MXPA97007282A - Stabilized reagent using the coenz reduction system - Google Patents

Abstract

The present invention relates to a reagent for the enzymatic determination of the concentration of an analyte in a patient where it measures the degree of oxidation of the coenzyme, characterized in that said reagent is stabilized against oxidation by a coenzyme reduction system that it comprises an enzyme pair and substrate selected in such a way as to allow the continuous regeneration of said coenzyme during the storage of said reagent. An improvement of an anzyme method of determining the concentration of an analyte in a body fluid sample where the degree of oxidation of a coenzyme is measured, the improvement comprises the stabilization of a reagent containing said coenzyme against oxidation is also shown. by a coenzyme reduction system comprising an enzyme pair and substrate selected in such a way as to allow the continuous regeneration of said coenzyme during the storage of said reagent. Reagents are also exposed for the determination of aspartate aminotransferase, alanine aminotransferase, ammonia and ur

Description

STABILIZED REAGENT USING THE COENZIMA REDUCTION SYSTEM This invention relates to the reagents used in the enzymatic methods of determining the concentration of analytes (substance to be analyzed) in a body fluid sample. In particular, this invention relates to the reagents used in the methods wherein the amount of an oxidized coenzyme in the reaction sample corresponds directly to the concentration of analyte present in the sample. The invention also relates to improved methods for carrying out the determination of the analyte concentration. Analytes that can be determined by the reagents of the invention include transaminases, ammonia, urea, lactate dehydrogenase, triglycerides and salicylate. Aspartate aminotransferase is an enzyme found at high levels in the heart, liver, erythrocyte cells and skeletal muscle. Catalyze the following reaction: aspar + 2-oxoglutatate ** • oxaloacetate + glu size Increases in serum levels of aspartate aminotransferase were found in many diseases of the REF: 25713 liver where there is cell destruction, especially in, for example, hepatitis. The levels are also improved after myocardial infarction and in muscle disease. The enzyme alanine aminotransferase was also found in high concentrations in the liver and to a lesser extent in the heart, kidney and skeletal muscle. Catalyze the following reaction: alanine + 2-oxoglutarate * pyruvate + glu tama ts Increases in serum level of this enzyme are usually found in liver diseases, especially hepatitis. Indirect quantification of enzymes, in particular, the transaminases aspartate aminotransferase and alanine aminotransferase in body fluid samples could include contrasting a "blank" sample against a sample in which the enzymatic conversion of an analyte associated with the analyte was carried out. enzyme of interest. To achieve the enzymatic conversion of the analyte, the specific substrate enzyme (transaminase) is allowed to act on known substrates of the enzyme for use in quantification of the enzyme of interest.

The change in the reaction composition with respect to White can be calculated by several methods which measure the change in absorbance of the composition. The change in absorbance directly correlates the amount of transaminase present in the sample. While traditional methods such as colorimetric determination have been proven adequate, enzyme analysis has been shown to be considerably more accurate, reliable and simpler than the other methods that come to determine transaminase levels. A transaminase quantification method commonly used in a sample is a kinetic method that uses a coupled enzyme reaction. In the case of aspartate aminotransferase (AST), the oxaloacetate formed by AST is converted to malate including malate dehydrogenase (MDH) in the assay system. This is accompanied by the oxidation of the coenzyme nicotinamide adenindinucleotide (NADH to NAD *) which can be followed spectrophotometrically at 340 nm. In this way the reaction sequence is commonly as follows: L - aspartate + 2-oxoglutarate AS * oxaloacetate + L oxaloacetate + NADH MDH • - L - malate + NAD * sample pyruvate + NADH - LDH • > lactate + NAD * The third reaction is required to eliminate the potential presence of high levels of pyruvate in patient samples. The previous theory that includes high levels of the enzyme lactate dehydrogenase (LDH) is that if high levels are included in the reagent, in the event that a patient sample has a high level of pyruvate, the presence of NADH and LDH will rapidly eliminate the pyruvate in the sample, converting it into a lactate which will not interfere with the reaction. The need to introduce the reagent with LDH to eliminate this side of the reaction can affect the stability of the reagent by introducing more contaminants. For alanine aminotransferase (ALT), the pyruvate formed by means of ALT is converted to lactate including lactate dehydrogenase in the reaction mixture. This is accompanied by the oxidation of the coenzyme NADH to NAD * which can be followed again spectrophotometrically at 340 nm. In this way the Reaction sequence is carried out using the reagent as follows: L-alanine + 2-oxoglutarate - A - - * > pyruvate + glutamate pyruvate + NADH - LDH »lactate to + NAD * sample pyruvate + NADH - LDH lactate + NAD * The theory and methodology of ALT measurement is similar to the AST reagent with the exception that with ALT only an endogenous enzyme, ie LDH is required for measurement (more than the requirement for LDH and MDH) with the AST). As such, fewer contaminants are introduced into the ALT reagents which in general means that their reconstituted shelf life may be a bit longer than that of the AST reagents. For both reactants the rate of NAD * formation correlates to the transaminase concentration originally present in the sample. Urea is the main metabolic product that it contains nitrogen of protein catabolism, being synthesized in the liver by means of hepatic enzymes of the liver and excreted predominantly through the kidneys. Elevated levels of urea in the serum could be a consequence of poor kidney function, liver disease, dietary changes, congestive heart failure, diabetes, and infections. The level of urea in human serum and urine could be detected by direct and indirect methods. Direct methods usually involve variations of the Fearon reaction. In this reaction system, diacetyl reacts with urea to form the chromogenic diazine, which could be measured spectrophotometrically by its high absorbance at 540 nm. The most common method of measuring urea in human serum and urine involves an indirect coupled enzyme reaction system. Urease, the first enzyme in the reaction system, is used to convert urea into ammonium and bicarbonate ions. Glutamate dehydrogenase (GLDH), the second enzyme in the reaction system, is coupled with NADH and the ammonium ion to produce NAD * and glutamate. This reaction is monitored spectrophotometrically at 340 nm, as NADH converts to NAD *. Alternatively, the ammonium ion could be quantified by means of potentiometry or conductimetry.

