AU622859B2 - Method and composition for the determination of potassium ions in fluids - Google Patents

Description

Signature of declarnt(s) (no aftestation required) Note' Initial all alterations, THE FLINDERS UNIVERISITY OF M)JTH AUSTRALIA H Buchan, Registrar DAVIES COLLISON, MELBOURNE and BOE IINGER MANNHEIM GMBH ppa- D PP Dr.a in Daum Dr flerbrt Fouquet'

CANBERRA

i K '7 yl~li" ""anrrir~lC r~-l I L i rr 62 2&59 AU-AI-15788/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 08137 G01N 33/84 //C12Q 1/00 Al (43) International Publication Date: 20 October 1988 (20.10.88) (21) International Application Number: PCT/EP88/00275 (22) International Filing Date: (31) Priority Application Numbers: 2 April 1988 (02.04.88) PI 1365 PI 2311 10 April 1987 (10.04.87) June 1987 (05.06.87) (32) Priority Dates: (33) Priority Country: AU (71) Applicants (for all designated States except US): THE FLINDERS UNIVERSITY OF SOUTH AUSTRAL- IA [AU/AU]; Sturt Road,, Bedford Park, S.A. 5042 BOEHRINGER MANNHEIM GMBH [DE/ DE]; Sandhofer Str. 116, D-6800 Mannheim 31 (DE).

(72) Inventors; and Inventors/Applicants (for US only) BERRY, Michael, Nathaniel [GB/AU]; 30 Wiilora Road, Eden Hills, S.A. 5050 TOWN, Michael-Harold [GB/DE]; Waldstrasse 45, D-8125 Oberhausen KRESSE, Georg-Burkhard [DE/DE]; Alpspitzstrasse 6, D-8122 Penzberg HERRMANN, Uwe [DE/DE]; Wettersteinstrasse 4, D-8139 Bernried (DE).

(74) Agents: FOUQUET, Herbert et al.; Boehringer Mannheim GmbH, Sandhofer StraBe 116, D-6800 Mannheim 31 (DE).

(81) Designated States: AT, AT (European patent), AU, BE (European patent), BR, CH, CH (European patent), DE, DE (European patent), DK, FI, FR (European patent), GB, GB (European patent), HU, IT (European patent), JP, KR, LU, LU (European patent), NL, NL (European patent), NO, SE, SE (European patent), SU, US.

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

A. 8 DEC 1988

AUSTRALIAN

4 NOV 1988 PATENT OFFICE (54) Title: r METHOD AND COMPOSITION FOR THE DETERMINATION

OF

POTASSIUM IONS IN FLUIDS".

(57) Abstract The invention concerns a process and a reagent for the determination of ions in fluids, wherein the influence of these ions on the activity of an enzyme is measured. The ions for example are sodium, potassium, calcium, magnesium, manganese, lithium, lead, zinc, copper, iron or other heavy metals or non-metallic ions comprising chloride, bicarbonate, protons, ammonium substances that give rise to ammonium. The enzymes which are used may be for example a transferase, a hydrolase, an oxidoreductase or a lyase. An essential part of the invention is a method to exclude interferences by ions by masking the interfering ions with a binding agent.

1A 1 1 1 1 11 11 1 1 1 l l 0TN 0 A0TE

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m mw I I C-UNNrq j ;ltII- :e i 1 I W0 88/0813 7 PCT/EP88/002 7 "METHOD AND COMPOSITION FOR THE DETERMINATION OF POTASSIUM IONS IN FLUIDS".

This invention is concerned with methods and reagents for the determination of ions, hereinafter also called analytes, in biological and non-biological fluids.

The invention is based on the ability of many analytes to stimulate or inhibit the activity of a sensitive enzyme. The analytes may-be cations or anions, metallic or non-metallic, simple or compound. In practice it is frequently found that the analyte is present in the sample at a concentration that lies outside the range of sensitivity of the relevant analytical indicator enzyme, or that interference is caused by the presence of other ions to which the enzyme is also sensitive. This invention aadresses and solves these problems in diverse ways.

In the practice of Clinical Biochemistry the measurement of serum electrolytes are the most common analytical tests performed within hospitals. These measurements are requested not only for routine investigations but frequently for emergency and life-threatening situations where speed of analysis is essential. Since a major source of delay in hospitals is the transport of specimens from the wards to the diagnostic laboratories, a method that is easily performed near the bed-side would be of particular value in emergency situations.

A common method of analysing potassium and sodium in clinical biochemistry practice is flame photometry. This process depends on WO 88/08137 P CT/EP88/00275 the principle that certain atoms when energizea by heat become excited and emit light of a characteristic wavelength when returning to ground state. The intensity of the characteristic wavelength of radiant energy proauced by the atoms in the flame is directly proportional to the number of atoms excited in the flame, which is directly proportional to the concentration of the substance of interest in the sample. The apparatus required is complex and relatively expensive and requires the use of combustible gases.

An alternative method especially for sodium, potassium and chloride makes use of ion-selective electrodes. Ideally, each electrode would possess a unique ion-selective property that would allow it to respond to only one ion. In. practice this is not the case and interfering ions exist for all ion-selective electrodes.

Moreover, ion-'specific electrodes are not absolutely specific although generally corrections are possible. The electrodes measure the potential developed in the presence of the specific ion. The instrumention is relatively expensive. Neither method can be performed spectrophotometrically and the clinical need for ion measurement therefore results in a substantial increase in the complexity of commercially available clinical analysers, most of which are designed primarily for spectrophotometric assays. Both methods require a considerable degree of skill and knowledge for their successful implementation.

Similarily, the routine determination of chloride by coulometric methods requires special instrumentation. The endpoint of this titration procedure is detected by an increase in electrical flux completion of formation of insoluble silver chloride product.

Alternatively, potentiometric determinations may be used which are also very time consuming and involve additional instrumentation.

41.

