CN104931326B - A kind of extracting method of zebra fish metabolin and its application - Google Patents
A kind of extracting method of zebra fish metabolin and its application Download PDFInfo
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Abstract
A kind of extracting method of zebra fish metabolin, step is as follows：1) by zebra fish sample with normal saline flushing it is clean after be immersed in liquid nitrogen save backup rapidly；2) sample is ground at a temperature of 80 DEG C and becomes homogeneous slurry；3) slurry shape sample is added in the extract solution of 20 DEG C of precoolings, carries out microwave abstracting twice, collected after centrifugation is added into sterile deionized water after supernatant is mixed twice, mixed solution is obtained after centrifuging, standing, dry；4) after mixed solution is dried in vacuo, performed the derivatization by methoxy semicarbazide hydrochloride pyridine solution, N methyl Ns (trimethyl silane) trifluoroacetamide two-step method, obtain zebra fish metabolin derivatization sample.It is an advantage of the invention that：The extracting method of the zebra fish metabolin is simple, accurate, it is easy to implement, and is tested for zebra fish exposed to low concentration nano material, can improve the sensitivity of zebra fish toxicity detection.
Description
Technical field
The present invention relates to Environmental Toxicological and biomedical, the extracting method of particularly a kind of zebra fish metabolin and its should
With.
Background technology
Metabolite analysis is a kind of important biology character and the analysis method of toxicological parameters, by organism intracellular metabolite
Thing carries out a series of analysis changes that can occur in vivo and the node changed qualitatively or quantitatively, contributes to
The analysis of situations such as judgement and environmental pollution of bio-toxicity.
Biological subject used in the present invention is zebra fish.Zebra fish have it is small, be easy to cultivation breeding and cost
Low advantage, the use in fields such as ecotoxicologicals is quite varied.And zebra fish is in terms of heredity, physiology and pharmacological reaction and people
There is the similitude of height in class, therefore can be used as good model animal.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned technical Analysis there is provided a kind of extracting method of zebra fish metabolin and its answer
With the extracting method technique is simple, easy to implement, the toxicity test exposed for nano material to zebra fish low concentration, relatively
In other detection method of toxicity it is sensitiveer and easy obtain more available data.
Technical scheme：
A kind of extracting method of zebra fish metabolin, step is as follows：
1) by the zebra fish sample clean rear correct amount of normal saline flushing, then it is immersed in rapidly in liquid nitrogen and preserves standby
With；
2) above-mentioned zebra fish sample is placed in the mortar for fill liquid nitrogen at a temperature of -80 DEG C and grinds 5 minutes to zebra fish
Tissue becomes homogeneous slurry；
3) above-mentioned slurry shape zebra fish sample is added in the extract solution of -20 DEG C of precoolings, the microwave abstracting at a temperature of 40 DEG C
30 minutes, microwave abstracting power was (sample jar number+2) × 100W, collected after centrifugation first time supernatant, then in solid residue
Same amount of above-mentioned -20 DEG C of extract solution is added in thing, continues microwave abstracting 30 minutes, collected after centrifugation at a temperature of 40 DEG C
Second of supernatant；
4) first time supernatant is mixed with second of supernatant, sterile deionized water, 4000rpm is added in mixed liquor
Lower centrifugation 5 minutes, solution is layered after standing 5 minutes, and upper solution is methanol/water phase, and lower floor's solution is chloroform phase, two-phase laminated flow
Chloroform is dried up with nitrogen afterwards, methanol/water phase transfer is then obtained into mixed solution into dry chloroform phase；
5) above-mentioned mixed solution is put into freeze drier after being dried in vacuo with -30 DEG C--50 DEG C, entered using two-step method
Row derivatization：Be firstly added concentration be 20mg/mL methoxy semicarbazide hydrochloride-pyridine solution, after sealing be vortexed 1 minute, after will
It centrifuges 1 minute, then warm bath 90 minutes at 30 DEG C at 3,000 rpm, then adds silylating reagent N- methyl-N- (front threes
Base silane)-trifluoroacetamide (MSTFA), warm bath 30 minutes, obtains zebra fish metabolin derivatization sample and is transferred at 37 DEG C
It is adapted to preserve in the internal lining pipe of GC-MS analyses.
