US3862012A - Quantitative determination of uric acid - Google Patents
Quantitative determination of uric acid Download PDFInfo
- Publication number
- US3862012A US3862012A US380020A US38002073A US3862012A US 3862012 A US3862012 A US 3862012A US 380020 A US380020 A US 380020A US 38002073 A US38002073 A US 38002073A US 3862012 A US3862012 A US 3862012A
- Authority
- US
- United States
- Prior art keywords
- uric acid
- solution
- ethanol
- reaction
- sample
- 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 - Lifetime
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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
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
Definitions
- ABSTRACT Uric acid is determined by contacting a sample suspected of containing uric acid with a phosphate buffer, an NADH solution, ethanol, alcohol dehydrogenase and catalase in the presence of air to form a reaction mixture containing 0.3 to 0.7 volume percent alcohol, measuring a first extinction value, adding uricase to the reaction mixture, measuring a second extinction value spectrophotometrically and taking the difference between the extinction values as a measure of the initial uric acid content in the sample.
- the present invention is concerned with a method for the quantitative photometric determination of uric acid. More specifically, the invention relates to such a determination which can be carried out completely enzymatically, the results obtained being such that they can be read off in the near ultra-violet range of a photometer.
- uric acid is oxidized by phosphotungstic acid and the phosphotungsten blue formed by the reaction is measured colorimetrically.
- Other reducing substances for example, medicaments or reducing metabolites, can, of course, have a disturbing effect on the reaction.
- uric acid is decomposed by means of uricase to give allantoin, the absorption decrease of the uric acid in the region of 293 mp. being a measure for its concentration.
- This process is, like most enzyme reactions, very specific but the inherent absorption of the protein component ofthe enzyme has a disturbing effect, in the same way as other proteinaceous materials present in the sample, so that false results can be obtained.
- German Pat. No. 2,149,675 describes a process in which methanol is oxidized to formaldehyde by the enzymatically-formed hydrogen peroxide in the presence of catalase and the formaldehyde then reacts, in the presence of an ammonium salt according to Hantzschs reaction, with acetylacetone to give 3,5- diacetyl-l ,4-dihydrolutidine.
- concentration of this yellow-colored material which is proportional to the concentration of the uric acid, can be determined colorimetrically at 410 mu.
- a bath thermostatically controlled at 37C. is necessary in which, for the development of the colored lutidine compound, it is necessary to incubate the test sample for at least 30 minutes but, practically speaking, for more than an hour. Furthermore, the last stage of the process does not take place enzymatically and can, therefore, be disturbed by reducing or oxidizing components present in the sample, which react with the formaldehyde.
- the present invention provides a method for the detection of uric acid which can be carried out completely enzymatically and which, in a short period of time, gives, without laborious incubation, a quantitative result which is not influenced by substances which disturb the previously known methods for determining uric acid.
- the method of the invention comprises reacting the hydrogen peroxide liberated by the allantoin reaction of uric acid with ethanol in the presence of catalase, reducing the acetaldehyde thereby formed back again to ethanol by reduced nicotinamide-adenine-dinucleotide (NADH) in the presence of alcohol dehydrogenase, and measuring the decrease of the NADH concentration spectrophotometrically in the favorable range of 340 or 366 my, as being proportional to the uric acid content of the sample.
- NADH nicotinamide-adenine-dinucleotide
- reaction (C) is disturbed by ethanol present in the sample.
- catalase is not an exclusively peroxidate-acting enzyme but that it, especially in the case of small concentrations of ethanol, preponderantly liberates oxygen directly from hydrogen peroxide which, as such, is not able to react with ethanol.
- reaction (B) requires, for a quantitative reaction of the hydrogen peroxide, a high ethanol concentration, whereas ethanol has an inhibiting action on the course of reaction (C) (cf. Methoden der enzymatischen Analyse, H. U. Bergmeyer, I962, p. 292)
- C course of reaction
- uricase and catalase act upon a uric acid-containing sample in the presence of ethanol and of air to form acetaldehyde, which is reduced back again to ethanol by the action of alcohol dehydrogenase with NADH, the decrease of the NADH concentration being measured spectrophotometrically, the process being-carried out at a pH of 6.3 to 7.1 and such a quantity of ethanol is added to the reaction mixture that the concentration thereof is within the limits of 0.3 to 0.7 vol.%.
- the optimum ethanol concentration for the reaction is about 0.5 vol.'/1.
- process step C is normally only carried out after a deproteinization of the sample. we have, unexpectedly, found that. in the case of the process according to the present invention. deproteinizing is unnecessary (see also FIG. 11 of the accompanying drawings).
- the extinction of the NADH can be measured in the near ultra-violet range at 340 or 366 mu.
- the process according to the present invention can be carried out at ambient temperature. a thermostatic control being unnecessary.
- the analysis results are obtained after about 20 minutes.
- the process according to the present invention only requires a very small amount of sample (0.1 ml. of serum) so that it can also be used for carrying out determinations with capillary plasma.
- HO. 1 represents a calibration curve produced with an aqueous solution of uric acid.
- FlG. 11 is a calibration curve which has been determined by means of a standard serum with a predetermined content of 5.9 mg.72 uric acid.
- Phosphate buffer 3.522 g. potassium dihydrogen phosphate and 7.268 g. disodium hydrogen phosphate dihydrate were dissolved in 1,000 ml. double distilled water.
- NADH solution 25 mg. NADH were dissolved in 10 ml. of a 1% aqueous sodium bicarbonate solution. This solution is stable for about 10 days.
- Alcohol dehydrogenase (Boehringer Mannheim, Germany): crystal suspension in 3.2 M ammonium sulphate solution. pH about 6; specific activity 200 U/mg. (25C.).
- Catalase Boehringer Mannheim, Germany: solution in 3071 glycerol/10% ethanol; specific activity 260,000 U/ml. (25C.).
- Uricase Boehringer Mannheim, Germany: solution in 50% glycerol. pH 10.2 50 mM glycine, 0.13 M sodium carbonate; specific acivity 4.5 U/mg. (25C.).
- the uric acid content was calculated as follows:
- Uricase Boehringer Mannheim, Germany: solution in 50% glycerol pH 10.2 50 mM glycine; 0.13M sodium carbonate; specific activity 4.5 U/mg. (C.).
- uric acid were dissolved in 0.lN aqueous sodium hydroxide solution and made up to I00 ml.
- the reagents used in Examples 1 and 2 can advantageously also be mixed for one or more tests in the volume ratios set out in section b).
- test volume taken from the mixture can then. in a single further pipetting step, be mixed with the sample to be tested (serum or urine or other uric acidcontaining solution) so that the laborious individual pipettings can be omitted.
- sample to be tested serum or urine or other uric acidcontaining solution
- Process for the quantitative determination of uric acid in a sample suspected of containing same comprises contacting said sample with a phosphate buffer, an NADH solution, ethanol, alcoholdehydrogenase, and catalase in the presence of air, wherein the ethanol concentration is from 0.3 to 0.7 volume percent of the resulting reaction mixture, whereby acetaldehyde is formed, and measuring a first extinction value, thereafter contacting the reaction mixture with uricase and measuring a second extinction value spectrophotometrically at 340 or 366 mu, and taking the difference between the extinction values as a measure ofthe initial uric acid content in said sample, wherein the process steps are carried out at a pH of from 6.3 to 7.l'.
- NADH solution is supplied in the form of aqueous solution containing about l'/r sodium bicarbonate.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE2237940A DE2237940C3 (de) | 1972-08-02 | 1972-08-02 | Verfahren zur quantitativen Bestimmung von Harnsäure |
DE2326756A DE2326756C3 (de) | 1972-08-02 | 1973-05-25 | Verfahren zur quantitativen Bestimmung von Harnsäure |
Publications (1)
Publication Number | Publication Date |
---|---|
US3862012A true US3862012A (en) | 1975-01-21 |
Family
ID=25763647
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US380020A Expired - Lifetime US3862012A (en) | 1972-08-02 | 1973-07-17 | Quantitative determination of uric acid |
Country Status (13)
Country | Link |
---|---|
US (1) | US3862012A (fr) |
JP (1) | JPS5441359B2 (fr) |
AR (1) | AR197053A1 (fr) |
AT (1) | AT330726B (fr) |
CA (1) | CA1000599A (fr) |
CH (1) | CH576640A5 (fr) |
DE (2) | DE2237940C3 (fr) |
FI (1) | FI52778C (fr) |
FR (1) | FR2231291A5 (fr) |
GB (1) | GB1395127A (fr) |
IT (1) | IT1007537B (fr) |
NL (1) | NL176873C (fr) |
SE (1) | SE388050B (fr) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4202938A (en) * | 1976-04-05 | 1980-05-13 | Human Gesellschaft mbH fur Biotopanalytic und Biotopschutz | Procedure for the quantitative determination of hydrogen peroxide concentration in aqueous solutions |
US5618686A (en) * | 1993-03-08 | 1997-04-08 | Nitto Boseki Co., Ltd. | Method of measuring the total ketone body and a sample reagent |
WO2008002911A2 (fr) * | 2006-06-26 | 2008-01-03 | University Of Florida Research Foundation, Inc. | Métabolites d'urate utilisés en tant que marqueurs diagnostiques pour une maladie cardiovasculaire et rénale |
US10302395B1 (en) | 2018-04-11 | 2019-05-28 | Darrell Holland | Quick aim reticle |
US10976135B1 (en) | 2018-04-11 | 2021-04-13 | Darrell Holland | Quick aim reticle |
US11041694B1 (en) | 2018-04-11 | 2021-06-22 | Darrell Holland | Quick aim reticle |
US11125533B1 (en) | 2020-04-08 | 2021-09-21 | Darrell Holland | Quick aim reticle |
CN114113062A (zh) * | 2021-12-17 | 2022-03-01 | 暨南大学 | 一种无酶、比色-光热双模式体外检测尿酸纳米金材料及其制备方法和应用 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS50151609A (fr) * | 1974-05-30 | 1975-12-05 | ||
JPS547685B2 (fr) * | 1974-05-30 | 1979-04-09 | ||
DE2718588C3 (de) * | 1977-04-26 | 1979-11-08 | Boehringer Mannheim Gmbh, 6800 Mannheim | Verfahren und Reagens zur Bestimmung von Harnsäure |
US4230798A (en) * | 1978-02-13 | 1980-10-28 | Miles Laboratories, Inc. | Unitized uric acid test composition and device |
JPS56501384A (fr) * | 1979-09-14 | 1981-09-24 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3335069A (en) * | 1964-12-14 | 1967-08-08 | Miles Lab | Composition and method for determining uric acid |
US3349006A (en) * | 1963-06-17 | 1967-10-24 | Miles Lab | Process and composition for the enzymatic determination of uric acid |
-
1972
- 1972-08-02 DE DE2237940A patent/DE2237940C3/de not_active Expired
-
1973
- 1973-05-25 DE DE2326756A patent/DE2326756C3/de not_active Expired
- 1973-07-17 US US380020A patent/US3862012A/en not_active Expired - Lifetime
- 1973-07-26 SE SE7310383A patent/SE388050B/xx unknown
- 1973-07-26 CA CA177,399A patent/CA1000599A/en not_active Expired
- 1973-07-27 AR AR249321A patent/AR197053A1/es active
- 1973-07-27 FI FI732377A patent/FI52778C/fi active
- 1973-07-30 IT IT27283/73A patent/IT1007537B/it active
- 1973-07-30 GB GB3615773A patent/GB1395127A/en not_active Expired
- 1973-07-30 FR FR7327814A patent/FR2231291A5/fr not_active Expired
- 1973-07-31 CH CH1116073A patent/CH576640A5/xx not_active IP Right Cessation
- 1973-08-01 NL NLAANVRAGE7310625,A patent/NL176873C/xx not_active IP Right Cessation
- 1973-08-01 AT AT675873A patent/AT330726B/de not_active IP Right Cessation
- 1973-08-02 JP JP8718273A patent/JPS5441359B2/ja not_active Expired
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3349006A (en) * | 1963-06-17 | 1967-10-24 | Miles Lab | Process and composition for the enzymatic determination of uric acid |
US3335069A (en) * | 1964-12-14 | 1967-08-08 | Miles Lab | Composition and method for determining uric acid |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4202938A (en) * | 1976-04-05 | 1980-05-13 | Human Gesellschaft mbH fur Biotopanalytic und Biotopschutz | Procedure for the quantitative determination of hydrogen peroxide concentration in aqueous solutions |
US5618686A (en) * | 1993-03-08 | 1997-04-08 | Nitto Boseki Co., Ltd. | Method of measuring the total ketone body and a sample reagent |
WO2008002911A2 (fr) * | 2006-06-26 | 2008-01-03 | University Of Florida Research Foundation, Inc. | Métabolites d'urate utilisés en tant que marqueurs diagnostiques pour une maladie cardiovasculaire et rénale |
WO2008002911A3 (fr) * | 2006-06-26 | 2009-04-02 | Univ Florida | Métabolites d'urate utilisés en tant que marqueurs diagnostiques pour une maladie cardiovasculaire et rénale |
US10302395B1 (en) | 2018-04-11 | 2019-05-28 | Darrell Holland | Quick aim reticle |
US10976135B1 (en) | 2018-04-11 | 2021-04-13 | Darrell Holland | Quick aim reticle |
US11041694B1 (en) | 2018-04-11 | 2021-06-22 | Darrell Holland | Quick aim reticle |
US11125533B1 (en) | 2020-04-08 | 2021-09-21 | Darrell Holland | Quick aim reticle |
CN114113062A (zh) * | 2021-12-17 | 2022-03-01 | 暨南大学 | 一种无酶、比色-光热双模式体外检测尿酸纳米金材料及其制备方法和应用 |
CN114113062B (zh) * | 2021-12-17 | 2023-12-12 | 暨南大学 | 一种无酶、比色-光热双模式体外检测尿酸纳米金材料及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CA1000599A (en) | 1976-11-30 |
FR2231291A5 (fr) | 1974-12-20 |
JPS4960290A (fr) | 1974-06-11 |
NL176873C (nl) | 1985-06-17 |
FI52778C (fi) | 1977-11-10 |
JPS5441359B2 (fr) | 1979-12-07 |
GB1395127A (en) | 1975-05-21 |
DE2237940C3 (de) | 1980-12-11 |
DE2326756B2 (de) | 1980-08-14 |
ATA675873A (de) | 1975-10-15 |
NL7310625A (fr) | 1974-02-05 |
SE388050B (sv) | 1976-09-20 |
DE2237940B2 (de) | 1980-04-17 |
AT330726B (de) | 1976-07-12 |
DE2326756C3 (de) | 1981-10-29 |
IT1007537B (it) | 1976-10-30 |
DE2326756A1 (de) | 1974-12-19 |
FI52778B (fr) | 1977-08-01 |
AR197053A1 (es) | 1974-03-08 |
DE2237940A1 (de) | 1974-02-07 |
CH576640A5 (fr) | 1976-06-15 |
NL176873B (nl) | 1985-01-16 |
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