WO1991006868A1

WO1991006868A1 - Bilirubin assay using electrochemical oxidation

Info

Publication number: WO1991006868A1
Application number: PCT/FR1990/000782
Authority: WO
Inventors: Fathi Moussa; François TRIVIN
Original assignee: Fathi Moussa; Trivin Francois
Priority date: 1989-10-31
Filing date: 1990-10-29
Publication date: 1991-05-16
Also published as: FR2653891B1; FR2653891A1

Abstract

A method for assaying bilirubin present in a biological medium in a conjugated and/or unconjugated form. A sample of the medium is placed in an electrochemical cell (121) containing a conductive aqueous solution (123) with a pH of between 3 and 10, then a suitable potential is applied to an active electrode (127), with respect to a reference electrode, to oxidize at least some of the bilirubin present in the sample, and the amount of bilirubin thereby oxidized is determined. Furthermore, a reagent, such as a diazoreaction accelerator, a surface active agent or an organic solvent, is added. It acts to break the BR-Alb bonds and/or to solubilize the bilirubin in the solution. However, this reagent is not necessary in some cases.

Description

Determination of bilirubins by electrochemical oxidation

The present invention relates to the assay of bilirubins present in biological samples such as blood serum samples.

The measurement of the various forms of bilirubin in human serum is of great importance for the diagnosis and treatment of certain diseases in children and in adults, in particular for assessing the risk of hyperbilirubinemic encephalopathy in newborns. .

Various methods of assaying bilirubin are known in blood samples which use chemical oxidation, enzymatic oxidation or diazoreeaetion, as described by W. J. Cashore et al in Journal cf Pediatrics, vol. 93, n ° 5, p. 827-833, November 1978; by P. Fossati et al, in Clin. Chem. 35/1, 173-176 (1989), and by B. T. Doumas et al in Clin. Chem. 33/8, 1349-1353 (1987).

Among these methods, the methods using diazoreaetion are very interesting because they allow the separate determination of the conjugated forms of bilirubin; but they are not very standardizable because the reagents are unstable. The most recent enzymatic oxidation assays pose certain problems associated with the use of enzymes and do not exhibit sufficient sensitivity at low concentrations.

So, none of the known processes today Lement does not allow to perform the assay quickly with good sensitivity, and in a standardized manner.

The subject of the present invention is precisely a method for assaying bilirubins based on electrochemical oxidation, which is easy and quick to implement, can be standardized easily and has good sensitivity.

It is known that bilirubin can be electrochemically oxidized when it is in an organic solvent such as dimethyl sulfoxide containing an electrolyte such as tetraethylammonium perchlorate, as described by F. Moussa et al in Analytical Chemistry, vol. 60, 11,

1179-1185 (1988). However, this rock electro oxidation cannot be carried out in a biological medium because bilirubin is not electro-oxidizable in these media when the bilirubin / albumin molar ratio is less than about 0.7-0.8.

The subject of the present invention is precisely a process which makes it possible to assay the bilirubins present in a biological sample in conjugated form and / or in non-conjugated form by electrochemical oxidation.

This process includes the following successive steps:

a) introducing a sample of the biological medium into an aqueous, electrically conductive solution having a pH of 3 to 10,

b) subjecting the solution thus obtained to electrochemical oxidation at a potential such that at least part of the bilirubin present in the sample is oxidized, and

c) determine the quantity of bilirubin thus oxidized. According to the invention, it is successful to oxidize the bilirubin eletrochemically present in a biological sample by diluting this sample in an aqueous solution having an appropriate pH.

Indeed, it has been discovered that the choice of the pH of the solution makes it possible to electrochemically oxidize certain forms or all forms of bilirubin at appropriate potentials, and thus to dose them.

In certain cases, it is however preferable to add to the aqueous solution a reagent capable of breaking the bonds of bilirubin with albumin and / or of dissolving the bilirubins to make them oxidizable by electrochemical route.

The aqueous solution may for example be an aqueous solution containing an electrolyte such as a 9% sodium chloride solution. 1000 or a buffer solution, for example a phosphate buffer having a pH of 5 to 8, a glycoeolle or lactate buffer having a pH less than 5.

By way of example, the reagents added can be chosen from diazoreaction accelerators, anionic and cationic surfactants, and organic solvents.

By way of example of diazoreaction accelerators, mention may be made of compounds such as caffeine, for example in solution in benzoic acid, diphylline, aniline derivatives such as dichloroaniline, and their mixtures.

By way of examples of an anionic action, mention may be made of sodium dodecyl sulphate and sodium cholate. As exe mp lesd 'ag in ttensi oa ctif cationic likely to be used, there may be mentioned hexadecyl trimethylammonium chloride.

As organic solvent which can be used, mention may be made of dimethyl sulfoxide. It is of course possible to use other organic solvents, but the amounts used must be such that there is no precipitation of the proteins in the sample.

The quantities of aqueous solution and of reagent added are respectively such that a sufficient degree of dilution of the biological medium is obtained in order to render the bilirubin oxidizable and that the reagent can fulfill its function, that is to say break all the connections of bilirubin with albumin and / or to dissolve the bilirubins to make them oxidizable.

Generally, 100 μl to 1 ml of solution is used for 5 to 50 μl of sample.

The reagent used must be in excess relative to the bilirubin, for example in an amount such that there is at least 5 to 10 mol of reagent per mol of bilirubin present in the sample.

