FR2508488A1 - AUTOMATIC FLOW CONTINUOUS PROCESS FOR THE ASSAY OF CREATININE BY IMMOBILIZED CREATININEDESIMIDASE - Google Patents

Abstract

THE INVENTION CONCERNS AN AUTOMATIC CONTINUOUS FLOW PROCESS FOR THE DOSAGE OF CREATININE IN BIOLOGICAL LIQUIDS BY ENZYMATIC TRANSFORMATION OF CREATININE BY CREATININEDESIMIDASE FOR GIVING AMMONIAC AND N-METHYLHYDANTOINE AND DETERMINATION OF THE QUANTIONTAINE PROIACATION 'HYDROLYSIS. THE PROCESS, ACCORDING TO THE INVENTION, IS CHARACTERIZED IN THAT THE CREATININEDESIMIDASE USED IS IMMOBILIZED, IN PARTICULAR ON A "NYLON" TUBE.

Description

The present invention relates to a novel self-

  continuous flow for the determination of creatinine in body fluids by enz-natal transformation of creatinine by creatinineimidase (EC 3 5 4 21) to give ammonia and N-methylhydantoin and determination of formed ammonia, characterized in that the creatine imidase is immobilized and the endogenous ammonia present in the

  biological fluids is previously eliminated.

  The present invention proposes a novel process

  continuous flow for the determination of creatinine with

  immobilized nidine-imidase and a series of materials and reagents

  for the implementation of the method.

  Creatinine is a product of the metabolism of creatinephosphoric acid, which is one of the energy sources of

muscle contraction.

  Under normal conditions, it is present in

  blood at concentrations of 0.5-1.5 mg / l O ml and it is eli-

  mined as the final metabolite in the urine.

  Quantitative determination of creatinine is known

  in the blood and urine provides an extreme diagnostic parameter

  useful for the assessment of renal function.

  In the diagnosis of renal diseases, the determination of creatinine is of a clinico-diagnostic importance far superior to that of other metabolites, such as urea, by

example.

  Hypercreatininaemia always indicates kidney damage and, since it is almost independent of the diet, it makes it possible to follow the course of diseases such as, for example, acute and chronic nephritis, nephrosis, urethrophyxia, mercurialism, etc.

  even in patients who are on special diets.

  The low concentration of creatinine in the blood and the absence of highly specific dosing procedures pocket

  still a 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 to say on the reaction

  known from Jaffé (Z Physiol Chem 10, p 391 (1886)).

-2508488

  It is known that the Jaffé method is not specific since many metabolites normally present in liquids

  biological causes interferences (Clin Chem 21, p 286 D-

  288 D (1975); Clin Biochem 11, 82 (1978); Ann Clin Biochem 12, p 219 (1975)). Many modifications of the Jaffé reaction are known today, but although they have increased

  accuracy, they only modified to a certain extent the speci-

  (Clin Clin Bitchem Clin 18, p 385 (1980).

  Attempts to eliminate the so-called chromium compounds

  positive Jaffe genes "did not find the application in the

  routine analyzes because of operational difficulties.

  The reaction of Jaffé after deproteinization and absorption on fuller's earth (Z Klin Chem und Klin Biochem 8, p 587 (1970); Clinm Chem 27, p 179 (198 D), usually considered as the reference technique, is similarly convenient for analyzes

routine.

  In order to obtain greater specificity in deter-

  In the production of creatinine, enzymatic methods based on creatinine-creatinine-creatinine-amido-hydrolase hydrolysis (EC 3 5 2 10) and on the subsequent determination of creatine obtained have been investigated (Scan J Clin Lab Invest 29, Suppl 126, p 1 (1972), Methods of Enzymatic Analysis, Section 4 HU Bergmeyer, Ed Academic Press, New York, NY 2nd Edition, 1974 p 1786-1790, Clin Chem 21, p 1422 (1975), British Patent

No. 1359402).

  Although these methods have good specificity, they are difficult to implement in laboratory practice

  routine, especially for use in

  (Clin Chem 25, 1665-1666 (1979), J Clin Chem Clin.

Biochem 17, p 633-638 (1979)).

  The present invention provides an enzymatic method for the determination of creatinine based on the use of a

  enzyme that catalyzes the conversion of cretinin to N-methyl-

  hydantoin and ammonia, and on the subsequent determination of

the ammonia formed.

  The enzyme used in the procedure according to the invention

  tion is the creatinine of imidase (E G 3 5 4 21).

  Creatinineimidase was discovered by J Szulmajster (J Bacteriol 75, p 633 (1958) and Biochim and Biophys Acta 30, p 154 (1958)) in microbial cells of Clostridium para-

  putrificum and isolated by chromatographic techniques.

  H Thompson (Anal Chem 46, 2, p 246 (1974)) and F Lim

  (Clin Chem 20, 7, p 871 (1974)) subsequently indicated the use of

  of the creatinineimidase for the quantitative determination of

  of creatinine, by the determination of ammonia produced in

the enzymatic reaction.