The step 3) extract solution be methanol, chloroform and water mixed liquor, the volume ratio of methanol, chloroform and water in mixed liquor
For 2.5:1:1, wherein methanol, chloroform and water is chromatographically pure；The amount ratio of zebra fish sample and extract solution is 0.3-0.5g：
15mL。
The step 4) in zebra fish sample and sterile deionized water amount ratio be 0.3-0.5g：500μL.
The step 5) in the amount ratio of zebra fish sample and methoxy semicarbazide hydrochloride-pyridine solution be 0.3-0.5g：50μ
The amount ratio of L, zebra fish sample and silylating reagent is 0.3-0.5g：80μL.
A kind of application of the extracting method of the zebra fish metabolin, for zebra fish nanometer toxicity detection.
It is an advantage of the invention that：The extraction of the zebra fish metabolin and analysis method are simple, accurate, it is easy to implement, be used for
Zebra fish is tested exposed to low concentration nano material, can improve the sensitivity of zebra fish toxicity detection.
Brief description of the drawings
Fig. 1 is the active comparison diagram of total number born of the zebra fish adult fish of same batch contamination.
Fig. 2 is the mda content comparison diagram of the zebra fish adult fish of same batch contamination.
Embodiment
Embodiment 1：
A kind of extracting method of zebra fish metabolin, step is as follows：
1) by the zebra fish sample clean rear correct amount of normal saline flushing, then it is immersed in rapidly in liquid nitrogen and preserves standby
With；
2) above-mentioned zebra fish sample is placed in the mortar for fill liquid nitrogen at a temperature of -80 DEG C and grinds 5 minutes to zebra fish
Tissue becomes homogeneous slurry；
3) above-mentioned slurry shape zebra fish sample is added in the extract solution of -20 DEG C of precoolings, extract solution extract solution is methanol, chlorine
The volume ratio of methanol, chloroform and water is 2.5 in the imitative mixed liquor with water, mixed liquor:1:1, wherein methanol, chloroform and water is color
Spectrum is pure, and the amount ratio of zebra fish sample and extract solution is 0.3g：15mL, microwave abstracting 30 minutes at a temperature of 40 DEG C, microwave extraction
It is (sample jar number+2) × 100W to take power, and then collected after centrifugation first time supernatant adds phase in solid residue
The extraction mixed liquor of above-mentioned -20 DEG C of same amount, continues on microwave abstracting 30 minutes at a temperature of 40 DEG C, collected after centrifugation second
Clear liquid；
4) first time supernatant is mixed with second of supernatant, sterile deionized water, zebra fish is added in mixed liquor
Sample and the amount ratio of sterile deionized water are 0.3g：Centrifuged 5 minutes under 500 μ L, 4000rpm, solution point after standing 5 minutes
Layer, upper solution is methanol/water phase, and lower floor's solution is to dry up chloroform with nitrogen after chloroform phase, two-phase laminated flow, then by first
Alcohol/aqueous phase is transferred in dry chloroform phase, obtains mixed solution；
5) above-mentioned mixed solution is put into freeze drier after being dried in vacuo with -30 DEG C--50 DEG C, entered using two-step method
Row derivatization：It is firstly added methoxy semicarbazide hydrochloride-pyridine solution that concentration is 20mg/mL, zebra fish sample and methoxy amino
The amount ratio of hydrochloride-pyridine solution is 0.3g：50 μ L, after sealing be vortexed 1 minute, after it is centrifuged to 1 point at 3,000 rpm
Clock, then warm bath 90 minutes at 30 DEG C, then add silylating reagent N- methyl-N- (trimethyl silane)-trifluoroacetamide
(MSTFA), the amount ratio of zebra fish sample and silylating reagent is 0.3g：80 μ L, warm bath 30 minutes, obtains zebra fish at 37 DEG C
Metabolin derivatization sample is simultaneously transferred to preservation in the internal lining pipe of suitable GC-MS analyses.
Above zebra fish sample is set to first group of sample.
Contrast experiment：
1) second group of sample
The extracting method of this group of zebra fish metabolin, step and first group of sample are essentially identical, and difference is：Will step
It is rapid 3) in microwave abstracting change into ultrasonic extraction, ultrasonic power is 100W.
2) the 3rd group of sample
The extracting method of this group of zebra fish metabolin, step and first group of sample are essentially identical, and difference is：Will step
It is rapid 3) in extraction mixed liquor change into chromatogram straight alcohol.
3) the 4th group of sample
The extracting method of this group of zebra fish metabolin, step and first group of sample are essentially identical, and difference is：
By step 3) in extraction mixed liquor change into chromatogram straight alcohol, while microwave abstracting changes into ultrasonic extraction, surpass
Acoustical power is 100W.
The analysis of four groups of samples of the above：
Analytical instrument is Agilent gas-chromatography tandem mass spectrum.Parameter setting is as follows：
Gas chromatographic sample introduction parameter is：Using automatic sampler sample introduction, the μ L of sample size 1,230 DEG C of injector temperature, zebra
Fish adult fish split sampling ratio is 1:10, juvenile fish does not shunt, carrier gas is helium, flow velocity 2mL/min.
Gas chromatograph parameters are：MDN-35 capillary chromatographic columns (30m), temperature program(me) are constant temperature 2min at 80 DEG C, then
325 DEG C are warming up to 15 DEG C/min speed, constant temperature 6min, transmission line temperature are 250 DEG C.
Mass spectrometry parameters are：Ion source temperature is 250 DEG C, mass scan range is 20 per second of 70-600m/z, acquisition rate
Scanning, mass spectrum electron bombardment ionization source filament opening time are after chromatographic solvent delay 170s, detector voltage 1700-1850V, mass spectrum
Loss is set to 0, filament bias current and tuned automatically for 70V, instrument.
The parameter setting of the spectrogram adjustment of data processing stations：
Spectrogram deconvolution parameter is：Business software Chroma TOF, baseline elimination (Baseline that Leco Corporation carries
Offset) 1 (0.5-1), spectrogram smooth (Smoothing) data point 5 (3-7), peak width (Peak Width) 3s (3s- are set
4s), (2-15) of signal to noise ratio S/N (Signal-Tonoise Ratio) 10
Analysis result is obtained by the analysis contrast of Nist.08 databases, table 1 is that four kinds of Different Extraction Methods extract situation
Table.
Table 1
Table 2
Table 3
Table 4
Table 5
It can be seen that from table 1- tables 5：The zebra fish metabolin extracted using the method for extracting mixed liquor-microwave abstracting is put down
There are 64 kinds, it includes amino acid, carbohydrate and carbohydrate derivative, oil substances and small molecule acid and part alkane, alkene
Hydrocarbon alcohols etc., Species distributing is more uniform, and the substance classes that its excess-three kind method is extracted are average within 60 kinds, and deviation is larger；
Amino acid, part lipid, carbohydrate and the small molecule acid difference in species that four kinds of methods are detected are little, but application method one
Can complete detection go out three kinds of carbohydrates：Glucose, mannose and fructose, remaining method are but and unstable；Other this method can be with
The presence of alkane and alkene is detected, its excess-three kind method is not detected；Additionally as the important substance lactic acid of biological metabolism, only
There is first method energy stable detection to analyze, remaining method the chance of lactic acid occurs at random or responded not high.
The application of the extracting method of the zebra fish metabolin, for zebra fish nanometer toxicity detection, method is as follows：Select
Healthy adult zebra fish, is uniformly divided into four groups, wherein three groups are separately added into 0.01mg/L graphene oxide, carbon nanometer
Contaminated in pipe, three kinds of nano materials of quantum dot graphene oxide solution, another group is put into the salt solution for being added without any nano material
In, water feeding, 27 DEG C of raising 21d are changed daily；Then respectively according to zebra fish metabolin extracting method five step operations,
Zebra fish metabolin derivatization sample will be obtained and be transferred in the internal lining pipe of suitable GC-MS analyses to preserve.By obtain four groups
Sample is analyzed according to method as hereinbefore.Table 2 is three kinds of different nano material contaminations and control group by extracting
Metabolin extracts situation (unit afterwards：Peak area/0.1g tissues).
Table 6
Table 7
Table 8
Table 9
Table 10
As can be seen from Table 4：Creatinine is changed significantly in the data obtained using the extracting method, available for assessing fish
The situation of class renal function；The significant changes of ornithine can also indicate the change of metabolic condition in zebra fish body in table 1；But
This species diversity is not fairly obvious in the result of the enzyme activity index measurement such as total number born and MDA.
Fig. 1 is shown in the active comparison diagram of total number born of the zebra fish adult fish of same batch contamination, figure：With
The zebra fish of control group is compared, and the total number born activity of the zebra fish by three kinds of nano material processing is not aobvious
The change of work..
Fig. 2 is shown in the mda content comparison diagram of the zebra fish adult fish of same batch contamination, figure：With the spot of control group
Horse fish is compared, and the mda content of the zebra fish by three kinds of nano material processing does not have significant change.
