WO2009135035A1 - Détermination enzymatique d'ions potassium utilisant des analogues de nad(p)h stables - Google Patents

Détermination enzymatique d'ions potassium utilisant des analogues de nad(p)h stables Download PDF

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Publication number
WO2009135035A1
WO2009135035A1 PCT/US2009/042361 US2009042361W WO2009135035A1 WO 2009135035 A1 WO2009135035 A1 WO 2009135035A1 US 2009042361 W US2009042361 W US 2009042361W WO 2009135035 A1 WO2009135035 A1 WO 2009135035A1
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Prior art keywords
reagent
diagnostic reagent
reagent kit
diagnostic
kit
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PCT/US2009/042361
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English (en)
Inventor
Richard A. Kaufman
Peter I. Mathe
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Specialty Assays, Inc.
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Publication of WO2009135035A1 publication Critical patent/WO2009135035A1/fr

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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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Definitions

  • the invention is directed to the use of NADH (Nicotinamide Adenine Dinucleotide) and NADPH (Nicotinamide Adenine Dinucleotide Phosphate) analogs used in an enzymatic potassium procedure to measure potassium ions in samples.
  • NADH Nicotinamide Adenine Dinucleotide
  • NADPH Nicotinamide Adenine Dinucleotide Phosphate
  • the first method used commercially to measure potassium ions in body fluids was flame emission spectroscopy.
  • the sample is diluted with a solution containing an internal standard (usually cesium or lithium) and then aspirated into a propane-air flame and the emission resulting from the excitation of potassium is measured at one of its emission lines e.g. at 589, 768, 671, or 852 run (Fundamentals of Clinical Chemistry, 3 rd ed., Norbert W. Tietz editor, p 618 1987).
  • an internal standard usually cesium or lithium
  • Potassium ion specific electrodes have largely replaced flame emission spectrophotometry for the measurement of potassium ions in clinical samples in hospital laboratories. These methods are precise and usually specific for potassium ions. However, the accuracy is somewhat effected by protein buildup on the electrode membrane surface which requires frequent cleaning and recalibration. Also for small inexpensive clinical chemistry analyzers, the hardware and software costs for incorporating ion specific electrodes can increase the cost of these analyzers substantially.
  • ADP adenosine diphosphate
  • NADH nicotinamide adenine dinucleotide (reduced form)
  • NAD nicotinamide adenine dinucleotide (oxidized form).
  • ADP and PEP are converted to pyruvate and ATP by the enzyme pyruvate kinase.
  • pyruvate is reduced by the NADH to lactate, and NADH is oxidized to NAD.
  • the reaction is conveniently followed by measuring the rate of change of absorbance (decreasing abs ⁇ rbance) at usually 340 nm.
  • the activity of pyruvate kinase is dependent on the potassium concentration in the reagent.
  • the activity of pyruvate kinase and the rate of oxidation of NADH, and thus the rate of change of absorbance at 340 nm will be proportional to the potassium ion concentration in a sample.
  • the assay is quite precise and correlates well with potassium ion specific electrode methods, the reagent is very expensive to produce and has very poor stability due to the instability of NADH.
  • the working reagent has a stability of only 1 week at 4° (Clin. Chem. 35, 817-820 (1989)).
  • the pyruvate kinase methods generally have poor reagent stability and thus poor shelf life and working stabilities for the reagents.
  • Methods using cryptands are usually stable reagents but the preparation of the cryptands involves multiple step syntheses and generally the cryptands are quite expensive to make.
  • the present invention includes a diagnostic reagent kit for measuring potassium ion in a sample wherein such kit contains at least one NAD(P)H analog.
  • the NAD(P)H analog can be selected from among AcNAD(P)H (3-acetylpyridine adenine dinucleotide and 3-acetylpyridine adenine dinucleotide phosphate), thio-N AD(P)H (thionicotinamide adenine dinucleotide and thionicotinamide adenine dinucleotide phosphate), and AId-NAD(P)H (3- pyridinealdehyde adenine dinucleotide and 3-pyridinealdehyde adenine dinucleotide phosphate), etc., and the like.
  • a method of quantifying the presence of potassium ion in a sample generally includes:
  • step a a) contacting a sample with an NAD(P)H analog such as one set forth above; and b) measuring the change in absorbance or fluorescence resulting from said contact (step a).
