CN106769299A - Glycine betaine as aspartate amino transferase stabilizer application - Google Patents
Glycine betaine as aspartate amino transferase stabilizer application Download PDFInfo
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- CN106769299A CN106769299A CN201611139402.5A CN201611139402A CN106769299A CN 106769299 A CN106769299 A CN 106769299A CN 201611139402 A CN201611139402 A CN 201611139402A CN 106769299 A CN106769299 A CN 106769299A
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- glycine betaine
- serum
- amino transferase
- aspartate amino
- stabilizer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to glycine betaine as the application to aspartate amino transferase AST enzyme stabilizers.Glycine betaine provided by the present invention has preferable stabilization to AST enzyme activities in human serum, the need for can very well meeting short-term transport serum at normal temperatures and the need for 20 DEG C long-term preservations.And it has no obvious collective effect in conventional detecting system, it is suitable for the preparation that AST standard substances, calibration object, correctness control thing.
Description
Technical field
The invention belongs to clinical biochemistry enzymatic assay and field of quality control, more particularly to glycine betaine is used as lucid asparagus
Histidine amino group shifts the application of enzyme stabilizers.
Background technology
Serum aspartate amino transferase (Aspartate Aminotransferas, AST) is most normal clinical biochemical
Liver function grade project, plays a positive role in hepatopathy preventing and treating.To ensure the accuracy of testing result, AST reference materials
Matter, calibration object, correctness control thing play an important role, and the stability of AST enzymatic activitys just turns into emphasis in these materials.But
The activity of AST is because being influenceed can significant change by many factors such as time and storage temperatures, and the enzyme activity holding time is shorter.
At present, the stabilizer of AST the more commonly used both at home and abroad is generally glycitols such as trehalose etc..But the addition of these stabilizers can make inspection
Examining system produces different degrees of matrix effect ((matrix effect), so as to influence testing result.Glycine betaine (betaine)
Also known as trimethylglycine, chemical name is betaine, as a kind of bleeding agent (osmolyte) in nature
It is widely present.The research of Stefan Knapp et al. (Stefan K, Extremophiles [J], 1999) shows glycine betaine energy shadow
The unfolding thermokinetic parameters of RNaseA are rung so as to stablize the natural folding state of protease, Dominic Lambert etc.
(Dominic Lambert, J.Mol.Biol [J], 2007) research finds that the bleeding agents such as glycine betaine can stablize two grades and three of RNA
Level structure, and stablize the interaction of RNA-Mg2+.
At present, usage ratio, Dan Shuan of the detection kit that domestic each company provides from reagent composition, serum and reagent
Reagent, detection program etc. all can be different, cause the inconsistent of patient samples testing result, diagnosis, treatment, treatment to disease
The work such as effect observation, prevention, scientific research bring detrimental effect.In order that enzymatic determination result is between method, laboratory monitoring has can
Than property, the multiple confusion of term of reference is eliminated, be beneficial to clinical practice, the standardization effort for carrying out enzymatic determination is imperative.
In the standardization effort of AST projects, the standard substance of AST, calibration object, correctness control thing play an important role, and country two
Level standard substance and correctness control product are prepared from using the people's pooled serum with patients serum's tool interchangeability, while by
In the presence of commercial calibration product matrix effect in detection architecture, generally acknowledged people's pooled serum is only best calibration object at present.By
AST enzymatic activitys is unstable in human serum, and the transport and preservation for giving people pooled serum form great inconvenience.Find one kind
AST concentration of enzymatic activity in energy stabilization human serum, and be to solve AST standards to AST stabilizers of each detecting system without matrix effect
The key that chemical industry is made.It is an object of the invention to provide glycine betaine as aspartate amino transferase stabilizer application.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of stabilizer glycine betaine of stabilization serum AST enzyme activity, i.e.,
Glycine betaine as aspartate amino transferase stabilizer application.It is a feature of the present invention that：Described application is beet
Alkali is used as stabilizer stabilised blood clear sky AST enzyme activity.
Addition concentration of the glycine betaine in serum is 0.8-1.0mol/L in described application.
Glycine betaine has preferable stabilization, 25 DEG C of seroenzyme activities of addition glycine betaine to AST enzyme activities in human serum
Can stablize 7 days, the need for such stablizing effect can very well meet short-term transport serum at normal temperatures；4 DEG C are added glycine betaine
Seroenzyme activity can stablize 24 days, -20 DEG C addition glycine betaines seroenzyme activities can stablize 24 months, -20 DEG C long can be met
The need for phase preserves.
Additive in serum can produce different degrees of matrix effect (matrix effect) to each detecting system, but
Matrix effect has the absoluteness also to have its relativity, as long as approve that certain matrix effect can be ignored through methods comparison disregarding or by base
The error that bulk effect is introduced to certain detecting system is it is believed that "None" matrix effect in acceptable level.In clinical conventional each inspection
Tested in survey system, the pooled serum that with the addition of the stabilizer of present invention offer is imitated in each detection architecture without obvious matrix
Should.
Glycine betaine stabilizer provided by the present invention has good stablizing effect, especially the effect steady in a long-term of -20 degree,
And it has no obvious matrix effect in conventional detecting system, it is suitable for standard substance, calibration object, correctness and controls thing
Prepare, the glycine betaine of 0.8-1.0mol/L is added into pooled serum, so as to solve the accurate of clinical AST standardized testings work
Sex chromosome mosaicism.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
Stabilization experiment
First prepare the glycine betaine storage liquid of 5mol/L；The serum of collection is carried out to be mixed to get former pooled serum, by part
The glycine betaine storage liquid of 5mol/L is added in pooled serum so that the stabilizer glycine betaine final concentration of 0.8 and 1mol/L of addition,
Obtain adding the serum of glycine betaine；Former pooled serum is filtered with the filter of 0.22um respectively with the serum of addition glycine betaine, by three
Kind serum is distributed into EP and manages and use parafilm film tube sealings with every pipe 300ul, and former serum is control serum.The serum tube of packing point
1-4d, 7d and 14d, 2-8 DEG C of refrigeration 7d, 14d, 24d and 32d, in -20 DEG C of freezing monitoring 24m are not placed in 25 DEG C of room temperatures.