  Patents were then published, on behalf of Kyowa Hakko Kogyo Co., for example British Patent No. 1515805,

  which describes not only a process for the production of creatinine

  deimidase from certain particular microbial sources, but also a method for the quantitative determination of creatinine by hydrolysis by the creatinine desimidas% followed by

  determination of ammonia or N-methylhydantoin formed.

  In the English patent mentioned above, as well as

  in the previous work, the possibility of applying

  quer the method to quantitative determinations

  In particular, there is no description of a technique

  automatic flow which allows the preliminary elimination of endogenous ammonia, always present in body fluids,

  and the resulting dosage of the single ammonia released by creatinine.

  It is known that it is commonly used in the field of

  automatic flow diagnoses.

  The present invention provides an automatic continuous flow method for the quantitative determination of creatinine by hydrolysis by creatinineimidase and assay

specific ammonia produced.

  The development of an automatic technique

  Continuous use for the determination of creatinine in body fluids using imidase creatinine involves the resolution of complex problems such as, for example, the need to limit the amount of enzyme used; using iminase creatinine free of other NH 3 -dependent enzymes, for example urease; to eliminate interferences due to endogenous ammonia which is always present in serum and urine and to use sensitive and specific systems to detect only the ammonia produced in the enzymatic transformation of creatinine. The method of analysis of creatinine that is the subject

  of the present invention overcomes all these problems.

  The need to limit the amount of enzyme used is obviously for economic reasons, a much larger problem in a continuous flow process than in

a manual process.

  The use of imidase creatinine in solution in a flowing system, besides a high consumption of this enzyme, has negative secondary phenomena, such as, for example, the increase in the blank value, a disturbance of the baselines, long durations of incubation and entry into the system of foreign substances of protein origin (eg enzyme proteins) that are not always compatible

  with the reagent used for the detection of ammonia.

  In the flow process according to the invention, the imidase creatinine is used in an immobilized form; the result is significantly reduced consumption, with the consequent possibility of obtaining enough enzyme, even from

  microbial sources of poor production.

  The use in the immobilized enzyme makes it possible not to circulate proteinaceous material (for example enzymatic proteins), but at the same time to guarantee a strong

  enzymatic activity that allows for short incubations with trans-

  complete formation of the substrate without disturbance of baseline or ammonia detection system from creatinine Immobilization of imidase creatinine may be

  performed according to known mobilization techniques.

  Such techniques may be, for example, those described for other enzymes in Biochem J 147, p 593-603 (1975); and

  British Patents Nos. 1470955 and 1485122.

  A particularly suitable method for immobilizing

  For example, the creation of the iminase creatine provides for the attachment of the enzyme to "nylon" tubes by (a) alkylation of the "nylon" tube with triethyloxonium tetrafluoroborate; b) introduction of a straight chain (spacer) obtained by acting on the alkylated yl solution of hexamethylenediamine or ethylenediamine; c) activation of the lilylodiécarteur by a bifunctional reagent which

  can be, for example, dimechyl suberimidate or glutamate

taraldéhyde; and

  d) attachment of the enzyme to the activated nylon -

  In another possible technique, after treatment as described above under b), the "N-ylon" spacer is reacted.

  with maleic anhydride or succinic anhydride.

  The "carboxyl-terminated" nylon-spacer is then treated with N-hydroxysuccinimide in the presence of N, N-dicyclohexylcarbodiimide and finally the enzyme is fixed in neutral buffered medium, for example phosphate buffer. on the

"Nylon" carboxylic acid thus activated.

  The immobilization techniques mentioned above make it possible both to bind a substantial amount of enzyme to Nylod, to obtain an enzyme in a particularly purified form and to

  especially an example of urease and / or other NH 3 -dependent enzymes.

  The enzyme immobilized by the process according to the invention is stable, active on the aqueous and physiological samples and acts in

continuous flow.

  Tables I and II below indicate the stability of the immobilized iminase creatine according to the described techniques.

  above Table III provides data on the function

the immobilized enzyme.

  In determining the stability at 40 C of creativity

  The suspension is immobilized on "nylon" tubes and the enzyme is immobilized on "nylon" tubes of 1 mm inner diameter and equilibrated at a temperature of 4 ° C. in 100 mM phosphate buffer solution. The immobilized activity values are expressed in U / m / min and indicated in the controls in% by

  relation to the initial activity (time 0).

TABLE I

  Immobilization A = 1 Activated by dimethyl suberimidate Immobilization B = 'Nylord activated by glutaraldehyde Immobilization C =' N 1 ylod 'activated by N-hydroxysuccinimide

  For the determination of stability at 35 C,

  The enzyme is read on 1 mm diameter nylon tubes and equilibrated at 35 ° C in 100 mM phosphate buffer solution at pH 7. Immobilized activity values are expressed in U / m / min and indicated

  in% compared to the initial activity (time 0).

