US3862012A - Quantitative determination of uric acid - Google Patents

Quantitative determination of uric acid Download PDF

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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
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Prior art keywords
uric acid
solution
ethanol
reaction
sample
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US380020A
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Harald Stork
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Roche Diagnostics GmbH
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Boehringer Mannheim GmbH
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    • 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)
US380020A 1972-08-02 1973-07-17 Quantitative determination of uric acid Expired - Lifetime US3862012A (en)

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

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US3862012A true US3862012A (en) 1975-01-21

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US (1) US3862012A (es)
JP (1) JPS5441359B2 (es)
AR (1) AR197053A1 (es)
AT (1) AT330726B (es)
CA (1) CA1000599A (es)
CH (1) CH576640A5 (es)
DE (2) DE2237940C3 (es)
FI (1) FI52778C (es)
FR (1) FR2231291A5 (es)
GB (1) GB1395127A (es)
IT (1) IT1007537B (es)
NL (1) NL176873C (es)
SE (1) SE388050B (es)

Cited By (8)

* Cited by examiner, † Cited by third party
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 (en) * 2006-06-26 2008-01-03 University Of Florida Research Foundation, Inc. Urate metabolites as diagnostic markers for cardiovascular and renal disease
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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS50151609A (es) * 1974-05-30 1975-12-05
JPS547685B2 (es) * 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
WO1981000725A1 (en) * 1979-09-14 1981-03-19 Charles Hospital Dev Method of determining a substrate in a sample

Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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 (en) * 2006-06-26 2008-01-03 University Of Florida Research Foundation, Inc. Urate metabolites as diagnostic markers for cardiovascular and renal disease
WO2008002911A3 (en) * 2006-06-26 2009-04-02 Univ Florida Urate metabolites as diagnostic markers for cardiovascular and renal disease
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
CH576640A5 (es) 1976-06-15
DE2237940B2 (de) 1980-04-17
JPS4960290A (es) 1974-06-11
DE2326756A1 (de) 1974-12-19
CA1000599A (en) 1976-11-30
DE2326756B2 (de) 1980-08-14
AR197053A1 (es) 1974-03-08
AT330726B (de) 1976-07-12
GB1395127A (en) 1975-05-21
ATA675873A (de) 1975-10-15
JPS5441359B2 (es) 1979-12-07
NL176873B (nl) 1985-01-16
FI52778C (fi) 1977-11-10
DE2326756C3 (de) 1981-10-29
NL176873C (nl) 1985-06-17
FI52778B (es) 1977-08-01
NL7310625A (es) 1974-02-05
DE2237940A1 (de) 1974-02-07
DE2237940C3 (de) 1980-12-11
SE388050B (sv) 1976-09-20
FR2231291A5 (es) 1974-12-20
IT1007537B (it) 1976-10-30

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