CA1119923A - Uric acid standard solutions - Google Patents
Uric acid standard solutionsInfo
- Publication number
- CA1119923A CA1119923A CA000324956A CA324956A CA1119923A CA 1119923 A CA1119923 A CA 1119923A CA 000324956 A CA000324956 A CA 000324956A CA 324956 A CA324956 A CA 324956A CA 1119923 A CA1119923 A CA 1119923A
- Authority
- CA
- Canada
- Prior art keywords
- solution
- uric acid
- buffer
- value
- component
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
Abstract
URIC ACID STANDARD SOLUTIONS
ABSTRACT OF
THE DISCLOSURE
Stable aqueous solutions useful as standards in the determination of uric acid are disclosed. The solutions have a pH of about 6-7 and contain a buffer and a known concentration of uric acid present in solution as a salt of the pH-controlling buffer component. They do not contain any strong base such as lithium carbonate. The standards can be used in connection with either enzymatic or non-enzymatic procedures.
ABSTRACT OF
THE DISCLOSURE
Stable aqueous solutions useful as standards in the determination of uric acid are disclosed. The solutions have a pH of about 6-7 and contain a buffer and a known concentration of uric acid present in solution as a salt of the pH-controlling buffer component. They do not contain any strong base such as lithium carbonate. The standards can be used in connection with either enzymatic or non-enzymatic procedures.
Description
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 . , . ... , .. ., ~ . ... ~ .. ..... ... . .... ... . . .
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 . , . ... , .. ., ~ . ... ~ .. ..... ... . .... ... . . .
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
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, 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.
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.
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, 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.
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.
Claims (8)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A stable solution useful as a standard in the determination of uric acid, said solution consisting essentially of water, a buffer sufficient to maintain the pH value of the solution at about 6-7, and a known concentration of uric acid present as a soluble salt of the pH-con-trolling buffer component, said component having a pK value of about 5-7.5.
2. The solution of Claim 1 which also contains one or more microbial growth inhibitors and metal chelatins agents.
3. The solution of Claim 2 wherein the pH-controlling buffer component has a pK value of 6-7.5.
4. The solution of Claim 3 wherein the buffer contains a weakly basic amine.
5. The solution of Claim 4 wherein the amine is imidazole, pyri-dine or collidine.
6. The solution of Claim 5 wherein the pH value is 6.5-7.
7. The solution of Claim 6 wherein the amine is imidazole.
8. The solution of Claim 7 wherein the acidic component of the buffer is benzoic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89673178A | 1978-04-17 | 1978-04-17 | |
| US896,731 | 1978-04-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1119923A true CA1119923A (en) | 1982-03-16 |
Family
ID=25406739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000324956A Expired CA1119923A (en) | 1978-04-17 | 1979-04-05 | Uric acid standard solutions |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA1119923A (en) |
| GB (1) | GB2018989B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2484093A1 (en) * | 1980-06-06 | 1981-12-11 | Farmatest | CHEMICAL REAGENT INDICATOR FOR "IN VITRO" DIAGNOSIS OF PREGNANCY |
| US5667983A (en) * | 1994-10-24 | 1997-09-16 | Chiron Diagnostics Corporation | Reagents with enhanced performance in clinical diagnostic systems |
-
1979
- 1979-04-05 CA CA000324956A patent/CA1119923A/en not_active Expired
- 1979-04-12 GB GB7913055A patent/GB2018989B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB2018989B (en) | 1982-07-21 |
| GB2018989A (en) | 1979-10-24 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |