GB2102568A - Automatic continuous flow method for the assay of creatinine - Google Patents

Abstract

An automatic continuous flow method for the assay of creatinine in a biological fluid comprises enzymatic transformation of the creatinine with an insolubilised creatinine desimidase, to give ammonia and N-methylhydantoin followed by quantitative determination of the resulting ammonia. The enzyme is preferably immobilized on a nylon tube by a technique involving: a) alkylation of the nylon with triethyloxonium tetrafluoroborate; b) introduction of a linear-spacer chain on the alkylated nylon by treatment with hexamethylenediamine or ethylenediamine; c) activation of the spaced-nylon with a two-functional reagent such as dimethylsuberimidate a and/or glutaraldehyde; and d) attachment of the enzyme to the activated nylon. The assay is carried out using a set of reagents comprising (1) a reagent to remove any endogenous ammonia from the sample, (2) a reagent to detect ammonia produced from the creatinine and (3) the immobilized enzyme.

Description

SPECIFICATION Automatic continuous flow method for the assay of creatinine The present invention relates to a new automatic continuous flow method for the assay of creatinine in biological fluids, by means of enzymatic transformation of creatinine using creatinine desimidase (EC 3.5.4.21) in immobilized form, giving ammonia and N-methyl-hydantoin, and then determining the ammonia formed. Any endogenous ammonia present in biological fluids is preferably removed beforehand.

Text of the invention The present invention affords a new continuous flow method for the assay of creatinine with immobilized creatinine desimidase and a series of materials and reagents for carrying out the method.

Creatinine is a product of the metabolism of creatinephosphoric acid, one of the sources of energy for muscular contraction. Under normal conditions it is present in the blood at concentrations of 0.5-1.5 mug/100 ml, and is excreted as a final metabolite in the urine.

It is known that the quantitative determination of creatinine in the blood and urine affords an extremely useful diagnostic parameter for the evaluation of renal function.

Determination of the creatinine is of greater clinical diagnostic significance in the diagnosis of kidney diseases than that of other metabolites, such as urea for instance.

Hypercreatininaemia always indicates damage to the kidneys, and since it is almost independent of the diet, it allows the course of diseases such as, for instance, acute and chronic nephritis, nephrosis, urethrophraxis, mercurialism, etc., to be followed even in patients on special diets.

The low concentration of creatinine in the blood and the lack of highly specific assay methods still hinder correct determination of this metabolite.

Most of the published methods for the determination of creatinine are based on the reaction of the latter with picric acid in alkaline medium, that is, on JaffB's test [Z. Physiol. Chem. 10,391 (1886)].

It is known that Jaffe's method is non-specific, since many metabolites normally present in biological fluids cause interference [Clin. Chem. 21,286D-288D(1975); Clin. Biochem, 11,82 (1978); Ann. Clin. Biochem.

12,219 (1975)].

Numerous modifications of Jaffe's test are nowadays known, but although they have greatly increased the precision they have modified the specificity to only a certain degree (J. Clin. Chem. Olin. Biochem. 18,385 (1980). The attempts made to eliminate "gaff6 positive chromogens" have not found application in routine analyses because of the operative difficulties. Jaff6's reaction after deproteinization and absorption on Fuller's earth [Z. Klin. Chem. und Klin. Biochem. 8; 587 (1970); Olin. Chem. 27, 179(1981)] which is usually taken as the reference method, is also little practical for routine analyses.

In order to achieve greater specificity in the determination of creatinine, enzymatic methods based on the hydrolysis of creatinine to creatine by means of the enzyme creatinine-amidohydrolase (E.C. 3.5.2.10) and successive determination of the creatine obtained, have been studied [Scan. J. Olin. Lab. Invest. 29, Suppl.

126,30.1(1972); Methods of Enzymatic Analysis, Section 4 H.U. Bergmeyer, Ed. Academic Press, New York, N.Y., 2nd Ed., 1974, pages 1786-1790; Olin. Chem. 21, (1975); British Patent No. 1359402].

Although possessing good specificity, these methods are difficult to carry out in routine laboratory practice, especially for utilization in automatic equipment [Clin. Ohem. 25,1665-1666(1978); J. J. Olin. Them.

Olin. Biochem. 17,633-638 (1979)].

The present invention affords an enzymatic method for the determination of creatinine based on the use of an enzyme which catalyzes the transformation of creatinine into N-methylhydantoin and ammonia, and successive determination of the ammonia thus formed.

The enzyme used in the procedure according to the present invention, is creatinine desimidase (E.C.

3.5.4.21). Creatinine desimidase was discovered by J. Szulmajster [J. Bacteriol. 75,633 (1958) and Biochim.

and Biophys, Acta 30, 154(1958)] in the microbial cells of Clostridium paraputrificum, and isolated by means of chromatography. H. Thompson [Anal. Them. 46,2,246(1974)] and F.Lim [Clin. Chem. 20,7,871(1974)] successively indicated the use of creatinine desimidase for the quantitative determination of creatinine, through assay of the ammonia produced in the enzymatic reaction.

Patents were subsequently published in the name of Kyowa Hakko Kogyo Co., for instance, the British Patent No. 1515805 which not only describes a method for the production of creatinine desimidase from some specific microbic sources, but also a method for the quantitative determination of creatinine by hydrolysis with creatinine desimidase and successive determination of the ammonia or N-methylhydantoin formed.