WO 88/08137 PCT/EP88/00275 For chloriae, in adaition, there are a number of photometric and titrimetric methods, which e.g. include: titrimetric determination of free Hg 2 ions via diphenylcarbazone complex colorimetric determination of the rhoaanide complex of iron, wnich is formed after dissociation of the mercury complexes upon precipitation of HgCI 2 (Skeggs, Clin. Chem. 10, 1964, 918f.; Schmidt, Zentralblatt Pharm. 124 1985, 527f) colorimetric determination of chloranilic acid from the respective mercury salt (Renschler) determination of the coloured Cu2+ complex of diethyldithiocarbaminic acid from the colourless mercury salt (German Offenlegungsschrift 2137146) the rather common TPTZ-method (tripyridile-s-triazine) (R.

Fried, Zeitschr. Klin. Chem., Klin. Bioch. 10, 1972, 280f; DOS 215 3387) which is similarily based on the formation of a coloured metal complex upon dissociation of a mercury complex.

A major drawback of these methods is the use of solutions containing highly toxic substances. Some of the methods are complicated ana imprecise the titration method). Many of the reagents are unstable and calibration curves are non-linear (e.g.

the rhodanide method). Some of these methods in addition need a pretreatment in order to eliminate interferences by the protein content of the sample.

An improved TPTZ-method is described in Wo. 83/002670, the use of toxic mercury compounas however is still a disadvantage; The only colorimetric method without use of mercury ions is the determination of hexachlorocomplexes of Fe(III) in a perchloric acid solution Hoppe., Ther. Ggw. 110(4), 1971, 554f.; WO 88/08137 P CT/EP88/00275 H. Mahner, Zeitschr. Klin. Chem., klin. Biochem. 11(11), 1973, 451f.; W. T. Law, Clin. Chem. 26 (13) 1980, 1874f.; US-4278440). A considerable limitation of this method is the use of strongly acid reagents which are corrosive and therefore not compatible with mechanical pitetting systems. A further disadvantage is the interference Dy bilirubin in the samples.

Calcium is a further example of an electrolyte which is routinely determined in the clinical laboratory. The concentration of this metal ion in boay fluids is regulated within a narrow range.

Pathologically hign or low concentrations can lead to life threatening disorders such as renal insufficiency, pancreatitis, tetany and congestive heart failure.

One of the earliest methods for the determination of calcium was that described by Tisdall Biol. Chem. 63, 461-465, 1925) in which calcium is precipitated by oxalic acid which is in turn estimated colorimetrically. The method involves a centrifugation step and is therefore very time consuming; it is not specific for calcium and depends on a careful handling of samples. The method has been succeeded in many laboratories by titrimetric and direct colorimetric procedures. The former also has the drawback of a complicated and cumbersome procedure and requires large sample volumes. In the latter procedure calcium affects the colour of a dye, for example orthocresolphthalein complexone, which can be measured in a photometer. Due to the simplicity of the method it lends itself to automation in the clinical laboratory. The method, however, involves the use of aggressive, highly alkaline solutions and toxic substances. It is particularly prone to interference by a number of serum components such as lipids, proteins, phosphate and bilirubin and as a result does not agree well with atomic absorption and flame photometric reference methods. A further disadvantage of the colorimetric procedure is that the calibration curves are non-linear and the colour is greatly dependent on temperature.

i 5 'WO 88/08137 PCT/EP88/00275 In Outlaw ana O.H. Lowry, Analytical Biochemistry 92, 370-374 (1979) an enzyme-mediated assay for measuring potassium ions in tissues is described. The method employs pyruvate kinase, from.

raobit muscle, which is activated by potassium ions and sodium ions, the former being about one forty-fold more effective.

Because of this non-specificity the method may be suitable for plant material in which potassium ions are the predominant cations, but it is unsuitable for measurements in body liquids like serum whicn contains a thirty-fold excess of sodium ions.

Therefore socium ions cause unacceptable interference when using the enzymatic photometric technique as described by Lowry et al.

to measure potassium in plasma or serum. A further problem is that ammonium ions give a similar activation to potassium ions. The above mentioned publication does not address or solve these critical problems in regard to the analysis of potassium ions in biological fluids such as serum, nor does it propose any method for the determination of sodium ions.

Therefore, it is an.object of the present invention to provide a process ana a reagent by which the above mentioned problems are avoided. The invention solves the problems by a process for the determination ions (analytes) in fluids wherein the influence of these ions on the activity of an enzyme is measured.

A key feature of this invention is the use of selective binding agents to bring the free concentration of the analyte within the optimal range for the analytical enzyme, particularly when dilution of tne fluid is not practicable. An additional element of the invention is the use of competitive inhibitors of the relevant analytical enzyme in order to reduce its sensitivity to the analyte, thereby permitting measurement of the latter at a higher concentration. This is especially useful, for example, where selective binding agents are not readily available or are unacceptably expensive.

Another feature of the invention is that selective binding agents are employed to reduce the free concentrations of .interfering ions

I

WO 88/08137 6 PCT/EP88/00275 to levels where interference is no longer significant. Use is also made of the fact that a competitive inhibitor may compete more effectively with interfering ions than with the analyte, thereby increasing the sensitivity of the enzyme to the analyte with respect to the interfering ion.

An important element is the choice of optimal reaction conditions, including the selection of an appropriate isoenzyme, such that the stimulatory or inhibitory effects of the analyte are substantially greater that those of the interfering ions. In addition, the action of the analyte and interfering ions on the activity of the analytical enzyme should be additive so that if the concentration of interfering ions is known, the concentration of the analyte can readily be determined by difference. Where an interfering ion is known to occur at a relatively constant concentration in the fluid under analysis-, allowance can be made for this by including an appropriate concentration of the interfering ion in standard (calibrating) solutions. Another method for assaying such analytes is the use of a competitive binding assay where the analyte displaces another ion from a binding agent and the effects of the released ion on the activity of an appropriate enzyme is determined.

These general principles can best be illustrated in detail by showing their application to the determination of potassium, sodium, calcium, chloride and bicarbonate ions in plasma or serum.

