GB2114290A

GB2114290A - Method and reagents for eliminating the interference from bilirubin in certain clinico-chemical determinations

Info

Publication number: GB2114290A
Application number: GB08302063A
Authority: GB
Inventors: Franco Grillo; Enzo Murador
Original assignee: Farmitalia Carlo Erba SRL
Current assignee: Pfizer Italia SRL
Priority date: 1982-01-28
Filing date: 1983-01-26
Publication date: 1983-08-17
Also published as: GB8302063D0; GB2114290B; FR2520381B1; FR2520381A1; IT8224881A0; IT1153913B

Abstract

Method for eliminating bilirubin interference in clinico-chemical determinations based on hydrogen peroxide production and quantitative determination by a chromogenic system in the presence of peroxidases, wherein a water-soluble complex containing a cyanomolybdate or cyanoruthenate ion is added to the system. This method is useful in assaying, for example, uric acid and triglycerides and specific reagents for this purpose are described.

Description

SPECIFICATION Method and reagent for eliminating the interference from bilirubin in certain clinicochemical determinations The present invention relates to a method for eliminating the interference from bilirubin in the clinico-chemical determinations based upon hydrogen peroxide production and quantitative assay thereof by a chromogenic system in the presence of peroxidases.

The invention also provides reagents for the realization of said method.

The use of certain chromogenic systems for determining the hydrogen peroxide produced from biological substrates by specific oxidases is known.

In Ann. Clin. Biochem., 6, 24, 1969, P. Trinder describes, for example, a method for the quantitative determination of glucose in blood, after deproteinization, which method involves the use of a chromogenic system constituted by 4-aminophenazone and phenol for determining, in the presence of peroxidase, the hydrogen peroxide produced from glucose by glucose oxidase.

Nowadays analogous techniques have been extended to the determination of many oxidizable substrates, including, for instance, cholesterol, uric acid, triglycerides and phospholipids, in biological fluids such as blood or urine.

Deproteinization is generally not considered necessary.

The reaction known as "Trinder's" can be set out schematically as follows, as it is usually understood: hydrogen peroxide (H202) forming as a result of the action of specific oxidases on certain metabolites causes, in the presence of peroxidases, oxidation of a system consisting of phenol or a phenol derivative, or an aromatic amine, and 4-aminoantipyrine (also called 4aminophenazone) or another substance from a heterogeneous group frequently referred to as "electron or radical acceptors" (for example, N,N-dimethyl-4-aminoantipirine, benzidine, 3methyl-2-benzothiazolone hydrazone, etc.).

The mechanism of the reaction is still a question of debate.

The product of the reaction in which 4-aminoantipyrine acts as acceptor is known as regards its basic structure: it is a quinonimine, which generally shows good absorption in the wavelength range between 500-600 nm. Since Trinder's system develops visible colour, determination of metabolites by this method is known as "colorimetric", and the phenol compound or aromatic amine, with the acceptor, form the "chromogenic system".

Since the colorimetric reaction results in stoichiometric formation of 0.5 moles of quinonimine for every mole of hydrogen peroxide reduced by the chromogenic system in the presence of peroxidases, the absorbance measured at equilibrium is proportional to the total concentration of H202 released in the system.

In addition, oxidation before the colorimetric reaction results in stoichiometric formation of 1 mole of H202 for every mola of specific substrate for oxidase, so the absorbance measured at equilibrium is proportional to the concentration of this substrate.

The so called Trinder's method permits quantitative determination of many substances of clinical interest in biological fluids, such as glucose (using glucose oxidase, EC 1.1.3.4.), cholesterol (after complete hydrolysis of esters, using cholesterol oxidase, EC 1.1.3.6.); uric acid (using urate oxidase, EC. 1.7.3.3.); triglycerides (for example after complete hydrolysis of ester bonds and transformation to glycerol, using glycerol oxidase, EC not yet assigned; for triglycerides there are other pathways requiring the use of other oxidases too); phospholipids (after hydrolysis of ester bonds and transformation to choline, using choline oxidase, EC 1.1.3.1 7.); non-esterified fatty acids (after activation to acyl-CoA, using-CoA oxidase, EC not yet assigned).

Determination of metabolites in biological fluids suffers from the main drawback of interference in measurement from many of the numerous components of such samples, sometimes present in high concentrations. The requirement until a short while ago to deproteinize serum or plasma before assaying certain constituents arose from the marked tendency of serum or plasma proteins, present at average concentrations around 70 g/l, to interfere in determination, on account of their reducing or complexing properties, or their ability to carry small molecules which in turn caused interference.

As micromethods have been developed, guaranteeing specific determinations through the use of high-purity enzymes, and offering sufficient sensitivity to permit very low volumetric ratios of sample to reagent ('1:40), the effect of protein interference as such has become minimal. Thus it has become habitual to determine metabolites directly, without first deproteinizing the sample.

Measurement of metabolites based on Trinder's system is also direct, usually. However, when serum or plasma is used as sample, a small molecule carried by albumin, i.e. bilirubin, remains in the reaction mixture.

Bilirubin can cause considerable interference in the final detectable absorbance. Normally this molecule is present in serum at concentrations below 1 7 Aemol/l, but in jaundice it can rise beyond 500 llmol/i.

It causes two types of interference: positive colorimetric interference and negative chemical interference.

(1) Positive colorimetric interference: biiirubin bound to albumin has an absorption spectrum with a maximum at 460 nm, and a tail to the right which overlaps a considerable part of the wavelength range of the absorption spectra of many quinomimines produced by the various "Trinder-type" systems. For example, this happens in systems which use phenol and its derivatives as chromogens. As a result part of the absorbance measured at the end of the determination is not due to quinonimine, the specific aim of the analysis, but to bilirubin present in the sample.

