JP3365091B2 - Lipid extraction method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a simple and rapid method for extracting lipids contained in blood components. In the present invention, the blood component is whole blood, plasma, blood cells and the like. Plasma is the supernatant of whole blood after centrifugation (3000 rpm). Blood cells are the precipitates after centrifugation (3000 rpm).

[0002]

2. Description of the Related Art Generally, as a method for extracting a lipid from a sample containing a lipid, the Folch method in which a solvent such as a chloroform-methanol mixture is added in an amount several times the volume of the sample, and the chloroform-methanol- A Bligh-Dyer method in which a solvent such as a water mixture is added in several volumes and extraction is performed is performed.

By the way, in the medical field, it is often necessary to extract lipids contained in blood components and to accurately quantify lipid peroxides in the lipids. The method described in "Lipid Research Method: Biochemistry Experimental Method 5" (Tamio Yamakawa et al., Published by Tokyo Kagaku Dojinsha Co., Ltd.) was usually used as the above.

That is, first, blood is put into an acidic citric acid-dextrose mixture and centrifuged, and then supernatant plasma is collected using a Pasteur pipette so as not to disturb the layer of the precipitated blood cells, and then the precipitated blood cells are collected. Layer is further suspended in physiological saline, centrifuged again, and the supernatant is carefully collected and combined with the above plasma. Further, the residue is resuspended in physiological saline to form a suspension, and a methanol-chloroform mixture is added separately from the above plasma to extract lipids, and these are combined to obtain lipids.

[0005]

In such a conventional method for extracting lipids from blood components, since water is present during the extraction operation, only the organic solvent layer in which the lipids are dissolved so that water is not mixed The operation such as taking out is complicated and the extraction operation is time-consuming because it is finally dehydrated by adding anhydrous sodium sulfate. Therefore, there is a demand for the development of a simple and rapid lipid extraction method in which there is no water during the operation. An object of the present invention is to provide a method for easily and efficiently extracting lipids contained in blood components, particularly a method for quantitatively extracting lipid peroxides.

[0006]

[Means for Solving the Problems] The present inventors have solved the drawbacks of these conventional extraction methods and obtained a method for extracting lipids contained in blood components simply, quickly and efficiently. , Furthermore, as a result of earnest research for the purpose of accurately quantifying lipid peroxide, first, by freeze-drying blood components to remove water that complicates the extraction procedure, and then by extracting with an organic solvent, Knowing that the object can be achieved, the present invention has been completed.

That is, the present invention relates to a method for extracting lipids, which comprises freeze-drying a blood component and then extracting it with an organic solvent.

In the present invention, the lipids to be extracted are long-chain hydrocarbons having 10 to 40 carbon atoms and 10 to 5 carbon atoms.
0 higher alcohols, C8-35 aldehydes, C6-24 fatty acids, glycerides and their derivatives, wax esters, phospholipids, glycolipids, sulphur lipids, fat-soluble vitamins A, D, E, K and its derivatives, carotenoids, sterols and the like.

As the organic solvent used in the present invention, a solvent having a low polarity such as hexane, petroleum ether, benzene and chloroform and a solvent having a high polarity such as acetone, ethanol, methanol and 2-propanol are used.
It is a mixture in which the volume ratio is (5: 1) to (1: 5). Among them, extraction from plasma was performed by mixing chloroform and methanol at a volume ratio of (2: 1), and extraction from blood cells or blood was performed by mixing chloroform and 2-propanol at a volume ratio (7:11). It is particularly preferable to use a mixture such that The amount of the organic solvent used may be equal to or more than that of the blood component, particularly 2 to
A 10-fold amount is preferable.

The antioxidant used in the present invention is added to an organic solvent in order to suppress the production of lipid peroxides generated during the extraction operation. As long as it is completely soluble in a solvent, for example, synthetic antioxidants such as phenolic compounds, natural antioxidants such as tocopherol,
Particularly preferred is t-butylhydroxytoluene. The amount of the antioxidant added is 0.001 to the organic solvent.
Although it is 0.01%, a range of 0.001 to 0.005% is particularly preferable.

The freeze-drying in the present invention is carried out by freezing the sample to -20 ° C or lower with liquid nitrogen or the like and then drying it under reduced pressure for 1 hour or more.

[0012]

EFFECT OF THE INVENTION According to the extraction method of the present invention, lipids can be extracted from blood components simply and quickly. Further, when an antioxidant is contained in the organic solvent, lipid peroxides are particularly produced. It can be used for extraction from a simple sample and its generation can be suppressed.

[0013]

EXAMPLES, COMPARATIVE EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples. In each of these examples, the subjects to be inspected were 5 healthy subjects A to E who were the same in all cases.

Example 1-1 (Extraction of Lipid from Plasma with Antioxidant-Free Organic Solvent) 5 ml of blood collected from each test subject was centrifuged for 10 minutes at 3000 rpm with a centrifuge. Separation was performed to obtain plasma as a supernatant. 1 ml of this plasma was placed in a test tube, frozen with liquid nitrogen, and then freeze-dried for 12 hours to completely remove water.

