Note: Descriptions are shown in the official language in which they were submitted.
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FIELD OF THE INVENTION
The present invention relates generally to the field of
diagnostic tests and, more particularly, to those tests
useful in qualitative and quantitative determination of uric
acid in body fluids such as urine or blood. More particularly
it relates to those tests in which the uric acid is con-
verted to an oxidizing substance, such as a peroxide.
BACX&ROUND O~ THE INVENTION
The oxidative coupling reaction between phenol and 4-
aminophenazone, also known as 4-aminoantipyrinel to give a
red quinoneimine dye has been known for a long time, ha~ing
been described by Emerson, J. Org. Chem. 8:417 (1943).
The reaction has gained popularity in clinical chem-
istry since the application made by Trinder, LAnn. Clin.
Biochem, 6:24 (1969)] to the enzymatic determination of
glucose, based on the reaction scheme:
glucose oxidase
glucose + 2 _gluconic acid + H202
peroxidase
2H202 + phenol + 4-aminophenazone ~uinoneimine dye
+ 4H20
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The chromogenic system phenol (including substituted phenols) -
+ 4-aminophenazone + peroxidase, referred to as the ~merson-
Trinder system, is now used ln the quantitative determina-
tion not only of glucose, but also of cholesterol and uric
acid in serum, plasma or other biological fluids. Use of
this system for the determination of glucose is disclosed in
Meiattini, U.S. Patent No. 3,886,045 now reissued as Re 29,498
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The general scheme of the reaction is the following:
specific oxidase
1) analyte + 2 oxidized analyte + ~IZ02
2)
C6H5
H3C- ~ ~ + ~ H peroxidase
H3C H2
l 6H5
3G~ ~ ~ ~
\ ~ /~=,\ ~colored product)
H3C~ = ~ = 0
Many phenols can be used in the Emerson-Trinder reaction.
S Examples of those most commonly used in clinical chemistry
are phenol; p-hydroxybenzoate; 2,4-dichlorophenol; 3,5-
dichloro-2-hydroxybenzenesulfonic acid. Likewise various
substituted and unsubstituted napthols can be used.
The sample constituents which can be determined include
glucose, cholesterol, uric acid or other metabolites which
can be oxidized by a specific oxidase with contemporaneous
formation of hydrogen peroxide. The oxidase is glucose
oxidase for determining glucose; cholesterol oxidase for ` `
determining cholesterol (cholesterol ester hydrolase is also ;~
added to hydrolyze esterified cholesterol); and uricase for
uric acid determinations.
The amount of dye formed is proportional to the concen-
tration of the hydrogen peroxide and, therefore, to the
concentration of the constituent in the sample. Thus, the
concentration of the constituent in the sample can be obtained
by a simple measurement of the absorbance of the reacted
solution and comparison of such measurement to that of a
known standard solution of the constituent.
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The dye formed can be measured in the visible range,
generally between 500 and 550 nanometers (nm) (depending on
the phenol used); requiring only a colorimeter or a visiblc
color range photometer.
The Emerson-Trinder chromogenic system suffers the
major disadvantage that the oxidative coupling reaction is
affected by reducing compounds and bilirubin, a metabolitc
which is usually present in serum in concentrations not
higher than 1 milligram per deciliter (mg/dl), but whic}l ca
reach very high levels (20 or more mg/dl) in some diseases.
Levels of bilirubin higher than normal affect the enzymatic
glucose, cholesterol and uric acid tests by decreasing the
color of the reaction. Interference increases with the
increase of the bilirubin level.
The explanation of negative interference of reducing
compounds (e. g. ascorbic acid) is quite obvious, sincc thcy
act chiefly as competitors with the chromogen in the peroxi-
dase catalyzed reaction with hydrogen peroxide, or as bleaching
agents on the color formed. The interference of reducing
substances, however, is not a real problem, at least in
serum, where ascorbic acid rarely exceeds 3 mg/dl.
In contrast, the interference by bilirubin is a signi-
ficant problem for the determination of metabolites in serum
through the Emerson-Trinder chromogenic system, and represent~
~5 a major negative aspect of this system in routine laboratory
practice, where hyperbilirubinemic samples are frequently
found.
The mechanism of reaction of bilirubin is quite coml~lcx
and, as yet, not fully understood. The best approach so far
aEforded to the problem is that of Witte [Clin. Chem. 24:1778
~1978)], who ascribes the interference of bilirubin to onc
or more of the following factors: simple spectral ef-fects,
acting as an alternative peroxidase substrate, or dcstruc-
tion of peroxidase reaction intermediates.
