> yellow</td><td> deep blue</td>

<td> 0.05</td><td> dark blue</td><td> orange</td><td> dark blue</td>

<td> 0.09</td><td> b lue</td><td> brown</td><td> blue</td>

<td> 0.13</td><td> light blue</td><td> red</td><td> light blue</td>

<td> 0.17</td><td> blue-green</td><td> blue-green</td>

<td> 0.22</td><td> green</td><td> green</td>

<td> 0.24</td><td> yellow-green</td><td> yellow-green</td>

<td> 0.26</td><td> yellow</td><td> ochre</td>

<td> 0.27</td><td></td><td> orange</td>

<td> 0.30</td><td></td><td> brown</td>

<td> 0.34</td><td> .»</td><td> red-brown</td>

<td> 0.38</td><td></td><td> red</td>

<td> U. 55</td><td></td><td> dark red</td>

The changes in the extinctions in each test solution are shown in Fig. 1.

While a visual determination of the concentration of NADH in the range up to a maximum of 0.25 mmol/l is pos15 sible with mixtures A and B, a determination up to 0.55 mmol/l is possible with mixture C.

Example 2

Determination of NADH

Various amounts of NADH are added to 2 ml of a solu tion containing

<td></td><td> 0.01</td><td> mmo I /1</td><td> of</td><td> MPMS</td>

<td></td><td> 0.2</td><td> mmol/L</td><td> of</td><td> INT</td>

<td></td><td> 0.03</td><td> mmol/l</td><td> of</td><td> thionine and</td>

<td> 25</td><td> 0.15</td><td> mo 1/ L</td><td> of</td><td> a citrate buffer, pH 5.2.</td>

After 10 minutes, the extinctions of each test solution at 460 and 600 nm are measured. The results obtained, as a function of the concentration of NADH used in the total test solution, are shown in Fig. 2.

The range of measurement for the determination of

NADH obtained for the test mixture containing two dyestuffs is about double that of corresponding test mixtures contain ing only one dyestuff.

Example 3

Determination of NADH

The following mixtures are prepared:

Mixture A 0.01 mmol/L Meldola’s blue 0.25 mmol/L DIP 0.025mmol/l titanium yellow

0.15 mol/l citrate buffer pH 5.8

Mixture B 0.01 mmol/L MeLdoLa's blue 0.25 mmol/L DIP 0.2 mmoL/L INT 0.025mmoL/L titanium yeLLow

0.15 moL/L citrate buffer pH 5.8

Various amounts of NADH are added to 2 ml of each of the mixtures. The colour impression of each test soLution is determined after 20 minutes in each instance. The foLLowing results are obtained for the coLour impression, depending on the concentration of NADH used in the total test solution:

NADH Resulting colour impression of the test solution

<td> (mmol/ I)</td><td> Mixture A</td><td> Mixture B</td>

<td> 0</td><td> deep blue</td><td> deep blue</td>

<td> 0.10</td><td> turquoise</td><td> turquoi se</td>

<td> 0.20</td><td> dark green</td><td> dark green</td>

<td> 0.25</td><td> pale green</td><td> pale green</td>

<td> 0.30</td><td></td><td> pale brown</td>

<td> 0.35</td><td></td><td> pale red-violet</td>

<td> 0.40</td><td></td><td> wine red</td>

<td> 0.50</td><td></td><td> dark red</td>

While visual determination of the concentration of NADH is possible in the range up to 0.25 mmol/L with mixture A, determination up to 0.5 mmol/L is possible with mix ture B.

Example 4

Determination of urea in serum μΐ serum samples, each having different contents of urea (adjusted by addition of urea, and checked with a standard method) are each added to 1.5 ml of reaction solution containing

0.03 mol/l of phosphate buffer, □H 7.6

2.2 mmol/L of ADP

4.0 mmol/l of ketoglutarate

0.5 mmol/L of NADH

KU/l of glutamate dehydrogenase and

KU/l of urease

After 10 minutes, 350 μΐ of a solution containing 0.35 mol/l of citrate buffer, pH 5.8 0.05־ mmol/l of PMS .34 mmol/l of DIP

1.1 mmol/l of INT

0.13 mmol/l of titanium yellow and

250 II11 of diaphorase is added to each, and after a further 5 to 10 minutes, the colour impression of the reaction solution is determined with through-illumination. The results obtained are as folLows:

U rea

Resulting colour impression of the test so Lut i on

<td> 0</td><td> red</td>

<td> 30</td><td> red-brown</td>

<td> 45</td><td> brown</td>

<td> 55</td><td> orange</td>

<td> 60</td><td> och re</td>

<td>⁷⁰</td><td> yellow-green</td>

<td> 80</td><td> green .</td>

<td> 100</td><td> blue-green</td>

<td> 120</td><td> pale blue</td>

<td> 150</td><td> dark blue</td>

<td> 180</td><td> deep blue</td>

While visual determinations of urea up to 70 mg/dl can be carried out with a test mixture containing one dyestuff, the system according to the invention permits determinations up to 180 mg/dl.

Example 5

Determination of uric acid in urine

200 μΐ urine samples containing different amounts

<td rowspan="2"></td><td colspan="2"> of uric acid (adjusted by addition of uric acid, and checked by a standard procedure) are added to 1,3 ml of a reaction solution containing</td>

<td> 0.03</td><td> mol/L of phosphate buffer, pH 8.5</td>

<td> 5</td><td> 42</td><td> mmol/l of KCI</td>

<td></td><td> 1.43</td><td> mol/l of ethanol</td>

<td></td><td> 0.5 -</td><td> mmol/L of NADP⁺</td>

<td></td><td> 1750</td><td> II/1 of catalase</td>

<td></td><td> 150</td><td> U/l of aldehyde dehydrogenase and</td>

<td> 10</td><td> 50</td><td> II / L of uricase.</td>

<td></td><td colspan="2"> After 20 minutes, 350 μΐ of a solution, which contains the</td>

<td></td><td colspan="2"> following constituents:</td>

<td></td><td> 0.5</td><td> mol/L of citrate buffer, pH 5.8</td>

<td></td><td> 0.05</td><td> mmol/l of PES</td>

<td> 15</td><td> 1.34</td><td> mmol/l of DIP</td>

<td></td><td> 1 .10</td><td> mmol/l of INT and</td>

<td></td><td> 0.13</td><td> mmol/l of titanium yellow</td>

<td></td><td> are added to</td><td> each test solution. After a further 10 min-</td>

<td></td><td colspan="2"> utes, the colour impression of the reaction solution is</td>

<td> 20</td><td colspan="2"> determined with through-illumination. The results obtained</td>

<td></td><td colspan="2"> are as follows:</td>

<td></td><td> Uric acid</td><td> Resulting colour impression of the test</td>

<td></td><td> (mg/d I)</td><td> solution</td>

<td></td><td></td><td></td>

<td></td><td> 0</td><td> deep blue</td>

<td></td><td> 9</td><td> dark blue</td>

<td></td><td> 15</td><td> blue</td>

<td></td><td> 21</td><td> light blue</td>

<td> 30</td><td> 29</td><td> blue-green</td>

<td></td><td> 37</td><td> green</td>

<td></td><td> 40</td><td> yellow-green</td>

<td></td><td> 42</td><td> ochre</td>

<td></td><td> 45</td><td> brown</td>

<td> 35</td><td> 50</td><td> red-brown</td>

<td></td><td> 57</td><td> red</td>

<td></td><td> 64</td><td> dark red</td>

While visual determinations of uric acid in a range up to about 40 mg/dl can be carried out with an appropriate test containing only one dyestuff/ up to more than mmol/L can still be reliably measured with the system according to the invention.

Example 6 Determination of Lactate dehydrogenase in serum 100 μΐ of serum samples containing different activities of LDH (adjusted by addition of LDH_Z and checked with a standard method) are added to 1.2 ml of a reaction solution containing 0.03 mol/l of phosphate buffer_z pH 7.5 0.6 mmol/l of pyruvate and 0.5 mmol/l of NADH.

After 10 minutes at 25°C_Z 300 μΐ of a solution containing 0.5 mol/L of citrate buffer_z pH 5.8 1.34 mmol/l of DIP 1.10 mmol/l of INT 0.05 mmol/l of PMS ¹ 0.13 mmol/L of titanium yellow and mmol/l of oxamic acid are added. After a further 10 minutes, the colour impression of each total test solution is determined. The results obtained are as follows:

LDH Resulting colour impression of the test (U/l) solution

<td> 32</td><td> red</td>

<td> 96</td><td> red-brown</td>

<td> 160</td><td> brown</td>

<td> 210</td><td> orange</td>

<td> 225</td><td> ochre</td>

<td> 255</td><td> yellow-green</td>

<td> 290</td><td> green</td>

<td> 370</td><td> b lue־g reen</td>

<td> 430</td><td> pa Le b Lue</td>

<td> 500</td><td> blue</td>

<td> 560</td><td> dark blue</td>

<td> 640</td><td> deep blue</td>

־ ¹³ ־

While LDH determinations with visual evaluation in a range to a maximum of 225 U/l can be carried out with an appropriate test containing only one dyestuff, determin ations up to more than 600 U/l are possible with the test 5 system according to the invention.