TABTEAIU TT

  in the controls Stability Immobilization A Immobilisation B immobilisation C month 35 C 35 C 350 C U / m / min U / m / min U / m / min%

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

  Immobilization A = "nylon" activated by dimethyl sub-imimidate Immobilization B = "Nylon" activated by glutaraldehyde Immobilization C = "Nylon" activated by N-hydroxysuccinimide Stability Immobilization A Inmobiization 3 I Mobilization C month 44 C 4 e C 4 CU / m / min U / m / min% U / m / min%

O 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

  For the determination of the functionality of the creation

  In a continuous flow immobilized forminimidase, immobilized iminase creatine is used on a nylon tube 1 mm in ID and 1 m long for the routine serum, plasma and urine creatinine assay. a system to

  flow of the type shown in Figure 3 below.

  Residual activities are controlled at

  values of 5,000, 10,000 and 20,000 analyzes and expressed in% of the

initial value in U / m / min.

TABLE III

  Immobilization A = "ylo'n" activated by dimethyl-suberimidate Immobilization B = Sylod Activated by glutaraldehyde Immobilization C = "Nylon" activated by N-hydroxysuccinimide The creatinine deimidase used for the enzymatic dissociation of creatinine by the method according to the invention can be obtained by known methods from various microbial sources and can be, for example, the enzyme mentioned in the references cited above: Biochimica and Biophysica Acta 30,

  154 (1958) and Analytical Chemistry, 46, 2, p 246 (1974).

  The problem of eliminating the interferences due to ammoniacendogen, which is always present in the serum and

  urine, is of fundamental importance in the analysis of creativity.

  based on the determination of the ammonia produced in the process of

  catalyzed by imidase creatine.

Activity after

  Fixed assets Activity 5000 ana 10000 ana 20000 ana-

  initial lyses lyses lyses U / m / min% U / m / min% U / m / min% t / m / min%

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

  The problem is relatively simple when using a manual process and can be solved by a differential assay performed on the same sample before and after incubation with imidase creatinine. In a continuous flow process, the differential dosage, if possible, is a much more complicated problem, especially in cases such as the creatinine assay, where the blank value is significant in relation to that

of the sample.

  In addition, in a continuous flow dialysis process, such as that proposed by the invention, the contribution of endogenous ammonia to the total response would be further enhanced by the high dialysis power of the ammonium ion relative to the creatinine.

  The method according to the invention makes it possible to eliminate the

  endogenous monile and predispose the catalysis process between

  immobilized creatine imidase and creatinine, in order to

  unambiguously proud of the ammonia produced in the enzymatic reaction

only.

  According to the invention, the quantitative determination of the creat-

  in the serum and in the urine is carried out by a continuous flow technique comprising: a) removing the endogenous ammonia in the sample by pre-dialysis incubation with a suitable reagent; b) continuous dialysis; c) reaction in the acceptor channel of creatinine with immobilized creatinine imidase to give ammonia and N-methylhydantol; and (d) the determination of ammonia produced by a system

appropriate detection.

  The reagent used for the removal of endogenous ammonia from the donor channel must be specific and not interfere with the evaluation of ammonia produced from creatinine in the acceptor channel. For example, this reagent may be a reagent NADH / GLDH acting on endogenous ammonia according to reaction N 3 (endogn + ac Gtoglutarate + NADH LDH Na D + O NII 3 (endogenous) + α-ketoglutarate + NADH Na D + glutamate + H 20 or a reagent based -aspartase (EC 4 3 11) which acts on the endogenous ammonia according to the equation aspartase NH 3 (endogenous) + fumarate aspartate

  The dialysis process makes it possible to eliminate the

  Teeth and corpuscular samples, allowing a correct analysis of creatinine, even in sera strongly

lipemic or icteric.

  The dialysed creatinine is substantially free of endogenous ammonia and encounters downstream dialysis the creatinine of imidase

  immobilized which catalyzes its hydrolysis to ammonia and N-medyl-dione.

  The determination of the free ammonia is preferably carried out by means of the reaction with a Berthelot reagent, a chemical reagent based on phenol and sodium hypochlorite, or else a reagent NADH / GLDH / "ketoglutarate qualitatively equal to the one used for

destroy endogenous ammonia.

  The fumarate / aspartase system used to remove endogenous ammonia can be coupled with the detection system of either the Berthelot type (Clin Chem Vol 8, p 130-132 (1962)) or

NADH-GLDH type.

  On the contrary, the use of the reagent NADH / GLDH / a-keto-

  glutarates to remove endogenous ammonia is a source of

  ferences with the downstream ammonia detection system

  analysis, when this system consists of the same reactive

NADH.

  To make this coupling possible, it is absolutely necessary to avoid that the residual NADH of the reagent used in the donor line, after destroying the endogenous ammonia, passes into the receiving line, abnormally varying the

  NADH content of the ammonia detection reagent.

  According to the technique of the invention, this can be achieved by bringing the medium to pH 2 so that the residual NADH, after removal of the endogenous ammonia and before passing through the dialysis,

  either completely eliminated as a result of its instability in an acid medium.

  In a preferred embodiment, the medium is acidified with phosphoric acid and the pH is brought to about 7.5 before entering the dialysis, for example by adding a

  tris-hydroxymethylaminomethane solution.

  In a particularly advantageous embodiment

  However, the use of the NADH / GLDH reagent for the removal of endogenous ammonia is coupled with the use of the reagent of

  Berthelot for the detection of ammonia derived from creatinine.

  In another preferred embodiment, the method of the invention provides the use of an aspartase / fumarate reagent to remove endogenous ammonia prior to dialysis, coupled with the use of the NADH / GLDH reagent for the assay. ammonia

  produced from creatinine.

  The conversion of creatinine to ammonia and N-methyl-

  Hydantoin is always obtained with the immobilized creatinineimidase

  Lisée. As already indicated, the immobilization is carried out on tubes and in particular on "nylon" tubes according to one of

  systems previously described.

  The nylon tube used may have an inside diameter

  varying from about 0.8 to 1.6 min, preferably from 1 to 1.2 mm.

  The length of the tube varies from approximately 0.5 to 1 m,

  it is close to 1 m, and the activity of the enzyme fixed

  should preferably not be less than 4-6 U / m / min for

  put a conversion close to 100% with a linear answer

  up to 15 mg / 100 ml creatinine.

  It is possible to carry out countless amounts of creatinine

  in serum and urine with a single tube of creatinine

immobilized midase.

  The good functionality of imidase creatinine

  immobilized and its good stability over time are also equal

  by the use of buffer substances that make it possible

  appropriate pH and ionic strength values.

  For this purpose, the acceptor stream is preferably buffered at pH 7-8, preferably at pH 7.5 with a phosphate buffer, for example 50 mM phosphate buffer, or at pH 7.8 with buffer, for example 50 m Tris / HCl buffer. The donor stream is preferably buffered with 200 mM phosphate at pH 7.5 or with 200 mM tris buffer at pH 7.2. 8 The phosphate buffer is used in combination with the Berthelot type chromogenic system;

  Tris buffer / HC 1 is used with NADH / GLDH type systems.

  The automatic flow method according to the invention is the result of an in-depth study based on various flow systems always designed for the purpose of eliminating or reducing

  as far as possible endogenous ammonia before dialysis, trans-

  to form creatinine after dialysis with imidase creatine

  immobilized and to detect ammonia produced by specific agents

  compatible with the rest of the system.

  The invention will be better understood on reading the

  description which follows with reference to the accompanying drawings in which

  FIG. 1 represents a flow diagram of a one-channel system that can be used for carrying out the method according to the invention; FIG. 2 represents a flow diagram of a two-channel system that can be used for carrying out the method according to the invention; and Figures 3 and 4 show flow patterns of two-channel flow systems with a single dialyzer that

  it is possible to use for carrying out the process according to the invention

  tion. The applicant has experimented, for example, with a one-channel system of the type shown in FIG. 1, in which the sample X is mixed with a reagent R consisting of NADH / GLDH / α-ketoglutarate and pumped into the fractionated system by reaction bubbles. A. The current enters the bath B where the endogenous ammonia is removed simultaneously with all other substances

influence on NADH.

  After incubation, the stream is acidified by the addition of P, for example by the addition of dilute phosphoric acid, maintained at pH 2 over the entire path S, and then reduced to about pH 7 by

  introduction into the T system, for example trishydroxymethyl-

  Aminomethane After this treatment, the current penetrates into the dialy-

  The non-dialysed portion is sent to the outlet port 0, while the dialysed portion containing the interference free creatinine enters the acceptor channel. The acceptor channel stream that contains the reagent F for the determination of ammonia (NADH / GLDH / cz-ketoglutarate), fractionated by air bubbles A, passes downstream of the dialysis and enters the reactor (nylon tube) containing immobilized creatinine-deimidase E. Creatinine It releases ammonia which, by reaction with the reagent F in the development coil Z. oxidizes NADH to NAD. A reading of the colorimeter C, for example at 340 nm, makes it possible to determine the concentration of creatinine and to record it on In one-channel systems as shown in FIG. 1, the recording apparatus V, the total elimination of the endogenous ammonia and all the substances acting on

  the NADH system, by means of dialysis, is a necessary condition

  to read the value of the sample in relation to the value

  blank with a constant baseline.

  These absolutely essential conditions always require a perfect efficiency of the reagents and can limit the application of the process, in particular in the controls of

routine in hospital.

  In addition to the one-channel system of the type shown in Figure 1, for example, two-channel systems have been used to implement the method of the invention; these provide for the elimination of endogenous ammonia, coupled with the reading of ammonia from creatinine in a differential phase, in which the blank value is automatically subtracted from

that of the sample.

  The answer is made safer in this way by removing the obligation of complete elimination of ammonia

  endogenous to have a constant control and equal to the baseline.

  At the same time, the method of removing endogenous ammonia allows in each case to see weak controls and to operate under optimal conditions for an analysis performed by

differential reading.

  An example of a two-channel system that can be used for carrying out the method according to the invention is shown in FIG. 2 in which the sample X is mixed with the reagent R which can be NADH / GLDH / acetoglutarate or aspartase / fumarate and pumped into the system, fractionated by air bubbles A The current

  enters bath B for the removal of endogenous ammonia.