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OBJECTS OF THE INVENTIO~
It is therefore an object of the present invention to
provide an improved test for the detection of uric acid
in a fluid sample.
A further object of the invention is to provide an
improved test for the detection of uric acid which is highly
resistant to the interfering effects of bilirubin.
Other objects and a fuller understanding of the invcn-
tion will be had by referring to the following description
and claims drawn to preferred embodiments thereof.
SUMMARY O~ T~E INVENTION
.
As part of the present invention it has been discovered
that bilirubin strongly interferes with chromogenic tests ol
the type having a substituted or unsubstituted phenol and 4-
aminophenazone, chiefly by two different mechanisms: (1) hy
overlapping the spectrum of the dye formed in the reaction,
thereby causing a positive interference, and ~2) by a chemical
mechanism, as previously discussed, which causes a negative
interference. The positive interference resulting from thc
first mechanism, can be reduced by reading the absorbance at
a wavelength of 520 nm or higher. However, such is not the
case with respect to the second mechanlsm. It plays an
extremely important role, causing inaccurate results in the
above-described tests when bilirubin is present in the
sample in abnormal concentrations.
In contrast to prior art compositions, that of the
present invention is highly sensitive to the presence of
uric acid in body fluids, while also being substantially
resistant to bilirubin interference. ;
.. ~
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This surprising result is achieved, in accordancc with
the present invention, by a composition -for the detection ol
uric acid in a fluid sample of the type comprising means
responsive to the presence of uric acid in the sample,
phenol and 4-aminophenazone and to which resistance to
interference by bilirubin is imparted by inclusion thercin
of reagent means comprising a ferrocyanide ion.
DESCR~PTION OF THE PRE~ERRED EMBODIMENTS
Although specific terms are used in the following `~
description for the sake of clarity, these terms are in-
tended to refer only to the particular embodiment of the
invention selected for exemplary illustration, and are not
intended to define or limit the scope of the invention.
The composition according to the invention can take
many physical forms and include a phenol, including sub-
stituted and unsubstituted phenols, well known for its
applicability of use with 4-aminophenazone indicator com-
positions, in combination with the reagent means which
comprises a ferrocyanide ion. These, along with materials
~0 such as stabilizing agents and other conventional additives, `
which can additionally be employed if desired, are describc~. `
Preferred reagent means comprising a ferrocyanide ion
include alkali metal salts of ferrocyanide, such as sod-ium
or potassium ferrocyanide, as well as any other source Or
~5 ferrocyanide ion, including any other salt or system con-
taining or capable of releasing Fe(CN) 6 ions.
The composition comprises along with the reagent mcans
according to the invention, mears responsive to the prcsonco
~"
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of uric acid in a fluid sample to produce an oxidizing
substance. Such uric acid responsive means are preferably
enzymatic in nature and preferably comprise uricase an~ a
peroxidatively active substance. The concentrations an~
types of reagents useful in the uric acid responsive means
are contemplated to include those known to the art.
The test means can be used as a solution for determina-
tion of uric acid. The solvents used in preparing the sol-
utions can be water, physiological solutions, organic sol-
vents, such as methanol, or mixtures thereof.
The composition is preferably used to detect uric .IC i~by adding it to a specimen such as urine, cerebrospinal
fl~lid, tissue culture supernatant and preferably, serum,
plasma or whole blood.
When the composition is used in solution form the
reagent means comprising the ferrocyanide ion is preferahly
used in concentrations of from about 1.0 micromol/liter
t~mol/l) to a saturated solution. The preferred range is
from about 5 ~mol/l to about 50 ~mol/l. When uricase is l)ar-t
2~ of the uric acid responsive means, concentrations thereo~
are perferably from about 10 International Units (I.U.)/
liter ~1) to about 200 I.U./l. When peroxidase is at least
one of the reagents comprising the uric acid responsive
means concentrations of the peroxidase are preferably from
~5 about 10 I.U./l to about 200 I.U./l.
The enzyme activity is expressed in International Units
(I.U.), one I.U. being the amount of enzyme activity re~u;rc~
to catalyze the conversion of 1 micromol ~mol) of substratc
per minute under specified conditions of pH and temperaturc.
Horseradish peroxidase and uricase used in the examples can
be obtained from The Research Products Division, Miles
Laboratories, Inc., Elkhart, Indiana.