In the process of the invention, the pH of the solution is chosen. the reagent possibly added and the oxidation potential so as to carry out the oxidation of the desired form or forms of bilirubin.

Indeed, in the blood, bilirubin (BR) is present in various forms which are mainly

- unconjugated bilirubin (BRnC),

- unconjugated bilirubin linked to proteins such as albumin,

- conjugated bilirubin (BRC), for example monoglucuronides and diglucuronides bilirubin, more or less linked to proteins like albumin, and

- conjugated bilirubin covalently linked to proteins such as albumin. (This possibility is encountered in certain pathological circumstances).

Also, given that all of these forms of bilirubin are not eletrooxidizable under the same conditions, it is possible according to the invention to choose the pH conditions and the reagent added if necessary so as to dose the desired form (s) ( s) bilirubin.

Thus, at a neutral pH (7 + 0.5) and for a ratio (BR / Alb <0.7), the bilirubin conjugated in the presence of albumin is not electroactive. At alkaline pH the BRC / Alb ratio for which the BRC is not electrooxidizable decreases (around 0.3 to 0.2). On the other hand, in an acid medium (pH 2.6 to 3.8) and in the presence of albumin, the conjugated bilirubin is oxidized at potentials of +0.4 V and + 0.6 V compared to DHW (electrode at saturated calomel).

Unconjugated bilirubin is not electroactive in the presence of albumin for report

BRnC / Alb <0.8 in the pH range from 2.6 to 7.56. For pH greater than 7.5, the electrooxidation of BRnC is obtained for BRnC / Alb ratios less than 0.8 but not quantitatively. On the other hand, in the presence of a reagent such as caféirç associated with a benzoate, all forms of bilirubin are oxidized at potentials of +0.25 and +0.45 V compared to DHW.

Thus, according to a first embodiment of the invention, the method is implemented so as to dose the total bilirubin present in the sample. In this case, it is possible to operate at a pH of 7.4 + 0.05 with a reagent constituted by a cationic surfactant or by a diazoreaction accelerator, and carry out step b) at a potential such that one oxidizes the conjugated bilirubin and the unconjugated bilirubin, which makes it possible to assay the total bilirubin.

Total bilirubin can also be determined by operating at a pH of around 5, with a potential such that unconjugated bilirubin and conjugated bilirubin are oxidized.

It is also possible to assay the total bilirubin by operating at a pH of 8 with an anionic surfactant at a potential such that the conjugated bilirubin and the unconjugated bilirubin are oxidized.

According to a second embodiment of the invention, suitable for assaying the conjugated bilirubin, the operation is carried out at a pH of approximately 5 and at a potential such that only the conjugated bilirubin is oxidized.

Alternatively, the conjugated bilirubin can be determined by operating at a pH of 7.4 + 0.5 with a reagent consisting of an anionic surfactant, choosing a potential such that only the conjugated bilirubin is oxidized.

According to a third embodiment of the invention suitable for assaying unconjugated, unbound bilirubin, the conjugated bilirubin is first removed from the sample by using a solution at a pH of about 5 and performing a electrochemical oxidation at a potential such that all of the conjugated bilirubin is oxidized, the pH of the solution is then adjusted to approximately 7.4, electrochemical oxidation is carried out at a potential such that only non-bilirubin is oxidized conjugate not linked to albumin, and the amount of unconjugated, unbound bilirubin thus oxidized is determined, for example by spectrophotometry or by differential spectrofluorescence.

Thus, the method of the invention has many advantages. In fact, the assay can be specific because the oxidation is a function of the pH of the reagent and of the imposed potential.

However, this specificity can only be obtained directly in the case where the electrochemical oxidation is carried out in the presence of a reagent capable of breaking the bilirubin-albumin bonds and / or of solubilizing the bilirubins in this solution.

In this case, the measurement can be made directly by amperometry or coulometry after an appropriate calibration.

On the other hand, in the case where one operates without addition of the reagent mentioned above, the electrochemical oxidation cannot be specific because other constituents of the sample can also be oxidized and interfere for a large part in the result. .

In fact, the bilirubins bound to albumin are not rooxidizable and the contents of unbound bilirubin are generally low compared to those of the other oxidizable constituents.

In the latter case, however, the specificity of the assay can be obtained by combining electrochemical oxidation with another differential measurement technique such as spectrophotometry, reflectometry, spectrofluorometry or amperometry. The assay is very sensitive because the various methods which can be used to determine the amount of oxidized bilirubin all have good sensitivity of the order of nmol / l. The process does not require the use of expensive reagents such as enzymes.

In addition, it is possible to use less than 10 μl of sample and carry out the assay in one to two minutes in a fully automated manner.

According to the invention, the quantity of oxidized bilirubin can be determined by amperometric measurements, coulometric measurements, and spectrophotometric measurements.

Amperometric and coulometric measurements can be carried out using existing conventional microdosing devices.

The quantity of oxidized bilirubin can also be determined by spectophotometric or spectrofluorometric measurements. In fact, during oxidation, bilirubin is transformed into biliverdin and then into other products with natural fluorescence. However, bilirubin absorbs with a maximum at 450nm while the maximum absorption of bi li ve rd i does not sit at 380nm. Also if, by measuring the increase in optical density at 380nm or the decrease in optical density at 450nm, one can determine the amount of bilirubin oxidized to biliverdin. The apparatuses used can be conventional spectrophotometers or spectrofluorimeters, this measurement can also be carried out by reflectometry. The method of the invention can also be combined with conventional techniques for assaying bilirubin, for example with assay techniques by enzymatic oxidation.