  • a preferred NAD(P)H analog is AcNAD(P)H, although the other analogs set forth above can also be used.
  • AcNAD(P)H and the other analogs set forth above are stable in aqueous sol ⁇ tions at pH values from about 6 and up to 11.
  • Potassium reagents using pyruvate kinase as the potassium ion concentration indicator can now be manufactured as stable liquid reagents with long shelf life compared with the same reagents using NAD(P)H.
  • the kit components of suitable potassium ion kits are inexpensive and most components are readily available.
  • kits prepared in accordance with the present invention include one or two reagent vial systems which contain the NAD(P)H analog, suitable buffers, etc, optionally, pyruvate kinase, lactate dehydrogenase, PEP, ADP, and an NAD(P)H- analog regenerating system.
  • concentration of the analogs in the reagent kits can range from 0.1 mmol/L to about 1.0 mmol/L.
  • some preferred diagnostic reagent kits for the enzymatic measurement of potassium ion concentration include a working reagent include an NAD(P)H analog.
  • Some preferred NAD(P)H analogs include, for example, 3-acetylpyridine adenine dinucleotide, 3-acetylpyridine adenine dinucleotide phosphate, 3-pyridinealdehyde adenine dinucleotide, 3- pyridinealdehyde adenine dimxcleotide phosphate, thionicotinamide adenine dinucleotide, and thionicotinamide adenine dinucleotide phosphate.
  • One preferred analog is 3-acetylpyridine adenine dinucleotide (phosphate).
  • the analogs can be prepared using known techniques or purchased from Specialty Assays Inc. of Hillsborough, NJ.
  • kits of the invention will also preferably include pyruvate kinase, and more particularly, pyruvate kinase obtained from Bacillus stearothermophilus.
  • the enzyme can be extracted using known techniques or purchased from Shinco American, Inc. of New York, NY.
  • the diagnostic reagent kits of the present invention can also include a divalent metal ion such as manganese or magnesium.
  • the diagnostic reagent kits of the present invention preferably include a 2 reagent system .
  • the first reagent, Reagent 1 will have a pH of from about 6 to about 9.5, and preferably about 8.25.
  • the second reagent, Reagent 2 has a pH of from about 5 to about 8.5 and is preferably about 7.0, so that when the reagents 1 and 2 are combined, the pH of thereof is from about 6 to about 9, and most preferably about 7.5.
  • One preferred buffering system includes L-alanine.
  • Other suitable buffers which can be used include those with a pKa around 6.5 - 8.5.
  • a non-limiting list is as follows: MOPS, TES, Methylimidazole, HEPES, Triethanolamine, Tricine, EPPS, TRIS, Glycineglycine, BICINE, Imidazole, ADA, and PIPES.
  • kits of the present invention include: a. ADP; b. PEP; and c. lactate dehydrogenase.
  • kits are designed to include two separate reagent containers; this is known as a two-reagent configuration.
  • the first reagent, Regent 1 preferably includes: a. lactate dehydrogenase; b. PEP c. ADP; d. AcNADH; and e. L-alanine buffer, pH 8.25.
  • the second reagent, Reagent 2 preferably includes: a. pyruvate kinase; b. manganese sulfate; and c. L-alanine buffer, pH 7.
  • a preferred method of the present invention to measure potassium ion concentration is the following series of coupled enzyme reactions:
  • pyruvate kinase is activated by potassium ions.
  • potassium ion concentrations in the reagent up to about 1/10 th of the Km of pyruvate kinase for potassium, the increase in activity will proportional to the potassium concentration in the reagent, and thus in the sample added to the reagent.
  • the activity of pyruvate kinase is then determined in the second enzymatic reaction using lactate dehydrogenase and an NADH analog, which in this case is AcNADH.
  • the rate of pyruvate formation is followed by determining the rate of oxidation of AcNADH, which can be followed by measuring the rate of decrease in absorbance at any wavelength between about 320 nm and 415 rnn.
  • the wavelength for maximum sensitivity is 363 nm, most clinical chemistry analyzers only offer measuring wavelengths of 340, 375, 380, and 405 nm. Any of these wavelengths is acceptable, but 340, 375, arid 380 nm wavelengths are preferred for sensitivity.
  • the pyruvate kinase from Bacillus stearothermophilus is one preferred enzyme to use in the assay.
  • the enzyme is quite stable at elevated temperatures and shows less sensitivity to sodium ions than does the rabbit muscle enzyme, for example.
  • Other sources of pyruvate kinase are well known to those of ordinary skill in the art.
  • Useful enzyme activities in the final reagent are activities in a range from 20 units/L to 1000 units/L, with a preferred activity of about 50 units/L.
  • a divalent metal ion is needed to bind the ADP and PEP to pyruvate kinase.