.. ..
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Also provided are test devices incorporating thc coml)o-
sition of the invention and a method of making such test
devices which comprises incorporating a carrier, such as a
matrix, with the composition. When this incorporation is l)y
impregnation with a solution of the composition accordin~ to
the invention the carrier so impregnated is then dried. In
addition to impregnation, the devices of the present inven-
tion can be made by other suitable incorporating tcchni~lucs,
such as printing or spraying the composition onto a sul)str.ltc
or matrix. Alternatively, the compositions of the invcntion
can be embodied in a carrier taking the form of a ~rcssc~l or
molded tablet containing conventional carrier material.
The term carrier refers to matrices which are insol-ll)lc
in and maintain there structural integrity when exposcd to
physiological or other liquid. Suitable matrices which may
be used include paper, cellulose, wood, synthetic resin
fleeces, glass fiber, nonwoven and woven fabrics, gelatin,
various organic polymers, such as polypropylene, and othcr
organic materials weli known as film formers to those skillc~
in the art. For convenience, the carrier or test dcvicc can
be associated with an insoluble support or handle membcr,
such as can be made with polystyrene.
When the test composition is to be used for detecting ~"`
uric acid in blood, the surface of the impregnated carrier
~5 matrix is advantageously covered with a semipermeable trans~
parent coating film of ethyl cellulose or other suitah]c
material. This can be accomplished by applying a laycr o r
ethyl cellulose dissolved in benzene, for example, to thc
surface of the impregnated carrier matrix and then remo~ing
the solvent by evaporative drying.
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Uric acid indicators in the form of treated carricr
matrices or test devices are often stored for considcrahlc
periods of time before use, and it is therefore desirablc
that the reagents chosen are not easily auto-oxidizable in
air. Advisably, the test devices should be protected from
exposure to light, and in some cases it is desirable to kcc
~hem sealed in a moisture repellent package which is opcncd
only for the purpose of removing one or more test devices
shortly before use thereof.
If desirable, a carrier matrix can be treated with a
background dye of a particular color, such as yellow, so
that the color produced by the test reaction blends with thc
background color to produce varying tints corresponding to
the concentration of the sample constituent.
The device is preferably prepared by a single dip
process. The concentrations of reagents used in the dip
range from about 10-3 mM up to a saturated solution. Most
`generally useful for the 4-aminophenazone is a concentration
of about 0.2 mM. Peroxidase concentration is from about
~0 0.1 mg/dl to about 20 mg/dl in the dip solution. The
solvents used in preparing the impregnating solution can hc
water, physiological solutions, organic solvents or com-
binations thereof.
The test device is advantageously used by momentarily
dipping it in a test sample or by otherwise introducing a
test sample onto the carrier matrix, whereby a detcctable
color change results thereon when uric acid is present. Thc
test device can be used in the same way whether samples of
plasma, serum or other body fluids are tested. However,
when testing whole blood it is preferred that a drop o~
blood be contacted with the surface of the device.
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The examples shown are merely illustrative and are not
to be con~trued as a limitation of the invention. One
skilled in the art will be able to make such variations,
substitutions and changes in the ingredients and parameters
as may seem desirable.
Example I
Determination of uric acid in serum with uricase
and peroxidase/3,5-dichloro-2-hydroxybenzenesulfonic
acid/4-aminophenazone
Test solutions were prepared according to the prior art
and the present invention and compared as to their resist- ;
ance to the interfering effects of bilirubin in the deter-
mination of uric acid.
Test solutions were prepared in accordance with the
1~ prior art having the following formulation:
phosphate buffer 150 mmol/l, pH 7.0
uricase 60 I.U./l
peroxidase 140 I.U./l ~2~`
4-aminophenazone 0.24 mmol/l
~0 3,5-dicholoro-2-hydroxy-
benzenesulfonic acid2.0 mmol/l
Test solutions incorporating the composition of the inven- `
tion were prepared exactly as above but with the addition O~r -
20 ~mol/l potassium ferrocyanide [K4Fe~CN)6]. ~ ~
A 2.0 ml aliquot of prior art test solution was pip- ~-
etted into each of a first group of test tubes and a 2. n ml
aliquot of the solution of the composition of the invention `
was pipetted into each of a second group of test tubes.
parallel series of samples was introduced to the test tubcs
of each group.
