CN117805044A - Blood ammonia measuring method - Google Patents
Blood ammonia measuring method Download PDFInfo
- Publication number
- CN117805044A CN117805044A CN202410182921.8A CN202410182921A CN117805044A CN 117805044 A CN117805044 A CN 117805044A CN 202410182921 A CN202410182921 A CN 202410182921A CN 117805044 A CN117805044 A CN 117805044A
- Authority
- CN
- China
- Prior art keywords
- reagent
- ammonia
- gamma
- lactate dehydrogenase
- nadh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 229910021529 ammonia Inorganic materials 0.000 title claims abstract description 52
- 210000004369 blood Anatomy 0.000 title claims abstract description 27
- 239000008280 blood Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 37
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims abstract description 31
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims abstract description 29
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims abstract description 29
- 210000002381 plasma Anatomy 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 claims abstract description 20
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 20
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 19
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims abstract description 13
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims abstract description 13
- 238000002835 absorbance Methods 0.000 claims abstract description 13
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 claims abstract description 11
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 claims abstract description 11
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 11
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 claims abstract description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 9
- NTFPDEDRMYYPAC-UHFFFAOYSA-N 2-amino-4-[[3-(carboxymethyl)phenoxy]-methoxyphosphoryl]butanoic acid Chemical compound OC(=O)C(N)CCP(=O)(OC)OC1=CC=CC(CC(O)=O)=C1 NTFPDEDRMYYPAC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007983 Tris buffer Substances 0.000 claims abstract description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229930195712 glutamate Natural products 0.000 claims abstract description 7
- 229940124186 Dehydrogenase inhibitor Drugs 0.000 claims abstract description 6
- 229940122503 Glutamyltranspeptidase inhibitor Drugs 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims abstract description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims abstract description 4
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 4
- 239000004220 glutamic acid Substances 0.000 claims abstract description 4
- 230000002008 hemorrhagic effect Effects 0.000 claims abstract description 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims description 8
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims description 8
- 229940009662 edetate Drugs 0.000 claims description 6
- 239000003755 preservative agent Substances 0.000 claims description 6
- 230000002335 preservative effect Effects 0.000 claims description 6
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 229910001424 calcium ion Inorganic materials 0.000 claims description 2
- 230000009088 enzymatic function Effects 0.000 claims description 2
- 239000002516 radical scavenger Substances 0.000 claims description 2
- 101000833181 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Glycerol dehydrogenase 1 Proteins 0.000 claims 2
- 230000008030 elimination Effects 0.000 claims 1
- 238000003379 elimination reaction Methods 0.000 claims 1
- SOEVVANXSDKPIY-UHFFFAOYSA-M sodium glyoxylate Chemical compound [Na+].[O-]C(=O)C=O SOEVVANXSDKPIY-UHFFFAOYSA-M 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000009877 rendering Methods 0.000 abstract 1
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical group CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 16
- 239000003112 inhibitor Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 229940107700 pyruvic acid Drugs 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 238000005259 measurement Methods 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- KKVFPVZIZAJCBZ-UHFFFAOYSA-N (2-hydroxyphenyl) hypochlorite Chemical compound OC1=CC=CC=C1OCl KKVFPVZIZAJCBZ-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004958 brain cell Anatomy 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 208000007386 hepatic encephalopathy Diseases 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- SOWBFZRMHSNYGE-UHFFFAOYSA-N oxamic acid Chemical compound NC(=O)C(O)=O SOWBFZRMHSNYGE-UHFFFAOYSA-N 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000022558 protein metabolic process Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 206010010075 Coma hepatic Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101000926208 Homo sapiens Inactive glutathione hydrolase 2 Proteins 0.000 description 1
- 206010020575 Hyperammonaemia Diseases 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- 101710141452 Major surface glycoprotein G Proteins 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- RFUGMORVSDZBLC-UHFFFAOYSA-N [Na].CC(N)=O Chemical compound [Na].CC(N)=O RFUGMORVSDZBLC-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 201000001059 hepatic coma Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 102000012194 human gamma-glutamyltransferase Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000013038 irreversible inhibitor Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- CMPGARWFYBADJI-UHFFFAOYSA-L tungstic acid Chemical compound O[W](O)(=O)=O CMPGARWFYBADJI-UHFFFAOYSA-L 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of blood detection, in particular to a blood ammonia determination method for eliminating interference of lactate dehydrogenase and gamma-glutamyl transpeptidase in blood plasma, which comprises the following steps: the reagent I Tris buffer solution contains a lactate dehydrogenase inhibitor, a gamma-glutamyl transpeptidase inhibitor, EDTA, alpha-ketoglutarate and NADH; reagent II contains only glutamate dehydrogenase and ADP, and when plasma is added into reagent I, the reagent II contains H 2 NCOCOO ‑ Inhibiting LDH activity, rendering it incapable of consuming NADH, and GGsTop inhibits gamma-glutamyltransferase from catalyzing glutamate to generate NH 3 When reagent II is added, ammonia in blood plasma is in the presence of sufficient alpha-ketoglutarate and NADH to produce glutamic acid and NAD by glutamate dehydrogenase + The instrument detects at 340nm wavelength, and calculates the content of hemorrhagic ammonia by taking absorbance generated by the reaction of the reagent I as a blank and absorbance generated by the reaction of the reagent II.
