Note: Descriptions are shown in the official language in which they were submitted.
The present invention relates to the determination of uric acid
in biological fluids and, more p æ ticularly, to an im?roved uric aci~ -
standard solution which can be llsed in such determinations.
In man urate is the final product or the metabolism of purines,
especially adenine and quanine which are constituents o~ all nucleic acids~
In most other mammals, urate is further broken down by the enzyme, uricase,
to allantoin, which is highly soluble~ But, since man does not possess
uricase, urate is not broken do~n further in the human body and this leads
to ~he possibility of hyperuricemia which is an elevated serum uric acid
concentration. Among other problems, hyperuricemia ~ay give rise to the
clinical syndrome of gout.
Clinical diagnostic determinations of uric acid in biological
samples such as serum rely on the fact that uric acid can be oxidized to
.. .. . .. . ..
allantoin. Either the enzyme, uricase, or a non-enzy~atic o~idant can be
used. One non-enzymatic procedure uses, as the oxidizin~ agent,
copper~II), in the presence of a color for~ing reagent such as 2,2'-bi-
cinchoninate. See Gindler U.S. patent 4,072,627. Through oxidation,
uric acid reduces co~per(XI) to coppertI) which, in tunl, Eorms ~ colored
--1--
. , . ... , .. ., ~ . ... ~ .. ..... ... . .... ... . . .
2~
complex with the bicinchoninate. The intensity of the complex, measured
at 562um, correlates with the amount of copper(I) formed during oxidation
of uric acid, and thus the concentration of uric acid present in the sam-
ple Other procedures using non-enzymatic oxidants are sho~n in Moran
U.S. patent 3,528,777 (phosphotungstate) and ~orin, et. al. U.S. patent
3,822,115 (ferric ions).
To quantitatively determine the concentration of uric acid in
the sample, it is customary to use the copper(II)-bicinchoninate system
with standard solutions containing known concentrations of uric acid.
Calibration graphs can then be constructed from standards containinq
different concentrations of uric acid for comparison with data obtained
from samples containing the unknown concentrations.
Standard solutions containing uric acid in known concen-trations
-- - have been available for a number of years. Gindler has observed that "A
solution of uric acid in aqueous tris (hydro~ymethyl~ aminomethane is a
rapidly prepared standard." Clin. Chem. 16, 536 (1970). Moreover, since
uric acid itself is usually thought to be soluble in water only at a high
pH, the solutions are ordinarily made under basic c~nditions with the use,
for example, of a strong base such as lithium carbonate. See Clinical
Diagnosis by Laboratory Methods, Clinical Chemistry, Todd-Sanford, Edn.
13, pp 451-452. Solutions so prepared are stable for an extended period
o~ time when re~rigerated. The necessity ~or refrigeration is, o~ course,
a drawback where the standard is not going to be immediately used such as
when initially formulated by a manu~acturer, and then shipped to a custo-
..... . .. ..
mer for ultimate use.
Stable standard solutions o~ uric acid can, however, exist underacidic conditions when the uric acid is present as the N-hydroxymethyl
adduct. See above identified Moran patent. First, an aqueous solution o~
the acid is prepared using lithium carbonate with heating at about 60C
Then the adduct is formed by reaction with formaldehyde lollowed by acidi-
fication with a strong acid such as sulfuric acid. The ormaldehyde adduct
does not precipitate in acid solution and, being acidic, the solution is
stable without refrigeration. Also, as conventionally repared, standard
solutions of uric acid contain various preservatives to inhibit microbial
growth and chelating agents to bind various metal ions which might other-
wise adversely affect stahility by causing oxidation of the uric acid
adduct.
While standard solutions based on ~he formaldehyde adduct of
uric acid are useful in diagnostic procedures relying u~on oxidation with
non-enzymatic oxidants, they are not a~plicable with respect to all analy- -
tical methods, and particularly enzymatic methods such as tshose using
uricase as the uric acid oxidant. Formaldehyde inhibits uricase and,
accordingly, stanaards prepared with formaldehyde are not useful with
some enzymatic procedures.
Scheibe, et. al. U.S. patent 3,920,400 is directed to a uric
acid sta~dard solution which is said to be useful in bo~h enzymatic and
chemical processes. The solution is descrlbed as "cors~rising an aqueous
buffered solution of uric acid in the Eorm of a lithium salt thereof,
with a pH value of between 6 and 8, and at least one cor~lexing agent for
the higher valence stage of polyvalent heavy metal ions and/or an alkali
aæide". The solution is stated to be "remarkably stable". To form the
solution, uric acid is dissolved in the presence of lithium carbonate
and the pH of the solution adjus-ted to the indicated range with a suit-
able buffer. A phosphate buffer is preferrèd. Other buffers rlentioned
are imidazole, trie~hanolamine and tris.
There are at least two apparent problems acco~anying the pre-
paration of standards using lithium carbonate even without thereafter
fonning the formaldehyde adduct. Scheibe, et al. identify one; na~lely,
-3
the inevitability that small amounts of catalytically active metal ions
will get into solution. The other is that to achieve solution either long
stirring times or heatiny is required. Heating in alkaline solution can
cause hydrolysis of uric acid while extended stirring time can be econo-
mically unattractive.
Accordingly, a principal object of the present invention is to
provide a standard solution of uric acid which can be easily prepared at
room temperature and which can be used in connection with both enzymatic
and non~enzymatic procedures for determination of uric acid. A further
object is to provide such a standard solution which is stable for extended
periods without refrigeration and which can be conveniently stored and/or
shipp~d.
In one of its aspects, the pxesent invention provides a stable
solution, useful as a standard in the determination of uric acid, which
consists essen-tially of water, a buffer sufficient to maintain the pH
value of the solution at about 6-7, and a known concentration of uric
acid pre~ent as a soluble salt of the pH-controlling buffer component,
the component having a pK value of about 5-7.5. The standard solution
can be used in connection with either enzymatic or non-enzym~tic proce-
dures for the determination of uric acid, it can be easily prepared, andit is stLlble at an ambient temperature Eor at least about six months.
A~ used herein, the term "consists essentially of" means tha~
the specified substances must be present, but that other substances which
do not prevent the advc~ltages of the in~ention from being realized can
....
also be present. Accordingly, metal ions such as lithium and bases with
a pK above c~out 8 such as tris and lithium carbonate are excluded from
the solutions of the present invention. ~owever, other substances such
as one or more microbial growth inhibitors and chelating agents to bind
metal ion impurities can and, in most instances, are present.
To prepare solutions of the present invention, a kno~ concen-
tration of uric acid can be dissolved in water in the presence of a buffer.
The buffer and the amount thereof are selected such that the pH value of
the solution is about 6-7 and, preferably, 6.5-7. Dissolution of uric
acid occurs rapidly at room temperature with the formation, in solution,
of a uric acid salt with the pH-controlling buffer çomponent. It is be-
lieved that the salt so formed is monobasic, i.e., only one of the hydroxyl
groups of the acid is ionized.
Useful buffers are those having a pH-controlliny component with
a pK value of about 5-7.5. As used herein, the pH-controlling buffer
component is that component of the buffer system which has the greates-t
buffer capacity at the pH value of the solution. Buffering capacity is a
function of both the buffer component concentration and the difference
between the solution pH value and the pK value of the co~ponent. It
reaches a maximum at pH = pK andr therefore, the use of buffers with a
component having a pK of about 6-7.5 is preferred. Buffers containing
weakly basic amines, such as imidazole, pyridine, and collidine, are use~
ful. In addition, organo-phosphorus compounds, such as trimethyl phos-
phorus, and conjugated oxygen compounds, such as pyrilium, are also useful.
So long AS pH control as above specified can be achieved, the selection
o~ the acidic component of the buffer is not particularly critical. Wealc
acids, such as benzoic acid, acetic acid, propionic acid and the like,
can be conveniently employed.
The following formulation illustrates a useful standard solu-
.. . . . .
tion of the present invention which is stable for an extended period at
room temperature and can be used in either enzymatic or non-enzymatic
procedures.
--5--
2~
2.000 gm uric acid
13.6 gm imidazole ~0.200 mole)
0.3 gm ethylene diamine tetraacetic acid tchelating agent)
26.88 gm phenoxyethanol ~preservative)
12.2 gm benzoic acid (0.100 mole for pH control through
buffer formation with imida~ole ana as preservative)
0.5 gm sodium a~ide (preservative)
500 ml isopropanol (preservative)
Sufficient deionized or distilled water to bring solution to
10, 2.00 liters.
The above solution has a~pH value of 6.85 and contains uric acid
in a concentration of 100 mg/dl. Standard so}utions with differant con-
centrations can be prepared by appropriately diluting this solution.
While the presPnt invention has been illustrated in connection
with certain preferred embodiments~, it is to be understood that the inven-
.. .
tion is not to be limited to those embodiments disclosed. On the contrary,it is intended that the invention cover all modifications and alternatives
falling within the sphere and scope of the invention as expressed in the
appended claims.