Beckman DXC600 automatic biochemistry analyzers and its matched reagent detect its AST enzyme activity respectively, and each detection asks equal three times
Value.
Criterion using 95% credibility interval as enzyme activity (A%) stably, i.e. enzyme activity are enzyme for 100 ± 5%
Vigor stabilization.Result shows that the activity of serum enzyme of 25 DEG C of addition glycine betaines can be stablized 7 days, and control serum can only be stablized 4 days；4℃
The activity of serum enzyme for adding glycine betaine can be stablized 24 days, and control serum enzymatic activity is only stablized 14 days；- 20 DEG C are added glycine betaine
Serum can stablize at least 24 months, control serum after 1 month enzymatic activity be reduced to 90%.It is shown in Table 1.
The glycine betaine of table 1 is in different temperatures to the stabilization of AST enzyme activities
Note：T represents that ＂ temperature ＂, t represent ＂ times ＂.
Embodiment 2
Matrix effect is tested
Prepare the glycine betaine storage liquid of 5mol/L；The serum of collection is carried out to be mixed to get former pooled serum, part is mixed
Close serum and press 4 with glycine betaine storage liquid：1 mixing so that the final concentration of 1mol/L of stabilizer glycine betaine of addition, while part is mixed
Close serum and press 4 with physiological saline (0.9%NaCl)：1 is mixed into normal saline dilution control sample；Three kinds of blood serum samples are used respectively
The filter filtering of 0.22um, often pipe 500ul packing.Three kinds of blood serum samples are issued to simultaneously with cold chain transportation (2-8 DEG C), special messenger
Extra large 32 clinical labororatories are detected that 32 clinical labororatories are related to 12 kinds of detection reagents of AST, totally 14 detecting systems,
The covering Shanghai clinical testing laboratories all conventional detecting systems of AST.
32 testing results of the three of laboratory kinds of blood serum samples are reclaimed, average, coefficient of variation statistics are carried out to result, and
Result according to pooled serum bulk samples calculates dilution theoretical value.Result shows that addition glycine betaine sample, physiological saline are dilute
The result for releasing sample is consistent average, with dilution theoretical value without obvious deviation；Add the variation situation of glycine betaine sample and mix
The result variation situation of serum bulk samples and normal saline dilution sample is consistent, 5% or so.Illustrate, add glycine betaine
Pooled serum sample often exist with having no obvious matrix effect in clinical detection system in AST.Concrete outcome is shown in Table 2.
Table 2 adds matrix effect of the glycine betaine sample in each detecting system
Claims (6)
1. glycine betaine as aspartate amino transferase stabilizer application, it is characterised in that：Described application is beet
Alkali is used as stabilizer stabilised blood clear sky AST enzyme activity.
2. glycine betaine according to claim 1 as aspartate amino transferase stabilizer application, its feature exists
In：Addition concentration of the glycine betaine in serum is 0.8-1.0mol/L in described application.
3. glycine betaine according to claim 2 as aspartate amino transferase stabilizer application, its feature exists
In：The serum aspartate amino transferase vigor that with the addition of glycine betaine is stablized 7 days at 25 DEG C.
4. glycine betaine according to claim 2 as aspartate amino transferase stabilizer application, its feature exists
In：The serum aspartate amino transferase vigor that with the addition of glycine betaine is stablized 7 days at 4 DEG C.
5. glycine betaine according to claim 2 as aspartate amino transferase stabilizer application, its feature exists
In：The serum aspartate amino transferase vigor that with the addition of glycine betaine is stablized 24 months at -20 DEG C.
6. glycine betaine according to claim 2 as aspartate amino transferase stabilizer application, its feature exists
In：Described application is the preparation that thing is controlled for AST standard substances, calibration object, correctness.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611139402.5A CN106769299A (en) | 2016-12-12 | 2016-12-12 | Glycine betaine as aspartate amino transferase stabilizer application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060233774A1 (en) * | 2005-04-19 | 2006-10-19 | Korea Yakult Co., Ltd | Composition for the improvement of liver function, the reduction of serum ethanol level and antioxidant activity enhancement |
| CN103800312A (en) * | 2012-11-14 | 2014-05-21 | 张晓元 | Application of betaine hydrochloride in preparing medicines and health foods for preventing and treating hepatic diseases |
-
2016
- 2016-12-12 CN CN201611139402.5A patent/CN106769299A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060233774A1 (en) * | 2005-04-19 | 2006-10-19 | Korea Yakult Co., Ltd | Composition for the improvement of liver function, the reduction of serum ethanol level and antioxidant activity enhancement |
| CN103800312A (en) * | 2012-11-14 | 2014-05-21 | 张晓元 | Application of betaine hydrochloride in preparing medicines and health foods for preventing and treating hepatic diseases |
Non-Patent Citations (3)
| Title |
|---|
| 朱定贵等: "甜菜碱对奥尼罗非鱼生长、体脂含量及血清生化指标的影响", 《上海海洋大学学报》 * |
| 欧元祝等: "甜菜碱对血清酶ALT活性的稳定作用及其基质效应的研究", 《中华检验医学杂志》 * |
| 鲁海军等: "蛋氨酸羟基类似物及甜菜碱在急性热应激下对肉鸡血清酶指标的影响", 《单胃动物营养》 * |
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