CN110346572A - It is coupled the method and assay kit of pyruvate oxidation enzymatic determination homocysteine (Hcy) - Google Patents
It is coupled the method and assay kit of pyruvate oxidation enzymatic determination homocysteine (Hcy) Download PDFInfo
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- CN110346572A CN110346572A CN201810299069.7A CN201810299069A CN110346572A CN 110346572 A CN110346572 A CN 110346572A CN 201810299069 A CN201810299069 A CN 201810299069A CN 110346572 A CN110346572 A CN 110346572A
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- Prior art keywords
- homocysteine
- reagent
- detection kit
- cystathionine
- kit according
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- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000002255 enzymatic effect Effects 0.000 title description 4
- 238000007254 oxidation reaction Methods 0.000 title description 3
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 title description 2
- 238000003149 assay kit Methods 0.000 title description 2
- 230000003647 oxidation Effects 0.000 title description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- ILRYLPWNYFXEMH-UHFFFAOYSA-N D-cystathionine Natural products OC(=O)C(N)CCSCC(N)C(O)=O ILRYLPWNYFXEMH-UHFFFAOYSA-N 0.000 claims abstract description 7
- ILRYLPWNYFXEMH-WHFBIAKZSA-N L-cystathionine Chemical compound [O-]C(=O)[C@@H]([NH3+])CCSC[C@H]([NH3+])C([O-])=O ILRYLPWNYFXEMH-WHFBIAKZSA-N 0.000 claims abstract description 7
- 102100034976 Cystathionine beta-synthase Human genes 0.000 claims abstract description 6
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- YPWSLBHSMIKTPR-UHFFFAOYSA-N Cystathionine Natural products OC(=O)C(N)CCSSCC(N)C(O)=O YPWSLBHSMIKTPR-UHFFFAOYSA-N 0.000 claims abstract description 5
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- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
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- ZYVIIEFODRWQGD-UHFFFAOYSA-N 2-piperazin-1-ylethanol;propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O.OCCN1CCNCC1 ZYVIIEFODRWQGD-UHFFFAOYSA-N 0.000 claims description 2
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 2
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- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
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- SVLRFMQGKVFRTB-UHFFFAOYSA-M sodium;3-(3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].COC1=CC(NCC(O)CS([O-])(=O)=O)=CC(OC)=C1 SVLRFMQGKVFRTB-UHFFFAOYSA-M 0.000 description 1
- HDARHUHTZKLJET-UHFFFAOYSA-M sodium;3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 HDARHUHTZKLJET-UHFFFAOYSA-M 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
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- 239000011715 vitamin B12 Substances 0.000 description 1
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
- G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Immunology (AREA)
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- Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plasma & Fusion (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The present invention is with cystathionine β-synthase and cystathionine beta-catabolic enzyme, coupling pyruvate oxidase, the method for measuring homocysteine.Principle is: with three (2- carboxyethyl) phosphorus hydrogen chloride, oxidized form homocysteine is set to be converted to sequestered homocysteine, L-cystathionine is generated in the catalysis of cystathionine β-synthase and serine reaction, L-cystathionine generates homocysteine, ammonia and pyruvic acid again under the catalysis of cystathionine beta-catabolic enzyme, pyruvic acid generates hydrogen peroxide under the catalysis of pyruvate oxidase, then carries out Trinder reaction solution.And the homocysteine generated in reaction process goes back to participate in reaction, forms a circular response.The present invention can be made into homocysteine detection reagent box simultaneously.
Description
Technical field
The invention belongs to laboratory medicine fields, are a kind of methods and assay kit for measuring homocysteine.
Background technique
Homocysteine is a kind of sulfur-containing amino acid, is important intermediate during Methionine metabolism.Half Guang of homotype
The detection of propylhomoserin, application clinically is mainly as cardiovascular disease, especially coronary atherosclerosis and myocardial infarction
Important indicator, its concentration elevated-levels are directly proportional to the risk of disease.Plasma homocysteine and cardiovascular disease
Property it is closely related, concentration elevated insult coronary artery and other blood vessels finally cause atherosclerosis and other cardiovascular diseases
Disease.Vitamin B6、B12And folic acid, by participating in homocysteine metabolism, plaing a part of to adjust and reducing homocysteine,
Thus the risk of cardiovascular disease is reduced.Homocysteine is the independent hazard factor of cerebrovascular disease, mostly by smoking and drinking
Cause.
There are many method of measurement homocysteine at present, there is high performance liquid chromatography, fluorescent marker method, enzyme linked immunological
Method, enzymic colorimetric etc., most commonly used in Routine Test Lab is enzymic colorimetric.Since homocysteine is in people's in-vivo content
Very low, the method sensitivity used in the past in Routine Test Lab is not high, has invented Enzymatic cycling again in recent years.And Enzymatic cycling
Divide ultraviolet method and Trinder method.