The Kyowa Patent, like the previous works, does not indicate the possibility of applying the method to quantitative continuous flow determinations. In particular, no automatic flow technique which permits preliminary elimination of endogenous ammonia, always present in biological liquids, and consequent assay of the ammonia released from the creatinine only, is described.

It is known that automatic continuous flow techniques are commonly used in mass routine practice, in the diagnostic field. The present invention affords an automatic continuous flow method for the quantitative determination of creatinine by means of hydrolysis with creatinine desimidase and specific assay of the ammonia produced.

Development of an automatic continuous flow technique for the assay of creatinine in biological fluids, using creatinine desimidase, involves solving complex problems such as, for instance, the need to limit the quantity of enzyme used; to use creatinine desimidase free from other NH3-dependent enzymes, urease for instance; to eliminate interferences due to endogenous ammonia which is always present in serum and urine, and to utilize sensitive, specific systems for detecting only the ammonia produced in the enzymatic transformation of creatinine.

The method for the analysis of creatinine which is the subject of the present invention, permits overcoming all these problems. The need to limit the quantity of enzyme used is obviously a much more important problem, for economic reasons, in a continuous flow method than in a manual one.

Use of creatinine desimidase in solution in a flow system involves not only high consumption of this enzyme but also negative side effects such as, for instance, increase of the blank value, long incubation times, and entry of foreign substances of protein origin (for instance, enzymatic proteins) into the system which are not always compatible with the reagent used for detection of the ammonia.

In the flow method forming the subject of the present invention, creatinine desimidase is used in an immobilized form; the result is greatly reduced consumption with the consequent possibility of obtaining sufficient enzyme even from poorly productive microbial sources.

Use of immobilized enzyme makes it possible not only to keep out of circulation protein material (for instance, enzymatic proteins), but at the same time to guarantee high enzymatic activity which allows short incubations with complete transformation of the substrate without disturbance of the basal line or of the system for detection of the ammonia derived from the creatinine. Immobilization of creatinine desimidase can be effected using known immobilization techniques.

Said techniques can, for instance, be those described for other enzymes in Biochem. J. 147,593-603 (1975); British Patent No. 1470955 and British Patent No. 1485122.

For instance, a method particularly suitable for the immobilization of creatinine desimidase provides for attachment of the enzyme to nylon tubes by means of: a) alkylation of the nylon tube with triethyloxonium tetrafluoroborate; b) introduction of a straight chain (spacer) obtained by allowing a solution of hexamethylenediamine or, alternatively, ethylenediamine, to act on alkylated nylon; c) activation of the nylon-spacer with a bifunctional reagent which, for instance, may be dimethylsuberimi date or glutaraldehyde; and d) attachment of the enzyme to the activated nylon.

In an alternative technique, after treatment as described above under point b), the nylon-spacer is allowed to react with maleic anhydride or succinic anhydride.

The nylon spacer with terminal carboxylic function is then treated with N-hydroxysuccinamide in the presence of N,N-dicyclohexylcarbodiimide and, finally, the enzyme in a medium buffered to a neutral pH, for instance, phosphate buffer, is bound to the carboxylic nylon so activated.

The immobilization techniques mentioned above make it possible, at one and the same time, to bind a large quantity of enzyme to the nylon, to obtain a particularly purified form of enzyme which, above all, is free from ureases and or other NH3-dependent enzymes. The enzyme immobilized according to the invention procedure is stable, active on aqueous and physiological samples and functions in continuous flow.

The flowing Tables (I and II) give the stability data of creatinine desimidase immobilized according to the techniques described above. Table Ill reports the data regarding functionality of the immobilized enzyme.

TABLE I Stability at 40C of creatinine desimidase immobilized on nylon tubes The enzyme, creatinine desimidase, is immobilized on nylon tubes of 1 mm internal diameter and placed in stability at the temperature of 4"C in a 100 mM phosphate buffer solution, pH 7. The immobilized activity values are expressed in Ulmimin and referred in the controls as % compared to the original activity (0 time).

Immobilization A = nylon activated with dimethyl-suberimidate Immobilization B = nylon activated with glutaraldehyde Immobilization C = nylon activated with N-hydroxy-succinimide Immobilization A Immobilization B Immobilization C Stability 4"C 4"C 4"C months Ulmimin % Ulmimin % Ulmimin 0 9.9 100 9.7 100 9.2 100 1 9.9 100 9.3 96 8.9 97 2 9.7 98 9.7 100 9.3 101 4 9.9 100 9.7 100 9.7 105 6 9.9 100 9.3 96 9.4 102 12 9.9 100 9.4 97 9.5 103 18 9.6 97 8.9 92 8.6 93 TABLE II Stability at 35"C of creatinine desimidase immobilized on tubes The enzyme, creatinine desimidase, is immobilized on nylon tubes of 1 mm internal diameter and placed in stability at the temperature of 35"C in 100 mM phosphate buffer solution, pH 7. The immobilized activity values are expressed in U/m/min and referred in the controls as % compared to the initial activity (0 time).