However, they are applicable to a wide spectrum of ions, for example cations such as magnesium, manganese, lithium, lead, zinc, copper, iron or other heavy metals. Examples of non-metallic ions that can be measured are protons or ammonium. Substances such as urea that give rise to ammonium can also be determined.

Suitable Enzymes Enzymes which may used can be for example Evans et al. Ann.

Rev. Plant Physiol. 17, 47, 1966): SWO 88/08137 7 PCT/EP88/00275 Transferases like phosphorous-containing group-transferring transferases. Sucn a transferase may be pyruvate kinase. In place of pyruvate kinase other kinases such as adenylate kinase or hexokinase, sensitive to magnesium ion or manganous ion may oe employed. Another transferase is acetate kinase (from E. coli).

Another example is pyridoxal kinase from brain which is sensitive to zinc ions.

Hydrolases like glycosidases, for exampleJ- or 16-D-galactosidase (from Escherichia coli), carboxypeptidase A (from bovine pancreas), collagenase (from Clostridium hy'stolicum), amylase (from saliva or pancreas) or phosphoglycollate phosphatase.

Also peptice hydrolases such as the cysteine or thiol dependent proteinases, specific examples of which are Calpain I and II (also called calcium activated neutral protease) described by Sasaki et al. in J. Biol. Chem. 259, 12489-12494, (1984). The latter enzymes can be isolatea and purified from a variety of animal tissues such as: rat liver and kidney, human and porcine erythrocytes, bovine brain, and rabbit skeletal muscle according to the method of A. Kitahara et al., described in J. Biochem. 95, 1759-1766 (1984).

A further example is dipeptidyl aminopeptidase I 3.4.14.1, Cathepsin J. Ken Mc Donald, Bioch. Biophys. Res. Communication 24(5), 66, 771f. Another source for the enzymes are proteins obtained by gene recombination techniques.

Oxidoreductases like glycerol dehydrogenase (from Enterobacter aerogenes), acetaldehydrogenase (from yeast) or tyrosinase (catechol oxidase).

Lyases like aldolase (from yeast) or carbonic anhydrase (from bovine erythrocytes).

Other suitable enzymes are various enzymes from halophilic organisms. Another source for the enzymes are proteins obtained by gene recombination techniques.

WO 88/08137 PCT/EP88/00275 Selective Binding Agents: A wide variety of binding agents are available for the binaing of analytes or interfering ions. Such binding or masking substances are cryptands, coronands, crown ethers, podands, spheranos, hemispnerands, calixarens ana combinations thereof, natural occurring ionophores, for example antibiotics, cyclic peptides like valinomycin, complexones and chelating agents, for example iminodiacetic acid, EDTA, nitrotriacetic acid and derivatives thereof. Such compounds are described in Kontakte (Merck), 1977, No. 1, p. 11 ff and p. 29 ff; Kontakte (Merck), 1977, No. 2, p. 16 ff; Konak'te (Merck), 1977, No. 3, p. 36 ff; Phase Transfer Catalysts, Properties and Applications (Merck-Schucharat) 1987, Thermodynamic and Kinetic Data for Cation-Macrocycle Interaction; R.M. Izatt et al., Chemical Reviews 85, 271-339 (1985); Data for Biochemical Research, 1986, R.M.C. Dawson et al., Eds., 3rd edit., 399-415 (Clarendon Press) Oxford; F. Vbgtle et al., Chem. Macrocycles, Springer Verlag, New York, 1985; G.W. Gokel et al., Eds., Macrocyclic Polyether Synthesis, Springer Verlag, New York, 1982; M. Hiraoka, Ed., Crown Compounds, Elsevier, Amsterdam, Oxford, New York, 1982; J.M. Lehn et al., J.Am. Chem. Soc. 97, 6700-6707 (1975); G. Schwarzenbach et al., Helv.Chim. Acta 28, 828 (1945); S.F.A. Kettle, Koordinationsverbindungen, Taschentext 3, Verlag Chemie, Weinheim/Bergstr. 1972; A.E. Martell et al., Die Chemie der Metallchelatverbindungen, Verlag Chemie, Weinheim/Bergstr.

1958; M. Becke-Goehring et al., Komplexchemie, Springer Verlag, 1970; F. Kober, Grundlagen der Komplexchemie, Otto Salle Verlag, Frankfurt/Main 1979; G. Schwarzenbach et al., Helv.Chim.Acta 31, 1029 (1948); R.G. Pearson et al., Science 151, 172 (1966).

Examples of chelators capable of binding multivalent ions, in particular bivalent cations are ethyleneglycol-bis-(2-aminoethyl ether)-N, N, N'-tetraacetic acid (referred to as EGTA) and (ethylenedinitrilo) tetraacetic acid (EDTA).

S!7

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1 i Lr I :_ii 6 i WO 88/08137 9 PCT/EP88/00275 l~hile many binding agents exist that can oind multivalent ions, e.g. EDTA and its derivatives, agents which bind monovalent ions are less common. Tetraphenylboron binds potassium ions. However-, a group of compounds with wider possibilities are cryptands which are examples of reagents that can selectively bind monovalent cations in aqueous solutions Izatt et al., Chem. Reviews 271-339). Special examples for cryptands are the Kryptofi"P compounds of Merck-Schuchardt, for example: 4,7 ,13,16 21-Pentaoxa-1,10-diazabicyclo[8.8.5 -tricosan, Kryptofix 221, page 438, Merck-Schuchardt catalogue, dated 1987/88, no. 810646 (K 221).

4 ,7,13,16 21 ,24-Hexaoxa-, 10-diazabicyclo 8 .8.8 hexacosan, Kryptofi 222, page 438, Merck-Schuchardt catalogue, dated 1987/1988, no. 810647 (K 222).

As masking compounds for the elimination of interfering anions the following classes of substances may potentially be used: anioncryptanaes, heterocyclophanes, catapinands and inorganic metalcomplexes or insoluble salts. Special examples of anion complexing compounds are described in the literature, e.g.

azamono- or azapolycycles, macrocyclic quarternary tetrahedron compounds, macrocyclic bis -metal-complexes, macrocycles with covalently incorporated lewis acid centers, protonated- or alkylated quarternary cryptands or catapinands P. Schmidtchen, Nachrichten Chem. Techn. lab. 36(1), 1988, S. 8f; E. Graf, J.