Ultraviolet method, using cystathionine β-synthase and cystathionine beta-catabolic enzyme, utilizes in patent 201410190202.7
The pyruvic acid generated in reaction process is coupled lactic dehydrogenase, so that reduced coenzyme Ⅰ is generated oxidized coenzyme I, in 340nm wave
Under length, the fall off rate by measuring absorbance acquires the content of homocysteine.In patent 200610041372.4, application
Cystathionine β-synthase and cystathionine beta-catabolic enzyme are coupled lactic dehydrogenase using the pyruvic acid and ammonia generated in reaction process
And glutamte dehydrogenase, so that reduced coenzyme Ⅰ is generated oxidized coenzyme I, under 340nm wavelength, by under measurement absorbance
Reduction of speed rate acquires the content of homocysteine.In addition, also useful homocysteine methyltransgerase, half Guang of S- adenyhomotype
Propylhomoserin hydrolase, the ammonia generated in coupled glutamate ldh assay reaction process make reduced coenzyme Ⅰ generate oxidized coenzyme
Ⅰ。
Trinder method, it is same using homocysteine methyltransgerase, S- adenosine in patent 201410195083.4
Type cysteine hydrolases, coupling adenosine deaminase, purine nucleotide phosphorylase, xanthine oxidase and peroxidase,
Carry out Trinder reaction.
Summary of the invention
The present invention provides a kind of coupling pyruvate oxidase, is recycled using the homocysteine of Trinder reaction solution
Enzyme process, and the homocysteine detection reagent box prepared with this method.Specific technical solution is as follows:
In reagent one, original pyruvic acid in sample is decomposed with pyruvate oxidase and peroxidase oxidization, simultaneously
Oxidized form homocysteine is reduced into sequestered, after reagent two is added, cystathionine β-synthase and Guang sulphur in reagent two
Ether β-catabolic enzyme starts to react, and reaction equation is as follows:
The homocysteine that the second step of reaction generates is returned to the first step and participates in reaction, forms a circulation, plays
Sensitivity can be improved in amplification, reduces the coefficient of variation, reproducible, stable reagent.Trinder method itself is than ultraviolet method spirit
Quick, the color developing agent of ultraviolet method is to use Reducing Coenzyme I, because Reducing Coenzyme I is easy to oxidize, the stability of reagent just compares
It is poor.
Agent structure:
Reagent one:
Reagent two:
The ratio of the reagent one and reagent two can be 1/1 to 8/1.
By the composition of reagent, reagent one: two=1~8 ︰ 1 of reagent different concentration ratios, preferred reagent one: reagent can be made into
Two=2~4 ︰ 1.Sample: reagent=1: 50~150, preferably 1 ︰ 100.
Buffer is glycinate, phosphate, trishydroxymethylaminomethane, 2-morpholine ethane sulfonic acid, 3- (N- morpholinyl) -2-
One or both of hydroxy-propanesulfonic acid, 4- hydroxyethyl piperazineethanesulfonic acid, 4- hydroxyethyl piperazine propane sulfonic acid etc..It is preferred that with three hydroxyl first
Base aminomethane, dosage are 20~120mmol/L.Reagent one, reagent two pH of buffer be 6.0~8.0.
Preservative is one of sodium azide, ProClin-300, P-hydroxybenzoic acid and esters etc. or a variety of, still,
It take sodium azide as preferred, what influence no on reaction system, price is also low.
Stabilizer is Bovine serum albumin, amino acids, carbohydrate, and polyalcohols etc. can be applied alone, can also be two kinds or more
Kind or more combination, preferred Bovine serum albumin.Mannitol, trehalose, polyalcohol can be selected in flavin adenine dinucleotide (FAD), carbohydrate
Optional spent glycol, PEG6000 etc..
It can be suitably added surfactant in reaction system, reaction can be accelerated, increase the stability of enzyme, including Tweens,
The nonionic surfactants such as polyethenoxy ether class, particularly preferably polyethenoxy ether class, use scope is in 0.5~10ml/
L, most preferably 0.5~1.5ml/L of selection.
Zymoexciter is Ca2+ or Mg2+.Dosage in 0.05~10mmol/L,
Color developing agent is 4-AA, phenol, phenyl amines.Phenol includes: phenol, 2,4- Dichlorophenol, 4- chlorine
Phenol, P-hydroxybenzoic acid, tribromo hydroxybenzoic acid, 3, the chloro- 2- sodium hydroxybenzenesulfonate of 5- bis- etc., Detection wavelength 500~
520nm；Phenyl amines includes: N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5- dimethoxyaniline sodium salt, N- (2- hydroxyl -3-
Sulfopropyl) -3,5- dimethoxyaniline sodium salt, N- ethyl-N- (the third sulfo group of 2- hydroxyl -3-) meta-aminotoluene, N- ethyl-N- (3-
Sulfopropyl) -3- aminoanisole sodium salt etc., very much, just seldom describe.Detection wavelength is sensitive between 540~600nm
Degree is higher than phenol, it is therefore preferable that using phenyl amines color developing agent.It is common in current biochemistry detection reagent.Use difference
Color developing agent just have corresponding wavelength.