Immobilization A = nylon activated with dimethyl-suberimidate Immobilization B = nylon activated with glutaraldehyde Immobilization C = nylon activated with N-hydroxy-succinimide lmmobilization A Immobilization B Immobilization C Stability 35"C 35"C 35"C months Ulmlmin % Ulmimin % Ulmimin 0 8.6 100 9.6 100 8.9 100 1 8.3 96 8.2 85 8.7 97 2 7.1 83 7.9 82 8.2 92 4 7.5 87 7.1 74 7.9 88 6 6.3 73 5.8 60 5.7 64 TABLE Ill Functionality ofimmobilized creatinine desimidase in continuous flow Creatinine desimidase immobilized on nylon tubing of 1 mm internal diameter and 1 metre in length, is used for routine analysis of creatinine in serum, plasma and urine, in a flow system of the type illustrated later in Figure 3.

The residual activity is controlled at intervals of 5000, 10,000 and 20,000 analyses and expressed as % of the initial U mimin.

Immobilization A = nylon activated with dimethyl-suberimidate Immobilization B = nylon activated with glutaraldehyde Immobilization C = nylon activated with N-hydroxy-succinimide Initial Activity at Activity at Activity at Immobili- activity 5000 anal. 10,000 anal. 20,000 anal.

zation U m min % U m min Oc U m min % U mimin % A 9 100 8.2 91 8.1 90 6.6 74 B 8.6 100 7.5 87 7.6 88 6.1 71 C 8.3 100 7.4 89 7 84 6.2 75 Creatinine desimidase utilized for the enzymatic splitting of creatinine according to the method of the invention, can be obtained by known methods from various microbic sources and, for example, can be the enzyme mentioned in the reference given above: Biochimica et Biophysica Acta 30, (1958) and Analytical Chemistry, 46, 2, 246, (1974).

The problem of elimination of interferences due to endogenous ammonia, which is always present in serum and urine, is of fundamental importance in the analysis of creatinine based on assay of the ammonia produced in the catalysis process with creatinine desimidase.

The problem is relatively simple when a manual method is used, and can be solved by differential assay effected on the same sample before and after incubation with creatinine desimidase.

With a continuous flow method, differential assay, even if possible, is a much more complicated problem, especially in cases where the value of the blank compared to the sample is great, as is the case of the assay of creatinine.

Moreover, in a continuous flow method with dialysis, such as that proposed in the present invention, the contribution of the endogenous ammonia to the total response would be increased still further by the high dialysis power of the ammonium ion compared to creatinine.

The method forming the subject of the invention makes it possible to eliminate the endogenous ammonia and to predispose the catalysis process between immobilized creatinine desimidase and creatinine, for the purpose of quantifying unequivocally, only the ammonia produced in the enzymatic reaction.

According to the invention, quantitative determination of creatinine in a biological fluid such as serum or urine, is carried out by means of a continuous flow method comprising: a) elimination of any endogenous ammonia in the sample by incubation with a suitable reagent in predialysis; b) continuous dialysis; c) reaction of the creatinine with immobilized creatinine desimidase, in the acceptor line, to give ammonia and N-methylhydantoin; and d) determination of the ammonia produced, using a suitable detection system.

The reagent used to eliminate any endogenous ammonia in the donor line, must be specific and not interfere with the evaluation of the ammonia produced from the creatinine in the acceptor line. For instance, this reagent may be an NADH;GLDH reagent acting on the endogenous ammonia according to the NH3 (endogenous) + a-ketoglutarate + NADH GLDH NAD + glutamate + H2O reaction, or else a reagent based on aspartase [E.C.4.3.1.1] which acts on endogenous ammonia according to the equation NH3 (endogenous) + fumarate aspartase aspartate The dialysis process makes it possible to eliminate the protein and corpuscular part of the samples, thus permitting correct analysis of the creatinine, even in the serum of high lipid or icteric content.

Creatinine dialyses substantially free from endogenous ammonia and downstream from the dialysis encounters immobilized creatinine desimidase which catalyses its hydrolysis to ammonia and N-methylhydantoin.

Assay of the free ammonia is preferably effected by means of reaction with a Berthelot reagent, a chemical reagent based on phenol or sodium hypochlorite or, alternatively, an NADH GLDH a-ketoglutarate reagent qualitatively equal to that used to destroy the endogenous ammonia.

The fumaratesaspartase system used to eliminate the endogenous ammonia may be coupled either to the Berthelottype detection system [Clin. Chem. vol.8, 130-132 (1962)] orto the NADH-GLDH one.

On the contrary, use of NADH/GLDH/a-ketoglutarate reagent to eliminate the endogenous ammonia, is a source of interference with the ammonia detection system which acts downstream from analysis, when said system consists of the same NADH-dependent reagent.

To make this coupling possible, it is absolutely necessary to avoid that the residual NADH of the reagent used in donor line, having destroyed the endogenous ammonia, passes into the recipient line, abnormally changing the NADH content of the ammonia detection reagent.

According to the technique of the invention, this can be brought about by bringing the pH of the medium to a value of 2, so that the residual NADH after elimination of the endogenous ammonia and before passage into dialysis, is eliminated totally in view of its instability in acid medium.

In a preferential method the medium is acidified with phosphoric acid and the pH is brought to about 7.5 before entry into dialysis, by the addition for instance, of a tris-hydroxymethylaminomethane solution.

In a particularly preferred form of realization of the method, use of NADH/GLDH reagent for elimination of the endogenous ammonia is coupled with use of Berthelot's reagent for detection of the ammonia derived from creatinine.

In another form of preferred realization, the invention method provides for the use of aspartase/fumarate reagent to eliminate the endogenous ammonia before dialysis, coupled with use of NADH/GLDH reagent for assay of the ammonia produced from the creatinine.