  The sample, freed from endogenous ammonia, is pumped by means of recirculation tubes C and C 1, which start from the separator T, into the stream upstream of the dialysis with respect to

  dialyzer D of the sample channel and dialyzer D 1 of the control channel.

  Currents upstream of the dialysis-are then rejected by means of

outlet ports S and 51.

  The currents downstream of the dialysis, T and Tj, fractionated by air bubbles A, enter the reactor N (Nylon tube with immobilized immobilized creatinine) and the blank reactor N 1

(white reference tube).

  The ammonia released by creatinine N is detected by means of a reagent based on phenol F and sodium hypochlorite I. Simultaneously, the blank value is evidenced on the reference channel with the same reagents pumped at through

F 1 and II.

  After incubation in Z and Z 11, the currents enter the differential scanning calorimeter L and read the value

  creatinine, for example at 650 nm, and is recorded on the

  In a particularly preferred embodiment, the continuous flow system used for the automatic analysis method according to the invention provides for the use of a two-channel system which makes it possible to eliminate ammonia. endogenous and perform the analysis uniquely with sample channel separation and blank channel only just before the catalytic process with

  immobilized creatinineimidase.

  The most characteristic points of this system are the following: a) the simplicity of the tube system compared to conventional two-channel and two-dialyzer systems; b) the possibility of obtaining a satisfactory sensitivity, even using a small sample volume, with good values of accuracy and precision; c) the use of a single dialyzer for both channels; (d) the vesicle recycling system which allows the dialysate to be dispensed both in the sample channel and in the blank channel simultaneously and in equal volumes; e) the use of immobilized iminase creatinine which, conveniently placed in the sample channel alone, makes it possible to differentially read the control; and f) the ease of phasing which involves only the

last part of the system.

  Two-channel flow systems with a single dialyzer having the above-mentioned characteristics are

  illustrated for example in Figures 3 and 4 appended hereto.

  In the system shown in FIG. 3, the sample X is mixed with the reagent R and pumped into the system, fractionated by air bubbles A. The stream enters the bath W for elimination

endogenous ammonia.

  After the incubation, the current passes into the dialysis zone before dialysis D and is removed through the outlet orifice S. The dialyzed solution passes into the acceptor stream F and, fractionated by the air bubbles A, it is transported by the tube to the mist separator T. Part of the current is recycled to the system> from the two-way mist eliminator by means of the tubes G and G 1 and, fractionated by air bubbles A, passes respectively into the reactors C (tube with immobilized creatinineimidase) and B (blank reference tube) The ammonia released by creatinine C is detected by a reagent based on phenol L and sodium hypochlorite M. The blank value is revealed simultaneously in the reference channel with the same agents pumped from L 1 and M 1. After incubation in Z and ZI, the currents enter the differential reading colorimeter U and the value of creatinine is read, for example at 650 nm. stored on a V recording device. When using the aspartase / fumarate reagent

* for the destruction of endogenous ammonia is coupled with the use of

  GLDH / NADH / c-ketoglutarate reagent for the detection of ammonia produced from creatinine, the flow system shown in Figure 4 is used. In this system, the flow of sample X is mixed. with the reagent R (aspartase / fumarate) and pumped into the system, fractionated by air bubbles A The current enters the bath W for the removal of endogenous ammonia and, after incubation, in the dialyser D d o It is discharged through the outlet orifice S. The dialysate passes into the acceptor stream which enters the dialysis, fractionated by air bubbles A. The acceptor stream F containing the reagent NADR / GLDH / a-ketoglutarate is sent by means of the tube d or the mist separator T. Part of the stream is recycled into the system from the two-way mist separator by means of tubes G and G 1 and, fractionated by air bubbles A, passes respectively into the reactors C ( tube containing imidin imidinimidase mobilized) and B (blank reference tube Ammonia is released in C from

  from creatinine and reacts Z with NADH.

  In the reference channel, and precisely in B and Z 1, the control is developed which is read by differential reading at 340 nm

  for example, in comparison with the respective sample, using

  reading a U colorimeter and recording the result by means of the recording apparatus V. In addition to the continuous flow enzymatic method described above for the determination of creatinine, the invention also proposes a series of materials and reagents for the determination of creatinine.

implementation of the method.

  This series of materials and reagents includes a) a reagent for the removal of endogenous ammonia in an appropriate buffer b) a reagent for the detection of ammonia from creatinine,

  c) Immobilized imidase creatine.

  The reagent for the removal of ammonia can be

  one of the reagents NADH / GLDH and aspartase / fumarate previously mentioned

  in a suitable neutral-basic buffer.

  The reagent for the detection of ammonia from creatinine may be a Berthelot reagent or a reagent

  FADH / GLDH as indicated above.

  Immobilized creatinineimidase is a creatinine

  immobilized deimidase on a tube, preferably a "nylon" tube,

  according to the techniques described above.

