A kind of detection method of serum cholesterol oxide
Technical field
The invention belongs to biomarker technical field of analysis and detection, be specifically related to a kind of cholesterol in serum oxygen
The detection method of compound.
Background technology
Cholesterol has an important meaning to the physiological function maintaining human normal, but too much cholesterol heap in vivo
The long-pending health that can affect again human body self.Numerous studies have shown that: the danger of cardiovascular disease (CVD) because of
Element is relevant with oxidative stress increase.Wherein, cholesterol oxidation product COPs is the non-of body oxidative stress status
One of mark of Chang Teshu.
In 1966, Brooks etc. detected that oxidized gallbladder is solid first in human atherosclerosis organizes
Alcohol, 26-oxycholesterol and 7-ketone group cholesterol.But, people think atherosclerotic important wind always
The danger factor is hypercholesterolemia, so concentrate in hypercholesterolemia the most always, right
Cholesterol oxidized forms does not draw attention rare report.In recent years, deep along with to low density lipoprotein, LDL (LDL)
Entering research, research finds first, and one of risks and assumptions that cardiovascular is the most serious is probably oxidized cholesterol,
I.e. cholesterol oxide (cholesterol oxidation products), this compounds is more solid than inoxidized gallbladder
Alcohol is more easily caused atherosclerosis, increases the risk of heart disease and stroke outbreak.Cholesterol oxide is a lot
Aspect shows its toxic action, such as, can increase carcinogenic probability, Cytotoxic, promote cell to occur
Sudden change, makes steroid compound metabolism disorder, substantially can work atherosclerosis, and harmful to human is good for
Health.At present, there is the cholesterol oxide in numerous food studied, including egg products, Animal fat
Deng.Scientific research result demonstrates, cholesterol and oxidation product thereof are the risk of cardiovascular disease to a certain extent
The factor, but cholesterol oxide and the research of cardiovascular disease generation potential mechanism indefinite.
The a series of of A ring, B ring or side chain are caused under the environmental conditions such as cholesterol illumination in vitro, heating
Automatic oxidation reaction, thus produce multiple oxidation product.Cholesterol can also be produced by autoxidation mode in vivo
Raw.In theory, cholesterol oxide can have more than 70 to plant, and some of them are not autoxidizable generation in cholesterol body
Table product, and some other cholesterol oxide is autoxidizable representative product in cholesterol body, can be interior
Source property produces or obtains from diet.
Summary of the invention
For prior art thinks little of the detection of cholesterol in serum oxide, the easy external autoxidation of cholesterol
The cholesterol oxide that internal autoxidation forms cholesterol oxide, cannot produce carries out classification and Detection, place
During reason, the loss of oxygen sterin causes defects such as being difficult to the most quantitatively, serum composition complex jamming is many, the present invention
The detection method of a kind of serum cholesterol oxide is provided.
Specifically, the application provides the detection method of a kind of serum cholesterol oxide, it is characterised in that bag
Include following steps:
(1) sample treatment: extract serum with chloroform-methanol
(2) saponification: with KOH solution, dehydrated alcohol, the concussion saponification of BHT room temperature;
(3) Solid-Phase Extraction: the solution after saponification crosses SPE solid-phase extraction column;
(4) derivatization: the product of step (3) is derived with Tri-Sil reagent;
(5) LC-MS analysis: liquid chromatograph uses RP-18e cation seperation column, mass spectrum uses API4000
Triple quadrupole mass spectrograph.
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (1) sample
Process includes: target step in first adding quantitatively in serum before extraction, described quantitative in be designated as non-internal
The cholesterol oxidation species that autoxidation produces, such as 19-oxycholesterol etc..
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (1) includes
Configure chloroform-methanol mixture by the volume ratio of 1:2, serum is diluted 10 with described chloroform-methanol mixed liquor
Times, vortex concussion 1min, stand extraction 3min, 4000g and be centrifuged 10min, draw chloroform layer.
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (2) saponification
Including: add the KOH solution of 60% (w/v), dehydrated alcohol, BHT room temperature concussion saponification 24h, its
The addition of middle KOH solution and dehydrated alcohol is 10 times of serum volume, and BHT's is final concentration of
5-50ng/mL。
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (3) solid phase
Extraction includes: by the solution ether extraction after saponification 3 times, then with distilled water wash 2 times, anhydrous slufuric acid
The dried 30 DEG C of rotations of sodium are evaporated off the ether of residual;After adding the n-hexane dissolution of serum volume 5 times, cross SPE
Solid-phase extraction column, through normal hexane: diethyl ether (95:5, v/v), normal hexane: diethyl ether (90:10, v/v),
After diethyl ether (80:20, v/v) washing, with acetone: methanol (60:20v/v) eluting.
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (4) derives
Change includes: taking the solution 1ml after saponification, nitrogen dries up, and adds 400 μ LTri-Sil reagent, 60 DEG C of insulations
45min, rotation steaming adds methanol after drying: in the solution of water (3:1, v/v).
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (5) liquid matter
In combination analysis, HPLC parameter is as follows: solution A is methanol: water (3:1, v/v), and solution B is isopropanol;
The solution A of 0-0.5min 60%, the solution B of 40%;0.5-3.5min linear elution, solution B is carried by 40%
High to 70%;> 3.5min, the solution B of 100%;Flow velocity is 1ml/min.
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described step (5) liquid matter
In combination analysis, mass spectrometry parameters is: source temperature 500, atomization electric current 3 μ A, gas curtain air pressure 25psi, atomization gas
Pressure 25psi.