Transformation of creatinine into ammonia and N-methylhydantoin is always obtained with immobilized creatinine desimidase. As already stated, the immobilization is effected on tubes and, in particular, on nylon tubes, according to one of the systems described previously.

The nylon tube used may have an internal diameter varying from approximately 0.8 to 1.6 mm, preferably from 1 to 1.2 mm.

The length of the tube varies roughly from 0.5 to 1 m, preferably around 1 m, and the activity of the bound enzyme should, preferably, be not less than 4-6 U/m/min. in order to permit close to 100% conversion with a linear response up to 15 mg/100 ml of creatinine.

A thousand analyses of creatinine in serum and urine can be carried out with a single tube of immobilized creatinine desimidase. Good functionality of immobilized creatinine desimidase and its good stability in time are guaranteed also by use of buffers which make suitable pH values and ionic force possible.

For this purpose, the acceptor flow is preferably buffered at pH 7-8, preferably at pH 7.5, with phosphate buffer, 50 mM phosphate buffer for instance, or at pH 7.8 with Tris buffer, for instance 50 mM Tris/HCI buffer.

The donor flow is preferably buffered with 200 mM phosphate, pH 7.5 or 200 mM Tris buffer, pH 7.8.

Phosphate buffer is used in conjunction with Berthelot type chromogen system; Tris/HCI buffer is used with NADH/GLDH type systems.

In the accompanying drawings: Figure 1 shows a flow diagram of a single channel/single dialyser system; Figure 2 shows a flow diagram of a two channel/two dialyser system; and Figures 3 and 4 show two variations of a two channel/single dialyser system.

The automatic flow method which is the subject of the present invention is the result of an in-depth study based on various flow systems conceived always with the aim of eliminating or reducing the endogenous ammonia as much as possible before dialysis, of transforming the creatinine after dialysis, with immobilized creatinine desimidase, and of detecting the ammonia produced with specific agents compatible with the rest of the system. For instance, one channel systems of the type illustrated in Figure 1 have been experimented, where sample X is mixed with reagent R, consisting of NADH/GLDH/a-glutarate, and pumped into the system fractionated with air bubbles A.

The flow enters bath B where the endogenous ammonia is eliminated along with all other substances interacting with NADH.

After incubation, the flow is acidified by the addition of P, for instance, by addition of dilute phosphoric acid, left at pH = 2 for the entire S tract, and the returned to pH = 7 by immission into the system of T, for instance, of Trihydroxymethylaminomethane.

After this treatment the flow enters dialyser D, through coil S1. The non-dialyzed portion is sent to the discharge outlet 0, while the dialyzed portion containing creatinine free from any interference enters the acceptor channel. The acceptor channel flow, which contains the ammonia determination reagent F (NADH/GLDH/ct-ketoglutarate) fractionated by air bubbles A, passes downstream from dialysis and enters the reactor (nylon tube) with immobilized creatinine desimidase E.

The creatinine present gives up ammonia which, reacting with reagent F in the developing coil Z, oxidizes NADH to NAD. A reading in the colorimeter C, at 340 nm for instance, makes it possible to determine the creatinine concentration and to record it on the recorder V. In one-channel systems, such as that of Figure 1, total elimination of the endogenous ammonia and of all substances interfering with the NADH system, by means of dialysis, is a necessary condition for reading of the sample value against the blank value at constant base line.

These absolutely indispensable conditions always require perfect efficiency of the reagents and can limit the applicability of the method, especially in hospital routines.

Apart from the one-channel system of the type reported in Figure 1 for instance, two channel systems have been utilized to achieve the invention method; these provide for elimination of the endogenous ammonia, coupled with reading of the ammonia derived from the creatinine in differential phase, where the value of the blank is automatically subtracted from that of the sample.

The response has been made more certain in this way, freeing it from the compulsory restraint of complete elimination of the endogenous ammonia in order to have a constant blank equal to the base line.

At the same time, the endogenous ammonia elimination process makes it possible to have low blanks in every case, and to operate under optimal conditions for an analysis effected with differential reading.

Figure 2 illustrates a two-channel system that can be used for realization of the method of the invention; in this Figure sample Xis mixed with reagent R which may be NADH GLDHSa-ketoglutarate or aspartasel fumarate, and pumped into the system, fractionated by air bubbles A. The flow enters bath B for elimination of the endogenous ammonia.

The sample, freed from the endogenous ammonia, is pumped by means of recycling tubes C and C1 which lead off from the debubbler T, into the flow upstream from dialysis relative to dialyzer D of the sample channel and dialyzer D, of the blank channel. The flows upstream from dialysis are then discharged by means of the outlets S and S1. The flows downstream from dialysis, T and T1, broken up by bubbles, are allowed to enter the reactor N (nylon tube with immobilized creatinine desimidase) and the blank reactor N1 (blank reference tube).

The ammonia released from the creatinine in N is detected by means of a reagent based on phenol F and sodium hypochlorite I.

At the same time, the value of the blank is shown in the reference column with the same reagents pumped through F1 and 11.

After incubation on Z and Z1, the flows enter the differential reading colorimeter Land the value of the creatinine is read, at 650 nm for instance, and recorded by recorder V.