Claims (4)
1. a kind of extracting method of zebra fish metabolin, it is characterised in that step is as follows：
1) by the zebra fish sample clean rear correct amount of normal saline flushing, then it is immersed in liquid nitrogen rapidly and saves backup；
2) above-mentioned zebra fish sample is placed in the mortar for fill liquid nitrogen at a temperature of -80 DEG C and grinds 5 minutes to zebra fish tissues
Become homogeneous slurry；
3) above-mentioned slurry shape zebra fish sample is added in the extract solution of -20 DEG C of precoolings, the extract solution is methanol, chloroform and water
Mixed liquor, the volume ratio of methanol, chloroform and water is 2.5 in mixed liquor:1:1, wherein methanol, chloroform and water is chromatographically pure；
The amount ratio of zebra fish sample and extract solution is 0.3-0.5g：15mL；Microwave abstracting 30 minutes, microwave abstracting at a temperature of 40 DEG C
Power is (sample jar number+2) × 100W, and then collected after centrifugation first time supernatant adds identical in solid residue
Above-mentioned -20 DEG C of extract solution of amount, continues microwave abstracting 30 minutes, second of supernatant of collected after centrifugation at a temperature of 40 DEG C；
4) first time supernatant is mixed with second of supernatant, added in mixed liquor under sterile deionized water, 4000rpm from
The heart 5 minutes, solution is layered after standing 5 minutes, and upper solution is methanol/water phase, and lower floor's solution is general after chloroform phase, two-phase laminated flow
Chloroform is dried up with nitrogen, and methanol/water phase transfer then is obtained into mixed solution into dry chloroform phase；
5) above-mentioned mixed solution is put into freeze drier after being dried in vacuo with -30 DEG C--50 DEG C, spread out using two-step method
It is biochemical：Be firstly added concentration be 20mg/mL methoxy semicarbazide hydrochloride-pyridine solution, after sealing be vortexed 1 minute, after by its
Centrifuged 1 minute under 3000rpm, then warm bath 90 minutes at 30 DEG C, then add silylating reagent N- methyl-N- (trimethyl silicanes
Alkane)-trifluoroacetamide (MSTFA), warm bath 30 minutes, obtains zebra fish metabolin derivatization sample and is transferred to suitable at 37 DEG C
Preserved in the internal lining pipe of GC-MS analyses.
2. the extracting method of zebra fish metabolin according to claim 1, it is characterised in that the step 4) in zebra fish sample
Product and the amount ratio of sterile deionized water are 0.3-0.5g：500μL.
3. the extracting method of zebra fish metabolin according to claim 1, it is characterised in that：The step 5) in zebra fish sample
The amount ratio of product and methoxy semicarbazide hydrochloride-pyridine solution is 0.3-0.5g：The use of 50 μ L, zebra fish sample and silylating reagent
Amount is than being 0.3-0.5g：80μL.
4. a kind of application of the extracting method of zebra fish metabolin as claimed in claim 1, for zebra fish nanometer toxicity detection.
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| CN105651983A (en) * | 2015-12-29 | 2016-06-08 | 湖南省植物保护研究所 | Method for determining toxicity of pesticide water dispersible granule by using zebra fish |
| CN106568931A (en) * | 2016-10-31 | 2017-04-19 | 首都医科大学 | Method for detecting cardiotoxicity of nano-material |
| CN115015450B (en) * | 2022-05-26 | 2024-05-14 | 贵州省烟草科学研究院 | Method for analyzing metabolites in soil by microwave derivatization-quasi-target gas chromatography-mass spectrometry |
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| CN101957346A (en) * | 2010-08-20 | 2011-01-26 | 江苏省中医药研究院 | Novel method for studying medicament metabolism by using model organism zebrafish |
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| CN102023199A (en) * | 2009-09-11 | 2011-04-20 | 华东理工大学 | Method for testing toxicity of nanomaterial by using transgenic zebra fish embryo model |
| CN101957346A (en) * | 2010-08-20 | 2011-01-26 | 江苏省中医药研究院 | Novel method for studying medicament metabolism by using model organism zebrafish |
| CN103623433A (en) * | 2013-11-22 | 2014-03-12 | 江苏省中医药研究院 | Modified zebra fish drug metabolism modeling method |
| CN104569191A (en) * | 2014-12-26 | 2015-04-29 | 上海大学 | Detection method for ornithine in plasma |
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