Bilirubin-resistant determination of uric acid and cholesterol

Page 1scan →

United States Patent (19)

Acquati et al.

(54) bilirubin-resistant determination

Of uric acid and cholesterol

75 Inventors: Giancarlo Acquati, Robbiate;

Giovanni Berti, Malgrate; Piero

Fossati, Lissone, all of Italy (73) Assignee: Miles Laboratories, Inc., Elkhart, Ind.

Related U.S. Application Data abandoned.

52 u.s. c. .................................... 435/10; 23/230 b;

58) Field of Search ....................... 23/230 B, 925, 40;

Re. 29,498 12/1977. Meiattini........................... 435/28 X

4,184,921 1/1980 Roeschlav ............................. 435/11 4,186,251 1/1980 Tarbutton ..... - ... 435/11 4,247,631 1/1981 Nix ................ - d - - - - - - -- - - - - - - - - - 435/10

4,251,629 2/1981 Yamanisi........................... 435/10 X

Other publications

"Hackh's Chemical Dictionary,” J. Grant, ed., 4th Edi tion, p. 507, McGraw-Hill, New York, 1969.

Primary Examiner-Sidney Marantz

Attorney, Agent, or Firm-Charles J. Herron

A composition, test device, method of making a test device and process for bilirubin-resistant determination of an analyte selected from uric acid or cholesterol in a fluid sample are disclosed. More particularly, the com position is of the type comprising means responsive to the presence of the particular analyte to be determined in the sample, a phenol and 4-aminophenazone to which resistance to interference by bilirubin is imparted by inclusion therein of reagent means comprising a ferro cyanide ion. The uric acid or cholesterol responsive means is preferably of the type which determines perox ides formed from enzymatic conversion of the analyte. The composition can optionally be incorporated with a carrier to provide a test device.

17 Claims, No Drawings 2 lites which can be oxidized by a specific oxidase with

BILIRUBIN-RESISTANT DETERMINATION OF contemporaneous formation of hydrogen peroxide. The

URIC ACID AND CHOLESTEROL oxidase is glucose oxidase for determining glucose;

CROSS-REFERENCE TO RELATED cholesterol oxidase for determining cholesterol (choles 5 terol ester hydrolase is also added to hydrolyze esteri

Application

fied cholesterol); and uricase for uric acid determina

This application is a continuation-in-part of U.S. Ser. tions,

No. 29,670, filed Apr. 13, 1979, abandoned. The amount of dye formed is proportional to the FIELD OF THE INVENTION 10 concentration of the hydrogen peroxide and, therefore, to the concentration of the constituent in the sample.

The present invention relates generally to the field of diagnostic tests and, more particularly, to those tests Thus, the concentration of the constituent in the sample useful in qualitative and quantitative determination of bance ofobtained can be by a simple measurement of the absor analytes selected from uric acid or cholesterol in body 15 measurement to that ofsolution the reacted and comparison of such a known standard solution of the fluids such as urine or blood. More particularly, it re constituent.

lates to those tests in which the analyte is converted to The dye formed can be measured in the visible range, an oxidizing substance, such as a peroxide. generally between 500 and 550 nanometers (nm) (de BACKGROUND OF THE INVENTION pending on the phenol used); requiring only a colorime The oxidative coupling reaction between phenol and ter or a visible color range photometer.

4-aminophenazone, also known as 4-aminoantipyrine, to The Emerson-Trinder chromogenic system suffers give a red quinoneimine dye has been known for a long the major disadvantage that the oxidative coupling time, having been described by Emerson, J. Org. Chem. reaction is affected by reducing compounds and biliru 8:417 (1943). bin, a metabolite which is usually present in serum in The reaction has gained popularity in clinical chemis 25 concentrations not higher than 1 milligram per deciliter try since the application made by Trinder, Ann. Clin. (mg/dl), but which can reach very high levels (20 or Biochem, 6:24 (1969)) to the enzymatic determination of more mg/dl) in some diseases. Levels of bilirubin higher glucose, based on the reaction scheme: than normal affect the enzymatic glucose, cholesterol 30 and uric acid tests by decreasing the color of the reac glucose + O2 glucose oxidase-gluconic acid + H2O2 tion, Interference increases with the increase of the bilirubin level.