In a particularly preferred form of realization, the continuous flow system used for the automatic method of analysis proposed by the invention provides for use of a specific two-channel system which permits elimination ofthe endogenous ammonia and execution of dialysis in an unequivocal manner with separation into sample channel and blank channel only just before the catalytic process with immobilized creatinine desimidase.

The most qualifying pointsofthissytem are: a) the greater simplicity of the tube system compared to the conventional two-dialyzertwo-channel systems; b) the possibility of obtaining satisfactory sensitivity even when using a small volume of sample, with good accuracy and precision values; c) the use of a single dialyzer for the two channels; d) the recycling system with debubbler which permits distribution of the dialysate itself in both the sample channel and that of the blank, simultaneously and in equal volume; eel the use of immobilized creatinine desimidase which, placed suitably in the sample channel only, makes differential reading of the blank possible; and f) the ease of phasing which involves only the last part of the system.

Two-channel flow systems with single dialyzer and possessing the above-mentioned characteristics are given, for example, in Figures 3 and 4.

In the system illustrated in Figure 3, sample Xis mixed with reagent R and pumped into the system, fractionated by air bubbles A.

The flow enters bath W for elimination of the endogenous ammonia. After incubation, the flow passes into the before dialysis zone of dialyzer D and is eliminated through outlet S.

The dialyzed solution passes into the flow acceptor F and, fractionated with air bubbles A, is conveyed by tube d to the debubbler T.

Part of the flow is recycled in the system from the two-way debubbler by means of tubes G and G1 and, fractionated with air bubbles A, passes respectively into reactors C (tube with immobilized creatinine desimidase) and B (blank reference tube). The ammonia released from the creatinine in C is detected by a reagent based on phenol Land sodium hypochlorite M.

The value of the blank is simultaneously revealed in the reference channel with the same agents pumped from L1 and M,.

After incubation in Z and Z1, the flows enter the differential reading colorimeter and the value of the creatinine is read, at 650 nm for instance, and recorded with recorder V.

When the aspartase fumarate reagent for the destruction of the endogenous ammonia is coupled to use of the GLDH:NADH;u-ketoglutarate reagent for detection of the ammonia produced from the creatinine, the flow system represented in Figure 4 is utilised.

In this system the flow of sample X is mixed with reagent R (aspartase,fumarate) and pumped into the system fractionated with air bubbles A. The flow enters bath W for elimination of the endogenous ammonia and, after incubation, enters dialyzer D where it is discharged from outlet S.

The dialysate passes into the acceptor flow which enters into dialysis fractionated with air bubbles A.

The acceptorflow F, containing the NADH GLDH,u-ketoglutarate reagent, is conveyed by means oftube d to debubbler T. Part of the flow is recycled into the system from the two-way debubbler by means of tubes G and G1 and, fractionated with air bubbles A, passes respectively into reactors C (tube containing immobilized creatinine desimidase) and B (blank reference tube). Ammonia is released in C from the creatinine and reacts in Z with NADH.

In the reference channel, and precisely in B and Z1, the blank is developed and read in differential reading at 340 nm for instance in comparison with the respective sample, using colorimeter U and recording the result with recorder V.

Besides the continuous flow enzymatic method for the determination of creatinine described above, the present invention also provides for a series of materials and reagents to carry out the method.

This series of materials and reagents comprises: a) a reagent for elimination of the endogenous ammonia in a suitable buffer; b) a reagent for the detection of ammonia from creatinine; c) immobilized creatinine desimidase.

The reagent for elimination of the ammonia may be one of the NADH/GLDH and aspartase/fumarate reagents previously mentioned in this Application, in a suitable neutral-basic buffer.

The reagent for the detection of ammonia from creatinine can be a reagent according to Berthelot or an NADH/GLDH reagent as those indicated previously.

The immobilized creatinine desimidase is creatine desimidase immobilized on a tube, preferably a nylon tube, according to the techniques described above.

When the reagent for elimination of endogenous ammonia, in the series of reagents supplied by the invention, is an NADH/GLDH reagent, the solution of said reagent used contains preferably: phosphate buffer 200 mM at pH 7.3-7.8, preferably 7.5; ct-ketoglutarate 8-12 mM, preferably 10 mM; NADH 0.6-1 mM, preferably 0.8 mM; GLDH 40-50 U/ml, preferably 45 U/ml.

When the reagent for the elimination of endogenous ammonia is an aspartase/fumarate reagent, the solution of said reagent used contains preferably: phosphate buffer 200 mM at pH 7.8-8.2, preferably 8; aspartase 18-22 U/ml, preferably 20 U/ml; fumarate 40-60 mM, preferably 50 mM.

When the reagent for the detection of ammonia from creatinine is a reagent according to Berthelot, the solution of said reagent used contains preferably: phenol 80-120 mM, preferably 100 mM; sodium nitroprusside 1.2-1.6 mM, preferably 1.4 mM; sodium hypochlorite 13-17 mM, preferably 15 mM; sodium hydroxide 0.15-0.19 mM, preferably 0.17 mM.

When the reagent for the detection of ammonia from creatinine is an NADH/GLDH reagent, the solution of said reagent used contains preferably: phosphate buffer 50 mM, at pH 7.3-7.8, preferably 7.5; ot-ketoglutarate 6-10 mM, preferably 8 mM; NADH 0.05-0.1 mM, preferably 0.075 mM; GLDH 10-20 U/ml, preferably 15 U/ml.