This type of interference depends on: (a) the bilirubin content in the sample; (b) the dilution of the sample for determination. This obviously depends on the sensitivity of the method, representing the optimized combination of two elements: the absorptivity of the final coloured product and the range of concentrations at which the metabolite may be found in the sample; (c) the wavelength at which the final absorbance is read.

This type of interference can be minimized more by procedural devices than by chemical additives, usually by -setting the volumetric ratio of sample to reagent as low as possible and -selecting a wavelength as far away as possible from the maximum of absorbance for bilirubin bound to albumin (460 nm).

It has now become established practice to overlook the part of absorbance due to bilirubin pigment. It is difficult to quantitate (Perlstein, M.T. et al, Microchem. J. 22, 403, 1977) because bilirubin partly reacts chemically in the system, so the absorbance of the pigment at equilibrium is anyway lower than that measurable initially.

In addition, this chemical reaction of bilirubin gives rise to negative interference, discussed below.

It is therefore usual to consider the residual spectral effect at equilibrium as "compensation" for the drop in response caused by other paths. Accurate determinations are so possible, with no particular special devices, of metabolites that are normally present in high concentrations in the blood even in samples with a fairly high concentration of bilirubin; examples are glucose and cholesterol (about 5.5 mmol/l in plasma).

However, for assays of substances whose concentrations in the blood are lower, such as triglycerides (about 1.1 mmol/l), or especially uric acid (about 0.3 mmol/l), the volumetric ratio of sample to reagent required, and the extreme sensitivity of the chromogens that have to be used result in the negative chemical interference from bilirubin becoming much greater than its positive colorimetric interference.

(2) Negative chemical interference: little knowledge is available to clarify the mechanism of this interference, partly because of the mechanism of Trinder's reaction, without interference, is only poorly understood. For example two contrasting hypotheses can be cited: -in Trinder's reaction, the non-phenolic and non-anilinic component of the chromogenic system gives rise to formation of an electrophilic intermediate which reacts with the other component, or with bilirubin (Witte D.L. et al., Clin. Chem. 24, 1778, 1978); -in Trinder's reaction the phenolic or anilinic component gives rise to formation of an intermediate radical which is picked up by the radical acceptor or by bilirubin (Brodersen R. et.

al., Scand. J. Clin. Lab. Invest. 39, 143, 1979).

Furthermore, it is to be noted that bilirubin can act as a hydrogen donor in the peroxidase reaction, and this also occurs in Trinder's system (Matsumoto K. et al., Rinsho Kagaku 8, 63, 1979).

Whatever the mechanism(s) of negative chemical interference by bilirubin, in practice it constitutes a serious problem in developing an analytical method based on Trinder's principle.

We have now found that said interference from bilirubin, particularly negative interference, can be eliminated by adding to the optimal composition of the chromogenic system a particular water-soluble complex with redox activity; this acts on the process of chromogens coupling, mediated by the action of peroxidases, and cancels out completely or, at least, to a large extent the interference due to bilirubin, particularly any negative chemical interference. More precisely, the present invention provides a method of eliminating the interference from bilirubin, in particular the negative interference, in the clinico-chemical determinations based upon hydrogen peroxide production and quantitative assay thereof by a chromogenic system in the presence of peroxidases, characterized in that a water-soluble complex containing a cyanomolybdate or a cyanoruthenate ion is added to the chromogenic system.

The addition of the ruthenium or molybdenum complexes to the chromogenic system according to the method of the invention, makes it possible to eliminate the interference from bilirubin in the clinical evaluations of any biological sample even though the better results are achieved on biological samples containing up to about a 350 ELmoi/l concentration of bilirubin.

The method provided by the invention may be applied to the analysis of any oxidisable biological substrate and is particularly useful in the determination of uric acid and of triglycerides.

Obviously, when the oxidisable substrate to be determined is in bound form it may be necessary to make it free before determination.

Thus, for example, in triglycerides assay the oxidisable substrate glycerol needs to be liberated from the lipidic moiety, e.g. by means of a lipase, before determination.

The cyanomolybdate or cyanoruthenate ion characterizing the water-soluble complex employed in the method of the invention is, preferably, an ion salified with an alkali or alkalineearth metal cation, preferably a sodium or potassium cation.

Among the cyanomolybdate ions, octacyanomolybdates of tetravalent molybdenum are preferred.

Among the cyanoruthenate ions hexacyanoruthenates of bivalent ruthenium are preferred.

Particularly preferred complexes are potassium octacyanomolybdate and potassium hexacyanoruthenate, preferably in their hydrated forms K4Mo(CN)8.2H20 and K4Ru(CN)6.3H20.

The said complexes are usually active in any, and of any concentration, buffer in a range of pH between about 5.5 and about 8.5, preferably between 6 and 8. They are preferably used at a concentration of 0.1-10 mmol/l in the reaction mixture.

Cyanomolybdate ions, in particular K4Mo(CN)8.2H20 are more preferably used at a concentration of about 0.5-1.5, in particular about 1, mmol/l in the reaction mixture preferably in the presence of a pH 5.5-8.5 buffer.

For cyanoruthenate ions, in particular K4Ru(CN)6.3H20, a concentration of about 1-3, preferably about 2, mmol/l in the reaction mixture is particularly preferred, always in the presence of a pH 5.5-8.5 buffer.

The basic chromogenic system (to which, according to the invention, a cyanomolybdate or a cyanoruthenate ion is added) usually comprises a first component, which may be any compound able to act as electron or radical acceptor, and a second component able to generate, by coupling to the first component, a coloured product.