6 ml of a chloroform-methanol mixture (volume ratio 2: 1) was added to the lyophilized product obtained, shaken for 30 seconds, centrifuged under the same conditions as above, and the supernatant was concentrated to dryness under a nitrogen stream. A first-time lipid extract was obtained. Similarly,
This operation was repeated twice more to obtain a second lipid extract and a third lipid extract, respectively. The obtained lipid extracts were dried under reduced pressure in a desiccator until the weight became constant and weighed. The total amount is shown in Table 1 as a lipid content.

Further, the amount of lipid peroxide in the obtained lipid extract was measured by chemiluminescence detection-high performance liquid chromatography, and the results are shown in Table 1 as phosphatidylcholine hydroperoxide.

The measuring method is as follows.
That is, the obtained lipid was dissolved in 50 μl of a chloroform-methanol mixture (volume ratio 1: 1), and 2
Inject 0 μl for high performance liquid chromatography. The conditions for chemiluminescence detection and high performance liquid chromatography are shown below. (Chemical luminescence detection) Luminescent reagent: Cytochrome c (1 μg / ml) and luminol (10 μg / ml) dissolved in 50 mM borate buffer (pH 9.3) Chemiluminescence detector: Tohoku Denshi Sangyo KK, Chemiluminescence Analyzer OX -7 type (high performance liquid chromatography) column: normal phase silica-NH ₂ , 4.6 × 250 mm,
JASCO Corporation mobile phase: hexane: 2-propanol: methanol: water = 5: 7: 2: 1

Example 2-1 (Extraction of Lipid from Plasma with Organic Solvent Containing Antioxidant) In Example 1-1, 6 ml of chloroform-methanol mixture (volume ratio 2: 1) was used as the organic solvent. ,
In addition to this formulation, t-butyl hydroxytoluene 0.0
The procedure was as in Example 1-1, except that 6 ml of the mixture containing 02% was used. The results are shown in Table 1.

Comparative Example 1 (Extraction of Lipid from Plasma by Organic Method Using Antioxidant-Containing Organic Solvent) 5 ml of blood collected from each test subject was centrifuged at 3000 rpm for 10 minutes by a centrifuge. Separation was performed to obtain plasma as a supernatant.

To 1 ml of this plasma was added 6 ml of a chloroform-methanol mixture (volume ratio 2: 1, containing t-butylhydroxytoluene 0.002%), and the mixture was shaken for 30 seconds and then centrifuged under the same conditions as above to form a chloroform layer. And water −
Separate the methanol layer and the residue, add anhydrous sodium sulfate to the chloroform layer, leave it for one day and leave it for dehydration, and then filter.
The filtrate was concentrated to dryness under a nitrogen stream to obtain a lipid extract for the first time. Further, the same operation was continued twice from the water-methanol layer and the residue to obtain a second lipid extract and a third lipid extract, respectively. The lipid extract obtained is from Example 1.
It processed according to -1, and the result is shown in Table 1.

[0021]

[Table 1]

Example 1-2 (Extraction of Lipid from Blood Cells with Organic Solvent without Antioxidant) 5 ml of blood collected from each test subject was centrifuged for 10 minutes at 3000 rpm with a centrifuge. Separation was performed to obtain blood cells as a precipitate. 2.5 ml of distilled water was added to 0.5 ml of these blood cells to hemolyze them, and then freeze-dried according to Example 1-1 to completely remove water.

2-Propanol (0.1 mM, EDTA solution) (11 ml) and chloroform (7 ml) were added to the obtained freeze-dried product, and the mixture was shaken at 4 ° C. for 30 minutes and then centrifuged under the same conditions as described above. The mixture was concentrated to dryness with to obtain a first lipid extract. Similarly, this operation was repeated twice more to obtain a second lipid extract and a third lipid extract, respectively. The obtained lipid extract was treated according to Example 1-1, and the results are shown in Table 2.

Further, the amount of lipid peroxide in the obtained lipid extract was measured according to Example 1-1, and the results were also shown as phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide. Shown in 2.

Example 2-2 (Extraction of Lipid from Blood Cells with Organic Solvent Containing Antioxidant) In Example 1-2, 11 ml of 2-propanol (0.1 mM, EDTA solution) as the organic solvent was used instead. ,
In addition to this formulation, t-butyl hydroxytoluene 0.0
Except using 11 ml of a mixture containing 02%
It carried out according to Example 1-2. The results are shown in Table 2.

Comparative Example 2 (Extraction of Lipids from Blood Cells by Organic Method Using Antioxidant-Containing Organic Solvent) 5 ml of blood collected from each test subject was centrifuged at 3000 rpm for 10 minutes by a centrifuge. Separation was performed to obtain blood cells as a precipitate.

2.5 ml of distilled water was added to 0.5 ml of the blood cells to cause hemolysis, and then 2-propanol (0.1 mM,
EDTA solution, t-butylhydroxytoluene 0.00
11 ml (including 2%) and 7 ml of chloroform were added, and 4
After shaking at ℃ for 30 minutes, it was centrifuged under the same conditions as above to separate into a chloroform layer, a water-2-propanol layer and a residue. First, anhydrous sodium sulfate was added to the chloroform layer to obtain 1
The mixture was left standing for 24 hours, dehydrated and filtered, and the filtrate was concentrated to dryness under a nitrogen stream to obtain a first lipid extract. Further, the same operation was continued twice from the water-2-propanol layer and the residue to obtain a second lipid extract and a third lipid extract, respectively. The obtained lipid extract was treated according to Example 1-1, and the results are shown in Table 2.

[0028]

[Table 2]

Example 1-3 (Extraction of Lipid from Blood by Organic Solvent without Antioxidant) 2.5 ml of distilled water was added to 0.5 ml of blood collected from each subject to be hemolyzed. After that, Example 1-1
Water was completely removed by freeze-drying according to.

2-Propanol (0.1 mM, EDTA solution) (11 ml) and chloroform (7 ml) were added to the obtained freeze-dried product, and the mixture was shaken at 4 ° C. for 30 minutes and then centrifuged under the same conditions as in Example 1-1 to obtain a supernatant. Was concentrated to dryness under a nitrogen stream to obtain a first lipid extract. Similarly, this operation was repeated twice more to obtain a second lipid extract and a third lipid extract, respectively. The obtained lipid extract was obtained from Example 1-1.
The results are shown in Table 3.

Further, the lipid extract thus obtained was measured for the amount of lipid peroxide in accordance with Example 1-1, and the results were also shown as phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide. Shown in 3.

Example 2-3 (Extraction of Lipid from Blood with Organic Solvent Containing Antioxidant) In Example 1-3, 2-propanol (0.1 mM, EDTA solution) as an organic solvent was replaced with 11 ml. ,
In addition to this formulation, t-butyl hydroxytoluene 0.0
Except using 11 ml of a mixture containing 02%
It carried out according to Example 1-3. The results are shown in Table 3.

Comparative Example 3 (Extraction of Lipid from Blood by Organic Solvent Containing Antioxidant by Conventional Method) 2.5 ml of distilled water was added to 0.5 ml of blood collected from each subject to be hemolyzed. Then, 11 ml of 2-propanol (0.1 mM, EDTA solution, containing 0.002% of t-butylhydroxytoluene) and 7 ml of chloroform were added, and the mixture was shaken at 4 ° C. for 30 minutes.
Centrifuge under the same conditions as above, and separate into chloroform layer, water-2-propanol layer and residue. First, add anhydrous sodium sulfate to the chloroform layer, leave it for 24 hours, dehydrate and filter,
The filtrate was concentrated to dryness under a nitrogen stream to obtain a lipid extract for the first time. Further, the same operation was continued twice from the water-2-propanol layer and the residue to obtain a second lipid extract and a third lipid extract, respectively. The obtained lipid extract is
The treatment was carried out according to Example 1-1, and the results are shown in Table 3.

[0034]

[Table 3]

From the results of Tables 1 to 3 above, the following is clear. (1) Regarding the lipid content in blood components (including plasma, blood cells, and blood components), when the method of the present invention in which the blood components are freeze-dried in advance does not contain an antioxidant in the organic solvent In both cases (Examples 1-1, 1-2, 1-3) and also in the case of containing an antioxidant (Examples 2-1-2-2, 2-3), conventional freeze-drying was performed. The effect of freeze-drying is clearly shown in comparison with the case of the comparative example in which the lipid content is much higher. In this case, no significant difference is observed between the case where the organic solvent does not contain the antioxidant and the case where the organic solvent contains the antioxidant.

(2) Regarding the content of lipid peroxide in blood components, both phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide were compared with the case where no antioxidant was contained in the organic solvent. In the case of containing an antioxidant, the content of lipid peroxide is much lower, and the effect of the antioxidant is clearly shown.

Further, comparing the cases of Examples 2-1, 2-2, and 2-3 with the corresponding cases of Comparative Examples 1, 2, and 3, these two are respectively an organic solvent and an antioxidant. Example 2 in spite of using the exact same agent
In the cases of -1, 2, 2 and 2-3, the content of lipid peroxide is obviously smaller, and the effect of freeze-drying is shown.

Further, regarding the effect of this freeze-drying, when the freeze-drying is performed, it can be seen from the fact that the values of the case where the antioxidant is not contained and the case of the comparative example which contains the antioxidant are almost the same. It can also be confirmed from the fact that the effect is almost the same as that of the comparative example even if the antioxidant is not included.

Claims (2)

(57) [Claims] 1. A method for extracting lipids, which comprises freeze-drying a blood component and then extracting it with an organic solvent. 2. The extraction method according to claim 1, wherein the organic solvent contains an antioxidant.