2H2O2 + phenol + 4-aminophenazone peroxidase The explanation of negative interference of reducing quinoneimine dye -- 4H2O compounds (e.g. ascorbic acid) is quite obvious, since

The chromogenic system phenol (including substituted 35 they act chiefly as competitors with the chromogen in phenols) + 4-aminophenazone--peroxidase, referred to the peroxidase catalyzed reaction with hydrogen perox as the Emerson-Trinder system, is now used in the ide, or as bleaching agents on the color formed. The quantitative determination not only of glucose, but also interference of reducing substances, however, is not a of cholesterol and uric acid in serum, plasma or other real problem, at least in serum, where ascorbic acid biological fluids. Use of this system for the determina rarely exceeds 3 mg/dl.

tion of glucose is disclosed in Meiattini, U.S. Pat, No. In contrast, the interference by bilirubin is a signfi 3,886,045, now reissued as U.S. Pat. No. Re 29,498. cant problem for the determination of metabolites in The general scheme of the reaction is the following: serum through the Emerson-Trinder chromogenic sys tem, and represents a major negative aspect of this sys 45 tem in routine laboratory practice, where hyper analyte + O2 Specific oxidase-oxidized analyte + H2O2 (1) bilirubinemic samples are frequently found.

The mechanism of reaction of bilirubin is quite com g-Hs (2) plex and, as yet, not fully understood. The best ap proach so far afforded to the problem is that of Witte

N oH peroxidase Clin. Chem. 24:1778 (1978)), who ascribes the interfer ence of bilirubin to one or more of the following factors:

H3C NH simple spectral effects, acting as an alternative peroxi dase substrate, or destruction of peroxidase reaction phs

N 55 intermediates.

H3cny 20 objects of the invention

It is therefore an object of the present invention to

H3C N provide an improved test for the detection of an analyte selected from uric acid or cholesterol in a fluid sample.

Many phenols can be used in the Emerson-Trinder A further object of the invention is to provide an reaction. Examples of those most commonly used in improved test for the detection of an analyte selected clinical chemistry are phenol; p-hydroxybenzoate; 2,4- from uric acid or cholesterol which is highly resistant to dichlorophenol; 3,5-dichloro-2-hydroxybenzenesul the interfering effects of bilirubin. fonic acid. Likewise various substituted and unsubsti 65 Other objects and a fuller understanding of the inven tuted napthols can be used. . . . . tion will be had by referring to the following descrip The sample constituents which can be determined tion and claims drawn to preferred embodiments include glucose, cholesterol, uric acid or other metabo thereof.

Page 3scan →

solutions can be water, physiological solutions, organic

SUMMARY OF THE INVENTION solvents, such as methanol, or mixtures thereof. As part of the present invention it has been discov The composition is preferably used to detect the ered that bilirubin strongly interferes with chromogenic analyte by adding it to a specimen such as urine, cere tests of the type having a substituted or unsubstituted brospinal fluid, tissue culture supernatant and prefera phenol and 4-aminophenazone, chiefly by two different bly, serum, plasma or whole blood. mechanisms: (1) by overlapping the spectrum of the dye When the composition is used in solution form the formed in the reaction, thereby causing a positive inter reagent means comprising the ferrocyanide ion is pref. ference, and (2) by a chemical mechanism, as previously erably used in concentrations of from about 1.0 mi discussed, which causes a negative interference. The O cromol/liter (umol/l) to a saturated solution. The pre positive interference resulting from the first mechanism, ferred range is from about 5 umol/l to about 50 umol/l. can be reduced by reading the absorbance at a wave When uricase is part of the analyte responsive means, length of 520 nm or higher. However, such is not the concentrations thereof are perferably from about 10 case with respect to the second mechanism. It plays an 15 International Units (I.U.)/liter (1) to about 200 I.U./1. extremely important role, causing inaccurate results in When cholesterol oxidase is part of the analyte respon the above-described tests when bilirubin is present in sive means, concentrations thereof are preferably from the sample in abnormal concentrations. about 10 I.U./1 to about 200 I.U./I. When cholesterol In contrast to prior art compositions, that of the pres ester hydrolase is used to obtain determinations of total ent invention is highly sensitive to the presence of an 20 cholesterol concentrations thereof are preferably from analyte selected from uric acid or cholesterol in body about 10 I.U./1 to about 200 I.U./1. When peroxidase is fluids, while also being substantially resistant to biliru atsponsive:

least one of the reagents comprising the analyte re means concentrations of the peroxidase are