The component acting as acceptor may be, for example, a compound chosen from the group consisting of 4-aminoantipyrine, N, N-dimethyl-4-aminoantipyrine, benzidine, 3-methyl-2-benzothiazolone hydrazone and similar.

Particularly preferred acceptor is 4-aminoantipyrine.

The second component, is preferably, a phenol derivative.

A phenol derivative may be phenol optionally substituted for example, by one or more halogens, chlorine in particular, and/or by one or more acid groups, carboxy or sulpho for instance.

Particularly preferred phenol derivatives are sulphonated dichlorophenols such as, for example, 3, 5-dichloro-2-hydroxy-benzene-sulphonic acid.

In the most preferred features the basic chromogenic system used in the method of the invention comprises 4-aminophenazone (also called 4-aminoantipyrine) and 3,5-dichloro-2hydroxy-benzene-sulphonic acid (here reported with the trivial name "sulphonated dichlorophe nol").

As already said, in addition to the above described method of eliminating the interference from bilirubin, the present invention provides also reagents for the realization of that method.

Each of the said reagents comprises, as essential components: (a) a chromogenic system able to detect hydrogen peroxide in the presence of peroxidases; (b) a water soluble complex containing a cyanomolybdate or a cyanoruthenate ion; (c) a specific oxidase; (d) a peroxidase; and (e) a buffer.

The chromogenic system able to reveal hydrogen peroxide in the presence of peroxidase may be any chromogenic system falling within the definition previously given in this specification, the combination 4-aminoantipyrine/sulphonated dichlorophenol being the most preferred one.

Also the water soluble complex containing a cyanomolybdate or cyanoruthenate ion may be any complex according to the definition given above.

As already said it is preferably a cyano-complex of tetravalent molybdenum or of bivalent ruthenium salified with an alkali or alkaline-earth metal cation, in particular sodium or potassium.

As already said too K4Mo(CN)8.2H20 and K4Ru(CN)6.3H20 are the preferred complexes.

The oxidase enzyme present in each reagent must be an oxidase specific to the substrate to be determined.

It is, for example, a urate-oxidase in a reagent used for the determination of uric acid, and a glycerol-oxidase or glycerol phosphate oxidase, in a reagent used for the determination of triglycerides. Preferably a specific oxidase of microbial origin is used. In particular, for example, in the determination of uric acid a urate oxidase of the type presented by Bassi et al. under the title "New microbial uricase" in the first Session-Poster N. 3 of the 9th International Symposium on Clinical Enzymology-Verona (April 17-20 th, 1980) may be used.

The peroxidase enzyme may be any enzyme with peroxidase activity.

It may be, for instance, horseradish peroxidase.

The buffer supplied as a further component of the reagents according to the invention, may be any, of any concentration, buffer having a pH comprised between about 5.5 and about 8.5 preferably between 6 and 8.

Particularly preferred buffers are a pH 6.6 imidazole/HCI buffer at a concentration of about 20-100, preferably about 50, mmol/l in the reaction mixture, a pH 7.0 sodium or potassium phosphate buffer at a concentration of about 20-200, preferably about 50, mmol/l, a pH 7.3 sodium ethylenediaminetetraacetate buffer at a concentration of about 10-30, preferably about 20, mmol/l, and a pH 8.0 Tris/HCI buffer at a concentration of about 20-100, preferably about 50, mmol/l. For different clinical determinations distinct reagents are provided mainly differing from one another in the nature of the oxidase specifically required.

Furthermore, for certain determinations, reagents are required which, in addition to the above mentioned essential components, may contain, either optionally or not, further components such as, e.g. other enzymes, cosubstrates, tensioactive substances and enzyme activators or accelerators. For instance, a reagent useful for the determination of triglycerides may contain, as additional components a lipase, a tensioactive substance and, optionally, if a glycerolphosphate oxidase is used, glycerol kinase, a nucleotide-cosubstrate and a glycerol kinase activator, as well as, if desired, a lipase accelerator. A lipase is preferably a microbial lipase, in particular a microbial lipoproteinlipase.It may be, for example, a lipase of the type presented by Bassi D., et al., in poster P 9-1 2 of the 1st African and Mediterranean Congress of Clinical Chemistry, Milan (1980) or a lipase from Mucor juvanicus or from Pseudomonas fluorescens or from Pseudomonas aeruginosa or from Staplhyococcus aureus. Tensioactive substances and enzyme activators or accelerators may be those usually employed in the diagnostic kits. A useful tensioactive substance may be for example a non-ionic detergent, such as Tritons or Nonidet(E or an ionic surfactant, such as, for example, a cholate or a dodecyl-sulphate, in particular, for instance, cholic acid-sodium salt.

An accelerator of the lipase enzyme may be, for instance, a protein, albumin for instance, bovine albumin in particular.

An activator of glycerol kinase may be, for example, a magnesium salt, in particular magnesium sulphate or acetate.

A nucleotide acting as cosubstrate with glycerol kinase may be, for instance, adenosine triphosphate, in particular adenosine triphosphate trihydrated disodium salt.

In the ambit of each reagent the nature of the components and their concentrations may vary in broad ranges. The activity of the enzymatic components may vary as well depending on the kind of enzyme used and on the clinical determination to be made.

Thus, for example, a preferred reagent useful for the clinical determination of the uric acid is a reagent comprising: (1) 4-amino antipyrine; (2) sulphonated dichlorophenol; (3) a water-soluble complex containing a cyanomolybdate or a cyanoruthenate ion; (4) urate oxidase; (5) peroxidase; and (6) a buffer.

Preferably in such a reagent the water-soluble complex containing a cyanomolybdate or a cyanoruthenate ion is K4Mo(CN)8.2H2O or, alternatively, K4(Ru)CN)6.3H20; the urate-oxidase is a microbial urate oxidase of the type indicated above; the peroxidase is horseradish peroxidase; the buffer is a pH 5.5-8.5, in particular a pH 6-8 buffer, for example a pH 6.6 imidazole/HCI buffer or a pH 7.3 sodium ethylenediaminetetraacetate buffer.

In particularly preferred feature, the reagent for the determination of uric acid according to the present invention has the following composition: (1) 4-amino antipyrine: about 0.2-2.0, preferably about 0.4, mmol/l; (2) sulphonated dichlorophenol: about 2-6, preferably about 4, mmol/l; (3) K4Mo(CN)8.2H20: about 0.5-1.5, preferably about 1, mmol/l; (4) microbial urate-oxidase: 2800 A/l, preferably about 2000 IL/I; (5) horseradish peroxidase: a5000 ,u/l, preferably about 20000 it/I; (6) pH 6.6 imidazole/HCI buffer: about 20-100, preferably about 50, mmol/l.

In another preferred feature the reagent for the determination of the uric acid comprises: (1) 4-amino antipyrine: about 0.2-2.0, preferably about 0.4, mmol/l; (2) sulphonated dichlorophenol: about 2-6, preferably about 4, mmol/l; (3) K4Ru(CN)6.3H2O: about 1.5-2.5, preferably about 2, mmol/l; (4) microbial urate-oxidase: m800 ,u/l, preferably about 2000 ,u/l; (5) horseradish peroxidase: > 5000 y/I, preferably about 20000 y/I; (6) pH 6.6 imidazole/HCI buffer: about 20-100, preferably about 50, mmol/l.

In a further feature the reagent for the determination of the uric acid comprises: (1) 4-amino antipyrine: about 0.2-2.0, preferably about 0.4, mmol/l; (2) sulphonated dichlorophenol: about 2-6, preferably about 4, mmol/l; (3) K4Ru(CN)6.3H20: about 1.5-2.5, preferably about 2, mmol/l; (4) microbial urate-oxidase: a800 it/1 preferably about 2000 it/1; (5) horseradish peroxidase: a5000 p/l, preferably about 30000 it/1; (6) pH 7.3 sodium ethylenediaminetetraacetate buffer: about 10-30, preferably about 20, mmol/l, and (7) a tensioactive substance, preferably cholic acid sodium salt at a 1 g/l concentration.

Analogous reagents, but containing different specific oxidases, are usually preferred also for clinical determinations of other biological substrates.

Thus for the determination of triglycerides analogous reagents containing a glycerol oxidase, are used.

These reagents may comprise, as further components, a lipase, preferably a microbial lipase, for liberating glycerol from the lipidic moiety and, possibly, another enzymatic component such as, e.g., glycerol kinase in the case the used glycerol oxidase is a glycerolphosphate oxidase.

A tensioactive substance, which may be, for instance, cholic acid-sodium salt, and a glycerol kinase activator, which may be, for example, magnesium acetate, may be present; a lipase accelerator such as, for example, a protein, albumin for instance, bovine albumin in particular, may be optionally present too.

When glycerol kinase is used, a nucleotide as cosubstrate needs to be present, which may be, for example, adenosine triphosphate, in particular adenosine triphosphate trihydrated disodium salt; Accordingly, in a preferred feature a reagent for the determination of triglycerides, contains: (1) 4-amino antipyrine: about 0.2-2.0, preferably about 0.4, mmol/l; (2) sulphonated dichlorophenol: about 2-6, preferably about 4, mmol/l; (3) K4Ru(CN)6.3H20: about 1.5-2.5, preferably about 2, mmol/l; (4) glycerolphosphate oxidase: a2000 y/I, preferably about 5000 it/1; (5) peroxidase: > 5000 y/I, preferably about 30000 it/1; (6) glycerol kinase: a200 y/I, preferably about 1000 it/1; (7) microbial lipase: a100 y/I, preferably about 500 it/1; (8) a tensioactive substance; (9) a lipase accelerator; (10) a glycerol kinase activator; (11) a glycerol kinase cosubstrate; and (12) a buffer.

In such a reagent the tensioactive substance may be, for example, cholic acid-sodium salt at a concentration about 5-10, preferably about 7, g/l in the reaction mixture.

The lipase accelerator may be, for example, bovine albumin at a concentration about 0.2-5.0, preferably about 1.0, g/l in the reaction mixture. The buffer may be, for instance, a pH 6.6 imidazole/HCl buffer at a concentration about 20-100, preferably 50, mmol/l.

The glycerol kinase is, preferably, a microbial glycerol kinase, from Candida mycoderma for example.

The glycerol kinase activator may be for example, magnesium acetate at a concentration about 0.2-5.0, preferably about 1.0, g/l in the reaction mixture.

The glycerol kinase cosubstrate may be, for instance, adenosine triphosphate trihydrated disodium salt, at a concentration about 0.1-2.0, preferably about 0.5, mmol/l in the reaction mixture.

The glycerol phosphate oxidase is, preferably, of microbial origin.

The peroxidase, may be, for instance, horseradish peroxidase; and the lipase may be, for example, a microbial lipase of the type reported by Bassi D., et al., in poster P 9-12 of the 1st African and Mediterranean Congress of Clinical Chemistry, Milan, 1 980.

The workability of the method and of the reagents of the invention has been verified both in determinations on biological samples to which variable amounts of bilirubin were previously added, and in determinations of biological samples containing only endogen bilirubin.

