CN1769481A - Blood ammonia content determination method and blood ammonia diagnosis kit - Google Patents
Blood ammonia content determination method and blood ammonia diagnosis kit Download PDFInfo
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- CN1769481A CN1769481A CN 200410065296 CN200410065296A CN1769481A CN 1769481 A CN1769481 A CN 1769481A CN 200410065296 CN200410065296 CN 200410065296 CN 200410065296 A CN200410065296 A CN 200410065296A CN 1769481 A CN1769481 A CN 1769481A
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Abstract
The invention relates to a method for determining the content of blood ammonia, and also the reagent kit for blood ammonia diagnosis. The reagent kit comprises cushioning solution, adenosine triphosphate, phosphoenolpyruvate phosphatase, deacidized type coenzyme, ammonia kinase, pyruvate kinase, lactate dehydrogenase, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the blood ammonia content can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.
Description
Technical field
The present invention relates to a kind of method of measuring blood ammonia content, the invention still further relates to the blood ammonia diagnostic kit simultaneously, belong to medical test determination techniques field.
Background technology
The ammonia method for measuring has microdiffusion, ion exchange method, enzyme process and ammonia electrode method etc.Use at present maximum methods and be enzyme process and based on the determination of blood ammonia instrument analytical method of ion selective electrode.
Diffusion process discharges NH after being the sample alkalization
3, the ammonia that discharges with acidometric titration, or form the two mercury amine of pale brown look iodate with the Nessler reaction and carry out colorimetric.These methods need alkalization, and endogenic ammonia forms and impacts, and its accuracy and precision are affected, and seldom use at present; Ion exchange method is more accurate than diffusion process, and CV is 8%--13%; Ion selective electrode method is to utilize NH
3Be diffused into electrode surface, the PH that causes electrode changes and measures, and the CV of this method is 3.5%--4.8%, rate of recovery height, but all need special instrument, be not suitable for the concrete reality of China.
The retrieval Chinese patent is only found No. 87105593.7 patent applications and is disclosed a kind of rapid freezing cup for blood ammonia determination, does not but find more satisfactory determination of blood ammonia method.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of blood ammonia Determination on content method that can overcome above prior art shortcoming, provide simultaneously in order to realize the blood ammonia diagnostic kit of the inventive method.Adopt the reagent in this test kit not only can measure, and finding speed is fast, accuracy is high, thereby can obtains practical applying at ultraviolet analyser or half, the enterprising promoting circulation of blood ammonia content of automatic clinical chemistry analyzer.
It is as follows that the present invention measures the method steps of blood ammonia content:
1), with sample and the reagent mix of mainly forming by adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, ammonia kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate+pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
2), detect the end reaction thing in predominant wavelength 340nm absorbancy decline scope, calculate the content of blood ammonia.
Common step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the degree that predominant wavelength 340nm absorbancy descends, calculate the content of blood ammonia.
The blending ratio of above sample and reagent is by volume 1/10 to 1/100, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.
This method is used ammonia kinase (EC 2.7.3.8) coupling pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.Ammonia is by the effect of ammonia kinase, adenosine triphosphate is become adenosine diphosphate (ADP), adenosine diphosphate (ADP) and phosphoenolpyruvic acid produce pyruvic acid under the effect of pyruvate kinase, effect by the coupling serum lactic dehydrogenase again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate the active size of content of ammonia.
The blood ammonia diagnostic kit of realizing the inventive method can be single agent, comprising:
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent following pair of agent be can be made into, inside and outside source adenosine diphosphate (ADP), pyruvic acid pollution more helped eliminating:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Ammonia kinase 500--50000U/l
Stablizer 10--80% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, pyruvate kinase, serum lactic dehydrogenase etc. can be placed on reagent II, reagent II composition wherein, ammonia kinase also can be placed on reagent I, so can form multiple formulations, not describe in detail one by one at this.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source adenosine diphosphate (ADP), pyruvic acid pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Stablizer 10--50mmo/l
Reagent II
Damping fluid 40--200mmol/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Ammonia kinase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, pyruvate kinase, serum lactic dehydrogenase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, ammonia kinase also can be placed among reagent I or the reagent II, so can form multiple formulations, do not describe in detail one by one.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
Above reduced coenzyme can be reduced form nicotinamide coenzyme or derivatives thereofs such as NADPH, NADH or thio-NADH.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10～80% or 10～50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine or the salt etc.