The detection method of serum cholesterol oxide of the present invention, it is characterised in that described serum cholesterol oxygen
Compound choosing freely 7 α-OH cholesterol, 7 β-OH cholesterol, 5 β, 6 beta epoxide cholesterol, 5 α, 6 α-
The component of epoxycholesterol, 7 ketone group cholesterol, 25-OH cholesterol composition.
What detection method of the present invention reached has the beneficial effect that:
(1) by sample treatment, saponification, Solid-Phase Extraction, cholesterol in serum class component is isolated;Pass through
The cholesterol oxide that derivatization makes structure similar more adapts to follow-up HPLC-MS/MS detection, it is to avoid blood
The interference of complicated ingredient in clear, improve accuracy of detection.
(2) by the internal non-existent cholesterol oxide of addition as quantitative internal standard, itself and internal cholesterol
Oxide physicochemical character, chemical constitution are similar, and the loss in reason step is similar, using it as internal standard throughout
It is obtained in that in described extracorporeal treatment step the cholesterol oxide ratio of loss, thus to the cholesterol recorded
Oxide content is proofreaded, and improves the accuracy that serum cholesterol oxide is quantitative.
(3) the method for the invention one-time detection i.e. can be simultaneously to generation autoxidizable in cholesterol in serum body
Table product, 7 α-OH cholesterol, 7 β-OH cholesterol, 5 β, 6 beta epoxide cholesterol, 5 α, 6 α-
Epoxycholesterol, 7 ketone group cholesterol, 25-OH cholesterol carry out detection by quantitative respectively.
Accompanying drawing explanation
Fig. 1 is cholesterol oxide HPLC-MS/MS collection of illustrative plates.
Wherein: 1:7 α-OH cholesterol, 2:7 β-OH cholesterol, 3:7-ketone group cholesterol, 4:5 β, 6
Beta epoxide cholesterol, 5:5 α, 6 α-epoxycholesterol, 6: cholesterol, 7:19-OH cholesterol, 8:
25-OH cholesterol.
Detailed description of the invention
Embodiment 1: the extraction of cholesterol oxide
1. take normal individual in blood bank, serum 1mL to be checked, be added thereto to 19-OH cholesterol to final concentration
10 μ g/mL, add 10mL chloroform-methanol mixture (chloroform: methanol=1:2, v/v).Vortex concussion 1min,
Room temperature stands extraction 3min, 4000g and is centrifuged 10min, draws chloroform layer.
2. add the KOH solution of 10mL 60% (w/v), 10mL dehydrated alcohol, 0.5 μ g BHT, room
Temperature concussion 24h, makes the oil component saponification in serum, prevents cholesterol oxidation simultaneously.
3., by non-saponification organic facies ether extraction 3 times, then with distilled water wash 2 times, anhydrous sodium sulfate is dried
Rear 30 DEG C of rotations are evaporated off the solvent of residual.Use 5mL n-hexane dissolution, cross SPE solid-phase extraction column, Jing Guozheng
Hexane: diethyl ether (95:5, v/v), normal hexane: diethyl ether (90:10, v/v), diethyl ether (80:20, v/v)
After washing, with acetone: methanol (60:20v/v) eluting.
4. taking the solution 1ml after Solid-Phase Extraction, nitrogen dries up, and adds 400 μ LTri-Sil reagent, 60 DEG C of guarantors
Temperature 45min, rotation steaming adds methanol after drying: in the solution of water (3:1, v/v).
Embodiment 2:HPLC-MS/MS is analyzed
Liquid chromatograph uses RP-18e cation seperation column (50 × 4.6cm).275 series injector automatically, 275
Series high-resolution binary pump.HPLC parameter is as follows: solution A is methanol: water (3:1, v/v), solution B
For isopropanol;The solution A of 0-0.5min 60%, the solution B of 40%, 0.5-3.5;0.5-3.5min linear elution,
Solution B is brought up to 70% by 40%;> 3.5min, the solution B of 100%;Flow velocity is 1ml/min.
Mass spectrum uses API4000 triple quadrupole mass spectrograph to carry out ACPI mass spectrum.Mass Spectrometry Conditions is set to: source temperature
Degree 500, atomization electric current 3 μ A, gas curtain air pressure 25psi, atomization air pressure 25psi.Mass Spectrometry Conditions such as table 1:
Table 1: Mass Spectrometry Conditions
Parameter index |
Numerical value |
Detector |
385.3/367.3 |
Solve bunch voltage |
70V |
Entrance potential |
10V |
Exit potential |
8V |
Impact energy |
28eV |
Above-mentioned HPLC-MS/MS method can complete the detection to six kinds of cholesterol oxides, institute in 6 minutes
Obtain collection of illustrative plates as it is shown in figure 1, measure its peak area.Addition according to 19-OH cholesterol obtains after proofreading
Obtain various cholesterol oxide content such as table 2 in serum.
Table 2: in serum, various cholesterol oxide content are as follows:
Numbering |
Cholesterol oxide |
Ng/mL serum |
1 |
7 α-OH cholesterol |
9.5 |
2 |
7 β-OH cholesterol |
22.0 |
3 |
7-ketone group cholesterol |
7.3 |
4 |
5 β, 6 beta epoxide cholesterol |
5.8 |
5 |
5 α, 6 α-epoxycholesterol |
5.6 |
6 |
25-OH cholesterol |
8.2 |
Should be appreciated that the above-described embodiment technology only for the explanation present invention is conceived and feature, its object is to allow
Person skilled in the art will appreciate that present disclosure and is carried out, and can not limit this with this
Bright protection domain, all equivalence changes made according to spirit of the invention or modification, all should contain at this
In the protection domain of invention.