  In the series of reagents proposed according to the invention, when the reagent for the elimination of endogenous ammonia is a NADH / GLDH reagent, the solution for using said reagent preferably contains a phosphate buffer 200 mM at pH 7, 3-7.8, preferably 7.5; a-ketogluearate 8-12 mi N, preferably 10 m M; NADH 0.6-1 me, preferably 0.8 m I;

  GIDH 40-50 U / ml, preferably 45 U / ml.

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

  When the reagent for the detection of ammonia

  from creatinine is a Berthelot reagent, the solution for use of said reagent preferably contains: phenol 80-120 Bi, preferably 100 nm; sodium nitroferricyanide 1.2-1.6 ml, preferably 1.4 m M; sodium hypochlorite 13-17 mM, preferably 15 m K; sodium hydroxide 0.15-0.19 nm, preferably 0.17 M. When the reagent for the detection of ammonia containing creatinine is a NADH / GLDH reagent, the solution for use of said reagent preferably contains 50 mM phosphate buffer at pH 7.3-7.8, preferably 7.5? -ketoglutarate 6-10 mM, preferably 8 to M; NADH 0.05-0.1 m K, preferably 0.075 m M;

  GLDH 10-20 U / ml, preferably 15 U / ml.

  In the series of reagents proposed according to the invention, the immobilized creatinine of imidase preferably has an activity

  about 6-10 U / m / min, preferably 8 U / m / min and it is im-

  mobilized preferably on nylon tubes 0.5-1 m long, preferably 0.7-0.8 m, about 0.8-1.2 mm inner diameter,

preferably 1 mm.

  In the preferred embodiments, the series of materials proposed according to the invention contains a reagent NADH / GLDH for the elimination of endogenous ammonia, coupled with a Berthelot reagent for the detection of ammonia from the

  creatinine, or an aspartase / fumarate reagent for the elimination of

  endogenous ammonia, coupled with a NADH / GLDH reagent for the detection of ammonia containing creatinine,

  reagents (NADH / GLDH, Berthelot, aspartase / fumarate) and creatinine-

  immobilized deimidase preferably have the compositions and

  preferred ratios indicated above.

  According to the invention the abbreviations NADH, NAD + and GLDH are used to designate nicotinamideadeninedinucletotide in reduced form, nicotinamideadeninedinucletotide in oxidized form and glutamate dehydrogenase respectively;

  The terms a-ketoglutarate, glutamate, fumarate and aspar-

  are used to designate a salt, preferably an alkaline salt,

  especially a salt of sodium or potassium, α-keto-

  glutaric, glutamic, fumaric and aspartic, respectively; The abbreviations EDTA and Tris respectively denote the disodium salt of ethylenediaminetetraacetic acid and trishydroxymethylaminomethane.

  The following examples illustrate the invention without any

limit its scope -

Example 1

  Creatinine is measured in the sera using the

  flow system shown in Figure 1 with creatinine-

  immobilized deimidase and using the Jaffé method (according to

  the procedure described by H Bartels et al in Clin Chim.

  Acta 37 p 193 (1972)) simultaneously with the Mercotest 3385 method

(brand name).

  Serum creatinine controls at 2 mg / dL were assayed for 60 sera using Technicon Autoanalyser II from Technicon Instruments Co. Tarrytown, NY: Samples X = 0.23 ml / min were analyzed for 60 analyzes / hour. The reagent R = 0.60 ml / min is a solution of tris / HC buffer 1 200 m M

  at pH 7.8 containing 0.6 mM NADH, 40 U / ml GLDH, a-keto-

  potassium glutarate 10 mM and air A = C, 23 ml / min.

  B = 5 ml is the incubation bath at 37 GC, P = 0.23 ml / min and T = 0.23 ml / min, respectively pump the acid

  phosphoric acid at 2.5% w / v and tris-hydroxymethyl-

  10% by weight by volume of aminomethane, with S and Si = 1.5 ml.

D = 61 cm is the dialyzer.

  The acceptor current F = 1.2 ml / min is a solution of 50 mM Tris / HCl buffer at pH 7.8 containing 0.05 mM NADH, 20 U / ml GLDH, and ccetoglutarate. of potassium 8 m M and air

A = 0.23 ml / min.

  E = 1 m is the tube of 'nylon' with the creatine of imidase at 8 U / m / min.

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

  C is the colorimeter that works at 340 nm with a cell

  1.5 cm in the linearity range 1-12 mg / dl of creatinine.

  The results obtained by the process according to the invention (Y) are compared with those obtained by the reference method of Jaffé (X). The correlation data are: regression y = 0.946 x + 0.065 correlation coefficient r = 0.994

Example 2

  Sixty sera, 60 anti-coagulant EDTAs, 60 anticoagulant-packed heparin plasmas and 60 urine specimens (diluted 1: 100 with water) were administered to aqueous creatinine samples at 2 mg / dl, using a Technicon Autoanalyzer II apparatus and the flow system shown in Figure 3: Samples X: 0.23 ml / min are analyzed at 60 analyzes per hour; the reagent R = 0.42 ml / min is a solution of 200 mM phosphate buffer at pH 7.5 containing 0.8 mM NADH, 40 U / ml GLDH, α-ketoglutarate, potassium 10 mn with

air bubbles A = 0.23 ml / min.