In this way, the reaction process commonly used to determine the urea concentration is as follows: Urea + H20 UreaS ^ 2NHi + C02 NHr3, +? -c? Tsglutarats + NADH - ^^ L-glutamate + NAD ' The decrease in absorbance is measured at 340 nm as NADH is converted to NAD * and this is proportional to the concentration of urea in the original sample. The main source of ammonia circulation is the gastrointestinal tract. Ammonia is metabolized in the liver, being converted to urea in the Krebs-Henseleit cycle. Elevated levels of ammonia in human serum are more frequently associated with the progression of liver disease. Hyperammonemia has a toxic effect on the central nervous system. The level of ammonia in human serum is most commonly measured by a direct method, an enzymatic step method, incorporating glutamate dehydrogenase. In this reaction, the conversion of ammonia, ar-ketoglutarate and NADH (or NADPH) to glutamate and NAD * (or NADP *) is measured spectrophotometrically at 340 nm. The reaction sequence commonly used to determine the ammonia concentration of a sample is as follows: NH, + a-ketoglutarate + NADPH G DHy L-gl u tama ts + NADP * The decrease in absorbance at 340 nm according to NADPH is converted to NADP * it is measured and this is proportional to the concentration of ammonia in the patient's sample. Historically, as mentioned above, the transaminase reagents have undergone low reconstituted stability. The stability of these reagents especially in a single-format vial is usually limited to approximately a maximum of one month under refrigeration conditions. The cause of this instability could be attributed to the deterioration of the endogenous ingredients in the reagents in addition to the instability of the NADH in solution. The main causes of the instability of NADH in solution are directly related to the presence of enzymes of endogenous reagents ie LDH and MDH in the reagent of AST and LDH in the reagent of the ALT. Commercial preparations of the endogenous enzymes MDH and LDH, both of animal or microbial origin, contain contaminants that lately affect the reconstituted stability of NADH and consequently the stability of the reagents. These contaminants are usually at low levels of AST and ALT, the enzymes to be measured and NADH oxidase, which initiate the oxidation of NADH in the reagent. The pH of the reagent may also have an effect on the stability of NADH since NADH will decompose rapidly in solution, especially in an acid medium. Most of the reagents for the determination of transaminase are formulated with a pH range of 7.3 to 8.0. The higher the alkalinity of the reagent, the greater the stability of NADH in solution. In addition, the reactants ammonia and urea also suffer from stability problems and the stability of these reagents in a single format vial and at refrigeration temperatures, has usually been limited to a maximum of one month in the case of ammonia and two months in the case of urea. The cause of this instability could be attributed to the deterioration of the endogenous ingredients in the reagents, the instability of NADH or NADPH in solution and the contamination by ammonia present in water used to reconstitute the powder reagent. The main causes of instability of NADH or NADPH in solution are directly related to the presence of endogenous reactive enzymes. The pH of the reagent can also have an effect on the instability of NADH or NADPH since NADH will decompose in solution, especially in an acid medium. NADPH is commonly employed in ammonia reagents (preferably NADH) to overcome the interference in the assay by endogenous lactate dehydrogenase in patient's serum. The endogenous lactate dehydrogenase and the pyruvate in the patient sample will react specifically with NADH in the following reaction sequence: sample pyruvate + NADH - • > lactate + NAD * The maintenance of? ADP? in solution will also be affected by the presence of contaminants of commercial preparations of glutamate dehydrogenase, which will initiate the oxidation of? ADP? in the ammonia reagent. Also, the presence of pollutants from Commercial preparations of urease and glutamate dehydrogenase will initiate the oxidation of NADH in the urea reagent. One means of overcoming this difficulty relating the stability of NADH and NADPH in solution has been to generate reduced coenzyme in the reagent just before use. One of these methods is described in Australian patent application AU-A-61906/90 by F. Hoffmann La Roche AG with particular attention to similar enzyme systems for the measurement of bicarbonate and serum ammonia. In this exhibition, the reduced coenzyme is generated in situ simultaneously with or before the reoxidation of the coenzyme by the analyte, substrate and specific enzymes. This is achieved by including in the reaction mixture an enzyme and substrate of the enzyme capable of reducing the oxidized coenzyme. The specific reaction exposed and favored by F. Hoffmann La Roche AG is: NAD + + Glucose-β-Phosphate > H * + NADH + 6-phospho- (G-ß-P) Glucose-6-glucolactopa phosphate dehydrogenases * (G-ß-P-DH) This makes it possible to reduce nicotinamide adenosindinucleotide. The problem associated with this form of generation of NADH is that a stable simple reagent vial configuration is not possible. To some extent F. Hoffmann La Roche AG have overcome this problem by dividing the reactive system into two vials. The first reagent comprises in the case of the ammonia quantification, NADP * and G-6-P, and the second reagent a-ketoglutarate, G6PDH and GLDH. The determination of the reaction therefore proceeds as follows: Reagent j Reagent 2 NADP + + G-ß-P -x-cßtoglutarate + G 6 P D + G L D H (A) Serum from patient added reduced coenzyme patient serum (B) added oxidized coepzyme where (A) and (B) represent alternative, equivalent routes.

The difficulties remain, however, with this reactive system. Apart from the fact that these two reactive ampoules are required, thus increasing cost, inventory and expense, very precise levels of glucose-6-phosphate are required and in addition, the system is limited to the use of specific chemical analyzers. As soon as the reagents are combined, the generation of NADH from NAD * occurs by the depletion of glucose-6-phosphate. Because glucose-6-phosphate is thus depleted the stability of the combined reagent could be severely impaired if the two reagents were to be combined and not used immediately. If excess or inappropriate levels of glucose-6-phosphate are present, the time associated with the incubation of the reagent becomes critical. The results could be falsely low in absorbance changes and extremely inappropriate. A premature solution also relates to the measurement of the levels of analyte described US Patent 4,394,449 by Modrovich using substrate / enzyme pairs to generate the reduced coenzyme as does the Roche solution, however, in this case glucose-6-phosphate is generated from glucose according to the following: D-glucose + ATP ^ ex0CÍnas ^ App + glucose- - phosphate The NAD * then reacts with the glucose-6-formed in the presence of the enzyme glucose-6-phosphate dehydrogenase to form NADH. Modrovich also includes NADH and NAD * in the formulation so that when NADH is oxidized or destroyed, the NAD * present in the reagent will facilitate the regeneration of NADH. This is also a vial of two reagents. An initial alternative is provided by Klose et al in US Patent 4,019,961. This invention is based on several separate reaction steps and an enzymatic system for regenerating NADH. This system has the disadvantage of depending on carrying out several reaction stages and separation steps making this test consume time. In addition, this reactive system is only suitable for substrates which can be phosphorylated. The general problem associated with the mechanism of generation of NADH and NADPH adopted for each of the inventions described above, this is NAD * + glucose-6 -fo fato - > AHJPH + 6-fosfsglucsnslactone + H is that a simple reaction stage using a single vial is not possible because as soon as the patient's serum is added to the reagent, two simultaneous reactions occur: (a) a decrease in absorbance due to NADH (or NADPH) being converted to NAD * (NADP *), (b) generation of NADH (NADPH) from NAD * (NADP *) resulting in an increase in absorbance. These two reactions occur at similar speeds with the end result being a falsely low change in absorbance and extremely inappropriate results.

Accordingly, it is an object of this invention to provide a reactive system for use in the determination of serum analyte levels that substantially alleviate the problems of the reactive systems of the prior art used in enzyme analysis of serum analyte levels depending on the oxidation of a coenzyme, particularly those problems that relate to the endogenous or exogenous contamination of the reagent. It is a further object of the invention to provide an improved method of determining the concentration of analyte levels in a patient sample, the method overcomes the problems associated with the methods of the prior art including premature oxidation of the coenzyme determinant and the need for a multi-loop system to minimize reagent degradation. For this purpose, a reagent is provided for the enzymatic determination of an analyte concentration in a patient wherein the degree of oxidation of the coenzyme is measured, characterized in that said The reagent is stabilized against oxidation by means of a coenzyme reduction system containing a selected substrate and enzyme pair to allow the continuous regeneration of said coenzyme during the storage of said reagent. A reagent for the enzymatic determination of the transaminase concentration in a patient is also provided wherein the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation is measured by means of a coenzyme reduction system containing a substrate and enzyme pair selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. A reagent for the enzymatic determination of the concentration of aspartate transaminase in a patient is also provided wherein the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation by means of a coenzyme reduction system containing a selected substrate and enzyme pair to allow continuous regeneration of said coenzyme during the storage of said reagent. A reagent for the enzymatic determination of alanine aminotransferase concentration in a patient is also provided wherein the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation is measured by means of a coenzyme reduction system containing a substrate and enzyme pair selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. A reagent is also provided for the enzymatic determination of the urea concentration in a patient wherein the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation is measured by means of a coenzyme reduction system containing a par substrate and selected enzyme to allow the continuous regeneration of said coenzyme during the storage of said reagent. A reagent is also provided for the enzymatic determination of ammonia concentration in a patient where the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation by means of a coenzyme reduction system containing a selected substrate and enzyme pair to allow the continuous regeneration of said coenzyme during the storage of said reagent. Preferably, the coenzyme reduction system comprises an enzyme and a substrate, said enzyme having incomplete specificity for said substrate resulting in this way at a reduced rate of cross-reactivity. The reagent is preferably in a simple tablet configuration. According to this specification the term "incomplete specificity" is used with respect to the enzyme and substrate pairs wherein the selected substrate is not the natural substrate of the selected enzyme and thus has less than 100% cross-specificity for the respective enzyme . This invention is based on the discovery that by coupling an enzyme and substrate having incomplete specificity for both, the rate of coenzyme reduction is considerably slow. By retarding the reduction reaction, the essential components of the reagent can be contained in an ampoule of storage, the contents are stabilized against contamination by the low level of continuous regeneration of the coenzyme. By delaying the process, the regeneration of NADH or NADPH may occur without affecting the measurement of the analytes. Regeneration can occur in the reagent when it is not in use and the rate at which regeneration occurs can be well adjusted by adjusting the nature of the selected enzyme / substrate pair and the levels thereof. In an alternative embodiment of the invention, a reagent is provided for use in an enzymatic determination of an analyte concentration in a patient wherein the degree of oxidation of a coenzyme characterized in that said reagent is stabilized against oxidation by means of a coenzyme reduction system containing an enzyme pair and substrate to allow the regeneration of said coenzyme at a rate of 0.01-0.9 mAbs / min at 340 nm. Preferably the rate of regeneration in a reagent according to this aspect of the invention is 0.05-0.4 mAbs / min and more preferably the regeneration rate is 0.05-0.4 mAbs / min at room temperature (18-25 ° C) and at 340 nm . In a preferred embodiment of the invention, the degree of specificity between the substrate and the enzyme of the system of coenzyme reduction is preferably less than 100% more preferably less than 50% and more conveniently less than 10% on an equimolar basis. Optimally, an enzyme / substrate pair having a cross reactivity of less than 5% on an equimolar basis could be used. The coenzymes preferably used in the reagent according to the invention are reduced nicotinamide adenyndiucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) although analogous coenzymes such as nicotinamide phosphate hypoxanthineaducleotide or thio-NADH could be appropriate. Surprisingly, it has been found that reagents of the invention provide an additional advantage, particularly for ammonia and urea reagents. Levels of NADH and / or NADPH will be reduced in the reactants of urea and ammonia by the presence of contaminating ammonia, introduced with the water used to reconstitute the powder reagent. Also, the ammonia in the air that dissolves in the liquid urea / ammonia reactants consumed will reduce the levels of NADH and / or NADPH. The presence of a contaminating ammonia in ammonia and urea reagents can not only lead to inappropriate determinations of urea and ammonia, but it may mean that the reaction with α-ketoglutarate and NADH in the presence of GLDH will occur earlier to the samples being added. This leads to reduced levels of NADH or NADPH and thus can lead to errors in the determination of ammonia and urea concentrations in samples. However, the present invention allows the removal of contaminating ammonia and also the regeneration of NADH or NADPH to allow the proper determination of ammonia and urea concentrations in patient samples. The enzymes preferably used in the coenzyme reduction system for the determination of the transaminase content of a serum sample could be glucose-6-phosphate dehydrogenase (G-6-P-DH) or glucose dehydrogenase. The enzymes preferably used in the coenzyme reduction system for the determination of the urea or ammonia content of a patient serum could be glucose-6-phosphate dehydrogenase (G-6-P-DH) or glucose dehydrogenase. Enzymes such as formate dehydrogenase, glycerol dehydrogenase, leucine dehydrogenase, L-Alanine dehydrogenase, 3of-Hydroxy-steroid Dehydrogenase, L-lactate dehydrogenase (from Lactobacillus sp.) Or 3-phosphate dehydrogenase may also be appropriate. The preferred enzyme that is used for the transaminase / ammonia and urea level determination reagents is glucose-6-phosphate dehydrogenase. This could be obtained from any appropriate source such as Leuconostoc meganteroides. Bacillus stearothermophilus. Zy QmQnas mousus or yeast. Such enzymes are preferably derived from microbial sources. The incorporation into the reagent of enzymes from microbial sources have been found to minimize the presence of endogenous contaminants such as NADH oxidase and protease that previously strongly affect the stability of the reagents. Microbial enzymes also have the additional advantage of being more thermostable in this way by improving their long-term stability in solution. The most preferred source of glucose-6-phosphate dehydrogenase is euconQStQC Meseateroides. If glucose-6-phosphate from Bacillus Stearothermophilus or Zymomonas Mobilus is used. the reaction rate is reduced. Similarly, if yeast is used as the source of glucose-6-phosphate dehydrogenase, the coenzyme NADPH has to be used as an alternative for NADH since the yeast glucose-6-phosphate dehydrogenase is only specific for NADP *. The appropriate amount of glucose-6-phosphate dehydrogenase present in the reagents according to the invention will vary according to the desired regeneration rate. Particularly preferred for the AST reagent, however, is an amount of about 200 U / L to allow for deterioration consumed in solution. For ALT the preferred concentration in particular is 2000 U / L. A preferred concentration for the urea reagent is 2000 U / L and a preferred concentration for the ammonia reagent is 3500 U / L. Having the purpose that the selection of substrate and enzyme should be such that in the coenzyme reduction system have incomplete specificity for one and the other, substrates suitable for use in the reagent according to the invention include ribose-5-phosphate, glucose -1-phosphate, 6-phosphogluconic acid, 2-deoxyglucose-6-phosphate, 2-deoxy-2-fluoroglucose-6-phosphate, 2-deoxy-2-chloroglucose-6-phosphate, 2-deoxy-2, 2- difluoroglucose-6-phosphate, 2-0-methylglucose-6-phosphate, mannose-6-phosphate, glucosamine-6-phosphate, 3-deoxyglucose-6-phosphate, 3-deoxy-3-fluoro-glucose-6-phosphate, 3-0-methylglucose-6-phosphate, allose-6-phosphate, arose-6-phosphate, 4-deoxy-4-fluoro-glucosyl-6-phosphate, galactose-6-phosphate, 5-thio-glucose-6-phosphate, phosphonate analogues, glucose- 6-stalate, β-D-glucose, D-galactose, 2-deoxyglucose, arabinose, xylose, 1-sorbose, D-mannose, D-fructose, D-lactose, D-sorbital, D-mannitol, sucrose, inositol, maltose. Using NADH as the preferred coenzyme in the reagent, the preferred enzyme / substrate combination is glucose-6-phosphate dehydrogenase (G-6-P-DH) / D-glucose. Preferred alternative substrates for D-glucose are those for which, in relation to the specificity between glucose-6-phosphate (G-6-P) and G-6-P-DH, the reaction rate between the G-enzyme -6-P-DH and the selected substrate is less than 50%, more preferably less than 10% and more preferably less than 5%. Again, one has in mind the required regeneration rate, the most appropriate D-glucose level for the reagents according to the invention, and therefore the preferred ones are approximately 100 mmol / L although levels of up to 1000 could be used. mmol / L. The solubility of D-glucose in the reagent becomes a problem at the highest concentration levels. Where the preferred combination of D-glucose / glucose-6-Phosphate dehydrogenase is used, the potassium phosphate ions could be introduced into the composition in the form of potassium dibasic phosphate. A level of variation of phosphate ions could be suitable depending on the speed of regeneration desired. However, when the concentration of D-glucose is approximately 100 mmol / L (but can vary between 20 and 200 mmol / L) for example, and the corresponding level of glucose-6-phosphate dehydrogenase is approximately 2000 U / L (but can vary between 500 and 3500 U / L), the level of phosphate ions that could be adequate would be in the range of 2.0 mmol / L up to 20 mmol / L. Increasing the concentration of phosphate ions will increase the rate of regeneration. The preferred level of phosphate ion addition is about 10 mmol / L for AST and about 5 mmol for ALT. For the urea reagent the preferred phosphate ion concentration is about 5 mmol and is also about 5 mmol for the ammonia reagent. When the preferred combination of D-glucose and glucose-6-phosphate dehydrogenase is used, or in any system where free phosphate is not actually generated, it is essential to incorporate free phosphate ions into the reagent. In particular, free phosphate ions are required to form a non-specific complex with D-glucose to initiate regeneration in the presence of glucose-6-phosphate dehydrogenase. A preferred alternative for the use of D-glucose / G-6-P-DH is the use of glucose dehydrogenase (GLD) according to the following reaction where D-glucose is 100% reactive substrate: D-glucose + NAD * CLD > D-glucan-b-1 &ctona + NADH + H * If glucose dehydrogenase is used as the enzyme, the preferred substrates for the reduction of coenzyme NAD and their relative degree of cross-reactivity when compared to D-glucose are: Substrate Relative activity xylose 8.9% L-sorbose 0.3% D-mannose 2.4% D-fructose 0.8% D-galactose 0.1% D-lactose 1.2% D-sorbitol 0.1% Inositol 0.2% Maltose 3.9% where the values represent the relative reaction rate for glucose dehydrogenase in the presence of the natural substrate lß-D-glucose. Alternatively, using glycerol dehydrogenase (GLY.DH) as the enzyme, appropriate substrates in the reaction glycerol + NAD * GLY-DH dihydxsxiacetona + NADH + H * and its relative activities for glycerol (100%) are: Substrate Relative activity glycerol-oí-monochlorohydrin 48.5% Ethylene glycol 7.8% 2, 3-butanediol 52.6% where leucine dehydrogenase (L.D) is used as the enzyme according to the reaction substrate + NAD * Hp ___ '__ tt-ketoisocaproate + NHy + NADH + H * The appropriate substrates and their relative activities for L-leucine (100%) are Substrate Relative activity L-valine 74% L-isoleucine 58% L-norvaline 41% L-norleucine 10% L-methionine 0. 6% L-cysteine 0. 3% If L-alanine dehydrogenase (A.D) is used as an enzyme in a similar reaction system for the use of leucine dehydrogenase, an appropriate substrate and its relative activity for L-alanine (100%) is Substrate Relative activity L-serine 5% The 3-hydroxysteroid dehydrogenase (H.DH) could also be used as an enzyme in combination with the substrates listed above. Its relative activities for cholic acid are also listed.