This surprising result is achieved, in accordance with preferably from about 10, I.U./1 to about 200 I.U./1. the present invention, by a composition for the detec 25 The enzyme activity is expressed in International tion of an analyte selected from uric acid or cholesterol Units (I.U.), one I.U. being the amount of enzyme activ ity required to catalyze the conversion of 1 micromol in a fluid sample of the type comprising means respon (umol) sive to the presence of said analyte in the sample, phenol tions ofofpHsubstrate per minute under specified condi and 4-aminophenazone and to which resistance to inter uricase and cholesterol oxidase Horseradish and temperature. peroxidase, used in the examples can ference by bilirubin is imparted by inclusion therein of 3O be obtained from Research Products Division, Miles reagent means comprising a ferrocyanide ion. Laboratories, Inc., Elkhart, Ind. DESCRIPTION OF THE PREFERRED Also, provided are test devices, incorporating the EMBODIMENTS composition of the invention and a method of making such test devices which comprises incorporating a car

Although specific terms are used in the following 35 rier, such as a matrix, with the composition. When this description for the sake of clarity, these terms are in incorporation is by inpregnation with a solution of the tended to refer only to the particular embodiment of the composition according invention selected for exemplary illustration, and are impregnated is then dried. to the invention the carrier so In addition to impregnation, not intended to define or limit the scope of the inven the devices of the present invention can be made by

40 other suitable incorporating techniques, such as printing

The composition according to the invention can take or spraying the composition onto a substrate or matrix. many physical forms and include a phenol, including Alternatively, the compositions of the invention can be substituted and unsubstituted phenols, well known for embodied in a carrier taking the form of a pressed or its applicability of use with 4-aminophenazone indicator molded tablet containing conventional carrier material. compositions, in combination with the reagent means 45 The term carrier refers to matrices which are insolu which comprises a ferrocyanide ion. These, along with ble in and maintain there structural integrity when ex materials such as stabilizing agents and other conven posed to physiological or other liquid. Suitable matrices tional additives, which can additionally be employed if which may be used include paper, cellulose, wood, desired, are described. synthetic resin fleeces, glass fiber, nonwoven and Preferred reagent means comprising a ferrocyanide 50 woven fabrics, gelatin, various organic polymers, such ion include alkali metal salts of ferrocyanide, such as as polypropylene, and other organic materials well sodium or potassium ferrocyanide, as well as any other known as film formers to those skilled in the art. For source of ferrocyanide ion, including any other salt or convenience, the carrier or test device can be associated system containing or capable of releasing Fe(CN)6-4 with an insoluble support or handle member, such as ions. 55 can be made with polystyrene. The composition comprises along with the reagent When the test composition is to be used for detecting means according to the invention, means responsive to uric acid or cholesterol in blood, the surface of the the presence of an analyte selected from uric acid or impregnated carrier, matrix is advantageously covered cholesterol in a fluid sample to produce an oxidizing with a semipermeable transparent coating film of ethyl substance. Such analyte responsive means are prefera 60 cellulose or other suitable material. This can be accom bly enzymatic in nature and preferably comprise a per plished by applying a layer of ethyl cellulose dissolved oxidatively active substance, uricase for the determina in benzene, for example, to the surface of the impreg tion of uric acid and cholesterol oxidase for the determi nated carrier matrix and then removing the solvent by nation of cholesterol. The concentrations and types of evaporative drying: - reagents useful in the analyte responsive means are 65 Indicators in the form of treated carrier matrices or contemplated to include those known to the art. test devices are often stored for considerable periods of The test means can be used as a solution for determi time before use, and it is therefore desirable that the nation of the analyte. The solvents used in preparing the reagents chosen are not easily autoxidizable in air. Ad 4 visably, the test devices should be protected from expo trations of 0.7, 1.4, 2.2, 3.6, 5.0, 6.5, 8.8, 12.1, 16.7 and sure to light, and in some cases it is desirable to keep 23.4 mg/dl. Other uric acid solution aliquots, having no them sealed in a moisture repellent package which is bilirubin, were used as a standard solution. opened only for the purpose of removing one or more Each group of test tubes was injected with a parallel test devices shortly before use thereof. series of 0.05ml sample aliquots containing the various If desirable, a carrier matrix can be treated with a bilirubin concentrations and the reaction in these tests background dye of a particular color, such as yellow, so tubes was allowed to go forward for 15 minutes at room that the color produced by the test reaction blends with temperature. The absorbance readings were performed the background color to produce varying tints corre at 520 nm against the corresponding sample blanks ob sponding to the concentration of the sample constituent. 10 tained by omitting uricase from the reagent formula The device is preferably prepared by a single dip process. The concentrations of reagents used in the dip tions.The results reporting the percent recovery of uric range from about 10-mM up to a saturated solution. acid in tests using the prior art and the inventive test Most generally useful for the 4-aminophenazone is a compositions at the various bilirubin concentrations are concentration of about 0.2 mM. Peroxidase concentra 15 shown in Table 1.

