CN101173936A - Kalium diagnosis/measuring reagent kit and method for measuring kalium concentration - Google Patents

Abstract

The invention relates to a diagnosing and determining reagent box for potassium by use of Enzymatic colorimetric method and Enzymic linkage Method, as well as to the principle for determining the concentration of potassium, the compositions and components of the reagent, belonging to the technical field of medicine/ foodstuff/ environment testing and determining. The main components of the reagent box comprise buffer solution, reduced coenzyme, tryptophan, 2- ketoglutarate, tryptophan enzyme, glutamic acid dehydrogenase and stabilizer. By mixing the sample and reagent according to specific volume ratio, a series of enzymatic (or enzyme-coupled) reactions will occur; after that, the reactants are placed under an ultraviolet/ visible light analyzer to detect the speed at which the absorbance reduces at the main wavelength 340 nm, and thus the concentration of the potassium can be derived. The invention has the advantage of acquiring the required determination results by means of an ultraviolet/visible light analyzer.

Description

Potassium diagnosis/determination kit and method for measuring kalium concentration

Technical field

The present invention relates to a kind of potassium diagnosis/determination kit, the invention still further relates to the method for measuring potassium concn simultaneously, belong to medical science/food/environmental test determination techniques field.

Background technology

The clinical meaning of three decisive levels of serum potassium: be lower than the reference value lower limit, the performance hypopotassaemia, its reason has renal tubular disease, high aldosterone disease, hyperinsulinism, metabolic alkalosis, potassium ion is transferred in the cell etc. when diuretic therapy, insulin for treating diabetes ketosis.Be higher than the reference value upper limit, may be weak, the poisoning of diabetes ketosis, hypoadrenocorticism etc. by renal glomerular disease, renal function.When blood potassium up to 7.5mmol/L or when above patient arrhythmia cordis will appear, should consider to determine suitable therapeutic scheme.When serum potassium surpasses 10mmol/L, ventricular fibrillation can take place, cardiac arrest and death.High potassium makes cardiac arrest diastole.

Potassium is measured method commonly used at present has: isotope dilution mass spectrometry, neutron activation method, flare photometer, chemical assay, ion selective electrode method, atom spectrophotometric method, enzyme kinetics method.

Flame photometry nineteen fifty is brought into use and the flame photometry always used till today detects the Na of serum, urine, cerebrospinal fluid and ascites pleural fluid ⁺And K ⁺, be a kind of emission spectrometry method.Assay method is divided into internal reference method and two kinds of outer standard laws.Outer standard law operate miss is bigger, does not generally adopt.The main now internal standard method of using, promptly sample and titer adopt internal standard element lithium or the caesium of adding same concentrations to measure.During operation, the solution that will contain lithium is measured the concentration of K, Na and Li simultaneously as dilution, with the Na/Li and the K/Li ratio of sample and titer, calculates Na, K concentration.Because serum diluting multiple is big, the influence of serum proteins viscosity almost can be ignored.

Chemical assay mainly utilizes multiple ring crown compound such as cave crown ether or spherical crown ether, also be called crown ether, being ionophore measures, owing in the macrocyclic structure hole is arranged, the intramolecule oxygen atom has not, and share electron pair can combine with metallic ion, according to the hole size, alternative metallic ion in conjunction with different-diameter, thus can reach the purpose of measuring ion concentration.Measure serum K, generally adopt common crown ether anionic dye to carry out colorimetric assay.

Ion selective electrode method ISE method is to adopt sensitive specific electrode special, at the K of body fluid such as the cleer and peaceful urine of the enterprising promoting circulation of blood of instrumentation ⁺, Na ⁺Mensuration, because of the sample consumption is few, quick and precisely, the trend that replaces additive method is arranged almost.Its Instrument measuring principle is that ion-selective electrode and contrast electrode combination are immersed in the sample solution to be measured, detects.Present existing type of electrodes is: 1. glass-membrane electrode, inductive material be glass film pH electrode, K arranged ⁺Electrode and Na ⁺Electrode; 2. immobilon-p electrode by the extrusion forming of slightly solubility metallics, has Cl with solid film or odd-numbered day film as the electrode of sense film ^-Electrode and F ^-Electrode; 3. liquid film electrode is with epoxy resin or the interior dress polyvinyl chloride Ca that is sense film ²⁺Electrode; 4. the K that makes with the valinomycins film ⁺Electrode.Above-mentioned electrode all has certain life-span, because electrode uses a period of time will automatic aging, the term of validity is different in size.

Atom spectrophotometric method atom spectrophotometric method can be used for detecting serum K+, Na+, trivial operations, and error is bigger, and is easy not as good as flame photometry.

The decisive method that potassium is measured is isotope dilution mass spectrometry and neutron activation method.The method that WHO recommends is a flare photometer.Ion selective electrode method application at present is comparatively general, but electrode cost costliness.