Substrate Relative activity Lithocholic acid 96% Etiocholic acid 60% Where, L-lactate dehydrogenase (LDH) from Lactobacillus sp. it is used as the enzyme in the following reaction, pyruvate + NADH + H * LDH > L-lactats + NAD ' The substrates and their relative activities for L-lactate are: Substrate Relative activity 2-oxoglutarate 0.09% oxoloacetate 36% Where NADP * is the coenzyme, for example yeast, the preferred substrate / enzyme combinations are: G-6-P-DH / galactose-6-P 25% G-6-P -DH / 2-deoxyglucose-6-P 18% G-6-P-DH / glucosamine-6-P 2% The values on the right side represent the relative reactivity for a pair of G-6-P-DH / G- 6-P. It is also possible to use NADP * as a coenzyme to combine the enzyme / substrate glycerol-3-phosphate dehydrogenase with dihydroxy acetone phosphate. As described in the preamble to this specification, the other requirements of a reagent according to the invention for use in the determination of serum AST levels are lactate dehydrogenase, nicotinamide adenindinucleotide, (NADH) reduced, malate dehydrogenase (MDH), aspartate and 2-oxoglutarate. In the case of ALT, malate dehydrogenase is not required and instead of aspartate, L-alanine is required. in the case of the urea reagent, urease and -ketoglutarate are also required, while of-ketoglutarate is also required in the ammonia reagent. Aspartate is available as a variety of salts, such as sodium and potassium salts. The preferred salt according to the invention is potassium salt since it appears to be more soluble and is less hydrated than the sodium salt. A range of concentration that could be acceptable in the reagents of the invention is 180-240 mmol / L. More preferred is a final concentration of about 200 mmol / L and it is noted that this is the recommended level of IFCC. The considered range of 2-oxoglutarate considered to be the one that is preferred for the reagents of the reaction is approximately 1-15 mmol / L, however it is observed that high concentrations of this substrate could limit the amount of NADH that can be added to the composition since 2-oxoglutarate absorbs at 340 nm causing absorbance that interferes with the absorbance of NADH. Limiting the amount of 2-oxoglutarate When added to the composition, the reagent can be used in more spectrum analyzers without difficulty. A preferred concentration of this substrate for the reactants of AST and ALT is approximately 12 mmol / L, which is again the level recommended by the IFCC. For the urea and ammonia reagents the preferred concentration is about 7.5 mmol / L. The amount of alanine present in the ALT reagent is governed to some extent by the solubility of this component. In particular, a preferred rengo is 200-500 mmol / L although at the highest end of the range there is an appreciable increase in catalytic activity. The most preferred concentration of this substrate is about 400 mmol / L for the solubility ratio of this substance. The level of the coenzyme in the reagent will vary according to the following factors: linearity required in the chosen wavelength measurement - sample for the volumetric ratio of the reagent photometric system of the selected analyzer. In general, increasing the sample volume improves the sensitivity but decreases the linearity of the reading obtained, while decreasing the volume of the sample improves the linearity at the expense of losing sensitivity. The preferred measurement wavelength is 320-400 nm, however, the level of coenzyme used should be adjusted such that the absorbance does not exceed 2.0 A. The preferred absorbance wavelength according to the invention is 340 nm. MDH is preferably obtained from microbial sources to limit the risk of endogenous contamination and because it exhibits superior characteristics with respect to thermal stability. Appropriate levels are in the range of 150-1500 U / L, more preferably 200-800 U / L. The most preferred level according to the invention is approximately 250 U / L. In the AST reagent, LDH participates in the removal of endogenous pyruvate from the sample. The level of LDH preferably incorporated in the reagent of the AST of the invention was such that a sample of 1O mmol / L of pyruvate was rinsed in one minute using a sample to reagent ratio of 1:10. This level was determined to be approximately 2000 U / L. In the ALT reagent, LDH participates in two reactions, (i) the reaction of the enzyme coupled for the measurement of ALT, and (ii) the removal of endogenous pyruvate from the sample. The level of the incorporated LDH, as with the reagent of the AST was such that the reagent It would remove up to 1.0 mmol / L of pyruvate from the sample in 1 minute. The main reaction can then be measured after 1 minute without interference of the endogenous pyruvate from the sample. The minimum level of LDH required to clear the endogenous pyruvate was determined to be approximately 1500 U / L. The preferred amount incorporated in the ALT reagent of the invention was about 2000 U / L. In the urea reagent, the minimum required urease activity at pH of 8.5, since the enzyme not limited in speed in the kinetic form of the test is approximately 5000 U / L. Excess amounts of this could be included in the increase in the long-term stability of the reagent. The preferred way of measuring the analyte in the urea and ammonia reagents is based on kinetic principles. Thus glutamate dehydrogenase (GLDH) is the enzyme that limits the speed in the formulation, the activity level of the GLDH included in the reagent is critical for the linearity of the test. The level of activity of the GLDH required will also vary as a function of the pH of the reactant system. An adequate range of GLDH activity could vary between 250-10000 U / L. The most suitable enzymes are those of microbial origin, since the preparation of the GLDH Commercial sources from animal sources are less stable and appear to contain higher levels of NADH oxidase activity as a contaminant. The activity of the preferred GLDH for the urea reagent at pH of 8.50 is about 500 U / L and for the ammonia reagent, at pH of 8.50 is about 8500 U / L. The reagents according to the invention could also include the reduction system of coenzyme and other essential substrates and enzymes necessary to determine the concentration of analyte, preservatives, chelating agents, surface activity agents, protease inhibitors, buffers, cofactors, antibacterials and other constituents that develop stability that improves the functions but does not materially affect the characteristics of the invention. The main criterion for selecting a buffer is such that it will have good buffering capacity at the selected pH with minimum bond of divalent cation. The pH and buffer system for the reagents of AST and ALT are selected according to the recommendations of the International Federation of Clinical Chemistry (IFCC) for the measurement of transaminases. A general heuristic rule is that a buffer could be considered effective if its pKa is ± 1.0 pH units from the chosen pH. A pH Preferred of the reagent according to the invention is 7-9. For aspartate transaminase, the optimal catalytic activity occurs at approximately pH 7.0-8.2 at 30 ° C. The most preferred pH for the AST reagent is about 8.1 ± 0.1 at 20 ° C while at this pH the NADH is stable. For the ALT reagent, the maximum catalytic activity occurs in a pH range of about 7.3-7.9 at 20 ° C. The most preferred pH for the ALT reagent is approximately 7.7 at 20 ° C. At these preferred pHs, an agreement is reached between the optimal activity of the enzyme and the stability of the enzymes and coenzyme in solution. A lower pH could result in the increased degradation of the coenzyme.