In this case, the enzymatic oxidation is carried out and the quantities of bilirubin in the medium are determined by the method of the invention by means of amperometric or coulometric measurements with electrochemical oxidation.

The enzyme (bilirubin oxidase) can either be in suspension in the aqueous solution used, or immobilized on the electrode.

The invention also relates to a measurement cell for implementing this method, which comprises:

- a parallelipipedic tank provided with two parallel faces forming an optical window for the passage of a light beam in order to measure the optical density of a solution present in the tank,

- a working electrode placed in the tank so as not to be located on the passage of a light beam passing through the optical window,

an auxiliary electrode and a reference electrode arranged so as to be in electrolytic contact with a solution present in the tank, and

- Means for connecting the electrodes to an electric generator.

Alternatively, the parallelepiped tank used for photometric reading can be replaced by optical fibers placed in the vicinity of or inside the working electrode and a cell can be used. of any shape.

Other characteristics and advantages of the invention will appear better on reading the description which follows, given of course by way of illustration and not limitation, with reference to the appended drawing in which:

- Figures 1 to 3 are curves representing the behavior of unconjugated bilirubin BRnC in the presence of albumin and caffeine (fig. 1), of bilirubin with addition of an anionic detergent (fig. 2), of conjugated bilirubin in presence of albumin (fig. 3).

FIG. 4 schematically represents an installation for measuring the oxidation potentials of bilirubin and / or biliverdin,

- Figure 5 shows schematically a measurement installation by reflectometry.

Figures 6 to 13 are curves representing the intensity which passes through the cell (3) of the installation of Figure 4 as a function of the potential imposed on the electrode for various solutions containing samples of bilirubins and / or biliverdin.

FIG. 14 represents the evolution of the optical density of a solution as a function of the potential applied.

FIG. 15 schematically represents a measurement cell suitable for implementing the method of the invention.

- Figure 16 shows the spectrophotometric curves obtained during the dosing of bilirubin by coulometry.

Referring to Figures 1 to 3, we can see what the electrochemical behaviors of conjugated or unconjugated bilirubin are in the presence of various compounds.

These figures are intensity-potential curves obtained using an electrochemical cell comprising:

- an indicator electrode constituted by a glassy carbon electrode with a rotating disc, EDI TACUSSEL type (apparent diameter 2 mm, speed of rotation = 3000 rpm).

- an auxiliary electrode constituted by a Pt wire.

- a reference electrode: Ag / AgCl type C10 TACUSSEL.

In FIG. 1, which illustrates the behavior of unconjugated BR in the presence of albumin and caffeine, the intensity curves (in μ A) are represented, as a function of the potential (in V) obtained with the following solutions:

- curve A = Voltammogram of a bovine albumin solution (A) at 139 μmol / l of phosphate buffer pH 7.4 (final volume = 55 ml).

- curve B: solution 1 = solution A + 5.1 μmol / 1 of unconjugated BR (BR / AlB = 0.7).

- curve C: solution 2 = solution A + benzoic acid (AB) + caffeine (C). Final concentrations = 50 g / l AB and 30 g / l C.

- curve D = solution 3 = solution 2 + 5.1 umol / l of BR final pH = 7.4.

- curve E = solution 3, final pH = 8.0.

- curve F = solution 3, final pH = 6.0. These curves show that for a ratio

BR-Alb of 0.07, BR is not electro-stainless

(curve B). If cefine is added to the medium, the BR becomes electroaetive (curve D). This electroaetivity is dependent on the pH (curves E and F).

In FIG. 2 which illustrates the effect of the addition of an anionic detergent, the intensity curves (in μA) as a function of the potential (in V) have been shown obtained with the following solutions:

- curve G (solution 4): voltammogram of a BR solution at 190 μmol / l in the phosphate buffer pH 7.8 containing bovine albumin at 139 pmol / l (final volume = 54 ml, BR / Alb ratio = 1.4)

- curve H: solution 5 = solution 4 + SDS (Na dodecyl sulfate) (final concentration 5 mol / l, final pH = 7.8).

- curve I: solution 6 = solution 5 at pH 7.4.

These curves show that at pH 7.8, the unconjugated BR is not electrooxidizable in the electrochemical system used (curve G). In the presence of excess SDS and at a slightly alkaline pH, the BRnC becomes electro-stainless (curve H). The electroaetivity of the pigment drops considerably at neutral pH (curve I).

In FIG. 3 which illustrates the electrooxidation of a conjugated BR (conjugated ditauro BR, disodium salt, in the presence of albumin, the intensity curves (in uA) as a function of the Cen V potential) have been represented obtained with the following solutions:

- curve J: solution 7 = phosphate buffer solution pH 7.4 (volume = 50 ml).

- curve K: solution 8 = solution 7 + 16.7 μmol / l of conjugated BR.

- curve L: solution 9 = solution 8 + 0.5 g of albumin (final pH 7.4, BR / AlB ratio = 2.3).

- curve M: solution 10 = solution 9 +

SDS (final pH = 7.4).

These curves show that, unlike the unconjugated BR, the conjugated BR is electro-stainless in phosphate buffer at neutral pH (curve K). In the same way as for BRnC, albumin added in the medium inhibits the electrochemical oxidation of conjugated BR (curve L). The SDS acts on the BR-alb complex to release the conjugated BR which becomes electrooxidizable again (curve M).

Figures 1 to 3 thus illustrate the principles of the process which is the subject of the invention. However, the electrochemical system used, operating in macromethod, is not sensitive enough to allow work on blood samples.

Also, modes of implementation of the method suitable for assays on blood samples will be described below.

As we saw earlier. the method of the invention can be implemented in different ways depending on whether one wishes to assay total bilirubin, free bilirubin, conjugated bilirubin and unconjugated bilirubin. These different possibilities depend in particular on the aqueous solution used to dilute the sample to be measured, the pH of this solution, the reagent added and the potential imposed.

The potential imposed depends on the aqueous solution and the reagent used.

Also, to implement the method of the invention, the oxidation potentials of the various forms of bilirubin and biliverdin are determined beforehand in the medium used, by means of the apparatus used.

In Figure 4, there is shown schematically an installation for measuring these oxidation potentials.

This installation includes a container

(1) containing the aqueous dilution solution which can be introduced into a coulometric cell

(3) consisting for example of an ESA 5011 coulometric cell controlled by a module

Coulochem 5100A sold by the ESA Company, via the line (5) fitted with the pump (7) of the Spectra - Physics 8700 type.

The sample is introduced into this solution by the Rheodyne 7125 injector (9), then the solution is treated in the filters (11) and (13) having respectively openings of 2um and 0.5um.

The coulometric cell is followed by a Spectra speetrophotometric detector - Physics

8490 (15). The cell (3) and the detector (15) are respectively associated with the recorders

(17) and (19) constituted by SEFRAH Servo trace devices.

The cell comprises a working electrode in the form of a porous filter, a reference electrode in electrolytic contact with the solution present in the cell and an auxiliary electrode also in electrolytic contact with the aqueous solution. The reference electrode is a palladium electrode. To carry out the measurements, proceed as follows:

- the pump is adjusted to obtain a flow rate of 0.5 to 0.8 ml / min.

-The blood sample (5 to 50 μl) is previously diluted in 0.5 to 1 ml of the aqueous dilution solution and 100 μl of this solution are injected into the circuit using the injector (9). When the sample reaches the cell (3), the pump (7) is stopped. Measurements can then be started. The measurement consists in applying increasing potentials to the cell (3) using the Coulochem 5100A module. This is also used to measure the variations in intensity passing through the cell (3) during the applied potential scan. The intensity-potential curves (voltammograms) thus obtained are recorded using the recorder (17).

The speetrophotometric measurements, after coulometric oxidation, are carried out using the spectrophotometric detector (15). In this case, the pump does not stop during the measurements which are carried out continuously. This involves setting the measurement wavelength of the spectrophotometer (15), and varying the potential applied to the cell (3), before each injection.

The increase and decrease in optical density during the analysis are recorded by (19).

In Figure 5, there is shown schematically a device for performing assays using ref lectometry to determine the amount of oxidized bilirubin.

In this figure, we see that this device comprises a working electrode (2) removable or in the form of an optically transparent metal minigrid, an auxiliary electrode (4) having the same surface as the working electrode (4) and a reference micro electrode (6) connected to a potentiostat (8). The sample to be dosed is introduced between the electrodes (2) and (4) via a support (10) for single use of very small thickness (a few μm) having the same surface as the electrodes (2) and (4), for example an area of 0.25 cm2. This support contains in the dry state the desired reagent and buffer and the blood sample (12) which has been previously diluted is deposited on this support (10) before its introduction into the cell.

The reflectometric measurements, after coulometric oxidation, are carried out using a light source (14) directed on the sample. The ray reflected by the sample is detected by a photoelectric cell (16) coupled to an amplifier (18) and a recorder (20).

We will examine below the results obtained with the installation of Figure 4 on different solutions used in the invention.

I. Aqueous solutions at pH 7.4.

This aqueous solution is for example a phosphate buffer at pH 7.4.

In FIG. 6, the intensity (μ A) - potential (V) curves are represented. by injecting 100 μl of bilirubin sample into 0.066M phosphate buffer. In this figure, curve 21 relates to a sample containing 401 μmol / l of conjugated bilirubin, free of protein. On this curve 21, we see 3 oxidation peaks which are located respectively at the potentials of approximately +0.03 (EpD, approximately + 0.19V (Ep2) and approximately + 0.49V (Ep3).

The first peak corresponds to the oxidation of bilirubin to biliverdin, the second peak corresponds to the oxidation of biliverdin and the third peak corresponds to the subsequent oxidation stages.

Thus, the conjugated bilirubin is oxidizable in this medium in the absence of proteins.

Curve 22 represents the results obtained when adding biliverdin to the sample in addition (361 μmol / l of conjugated bilirubin and 76umol / l of biliverdin). In this case, we note that the oxidation peak (Ep2) of biliverdin is much greater than in the case of curve 21.

If the same experiment is carried out using a sample containing albumin with a molar ratio of conjugated bilirubin to albumin of 0.7 or less, the oxidation of conjugated bilirubin is inhibited and only observes the oxidation of biliverdin combined with the Ep2 potential of approximately + 0.19V.

If this experiment is repeated using a sample containing unconjugated bilirubin, in the presence of albumin, with a molar ratio of unconjugated bilirubin to albumin of 0.8 or less, unconjugated bilirubin n is not electro-oxidized. In contrast, if the molar ratio of unconjugated bilirubin to albumin exceeds about 0.8, there are two oxidation peaks at potentials of approximately -0.05V and approximately + 0.19V respectively.