Amer. Chem. Soc. 98 1976, 6403f; C. H. Park, J. Amer. Chem.

Soc. 90 1968, 2431f) as well as e.g. the hexachloro complex of Fe (III) or silver nitrate.

Function of Binding Agents: These binding agents are used for the following purposes: 1. The selectivce binding of interfering ions.

r, r .I WO 88/08137 10 PCT/EP88/00275 2. To reduce the concentration of the analytes to optimal measuring levels, if dilution of the sample is not feasible.

3. An embodiment of the invention is a process, wherein the binding agent is present and forms a complex with "indicator" ions, from which complex the indicator ions are displaced stoichiometrically by the analyte ions, and wherein the influence of the displaced indicator ions on the activity of an enzyme is assayed, thereby giving an indirect measure of the cohcentration of analyte ions. For example in such a process the enzyme is pyruvate kinase, the indicator ions are potassium, the binding agent is Kryptofixo 221 and the ion to be determined is sodium; or the enzyme is a kinase, the indicator ion is IMg the binding agent is a chelating agent, e.g. EDTA, and the ion is a metal or the enzyme is pyridoxal 'kinase, the indicator ion is Zn 2 the binding agent is Kryptofixo 221, and the ion is a heavy metal; or the enzyme is J.-amylase, the binding agent is Ag or Hg and the ion to be determined is chloride; or the enzyme is collagenase the binding agent is a chelating agent such as EDTA and the ion to be determined is calcium.

Fluids for Analysis: The biological fluids in which the measurement of analytes is made are blood, serum, plasma, sweat, transudates or exudates or urine for example. Other examples of fluids are tap water or extracts of foodstuffs or fruits or fermented liquids such as wine.

Application of General Principles to the Determination of Potassium and Sodium Ions The essential requirements for a satisfactory method for'the determination of potassium ions in serum or plasma, on the basis of the sensitivity of pyruvate kinase to potassium ions, is the WO 88/08137 1 PCT/EP88/00275 overcoming of the interference by sodium and ammonium ions.

According to the general principles embodied in this invention this can be achieved by one or more of the following procedures: 1. The selective binding of sodium ions with a suitable binding agent, for example Kryptofix 221.

2. The selection of Bacillus stearothermophilus, rather than rabbit muscle, as the source of pyruvate kinase since the bacterial enzyme has a sensitivity for potassium ions in relation to sodium ions twice as great as the muscle enzyme.

3. Inclusion of ions which are competitive inhibitors of the sensitive indicator enzyme in the assay, for example the use of lithium ions to compete with sodium ions and potassium ions.

4. Enzymatic removal of ammonium ions.

Since lithium ions are less effective as a competitor against potassium ions, the net effect is to increase the relative sensitivity of pyruvate kinase towards potassium ions a further as compared with sodium ions. Moreover, in the presence of lithium ions the effects of potassium and sodium ions on the activity of pyruvate kinase become additive, rather than co-operative. This allows the possibility of mesurement of the concentration of either potassium or sodium ions, provided that the concentration of the other ions is known.

By the use of procedure 2 and 3 in the absence of a binding agent it is possible to obtain a relative sensitivity of pyruvate kinase for potassium versus sodium ions in the order of 100:1. This means that even at extremely abnormal sodium ion concentrations of either 110 or 170 mmol/l, the error in measured potassium ion concentration will not exceed 0.3 mmol/l relative to a normal 1 1

M

WO 88/08137 12 PCT/EP88/00275 plasma socium ion concentration of 140 inmol/l (Example This is not regarded as sufficiently accurate for many clinical purposes.

However, if the true concentration of sodium ions in the plasma is known, accurate measurement of potassium ions down to 0.05 mmol/l is feasible (Example If procedure 1-3 are combined and a binding agent, e.g. Kryptofix 221 is included, the relative sensitivity of pyruvate kinase for potassium ions with respect to sodium ions can be increased to 7 500:1. Under these circumstances it is not necessary to know the sodium ion concentration to determine the plasma potassium ion concentration down to 0.05 mmol/l (Example C).

These methods for potassium ion determination demonstrate the applicability of the general principles embodied in the invention in regard to reduction of interferences. On the other hand the measurement of sodium ions in serum or plasma best illustrates the application of these principles in regulating effective analyte concentration. One means of measuring sodium ions as embodied in this invention is to use an enzyme whose activity is sensitive to sodium ions. An example of such an enzyme is B-galactosidase (Kuby et al., J. Am. Chem. Soc. 75, 890, 1953). However, the range of sodium ion concentration to which this enzyme is most sensitive is much lower than can conveniently be obtained in a plasma sample, without a dilution step.

In keeping with the principles embodied in this invention the following procedures are employed to lower the effective sodium ion concentration to optimal levels when dilution of the sample is not feasible.

1. Use of a sodium ion binding agent such as Kryptofix© 221.

2. Use of lithium ions as a competitive inhibitor of 6-galactosidase, thereby decreasing the sensitivity of the enzyme to sodium ions.

1 rne ions to be determined are reduced in concentration to optimal levels for measurement by means of a binding agent, if dilution of the sample is not feasible an3-t b the affinity of the enzyme to the potassium ions ne4a, 'I i to be decreased.

WO 88/08137 13 PCT/EP88/00275 The combination of procedures 1 and 2 readily allows the determination of sodium ions in plasma or serum, using 16-galactosidase, and the amount of binding aget can be manipulatea to minimise the signal for sodium ion concentrations below 110 mmol/l while enhancing the signal in the usual analytical range (110-170 mmol/l) (Example D).

Sodium ions may also be measured by means of pyruvate kinase, provided that conditions are chosen whereby the stimulation of enzyme activity by potassium ions is reduced, and a potassium ion-binding agent, e.g. Kryptofix 222, is included in the reaction mixture (Example E).