Reducing agent is in three (2- carboxyethyl) phosphonium salt hydrochlorates, dithiothreitol (DTT), two sulphur erythrose alcohol, beta -mercaptoethanol etc.
One or more, dosage is in 0.10~1.00mmol/L, preferably with 0.40~0.6mmol/L.
Detailed description of the invention
Fig. 1 is detection kit testing result linear graph of the present invention；
Fig. 2 is that detection kit of the present invention and outsourcing homocysteine detection reagent box detect clinical comparison figure.
Specific embodiment
Embodiment one
Reagent one:
Reagent two:
TOOS:N- ethyl-N- (the third sulfo group of 2- hydroxyl -3-) meta-aminotoluene
Reagent Yi ︰ reagent two is 2 ︰ 1, and performance rate method, positive reaction, wavelength: 546nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
3.0 ︰, 180 ︰ 90,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the suction of detection 2 minutes
Light varience rate.
Embodiment two
Reagent one
Reagent two:
Reagent Yi ︰ reagent two is 3 ︰ 1, and performance rate method, positive reaction, wavelength: 510nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
2.8 ︰, 210 ︰ 70,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the suction of detection 2 minutes
Light varience rate.
Embodiment three
Reagent one:
Reagent two:
ADPS:N- ethyl-N- (3- sulfopropyl) -3- aminoanisole sodium
Reagent Yi ︰ reagent two is 4 ︰ 1, and performance rate method, positive reaction, wavelength: 546nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
2.6 ︰, 240 ︰ 60,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the suction of detection 2 minutes
Light varience rate.
By taking embodiment three as an example, detection data is as follows:
The linear result of detection kit is
Linear equation: y=0.975x+0.613, R2=0.999.See Figure of description 1.
It detects outsourcing quality-control product (8.7~12.2~15.7mmol/L), repeats detection 20 times, as a result as follows:
SD=0.1598, CV=1.323%.
50 parts are detected with the detection kit (Trinder method) and outsourcing homocysteine detection reagent box (ultraviolet method)
Clinical serum does comparative test, and comparative test result is as follows:
The comparative test of 50 clinical samples
The resulting results relevance of two methods is good, r2=0.999.See Figure of description 2.
Detection of Stability: it detects outsourcing quality-control product (7.8~12.2~15.7 μm of ol/L), monthly standard items, quality-control product weight
New to redissolve, reagent opens again detect once, is repeated detection 10 times every time, is averaged, and 13 totally months.As a result as follows:
Quality-control product definite value are as follows: 1.68~2.10~2.52mmol/L, the reagent validity period formulated according to national regulation, enterprise
Be 12 months, should be more than one month validity period in it is still stable, just meet the requirements.
The above is only a preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that for the art
Those of ordinary skill for, several improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of method for measuring homocysteine, steps are as follows:
1), the reaction of reagent one:
For removing original pyruvic acid in sample；
2) after reagent two, is added, cystathionine β-synthase and cystathionine beta-catabolic enzyme in reagent two start to react, oxidative color-developing
Agent generates red quinone imines, detects absorbance, at a particular wavelength to calculate the content of homocysteine；
3), detection end reaction object calculates the content of homocysteine in wavelength 500~600nm absorbance.
2. measuring the method for homocysteine according to claim 1, it is characterised in that: the ratio of sample and reagent
Control is 1/50 to 1/100.
3. a kind of homocysteine detection kit, it is characterised in that: including reagent one and reagent two；
Reagent one:
Reagent two:
The ratio of the reagent one and reagent two is 1/1 to 8/1.
4. homocysteine detection kit according to claim 3, it is characterised in that: the buffer is glycine
Salt, phosphate, trishydroxymethylaminomethane, 2-morpholine ethane sulfonic acid, 3- (N- morpholinyl) -2- hydroxy-propanesulfonic acid, 4- ethoxy piperazine
One of piperazine ethanesulfonic acid, 4- hydroxyethyl piperazine propane sulfonic acid are a variety of, and the pH of buffer is 6.5~8.5.
5. homocysteine detection kit according to claim 3, it is characterised in that: the preservative is Azide
One of sodium, ProClin-300, P-hydroxybenzoic acid and esters are a variety of.
6. homocysteine detection kit according to claim 3, it is characterised in that: the stabilizer is calf serum
Albumin, amino acid, one of carbohydrate derivative, polyalcohol, flavin adenine dinucleotide (FAD) or a variety of.
7. homocysteine detection kit according to claim 3, it is characterised in that: the surfactant is tween
PEGlike coating ethers or polyethenoxy ether class nonionic surfactant.
8. homocysteine detection kit according to claim 3, it is characterised in that: the zymoexciter is Ca2+Or
Mg2+。
9. homocysteine detection kit according to claim 3, it is characterised in that: the color developing agent is 4- amino peace
For than woods, phenol, phenyl amines.
10. homocysteine detection kit according to claim 3, it is characterised in that: the reducing agent is three (2- carboxylics
Ethyl) microcosmic salt hydrochlorate, dithiothreitol (DTT), two sulphur erythrose alcohol, one of beta -mercaptoethanol or a variety of.
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