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

  The acceptor current F = 1 ml / min is a 50 ml phosphate buffer solution at pH 7.5. The recycle tubes G and G 1 are at 0.42 ml / min and C = 1 m is

  the Nylon tube containing iminase creatinine at 6 U / m / min.

  The phenol reagent L / L 1 enters the system at a rate of 0.42 ml / min

  and the sodium hypochlorite reagent M / M 1 at the rate of 0.42 ml / min.

  After incubation in Z and Z 1 = 6 ml, one reads on a colorimeter to

two channels at 650 nm.

  Yatats (Y are obtained by the process of the invention are compared with those (X) obtained with the reference method

  (Kinetic method of Jaffé used in Example 1).

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

  correlation coefficient r = 0.996.

Example 3

  60 sera are analyzed using the procedure of Example 2,

  but using as reagent R a solution of potassium fumarate

  50 mM sium and 20 U / ml aspartase in 200 m M phosphate buffer

at p H 8.

  The results obtained by the process of the invention (Y) are compared with those (X) obtained by the reference method

  (Kinetic method of Jaffé indicated in Example 1).

  The correlation data are the following regression: y = 1.032 x 0, 159;

  correlation coefficient r = 0.979.

Example 4

  Using the Technicon Autoanalyser II apparatus and the flow system of Figure 3, a series of

  serum sera at different concentrations of creatinine.

  Table IV below gives the brand name of the serum,

  lot number, the creatinine values determined according to the

  the average value indicated by the manufacturers, using the Jaffé method.

TABLE IV

Example 5

  To human iium with a creatinine titer of 1 mg / dl, serial quantities of creatinine standard are added. The various fractions are analyzed on the Technicon Autoanalyzer II instrument.

  with the flow system of Figure 3 The recovery values

  of creatinine are given in Table V below and expressed

  in X with respect to the calculated theoretical value.

TABLE V

  n Creatinine% in mg / dl Serum Lot Found Theoretical Pathonorm L "16 1, 26 1.40 Pathonorm 16 7.60 7.80 Seronoru I 142 1.41 1.52 Monitrol P '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" k Ortho Norm "7 T 025 0.93 1.05 Ortho Abn" 7 T 113 8.70 9.05

  Serum: Creatinine Creatinine Creatine Retrieval

  creatinine theoretical added concentration,% (mg / dl) (mg / dl) (mg / dl) (mg / dl)

1 O 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 having a creatinine content of 1.33 mg / dl is treated with serial amounts of G 5 to 2 ml

  a second human serum with a creatinine content of 7.86 mg / dl.

  The various fractions are analyzed on the device

  Technicon Autoanalyzer II with the flow system of Figure 3.

  The creatinine values found are shown in Table VI below and expressed as a percentage of recovery

  compared to the calculated theoretical value.

TABLE VI

  Creatinine Values Serum Theoretical Serum Found Recovery at 1.33 mg / dL 7.86 mg / dL mg / dL mg / dL X 1 mL + 0.5 mL 3.50 3.61 103 1 mi + 1 mL 4, 59 4.77 104 1 ml + 2 ml 5.68 5.85 103

Example 7

  20 analyzes of 3 human sera of concentration were carried out

  different creatinine levels on the Technicon Auto-

  analyzer II with the flow system of Figure 3.

  The results relating to the average value and the coef-

  variations are given in Table VII below.

TABLE VII

  Concentration Mean value Coefficient of creatinine mg / dl variation (C V) Low 1.3 1.98 Mean 2.4 1.10 High 7.1 0.79 It is understood that the invention is not limited to

  preferred embodiments described above as illustrative

  and that the person skilled in the art may make various modifications and changes thereto without departing from the framework and the spirit.

of the invention.

Claims (22)