In another method of determining plasma sodium ion concentration, the sodium ions are allowed to displace potassium ions from Kryptofix221, the released potassium ions stimulating the activity of pyruvate kinase in proportion to the plasma sodium ion concentration (Example F).

Other embodiments of the invention are compositions and reagents for the determination of ions in biological and non-biological fluids.

The reagent according to the present invention can be present in dissolved or dry form. It can be present impregnated on an appropriate carrier. A diagnostic agent in the form of a test strip can be produced by impregnating a carrier material, preferably filter paper, cellulose or synthetic fibre fleece, with solutions of the necessary reagents conventionally used for the production of test strip in readily volatile solvents, such as acetone. This can take place in one or more impregnation steps.

The finished test papers can be used as such or stuck in known manner on to handles or preferably sealed between synthetic resins and fine meshes.

LL i WO 88/08137 14 PCT/EP88/00275 The embodiments of the invention are described hereunder in some detail but it will be seen that the invention need not necessarily be limited in any one or in a combination of the details described, and in particular, mechanical or chemical variations can be utilized besides those described in this embodiment.

Detailed Description of Analytical Methods for Potassium Ions This section describes in more detail methods for potassium ion determination embodying the principles described in this invention.

For the determination of potassium ions, a fluid, for example blood plasma, is incubated with a buffered mixture containing adenosine diphosphate (ADP), phosphoenolpyruvate (PEP), reduced nicotinamide adenine dinucleotide (NADH), pyruvate kinase (PK) and lactate dehydrbgenase (LDH). The formation of pyruvate, and subsequently lactate in this mixture, in reactions catalysed by PK and LDH, is entirely dependent on the presence of appropriate cations, in the absence of which PK is virtually inactive. NADH absorbs strongly at 340 nm, whereas NAD does not.

Under the conditions chosen for analysis which include the presence of manganese ions which are required by the bacterial PK, tlhe rate of NADH oxidation is proportional to the concentration of potassium ions (see Examples A-C).

Under these conditions the following reactions take place: PEP ADP H PKPyruvate ATP Pyruvate NADH Ht L D H Lactate NAD The rate of reaction is determined by the concentration of potassium ions present in the system, and this in turn limits the rate of reaction There are several ways in which the rates of these reactions can be measured. A standard approach is the P:~1 'WO 88/08137 15 PCT/EP88/00275 spectrophotometric measurement of the rate of disappearance of NADH in reaction NADH absorbs strongly at 340 nm, whereas NAD does ot. Accordingly, the fall in absorbance of the reaction mixture at 340 nm (or an alternative wavelength) provides a direct measure of the rate of the reaction and from this the concentration of potassium ions present in the mixture can be derived. Alternatively, advantage can be taken of the fact that both reaction and consume H thus lowering the proton concentration of the reaction mixture. The rate of fall in proton concentration can be measured with a pH meter, or by means of a titration procedure. In these latter cases the concentration of buffer employed will be much less than in the spectrophotometric technique. Other equipment such as fluorimeters or luminometers can be used to monitor the activity of pyruvate kinase.

There are a number of other methods of detecting the accumulation of pyruvate associated with PK activity. These include any method for measuring the inorganic phosphate or oxygen consumed or the hydrogen peroxide; acetyl phosphate or carbon dioxide generated by the enzymatic action of pyruvate oxidase; the formation of the hydrazone with 2,4-dinitrophenylhydrazine; the measurement of the reactants or products of the enyzmatic action of pyruvate carboxylase; pyruvate decarboxylase or pyruvate dehydrogenase; the use of fl'avine coupled systems; and isotopic methods for measuring minute concentrations of substrates Berry et al., Analytical Biochem. 118, 344-352 (1981)).

In a survey of 200 serum samples good agreement has been obtained with other methods such as flame photometric or ion-selective electrode measurements. A significant interference with the method is ammonium ions which are generally present in serum or accumulate on standing. The possibilly of ammonium ion interference can be completely avoided by including :.-ketoglutarate (KG) and glutamate dehydrogenase (GDH) in the reaction mixture. Ammonium ions are removed in a preincubation according to the reaction: r .tr 7 i r ~s k 1" I io WO 88/08137 PCT/EP88/00275 NH KG NADH glutamate NAD+ In solutions such be high a coupled NH4 KG NADPH 4 as urine in which the ammonium ion content may reaction can be used.: glutamate NADP glucose-6-P NADP 6-phosphogluconate NADPH The coupled method employs glucose-6-phosphate dehydrogenase.

Provided that the added glucose-6-P and -KG are in excess of any ammonium ions present, all ammonium ions will be removed while preserving the NADH in the reagent.

Typical concentration ranges of enzymatic determination at 37 0

C

sample of plasma or serum are: PK stearothermophilus PEP (neutralized Tris salt) Kryptofix 221

NADH

buffer, pH 7-8 Mn 2 or Mg 2 LiC1 ADP (free acid) LDH (assayed at 25 C) Serum albumin GDH (assayed at 25 0

C)

KG (free acid) the main reagents of potassium. ions 50 U/i 0.3 mmol/1 0 mmol/1 0.01 mmol/l1 50 mnmol/l 1 mmol/1 2 mmol/1 0.5 mmol/1 5 000 U/1 0 g/1 2 500 U/1 1 mmol/1 for the using a 10 ul 10 000 U/1 30 mmol/1 30 mmol/1 0.8 mmol/1 500 mmol/1 10 mmol/1 100 mmol/1 10 mmol/1 100 000 U/1 5 g/l 20 000 U/1 10 mmol/1 lr- acid solution Hoppe., Ther. Ggw. 110(4), 1971, 554f.;

K

I 1 1 1 ft *i 1 1 I WO 88/08137 PCT/EP88/00275 Another example sensitive to potassium ions is glycerol dehydrogenase Lin et al., B 235, 1820, 1960). Typical concentration ranges of the main reagents for the enzymatic determination at 370 of potassium ions using glycerol dehydrogenase are: Glycerol dehydrogenase Glycerol Kryptofie 221

NAD

buffer, pH 9 Serum albumin GDH (assayea at 25 0

C)

KG (free acid) 50 U/1 to 0.3 mol/1 to 0 mmol/l to 0.1 mmol/l to 20 mmol/1 to 0 g/1 to 2 500 U/l to 1 mmol/l to 1000 U/1 3 mol/1 30 mmol/1 5.0 mmol/1 500 mmol/1 5 g/l 20 000 U/1 10 mmol/l Another enzyme sensitive to potassium ions is acetaldehyde dehydrogenase Black, Arch. Biochem. Biophys. 34, 86, 1951).