tion is from about 0.1 mg/dl about 20 mg/dl in the dip solution. The solvents used in preparing the impregnat TABLE 1 ing solution can be water, physiological solutions, or Uric acid Observed ganic solvents or combinations thereof. (percent recovery) - The test device is advantageously used by momen 20 Bilirubin With Without tarily dipping it in a test sample or by otherwise intro (mg/dl) K4 Fe(CN)6 K4 Fe(CN)6 ducing a test sample onto the carrier matrix, whereby a 0.7 100.0 - 1.2 100.0 - 1.2 detectable color change results thereon when the ana 1.4 lyte is present. The test device can be used in the same 3.6 98.0 83.6 way whether samples of plasma, serum or other body 25 5.0 97.0 78.5 fluids are tested. However, when testing whole blood it 6.5 95.9 7.5 is preferred that a drop of blood be contacted with the 8.8 surface of the device.

The examples shown are merely illustrative and are 23.4 89.0 - not to be construed as a limitation of the invention. One 30 skilled in the art will be able to make such variations, : substitutions and changes in the ingredients and parame A remarkable chemical interference (about 6%) is ters as may seem desirable. noticed in the uric acid test in the absence of ferrocya

Example 1

nide even at levels of bilirubin as low as 2 mg/dl, and 35 this is even more dramatic (more than 20%) at the 5

Determination of uric acid in serum with uricase and . mg/dl level.

peroxidase/3,5-dichloro-2-hydroxybenzenesulfonic When ferrocyanide is used, the chemical interference acid/4-aminophenazone from bilirubin is strongly reduced (statistically not sig Test solutions were prepared according to the prior nificant at 2 mg/dl bilirubin; 3% at 5 mg/dl; around art and the present invention and compared as to their 10% at levels of bilirubin as high as 15–20 mg/dl). resistance to the interfering effects of bilirubin in the EXAMPLE II determination of uric acid.

Test solutions were prepared in accordance with the Test device for determination of uric acid in urine with prior art having the following formulation: uricase and phosphate buffer 150 mmol/l, pH 7.0 Test devices incorporating compositions according peroxidase 140 E.U.A. to the prior art and the present invention were prepared

0.24 mmol/ and compared as to their resistance to the interfering 50 effects of bilirubin in testing for the presence of uric benzenesulfonic acid. 2.0 mmol/ acid in urine.

An impregnation solution according to the prior art

Test solutions, incorporating the composition of the was prepared to have the following formulation: invention were prepared exactly as above but with the addition of 20 umol/l potassium ferrocyanide 55 phosphate buffer 150 mmol, pH 7.0 K4Fe(CN)6). uricase 150 I.U. A 2.0 ml aliquot of prior art test solution was pipetted peroxidase 860 I.U. into each of a first group of test tubes and a 2.0 ml 4-aminophenazone 0.24 mmol aliquot of the solution of the composition of the inven 3,5-dichloro-2-hydroxy- , tion was pipetted into each of a second group of test benzenesulfonic acid