Description
Technical Field
The invention relates to the technical field of blood detection, in particular to a blood ammonia determination method for eliminating interference of lactate dehydrogenase and gamma-glutamyl transferase in blood plasma.
Background
Ammonia is a substance harmful to human body, liver is the main metabolism place, normal human blood contains trace free ammonia, endogenous ammonia is decomposed by deamination of amino acid generated in the process of protein metabolism in human body, and is the main source of ammonia in blood. Exogenous ammonia is produced by bacterial decomposition of proteinaceous foods in the intestinal tract. Ammonia in brain and kidney acts on glutamate to form glutamine, which is transported to liver, where it is converted to urea or other nitrogen-containing compounds, which are then excreted by kidney or ammonium salts are formed and excreted with urine. The increase of blood ammonia sources and decrease of blood routes can cause blood ammonia increase, which can affect the functions of neurons and the metabolism of nerve cells, and the blood ammonia increase is common in hepatic encephalopathy, hepatic coma, liver cirrhosis, some congenital hyperammonemia and the like. When a liver disease is serious, ammonia cannot be cleared from circulation to cause the rise of blood ammonia concentration, excessive ammonia can cause the energy metabolism disorder of brain cells, the energy supply of brain tissues influences the generation and balance of central neurotransmitters, and when the liver disease is serious, the brain cells are poisoned, so that the blood ammonia detection is emphasized, the treatment measures are timely taken, the life quality of a patient can be improved, the death rate of the patient is reduced, and the liver disease has important clinical significance.
The method for detecting the blood ammonia in the clinical laboratory comprises the following steps: (1) direct chromogenic assay: 2ml venous blood was taken at the bedside, after precipitation of the protein with tungstic acid, color development with phenol-hypochlorite was performed and ammonia content was calculated with reference to the standard. (2) glutamate dehydrogenase (GLDH) method: ammonia in plasma is present in sufficient amounts of alpha-ketoglutarate and NADH to produce glutamate by glutamate dehydrogenase and to consume NADH, which is produced in direct proportion to the ammonia concentration in the plasma. (3) ion exchange method: ammonia in deproteinized plasma samples is adsorbed by cation exchange resin, then separated, and the ammonia content is determined by phenol-hypochlorite color reaction. (4) Ammonia electrode method: the ammonia electrode method has high selectivity, so the method has good specificity and high accuracy, but has poor durability, and the stability of the electrode is influenced by various factors such as temperature, osmotic pressure, intermediate fluid and the like. The common measurement method in clinical laboratories is the glutamate dehydrogenase method, and the method has the following problems in the report of the 4 th edition of the national clinical examination protocol: lactate Dehydrogenase (LDH) in plasma can also utilize NADH; high levels of gamma-glutamyl transferase lead to NH production by glutamate decomposition 3 Diseases that affect the accuracy of plasma ammonia measurements, especially elevated lactate dehydrogenase, gamma-glutamyl transferase, severely affect blood ammonia measurements.