The latter evaluations were made in order not to alter in anyway, e.g. by additions and/or dilutions, the real conditions of the biological samples.

The abbreviations 'EC' and 'Tris' used in this patent application indicate, respectively, 'Enzyme Commission' and 'Tris(hydroxymethyl)aminomethane'.

The units referring to the peroxidase activity are O-dianisidine units.

The following examples illustrate but do not limit the invention.

Example 1 Determination of uric acid using a reagent with the following basic composition: sulphonated dichlorophenol 4 mmol/l 4-aminoantipyrine 0.4 mmol/l imidazole/HCI buffer pH 6.6 50 mmol/l urate oxidase 2000 ssA/l peroxidase 20000 it/1 Three (numbered from 1 to 3) non-jaundiced human serum samples were selected, and five (numbered from 4 to 8) with higher-than-normal bilirubin contents. Uric acid was assayed in these using the above basic reagent.

In comparison, the basic reagent was used with 1 mmol/l potassium octacyanomolybdate dihydrate added. Readings were taken at 515 nm by a Beckman Acta C 111 spectrophotometer.

The true uric acid concentrations in the samples were established using the uricase UV method (Praetorius E. et al., Scand. J. Clin. Lab. Invest., 5, 273, 1953).

TABLE 1 uric acid uric acid true uric found without found with Bilirubin acid K4Mo(CN)8 K4Mo(CN)8 Serum itmol/l mg/dl mg/dl mg/dl 1 9.2 5.68 5.63 5.69 2 16.8 4.02 4.11 4.17 3 13.2 7.86 7.79 7.85 4 68.6 5.34 4.24 5.42 5 123.1 4.32 3.60 4.31 6 189.8 9.18 7.78 9.30 7 263.4 4.25 3.00 4.17 8 340.9 6.11 4.08 5.98 Example 2 Determination of uric acid using a reagent with the same basic composition as set out for example 1. The samples used in example 1 were repeated for uric acid determination with the basic reagent and, as comparison, with the basic reagent containing 2 mmol/l potassium hexacyanoruthenate trihydrate.

Readings were taken at 51 5 nm by a Beckman Acta C 111 spectophotometer.

The true uric concentrations in the samples were established using the uricase-UV method (see ref. example 1).

TABLE 2 uric acid uric acid true uric found without found with Bilirubin acid K4RU(CN)6 K4RU(CN)6 Serum itmol/l mg/dl mg/dl mg/dl 1 9.2 5.68 5.63 5.74 2 16.8 4.02 4.11 4.09 3 13.2 7.86 7.79 7.84 4 68.6 5.34 4.24 5.41 5 123.1 4.32 3.60 4.40 6 189.8 9.18 7.78 9.12 7 263.4 4.25 3.00 4.25 8 340.9 6.11 4.08 6.05 Example 3 Determination of uric acid. Sample: pool of non-jaundiced human sera, with a uric acid content of 5.05 mg/dl (uricase-UV method, see ref. in example 1).

To portions of this sample, bilirubin was added in alkaline solution containing 40 g/l human albumin.

When necessary physiological saline was added to give a series of samples all diluted 4:5 compared to the original pool and containing the following additions of bilirubin: 0, 50, 100, 150, 200, 250, 300 and 350 itmol/l.

Uric acid was determined twice on these samples using: - basic reagent - basic reagent + k4Mo(CN)8.2H20 1 mmol/l - basic reagent + K4Ru(CN)8.3H20 2 mmol/l The mean of the two determinations is given.

Readings were taken at 515 nm by a Beckman Acta C 111 spectrophotometer.

TABLE 3 uric acid uric acid uric acid found Basic found [basic Bilirubin found [basic reagent + reagent + added reagents K4Mo(CN)8] K4Ru(CN)6] pmol/l mg/dl mg/dl mg/dl 0 5.10 5.15 5.18 50 4.32 5.07 5.09 100 3.65 4.97 4.90 150 3.15 4.78 4.72 200 3.03 4.85 4.81 250 3.34 5.00 5.06 300 3.68 5.25 5.28 350 3.92 5.52 5.62 Example 4 Determination of triglycerides using a reagent with the following basic composition: pH 6.6 imidazole/HCI buffer 50 mmol/l cholic acid-sodium salt 7 9/1 lipase (microbial) 500 y/l bovine albumin 1 g/l glycerolkinase 1000 it/1 magnesium acetate 1 g/l adenosine triphosphate trihydrated disodium salt 0.5 mmol/l glycerolphosphate oxidase 5000 it/1 sulphonated dichlorophenol mmol/l 4-aminoantipyrine 0.4 mmol/l peroxidase (horseradish) 30000 y/l The samples used in example 1 were repeated for triglycerides determination with the basic reagent and, as comparison, with the basic reagent containing 2 mmol/l potassium hexacyanoruthenate trihydrate. Readings were taken at 515 nm by a Beckman Acta C 111 spectrophotometer.

The true triglycerides concentrations in the samples were established using the enzymatic-UV method [Wahlefeld, A.W., in Bergmeyer, H.U., Methods of Enzymatic Analysis, 2nd ed., Academic Press, New York and London, 1974, p. 1831].

TABLE 4 triglycerides triglycerides true tri- found without found with bilirubin glycerides K4RU(CN)6 K4RU(CN)6 Serum itmol/l mg/dl mg/dl mg/dl 1 9.2 98 100 101 2 16.8 112 115 117 3 13.2 87 84 88 4 68.6 142 122 142 5 123.1 264 220 262 6 189.8 198 148 199 7 263.4 308 257 318 8 340.9 150 95 146

Claims

1. Method for eliminating the interference from bilirubin in the clinico-chemical determinations based upon hydrogen peroxide production and quantitative assay thereof by a chromogenic system in the presence of peroxidases, characterized in that a water-soluble complex containing a cyanomolybdate or a cyanoruthenate ion is added to the chromogenic system.