  • Magnesium and manganese are both acceptable, with manganese preferred. Others will be apparent to those of ordinary skill in the art.
  • the ADP and PBP can be either in the free acid, the non-potassium salt, or the non-sodium salt forms. They can be added to the reaction buffer and neutralized with, e.g., lithium hydroxide, or other non-potassium or non-sodium bases or liquid substituted ammonia bases such as triethylamine.
  • Any buffer can be used to maintain the pH of the reagent in a range of about 6 to 9.
  • one particularly preferred buffer is L-alanine, which was found to enhance the activity of pyruvate kinase.
  • the concentration of L-alanine can be from about 0.006 to 0.3 mol/L, and the preferred concentration is 0.05 mol/L.
  • the reagent solution containing the NADH analog has a pH of about 6 to 9.
  • a pH of about 7.5 to 8.5 is more preferable, with a still more preferred pH of about 8.25.
  • kits of the present invention can be prepared in either wet or dry form, including lyophilized form, depending upon the needs of the user. If desired, the kits can be prepared to include a suitable antimicrobial such as sodium azide, Kathon, Bronopol or parabens. Such antimicrobials can be present in amounts ranging from about 0.01 to about 0.5% by weight
  • kits of the present invention can also be prepared to include enzyme stabilization ingredients or reagent systems, i.e. enzymes and substrates, such as those set forth in U.S. Pat. No. 4,394,449 where an oxidized cofactor can be reduced back to its original reduced form by using an appropriate enzyme and substrate.
  • enzyme stabilization ingredients or reagent systems i.e. enzymes and substrates, such as those set forth in U.S. Pat. No. 4,394,449 where an oxidized cofactor can be reduced back to its original reduced form by using an appropriate enzyme and substrate.
  • the reagent is configured as a two-component liquid- stable reagent
  • the following is an example of a two-component reagent for measuring potassium ion concentration in samples.
  • this formulation There are many variations of this formulation which would be acceptable, as those skilled in the art of developing reagents will recognize.
  • Reagent 1 Acceptable Range Buffer having a pH 8.25 (L-alamne preferred at 83 mmol/L) pH range 6 - 9
  • BSA bovine serum albumin
  • an AcNADH regeneration system using glucose and glucose dehydrogenase, has been included. IfAcNADH becomes oxidized to AcNAD by, e.g., dissolved molecular oxygen in the reagent, it is easily reduced back to AcNADH in the following reaction.
  • the potassium ion assay is run by adding 1 volume of sample to 12 volumes of Reagent 1 plus 2 volumes of diluent (distilled water) to flush out the dispensing needle on most clinical chemistry analyzers. After a short incubation, e.g., 5 minutes, to metabolize any pyruvate present in the clinical sample, 3 volumes of Reagent 2 are added plus 2 volumes of diluent to flush out Reagent 2 from the dispensing needle. After a short lag phase absorbance readings are taken over a given time period, e.g., 5 minutes, and the ⁇ absorbance/minute at a wavelength discussed above is calculated.
  • the working reagent has the following concentrations.
  • the assay is calibrated using two or more standards.
  • a zero standard which contains a physiological amount of NaCl, e.g., 140 mmol/L, is needed for most analyzers.
  • the potassium ion standards should be protein based and preferably prepared in human serum or BSA. The following results were obtained using the reagents prepared in Example 1 and the protocol mentioned above. Several human control sera were assayed for potassium ion concentration.
  • Potassium ion concentration can also be determined with the following procedure, using the NAD(P)H analogs and the enzyme urea amidolyase. Urea amidolyase activity is also activated by potassium ions.
  • the activity of urea amidolyase will be proportional to the potassium ion in the reagent and thus proportional to the potassium ion concentration in the sample.
  • a divalent metal ion, magnesium or manganese, is also required for urea amidolyase to be active.
  • a suitable two reagent system for measuring potassium ion using the above procedure is as follows.
  • the assay is made by adding the sample containing potassium ion to Reagent 1 and after a short incubation, e.g., 5 minutes, to metabolize any endogenous ammonia in the sample, Reagent 2 is added.
  • the increase in the activity of urea amidolyase and thus the increase in the - ⁇ A/min at any wavelength between 340 to 415 run would be proportional to the potassium ion concentration in the sample.
  • the preferred wavelength is between 340 and 380 nm where the sensitivity of AcNADH and AcNADPH are highest