2 mmol tubes. A parallel series of samples was introduced to the

Serum samples were obtained, pooled and tested for Impregnation solutions incorporating a composition contents of uric acid and bilirubin. Uric acid was added of the present invention were prepared exactly as above to the pooled sera to a concentration of 6.0 mg/dl and 65 but with the addition of 20 umol/of potassium ferrocya the solution was separated into aliquots. Amounts of nide K4Fe(CN)6).

bilirubin were added so as to provide uric acid test Sheets of Whatman No. 17 filter paper (Whatman, solution samples respectively having bilirubin concen Inc. Clifton, N.J.) were impregnated to saturation with 5 the impregnation solutions and dried at 60 Centigrade C. The absorbance readings were performed and the (C.). These sheets containing the dried residue of the cholesterol concentrations calculated using the same impregnating solutions were cut to 2.5 millimeters method and formula as was used for uric acid in Exam (mm)x2.5 mm to form devices. The devices were then ple I.

backed with double-faced adhesive tape and fixed The results reporting the amount of cholesterol ob thereby to plastic handles. served in tests using the prior art and the inventive test Urine samples were obtained, pooled and tested for systems at the various bilirubin concentrations are contents of uric acid and bilirubin. The pooled urine shown in Table 2. * : was then diluted with 10 volumes of distilled water. TABLE 2

Uric acid was added to the diluted urine pool until a 10 concentration of 6 mg/dl of uric acid was achieved. Cholesterol Observed (percent recovery) Then, bilirubin was added to the diluted urine pool up Bilirubin With Without to a concentration of 10 mg/dl., thus providing a test (mg/dl) K4Fe(CN)6 K4Fe(CN)6 solution. This test solution was divided into aliquots. 1.3 100.0 - 1.7 OO.0 . .7 Test devices according to the invention were momen 15 3.8 tarily immersed in one aliquot of the uric acid test solu 9.8 97.8 87.1 tion and test devices according to the prior art were 2.0 97.9 85.4 momentarily immersed in another aliquot thereof. The 13.5 97.6 85.8 test devices were visually examined for color change 18.3 97.9 . 80.5 after about 10 minutes.

Devices prepared in accordance with the invention showed a distinct change in color indicating the pres theThe chemical intereference in the cholesterol test in ence of uric acid whereas the test devices containing the the presenceof of absence ferrocyanide is remarkable. However, in ferrocyanide the interference is so prior art composition did not change color, thereby stongly reduced that it can, for practical purposes, be reporting a false negative for uric acid. 25 considered as negligible up to at least 18 mg biliru

Determination of cholesterol with cholesterol EXAMPLE IV oxidase/cholesterol ester hydrolase and

Test device for determination of cholesterol in serum with cholesterol oxidase and

Test solutions were prepared according to the prior peroxidase/3,5-dichloro-2-hydroxy-benzenesulfonic art and the present invention and compared as to their acid/4-aminophenazone resistance to the interfering effects of bilirubin in the Test devices incorporating compositions according determination of cholesterol. w

Test solutions were prepared in accordance with the 35 to the prior art and the present invention were prepared prior art having the following formulaton: and compared as to their resistance to the interfering effects of bilirubin in testing for the presence of choles terol in serum.

phosphate buffer 100 mmol/l, pH 7.7 An impregnation solution according to the prior art cholesterol ester hydrolase 80 I.U.M1 was prepared to have the following formulation: cholesterol oxidase 40 I.U.M.

4-aminophenazone 0.5 mmol/ phosphate buffer 100 mmol/l, pH 7.7 phenol 10 mmol/ cholesterol ester sodium cholate 3 mmol/ hydrolase 240 I.U.M. Triton X-100 0.5% volume/volume (v/v) 45 cholesterol oxidase 120 I.U.A.

Test solutions incorporating the composition of the 3,5-dichloro-2- invention were prepared exactly as above but with the hydroxybenzene addition of 12 mmol/l potassium ferrocyanide sulfonic acid 10 nmol/ SO sodium cholate 3 mmol/

K4Fe(CN)6). Triton X-100 0.5% volume/volume (v/v) A 2.5 ml aliquot of the prior art test solution was pipetted into each of a first group of test tubes and a 2.5 Impregnation solutions incorporating a composition ml aliquot of the solution of the composition of the invention was pipetted into each of a second group of of the present invention were prepared exactly as above test tubes. Aparallel series of samples was introduced to 55 but with the addition of 20 umol/l of potassium ferrocy the test tubes of each group. r anide KAFE(CN)6).