Thus, in an aqueous solution having a pH of 7.4, the conjugated biLirubin can only be oxidized in the absence of proteins and the same is true for unconjugated bilirubin. Also, to carry out assays in such a solution, it is preferable to add a suitable reagent, for example an anionic surfactant, a cationic surfactant or a diazoreaction accelerator.

The results obtained in this case are given in FIG. 7.

In this figure, curve 31 represents the results obtained when using the phosphate buffer at pH 7.4, 0.066M, containing 40 g / l of albumin to which sodium dodecyl sulfate (DSS) is added at a rate of 10 mmol / l. On this curve 31, there appears no oxidation peak at the potentials less than 0.2V.

Curve 32 illustrates the results obtained when adding to the phosphate buffer containing DSS 100 μl of a sample of unconjugated bilirubin (401umol / l) with 36g / l of albumin, ie a BRnC / albumin ratio of 0.77. On this curve, only one seμl oxidation peak appears at around + 0.1V. Thus, unconjugated bilirubin oxidizes in a single step.

Curve 33 represents the results obtained when the sample contains only conjugated bilirubin at the rate of 379umol / l and 39g / l of albumin, ie a conjugated bilirubin / albumin ratio of 0.67. On curve 33, there are two oxidation peaks, a first peak at around -0.05V and a second peak at around + 0.16V. Thus, conjugated bilirubin oxidizes in two separate stages, at potentials different from those observed in the case of unconjugated bilirubin.

Also, in a solution containing albumin, unconjugated bilirubin and conjugated bilirubin, it will be possible to selectively oxidize the conjugated bilirubin by operating at a potential lower than -0.05V so as not to oxidize the unconjugated bilirubin.

In Figure 8, the results obtained are shown when adding to the phosphate buffer hexadecyl trimethylammonium chloride (HTHACl) at a rate of 5mmol / l. In this figure, curve 41 represents the results obtained with the phosphate buffer containing only HTMACl, curve 42 represents the results obtained when a sample of unconjugated bilirubin containing 379umol / l of unconjugated bilirubin and 36g / l of proteins is introduced , or a BRnC / albumin ratio of 0.73; and curve 43 illustrates the results obtained when a sample containing conjugated bilirubin containing 531 μmol / l of conjugated bilirubin and 36 g / l of proteins is introduced, ie a conjugated bilirubin / albumin ratio of 1.02. Under these conditions, the two forms of bilirubins are oxidized in two stages at the same potentials which are + 0.1V and + 0.22V respectively.

FIG. 9 shows the results obtained when a phosphate buffer at pH 7.4 is used as the aqueous solution to which 50 g / l of benzoic acid and then 30 g / l of caffeine are added after the benzoic acid has been dissolved. . In this figure, curve 51 relates to the results obtained with the aqueous solution alone containing caffeine and benzoic acid; curve 52 relates to the results obtained when a sample containing the aqueous solution is added 313μmol / l of unconjugated bilirubin and 35g / l of albumin, i.e. a BRnC / albumin ratio of 0.62. Curve 53 refers to the results obtained when a sample containing 313 μmol / l of conjugated bilirubin and 35 g / l of albumin is added to the solution, i.e. a BRC / albumin ratio of 0.62, and curve 54 refers to results obtained when a sample containing 313 μmol / l of conjugated bilirubin alone is added to the aqueous solution. By comparing these curves, we note that all forms of bilirubin oxidize in two stages at the respective potentials of -0.02V, and + 0.16V.

The following examples illustrate the use of an aqueous solution at pH 7.4 for the determination of total bilirubin and of conjugated bilirubin.

Example 1: Determination of total bilirubin in a solution at pH 7.4.

In this example, use is made of a 0.066 M phosphate buffer at pH 7.4 to which 50 g / l of benzoic acid and then 30 g / l of caffeine are added, and a microcell of measurement similar to that of FIG. 1, and one chooses as oxidation potential the Epi potential of -0.02V. First of all, the intensity I ₁ obtained with this potential is determined when the aqueous solution consists solely of the phosphate buffer, and the intensity I ₂ obtained at this potential when the aqueous solution is the phosphate buffer containing caffeine and benzoic acid.

The intensity X ₁ obtained at this same potential is then measured when 5 μl of serum to be tested is added to 1 ml of phosphate buffer alone, then the intensity X ₂ obtained at this potential when 5 μl of serum is added to 1 ml of phosphate buffer containing caffeine and benzoic acid.

We thus deduce the oxidation intensity bilirubin at the applied potential, which is: l _BRTOT = (X ₂ -I ₂ ) - (X ₁ -I ₁ )

Since the intensity (I) is proportional to the bilirubin concentration (C), I = KC, this bilirubin concentration can be determined after calibrating the device to determine the constant K which depends on the material, reagents and of the operating conditions used. Example 2: Measurement of the conjugated bilirubin in a solution at pH 7.4 containing an anionic surfactant.

In this example, the aqueous solution consists of the same phosphate buffer at pH 7.4 as in Example 1 to which 10mmol / l of sodium dodecyl sulfate is added.

Under these conditions, as shown in FIG. 7, the conjugated bilirubin and the unconjugated bilirubin oxidize at different potentials. We can therefore determine the concentration of conjugated bilirubin alone in a sample by operating at a potential below -0.02V.