Typical concentra'tion ranges of the main reagents enzymatic determination at 37 0 C of potassium ions acetaldehyde dehydrogenase are: for the using Acetaldehyde dehydrogenase Glycolaldehyde KryptofiQ 221

NAD

buffer, pH 7-8 Dithiothreitol Serum albumin GDH (assayed at 250C) KG (free acid) 50 U/1 0.3 mmol/1 0 mmnol/1 0.05 mmol/1 50 mmol/l 0.1 mmol/l 0 g/l 2 500 U/I 1 mmol/l to to to to to to to to to 10 000 U/1 30 mmol/1 30 mmol/1 2.0 mmol/1 500 mmol/1 2 mmol/1 5 g/1 20 000 U/1 10 mmol/1 Acetaldehyde (0.02 mmol/l to 1 mmol/l) may be glycolaldehyde.

substituted for :j iI

I

P1"' ,I b r 18 Acetaldehyde dehydrogenase also exhibits esterase activity so that potassium ion concentration can be determined by monitoring the release of 4-nitrophenol from 4-nitrophenyl acetate. Typical concentration ranges of the main reagents for the enzymatic determination at 37 0 C of potassium ions based on the esterase activity of acetaldehyde dehydrogenase are: Acetaldehyde dehydrogenase 4-nitrophenyl acetate Kryptofix 221

NADH

buffer, pH 7-8 Dithiothreitol Serum albumin GDH (assayed at 250C) KG (free acid) 50 U/l 0.1 mmol/1 0 mmol/1 0.001 mmol/1 50 mmol/1 0.1 mmol/1 0 g/l 2 500 U/l 1 mmol/1 10 000 U/1 2 mmol/1 30 mmol/1 0.1 mmol/1 500 mmol/l 2.0 mmol/1 5 g/l 20 000 U/1 10 mmol/1 r r r !ii 9

K

areM Lemploed UL Leduce the fee c entrat L L ioUn I V s Uof.int ring i on are employed to reduce the free concentrations of interfering ions 1. -g C~ I rr3llll i Yc WO 88/08137 Example A PCT/EP88/00275 Measurement of Potassium Ion without a Sodium-Ion-Binding Concentration Using Pyruvate Kinase m Agent, Sodium Ion Concentration Unknown.

The final incubation mixture contains: 175 mmol/l mmol/l mmol/i 2.6 mmol/l 2.9 mmol/l 0.4 mmol/l 17000 U/1 890 U/1 mmol/l 8600 U/1 140 mg/l Tris-HCl buffer, pH 7.4 Li [17 mmol/l LiOH, 3 mmol/l LiC1] MnC 12 ADP (free acid) PEP (neutralized tris salt)

NADH

LDH (assayed at 25 0

C)

PK from Bacillus stearothermophilus

KG

GDH (in glycerol; assayed at 25 0

C)

Human serum albumin Potassium ion standards (calibrating solutions) contain 140 mmol/l sodium ions to compensate for the stimulatory effect of sodium ions, present in plasma, on pyruvate kinase.

4,;sr r. ct 4 .4 4. \E .9 L I Kev. Flant Pnysiol. 17, 47, 1966): "7 WO 88/08137 PCT/EP88/00275 Example

B

Measurement of Potassium Ion Concentration Using Pyruvate Kinase, without a Sodium Ion Binding Agent, Sodium Ion Concentration Known.

The incubation mixture and calibrating solution are the same as for Example A.

A correction may be made for the sodium ion concentration of the mixture by adding (or subtracting) 0.1 mmol/l potassium for every mmol/l the sodium ion concentration is below (or above) 140 mmol/l sodium ions. However, this correction should be verified by analysing aqueous solutions containing known sodium and potassium concentrations.

Example C Measurement of Potassium Ion Concentration Using Pyruvate Kinase in the Presence of a Sodium Ion-Binding Agent.

As for Example B, but human serum albumin is omitted and the medium contains in addition 6 pmol of Kryptofix 221 per assay.

A pH of 7.8 is selected to minimise variations in displacement of sodium ions from Kryptofi" 221 due to the differing potassium ion content of individual specimens.

[i

I~

sAnL,- I cIuiauu ia Li on tecnniques

I>

U

.WO 88/08137 PCT/EP88/00275 Example G Measurement of Potassium and Sodium Ion Concentration in the Same Cuvette (Twin-Test) Sodium ion concentration is assayed first as in Example D except that the assay also contain.s: 2.6 mmol/I 2.9 mmol/l 0.4 mmol/l mmol/1 8 600 U/1 ADP (free acid) PEP (neutralized Tris salt)

NADH

KG

GDH

Following the measurement of sodium ions by means of determination fo the reaction rate, the pH of the incubation mixture is lowered to pH 7.4 with a hydrochloric acid aliquot.

Then the following ingredients are added to achieve the final concentrations indicated: 17 000 U/1 890 U/1 mmol/1 20.0 mmol/1

LDH

PK from Bacillus stearothermophilus MnC12 LiCI The reaction rate may then be monitored at 340 nm but may also be measured at a slightly higher wavelength to minimise possible interference by the 2-nitrophenol liberated in the sodium ion indicator reaction.

!i e j

I

1- I M l MI

Claims (18)

1. Process for the determination of potassium ions in body fluids, wherein the influence of the potassium ions on the activity of a microbial transferase or a microbial oxidereductase is measured.

2. Process as claimed in claim 1, wherein the microbial transferase is a pyruvate kinase from Bacillus Stearothermophilus and the microbial oxidereductase is a glycerol dehydrogenase or an actealdehyde Sdehydrogenase.

3. Process as claimed in claim 1 or claim 2, wherein interfering ions are masked with a binding agent. e

4. Process as claimed in any one of claims 1 3, wherein :the ions to be determined are reduced in concentration to optimal levels for measurement by means of a binding agent, if dilution of the sample is not feasible and-or the affinity of the enzyme to the potassium ions needs to be decreased. Process as claimed in claim 3 or claim 4, wherein the interfering ions are bound by cryptands, coronands, podands, crown ethers, spherands, hemispherands, calixarens and combinations thereof, natural occuring ionophores, cyclic peptides, complexones and chelating agents, and derivatives thereof.

6. Process as claimed in any one of claims 1 5, wherein interfering sodium ions are bound by Kryptofix 221. 23

7. Process as claimed in claim 4 or claim 5, wherein the binding agent. is Kryptofix 222.

8. Process as claimed in any one of claims I 7, wherein the binding agents form a complex with indicator ions and the indicator ions are displaced stoichiometrically from the complex by the potassium ions and wherein the influence of the displaced indicator ions on the activity of the enzyme is assayed, thereby giving an indirect measure of the concentration of the potassium ions.

9. Process as claimed in any one of claims 1 8, wherein ions which are competitive inhibitors of the indicator enzyme are included in the assay, when the affinity of the enzyme to the potassium ions needs to be decreased. Process as claimed in claim 9, wherein the competitive inhibitors are lithium ions.

11. Composition for the determination of potassium ions in body fluids by a process as claimed in any one of claims 7 to comprising a microbial transferase or a microbial oxide- reductase the activity of which is influenced by the potassium ions.

12. Composition for the determination of.potassium ions in body fluids as claimed in claim 11, comprising microbial transferase or microbial oxidereductase and a binding agent which binds the interfering ions in the fluid and/or lowers the concentration of the potassium ions to optimal levels for measurement and/or decreases the affinity of the microbial transferase or oxidereductasato the potassium ions. 4-i: I -24-

13. Composition as claimed in claim 12 wherein the binding agent is a cryptand.

14. Composition as claimed in claim 13, wherein the cryptand is Kryptofix 221 to bind interfering ions and Kryptofix 222 to lower the concentration of the potassium ions and/or to decrease the affinity of the microbial transferase or the microbial oxidereductase. Composition for the determination of potassium ions in body fluids, as claimed in claim 11, which is used preferably at 37 0 C, comprising: S S S J S S. S a S.. S S S S.: S 5I*S 6 0@ S S 0 PK stearotherm- philus) PEP (neutralized Tris salt) Kryptofix 221 NADH Buffer, pH 7 8 Mn 2 or Mg 2 LiCl ADP (free acid) LDH (assayed at 25 0 C Serum albumin GDH (assayed at 25 0 C) KG (free acid) 50 U/1 to 0.3 mmol/1 to 0 mmol/l to 0.01 mmol/l to 50 mmol/1 to 1 mmol/l to 2 mmol/1 to 0.5 mmol/l to 5000 U/1 to 0 g/l to 2500 U/1 to 1 mmol/l to 10000 U/1 30 mmol/l 30 mmol/l 0.8 mmol/l 500 mmol/1 10 mmol/1 100 mmol/l 10 mmol/l 100000 U/1 5 g/1 20000 U/1 10 mmol/1 L _r I i I- -e

16. Composition for the determination of potassium ions in body fluids as claimed in claim 11, which is used preferably at 37 0 C, comprising: Glycerol dehydrogenase 50 U/1 to 1000 U/1 Glycerol 0.3 mol/l to 3 mol/l Kryptofix* 221 0 mmol/l to 30 mmol/l NAD 0.1 mmol/l to 5.0 mmol/l Buffer, pH 9 20 mmol/l to 500 mmol/l Serum albumin 0 g/l to 5 g/l GDH (assayed at 25 0 C) 2500 U/1 to 20000 U/1 KG (free acid) 1 mmol/l to 10 mmol/l

17. Composition for the determination of potassium ions in body fluids as claimed in claim 11, which is used preferably at 37 0 C, comprising: r oo o oo *oo*o* i *o o *o Acetaldehyde dehydrogenase Glycolaldehyde Kryptofix® 221 NAD Buffer, pH 7 8 Dithiothreitol Serum albumin GDH (assayed at 25 0 C) KG (free acid) 0. 0.0 5 0. 50 U/1 3 mmol/l 0 mmol/l 5 mmol/l i0 mmol/1 1 mmol/l 0 g/l 2500 U/1 1 mmol/l 10000 U/i 30 mmol/l 30 mmol/l 2.0 mmol/1 500 mmol/1 2 mmol/l 5 g/l 20000 U/1 10 mmol/l oooe eooo r -26-

18. Composition for the determination of potassium ions in body fluids as claimed in claim 11, which is used preferably at 37 0 C, comprising: Acetaldehyde dehydrogenase 50 U/1 4-Nitrophenyl acetate 0.3 mmol/1 Kryptofix® 221 0 mmol/l NAD 0.001 mmol/1 Buffer, pH 7 8 50 mmol/l Dithiothreitol 0.1 mmol/l Serum albumin 0 g/l GDH (assayed at 25 0 C) 2500 U/1 KG (free acid) 1 mmol/l 10000 U/i 30 mmol/l 30 mmol/1 0.1 mmol/l 500 mmol/1 2 mmol/l 5 g/l 20000 U/1 10 mmol/l o r r

19. Process as claimed in claim 1, substantially as herein described with reference to the Examples.

20. Composition as claimed in claim 11, substantially as herein described with reference to the Examples. Dated this 30th day of January, 1992 THE FLINDERS UNIVERSITY OF SOUTH AUSTRALIA and BOEHRINGER MANNHEIM GmbH By their Patent Attorneys DAVIES COLLISON CAVE i. I I: INTERNATIONAL SEARCH REPORT Internatlonal Application No PCT/EP 88/00275 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC IPC 4 G 01 N 33/84,//C 12 Q 1/00 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Clasfification Symbols iPC 4 G 01 N 33/00; C 12 Q 1/00 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched II. DOCUMENTS CONSIDERED TO tL RELEVANT* Category Citation of Document, I with Indication, where aptropriate, of the relevant passages 1 I Relevant to Claim No. 1 X Chemical Abstracts, vol. 90, no. 13, 26 1-5,7,12,15 March 1979 (Columbus, Ohio, US) W.M. Outlaw et al.: "Measurement of 7 to 10 12 mol of potassium by stimulation of pyruvate kinase" see page 230, abstract no. 99592 Anal. Biochemistry 92, 1979, 370-374 (cited in the application) X Clinical Chemistry, vol. 32, no. 4, April 1-5,7,12 1986 (New York, US) M.C. Wimmer et al.: "A kinetic colorimetric procedure for quantifying magnesium in serum", pages 629-632, see abstract; pages 629,630 X DE, A, 3614470 STEINMAN) 20 November 1-5,7,12, 1986, see claims 1-5,9-11; page 15, 15,16,31 lignes 7-10 A 32,33,39-42, i 44 SSpecial categorie of cited documents: e later document published nter the Intrnational filing date or priority date and not in conflict with the eapplication but document defining the general state of the art which i not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international X 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 establish the publication date of another document of particular relevnce;' the claimed Invention citation or other special reason (as specified) cannot be considered to involve an inventive stpo when the 0 document referring to an oral disclosure, use, exhibltlon or document is combined with one or more other such docu- other means menta, such combination being obvious to a person skilled document published prior to the international filing dat but in the art. later than the priority date claimed "A 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 24th August 1988 I 7 nrPT tnr International Searching Authority EUROPEAN PATENT OFFICE Form PCTIISAI210 (second sheet) (January 1985) i 9 Internatlona Application No. PCT/EP 88/00275 III. DOCUMENTS CONSIDERED TO IE RELEVANT (CONTINUED FROM THE SECOND SHEET Category I Citation of ocunO t, with indication, where approprite, of the rvalnt pisages j Relevant to Claim No -2- X Journal of Analytical Chemistry of USSR, vol. 35, no. 8, Part 2, August 1980 (New York, US) I.F. Dolmanova et al.: "Enzymatic methods of analysis", pages 1042-1081, see pages 1063-1065, 1076,1077 X,P US, A, 4657854 WEGFAHRT) 14 April 1987, see column 7, table I; claims 1-14 A EP, A, 0207392 (MILES LABORATORIES, INC.) 7 January 1987 see page 5, lines 13-15; page 6, lines 1-10; page 12, line 21 page 14, line 1, A Chemical Abstracts, vol. 84, no. 3, 19 January 1976 (Columbus, Ohio, US) G. Czerwenka et al.: "Analytical use of cryptates for the volumetric determi- nation of lithium, sodium, and potassium in aqueous solutions" see page 585, abstract no. 25385b Fresenius'Z. Anal. Chem. 1975, 276(1)37-40 Y US, A, 2791533 SEGAL et al.) 24 February 1951, see column 2, lines 15-18, 34-48; column 3, lines 30-32,

40-52 A Y Journal of Analytical Chemistry OF USSR, vol. 35, no. 8, Part 2, August 1980 (New York, US) I.F. Dolmanova et al.: "Enzymatic methods of analysis" pages 1042-1081, see page 1076, lines 28-36 A Chemical Abstracts, vol. 84, no. 3, 19 January 1976 (Columbus, Ohio, US) C. Czerwenka et al.: "Analytical use of'cryptates for the columetirc determi- nation of lithium, sodium, and potassium in aqueous solutions", see page 585, abstract no. 25385b Fresenius'Z. Anal. Chem. 1975, 276(1)37-40 1-8,12,13 1-5,7,12,31 1,12,13,17 1,12,17,18, 32,34,36,38 39-44 14 21 14 15-17,20, 22,24,26,32 34,36,38 Form PCT ISA 210 (extra sheet) (January 1915) International Apolication No. PCT/EP 88/00275 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V. OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This International search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1.E Claim because they relate to subject matter not required to be searched by this Authority, namely: 2. Claim because they relate to parts of the International sapllcatlon that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out. spe tficany: 3. Claim because they are dependent laims and are not drafted In accordance with the second and trd sentences of PCT Rule 6.4(a). I VIl. OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING I This International Searching Authority found multiple inventions In this International applicaton as follows: Claims 1-12, 31-48 Claims 13,15-18, 29 30,32-48 Claims 14-17, 19-28, 32-38 Please refer to Form PCT/ISA 206 dated 15th July 1988 1. As all required additional search fees were timely paid by the applicant, this international search report covers all searchable clalms of the international application. 2. As only some of the required additional search fee were timely paid by the applicant, this International search report covers only those claims of the International appllic'lln for which fees were paid, specifically claims: 31 No required additional search fees were timely paid by the applicant. Consequently, this International search report is restricted to the invention first mentioned in the claims: it is covered by claim numbers: 4. As all lealrchbl climl could be searched without effort justifying an additional fe. the International Searching Authority did not invite payment of any additional foe. Remark on Protest The additional search fees were accompanlid by applicant's protest. C No protest accompanied the payment of additional search fees. Form PCTIISA/210 (supplemental sheet (January 19S) :\c a P B il e ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 8800275 SA 21679 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 27/09/88 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date DE-A- 3614470 20-11-86 None US-A- 4657854 14-04-87 None EP-A- 0207392 07-01-87 AU-A- 5946386 08-01-87 JP-A- 62009271 17-01-87 US-A- 4645744 24-02-87 AU-B- 573449 09-06-88 US-A- 4649123 10-03-87 US-A- 2791533 None i SFor more details abouthis annexsee Official Journal of the European Patent Office, 2/82 For more details about this annex see Official Journal of the European Patent Office, No. 12/82 LI i;l