Gamma-glutamyl transferase (GGT) is a membrane-bound glycoprotein and the molecular weight of human GGT is 76-87kD. Its main function is to participate in protein metabolism in vivo and is mainly distributed in the liver, kidney, pancreas and other parenchymal organs. GGT in serum of normal people mainly comes from liver, and GGT in liver is mainly limitedIn the capillary bile duct and hepatocyte microsomes, GGT plays an important role in the metabolism of plasma Glutathione (GSH) and its S-conjugates, cleaving the gamma-glutamine bonds by hydrolysis and/or peptide transfer. GGsTop is a novel, highly selective and irreversible gamma-glutamyl transpeptidase (GGT) inhibitor having Km value of 0.17mM, molecular weight of 331.26, molecular formula of C 13 H 18 N0 7 P, the structural formula is (S) -2-amino-4- ((S) - (3- (carboxymethyl) phenoxy) (methoxy) phosphorylated butyric acid), and the solubility is more than 10mM.
The substrate for lactate dehydrogenase is pyruvic acid (formula CH 3 COCOOH) and NADH double substrate, H 2 NCOCOOH is-H 2 N-group replaces-CH in pyruvic acid molecule 3 The chemical name is the ethanamic acid or the oxamic acid, which is the molecular piece-CH in the substrate pyruvic acid 3 Equivalent molecular fragment-NH 2 Substituted by occurrence of a slave-CH 3 The change to-NH 2 breaks down the pyruvate-induced fit and thus the subsequent inhibition is caused, the charge distribution of the substrate and the inhibitor is greatly changed, but at the same time, the great change in structure is not brought, the great change in charge distribution and the great structural consistency lead to the inhibitor entering the enzyme active center more easily than the substrate to be combined with the enzyme, after the inhibitor enters the active center, the partial structure of the active center of the enzyme is changed to a certain extent through the induced fit to the enzyme, the influence on the reaction is very great, the stable conformation of the inhibitor and the substrate is very similar in structure, the lactic dehydrogenase can not effectively recognize the substrate, the advantage of net charge brought by each atom of the inhibitor leads to the inhibitor to be combined with the enzyme active center more easily, the space of the enzyme active center is reduced through the induced fit to the enzyme, therefore, the acetamidate inhibits the action of lactic dehydrogenase on oxalate, and experiments of White et al prove that HN-CO-COO - Is an effective competitive inhibitor of LDH and produces LDH.H 2 N-CO-COO - NADH ternary complex crystals.
In the prior art, in order to eliminate the interference of endogenous lactate dehydrogenase on blood ammonia measurement, the Biochemical share of Beijing Lidammann is limitedThe company, shanghai Fuxing Chang Xie company etc. mostly adopts the addition of high concentration LDH into reagent I to make LDH catalyze pyruvic acid and NADH reaction in the first step reaction, after adding reagent II, ammonia in blood plasma can produce glutamic acid and NAD under the action of glutamate dehydrogenase in the presence of sufficient quantity of alpha-ketoglutarate and NADH + And NADH is consumed, the instrument detects at 340nm wavelength, and calculates the content of hemorrhagic ammonia by taking absorbance generated by the reaction of the reagent I as a blank and absorbance generated by the reaction of the reagent II.
Disclosure of Invention
The invention aims to provide a blood ammonia determination method for eliminating interference of lactate dehydrogenase and gamma-glutamyl transferase in blood plasma, wherein the lactate dehydrogenase takes pyruvic acid and NADH as substrates, and H is added into a kit 2 NCOCOONa，H 2 NCOCOO - is-H 2 N-group replaces-CH in pyruvic acid molecule 3 The method has the advantages that the matching effect of the lactate dehydrogenase and the pyruvic acid is destroyed, the partial structure of the active center of the enzyme is changed to a certain extent, the advantage of the net charge carried by each atom of the inhibitor enables the inhibitor to be combined with the active center of the enzyme more easily, so that the lactate dehydrogenase can not effectively identify the pyruvic acid, and the interference reaction of the lactate dehydrogenase is inhibited; GGsTop is a novel, highly selective and irreversible inhibitor of gamma-glutamyl transpeptidase (GGT) capable of inhibiting gamma-glutamyl transferase from catalyzing glutamate to produce NH 3 Without affecting the accuracy of the plasma ammonia assay results.