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 blood ammonia diagnostic kit of the present invention of following system component relation is comparatively desirable:
Damping fluid 80--120mmol/l
Adenosine triphosphate 2--8mmol/l
Phosphoenolpyruvic acid 2--8mmol/l
Reduced coenzyme 0.2--0.3mmo/l
Ammonia kinase 4000--10000U/l
Pyruvate kinase 1000--10000U/l
Serum lactic dehydrogenase 5000--20000U/l
Stablizer 20--50% (cumulative volume)
Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.
In addition, one of striking features of the present invention is the pollution that can eliminate inside and outside source adenosine diphosphate (ADP), pyruvic acid, the effect of eliminating inside and outside source adenosine diphosphate (ADP), pyruvic acid occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source adenosine diphosphate (ADP), pyruvic acid, and all be the content that results from blood ammonia at the needed adenosine diphosphate (ADP) of second half section time test blood ammonia content, pyruvic acid.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The blood ammonia diagnostic kit of present embodiment comprises:
Damping fluid 80mmol/l
Adenosine triphosphate 2mmol/l
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.2mmo/l
Ammonia kinase 4000U/l
Pyruvate kinase 1000U/l
Serum lactic dehydrogenase 5000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 1/25, the Direction of Reaction is negative reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate+pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect predominant wavelength 340nm absorbancy decline scope, thereby calculate the content of blood ammonia.
Present embodiment is used ammonia kinase coupling pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.Ammonia is by the effect of ammonia kinase, adenosine triphosphate is become adenosine diphosphate (ADP), adenosine diphosphate (ADP) and phosphoenolpyruvic acid produce pyruvic acid under the effect of pyruvate kinase, effect by the coupling serum lactic dehydrogenase again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate the active size of content of ammonia.
Embodiment two (two agent)
The blood ammonia diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Adenosine triphosphate 5mmol/l
Phosphoenolpyruvic acid 5mmol/l
Reduced coenzyme 0.25mmo/l
Pyruvate kinase 6000U/l
Serum lactic dehydrogenase 12000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Ammonia kinase 7000U/l
Stablizer 50% (cumulative volume)
When measuring blood ammonia content, temperature is controlled at 30 ℃, 15 minutes reaction times, and test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 1/25, the Direction of Reaction is negative reaction.
Concrete determination step is:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate+pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect predominant wavelength 340nm absorbancy decline scope, thereby calculate the content of blood ammonia.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The blood ammonia diagnostic reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
Adenosine triphosphate 8mmol/l
Phosphoenolpyruvic acid 8mmol/l
Reduced coenzyme 0.3mmo/l
Stablizer 20mmo/l
Reagent II
Damping fluid 120mmol/l
Pyruvate kinase 10000U/l
Serum lactic dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Ammonia kinase 10000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 1/25, the Direction of Reaction is negative reaction.
Concrete determination step is:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate+pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect predominant wavelength 340nm absorbancy decline scope, thereby calculate the content of blood ammonia.
The reaction times of each reactions steps was controlled at 20 minutes.
Embodiment four
The blood ammonia diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.3mmo/l
Pyruvate kinase 10000U/l
Serum lactic dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine triphosphate 3mmol/l
Ammonia kinase 8000U/l
Stablizer 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 1/25, the Direction of Reaction is negative reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate+pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect predominant wavelength 340nm absorbancy decline scope, thereby calculate the content of blood ammonia.
In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.
Claims (9)
1. method of measuring blood ammonia content, step is as follows:
1), with sample and the reagent mix of mainly forming by adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, ammonia kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic acid+oxidized coenzyme
2), detect the end reaction thing in predominant wavelength 340nm absorbancy decline scope, calculate the content of blood ammonia.
2. according to the method for the described mensuration blood ammonia of claim 1 content, it is characterized in that: described step 2) for the end reaction thing being placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbancy descends, calculate the content of blood ammonia.
3, according to the method for claim 1 or 2 described mensuration blood ammonia content, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.
4, according to the method for claim 1 or 2 described mensuration blood ammonia content, it is characterized in that: the ratio control of sample and reagent is 1/10 to 1/100.
5. blood ammonia diagnostic kit is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
6. according to the described blood ammonia diagnostic kit of claim 2, it is characterized in that: described reagent is made into single agent, two agent or three doses.
7. according to claim 5 or 6 described blood ammonia diagnostic kits, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate or the salt.
8. according to claim 5 or 6 described blood ammonia diagnostic kits, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, phosphate buffered saline buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
9, according to claim 5 or 6 described blood ammonia diagnostic kits, it is characterized in that: described reduced coenzyme is a kind of in NADPH, NADH or the thio-NADH reduced form nicotinamide coenzyme or derivatives thereof.
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