WO2016208741A1 - Simple analysis method for lipids in trace biological sample - Google Patents

Abstract

The present invention pertains to a method for identifying a change in lipid pattern, the method comprising: (a) causing a trace biological sample to directly adsorb onto a TLC plate; (b) separating at least one lipid in a trace biological sample by TLC; and (c) detecting at least one lipid separated on the TLC.

Description

Simple lipid analysis method for trace biological samples

The present invention is a method for identifying changes in lipid patterns, wherein (a) a trace biological sample is directly adsorbed on a TLC plate, and (b) one or more lipids in the trace biological sample are separated by TLC. And (c) a method comprising detecting one or more lipids separated on TLC.

Through signal transduction and receptor-mediated transactivation studies, lipids in cell membranes are widely involved in mediating or stimulating cell motility, including cell proliferation, adhesion and differentiation, and changes in lipids are the onset and progression of disease Has been shown to be deeply involved in In addition, “lipid rafts”, which are microregions of cell membranes obtained as surfactant-insoluble fractions, are a topic of recent biological research.

Cholesterol and sphingolipids (including gangliosides etc.) are the main lipid components, and lipid rafts play an important role in agonist-receptor interactions and subsequent trans-signaling. However, studies on the function and structure of lipids involved complicated processes including lipid extraction, separation, purification, and concentration, and so far, skilled techniques were required.

In Patent Document 1, as a method for extracting sphingosine from cultured cells, cells (1 × 10 ⁶ to 1 × 10 ⁷ ) are collected, washed with phosphate buffered saline (PBS), 0.1 ml Resuspend in PBS, add 3 ml chloroform / methanol (1/2, v / v), sonicate, separate by adding 2 ml each of chloroform and 1M NaCl, discard upper layer and methanol 0.2N NaOH in the solution was added to the chloroform lower layer and separated by adding 3 ml each of chloroform and 1M NaCl, and the chloroform lower layer was washed three times with chloroform / methanol / water (3/48/47). It is described that the sample from the layer was dried. Further, it is described that a dried sample is treated with a 10 mM [ ³ H] acetic anhydride solution to tritize sphingosine and analyzed using TLC.

In Patent Document 2, as a method for extracting total lipid from mouse kidney, about 1 ml of chloroform: methanol = 2: 1 solution is added to 10 mg of mouse kidney tissue wet weight, and the supernatant is obtained by homogenization and centrifugation. In addition, after adding water for injection and mixing, the mixture was centrifuged again to recover the lower layer (organic layer) and dried with a centrifugal concentrator. Further, after the dried sample was treated with sphingolipid ceramide deacylase (SCDase), the enzyme was deactivated to obtain a sample, which was analyzed by high performance liquid chromatography tandem mass spectrometry (LC / MS / MS method). Are analyzed as lyso forms.

In Patent Document 3, 1000 μl of an extraction solution (methanol, chloroform, water (2: 1: 0.6) is added to each tube to a hair sample, the hair is ground in a low temperature crushing mill, centrifuged, and the supernatant (500 μl The dried sample was resuspended in 100 μl n-butanol and methanol (v / v, 1: 1) and centrifuged to obtain a supernatant, Analysis of phospholipids in the supernatant by liquid chromatography mass spectrometry (LC / MS) is described.

Patent Document 4 describes a method of measuring a PVDF membrane on which a TLC plate is transferred after separating phospholipids by TLC using a mass spectrometer. However, Patent Document 4 does not describe any method for extracting phospholipids from human (normally aging) brains.

Non-Patent Document 1 describes that after extracting a lipid from a biological sample with an organic solvent, filtering the residue, and then subjecting the lipid extract obtained through a multistage operation necessary for deproteinization to analysis. However, there is no description of directly adsorbing a small amount of biological sample to a TLC plate.

Japanese National Patent Publication No. 11-513110 International Publication No. WO2012 / 081508 Special table 2012-529630 gazette JP 2010-271157 A

The problem to be solved by the present invention is to provide a simple method for identifying changes in lipid patterns without going through complicated processes including extraction, separation, purification and concentration of lipids.

As a result of extensive research, the present inventors have, in a method for identifying changes in lipid patterns, (a) directly adsorbing a trace biological sample to a TLC plate, and (b) one or more lipids in the trace biological sample. Analysis of lipids in biological samples, including extraction, separation, purification, concentration of lipids from biological samples by detecting one or more lipids separated on TLC and (c) detecting the one or more lipids separated on TLC Thus, it is possible to omit the complicated process that has been necessary in the past, and the present invention has been completed.