In the series of reagents supplied by the invention, the immobilized creatinine desimidase preferably has an activity of about 6010 Ulmlmin, preferably 8 Ulmlmin, and is immobilized, for preference, on nylon tubes 0.5-1 m long, preferably 0.7-0.8 m, of internal diameter about 0.8-1.2 mm, preferably 1 mm.

In the preferred form of realization, the series of materials supplied by the invention contains an NADH/GLDH reagent as reagent for the elimination of endogenous ammonia, coupled to a reagent according to Berthelot for the detection of ammonia from creatinine or, alternatively, an aspartase/fumarate reagent for elimination of endogenous ammonia, coupled to an NADH/GLDH reagent for the detection of ammonia from creatinine, where the various reagents (NADH/GLDH, Berthelot, aspartase/fumarate) and the immobilized creatinine desimidase preferably have the compositions and characteristics indicated as preferential, above.

In the present invention, the abbreviations NADH, NAD+ and GLDH are used to indicate nicotinamideadeninedinucleotide in reduced form, nicotinamide-adeninedinucleotide in oxidized form and glutamate dehydrogenase, respectively; the terms a-ketoglutarate, glutamate, fumarate and aspartate are used to indicate a salt, preferably alkaline, of sodium or potassium in particular, of a-ketoglutaric, glutamic, fumaric and aspartic acids, respectively; the abbreviations EDTA and Tris respectively indicate the disodium salt of ethylenediaminotetraacetic acid and trihydroxymethylaminomethane.

The following examples illustrate the invention.

Example 1 Creatinine is assayed in sera using the flow system illustrated in Figure 1 with immobilized creatinine desimidase and using Gaffes method (according to the procedure described by H. Bartels etal. in Olin. Chim.

Acta 37, (1972)) simultaneously with the Mercotest 3385 method.

60 sera were assayed against aqueous standards of creatinine 2 mg dl, utilizing a Technicon Autoanalyzer llDR of Technicon Instruments Co. Tarrytown, N.Y.: the samples X = 0.23 ml min are analysed at the rate of 60 analyses. hour: the reagent R = 0.60 ml min is a solution of 200 mM Tris HCI buffer at pH 7.8 containing 0.6 mM NADH, GLDH 40 U ml, 10 mM potassium u-ketoglutarate and air A = 0.23 mlimin.

B = 5 ml is the incubation bath at 37"C.

P = 0.23 ml;min and T = 0.23 ml min. pump respectively 2.5% w v phosphoric acid and 10% wiv tris-hydroxymethylaminomethane, Sand S1 being = 1.5 ml.

D = 24 inches (61 cm) is the dialyser The acceptor flow F = 1.2 ml min is a solution of 50 mM Tris HCI buffer, pH 7.8, containing 0.05 mM NADH, GLDH 20 U;ml, 8 mM potassium a-ketoglutarate, and airA = 0.23 ml min.

E = 1 m is the nylon tube with creatinine desimidase at 8 U m min.

Z = 5 ml is the incubation coil at room temperature.

C is the colorimeter which operates at 340 nm with cell of 1.5 cm optical pathway in linearity field 1-12 mg/dl of creatinine.

The results obtained with the invention method (Y) have been compared with those obtained with Jaff6's reference method (X).

The correlation data obtained are: regression Y = 0.946x + 0.065 coefficient of correlation r = 0.994 Example 2 60 sera, 60 plasmas with EDTA anticoagulant, 60 plasmas with heparin anticoagulant, and 60 samples of urine (diluted 1:100 with water), are assayed against aqueous standards of creatinine at the concentration of 2 mg dl, using a Technicon Autoanalyzer II- and the flow system illustrated in Figure 3: the samples X = 0.23 ml min are analysed at the rate of 60 analysesihour; reagent R = 0.42 mI/min is a solution of 200 mM phosphate buffer, pH 7.5, containing 0.8 mM NADH, GLDH 40 U'ml, 10 mM potassium ct-ketoglutarate,with airA = 0.23 ml min.

W = 5 ml is the incubation bath at 37 C.

The acceptor flow F = 1 ml min is a 50 mM phosphate buffer solution, pH 7.5.

The recycling tubes G and G1 are of 0.42 ml min and C = 1 m is the nylon tube containing creatinine desimidase 6 U m min.

The reagent phenol L L1 enters the system at the rate of 0.42 ml min and the reagent sodium hypochlorite M'M1 at the rate of 0.42 ml min. After incubation in Z and Z1 = 6 ml, the solutions are read with a two-channel colorimeter at 650 nm.

The results (Y) obtained with the invention method have been compared with those (X) obtained with the reference method (Jaffe's kinetic method used in Example 1).

The correlation data obtained are: for the sera; regression y = 1.045x - 0.132; coefficient of correlation r = 0.998; forthe plasma: EDTA: regression y = 1.012x - 0.137; coefficient of correlation r = 0.998; for the heparinized plasma: regression y = 1.032x - 0.164; coefficient of correlation r = 0.995; for the urine: regression y = 0.997x - 0.137 coefficient of correlation r = 0.996.

Example 3 60 sera are analysed as in example 2, but using as reagent R a solution of potassium fumarate 50 mM and aspartase 20 u ml in 200 mM phosphate buffer at pH 8. The results obtained with the invention method are compared with those (X) obtained by the reference method (Jaffé's kinetic method indicated in example 1).