The preferred buffer system for the urea reagent is Tris at pH 8.50, although the range of pH 7.5-9.5 could be considered acceptable. The preferred buffer concentration for the effective buffering capacity is 100 mM Tris, although the Tris range of 20-200 mM could be used. A wide range of alternative dampers could be used in this reaction system, which provide effective damping capacity in the pH range of 7.5-9.5.

The preferred buffer system for the ammonia reagent is Tris at pH 8.5-9.0, although anyone in the range of pH 7.5-9.5 could be considered acceptable. The The preferred buffer concentration for the effective buffering capacity is 100 mM Tris, although any could be used in the 20-200 mM Tris range. Suitable buffers for the AST and ALT reagents include HEPES, 4-morpholine propanosulphonic acid (MOPS) or 2- [tris (hydroxymethyl) methylamino] -1-ethanesulfonic acid (TES) or diethanolamine or other shock absorbers GOOD, tricine, bicina, TEA and TAPS, TAPSO and POPSO. The preferred buffer according to the invention is TRIS which preferably has a total concentration of 30-150 mmol / L, and more preferably about 70-100 mmol / L, although about 80 mmol / L is preferred. At higher buffer concentrations the AST is increasingly inhibited. It is observed that the phosphate buffers seem to increase the decomposition rate of NADH and inhibit the association of pyridoxal-5-phosphate (P-5-P) with the apoenzyme transaminase. The sample to be tested could be diluted with any suitable diluent if desired, such as deionized water or saline. In addition to the appropriate shock absorbers mentioned above for the reactants of AST and ALT, the following GOOD shocks are also suitable for ammonia and urea reagents: CAPSO, CHES and AMPSO. Condoms such as azide are appropriate of sodium (NaN3), hydroxybenzoic acid, gentamicin, thymol and mercury-free preservatives available from Boehringer Mannheim such as methylisothiazolone. The appropriate level is such that it maintains its preservative properties for at least 6-8 months when stored at 2-8 ° C without inhibiting the enzymes present in the reagent. An adequate range that meets these criteria is 0.1-1.0 g / L. A variety of chelating agents such as EDTA, EGTA, N- (2-hydroxyethyl) ethylenediamine triacetic acid (HEDTA), etc. they are also suitable as non-specific stabilizers. In the reagents of the AST and ALT of the invention, EDTA was preferably used at a level of approximately 2.0-10.0 mmol / L to stabilize the 2-oxoglutarate. This is available as a tetrasodium salt in addition to a potassium salt, but the preferred salt according to the invention is the disodium salt. In the urea and ammonia reagents, EDTA is present in the range of 0.2 to 10 mM. A particularly preferred concentration of EDTA is 1 mM. Enzyme stabilizers could also be incorporated into the reagents of the invention. A preferred stabilizer is Bovine Serum Albumin, free grade of protease. Others that are appropriate may include gamma globulin of bovine, N-acetyl cysteine and glycerol.

Suitable antifoaming agents could also be added if desired. Surfactants that could be used include Zwitterionic surfactants and non-ionic surfactants at levels that do not inhibit the enzymes present in the reagents. In another aspect of the invention there is provided an improvement in an enzymatic method of determining the concentration of an analyte in a sample of body fluid wherein the degree of oxidation of a coenzyme against oxidation is measured by a coenzyme reduction system that it contains an enzyme pair and substrate selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. An improvement in an enzymatic method of determining the transaminase concentration in a body fluid sample is also provided wherein the degree of oxidation of a coenzyme is measured, the improvement comprising the stabilization of a reagent containing said coenzyme against oxidation by a coenzyme reduction system containing an enzyme pair and substrate selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. Improvement in one method is also provided Enzymatic determination of aspartate aminotransferase in a body fluid sample in which the degree of oxidation of a coenzyme is measured, the improvement comprising the stabilization of a reagent containing said coenzyme against oxidation by a coenzyme reduction system containing an enzyme pair and selected substrate to allow the continuous regeneration of said coenzyme during the storage of said reagent. An improvement is also provided in an enzymatic method of determining alanine aminotransferase in a body fluid sample wherein the degree of oxidation of a coenzyme is measured, the improvement comprising the stabilization of a reagent containing said coenzyme against oxidation by a Coenzyme reduction system containing an enzyme pair and substrate selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. An improvement is also provided in an enzymatic method of determining the concentration of urea in a body fluid sample wherein the degree of oxidation of a coenzyme is measured, the improvement comprising the stabilization of a reagent containing said coenzyme against oxidation through a system of reduction of coenzyme containing an enzyme pair and selected substrate to allow the continuous regeneration of said coenzyme during the storage of said reagent. An improvement is also provided in an enzymatic method of determination of ammonia concentration in a body fluid sample where the degree of oxidation of a coenzyme is measured, the improvement comprising the stabilization of a reagent containing said coenzyme against oxidation by means of a coenzyme reduction system containing an enzyme pair and substrate selected to allow the continuous regeneration of said coenzyme during the storage of said reagent. In a preferred method according to this aspect of the invention, the enzyme of the enzyme pair and substrate has incomplete specificity for said substrate in this way by reducing the rate of cross-reactivity between the enzyme and the substrate. In a preferred embodiment according to this aspect of the invention, the coenzyme reduction system contains an enzyme and a substrate having a specificity for both, relative to the specificity of the enzyme for its natural substrate, is less than 100. %, preferably less than 50% and more conveniently less than 10%. More conveniently, the specificity of the enzyme / substrate pair for both relative to the specificity of the enzyme for its natural substrate is less than 5%, approximately 2% desirable. The selection of coenzyme, substrate and enzyme could be made from those mentioned above in relation to the reagents of the invention, depending on the analyte to be calculated. In one embodiment of this aspect of the invention, the components of the coenzyme reduction system used for the determination of the analyte concentration are NADH, G-6-P-DH and D-glucose such that the regeneration reaction takes place is D-glucose + NAD * g "6 ~ p ~ Di ^ NADH + gluconolactone Due to the low specificity of G-6-P-DH for D-glucose, this regeneration reaction is slow and thus is not competitive with the main reactions involved in the determination of analyte levels.