The technical scheme of the invention is as follows: the reagent I Tris buffer solution contains a lactate dehydrogenase inhibitor, a gamma-glutamyl transpeptidase inhibitor, EDTA, alpha-ketoglutarate and NADH; reagent II contains only glutamate dehydrogenase and ADP, and when plasma is added into reagent I, the reagent II contains H 2 NCOCOO - Inhibiting LDH activity to make it incapable of consuming NADH, GGsTop inhibiting gamma-glutamyltransferase to catalyze glutamate to generate NH 3 When reagent II is added, ammonia in blood plasma is in the presence of sufficient alpha-ketoglutarate and NADH to produce glutamic acid and NAD by glutamate dehydrogenase + Detecting at 340nm wavelength by the instrument, calculating hemorrhagic ammonia content by using absorbance generated by the reaction of the reagent I as blank and absorbance generated by the reaction of the reagent II,the interference reaction of lactic dehydrogenase and gamma-glutamyl transpeptidase is eliminated, and the following formulas are an ammonia determination reaction equation (1), a lactic dehydrogenation interference reaction equation (2) and a gamma-glutamyl transpeptidase interference reaction (3).
Description of the drawings:
FIG. 1 is a real-time reaction diagram of a hemoammonoassay.
The specific embodiment is as follows:
example 1
1. Blood ammonia assay kit configuration: reagent I: tris buffer solution 50mmol/L pH 8.0 contains 16.0mmol/L alpha-ketoglutarate, 0.16mmol/L NADH, H 2 NCOCOONa 0.4mmol/L, GGsTop0.5mmol/L, EDTA 2.0mmol/L, proclin-300 preservative 200. Mu.L; reagent II: tris buffer 50mmol/L containing GLDH at pH 8.0>20.0KU/L, ADP 0.5mmol/L, proclin-300 preservative 200. Mu.L.
Wherein sodium acetamidate (H) 2 Ncoona) is a lactate dehydrogenase inhibitor, GGsTop is a gamma-glutamyl transpeptidase inhibitor, disodium Edetate (EDTA) is a calcium ion scavenger, proclin-300 is a high-efficiency preservative, adenosine Diphosphate (ADP) is an activator of GLDH, and can stabilize the conformational function of enzymes.
Example 2
Taking Beckmann AU5800 full-automatic biochemical analyzer as an example, the measurement parameters of the invention are as follows:
OD ammonia ＝OD 2 -OD 1 ×[(SV+R 1 V 1 )/(SV+R 1 V 1 +R 2 V 2 )]
Ammonia concentration = F x OD Ammonia
Wherein OD Ammonia Is the absorbance, OD, actually generated in the ammonia measurement reaction 1 Is the absorbance, OD, measured after the reaction of the sample with the reagent I 2 The absorbance measured after the addition of reagent II, SV is the volume of plasma, R 1 V 1 Is the volume of reagent I, R 2 V 2 Is the volume of reagent II. F is a correction factor, which is a negative reaction, and the absorbance OD generated by ammonia Ammonia All negative values.
Example 3
The experimental performance parameters of the invention are as follows: the normal reference value is less than or equal to 15 mu mol/L; blank absorbance>0.55ABS (0.5 cm optical path, 340nm,37 ℃ C.), linear: 0.21-50 mu mol/L, and precision in batch<8, between batches<15 percent, and the recovery rate is between 95 and 105 percent. Lactate dehydrogenase<1600U/L and gamma-glutamyl transpeptidase<800U/L no obvious measurement interference, and correlation analysis with Beijing Lidammann Biochemical Co., ltd: y is Y The invention is that ＝1.021X Lidemann -0.1535；R 2 ＝0.9910。
Claims (5)
1. A blood ammonia determination method for eliminating interference of lactate dehydrogenase and gamma-glutamyl transferase in blood plasma comprises the following technical scheme: the reagent I Tris buffer solution contains a lactate dehydrogenase inhibitor, a gamma-glutamyl transpeptidase inhibitor, EDTA, alpha-ketoglutarate and NADH; reagent II contains only glutamate dehydrogenase and ADP, and when blood plasma is added into reagent I, lactate dehydrogenase inhibitor inhibits LDH activity, so that NADH cannot be consumed; GGsTop inhibits gamma-glutamyl transferase from catalyzing glutamate to generate NH 3 When reagent II is added, ammonia in blood plasma is in the presence of sufficient alpha-ketoglutarate and NADH to produce glutamic acid and NAD by glutamate dehydrogenase + The instrument detects at 340nm wavelength, takes absorbance generated by the reaction of the reagent I as blank, and takes absorbance generated by the reaction of the reagent II as meterCalculating the content of hemorrhagic ammonia.