Enzyme assay and flare photometer or ion selective electrode method all have consistance preferably, and strong interference immunity, and good stability has remedied the deficiency that Biochemical Analyzer can not measured potassium sodium simultaneously.Analytical performance is preferably arranged, be easy to robotization, must replace flare photometer in the clinical chemistry analysis becomes the conventional analysis project.

Summary of the invention

The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for potassium concn, simultaneously, the present invention also will provide in order to realize the potassium diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out measuring kalium concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Method for measuring kalium concentration principle of the present invention is as follows:

Tryptophane+water TryptophanasePyruvic acid+ammonium ion+indoles

Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme Glutamte dehydrogenaseL-glutamic acid+water+coenzyme

Tryptophanase (the tryptophanase that this method utilization needs potassium ion to activate; EC4.1.99.1) enzyme (idol) connection glutamte dehydrogenase (Glutamate dehydrogenase; EC 1.4.1.2; EC 1.4.1.3) enzymatic reaction continuous monitoring method/speed ratio color method.The tryptophan reaction of tryptophanase enzymolysis produces ammonium ion, the effect of uniting glutamte dehydrogenase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of potassium.

Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the potassium diagnosis/determination kit of the present invention of following composition relation is comparatively desirable:

Damping fluid 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Tryptophane 20mmol/L

2-oxoglutaric acid ester 15mmol/L

Tryptophanase 12000U/L

Glutamte dehydrogenase 20000U/L

Potassium diagnosis/determination kit of the present invention can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, glutamte dehydrogenase.

Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.

Also above-mentioned single agent reagent can be made into following pair of agent reagent:

Reagent 1

Damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester.

Reagent 2

Damping fluid, stabilizing agent, tryptophanase, glutamte dehydrogenase.

Reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, the position of glutamte dehydrogenase in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.

Above-mentioned single agent reagent can also be made into following three doses of reagent:

Reagent 1

Damping fluid, stabilizing agent, reduced coenzyme.

Reagent 2

Damping fluid, tryptophane, 2-oxoglutaric acid ester.

Reagent 3

Damping fluid, stabilizing agent, tryptophanase, glutamte dehydrogenase.

Reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, the position of glutamte dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.

No matter be single agent, two agent or three doses, the present invention measures the method for potassium concn, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.

Embodiment

The present invention is further illustrated below in conjunction with examples of implementation.

Embodiment one

The potassium diagnosing/determining reagent of present embodiment is single reagent, comprising:

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Tryptophane 20mmol/L

2-oxoglutaric acid ester 15mmol/L

Tryptophanase 12000U/L

Glutamte dehydrogenase 20000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.

On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested potassium sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay is about 2 minutes detection times.

After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of potassium.

Embodiment two

The potassium diagnosing/determining reagent of present embodiment is double reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme is sent out 0.25mmol/L

Tryptophane 12mmol/L

2-oxoglutaric acid ester 12mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Tryptophanase 8000U/L

Glutamte dehydrogenase 24000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.

On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested potassium sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay is about 2 minutes detection times.

After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of potassium.

Embodiment three

The potassium diagnosing/determining reagent of present embodiment is three reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Tryptophane 12mmol/L

2-oxoglutaric acid ester 18mmol/L

Reagent 3

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Tryptophanase 12000U/L

Glutamte dehydrogenase 40000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.

When measuring potassium concn, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested potassium sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 1 minute of time delay is about 2 minutes detection times.

After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of potassium.

The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.

In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.

Claims (6)

1. method for measuring kalium concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Tryptophane+water TryptophanasePyruvic acid+ammonium ion+indoles

Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme Glutamte dehydrogenase

L-glutamic acid+water+coenzyme

The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the concentration measurement result of potassium.

2. potassium diagnosis/determination kit, principal ingredient comprises:

Damping fluid 20-500mmol/L

Stabilizing agent 1-50mmol/L

Reduced coenzyme 0.1-0.35mmol/L

Tryptophane 1-50mmol/L

2-oxoglutaric acid ester 1-50mmol/L

Tryptophanase 1000-80000U/L

Glutamte dehydrogenase 1000-80000U/L

It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.

3. according to the described potassium diagnosis/determination kit of claim 2, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, glutamte dehydrogenase.

4. according to the described potassium diagnosis/determination kit of claim 2, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, glutamte dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester; Reagent 2 is made up of damping fluid, stabilizing agent, tryptophanase, glutamte dehydrogenase.Reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, the position of glutamte dehydrogenase in reagent 1 or reagent 2 can not limit.

5. according to the described potassium diagnosis/determination kit of claim 2, it is characterized in that: form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, glutamte dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, tryptophane, 2-oxoglutaric acid ester; Reagent 3 is made up of damping fluid, stabilizing agent, tryptophanase, glutamte dehydrogenase.Reduced coenzyme, tryptophane, 2-oxoglutaric acid ester, tryptophanase, the position of glutamte dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described potassium diagnosis/determination kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.