To determine the concentration of conjugated bilirubin in a sample, the procedure is the same as in Example 1, first determining the intensities I ₁ and l ₂ corresponding respectively to the phosphate buffer alone and to the phosphate buffer supplemented with dodecyl. sodium sulfate, then the intensity X ₁ corresponding to the phosphate buffer alone containing the test sample and the intensity X ₂ corresponding to the phosphate buffer supplemented with sodium dodecyl sulfate and containing the sample lon to test. Under these conditions, the oxidation intensity of the conjugated Br is equal to

(X ₂ -I ₂ ) - (X ₁ -I ₁ )

This intensity is proportional to the concentration of conjugated bilirubin, the constant also depending on the material and the solutions used.

Il.Use of a solution at pH 8.

The aqueous solution used can be a Sorensen's phosphate buffer at pH 8. Under these conditions, when the two forms of bilirubins are present in a sample with albumin and a molar ratio of bilirubins to albumin less than or equal to 0 , Approximately 6, the electrochemical oxidation intensity of bilirubins is 3 to 4 times less than in the absence of albumin.

When the molar ratio is greater than approximately 0.6, an oxidation of the unconjugated bilirubin and of the conjugated bilirubin is obtained in two stages.

FIG. 1 shows the results obtained with this solution. In this figure, curves 61 and 62 relate respectively to the buffer at pH 8 and to the buffer at pH 8 containing albumin. Curve 63 relates to the buffer at pH 8 to which 100 μl of a sample containing 400 μmol / l of bilirubin and 36 g / l of albumin is added, ie a BrnC / albumin ratio of 0.77. Curve 64 refers to the results obtained when 100 μl of a sample containing only 400 μmol / l of bilirubin are added to the aqueous solution. Curve 65 refers to the results obtained when add to the buffer at pH 8, 100 μl of a 400 μmol / l solution of conjugated bilirubin. Note on curves 63, 64 and 65 that the unconjugated bilirubin and the conjugated bilirubin oxidize in two stages which have the potentials of approximately + 0.04V and +0.19V respectively.

FIG. 11 shows the results obtained with this same buffer at pH 8 when an anionic surfactant consisting of sodium dodecyl sulfate (DSS) is added to it at a rate of 10 mmol / l.

In this figure, curve 71 refers to the buffer alone containing sodium dodecyl sulfate, curve 72 refers to the buffer containing DSS to which 100 μl of a sample of bilirubin unconjugated to 512 μmol / l containing 39 g / l d is added. albumin, i.e. a BrnC / albumin ratio of 0.9. Curve 73 refers to the results obtained when the added sample contains only unconjugated bilirubin. Curve 74 refers to the results obtained when the sample contains albumin alone in the buffer with DSS.

By comparing these curves, we note that the two forms of bilirubins oxidize at the following potentials: from -0.02V and from + 0.16V.

III.Use of a solution at pH 5.

In this case, it is possible to use as aqueous solution, a phosphate buffer solution from Sorensen.

FIG. 12 shows the results obtained with such a solution. In this figure, curve 81 refers to the aqueous solution of phosphate buffer at pH 5 containing 40 g / l albumin. Curve 82 refers to the same aqueous solution additionally containing 100 μl of a sample containing 271 μmol / l of unconjugated bilirubin and 39 g / l of albumin, i.e. a BRnC / albumin ratio of 0.48, curve 83 refers to the same solution containing 100 μl of a sample at 426 μmol / l of unconjugated bilirubin, ie a BRnC / a lb ratio of 0.77, and curves 84, 85 and 86 refer to the results obtained when the sample is bilirubin conjugated to 345 μmol / l for curve 84, 500 μmol / l for curve 85 and 1000 μmol / l for curve 86, with 39 g / l of albumin, i.e. a BRC / alb ratio of 0.63 for curve 84 , 0.91 for curve 85 and 1.81 for curve 86.

By comparing all these curves, it is noted that the unconjugated bilirubin oxidizes in a single step at a potential of about 0.16 V with a very low limiting intensity and that the conjugated bilirubin oxidizes in two steps at respective potentials of + 0.1V and + 0.22V. It is thus possible to use a solution at pH 5 to assay separately the conjugated bilirubin and the unconjugated bilirubin not linked to albumin and the total unconjugated bilirubin.

Examples 3, 4 and 5 which follow illustrate such dosages.

Example 5: Coulometric determination of conjugated bilirubin.

In this example, an acid solution at pH 5 consisting of a 0.066M phosphate buffer is used as the aqueous solution.

From the curves of Figure 12, we know that at this pH the conjugated bilirubin oxidizes in two stages at potentials of + 0.1V and + 0.2V, while unconjugated bilirubin oxidizes very weakly to a different potential (+ 0.16V).

A potential of + 0.06 V is therefore chosen for the assay to oxidize only the conjugated bilirubin.

5 μl of sample previously diluted in 1 ml of pH5 buffer are introduced into the coulometric cell (see FIG. 11).

A first photometric measurement is carried out at 450nm (l ₁ ) or 380nm (I ' ₁ ). A potential of + 0.06V is then applied to the working electrode 127 for a time sufficient to carry out a total electrolysis of the sample. After this electrolysis, a second photometric reading is carried out at 450nm (I ₂ ) or 380nm ('l ₂ ) as the case may be.

The difference between the two measurements (l ' ₂ -l' ₁ ) or (l ₁ -l ₂ ) is directly proportional to the amount of BRC electrolyzed. After an appropriate calibration of the apparatus, the concentration of conjugated bilirubin is obtained from the initial sample.

We could also carry out the measurement by differential spectrofluorescence after electrolysis at + 0.22V.