2. A method of assaying blood ammonia for the elimination of interference from lactate dehydrogenase and γ -glutamyl transferase in plasma according to claim 1, characterized by the fact that the kit of assaying blood ammonia comprises kit reagent I: tris buffer solution reagent 50mmol/LpH 8.0.0 contains alpha-ketoglutarate 12.0-24.0 mmol/L, NADH 0.12-0.20 mmol/L, H 2 NCOCOONa 0.2-0.6 mmol/L, GGsTop 0.1-0.9 mmol/L, EDTA 1.0-3.0 mmol/L, proclin-300 preservative 100-300 μL; reagent II: tris buffer 50mmol/L containing GLDH at pH 8.0>20.0KU/L, 0.3-0.7 mmol/L of ADP and 100-300 mu L of Proclin-300 preservative.
3. The method for assaying blood ammonia for eliminating interference of lactate dehydrogenase and gamma-glutamyltransferase in blood plasma according to claim 2, wherein the method for assaying blood ammonia is characterized by comprising the steps of (1) measuring the concentration of sodium glyoxylate (H 2 Ncoona) is a lactate dehydrogenase inhibitor, GGsTop is a gamma-glutamyl transpeptidase inhibitor, disodium Edetate (EDTA) is a calcium ion scavenger, proclin-300 is a high-efficiency preservative, adenosine Diphosphate (ADP) is an activator of GLDH, and can stabilize the conformational function of enzymes.
4. A method of assaying blood ammonia to eliminate interference of lactate dehydrogenase and gamma-glutamyl transferase in plasma according to claim 2, wherein the pH of Tris buffer in reagent I and reagent II is 7.8±8.2.
5. The method for assaying blood ammonia for eliminating interference of lactate dehydrogenase and gamma-glutamyl transferase according to claim 2, wherein the volume ratio of the sample to the reagent I to the reagent ii=1:5 to 15:1.5 to 2.5 is determined.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410182921.8A CN117805044A (en) | 2024-02-19 | 2024-02-19 | Blood ammonia measuring method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410182921.8A CN117805044A (en) | 2024-02-19 | 2024-02-19 | Blood ammonia measuring method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117805044A true CN117805044A (en) | 2024-04-02 |
Family
ID=90432164
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410182921.8A Pending CN117805044A (en) | 2024-02-19 | 2024-02-19 | Blood ammonia measuring method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117805044A (en) |
-
2024
- 2024-02-19 CN CN202410182921.8A patent/CN117805044A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0034213B1 (en) | A stabilized aqueous coenzyme solution for use in a clinical assay, a method of stabilizing a labile coenzyme in an aqueous clinical assay solution and a kit for use in said clinical assay | |
| US3929581A (en) | Quantitative determination of blood ammonia | |
| Ishihara et al. | Enzymatic determination of ammonia in blood plasma | |
| US5780256A (en) | Method and composition for quantitative determination of ammonia, α-amino acid, or α-keto acid | |
| Pinder et al. | [102] The assay of intermediates and enzymes involved in the synthesis of the nicotinamide nucleotides in mammalian tissues | |
| CN102154444B (en) | Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit | |
| CN117805044A (en) | Blood ammonia measuring method | |
| Bruce et al. | Two-point determination of plasma ammonia with the centrifugal analyzer. | |
| EP0207493B1 (en) | Method of terminating isocitrate dehydrogenase reaction | |
| JPH0675515B2 (en) | Method for quantifying biological substances using ammonia as reaction product | |
| US5888828A (en) | Kit for measuring urea nitrogen | |
| US4575488A (en) | Interference free transaminase assay | |
| JP2000253898A (en) | Quantitative determination of substance or enzyme, and quantitative determining reagent | |
| JPH0773517B2 (en) | Method for quantifying creatine kinase | |
| CN111321199B (en) | Glutamic acid concentration determination reagent | |
| CN115201134B (en) | Creatinine detection kit resistant to piceatannol interference and application thereof | |
| JPH05111396A (en) | Method for measuring activity of creatine kinase and reagent used therefor | |
| CN100359008C (en) | Reagent for determining analyte concentration of patient by enzyme method | |
| JPH047200B2 (en) | ||
| Racek et al. | The use of N-ethylmaleimide in the calorimetric assay of activated creatine kinase with a tetrazolium | |
| JPH02255098A (en) | Method for determining guanidinoacetic acid | |
| CN118006725A (en) | Creatinine detection kit and method | |
| CN115420694A (en) | Glutamate dehydrogenase determination kit | |
| JPH0573400B2 (en) | ||
| JPS63233799A (en) | Method for measuring pyruvate kinase and reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |