AU611684B2 - Method for the estimation of reduced pyridine nucleotides and use of the method for estimation of co-a and antibiotics - Google Patents

Description

r I In.

AUSTRALIA (51)

PATENT

(43) 6.12.88 AU-A-17065/88 P\CT LITELL U ROPTY OaANIZATION INTERNATIONAL APPLICATION PBLIS ED ND HE ATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 08882 C12Q 1/00, 1/32, 1/48 Al (43) International Publication Date: 17 November 1988 (17.11.88) (21) International Application Number: PCT/GB88/00353 PRINCE, Christopher, Philip [GB/GB]; Church End, 36 Mingle Lane, Stapleford, Cambridgeshire CB2 58G (GB).

(22) International Filing Date: 5 May 1988 (05.05.88) (74) Agent: BECKHAM, Robert, William; Procurement Executive, (31) Priority Application Number: 8710882 Ministry of Defence, Patents 1A(4), Room 2014, Empress State Building, Lillie Road, London SW6 ITR (GB).

(32) Priority Date: 8 May 1987 (08.05.87) (81) Designated States: AT (European patent), AU, BB, BE (Euro- (33) Priority Country: GB pean patent), BG, BJ (OAPI patent), BR, CF (OAPI patent), CG (OAPI patent), CH (European patent), CM (OAPI pa- (71) Applicant (for a!l designated States except US): THE PUBIIC tent), DE (European patent), DK, FI, FR (European pa- HEALTH LABORATORY SERVICE BOARD [GB/3B]; tent), GA (OAPI patent), GB, GB (European patent), HU, 61 Colindale Avenue, London NW9 5DE IT (European patent), JP, KP, KR, LK, LU (European patent), MC, MG, ML (OAPI patent), MR (OAPI patent), (72) Inventors; and MW, NL (European patent), NO, RO, SD, SE (European Inventors/Applicants (for US only) ATKINSON, Anthony patent), SN (OAPI patent), SU, TD (OAPI patent), TG (OA- [GB/GB]; Twingley, Mill Corner, Winterbourne Gunner, PI patent), US.

Salisbury, Wiltshire SP4 6JT MILLER, Julie [GB/GB]; 12 Hartington Road, Salisbury, Wiltshire SP2 7LG Published SCAWEN, Michael, Denis [GB/GB]; 14 Paddock Close, With international search report.

Winterbourne Dauntsey, Salisbury, Wiltshire SP4 6EL (GB).

CAMPBELL, Robert, Stewart [GB/GB]; 28A Hillington Road, Cambridge, Cambridgeshire CB3 9HB MOR- RIS, Christine, Helen [GB/GB]; 165 Hills Road, Cambridge, Cambridgeshire CB2 2BJ HAMMOND, Peter, Michael [GB/GB]; Agneash, 10 The Butts, Shrewton, Wiltshire SP3 4LD (GB).

(54)Title: METHOD FOR THE ESTIMATION OF REDUCED PYRIDINE NUCLEOTIDES AND USE OF THE METHOD FOR ESTIMATION OF CO-A AND ANTIBIOTICS (57) Abstract Method for the estimation of an analyte which can be acylated which includes acylating the analyte with an acylcoenzyme A to liberate Co A, causing the Co A to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form NADH, and causing the NADH to reduce a further compound, the amount of reduction being measured. Preferably the acylation is an acetylation and the further compound is a tetrazolium salt which is reduced to a formazan. The method is suitable for the estimation of antibiotics which can be acetylated, e.g. thiamphenicol, chloramphenicol and gentamicin. A preferred tetrazolium salt is 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazolium chloride reduced in the presence of 1-methoxy-phenazinium methosulphate.

A. O. .p 27 JAN 1989

AUSTRALIAN

-6 DEC1988 PATENT OFFICE W~88/08882 PCT/GB88/00353 1 Method for the Estimation of Reduced Pyridine Nucleotides and use of the Method for Estimation of Co-A and Antibiotics.

This invention relates to methods for the estimation, ie the detection and quantitative analysis of reduced pyridine nucleotides, especially NADH (the reduced form of NAD, nicotinamide adenine dinucleotide) and NADPH (the reduced form of NAD-phosphate, NADP), the use of these methods in the estimation of coenzyme A (Co A) and compounds whic can be acylated by an acyl-Co A with the consequent liberation of Co A, in particular the antibiotics chloramphenicol, thiamphenicol and gentamicin. In particular the invention relates to the estimation of these antibiotics in blood plasma.

It is frequently necessary in biochemistry and medicince to estimate the reducing coenzymes NADH and NADPH, and Co A, as the presence or amount of these is often a good indication of the progress of a biochemical reaction, eg some metabolic step. As NADH plays a part in many such reactions, being oxidised to NAD in the process, it is often possible to use NADH estimation as a method of indirect estimation of other materials involved in the reaction.

One known method of estimation of NADH uses the quantitative reduction by NADH of tetrazolium salts, ie salts containing the nucleus: N N to a coloured formazan dye which contains the reduced nucleus:

NH

N N The amount of reduction may then be measured for example by measuring the change in spectral absorbance.

A number of tetrazolium salts are known for use in such methods.

Japanese. J. Clin. Chem. 14 June 19S5, p1 4 7-154 describes the use of 2-(p-icdophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), and EP-0149853-A describes the use of INT, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT); it nr WO 88/08882 PCT/GB88/00353 2 -biphenylene)-bis(2,5-diphenyl-2H tetrazolium cloride)(Neo-TB): 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis(2-(p-nitrophenyl)- 5-phenyl-2H tetrazolium chloride) (Nitro-TB); 3,3'-(3,3'-dimethoxy- 4 ,4'-biphenylene)-bis(2,5-diphenyl-2H tetrazolium chloride) 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis(2,5-bis(p-nitropheny')-2H tetrazolium chloride) (TNTB) and water soluble tetrazolium salts such as 2-(2-benzothiazolyl)-3-(o-carboxyphenyl)-5- (p-(hydroxy poly'oxy-l,2-ethanediyl))phenyl)-2H tetrazolium chloride.

Salts of 2-(2'-benzothiazolyl)-5-styryl-3-( h -phthalhylrazi-dyl)-tetrazolium (BSPT) have been used, eg the chlori'e, for detecting succinate lehydrogenase activity in cells, when the formazan formed is reacted with osmium tetroxide vapour for enhance" staining. This enhacement is not achievable in solution.

Such methods sometimes use the enzyme diaphorase or the electron acceptors phenazine methosulphate (PMS) or phenazine ethosulphate (PES) to accelerate the reaction, but even then they are slow, and PMS and PES are unstable towards light. Also a high pH is normally requires to increase the solubility of the formazans and to increase speed. Therefore there is a need for an improved method of estimating NADH.

Coenzyme A is another important adenine nucleotide which participates in many biochemical reactions, often acting in cOoperation with the redox couple NAD-NADH, to transfer acyl groups to and. from molecules, an acyl-Co A being destroyed or formed in the process, in particular acetyl-Co A. Often an appropriate acyl transferase enzyme is also involved in the transfer. It is an object of the invention to provide an improved method for the estimation of Co A.

1' WO'88/08882 PCT/GB88/00353 Chloramphenicol and thiamphenicol have structures below.

In chloramphenicol X is 0 2 and in thiamphenicol X is CH 3

SO

2 The gentamicins have the structure (II) below: S OH X CH.CH (I) R NHCOCHC1 2 11

CH-R

0 (ii) HO 7 CH NH R 6 The substituents R 1

-R

7 may vary, and various Gentamicins are identified below.

R1 R2 3 R4 5 6 R7 Gentamicin A H OH OH OH NH 2 H OH Gentamicin B H NH H OH OH OH CH 3 Gentamicin Ca H NH 2 H H NH 2 OH CH 3 Gentamicin C 2

CH

3

NH

2 H H NH 2 OH CH 3 Gentamicin C NHCH H H H NH OH CH 3 1 1 1 3 2 1 3 The term Gentamicin includes all Gentamicins and also includes Sisomicin, ie When these anti biotics are used therapeutically it is necessary to maintain their level in blood serum within a closely defined range to maintain a sufficient concentration for the antibiotic to have effect without reaching the concentration at which it may be toxic.

Chloramphenicol and Gentamicin are generally used in therapy at concentrations of 30-71 and 4-26 pmol/L respectively, and may prove toxic at concentrations above 75 and 26 u mol/L respectively.

It is therefore desirable to have methods by which the presence of these antibiotics in blood may be detected and their quantity measured. Ideally such methods should be rapid, convenient, and use cheap and safe reagents and equipment. Many currently a.vailable methods, eg microbiological methods, HPLC and GC, radiommunoassay or enzyme immunoassay do not meet these criteria.

17 1 i WO 88/08882 PCT/GB88/00353 4 A colo rimetric method for Chloramphenicol estimation has been previously described. (S P Bessman, S Stevens, J Lab Clin Med (1950).

129). This method is based on the reduction of the nitro group of chloramphenicol to an amino group. This amine form is then diazotised and coupled with ammonium sulphamate to produce a red complex. This method will detect all aryl nitro and aryl amine compounds and is therefore not specific for chloramphenicol. Also, this method is lengthy, taking at least 65 minutes to complete.

Estimation of Chloramphenicol using the enzyme Chloramphenicol acetyl transferase (CAT) hrs been described, but this employs radiolabelled acetyl coenzyme A, detecting the resulting labelled chloramphenicol-3-acetate by a liquid scintillation counter.

(P S Lietman et al Antimicrob.Agents Chemocher,(1976) 10 347), (R Daigneault, M Guitard, J.Inf. Dis.(1976) 133 515). These assays require the use of radioactive materials with their consequent hazards and need for special procedures.

Similar problems are encountered with presently used methods for estimation of Gentamicin. Enzyme mediated immunoassay is frequently used but requires a complex multi point calibration procedure.

It is therefore a major object of this invention to provide improved methods for the estimation of chloramphenicol and gentamicin and which by virtue of the chemical routes involved may be suitable for estimation of other, chemically related antibiotics.Other objects and advantages of the invention will be apparent from the following description.

According to a first aspect of the invention a method for the estimation of a reduced pyridine nucleotide in solution includes the step of causing the nucleotide to reduce a salt of BSPT to a formazan in the presnece of 1-methoxy phenazinium methosulphate (MPMS) and a non-ionic detergent.

The presence and/or amount of formazan produced may then be determined by known means, for example spectrophotometrically or colorimetrically at 575 nm, at which wavelength the formazan has a high extinction coefficient in some cases as high as 45000. The amount of formazan may then be related quantitatively to the amount of the nucleotide originally present. OCH, MPMS has the structure: N

CHHSO

CH

3 The method is tarticularly suitable for estimation of NADH or NADPH. which in the couse of the reaction are oxidised to NAD and NADP respectively.

Preferably the concentration of BSPT used in the reaction solution is 0.008 0.75 especially 0.09 mmol/L.

The use of MPMS in the method increases the speed of the reaction so that under optimum conditions the formazan may be determined within about 2 minutes. Additionally the MPMS is quite photostable. The method therefore provides substantial improvements over prior art methods of NADH estimation. A concentration of IPMHS in the solution of 0.001 to 10.0, especially 0.01 mmol L is suitable.

A preferred non-ionic detergent is Nonidet P-40, and other suitable detergents include Brij (polyoxyethylene (23) lauryl ether C, 1

E

23 CAS Registry No. 9002-92-0), Brij 58 (polyoxy-ethylene (20) cetyl ether C 16

E

20 CAS Registry No. 9004-95-9), Nonident S (Octylphenol ethylene oxide condensate; CAS Registry No.

S 9036-19-5), all three compounds are available from Sigma Pharmaceutical Company, St Louis, USA; lubrol (polyoxyethylene ether; Imperial Chemical Industries; Sand Triton X100 (Octylphenol ethoxylate nonionic S surfactant; Rohm and Haas). The presence of detergent is believed to create micelles and thus increase the solubility of the formazen. A preferred concentration range for the detergent in the solution is 0.001-0.5 wt%, an optimum being 0.09% of Nonidet a 0 T O -i i 5A The method is preferably used at a solution pH of between 6.0-11.0, with an optimum of 7.5. This optimum pH is preferably achieved using glycyl glycine buffer, 0.2 mol/L, pH 8.0. Suitable buffers of this pH also include sodium phosphate, sodium bicarbonate, imidazole-HCl, MOPS. HEPES, Tricine, Tris-HCl, potassium phosphate and limethyl glutarate (10 500 mmol/L).

The addition of serum to the solution (1 in 10 dilution) has been found to be advantageous in enabling the reaction of the method to take place at pH 7.5 without decreasing the rate of reaction or affecting optical absorbance. The maximum solubility of formazans normally occurs at higher pH.

By carrying out the method of the invention using known amounts of NADH in the -solution under standard conditions a calibration S. relationship can be established and the amount of NADH present in a sample to be analysed may be estimated by comparison.

Conveniently a reagent may be prepared for use in the method of the invention containing some or all of the necessary compounds, eg in aqueous solution. The tetrazolium salt, MIPMS and detergent may for example be made up in an acid solution (for stability) eg 0 0 0 0* 0, 00 0 0

'I'

t WO 88/08882 PCT/GB88/00353 6 containing hydrochloric acid 0.01-0.2 mol/L, malic acid 0.01-0.2 mol/L or preferably citric acid 0.01-0.2 mol/L (especially 0.012 mol/L), at a pH of 3-6. In use this reagent may be adjusted to an appropriate pH as described above, or may be mixed with a solution containing the NADH to be estimated under conditions which result in an appropriate pH in the solution-reagent mixture.

This reagent may be provided in the form of or as part of a kit for the estimation of reduced pyridine nucleotides eg NADH or NADPH.

Conveniently such a kit may contain the reagent held in an immobilised form, for example in the form of an indicator strip containing or impregnated with the immobiliesd reagent, or in the form of a. biosensor.

The method of this aspect of the invention appears to be relatively unaffected by other components of the solution (provided the conditions described above are observed). The method is thus applicable to the estimation of NADH in a wide variety of solutions, eg reaction mixtures from chemical or biochemical reactions or bodily fluids such as blood serum or plasma.

The method may for example be used for estimation of NADH which is already present in the solution to be analysed, or for estimation of NADH which is formed or consumed in the course of a reaction the progress of which it is desired to monitor, or for the indirect estimation of another compound which takes part in a reaction which produces NADH, one example of which is of course NAD itself.

Two uses of the method of estimation of NADH of the invention for the indirect estimation of other compounds are described below in further aspects of the invention.

According to a second aspect of this invention there is provided a method for the estimation of Coenzyme A in solution, which includes the steps of: Causing the coenzyme A present in the solution to react with 2-oxoglutarate (HOOC..CO.CH 2 ,C .COO) in the presence cf NAD and 2-oxoglutarate dehydrogenase to form succinyl coenzyme A and NADH, (ii) estimating the NADH formed in step using a method which includes causing the NADH formed to reduce a tetrazolium salt to a formazan.

C

WO 88/08882 PCT/GB88/00353 7 The amount of reduction of the tetrazolium salt which has occurred, or the amount of formazan which is formed may be determined by known means, eg spectrophotometrically or colorimetrically, and then related to the presence and/or amount of NADH and the presence and/ or amount of coenzyme A.

Various tetrazolium salts may be used in step for example INT, MTT, Neo-TB, Nitro-TB, TB and TNTB, but preferred salts are those of BSPT, eg the chloride.

Preferably step (ii) is carried out in the presence of an electron carrier, for example diaphorase, PMS, PES or MPMS, in particular when BSPT salts are used MPMS. The solution used in step (ii) should preferably also contain a detergent, preferably non-ionic.

The reaction between the Co A and the 2-oxoglutarate in step (ii) may be carried out in aqueos solution at room temperature. In step the solution should initially contain 0.05 50 especially 0.6 mmol/L of 2-oxoglutarate, 0.05 50, especially 0.5 mmol/L of NAD, and 2-200, especially 4 0 units/L of 2-oxoglutarate dehydrogenase, a preferred range in each case being 20% of these specific values. The solution may also advantageously contain 0.01 100 mmol/L especially 0.2 mmol/L of thiamine pyrophosphate (TPP) chloride, 0.01 100 mmol/L especially 1 mmol/L of MgC12 and a surfactant eg GAFAC R2610 (0.002 1.0% especially 0.03 Step operates within the pH range 6 10 with no specific buffer requirement, but optimum conditions are achieved using potassium phosphate buffer (pH 8.0, 0.05 mol/L).

When the solution in which CoA is to be estimated contains serum or plasma then endogenous NADH generating systems must be inhibited, by for example inclusion of urea or preferably oxamic acid (0.1 100 mmol/L) in the solution during step In step (ii) a suitable concentration for the electron carrier is 0.001 10.0, preferably 0.001 0.05, especially 0.01 mmol/L, MPMS being preferred for speed and convenience. A suitable concentration for the tetrazolium salt in step (ii) is 0.008 0.75 mmol/L especially 0.09 mmol/L 20 Suitable detergents, their concentrations, pH and other reaction conditions are as specified in the first aspect of the invention, whichever tetrazolium salt is used.

Preferably steps and (ii) are carried out sequentially, ie step is performed, and then all or a sample of the reaction i- WO 88/08882 PCT/GB88/00353 8 mixture from step may be mixed with reagents suitable for performing sten (ii).

Conveniently the method may be carried out by preparing a solution which contains all of the reagents for step and mixing a sample of this solution with a sample of the material, eg serum or a solution, in which CoA is to be estimated. After a suitable time, eg 3 minutes, a second solution may be added containing all the reagents necessary for step eg a reagent as discussed above. After a suitable time the amount of formazan present in the mixture may be determined, for example colorimetrically at 575 nm as described above.

The method may however be carried out as a one-step process, for example by using a solution which contains the reagents necessary for steps and (ii) at a suitable concentration, mixing this with the solution containing the CoA to be estimated, and detecting or measuring the amount of formazan formed.

By comparison of the colourimetric absorbance caused by known amounts of CoA in the material under standard estimation conditions, a calibration relationship can be established and the amount of CoA present in a sample to be analysed may be estimated by comparison.

Conveniently all the reagents necessary for step and/or (ii) may be in the form of or as part of a kit for the estimation of Coenzyme A, for example together with a reagent for the estimation of NADH as described above.

Conveniently such a kit may contain the reagents held in an immobilised form, for example in the form of an indicator strip containing the reagents in an impregnated or immobilised form, or in the form i of a biosensor.

SThe method of this aspect of the invention is applicable to the estimation of coenzyme A in a wide variety of solutions, eg reaction mixtures from chemical or biochemical reactions or bodily fluids such as blood serum or plasma.

The method may for example be used for estimation of coenzyme A which is already present in the solution to be analysed, or for estimation of coenzyme A which is formed or consumed in the course of a reaction the progress of which it is desired to monitor, or for the indirect estimation of another compound a reaction involving which forms or consumes coenzyme A.

I i WO 88/08882 PCT/GB88/00353 9 A particularly important use for a method of estimation of Co A is in the estimation of compounds which may be acylated by the mediation of an acyl-Co A which is converted into Co A in the process, and which can then be estimated.

Therefore according to a third aspect of this invention, there is provided a method for the estimation of an analyte which can be acylated, which includes the steps of: acylating the analyte in solution using an acyl-Co A, to form Co A, (ii) causing the Co A formed in step to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase, to fcrm NADH, (iii) causing the NADH formed in step (ii) to reduce a further compound and.

(iv) measuring the amount of further compound reduced or the amount of its reduction product formed.

The measurement made in step (iv) may then be quantitatively related to the amount of analyte originally present.

The acylation in step is preferably an acetylation using acetyl-Co A. It may be necessary to use acyl transferase enzymes to catalyse the acylation in step The method of this aspect of the invention is particularly suited to estimation of antibiotics which may be acetylated using acetyl Co A. Chloramphenicol and thiamphenicol are readily acetylated by acetyl-Co A in the presence of Chloramphenicol acetyl transferase and this enzyme may also acetylate other antibiotics having a related structure, ie structure with different groups X. Similarly gentamicin is readily acetylated by acetyl- Co A in the presence of Gentamicin acetyl transferase an enzyme which will also acetylate the antibiotics neomycin, soframycin, apramycin, kanamycin and to a lesser extent tobramycin and amikacin in the presence of acetyl Co A. In the case of the amphenicols, the 3- acetates are foremed, and in the case of gentamicin, acetyl gentamicin. In each case Co A is formed.

4' i, WO 88/08882 PCT/GB88/00353 In step (iii) it is preferred that the further compound reduced by the NADH is a chromogen, and that the reduction alters the properties of the chromogen in some way that can easily be measured in step (iv) by some physical method.

A preferred chromogen is a tetrazolium salt. which can be reduced to a formazan. with a consequent change in spectral absorbance.

Therefore according to a preferred embodiment of this third aspect there is provided a method for the estimation of an antibiotic which can be acetylated which includes the steps of: causing the antibiotic in solution to react with acetyl Co A in the presence of CAT or GAT to form Co A and an acetylated antibiotic.

(ii) causing the Co A formed in step to react with 2- oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form succinyl-Co A and NADH, (iii) estimating the amount of NADH formed in step (ii) using a method which includes causing the NADH formed in step (ii) to reduce a tetrazolium salt to a formazan.

The amount of NADH formed may then be quantitatively related to the amount of antibiotic originally present.

This embodiment is particularly suitable for estimation of cloramphenicol, thiamphenicol and gentamicin. In step the pH should be in the range 6-10 for chlor- and thi- amphenicol. and 5.5 10.5 for gentamicin. There appears to be no restriction on the type of buffer used, but for chlor- and thi- amphenicol buffer concentrations should be in the range 10-500 mmol/L. Tris-HCl has been found suitable, at 100 mmol/L, pH 7.8 for chlor- and thi- amphenicol, pH 8.4 for gentamicin. To produce optimum conditions for steps (ii) and (iii) a preferred buffer is glycyl glycine, 200 mmol/L at pH Initially the solution should contain 0.005 50, preferably about 0.09 20%) mmol/L acetyl-Co A, and 5 500 preferably about 20 units/L of CAT or 5 750 preferably about 240 20 units/L of GAT. For gentamicin 0.01 100 preferably about 1 mmol/L of magnesium chloride should preferably be present. Step may be carried out at room temperature.

0 WO,88/08882 PCT/GB88/00353 u.~ 11 i 0 Suitable and preferred conditions for performing steps (ii) and (iii) are as described above with respect to the first and second aspects of the invention.

Preferably the solution used in step (ii) should also contain glycyl glycine, eg 200 mmol/L at pH 8.0 to improve stability.

Steps (ii) and (iii) may be carried out sequentially, eg step may be performed, then all or a sample of the reaction 0 mixture from step may be mixed with the reagents necessary for step (ii) and then all or a sample of the reaction mixture from step (ii) may be mixed with the reagents necessary for step (iii).

Preferably steps and (ii) may be carried out simultaneously by mixing with the solution containing the chlor- or thi- amphenicol or gentamicin to be estimated reagents which are necessary for both steps and eg glycyl glycine, acetyl CoA, CAT or GAT, 2-Oxoglutarate, NAD, 2-oxoglutarate dehydrogenase, thiamine pyrophosphate magnesium chloride and a surfactant eg GAFAC R2610. Suitable and preferred concentrations for these reagents in the solution are those described above with respect 0 to the separate reactions.

Steps (ii) and (iii) may however be carried out simultaneously by for example mixing with the solution containing the chloror thi- amphenicol or gentamicin to be estimated all the reagents which are necessary for steps (ii) and (iii). These re gents may be conveniently be made up in a single solution, containing for example the reagents for steps and (ii) as described above, a tetrazolium salt, MPMS, diaphorase, PMS or PES, a detergent and a buffer.

When a single solution is prepared which is suitable for performing steps (ii) and (iii) simulataneously, then this solution should be used rapidly as it is likely to be less stable on long term storage than separate solutions for steps and (ii) and (iii), Conveniently a mixture containing reagents for step (ii) and (iii) separately or for two or three of these steps simultaneously may be prepared containing the reagents at an appropriate concentration. Such a mixture may form, or Ie part of, an analytical kit for the estimation of the antibiotics, and in such a kit the reagents may be present in an immobilised form, eg in an iniicator strip in an immobilised or impregnated form or as a biosensor.

i liT-tllY WO 88/08882 PCT/GB88/00353 12 In another form of such an analytical kit, the reagents for steps and and (iii) may be present in the form of an "enzyme reagent" containing 2-oxoglutarate. CAT or GAT, NAD. 2-oxoglutarate dehydrogenase, acetyl-Co A and suitable buffers, and a "colour reagent" containing a tetrazolium salt (eg BSPT chloride), MPMS, a non-ionic deterLent and suitable buffers. The enzyme reagent may conveniently be present in a freeze dried (lyophilised) form. ani the kit may then contain an appropriate liquid for reconstituting the enzyme reagent. These reagents may be provided in sealed vials containing sufficient for one or more estimations, or preferably also for a reagent blank and a standard estimation to be performe- as well.

i i C~P; WQ 88/08882s PCT/GB88/00353 The invention will now be described Example 1 Estimation of Coenzyme A by way of example only.

This example illustrates the use of the method of estimation of NADH by reduction of a tetrozolium salt to estimate Coenzyme A in a solution, ie both the first and second aspects of the invention.

Reagents Enzyme reagent for step (i) Potassium phosphate buffer pH 8.0 (0.05 mol/L), Magnesium chloride (1 mmol/L), oxamic acid (7 mmol/L), NAD (0.5 mmol/L), thiamine pyrophosphate (TPP) (0.2 mmol/L), 2-oxoglutarate (0.6 mmol/L), 2-oxoglutarate dehydrogenase (40 units Colour reagent for step (ii) estimation of NADH Citric acid (0.012mol/L), BSPT chloride (0.2 mmol MPMS (0.02 mmol L) and Nonidet P-40 (0.04 'Method A sample of serum (0.1 ml) containing coenzyme A was incubated with enzyme reagent (0.5 ml) for three minutes at room temperature.

Colour reagent (0.5 ml) was added and the absorbance of the formazan at 575 nm was determined after two minutes. The absorbances obtained for various coenzyme A concentrations in the serum are given in Table 1, where A A indicates the change in absorbance at the wavel- -egth shown.

Table 1.

Concentration of Coenzyme A in Serum (pmol/L) 4 A575 nm 0.110 0.230 75 0.342 100 0.467 125 0.538 150 0.641 175 0.728 200 0.820 WO 88/08882 WO 8/0882PCT/GB88/06353, Example 2 Estimation of chioramphenicol Reagent~s Enzyme reagent: (for carrying out steps and (ii) simultaneously) Glycyl glycine buffer pH8.0 (0.2 mol/L), magnesium chloride mmol/L), oxamic acid (7 mmol/L), acetyl coenzyme A (0.09 mmol/L), chioramphenicol acetyl transferase (60 units/L), NAD (0.5 mmol/L), thiamine pyrophosphate (0.2 mmol/L), 2-oxoglu1t.rate (0.6 mmol/L), 2-oxoglutarate dehydrogenase (20 unitrG/L), GAFAC R2610 (0.02 1) Colour reagent for step (iii) Citric acid (0.012 mol/L) BSPT chloride 2 nimol MPM4S 02 mmrol L) andI Nonidet P-40 (0.0 4 ,lethod A sample of serum (0.1 ml) containing chloramphenicol was incubated with enzyme reagent (0.5 ml) for four minutes at room temperature. Colour reagent 5m1) was added, After two minutes the absorbance at 575 nm was determined, The results for various chloramphenicol concentrations in the serum are given in table 2.

Table 2 Concentration of Chloramphenical in serum (jimol/L) 100 125 150 175 200 AA575nm 0.047 0.098 0.175 0.265 0.367 0.445 0.530 0.620 0.704 WO 88/08882 PCT/GB88/00353 Example 2 Estimation of Chloramphenicol and Thiamphenicol.

Enzyme Reagent (for steps and (ij) simultaneously).

Glycyl glycine buffer pH 9.0 (0.2 hiol/L), magnesium chloride (1 rnmol oxamic acid (7 mmol/L), acetyl coenzyme A (0.09 mmol/L), chiorarnphenicol acetyl transferase (60 units/L), NAD (0.5 mmol/L), thiamine pyrophosphate (TPP) (0.2 mmol/L), 2-oxoglutarate (0.6 mmol/L), 2-oxoglutarate dehydrogenase (40 units/L), CAFAC R2610 ClReagent for step (iii) Citric acid (0.012 mol/L), BSPT chloride (0.2 mmol/L), MPMS (0.02 mmol/L), Nonidet P-40 Method A sample of serum (0.1 ml) containing chloramphenicol was incubate1 with enzyme reagent (0.5 ml) for four minutes at room temperature. Colour reagent (0.5 ml) was added. After two minutes the absorbance at 575 nm was determined. The results for various serum chloramphenicol concentrations are given in table 3.

An identical procedure was followed with a sample of serum containing thiamphenicol, and the results for various serum thiamphenicol concentrations are shown in table 4 1 Table 3 Table 4 Concn. of chloramphenicol (pimol/L) 100 125 150 175 200 ,LA575 rim 0.047 0.09,3 0.175 0. 26_5 0.367 o.445 0.530 o.620 0.704 Cdoncn. of thiamphenicol (p1mol/L) 0 25 50 100 150 200 1LsA575 nm 0 0.099 0.204 0.375 o.496 0.594 WO 88/08882 PCT/GB88/00353 16 ExamoDle3 Estimation of Gentarnicin Reag-ents Enzyme 'reagent for carryinr, out stenps andi (ii) sirnulta--neouslV,.

Glycyl glycine buffer -pH 8.0 (0.2 mol magnesium chloride (1 minol oxamic acid (7 rnmol acetyl coenzyme A (0.09 mmol gentLamicin acetyl transferase 240 units NAD mmol thiamine pyrophosphate (0.2 mmol 2-oxo- Flutarate (0.6 mmol 2-oxoglutarate dehydrogenase (40 units GAFAC R261o (0.02 Colour reagent for stenLMIi Citric acid (0.012 mol BSPT chloride 2 mmol rTMS 0.O 2 rnmol Noni-iet P-40 Method A sample of serum (0.1 ml) containing gentamicin was incubated with enzyme reagent (0.5 ml) for four minutes at room temperature.

Colour reagent (0.5 ml) was added. After two minutes the absorbance at 575 nm was determined. The results for various gentamicin concentrations in the serum are given in table Concentration of gentamicin in serum (,iumol/L) &A575nm 0.075 0.160 0.256 30100 0.329 125 0.418 150 0.523 j 1750.615 200 0.707 From tables 1 to 5 calibration curves were plotted which could be used for estimation of CoA, chloramphenicol, thiamphenicol and gentamicin in samples of serum to be analysed.

SWO.88/08882 PCT/GB88/00353 17 Example 4 A suitable combination of reagents to be used as a kit for the estimation of chloramphenicol or thiamphenicol in serum.

Reagents.

Enzyme Reagent.

Glycyl glycine buffer pH 8.0 (30 mmol/L), magnesium chloride (1 mmol NAD (0.5 mmol/L), TPP (0.2 mmol/L), 2-oxoglutarate (0.6 mmol/L), chloramphenicol acetyl transferase (60 units/L), 2-oxoglutarate dehydrogenase (40 units/L), Acetyl Co A (0.9 mmol/L), all made up in distilled water. Quantities sufficient to make up to 2ml of reagent were freeze dried and sealed into vials, this quantity being enough for a reagent blank. a standard and two sample tests.

Diluent for Enzyme Reagent.

Glycyl glycine buffer pH 8.0 (200 mmol/L), oxamic acid (7 mmol/L), GAFAC RE610 (surfactant) (0.02 sodium azide (0.020%).

Colour Reagent.

Citric acid (12 mmol/L), BSPT chloride (0.2 mmol/L), MPMS (0.02 mmol Nonidet P-40 (0.04 sodium azide Method.

Freeze dried vials of enzyme reagent were reconstituted with diluent to 2 ml. A sample of serum (0.1 ml) containing chloramphenicol was incubated with the made up enzyme reagent for 4 minutes at room temperature. Colour reagent (0.5 ml) was added. After 2 minutes the absorbance at 575 nm was measured.

Example Steps (ii) and (iii) of the method for estimation of chloramphenicol were performed simultaneously as follows.

C;ombined Colour and Enzyme Reagent.

Glycyl glycine buffer pH 8.0 (100 mmol/L), magnesium chloride (0.05 mmol/L), NAD (0.25 mmol/L), TPP (0.1 aml/L), 2 -oxoglutarate (0.3 mmol chloramphenicol acetyl transferase (60 units/L), 2 -oxcglutarate dehydrogenase (40 units/L), acetyl CoA (0.45 mmol/L), oxamic acid (0,35 mmol/L), GAFAC RE610 (0.01 citric acid (6 mmol/L), BSPT chloride (0.01 mmol/L), MPMS (o.01 mmo?/L), Nonidet P-40 (0.02 Method.

A sample of serum (0.1 ml) containing chloramphenicol was incubated pith the combined colour and enzyme reagent (1.0 ml) for 6 minutes at room temperature. After this time the absorbance at 575 nm was measured.

Claims (12)

1. A method for the estimation of an analyte 4hich can be acylated characterised in that it includes the steps of: acylating the analyte in solution using an acyl-Coenzyme A to form Co A (ii) causing the Co A formed in step to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form NADH (iii) causing the NADH formed in step (ii) to reduce a further compound and (iv) measuring the amount of further compound reduced or its reduct- ion product formed.

2. A method as claimed in claim 1, characterised in that the analyte is an antibiotic, the acylation is an acetylation and the further com- pound is a chromogen the properties of which are altered by the reduct- ion in a way that can be measured by a physical method. A method for the estimation of an antibiotic which can be acet- ylated, characterised by the steps of: causing the antibiotic present in solution to react with acetyl- CoA in the presence of chloramphenicol acetyl transferase or gentamycin acetyl transferase, to form CoA and an acetylated antibiotic (ii) causing the CoA formed in step to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form NADH (iii) estimating the amount of NADH formed in step (ii) using a method which includes causing the NADH formed in step (ii) to reduce a tetra- zolium salt to a formazan then measuring the amount of formazan formed spectrophotometrically or colorimetrically.

4. A method as claimed in claim 3 characterised in that the antibiotic L is chloramphenicol oc thiamphenicol and chloramphenicol acetyl transfer- ase is used. A method as claimed in claim 5 characterised in that the antibiotic is gentamycin and gentamycin acetyl transferase is used.

6. A method as claimed in claim 5 characterised in that the antibiotic is selected from neomyoin, soframycin, apramycin, kanamycin, tobramycin _UBSTITUTE C. SUBSTITUTE E. United Ii;u5m st rt Oflica" S- .,o I r~ PCT/GB 8 8 0 53 24 Auumst 1989 24 08 89 19 6. (contd.) and amikacin, or from antibiotics containing the nucleus: OH CH2OH CH-CH 2 S CH-C -NHCOCHCl12

7. A method as claimed in any one of claims 3, 4, 5 or 6 characterised in that the tetrazolium salt is a salt of 2-(2'-benzothiazolyl)-5-styryl- 3-(4'-phthalhydrazidyl)-tetrazolium ("BSPT").

8. A method as claimed in any one of claims 3, 4, 5 or 6 characterised in that steps and (ii) are carried out simultaneously.

9. A method as claimed in claim 9 characterised in that steps (ii) and (iii) are carried out simultaneously. An analytical kit for the estimation of an antibiotic which can be acetylated, characterised in that it contains reagents for performing of the following steps: causing the antibiotic to react with acetyl-CoA in the presence of chloramphenicol acetyl transferase or gentamycin acetyl transferase to form CoA and the acetylated antibiotic (ii) causing the CoA formed in step to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form NADH (iii) causing the NADH formed in step (ii) to reduce a tetrazolium salt to a formazan.

11. A method for the estimation of a reduced pyridine nucleotide in solution, characterised by the step of causing the nucleotide to reduce a salt of BSPT bo a formazan in the presence of I'IPHS and a non-ionic detergent.

12. A method for the estimation of CoA in solution characterised by the steps of: causing the CoA present in solution to react with 2-oxoglutarate in the presence of NAD and 2-oxoglutarate dehydrogenase to form NADH (iii) estimating the NADH formed in step using a method which includes causing the NADH to reduce BSPT.

13. A method as claimed in any ohe of claims 1 to 9 or 11 or 12, characterised that the solution is a bodily fluid. A IUnited Kir~-dm Potent Office SUBSTITUTE SC -E PCT intelPatoolication SUBSTITUTE Snco. p 24August 1939 2 4 08 8 9

14. A method as claimed in claim 139 characterised in that the bodily fluid is blood serum or plasma. ij -00, Unied r: ci ?tont Office IICT Amlirionn SJBSTITUTE o:E 1 INTERNATIONAL SEARCH REPORT International Application No PCT/GB 88/00353 1. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to International Patent Classification IPC) or to bo h National Classificationn IPC 4 C 12 Q 1/00; C 12 Q 132; C 12 Q 1/48 IPC II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols IPC 4 C 12 Q Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT' Category f Citation of Document, with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 13 Y WO, A, 81/01419 (WISCONSIN ALUMNI 1-14 RESEARCH FOUNDATION) 28 May 1981, see page 4, lines 1-19; claims 1,3 Y US, A, 4318980 (ROBERT C. BOGUSLASKI etal.) 1-14 9 March 1982, see column 12, lines

59-68; claims 14,22,28 Y EP, A, 0149853 (WAKO PURE CHEMICAL 1-14 INDUSTRIES, LTD) 31 July 1985, see abstract; page 7, lines 11-18 Y EP, A, 0034213 (MODROVICH, 26 August 1-14 1981, see page 4, lines 14-20 A EP, A, 0007058 (BOEHRINGER MANNHEIM GmbH) 23 January 1980 SSpecial categories of cited documents: o later document published after the International filing date document defiing the general tte o the art which is not or piority date and not in conflict with the application but particular relevnce cited to understand the principle or theory underlying the considered to be of particular r1ancinvention earlier document but published on or ater the international document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establiah the publication date of another document of particular relevance; the claimed Invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report July 1988 0 9 AUAG 988 International Searching Authority Signature of Authorized 0 er EUROPEAN PATENT OFFICE n VAN MOL Form PCT/ISA/210 (second sheet) (January 1985) ANNEX TO THE INTERNATIONAL SEARCH- REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 8800353 SA 22101 This -annex lists the patent family members relating to the patent documents cited in the above-mcntioned international search report. -The members are as contained in the European Patent Office EDP file on 04/08/88 The European Patent Office is in no way liable for these particulars ihich are merely given for the purpose of information. Patent document I Publication Patent family Publication cited in search report date member(s) Idate W0-A- 8101419 28-05-81 EP-A- 0040246 25-11-81 AU-A- 664-1681 03-06-81 US-A- 4330621 18-05-82 US-A- 4318980 09-03-82 US-A- 4230797 28-10-80 US-A- 4380580 19-04-83 US-A-- 4492751 08-01-85 EP-A- 0149853 31-07-85 JP-A- 6014-1299 26-07-85 US-A- 4622296 11-11-86 *EP-A- 0034213 26-08-81 JP-A- 56124395 30-09-81 US-A- 43944-49 19-07-83 AT-B- E10208 15-11-84- CA-A- 1178875 04-12-84 EP-A- 0007058 23-01-80 IDE-B- 2831580 24-01-80 JP-A- 55019095 09-02-80 US-A- 4273870 16-06-81 AT-B- 362533 25-05-81 0 w For more details about this annex :see Official Journal of the European Patent Office, No. 12/82