Example 4: Coulometric determination of unconjugated bilirubin, not linked to albumin.

In this example, first of all a total electrolysis of the aqueous solution containing the sample to be assayed is carried out in a buffer at pH 5 as before at a potential of + 0.06 V to permanently rid the sample of the bilirubin in the form conjugate. After this step, the pH of the medium is reduced to 7.4 by adding concentrated sodium hydroxide 2 to 10N. In such a medium, only the fractions of unconjugated bilirubin, not linked to albumin are oxidized, as indicated above, the oxidation reaction taking place in two stages at potentials of -0.03V and + 0.19V.

Also, after bringing the pH of the medium to 7.4 by adding sodium hydroxide, a potential of 0.0V is applied to the electrode (9) to oxidize the unconjugated bilirubin, not linked to albumin. It is thus possible to determine the concentration of unconjugated bilirubin, not linked to proteins (BrnC / alb ratio> 0.8).

Example 5: Determination of unconjugated bilirubin.

In this example, the same procedure is followed as in Example 4 to first rid the sample of bilirubin in conjugated form by carrying out the electrolysis at a potential of 0.06 V for example. After this operation, the pH of the medium is brought to 7.4 using concentrated sodium hydroxide as in Example 4, then an agent capable of breaking the bilirubin-albumin bonds is introduced, for example sodium dodecyl sulfate or hexadecyl triethylammonium chloride and then performs the electrochemical oxidation of bilirubin conjugated to the desired potential which depends on the reagent added.

The concentration of unconjugated bilirubin present in the sample is then determined using one of the measurement methods mentioned above.

IV. Use of an aqueous solution with a pH of 3.7.

The aqueous solution used is a glycoeolle buffer at pH 3.7. The intensity curves are determined as before as a function of the applied voltage. The results obtained are given in FIG. 13.

In this figure, curve 101 relates glycoeolle buffer containing bilirubin unconjugated to 427umol / l bilirubin and albumin with a BRnC / albumin ratio of 0.77. Curve 102 relates to the glycoeolle buffer containing unconjugated biliverdin alone (500 μmol / l), curve 103 relates to the glycoeol buffer containing albumin and 345 μmol / l of conjugated bilirubin (BRC / Alb ratio = 0.63 ), Curve 104 relates to the glycoeol buffer containing albumin and 500 μmol / l of conjugated bilirubin (BRC / alb ratio = 0.91), curve 105 relates to the glycoeolle buffer containing 1000 μmol / l of conjugated bilirubin albumin (BRC / Alb ratio = 1.81).

By comparing these curves, it is noted that only the conjugated bilirubin is oxidized in two stages (curves 103, 104 and 105). The oxidation potentials are respectively -0.02V and + 0.13V.

The results obtained with the various solutions described above are grouped in the following table which gives the values of the oxidation potentials observed.

Although in the examples described above, the concentration measurements have almost always been made by coulometry or amperometry, these measurements can be carried out spectrophotometrically.

By way of example, the results obtained by circulating in a cell similar to that of FIG. 4 are given below, a mobile phase constituted by phosphate buffer at pH 7.4 containing benzoic acid (50 g / l) and caffeine (30 g / l) and by injecting 100 μl of a serum sample containing 5.1 μmol / l of bilirubin for a BRnC / Alb ratio of 0.35, at a flow rate of 0.8ml / min by applying variable potentials to the electrode. The optical density of the medium is measured continuously at the cell outlet at 440 nm.

FIG. 14 represents the evolution of the optical density of the solution continuously passing through the installation of FIG. 4 as a function of the potential applied. In this figure, we note that at + 0.7V, the drop in optical density at 440nm reaches 90% of the initial optical density.

In this specific case, a potential of 0.7V is necessary to oxidize 90% of the BR present in the blood sample. In fact, at a lower voltage, the surface of the cell electrode (3) of FIG. 4 and the passage time of the sample (0.5 ml / min) are not sufficient to obtain total electrolysis. The specificity of the assay is nevertheless obtained by highlighting the drop in optical density to 440nm.

When it is desired to carry out the measurement by spectrophotometry, it is possible to use as a measuring cell, a coulometric cell with optical faces to carry out the optical density measurement directly in the electrolysis cell.

In Figure 15, there is shown a cell of this type. This comprises a parallelepiped tank (121) into which the sample solution (123) to be assayed can be introduced. A magnetic stirrer (125) is arranged at the bottom of the tank and a working electrode (127) is pressed against the side wall of the tank.

A reference microelectrode (129) constituted for example by an Ag / AgCl electrode, is placed in a tube (130) in electrolytic contact with the solution (123) by through a sintered glass. An auxiliary electrode (131) constituted for example by a platinum wire, is arranged in a tube (132) also in electrolytic contact with the solution (123).

The tank (121) comprises two parallel optical faces forming an optical window for the passage of a light beam in order to be able to directly determine the optical density of the solution (123) present in the tank. The three electrodes are connected to an electric generator not shown in the drawing in order to apply to the working electrode (127) a controlled potential with respect to the reference electrode.

In this cell, the plated electrode (127) could be replaced by a wire immersed in the solution, as long as it is arranged so as not to interfere with the light beam which passes through the container to measure the optical density.

As seen above. the method of the invention can be implemented for carrying out different assays and the results obtained by coulometry with potential imposed at + 0.65V (Coulochem, ESA) on icteric serum will be described below. Determination of bilirubin in icteric serum.