PREFERRED MODALITIES In a preferred embodiment of the invention, the ALT reagent essentially contains G-6-P-DH reduction system D-glucose coenzyme L-alanine substrate LDH specific substrate enzymes NADH coenzyme K2HP04 activator 2-oxoglutarate substrate In addition, TRIS buffer, TRIS-HCl, EDTA-disodium, Bovine Serum Albumin and sodium azide are preferably included. An ALT reagent formulated according to the invention is as follows: TABLE 1 In particular, a preferred reagent of ALT according to the invention is formulated as follows: TABLE 2 The formulation is concentrated 10% to allow the dilution of the sample.

In a preferred embodiment of the invention, the AST reagent essentially contains G-6-P-DH reduction system D-glucose coenzyme L-aspartate substrate LDH substrate specific MDH enzymes NADH coenzyme K2HP04 activator 2-oxoglutarate substrate In addition, TRIS buffer, TRIS-HCl, EDTA-disodium, Bovine Serum Albumin and sodium azide are preferably included. An AST reagent formulated according to the invention is as follows: TABLE 3 TABLE 3 (continued) In particular, a preferred reagent of the AST according to the invention is formulated as follows: TABLE TABLE 4 (continued) The formulation is concentrated 10% to allow the dilution of the sample. In a preferred embodiment of the invention, the urea reagent essentially comprises: G-6-P-DH reduction system D-glucose coenzyme Urease enzyme specific analyte a-ketoglutarate substrate NADPH coenzyme K2HPOt activator GLDH specific substrate enzyme A reagent formulated according to the invention is as follows: TABLE 5A In particular, a preferred urea reagent of according to the invention is formulated as follows: TABLE 5B In a preferred embodiment of the invention the ammonia reagent contains essentially: G-6-P-DH reduction system D-glucose coenzyme a-ketoglutarate substrate NADPH coenzyme K2HP04 activator GLDH specific substrate enzyme In addition, TRIS buffer, TRIS HCl, EDTA-disodium, ADP-K, bovine serum albumin and sodium azide are preferably included. An ammonia reagent formulated according to the invention is as follows: TABLE 6A In particular, a urea reagent according to the invention is formulated as follows TABLE 6B Although it is preferred that the reagents of the invention be formulated in a simple ampoule configuration, it is also possible that they are formulated in a configuration of two ampoules. The regeneration component of the formulation only needs to be incorporated in one of the two vials. In particular, the IFCC recommends that for ALT and AST reagents, 2-oxoglutarate is formulated as a separate component for the remainder of the formulation. Reagent A (excluding 2-oxoglutarate) could be incubated with the patient's serum for a period of 5-10 minutes during this time, all side reactions are allowed to complete. After the incubation period, the 2-oxoglutarate can be added to start the main reaction. As an alternative to using 2-oxoglutarate as the initiator component, it may also be possible to use aspartate or alanine in the same manner since the presence of 2-oxoglutarate protects AST or ALT from inactivation during side reactions. The regeneration system comprising unpaired pair of enzyme and substrate should be added to the component of the two-vial system including NADH. If a two-bulb system is used, it is recommended that the formulation include P-5-P since During the incubation period, in the presence of the sample, the addition of P-5-P to the serum activates the apo-enzymes and allows the measurement of the concentrations by the total catalytic activity of the AST and ALT in the serum allowing it to be complete saturation with P-5-P. The preferred level of P-5-P for use in a two-vial system is approximately 80-120 μmol / L, with a more preferred level being 100 μmol / L.

The stability of four particular reagents formulated according to the invention was tested as follows: FORMULATION: Reagent of the AST: TABLE 7A TABLE 7B Reagent of the AST: TABLE 8A TABLE 8B STORAGE CONDITIONS: covered and refrigerated (2-80C) SPECTROPHOTOMETRIC PARAMETERS (Shimadzu PC2101): reaction temperature 37 ° C sample volume to reagent 1:10 to 1:25 wavelength 340 nm length of the path in the vessel 1 cm The phase of delay of the measurement is approximately 1 minute or less and the time for measurement is up to 3 minutes after the delay phase. These spectrophotometric parameters were used to determine the following Initial absorbance of the reagent at 340 nm regeneration rate at 20 ° C (expressed in mAbs / min) The Cobas Mira was used to determine the recoveries of the control standards. The following results were obtained: TABLE 9A: RESULTS OF ABSORBANCE FOR THE DB AST AND ALT REAGENTS SHOWN IN TABLES 7A and 8A TABLE 9B: RESULTS DB ABSORBANCE FOR THE REAGENTS OF AST AND ALT SHOWN IN TABLES 7B and 8B The following Tables (Table 10A and 10B) provide evidence of the continuous functionality of the AST and ALT reagents for 7 months. For each run, a high and low serum content of AST and ALT was run using fresh prepared reagent and reagent stored at 8 ° C at different time intervals.

TABLE 10A: RECOVERY OF THE SERUM CONTROL DB THE REAGENTS OF AST AND ALT SHOWN BN TABLE 7A AND 8A TAP TO 10B.:. RECOVERIES OF THE CONTROL SOURCE OF THE REAGENTS OF AST AND ALT SHOWN IN TABLE 7B AND SB - * - * 'n • "- i I? R = IT.19" tC Rea: t:, c • teactiv 'Baztivo -saetí ,; a. *! sre "a] 3 c'd.e oQG a aííí z £ j_ ú? l *? a: epa; c a" ': c: rtep * dG ie s-e-c Sajo Sajo high a .0? o sajo alte 4'L 40 136 19! 1"'182 4 *; 184 120 40 184 42 1S3 60 tflfi 45 125 44 18? 36 4! 188 44 IB "- TABLE HA: STUDIES DB LINEARITY DB THE REAGENTS OF AST AND ALT SHOWN IN TABLES 7A and 8A TABLE 11B: LINEARITY STUDIES OF THE AST AND ALT REAGENTS SHOWN IN TABLES 7B and 8B Note: The test reagent has been stored at 8QC for a period of 31 weeks. The standard batch was reconstituted fresh for this study.

From the results presented it is clear that the regeneration of the AST and ALT reagents are exhibiting at least stability for 6-7 months when stored capped at 2-8 ° C. The reagent must have an initial absorbance of 1.0 A to be functional. After 7 months the reagent still has an absorbance of at least 1.0 A. From the results obtained in Table 10A and 10B it is clear that there are no significant differences in the control serum recoveries obtained with fresh reagent as opposed to the reagent stored at 8o for up to 29 weeks. The results presented in Tables HA and 11B indicate that the AST and ALT reagents incorporating coenzyme regeneration technology are still able to meet the linearity specifications after 31 weeks of storage at 8CC. The incorporation of the refrigeration system according to the invention has resulted in an increase in the reconstituted stability of a serum of AST and ALT by measuring the capped reagent for 1 month at 2-8 ° C for at least 6-8 months at 2- 8 ° C.

Example 2 Urea reagents were prepared with ingredients as described in the above Table 5B, except that NADH 0.33 M was incorporated into the formulations, and was reduced the D-glucose level at 20 mM for one of the reagents.

The formulations thus prepared were at pH 8. 5. A conventional urea reagent formulation was used as a control. The reagents were prepared with an ammonia level of 0.15 mM introduced in the reactive system (final concentration) as a contaminant. This level of ammonia pollutant is sufficient to consume 0.15 M NADPH in the respective reagents - equivalent to 0.93 absorbance units at 340 nm. After the reaction was completed (eg clearance of ammonia in the reactive formulation), the absorbance of several solutions (located in sealed containers) was monitored all the time in a Shimadzu spectrophotometer at 340 nm, and at a temperature of 20 ° C. The results presented in Figure 1 show the time-dependent regeneration of NADH from NAD * in the formulation of the urea reagent of the invention after contamination with 0.15 mM ammonia. In Figure 1: Section A shows the regeneration of NADH for a conventional urea reagent; Section B shows the regeneration of NADH for a urea reagent containing 5 mM sodium phosphate, G-6- PDH 2000 U / L and 100 mM D-glucose. After 48 hours at 20 ° C, the conventional reagent failed to regenerate any NADH consumed after reagent contamination with ammonia. During the same period of time, the urea reagent of the invention with 20 mM D-glucose regenerated 0.23 absorbance units, or 0.04 mM NADH. For 48 hours, the urea reagent of the invention with 100 mM D-glucose regenerated 0.70 absorbance units, or 0.11 mM NADH. These results indicate the ability of the reagent described herein to overcome the reduction of NADH in the urea reagent followed by contamination of the reactant with ammonia.

TABLE 12 MAXIMUM NADH REGENERATION SPEEDS FOR THE REAGENT OF UREA AT 20 ° C MEASURED AT 340 NM.

Reagent conditions Urea regeneration rate (m / Abs / min) Conventional reagent -0.02 Reagent according to the +0.088 invention containing 5 mM Na-P04, G-6-PDH 2000 U / L and 2 mM D-Glucose Reagent according to the +0.386 invention containing 5 mM K-P04, G-6-PDH 2000 U / L and 100 M D-Glucose The maximum rates of NADH regeneration in the respective urea reagents are presented in Table 12. In the conventional urea reagent, there was a slow loss in absorbance throughout the time, followed by liquidation of the ammonia contaminant. In the formulation of the invention the rate of regeneration of NADH was increased with an increase in the concentration of D-glucose in the reagent. Ammonia reagents were prepared with ingredients as described in Table 6B above, except that the ratio of Tris / buffer Tris HCl (100 mM total buffer) was varied such that the pH values of the reagent were obtained in a range of pH 8.0-9.3. A final concentration of 0.2 mM NADPH was also used in the prepared ammonia reagents. An ammonia control reagent without D-glucose, potassium phosphate or G-6-PDH was also prepared. Reagents with a level of 0.1 mM ammonia introduced into the reagent system (final concentration) were prepared as a contaminant. This level of ammonia pollutant is sufficient to consume 0.1 mM NADPH in the respective reagents - equivalent to 0.62 absorbance units at 340 nm. After the reaction was completed (eg, complete ammonia clearance of the reagents), the absorbance of several solutions (located in sealed containers) was monitored on a Shimadzu spectrophotometer at 340 nm all the time, at a temperature of 20 ° C. The results presented in Figure 2 show the time-dependent regeneration of NADPH from NADP in ammonia reagent formulations between pH 8.0-9.3 according to the invention after contamination with 0.1 mM ammonia. The regeneration of NADPH followed by the liquidation of the polluting ammonia was completed in 24 hours after the contamination of Ammonia at 20 ° C. The maximum rates of NADPH regeneration in the respective ammonia reagents are presented in Table 13. In the conventional ammonia reagent at pH 8.0, the absorbance of the solution remained between 0.6-0.65, with a slow loss in absorbance. In the formulations of the invention at all tested pH values, the rate of NADPH regeneration was quite similar. The maximum regeneration rate of NADPH was at a pH of 8.50. These results clearly indicate the ability of the invention described herein to overcome the reduction of NADPH in the ammonia reagent followed by contamination of the reactant with ammonia.

TABLE 13 MAXIMUM DB SPEEDS REGENERATION DB NADPH FOR BL REAGENT OF AMMONIA AT 20 ° C MEASURED AT 340 nm.

Example 1 DB SPEEDS LOST DB NAD (P) H AMMONIA REAGENTS AND OREA A UREA REAGENT The urea reagents were prepared with a formulation composition as described in Table 5B, with the following variations. The final concentration of NADH in the reagent formulations was 0.25 mM. The pH of the urea reagent was adjusted by varying the ratio of Tris / Tris HCl (100 mM total buffer). The control urea reagent solutions were prepared in the absence of D-glucose, phosphate and G-6-PDH. The absorbance of the sample solutions of the reagents stored and sealed in containers at 20 ± 20C, were monitored at 340 nM. The decrease in absorbance of the solutions of the urea reagent during the storage time at 20 ± 2 ° C, monitored at 340 nm, are presented in Table 14. It is apparent that in the presence and absence of the coenzyme regeneration system of the invention, the solutions lost absorbance more rapidly at pH 8.0 than at pH 8.5. In the formulation of the invention, however, the absorbance loss rate of the reagent solution was also significantly less rapid. In other words, the high pH and incorporation of the coenzyme regeneration system of the invention significantly reduced the rate of disappearance of NADPH from the urea reagent solution. It should be noted that while an increase in pH Above 8.5 will also promote the stability of NADH, commercially available urease and glutamate dehydrogenase enzymes will become less stable and less active in the reagent formulation. Consequently, a balance in the pH of the reagent formulation is required, such that the adequate activity and stability of the enzyme are maintained, while also providing reasonable stability of NADH. In this base, a pH reagent formulation of about 8.5 is preferred.

TABLE 14 ABSORBANCE (340 NM) DB DB UREA REAGENT SOLUTIONS STORED AT 20 ° C AS A TIME DBL FUNCTION B AMMONIA REAGENT The ammonia reagents were prepared with a formulation composition as described in Table 6B, with the following variations. The pH of the ammonia reagent was adjusted by varying the ratio of Tris / Tris HCl (100 mM total buffer). The final concentration of NADPH in the ammonia reagent solutions was 0.2 mM. The control ammonia reagent solutions were prepared without D-glucose, phosphate and G-6-PDH. The absorbance of the sample solutions of the reagents stored and sealed in containers at 20 ± 2 ° C, were monitored at 340 nm. The decrease in absorbance of the ammonia reagent solutions during the storage time at 20 ° C, monitored at 340 nm, is presented in Table 15. It is apparent that in the presence and absence of the coenzyme regeneration system of the invention, the solutions lost absorbance more rapidly with a reduction in pH. In the formulations of the present invention, however, the rate of absorbance loss of the reagent solution was also significantly less rapid. In other words, the high pH and the incorporation of the coenzyme regeneration system of the invention significantly reduced the rate of disappearance of NADPH from the ammonia reagent solution. It should be noted that while an increase in pH will promote the stability of NADPH, commercially available glutamate dehydrogenase will become increasingly less stable and less active in the reagent formulation above pH 8.5. Consequently, a balance in the pH of the reagent formulation is required, such that the adequate activity and stability of the enzyme are maintained, while also providing reasonable stability of NADPH. On this basis, a pH reagent formulation of about 8.5-9.0 is preferred.

TABLE 15 ABSORBANCE (340 NM) DB AMMONIA REAGENT SOLUTIONS STORED AT 20 ° C AS A TIME DBL FUNCTION EXAMPLE 4 DB FUNCTIONALITY AMMONIA AND UREA REAGENTS TO UREA REAGENTS The urea reagent was prepared according to the ingredients listed in Table 5B, in the presence and absence of D-glucose, phosphate and G-6-PDH. However, the final concentration of NADH in the formulations Reactive was 0.25 mM. The linearity of the reagents was compared against Verichem / Normal / Abnormal controls, on a ROCHE * 1 COBAS MIRA ™ instrument, using the parameters specified below. Reaction temperature: 37 ° C Sample reagent volume: 1: 100 Wavelength 340 nm The results presented in Table 16 indicate that the urea reagent at pH 8.50 of the present invention is linear up to the highest level of the tested sample of urea control (Verichem E level, with urea 37.4 mM), with the variation of the values measured at 5% of the specified concentration. The urea reagent of the invention was also faithfully read in the deviation of the value for normal and abnormal urea control samples.