'' 10 ` ~"'. `
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Serum samples were obtained, pooled and tested for
contents of uric acid and bilirubin, Uric aci~ was added to
the pooled sera to a concentration of 6.0 mg/dl and the
solution was separated into aliquots. Amounts of bilirubin
were added so as to pro~ide uric acid test solution samplcs
respectively having bilirubin concentrations o 0.7, 1.4,
2.2, 3.6, 5.0, 6.5, 8.8, 12.1, 16.7 and 23.4 mg/dl. Other
uric acid solution aliquots, having no bilirubin, were used
as a standard solution.
Each group of test tubes was injected with a parallcl
series of 0.05 ml sample aliquots containing the various
bilirubin concentrations and the reaction in these tests
tubes was allowed to go forward for 15 minutes at room
temperature, The absorbance readings were performed at
lS 520 nm against the corresponding sample blanks obtained
by omitting uricase from the reagent formulations.
The results reporting the percent recovery of uric acid
in tests using the prior art and the inventive test compo-
sitions at the various bilirubin concentrations are shown in
Table 1.
Ir . .. . , ~ , `:.. , '` `.
Table 1
Uric acid Observed
(percent recovery)
Bilirubin With Without
~mg/dl) K4 Fe(CN)6 K4 Fe(CN)6
0.7 100.0 + 1.2 100.0 ~ 1.2
1.4 100.0 97.2
2.2 99.2 93.5 ':
3.6 98.0 83.6
5.0 97.0 78-5 :
6.5 95.9 71.5
8.8 93.4 59.0
12.1 91.0 43.5
1~.7 90.0 21.0
23.4 89.0
A remarkable chemical interference (about 6%) is `
noticed in the uric acid test in the absence of ferrocyanide
even at levels of bilirubin as low as 2 mg/dl, and this is ~
even more dramatic (more than 20%) at the 5 mg/dl level. -
When ferrocyanide is used, the chemical interference ~::
from bilirubin is strongly reduced (statistically not `~
significant at 2 mg/dl bilirubin; 3~ at 5 mg/dl; around ~-
10~ at levels of bilirubin as high as 15-20 mg/dl). `.`
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Example II ~.
Test device for determination of uric acid in urine ~:
with uricase and peroxidase/3,5-dichloro-2-hydroxy-
benzenesulfonic acid/4-aminophenazone ~ .
"':
- 12 -
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Test devices incorporating compositions according to
the prior art and the present invention were preparecl and
compared as to their resistance to the interfering cffects
of bilirubin in testing for the presence of uric acid in
s urine.
An impregnation solution according to the prior art
was prepared to have the following formulation:
phosphate buffer ]50 mmol, pH 7.0
uricase 150 I.U.
peroxidase 860 I.U.
~-aminophenazone 0.24 mmol
3,5-dichloro-2-hydroxy-
benzenesulfonic acid 2 mmol
H20 to 1000 ml
15Impregnation solutions incorporating a composition ot~
the present invention were prepared exactly as above but
with the addition of 20 ~mol/of potassium ferrocyanide
[K~Fe(CN)6].
Sheets of Whatman No. 17 filter paper ~Whatman, Inc.
Clifton, N.J.) were impregnated to saturation with the
impregnation solutions and dried at 60 Centigrade (C).
These sheets containing the dried residue of the imprcgnatin~
solutions were cut to 2.5 millimeters ~mm) x 2.5 mm to form `~
devices. The devices were then backed with double-faced
2S adhesive tape and fixed thereby to plastic handles.
Urine samples were obtained, pooled and tested for - `
contents of uric acid and biIirubin. The pooled urine was
then diluted with 10 volumes of distilled water. Uric aci~
was added to the diluted urine pool until a concentrat;on o r :
6 mg/dl of uric acid was achieved. Then, bilirubin was
added to the diluted urine pool up to a concentration of lO
mg/dl, thus providing a test solution. This test solution
was divided into aliquots.
13
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Test devices according to the invention were momentari]y
immersed in one aliquot o~ the uric acid test solution an~
test devices according to the prior art were momentarily
immersed in another aliquot thereo. The test devices were
visually examined for color change after about 10 minutes.
Devices prepared in accordance with the invention
sho~ed a distinct change in color indicating the presence
of uric acid whereas the test devices containing the prior
art composition did not change color, thereby reporting a
alse negative for uric acid.
Although the invention has been described with a certain
degree of particularity, it is understood that the present
disclosure has been made only by way o example and that
numerous changes in the details may be resorted to without
departing from the scope o the invention.
14 ~
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