The correlation data obtained are: regression: y = 1.032x - 0.159; coefficient of correlation; r = 0.979.

Example 4 Using the Technicon Autoanalyzer lle and the flow system illustrated in Figure 3, a series of standard sera at different creatinine concentrations have been analyzed. Table IV: which follows, gives the commercial name of the serum, its batch number, the creatinine values determined according to the invention and the mean value stated by the manufacturers, with Jaff6's method.

TABLE IV Creatinine in mgidl Serum Batch Found Theoretical Pathonorm L 16 1.26 1.40 Pathonorm H 16 7.60 7.80 Seronorm 142 1.41 1.52 Monitroll 147 1.36 1.40 Precilip 09367 1.33 1.24 Precinorm U 08543 1.65 1.81 Precipath U 09510 3.28 3.56 Ortho Norm. 7T025 0.93 1.05 Ortho Abn. 7T113 8.70 9.05 Example 5 Serial quantities of creatinine standard were added to human serum with a creatinine content of 1 mg/dl.

The various fractions were analyzed with a Technicon Autoanalyzer Il, using the flow system of Figure 3.

The creatinine recovery values are reported in Table (V) and expressed as percentages of the theoretical value calculated.

TABLE V Serum: Creatinine Theoretical Creatinine creatinine added creatinine found Recovery (mgldll (mgldl) {mgidl) {mgidl) 1 0 1 1 100 1 1 2 2.03 101 1 2 3 3.07 102 1 4 5 5.10 101 1 6 7 7.10 102 1 8 9 9.14 102 Example 6 1 ml of human serum with a creatinine content of 1.33 mg/dl was treated with serial quantities of 0.5 to 2 ml of a second human serum with a creatinine content of 7.86 mg/dl.

The various fractions were analyzed with a Technicon Autoanalyzer using the flow system illustrated in Figure 3.

The creatinine values obtained are given in Table (VI) expressed as percentage recovery with regard to the calculated theoretical value.

TABLE VI 0reatinine values Serum Serum Theoret Found Recovery 1.33 mgldl 7.86 mgldl mgidl mgidl % 1 ml + 0.5 ml 3.50 3.61 103 1 ml + 1 ml 4.59 4.77 104 1 ml + 2ml 5.68 5.85 103 Example 7 Three human sera of different creatinine concentrations were analyzed for 20 replications with a Technicon Autoanalyzer lls using the flow system illustrated in Figure 3.

The following Table (VII) gives the results relative to the mean value and the coefficient of variation.

TABLE VII Concentration Mean value Coefficient of ofcreatinine mgidl variation [c. v.] Low 1.3 1.98 Medium 2.4 1.10 High 7.1 0.79

Claims (40)

1. An automatic continuous flow method for the assay of creatinine in a biological fluid which comprises enzymatic transformation of the creatinine with an insolubilised creatinine desimidase, to give ammonia and N-methylhydantoin followed by quantitative determination of the resulting ammonia.

2. A method according to claim 1, in which the enzymatic transformation is preceded by elimination of any endogenous ammonia present in the biological fluid.

3. A method according to claim 2 comprising a) elimination of the endogenous ammonia by treatment with a suitable reagent, in predialysis; b) continuous dialysis; c) reaction of creatinine after dialysis with immobilized creatinine desimidase; and d) quantitative determination of the ammonia produced.

4. A method according to any one of the preceding claims, in which the immobilization of creatinine desimidase is effected on a nylon tube inserted in the flow line.

5. A method according to any one of claims 2 to 4 in which elimination of endogenous ammonia is effected automatically within the flow system.

6. A method according to any one of claims 2 to 5 in which the elimination of endogenous ammonia is achieved by treatment with an NADH GLDH reagent.

7. A method according to claim 6, in which the NADH GLDH reagent contains in the solution for use: phosphate buffer 200 mM at pH 7.3-7.8 ct-ketoglutarate 8-12 mM NADH 0.6-1 mM GLDH 40-50 U ml.

8. A method according to claim 7, in which the solution for use contains: phosphate buffer 200 mM at pH 7.5 ut-ketoglutarate 10 mM NADH 0.8 mM GLDH 45 U ml.

9. A method according to any one of claims 2 to 5, in which elimination of the endogenous ammonia is achieved by treatment with an aspartase fumarate reagent.

10. A method according to claim 9, in which the aspartase fumarate reagent contains in the solution for use: phosphate buffer 200 mM at pH 7.8-8.2 aspartase 18-22 U,ml fumarate 40-60 mM.

11. A method according to claim 10, in which the solution for use contains: phosphate buffer 200 mM at pH 8 aspartase 20 U ml fumarate 50 mM.

12. A method according to any one of the preceding claims, in which the resulting ammonia is determined using a reagent based on phenol and sodium hypochlorite as the chromogen detection system.

13. A method according to claim 12, in which the reagent based on phenol and sodium hypochlorite contains in the solution for use: phenol 80-120 mM sodium nitroprusside 1.2-1.6 mM sodium hypochlorite 13-17 mM sodium hydroxide 0.15-0.19 M.

14. A method according to claim 13, in which the solution for use contains: phenol 100mM sodium nitroprusside 1.4 mM sodium hypochlorite 15 mM sodium hydroxide 0.17 mM.

15. A method according to any one of claims 1 to 11, in which the resulting ammonia from creatinine is determined using an NADH/GLDH reagent as the detection system.

16. A method according to claim 15 in which the NADH/GLDH reagent for ammonia determination contains in the solution for use: phosphate buffer 50 mM at pH 7.3-7.8 a-ketoglutarate 6-10 mM NADH 0.05-0.1 mM GLDH 10-20 U/ml.

17. A method according to claim 16, in which the solution for use contains: phosphate buffer 50 mM at pH 7.5 ce-ketoglutarate 8 mM NADH 0.075 mM GLDH 15U/ml.

18. A method according to any one of the preceding claims, in which the flow system is a two-channel system with differential reading.

19. A method according to claim 18, in which the system comprises a single dialyzer.

20. A method according to any one of claims 3 to 19, in which the flow system consists of a tube in which the following are inserted in the order of the flow route: a) a bath for elimination of endogenous ammonia; b) a dialyzer; c) a recycling system with degassing which permits simultaneous distribution in the blank channel and the sample channel of the single dialyzer; d) a blank reactor inserted in the blank channel; e) a reactor containing immobilized creatinine desimidase inserted in the sample channel forthe enzymatic reaction with creatinine; and f) two coils, inserted respectively in the blank line and in the sample line, for developing the ammonia detection reaction.

21. A method for the assay of creatinine according to claim 1 substantially as hereinbefore described with reference to any one of the Examples.

22. A method for the assay of creatinine according to claim 1 substantially as hereinbefore described with reference to the flow sheet of the accompanying drawing.

23. Apparatus for carrying out the method of claim 20, consisting of: a) an automatic device for taking the samples; b) a system for inlet of the samples into the flow; c) a flow system; and d) a detection system, where the flow system is as defined in claim 20.

24. Set of materials and reagents for carrying out a method according to any one of claims 3 to 22 comprising: a) a reagent for elimination of endogenous ammonia in a suitable buffer; b) a reagent for detection of ammonia produced by enzymatic degradation of creatinine; and c) immobilized creatinine desimidase.

25. Set of materials and reagents according to claim 24, in which the reagent for elimination of endogenous ammonia consists of NADH/GLDH.

26. Set of materials and reagents according to claim 25, in which the NADH GLDH reagent contains, in the solution for use: phosphate buffer 200 mM at pH 7.3-7.8 ut-ketoglutarate 8-12 mM NADH 0.6-1 mM GLDH 40-50 U ml.

27. Set of materials and reagents according to claim 26, in which the solution for use contains: phosphate buffer 200 mM at pH 7.5 ce-ketoglutarate 10 mM NADH 0.8mM GLDH 45Uml.

28. Set of materials and reagents according to claim 24, in which the reagent for elimination of endogenous ammonia consists of aspartase;fumarate.

29. Set of materials and reagents according to claim 28, in which the aspartase'fumarate reagent contains in the solution for use: phosphate buffer 200 mM at pH 7.8-8.2 aspartase 18-22 U ml fumarate 40-60 mM.

30. Set of materials and reagents according to claim 29, in which the solution for use contains: phosphate buffer 200 mM at pH 8 aspartase 20 U ml fumarate 50 mM.

31. Set of materials and reagents according to any one of claims 24 to 30, in which the reagent for detection of ammonia is a reagent based on phenol and sodium hypochlorite.

32. Set of materials and reagents according to claim 31, in which the reagent based on phenol and sodium hypochlorite contains in its solution for use: phenol 80-120mM sodium nitroprusside 1.2-1.6 mM sodium hypochlorite 13-17 mM sodium hydroxide 0.15-0.19 mM.

33. Set of materials and reagents according to claim 31, in which the solution for use contains: phenol 100mM sodium nitroprusside 1.4 mM sodium hypochlorite 15 mM sodium hydroxide 0.17 M.

34. Set of materials and reagents to any one of claims 24 to 30, in which the reagent for detection of ammonia is an NADH GLDH reagent.

35. Set of materials and reagents according to claim 34, in which the NADHGLDH reagent for detection of ammonia contains, in the solution for use: phosphate buffer 50 mM at pH 7.3-7.8 ce-ketoglutarate 6-10 mM NADH 0.05-0.1 M GLDH 10-20Uml.

36. Set of materials and reagents according to claim 35, in which the solution of the NADH/GLDH reagent for use contains: phosphate buffer 50 mM at pH 7.5 ce-ketoglutarate 8 mM NADH 0.075 mM GLDH 15 Uxml.

37. Set of materials and reagents according to any one of claims 24 to 36, in which the immobilized creatinine desimidase is immobilized on a nylon tube of length 0.2 to 2 m and of internal diameter 0.8 - 1.6 mm.

38. Set of materials and reagents according to claim 37, in which the nylon tube is 0.8-1.2 m in length, internal diameter 1-1.2 mm.

39. Set of materials and reagents according to claim 37 or 38, in which the nylon tube containing the immobilized creatinine desimidase is obtained with an immobilization technique consisting of: a) alkylation of the nylon with triethyloxonium tetrafluoroborate; b) introduction of a linear-spacer chain on the alkylated nylon by treatment with hexamethylenediamine or ethylenediamine; c) activation of the spaced-nylon with a two-functional reagent such as dimethylsuberimidate and/or glutaraldehyde; and d) attachment of the enzyme to the activated nylon.

40. Set of materials and reagents according to claim 24 substantially as hereinbefore described with reference to any one of the Examples.