  1 Continuous flow automatic method for the determination of creatinine in body fluids by enzymatic transformation of creatinine by creatinine deimidase to give   ammonia and N-methylhydantol and quantitative determination of   ammonia resulting from the hydrolysis, characterized in that   creatinine imidase is immobilized.   Process according to Claim 1, characterized in that   enzymatic transformation of creatinine by creatinine   immobilized deimidase to give ammonia and N-methyl-   hydantoin is preceded by the elimination of endogenous ammonia   present in biological fluids.   3 Automatic continuous flow process for dosing   creatinine in body fluids by means of trans-   enzymatic formation of creatinine by creatinine deimidase   to give ammonia and N-methylhydantoin and determine   subsequent formation of the formed ammonia, characterized in that   takes: a) the elemination of endogenous ammonia present in body fluids by treatment with a suitable reagent, in predialysis; b) continuous dialysis;   c) the reaction of creatinine after dialysis with creatinine   immobilized deimidase; and (d) the quantitative determination of ammonia produced by means of a appropriate detection system.   4 Process according to claims 1 and 3, characterized   in that the immobilization of the imidase creatinine is carried out   on a nylon tube introduced into the flow line.   Method according to one of Claims 2 to 4,   characterized in that the elimination of the endogenous ammonia is   performed automatically in the flowing system.   Process according to any one of claims 2 to 5,   characterized in that the elimination of the endogenous ammonia is   obtained by treatment with a reagent NADE / GLDH.   7 Process according to claim 6, characterized in that the reagent NADH / GLDH contains in the solution of use: phosphate buffer 200 m M at pH 7.3-7.8; a-ketoglutarate 8-12 m I, NADH 0.6-1 m Ni, GLDH 40-50 U / ml,   Process according to claims 2 to 5, characterized in   the elimination of endogenous ammonia is achieved by treatment   with aspartase / fumarate reagent.   9 Process according to claim 8, characterized in that the reagent aspartase / fumarate contains in the solution of use: phosphate buffer 200 m M to p H 7.8-8.2, aspartase 18-22 U / ml, fumarate 40 -60 m. M.   Process according to any one of claims 1 & 9,   characterized in that the quantitative determination of ammonia from creatinine is carried out using a reagent of phenol and sodium hypochlorite as a chromogenic system detection.   Process according to claim 10, characterized in that the phenol and sodium hypochlorite reagent contains in the use solution: phenol 80-120 m M, sodium nitroferricyanide 1.2-1.6 m M sodium hypochlorite 13-17 mM, sodium hydroxide 0.15-0.19 M.   Process according to one of Claims 1 to 7,   characterized in that the quantitative determination of ammonia containing creatinine is performed using a reagent   NADH / GLDH as detection system.   13 Process according to claim 12, characterized in that the reagent NADH / GLDH contains in the solution of use: 50 mM phosphate buffer at pH 7.3-7.8, α-ketoglutarate 6-10 ml, NADH 0 , 05-0,1 me, GLDH 10-20 U / ml.   Process according to any one of claims 1 to   13, characterized in that the flow system is a system with   two channels with differential reading.   Method according to Claim 14, characterized in that the two-channel flow system with differential reading includes a single dialyzer.   Method according to any one of claims 3 to   , characterized in that the flow system consists of a tubular system into which the following elements are introduced, in order of flow: a) a bath for the removal of endogenous ammonia; b) a dialyzer; c) a degassing recycling system that allows the distribution   simultaneously in the control channel and the sample channel of the   single lyser; d) a control reactor introduced into the control channel; e) a reactor containing the immobolized imidase creatinine intro duced in the sample channel for enzymatic reaction with creatinine; and f) two coils introduced respectively into the control line and the sample line for the conduct of the   Ammonia detection reaction.   Apparatus for carrying out the method according to claim 16, characterized in that it consists of a) an automatic device for taking samples; b) a system for introducing samples into the flow; (c) a flow system; and d) a detection system in which the flow system is as described in claim 16; 18 Set of Materials and Reagents for Implementation   process of any one of claims 3 to 16,   characterized in that it comprises: a) a reagent for the removal of endogenous ammonia in a suitable buffer;   (b) a reagent for the detection of ammonia containing nine; and   c) immobilized imidase creatine.   19 Set of materials and reagents according to the claim   18, characterized in that the reagent for the removal of the endogenous ammonia consists of a reagent, AD 1 GLDH as defined in claim 6 or 7.   20 Set of materials and reagents according to the   cation 18, characterized in that the elimination of the endometrial ammonia   gene is obtained by treatment with a reagent aspartase / fumarate   as defined in claim 8 or 9.   21 Set of materials and reagents according to the   cation 17, characterized in that the quantitative determination of ammonia from creatinine is carried out using a phenol and sodium hypochlorite reagent as defined in claim 10 as a color detection system. or 11.   22 Set of materials and reagents according to the   cation 18, characterized in that the quantitative determination of ammonia containing creatinine is carried out using as a detection system a reagent NADH / GLDH as defined in claim 12 or 13.   23 Set of materials and reagents according to the   cation 18, characterized in that the immobilized creatinineimidase   is immobilized on a nylon tube with a length of 0.2 to   2 m and with an inside diameter of between 0.8 and 1.6 mm.   24 Set of materials and reagents according to the claim   23, characterized in that the nylon tube containing the   immobilized imidase is obtained by a technique of   mobilization consisting of a) alkylation of the nylon with triethyloxonium tetrafluoroborate; b) introducing a linear chain spacer element onto the alkylated nylon by treatment with hexamethylenediamine or ethylenediamine; c) activating the Nylon-retriever with a bifunctional reagent such as dimethyl suberimidate, glutaraldehyde or a mixture thereof; and   d) the attachment of the enzyme to the activated nylon.   Set of materials and reagents according to the   cation 18, characterized in that a) the reagent for the removal of endogenous ammonia is the reagent mentioned in claim 19; b) the ammonia detection reagent from creatinine is that indicated in claim 21; and c) the immobilized iminase creatine is obtained in the art described in claim 24 on a nylon tube as described in claim 23.