2. Method for eliminating the interference from bilirubin, according to claim 1, wherein the interference from bilirubin is the negative chemical interference.

3. Method, according to claim 1 or 2, for eliminating the interference from bilirubin in clinico-chemical determinations on biological samples containing bilirubin at a concentration of up to 350 itmol/l.

4. Method according to claim 3 wherein the concentration of bilirubin in the biological samples is up to 250 ssbmol/l.

5. Method according to any one of the preceding claims for the determination of uric acid in biological samples.

6. Method according to any one of claims 1 to 4 for the determination of triglycerides in biological samples.

7. Method according to claim 6 characterized in that the triglycerides are previously hydrolyzed to glycerol and free fatty acids and the glycerol is determined.

8. Method according to claim 7 wherein the hydrolysis of the triglycerides is carried out enzymatically by means of a lipase.

9. Method according to claim 8 wherein the lipase is microbial.

10. Method according to claim 9 wherein the microbial lipase is a microbial lipoprotein lipase.

11. Method according to claim 10 wherein the microbial lipoprotein lipase is from Mucor juvanicus or from Pseudomonas fluorescens or from Pseudomonas aeruginosa or from Staphylococcus aureus.

1 2. Method according to any one of the preceding claims wherein the water-soluble complex added to the chromogenic system contains a cyanomolybdate or a cyanoruthenate ion salified with an alkali or alkaline-earth metal.

1 3. Method according to claim 1 2 wherein the cyanomolybdate or cyanoruthenate ion is salified with sodium or potassium.

14. Method according to any one of the preceding claims wherein the water soluble complex added to the chromogenic system is potassium octacyanomolybdate.

1 5. Method according to claim 14 wherein the potassium octacyanomolybdate is in the hydrated form K4Mo(CN)8.2H20.

16. Method according to any one of claims 1 to 1 3 wherein the water soluble complex added to the chromogenic system is potassium hexacyanoruthenate.

1 7. Method according to claim 1 6 wherein the potassium hexacyanoruthenate is in the hydrated form K4Ru(CN)6.3H20.

1 8. Method according to any one of the preceding claims wherein the chromogenic system comprises a first component able to act as electron or radical acceptor and a second component able to generate, by coupling with the first one, a coloured product.

1 9. Method according to claim 1 8 wherein the component able to act as electron or radical acceptor is a compound chosen from 4-aminoantipyrine, N,N-dimethyl-4-aminoantipyrine, benzidine and 3-methyl-2-benzothiazolone hydrazone.

20. Method according to claim 1 9 wherein the component able to act as electron or radical acceptor is 4-aminoantipyrine.

21. Method according to claim 18 wherein the second component is a phenol derivative.

22. Method according to claim 21 wherein the phenol derivative is phenol optionally substituted by one or more halogens and/or one or more acidic groups chosen from carboxy and sulpho.

23. Method according to claim 22 wherein the phenol derivative is sulphonated dichlorophenol.

24. Method according to claim 1 8 wherein the chromogenic system comprises 4-aminoantipyrine and sulphonated dichlorophenol.

25. Reagent for the realization of a method according to any one of claims 1 to 24 comprising: (a) a chromogenic system able to detect hydrogen peroxide in the presence of peroxidases; (b) a water-soluble complex containing a cyano-molybdate or a cyanoruthenate ion; (c) a specific oxidase; (d) a peroxidase; and (e) a buffer.

26. Reagent according to claim 25 containing, as additional components, one or more enzyme activators or accelerators or cosubstrates and/or tensioactive substances.

27. Reagent according to claim 25 or 26 for the determination of uric acid wherein the specific oxidase is an urate oxidase.

28. Reagent according to claim 27 wherein the urate oxidase is microbial.

29. Reagent according to claim 25 or 26 for the determination of triglycerides wherein the specific oxidase is a glycerol oxidase.

30. Reagent according to claim 29 wherein the glycerol oxidase is microbial.

31. Reagent according to claim 25 or 26 for determination of triglycerides according to claim 29, containing, in addition to the components (a)-(e) of claim 25, a lipase and a tensioactive substance.

32. Reagent according to claim 31 wherein the lipase is microbial.

33. Reagent according to claim 32 wherein the microbial lipase is a lipoprotein lipase.

34. Reagent according to claim 33 wherein the microbial lipoprotein lipase is from Mucor juvanicus or from Pseudomonas fluorescens or from Pseudomonas aeruginosa or from Staphylococcus aureus.

35. Reagent according to any one of claims 31 to 34 wherein the tensioactive substance is an ionic surfactant.

36. Reagent according to claim 35 wherein the ionic surfactant is a salt of cholic acid.

37. Reagent according to claim 36 wherein the salt of cholic acid is the sodium salt.

38. Reagent according to any one of claims 31 to 37 containing a lipase accelerator as additional component.

39. Reagent according to claim 38 wherein the lipase accelerator is a protein.

40. Reagent according to claim 39 wherein the protein is an albumin.

41. Reagent according to claim 40 wherein the albumin is bovine albumin.

42. Reagent according to any one of claims 31 to 41 containing glycerolphosphate oxidase, as glycerol oxidase, and glycerol kinase as a further enzymatic component.

43. Reagent according to claim 42 wherein the glycerolphosphate oxidase is microbial.

44. Reagent according to claim 42 wherein the glycerol kinase is microbial.

45. Reagent according to any one of claims 42 to 44 contaning, as additional component, a glycerol kinase cosubstrate.

46. Reagent according to claim 45 wherein the glycerol kinase cosubstrate is a nucleotide.

47. Reagent according to claim 46 wherin the nucleotide is adenosine triphosphate.

48. Reagent according to claim 47 wherein the adenosine triphosphate is in the form of trihydrate disodium salt.

49. Reagent according to any one of claims 42 to 48 containing, as additional component, a glycerol kinase activator.

50. Reagent according to claim 49 wherein the glycerol kinase activator is a magnesium salt.

51. Reagent according to claim 50 wherein the magnesium salt is magnesium acetate.

52. Reagent according to claim 25 wherein the chromogenic system comprises a first component able to act as electron or radical acceptor and a second component able to generate, by coupling with the first one, a coloured product.

53. Reagent according to claim 52 wherein the component able to act as electron or radical acceptor is a compound chosen from 4-aminoantipyrine, N,N-dimethyl-4-aminoantipyrine, benzidine and 3-methyl-2-benzothiazolone hydrazone.

54. Reagent according to claim 53 wherein the component able to act as electron or radical acceptor is 4-aminoantipyrine.

55. Reagent according to claim 52 wherein the second component is a phenol derivative.

56. Reagent according to claim 55 wherein the phenol depivative is phenol optionally substituted by one or more halogens and/or one or more acidic groups chosen from carboxy and sulpho.

57. Reagent according to claim 56 wherein the phenol derivative is sulphonated dichlorophenol.

58. Reagent according to any one of claims 25 to 57 wherein the chromogenic system comprises 4-aminoantipyrine and sulphonated dichlorophenol.

59. Reagent according to any one of claims 25 to 58 wherein the water-soluble complex contains a cyanomolybdate or a cyanoruthenate ion salified with an alkali or alkaline-earth metal.

60. Reagent according to claim 59 wherein the cyanomolybdate or cyanoruthenate ion is salified with sodium or potassium.

61. Reagent according to claim 60 wherein the water-soluble complex is potassium octacyanomolybdate.

62. Reagent according to claim 61 wherein the potassium octacyanomolybdate is in the hydrate form K4Mo(CN)8.2H2O.

63. Reagent according to claim 60 wherein the water-soluble complex is potassium hyxacyanoruthenate.

64. Reagent according to claim 63 wherein the potassium hexacyanoruthenate is in the hydrated form K4Ru(CN)8.3H20.

65. Reagent according to any one of claims 25 to 64 wherein the peroxidase is horseradish peroxidase.

66. Reagent according to any one of claims 25 to 65 wherein the buffer is a pH 5.5-8.5 buffer.

67. Reagent according to claim 66 wherein the pH of the buffer is comprised between 6 and 8.

68. Reagent according to any one of claims 25 to 66 wherein the buffer is an imidazole/HCI buffer or a phosphate buffer or a sodium ethylenediaminetetraacetate buffer or a Tris/HCI buffer.

69. Reagent according to claim 68 wherein the buffer is a pH 6.6 imidazole/HCI buffer.

70. Reagent according to claim 68 wherein the buffer is a pH 7.0 sodium or potassium phosphate buffer.

71. Reagent according to claim 68 wherein the buffer is a pH 7.3 sodium ethylenediamine tetraacetate buffer.

72. Reagent according to claim 68 wherein the buffer is a pH 8.0 Tris/HCI buffer.

73. Reagent for the determination of uric acid in biological samples comprising: (1) 4-amino-antipyrine; (2) sulphonated dichlorophenol; (3) a water-soluble complex containing a cyanomolybdate or a cyanoruthenate ion; (4) microbial urate oxidase; (5) a peroxidase; and (6) a buffer.

74. Reagent according to claim 73 wherein the water-soluble complex is K4Mo(CN)8.2H2O.

75. Reagent according to claim 73 wherein the water-soluble complex is K4Ru(CN)6.3H20.

76. Reagent according to any one of claims 73 to 75 wherein the peroxidase is horseradish peroxidase.

77. Reagent according to any one of claims 73 to 76 wherein the buffer is an imidazole/HCI buffer.

78. Reagent according to claim 77 wherein the pH of the buffer is 6.6.

79. Reagent according to any one of claims 73 to 76 wherein the buffer is a sodium ethylenediaminetetraacetate buffer.

80. Reagent according to claim 79 wherein the pH of the buffer is 7.3.

81. Reagent according to any one of claims 73-80 containing a tensioactive substance as additional component.

82. Reagent according to claim 81 wherein the tensioactive substance is cholic acid sodium salt.

83. Reagent according to claim 73 comprising: (1) 4-aminoantipyrine: 0.2-2.0 mmol/l; (2) sulphonated dichlorophenol: 2-6 mmol/l; (3) K4Mo(CN)8.2H20: 0.5-1.5 mmol/l; (4) microbial urate oxidase: 800 it/I; (5) horseradish peroxidase: a5000 it/1; (6) pH 6.6 imidazole/HCI buffer: 20-100 mmol/l.

84. Reagent according to claim 83 comprising: (1) 4-aminoantipyrine: 0.4 mmol/l; (2) sulphonated dichlorophenol: 4 mmol/l; (3) K4Mo(CN)8.2H20: 1 mmol/l; (4) microbial urate oxidase: 2000 it/1; (5) horseradish peroxidase: 20000 it/I; (6) pH 6.6 imidazole/HCI buffer: 50 mmol/l.