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Abstract

L’invention concerne un kit et un procédé pour mesurer des ions potassium en utilisant les analogues de NAD(P)H Ac-NAD(P)H, thio-NAD(P)H et Ald-NAD(P)H. Les analogues permettent de préparer des réactifs enzymatiques de potassium liquides et stables, qui présentent des stabilités considérablement prolongées dans des solutions aqueuses en comparaison des mêmes réactifs utilisant NAD(P)H.
PCT/US2009/042361 2008-05-02 2009-04-30 Détermination enzymatique d'ions potassium utilisant des analogues de nad(p)h stables WO2009135035A1 (fr)

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US61/049,889 2008-05-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120241335A1 (en) * 2009-08-20 2012-09-27 Carina Horn Simplified storage of integrated systems
CN104726536A (zh) * 2013-12-24 2015-06-24 上海复星医药（集团）股份有限公司 一种稳定的酶法测定镁离子的试剂
CN105671127A (zh) * 2016-03-07 2016-06-15 王爰 一种稳定的酶法血清镁离子检测试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5801006A (en) * 1997-02-04 1998-09-01 Specialty Assays, Inc. Use of NADPH and NADH analogs in the measurement of enzyme activities and metabolites
US6068971A (en) * 1987-04-10 2000-05-30 Roche Diagnostics Gmbh Process for determination of ions in fluids by masking of interfering ions
US6380380B1 (en) * 1999-01-04 2002-04-30 Specialty Assays, Inc. Use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucliotide phosphate (NADP) analogs to measure enzyme activities metabolites and substrates
US7022494B2 (en) * 2003-09-19 2006-04-04 General Atomics Detection of potassium ions using ion-sensitive enzymes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6068971A (en) * 1987-04-10 2000-05-30 Roche Diagnostics Gmbh Process for determination of ions in fluids by masking of interfering ions
US5801006A (en) * 1997-02-04 1998-09-01 Specialty Assays, Inc. Use of NADPH and NADH analogs in the measurement of enzyme activities and metabolites
US6380380B1 (en) * 1999-01-04 2002-04-30 Specialty Assays, Inc. Use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucliotide phosphate (NADP) analogs to measure enzyme activities metabolites and substrates
US7022494B2 (en) * 2003-09-19 2006-04-04 General Atomics Detection of potassium ions using ion-sensitive enzymes

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120241335A1 (en) * 2009-08-20 2012-09-27 Carina Horn Simplified storage of integrated systems
US9528977B2 (en) * 2009-08-20 2016-12-27 Roche Diabetes Care, Inc. Simplified storage of at least one test element including a dry reagent layer having an enzyme and a stabilized coenzyme
US20170059506A1 (en) * 2009-08-20 2017-03-02 Roche Diabetes Care, Inc. Diagnostic products having an enzyme and a stabilized coenzyme
CN104726536A (zh) * 2013-12-24 2015-06-24 上海复星医药（集团）股份有限公司 一种稳定的酶法测定镁离子的试剂
CN105671127A (zh) * 2016-03-07 2016-06-15 王爰 一种稳定的酶法血清镁离子检测试剂盒
CN105671127B (zh) * 2016-03-07 2019-01-04 王爰 一种稳定的酶法血清镁离子检测试剂盒

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