An aqueous cholesterol solution was prepared to Sheets of Whatman No. 17 filter paper (Whatman, have a concentration 200 mg/dl and the solution was . . Inc. Clifton, N.J.) were impregnated to saturation with separated into aliquots. Amounts of bilirubin were the impregnation solutions and dried at 60 Centigrade added so as to provide cholesterol solution samples 60 (C.). These sheets containing the dried residue of the respectively having bilirubin concentrations of 1.3, 3.8, impregnating solutions were cut to 2.5 millimeters 6.3, 9.8, 12.0, 13.5, and 18.3 mg/dl. Other cholesterol (mm)x2.5 mm to form devices. The devices were then solution and the working solution was used to calibrate backed with double-faced adhesive tape and fixed a blank value. thereby to plastic handles. Each group of test tubes was injected with a parallel 65 Serum samples were obtained, pooled and tested for series of 0.02 ml sample aliquots containing the various contents of cholesterol (which was around 200 mg/dl) bilirubin concentrations and the reaction in these test and bilirubin. The pooled serum was then diluted with tubes was allowed to go forward for 15 minutes at 37 10 volumes of distilled water. The bilirubin was added 6 to the diluted serum pool up to a concentration of 20 8. A composition for the determination of uric acid in mg/dl., thus providing a test solution. This test solution a fluid sample of the type comprising means responsive was divided into aliquots. to the presence of uric acid on the sample, unsubstituted Test devices according to the invention were momen phenol and 4-aminophenazone wherein the improve tarily immersed in one aliquot of the cholesterol test ment comprises reagent means comprising potassium solution and test devices according to the prior art were ferrocyanide.

momentarily immersed in another aliquot thereof. The 9. The composition of claim 1 wherein the analyte is test devices were visually examined for color change cholesterol.

after about 10 minutes. 10. A composition for the determination of choles Devices prepared in accordance with the invention 10 terol in a fluid sample of the type comprising means showed a distinct change in color indicating the pres responsive to the presence of cholesterol in the sample, ence of cholesterol whereas the test devices containing unsubstituted phenol and 4-aminophenazone wherein the prior art composition did not change color, thereby the improvement comprises reagent means comprising reporting a false negative for cholesterol. sodium ferrocyanide.

Although the invention has been described with a 15 11. A test device which comprises a carrier incorpo certain degree of particularity, it is understood that the rated with the composition of claim 1. present disclosure has been made only by way of exam 12. A method of making a test device which com ple and that numerous changes in the details may be prises incorporating a carrier with the composition of resorted to without departing from the scope of the 20 claim 1.

invention. 13. The method of claim 12 wherein the composition What is claimed is: is incorporated with the carrier by impregnating the 1. Composition for the determination of an analyte carrier drying with a solution of said test means, followed by of the impregnated carrier. - selected from uric acid and cholesterol in a fluid sample of the type comprising means responsive to the pres 25 sample which comprises contacting said samplea with 14. A process for determination of uric acid in fluid ence of said analyte in said sample, a phenol or napthol and 4-aminophenazone wherein the improvement com the composition of claim 1 and observing any resultant color formed.

prises reagent means comprising a ferrocyanide ion. 15. A process for determination of uric acid in a fluid 2. The composition of claim 1 wherein the phenol is sample which comprises contacting said sample with - an unsubstituted phenol. 30 the test device of claim 11 and observing any resultant 3. The composition of claim 1 wherein the phenol is color formed thereon.

3,5-dichloro-2-hydroxybenzenesulfonic acid. 16. A process for determination of cholesterol in a 4. The composition of claim 1 wherein the reagent fluid sample which comprises contacting said sample means is a ferrocyanide ion salt. with the composition of claim 1 and observing any 5. The composition of claim 4 wherein the ferrocya 35 resultant color formed, nide ion salt is sodium ferrocyanide. 17. A process for determination of cholesterol in a 6. The composition of claim 4 wherein the ferrocya fluid sample which comprises contacting said sample nide ion salt is potassium ferrocyanide. with the test device of claim 11 and observing any resul 7. The composition of claim 1 wherein the analyte is tant color formed thereon.

uric acid. 40 + k is is sk