CN1749756A - Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit - Google Patents
Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit Download PDFInfo
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- CN1749756A CN1749756A CN 200410041963 CN200410041963A CN1749756A CN 1749756 A CN1749756 A CN 1749756A CN 200410041963 CN200410041963 CN 200410041963 CN 200410041963 A CN200410041963 A CN 200410041963A CN 1749756 A CN1749756 A CN 1749756A
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Abstract
The present invention belongs to the field of medical detection technology. The reagent kit for blood ammonia diagnosis includes buffering solution, adenosine triphophate, glutamic acid, pyruvic acid, alcohol, oxidized coenzyme, glutamine synthetase, pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase and stabilizer. Through mixing the sample and reagent in certain volume ratio to produce enzyme coupling reaction, and detecting in biochemical analyzer the main wavelength absorbency change speed, the blood ammonia content is measured. The present invention can obtain the measurement result in biochemical analyzer in high sensitivity, high precision and no contamination of various foreign and internal matters.
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 process, 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 analytic approach of ion-selective electrode.
Diffusion method 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 process is more accurate than diffusion method, 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%, recovery height.In conjunction with the concrete reality of China, should be practical with the enzymatic assays.
The retrieval Chinese patent is only found No. 87105593.7 patented claims 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 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.
The step that the present invention measures blood ammonia content is as follows:
1), with sample and the reagent mix of mainly forming by adenosine triphosphate, glutamic acid, pyruvic acid, ethanol, oxidized coenzyme, glutamine synthelase, pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbance descends, 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 absorbance descends, calculate the content of blood ammonia.
The blending ratio of above sample and reagent is by volume 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction time was controlled at conventional 2-30 minute.
The present invention uses enzyme coupling reaction systems such as glutamine synthelase coupling pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase, oxidized coenzyme is reduced into reducibility coenzyme the most at last, because reducibility coenzyme has absorption peak at wavelength 340nm, the increase degree of therefore testing wavelength 340nm absorption peak can directly reflect the size of ammonia content.The advantage of this enzyme coupling reaction system has: one, its utilize to measure the size that growing amount that oxidized coenzyme is reduced into reducibility coenzyme reflects ammonia content, the stability of oxidized coenzyme in solution is high more a lot of than reducibility coenzyme, so this system is stable fine.Two, the needs of this enzyme coupling reaction system destroy the phosphate radical that contained originally in body (blood) liquid earlier, otherwise the result understands the not influence of the phosphate radical of equal size in acceptor (blood) liquid.The step of the endogenous phosphate radical of this elimination can be by the allotment double reagent, allows blood sample earlier and in the reagent after second, third and the 4th reaction effect earlier, adds the effect of glutamine synthelase startup ammonia again.
Be used to realize that the blood ammonia diagnostic kit of the inventive method can be single agent, comprise:
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--20mmol/l
Glutamic acid l--30mmol/l
Pyruvic acid 1--30mmol/l
Ethanol l--20mmol/l
Oxidized coenzyme 0.5--5mmol/l
Glutamine synthelase 1000--50000U/l
Pyruvate oxidase 1000--50000U/l
Hydrogen peroxidase 1000--50000U/l
Aldehyde dehydrogenase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent, more help eliminating inside and outside source phosphate radical and pollute:
Reagent I
Damping fluid 40--200mmol/l
Glutamic acid 1--30mmol/l
Pyruvic acid 1--30mmol/l
Ethanol 1--20mmol/l
Oxidized coenzyme 0.5--5mmol/l
Glutamine synthelase 1000--50000U/l
Pyruvate oxidase 1000--50000U/l
Hydrogen peroxidase 1000--50000U/l
Aldehyde dehydrogenase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--20mmol/l
Stabilizing agent 10--50mmol/l
The prescription of two agent is not limited only to above-mentioned prescription, and the composition of reagent I wherein, can be placed on reagent II at glutamic acid, reagent II composition wherein, and adenosine triphosphate also can be placed on reagent I, so can form multiple formulations, does 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 phosphate radical and pollute, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Glutamic acid 1--30mmol/l
Pyruvic acid 1--30mmol/l
Ethanol 1--20mmol/l
Oxidized coenzyme 0.5--5mmol/l
Stabilizing agent 10--8050mmol/l
Reagent II
Damping fluid 40--200mmol/l
Glutamine synthelase 1000--50000U/l
Pyruvate oxidase 1000--50000U/l
Hydrogen peroxidase 1000--50000U/l
Aldehyde dehydrogenase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--20mmol/l
Stabilizing agent 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, glutamic acid can be placed among reagent II or the reagent III, other compositions of reagent I, pyruvic acid, ethanol, oxidized coenzyme can be placed among the reagent II, reagent II composition wherein, glutamine synthelase, pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase etc. also can be placed among the reagent I, the composition of reagent III, adenosine triphosphate can be placed among reagent I or the reagent II, so can form multiple formulations, not describe in detail one by one at this.
The pH scope of buffering agent can be 7.0-11.0.Buffering agent can be three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, triethanolamine (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 oxidized coenzyme can be NADP
+, NAD
+Or thio-NAD
+Deng oxidized form nicotinamide coenzyme or derivatives thereof.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent 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, stabilizing agent can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine, salt or the adenosine diphosphate 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 formula components relation is comparatively desirable:
Damping fluid 80--120mmol/l
Adenosine triphosphate 1--5mmol/l
Glutamic acid 4--10mmol/l
Pyruvic acid 4--10mmol/l
Ethanol 2--8mmol/l
Oxidized coenzyme 1--3mmol/l
Glutamine synthelase 10000--20000U/l
Pyruvate oxidase 10000--20000U/l
Hydrogen peroxidase 10000--20000U/l
Aldehyde dehydrogenase 10000--20000U/l
Stabilizing agent 10--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 operating 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 phosphate radical, the effect of eliminating inside and outside source phosphate radical occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source phosphate radical, and all be the effect that results from blood ammonia at the needed phosphate radical of second half section time test blood ammonia content.
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 1mmol/l
Glutamic acid 4mmo/l
Pyruvic acid 4mmol/l
Ethanol 2mmol/l
Oxidized coenzyme 1mmol/l
Glutamine synthelase 10000U/l
Pyruvate oxidase 10000U/l
Hydrogen peroxidase 10000U/l
Aldehyde dehydrogenase 10000U/l
Stabilizing agent 50% (cumulative volume)
On full-automatic analyser, set: 37 ℃ of temperature, 10 minutes reaction time, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 2/25, the Direction of Reaction is positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the degree that predominant wavelength 340nm absorbance rises, thereby calculate the content of blood ammonia.
Present embodiment is used enzyme coupling reaction systems such as glutamine synthelase coupling pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase, oxidized coenzyme is reduced into reducibility coenzyme the most at last, because reducibility coenzyme has absorption peak at wavelength 340nm, the increase degree of therefore testing wavelength 340nm absorption peak can directly reflect the size of ammonia content.
Embodiment two (two agent)
The blood ammonia diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Glutamic acid 7mmol/l
Pyruvic acid 7mmol/l
Ethanol 5mmol/l
Oxidized coenzyme 2mmol/l
Glutamine synthelase 15000U/l
Pyruvate oxidase 15000U/l
Hydrogen peroxidase 15000U/l
Aldehyde dehydrogenase 15000U/l
Stabilizing agent 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine triphosphate 3mmol/l
Stabilizing agent 10mmol/l
When measuring blood ammonia content, temperature is controlled at 30 ℃, 15 minutes reaction time, 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 positive reaction.
Concrete determination step is:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the degree that predominant wavelength 340nm absorbance rises, thereby calculate the content of blood ammonia.
The reaction time 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
Glutamic acid 10mmol/l
Pyruvic acid 10mmol/l
Ethanol 8mmo l/l
Oxidized coenzyme 3mmol/l
Stabilizing agent 50mmol/l
Reagent II
Damping fluid 120mmol/l
Glutamine synthelase 20000U/l
Pyruvate oxidase 20000U/l
Hydrogen peroxidase 20000U/l
Aldehyde dehydrogenase 20000U/l
Stabilizing agent 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine triphosphate 5mmol/l
Stabilizing agent 50mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction time, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested blood ammonia sample and reagent is 1/50, the Direction of Reaction is positive reaction.
Concrete determination step is:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the degree that predominant wavelength 340nm absorbance rises, thereby calculate the content of blood ammonia.
The reaction time 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
Pyruvic acid 5mmol/l
Ethanol 2mmol/l
Oxidized coenzyme 3mmol/l
Glutamine synthelase 20000U/l
Pyruvate oxidase 15000U/l
Hydrogen peroxidase 20000U/l
Aldehyde dehydrogenase 10000U/l
Stabilizing agent 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine triphosphate 5mmol/l
Glutamic acid 8mmol/l
Stabilizing agent 20mmol/l
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, 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 positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the degree that predominant wavelength 340nm absorbance rises, thereby calculate the content of blood ammonia.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, degree of accuracy 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, glutamic acid, pyruvic acid, ethanol, oxidized coenzyme, glutamine synthelase, pyruvate oxidase, hydrogen peroxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Ammonia+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvate oxidaseAcetyl phosphate+
Carbon dioxide+hydrogen peroxide
Hydrogen peroxide+ethanol
Hydrogen peroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbance descends, 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) be: the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance rises, 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 time 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 proportional control of tested blood ammonia 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--20mmol/l
Glutamic acid 1--30mmol/l
Pyruvic acid 1--30mmol/l
Ethanol 1--20mmol/l
Oxidized coenzyme 0.5--5mmol/l
Glutamine synthelase 1000--50000U/l
Pyruvate oxidase 1000--50000U/l
Hydrogen peroxidase 1000--50000U/l
Aldehyde dehydrogenase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
6. according to the described blood ammonia diagnostic kit of claim 5, 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 stabilizing agent is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate, salt or the adenosine diphosphate.
8. according to claim 5 and 6 described blood ammonia diagnostic kits, it is characterized in that: the pH scope of described buffering agent can be 7.0-11.0, and described buffering agent is a kind of in three (ethyloic) aminomethane-hydrochloride buffer, triethanolamine 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 oxidized coenzyme is NADP
+, NAD
+, thio-NAD
+A kind of in the oxidized form nicotinamide coenzyme or derivatives thereof.
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|---|---|---|---|
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|---|---|---|---|
| CN 200410041963 CN1749756A (en) | 2004-09-14 | 2004-09-14 | Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit |
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