TABLE 16 LINEARITY STUDIES WITH BL REAGENT DB UREA A PH 8.50 B AMMONIA REAGENTS The ammonia reagent was prepared in the presence and absence of D-glucose, phosphate and G-6-PDH according to the ingredients listed in Table 6B. The linearity of The reagents were compared against aqueous ammonia controls, on a ROCHER COBAS MIRA ™ instrument, using the parameters specified below. Reaction temperature: 37 ° C Sample volume of reagent: 1:10 Wavelength 340 nm The results presented in Table 17 indicate that the ammonia reagent at pH 8.50 of the invention is linear up to the highest level of the sample tested ammonia control (1200 μM ammonia), with the variation of the measured values at 5% of the specified concentration.

TABLE 17 LINEARITY STUDIES WITH AMMONIA REAGENT AT PH 8.50 Example 5 The urea reagent can be configured in a two-bulb format, according to the formulation configuration specified below. The relative volume of addition of ampoule reagent A and B required to obtain the combined reagent is 5: 1 for ampule A: ampule B.

Ampoule of urea A Ampoule of urea B Ammonia reagent. Format of flof- AlWK? Nfítfüff The ammonia reagent can be configured in a two-bulb format, according to the formulation configuration specified below. The relative volume of addition of ampoule reagent A and B required to obtain the combined reagent is 5: 1 for ampoule A: ampoule B. In the formulation of the For example, NADH is used instead of NADPH. As a result, LDH is included in the ampule A for the elimination of pyruvate interference from the patient sample before the start of the main assay reaction.

Ammonia ampoule A Ammonia bulb B Other main advantages of the reagent and the method according to the invention are that the reagent is in its most preferred form, a simple vial reagent therefore reduces space and inventory problems associated with reagents of the prior art, and that it is adaptable to several instrumentation systems. It should be appreciated that there are numerous pairs of "non-specific" substrate / enzyme that could be used for the slow regeneration of the coenzyme used in the reagent and method of the invention. In addition to those mentioned here, there are others that are not commercially or that are prohibitively expensive. It will also be appreciated that this invention will be applicable to the stabilization of reagents other than AST, ALT, ammonia and urea, for example, LDH (pyruvate or lactate), triglyceride and salicylate. The invention should not be considered limited by the exemplification thereof in this specification with specific reference to AST, ALT, ammonia and urea.

Claims (33)

1. A reagent for the enzymatic determination of the concentration of an analyte (substance to be analyzed) in a patient in which the degree of oxidation of a coenzyme is measured, characterized in that said reagent is stabilized against oxidation by means of a coenzyme reduction system comprising an enzyme pair and substrate selected in such a way as to allow the continuous regeneration of said coenzyme during the storage of said reagent.

2. A reagent as claimed in claim 1, characterized in that said continuous regeneration occurs at a speed in the range of 0.01 to 0.9 mAbs / min from 18 to 25 ° C.

3. A reagent as claimed in claim 1 or claim 2, characterized in that said coenzyme reduction system contains an enzyme and a substrate, said enzyme having incomplete specificity for said substrate.

4. A reagent as claimed in any of claims 1 to 3, characterized in that said pair of enzyme / substrate is glucose-6-phosphate dehydrogenase / D-glucose.

5. A reagent as claimed in claim 3 or claim 4, characterized in that the degree of specificity between said enzyme and said substrate is less than 50% on an equimolar basis.

6. A reagent as claimed in any of claims 3 to 5, characterized in that the degree of specificity between said enzyme and said substrate is less than 10% in an equimolar base.

7. A reagent as claimed in any of claims 1 to 6, characterized in that said analyte is a transaminase.

8. A reagent as claimed in claim 7, characterized in that said transaminase is aspartate transaminase.

9. A reagent as claimed in claim 7, characterized in that said transaminase is alanine transaminase.

10. A reagent as claimed in any of claims 1 to 6, characterized in that said analyte is urea.

11. A reagent as claimed in any of claims 1 to 6, characterized in that said analyte is ammonia.

12. A reagent for the enzymatic determination of the concentration of an analyte in a patient in which the degree of oxidation of a coenzyme is measured, characterized in that said reagent is configured as an ampoule simple and is stabilized against oxidation by a coenzyme reduction system comprising an enzyme pair and substrate selected in such a way as to allow the continuous regeneration of said coenzyme during the storage of said reagent.

13. A reagent as claimed in claim 12, characterized in that said continuous regeneration occurs at a rate in the range of 0.01 to 0.9 mAbs / min from 18 to 25 ° C.

14. A reagent as claimed in claim 12 or claim 13, characterized in that said coenzyme reduction system contains an enzyme and a substrate, said enzyme has incomplete specificity for said substrate.

15. A reagent as claimed in any of claims 12 to 14, characterized in that said pair of enzyme / substrate is glucose-6-phosphate dehydrogenase / D-glucose.

16. A reagent as claimed in claim 14 or claim 15, characterized in that the degree of specificity between said enzyme and said substrate is less than 50% in an equimolar base.

17. A reagent as claimed in any of claims 14 to 16, characterized in that the degree of specificity between said enzyme and said substrate is less than 10% on an equimolar basis.

18. A reagent as claimed in any of claims 12 to 17, characterized in that said analyte is a transaminase.

19. A reagent as claimed in claims 12 to 17, characterized in that said transaminase is aspartate transaminase.

20. A reagent as claimed in claims 12 to 17, characterized in that said transaminase is alanine transaminase.

21. A reagent as claimed in claims 12 to 17, characterized in that said analyte is urea.

22. A reagent as claimed in claims 12 to 17, characterized in that said analyte is ammonia.

23. An improvement in an enzymatic method of determining the concentration of an analyte in a body fluid sample wherein the degree of oxidation of a coenzyme is measured, characterized in that the improvement comprises the stabilization of a reagent containing said coenzyme against the coenzyme. oxidation by a coenzyme reduction system comprising an enzyme pair and substrate selected in such a way as to allow the continuous regeneration of said coenzyme during the storage of said reagent.

24. An improvement as claimed in claim 23, characterized in that said continuous regeneration occurs at a speed in the range of 0.01 to 0.9 mAbs / min from 18 to 250C.

25. A reagent as claimed in claim 23 or claim 24, characterized in that said coenzyme reduction system contains an enzyme and a substrate, said enzyme has incomplete specificity for said substrate.

26. An improvement as claimed in any of claims 23 to 25, characterized in that said pair of enzyme / substrate is glucose-6-phosphate dehydrogenase / D-glucose.

27. An improvement as claimed in claim 25 or claim 26, characterized in that the degree of specificity between said enzyme and said substrate is less than 50% on an equimolar basis.

28. An improvement as claimed in any of claims 25 to 27, characterized in that the degree of specificity between said enzyme and said substrate is less than 10% on an equimolar basis.

29. A reagent as claimed in any of claims 23 to 28, characterized in that said analyte is a transaminase.

30. A reagent as claimed in any of claims 23 to 28, characterized in that said analyte is aspartate transaminase.

31. A reagent as claimed in any of claims 23 to 28, characterized in that said analyte is alanine transaminase.

32. A reagent as claimed in any of claims 23 to 28, characterized in that said analyte is urea.

33. A reagent as claimed in claims 23 to 28, characterized in that said analyte is ammonia.