85. Reagent according to claim 73 comprising: (1) 4-aminoantipyrine: 0.2-2.0 mmol/l; (2) sulphonated dichlorophenol: 2-6 mmol/l; (3) K4Ru(CN)8.3H20: 1.5-2.5 mmol/l; (4) microbial urate oxidase: a800 y/I (5) horseradish peroxidase: a5000 it/I; (6) pH 6.6 imidazole/HCI buffer: 20-100 mmol/l.

86. Reagent according to claim 85 comprising: (1) 4-aminoantipyrine: 0.4 mmol/l; (2) sulphonated dichlorophenol: 4 mmol/l; (3) K4Ru(CN)6.3H20: 2 mmol/l; (4) microbial urate oxidase: 2000 it/1; (5) horseradish peroxidase: 20000 y/I; (6) pH 6.6 imidazole/HCI buffer: 50 mmol/l.

87. Reagent according to claim 73 and 79 to 81 comprising: (1) 4-aminoantipyrine: 0.2-2.0 mmol/l; (2) sulphonated dichlorophenol: 2-6 mmol/l; (3) K4Ru(CN)6.3H20: 1.5-2.5 mmol/l; (4) microbial urate oxidase: a800 su/l; (5) horseradish peroxidase: a5000,u/l; (6) pH 7.3 sodium ethylenediaminetetraacetate buffer: 10-30 mmol/l; and (7) a tensioactive substance.

88. Reagent according to claim 87 wherein the pH 7.3 sodium ethylenediaminetetraacetate buffer is at a concentration of 20 mmol/l in the reaction mixture.

89. Reagent according to claim 87 or 88 wherein the tensioactive substance is cholic acid sodium salt.

90. Reagent according to claims 87 to 89 comprising: (1) 4-aminoantipyrine: 0.4 mmol/l; (2) sulphonated dichlorophenol: 4 mmol/l; (3) K4Ru(CN)6.3H20: 2 mmol/l; (4) microbial urate oxidase: 2000 it/1; (5) horseradish peroxidase: 30000 y/I; (6) pH 7.3 sodium ethylenediaminetetraacetate buffer: 20 mmol/l; and (7) cholic acid sodium salt: 1 g/l.

91. Reagent for the determination of triglycerides in biological samples comprising: (1) 4-aminoantipyrine: 0.2-2.0 mmol/l; (2) sulphonated dichlorophenol: 2-6 mmol/l; (3) K4Ru(CN)6.3H20: 1.5-2.5 mmol/l; (4) glycerolphosphate oxidase: a2000 tl/l; (5) peroxidase: a5000 it/1; (6) a glycerol kinase: a200 y/I; (7) a lipase: a100 y/I; (8) a tensioactive substance; (9) a lipase accelerator; (10) a glycerol kinase activator; (11) a glycerol kinase cosubstrate; and (12) a buffer.

92. Reagent according to claim 91 wherein the peroxidase is horseradish peroxidase.

93. Reagent according to claim 91 or 92 wherein the glycerol-phosphate oxidase and the glycerol kinase are microbial.

94. Reagent according to claim 91, 92 or 93 wherein the lipase is microbial.

95. Reagent according to claim 94 wherein the microbial lipase is a microbial lipoprotein lipase.

96. Reagent according to claim 95 wherein the microbial lipoprotein lipase is from Mucor juvanicus or from Pseudomonas fluorescens or from Pseudomonas aeruginosa or from Staphylococcus aureus.

97. Reagent according to any one of claims 91 to 96 wherein the tensioactive substance is cholic acid-sodium salt.

98. Reagent according to claim 97 wherein the concentration of cholic acid sodium salt is 5-10 g/l in the reaction mixture.

99. Reagent according to any one of claims 91 to 98 wherein the lipase accelerator is bovine albumin.

100. Reagent according to claim 99 wherein the concentration of bovine albumin is 0.2-5.0 g/l in the reaction mixture.

101. Reagent according to any one of claims 91-100 wherein the glycerol kinase activator is magnesium acetate.

102. Reagent according to claim 101 wherein the concentration of magnesium acetate is 0.2-5.0 g/l in the reaction mixture.

103. Reagent according to any one of claims 91 to 102 wherein the glycerol kinase cosubstrate is adenosine triphosphate trihydrated disodium salt.

104. Reagent according to claim 103 wherein the concentration of adenosine triphosphate trihydrate disodium salt is 0.1-2.0 mmol/l in the reaction mixture.

105. Reagent according to any one of claims 91 to 104 wherein the buffer is a pH 6.6 imidazole/HCI buffer.

106. Reagent according to claim 105 wherein the concentration of the pH 6.6 imidazole/HCI buffer in the reaction mixture is 20-100 mmol/l.

107. Reagent for the determination of triglycerides in biological samples comprising: (1) 4-aminoantipyrine: 0.4 mmol/l; (2) sulphonated dichlorophenol: 4 mmol/l; (3) K4RU(CN)6.3H2O: 2 mmol/l; (4) microbial glycerol phosphate oxidase: 5000,u/l; (5) horseradish peroxidase: 30000 it/1; (6) microbial glycerol kinase: 1000,u/l; (7) microbial lipase: 500 it/1; (8) cholic acid sodium salt: 7 g/l; (9) bovine albumin: 1 g/l; (10) magnesium acetate: 1 g/l; (11) adenosine triphosphate trihydrated disodium salt: 0.5 mmol/l; (12) pH 6.6 imidazole/HCI buffer: 50 mmol/l.

108. Method according to claim 1 substantially as hereinbefore described with reference to any one of the Examples.

109. Reagent according to claim 25 substantially as hereinbefore described with reference to any one of the Examples.