For this assay, the cell shown in FIG. 15 is used which is placed in the rack with a photophotometric spectrometer UVIKON 810 (KONTRON Instruments) and the potential applied to the working electrode is controlled by a module 5 100 A (Coulochem ESA).

In the tank 121 installed in the spectrophotometer, 5 ml of solution are delivered conductive and 200 μl of icteric serum. The solution being continuously agitated, the potential is imposed at + 0.65V and the spectral evolution of the solution is monitored over time.

FIG. 16 shows the curves obtained with different solutions, before and after coulometry, which illustrate the optical densities of the solutions as a function of wavelengths (in nm).

In this figure, curve 151 refers to the results obtained with 4.8 ml of phosphate buffer (pH = 7.4).

Curve 152 relates to the solution

11 which is obtained by adding 200 μl of icteric serum to 4.8 ml of the phosphate buffer used previously, the serum containing 200 μmol / l of total BR determined by diazoreaction and 37 g / l of albumin determined by nephelometry.

Curve 153 refers to solution 11 after 30 minutes of coulometry at + 0.65V.

Curve 154 refers to the solution

12 which is obtained by adding 50 g / l of benzoic acid and 30 g / l of caffeine to solution 11.

Curve 155 refers to solution 12 after 30 minutes of coulometry at + 0.65V.

These curves show that the BR is not electro-stainless in the presence of serum albumin under the operating conditions used (curve 153). In the presence of caffeine, the BR oxidizes rapidly as evidenced by the drop in the OD of the solution at the maximum absorption of the pigment (curve 155).

In the examples described above, the corresponding measured potentials were used to the figures to choose the potential to be applied.

Since the value of this potential depends on the apparatus, the solution and the reagents used, it is advisable in each case to determine beforehand the potentials which correspond to the oxidation peaks with the apparatus and the solution used.

Claims

1. Method for assaying bilirubin present in a biological medium in conjugated form and / or in non-conjugated form, characterized in that it comprises the following successive stages:

c) determine the quantity of bilirubin thus oxidized.

2. Method according to claim 1, characterized in that the aqueous electrically conductive solution is a sodium chloride solution.

3. Method according to claim 1, characterized in that the electrically conductive aqueous solution is a buffer solution chosen from phosphate buffers having a pH of 5 to 8 and glycoeolle or lactate buffers having a pH of less than 5.

4. Method according to any one of claims 1 to 3, characterized in that the solution further comprises a reagent capable of breaking the bilirubin-albumin bonds and / or of dissolving the bilirubins in this solution.

5. Method according to claim 4, characterized in that said reagent is chosen from diazoreaction accelerators, agents anionic and cationic surfactants, and organic solvents.

6. Method according to claim 5, characterized in that the diazoreaction accelerator consists of at least one compound chosen from caffeine and diphylline.

7. Method according to any one of claims 1 to 6, characterized in that, in step c), the amount of bilirubin oxidized is determined by differential amperometry and in that the amount of bilirubin oxidizable under these conditions, initially present in the biological medium.

8. Method according to any one of claims 1 to 6, characterized in that, in step c), the amount of bilirubin oxidized is determined by differential coulometry.

9. Method according to any one of claims 1 to 6, characterized in that, in step c), the amount of bilirubin oxidized is determined by differential speetrophotometry.

10. Method according to any one of claims 1 to 9, characterized in that the aqueous solution has a pH of 7.4 + 0.5, in that it contains a reagent consisting of an anionic surfactant, and in what is done in step b) at a potential such that only the conjugated bilirubin is oxidized.

11. Method according to any one of claims 1 to 9, characterized in that the aqueous solution has a pH of approximately 8, in that it contains a reagent consisting of an anionic surfactant and in that one operates in stage b) at a potential such that the conjugated bilirubin and the unconjugated bilirubin are oxidized.

12. Method according to any one of claims 10 and 11, characterized in that the anionic surfactant is sodium dodecylsulfate or sodium cholate.

13. Method according to any one of claims 1 to 9, characterized in that the aqueous solution has a pH of 7.4 + 0.5, in that it contains a reagent consisting of a cationic surfactant or by a diazoreaction accelerator, and in that one operates in step b) at a potential such that the conjugated bilirubin and the unconjugated bilirubin are oxidized.

14. Method according to any one of claims 1 to 9, characterized in that the aqueous solution has a pH of approximately 5 and in that one operates at a potential such that one oxidizes is the conjugated bilirubin alone , i.e. unconjugated bilirubin and conjugated bilirubin.

15. Method according to any one of claims 1 to 9, characterized in that the aqueous solution has a pH of approximately 3 and in that one operates at a potential such that only bilirubin is oxidized conjugate.

16. Method according to any one of claims 1 to 9, characterized in that the conjugated bilirubin is first removed from the sample by using a solution at a pH of approximately 5 and by carrying out an electrochemical oxidation at a potential such that all of the conjugated bilirubin is oxidized, the pH of the solution is then adjusted to approximately 7.4, an electrochemical oxidation is carried out at a potential such that one oxidizes only the unconjugated bilirubin, not linked to albumin, and the amount of unconjugated, unbound bilirubin is determined, thus oxidized by speetrophotometry or by differential spectrofluorescence.

17. Measuring cell for implementing the method according to claim 9, characterized in that it comprises:

a parallelipipedic tank provided with two parallel faces forming an optical window for the passage of a light beam in order to measure the optical density of a solution present in the tank,

- Means for connecting the electrodes to an electric generator.