CN1778937A - Determination of blood ammonia content and blood ammonia diagnostic reagent kit - Google Patents
Determination of blood ammonia content and blood ammonia diagnostic reagent kit Download PDFInfo
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- CN1778937A CN1778937A CN 200410065557 CN200410065557A CN1778937A CN 1778937 A CN1778937 A CN 1778937A CN 200410065557 CN200410065557 CN 200410065557 CN 200410065557 A CN200410065557 A CN 200410065557A CN 1778937 A CN1778937 A CN 1778937A
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Abstract
The invention is about a method of measuring the content of blood ammonia, and it also concerns the reagent box of blood ammonia diagnosis. This invention belongs to the field of medical testing and measuring technology. The reagent box is consisted of buffer solution, glutamate, 2-ketoglutarate, reduced coenzyme, adenosine triphosphate, glutamine synthetase, glutamate synthetase and stabilizer. Firstly, we cause an enzyme-coupled reaction through mixing the sample and the reagent according to a certain proportion of volume; secondly, put the final reactant under the biochemical analyzer and test the absorbance variational situation (speed) of dominant wavelength; then we can get the content of blood ammonia. By using this invention, we can get the necessary measuring result with high sensitiveness and fine precision through biochemical analyzer, and the result would not be contaminated by material of internal and exogenous sources. Thus, this method can be conveniently promoted and applied.
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 L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, glutamine synthetase, NADPH-linked glutamate synthase, make it to take place the reaction of following principle:
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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 1/250, 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 glutamine synthetase coupling NADPH-linked glutamate synthase reaction continuous monitoring method.Ammonia is by the effect of coupling glutamine synthetase, generate glutamine with adenosine triphosphate and L-glutamic acid, glutamine is the effect by the coupling NADPH-linked glutamate synthase 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 adenosine deaminase.
Be used to realize that the blood ammonia diagnostic kit of the inventive method can be single agent, comprise:
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
20-ALPHA-KG 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent:
Reagent I
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Glutamine synthetase 1000--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, L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, NADPH-linked glutamate synthase etc. can be placed on reagent II, reagent II composition wherein, glutamine synthetase 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, more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Stablizer 10--50mmol/l
Reagent II
Damping fluid 40--200mmol/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Glutamine synthetase 1000--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 L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, NADPH-linked glutamate synthase also can be placed among reagent I or the reagent III, and reagent III composition wherein, glutamine synthetase also can be placed among reagent I or the reagent II, so can form multiple formulations, do not describe in detail one by one at this.
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., (for example can also be imidazoles/hydrochloride buffer 6.2-7.8 but be not limited only to these damping fluids; Sodium phosphate dibasic citrate buffer solution 5.0-8.0; Citric acid sodium citrate buffer solution 5.0-6.6; Phosphate buffered saline buffer 6.0-8.0; Sodium phosphate dibasic-potassium phosphate buffer 6.0-8.0; Potassium primary phosphate sodium hydrate buffer solution 6.0-8.0; Veronal sodium-hydrochloride buffer 6.8-9.6; Tris hydrochloride buffer 7.0-9.0; Boric acid borate buffer solution 7.4-9.0; Glycine-sodium hydrate buffer solution 8.6-10.6; Sand-sodium hydrate buffer solution 9.2-10.0; Yellow soda ash-sodium bicarbonate buffer liquid 8.8-10.6 (Ca
2+, Mg
2+Must not use when existing); PBS damping fluid 7.0-7.6 etc.).
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 that keeps reagent, so that standing storage, above single agent, the reagent I of two agent, the reagent I of reagent II or three doses, reagent II, usually add stablizer 10～80% or 10～50mmol/l among the reagent III, stablizer can be an ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, in sulfuric acid amine or the salt etc. one or more can also be at thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glucose, glutaminate, lactose, N.F,USP MANNITOL, sucrose, sodium-chlor, select in the succinate 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
L-glutamic acid 5--10mmol/l
2-oxoglutaric acid ester 5--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Adenosine triphosphate 2--8mmol/l
Glutamine synthetase 4000--10000U/l
NADPH-linked glutamate synthase 4000--10000U/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.
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
L-glutamic acid 5mmol/l
2-oxoglutaric acid ester 5mmol/l
Reduced coenzyme 0.2mmo/l
Adenosine triphosphate 2mmol/l
Glutamine synthetase 4000U/l
NADPH-linked glutamate synthase 4000U/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+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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 glutamine synthetase coupling NADPH-linked glutamate synthase reaction continuous monitoring method.Ammonia is by the effect of coupling glutamine synthetase, generate glutamine with adenosine triphosphate and L-glutamic acid, glutamine is the effect by the coupling NADPH-linked glutamate synthase 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 adenosine deaminase.
Embodiment two (two agent)
The blood ammonia diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
L-glutamic acid 8mmol/l
2-oxoglutaric acid ester 12mmol/l
Reduced coenzyme 0.25mmo/l
Adenosine triphosphate 5mmol/l
NADPH-linked glutamate synthase 7000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Glutamine synthetase 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+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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
L-glutamic acid 10mmol/l
2-oxoglutaric acid ester 20mmol/l
Reduced coenzyme 0.3mmo/l
Adenosine triphosphate 8mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
NADPH-linked glutamate synthase 10000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Glutamine synthetase 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+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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
L-glutamic acid 5mmol/l
2-oxoglutaric acid ester 12mmol/l
Reduced coenzyme 0.3mmo/l
Adenosine triphosphate 2mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 100mmol/l
Glutamine synthetase 10000U/l
NADPH-linked glutamate synthase 10000U/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+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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 L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, glutamine synthetase, NADPH-linked glutamate synthase, make it to take place the reaction of following principle:
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic 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 1/250.
5. blood ammonia diagnostic kit is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 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 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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|---|---|---|---|
| CN 200410065557 CN1778937A (en) | 2004-11-23 | 2004-11-23 | Determination of blood ammonia content and blood ammonia diagnostic reagent kit |
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|---|---|---|---|
| CN 200410065557 CN1778937A (en) | 2004-11-23 | 2004-11-23 | Determination of blood ammonia content and blood ammonia diagnostic reagent kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102298036A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
-
2004
- 2004-11-23 CN CN 200410065557 patent/CN1778937A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102298036A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
| CN113075139B (en) * | 2021-03-29 | 2022-10-11 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
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