That is, the present invention relates to the following.
(1) A method for identifying a change in lipid pattern, comprising the following steps:
(A) directly adsorbing a small amount of biological sample to a TLC plate;
(B) separating one or more lipids in a trace biological sample by TLC, and (c) detecting one or more lipids separated on TLC.
(2) The method according to (1) above, wherein the trace biological sample is a supernatant obtained by adding a trace amount of water and / or an organic solvent to a trace amount of body fluid, tissue, or cell and allowing to stand.
(3) The trace biological sample is a supernatant obtained by adding a trace amount of water and / or an organic solvent to a trace amount of body fluid, tissue, or cell, and further adding a trace amount of an inorganic base, and allowing to stand. (1) The method as described.
(4) Any of (1) to (3) above, wherein the trace biological sample is 0.1 to 2000 μl of body fluid, 0.1 to 200 mg of tissue, or 1.0 × 10 ⁵ to 1.0 × 10 ⁸ cells. The method according to claim 1.
(5) The trace biological sample is a trace amount of bodily fluid, tissue, or cell, 0.5 to 400 μl of bodily fluid, 5 × 10 ⁵ to 5.0 × 10 ⁷ cells, or 5.0 to 150 mg of tissue, The method according to any one of (1) to (4).
(6) (c) is the following process:
(D) transferring one or more lipids separated on the TLC to a membrane; and (e) attaching the membrane to which one or more lipids have been transferred to an analysis plate, The method according to any one of (1) to (5), comprising subjecting to analysis.
(7) The lipids are one or more lipids selected from the group consisting of neutral lipids, sphingophospholipids, glycerophospholipids, glycosphingolipids, glyceroglycolipids, and sulfolipids (1) to (6) ).
(8) The lipid according to (1) to (6), wherein the lipid is one or more lipids selected from the group consisting of neutral fat, plasmalogen, lysophosphatidylcholine, lysophosphatidic acid, ganglioside, and galactosylceramide. The method according to any one of the above.
(9) The method according to any one of (1) to (8), wherein the trace biological sample is cerebrum, cerebellum, hippocampus, whole blood, plasma, serum, or cells.
(10) Further, the following steps:
(F) The method according to any one of (1) to (9), comprising comparing a predetermined lipid pattern standard with one or more lipid patterns in a trace biological sample.

That is, the present invention also relates to the following.
(11) A method for identifying a symptom that changes a lipid pattern, comprising the following steps:
(A) directly adsorbing a supernatant obtained by adding water to blood to a TLC plate;
(B) separating one or more lipids contained in the supernatant by TLC, and (c) detecting the separated one or more lipids.
(12) A method for identifying a symptom that changes a lipid pattern, comprising the following steps:
(A) directly adsorbing a supernatant obtained by adding an organic solvent to brain tissue to a TLC plate;
(B) separating one or more lipids contained in the supernatant by TLC, and (c) detecting the separated one or more lipids.
(13) A method for identifying Alzheimer's disease, comprising the following steps:
(A) directly adsorbing the supernatant obtained by adding an organic solvent to the hippocampal white matter extracted from the subject or the subject and further adding an inorganic base to the TLC plate;
(B) separating one or more lipids contained in the supernatant by TLC; (d) transferring one or more lipids separated on the TLC to a membrane; and (e) one or more. A membrane to which the lipids of 1 are transferred is attached to an analysis plate, and the analysis plate is subjected to mass spectrometry to detect plasmalogen. (F) A predetermined lipid pattern standard and a subject or test subject Comparing the amount of one or more plasmalogens in a person's sample.
(14) A method for identifying neuroblastoma, comprising the following steps:
(A) directly adsorbing a supernatant obtained by adding an organic solvent to cells extracted from a subject or a subject and further adding an inorganic base to a TLC plate;
(B) separating one or more lipids contained in the supernatant by TLC; (d) transferring one or more lipids separated on the TLC to a membrane; and (e) one or more. A method comprising: adhering a membrane to which a lipid is transferred to an analysis plate, and subjecting the analysis plate to mass spectrometry to detect GD2 and GD3.

According to the present invention, since a lipid pattern in a biological sample can be visually compared and observed, a simple method for identifying a change in a lipid pattern can be provided.

FIG. 1 shows the results of separating and analyzing gangliosides of mouse cerebrum and cerebellum by TLC. Lanes 1, 3, 5, and 7 are samples derived from rat cerebrum. Lanes 2, 4, 6, and 8 are samples from rat cerebellum. Lane 9 is a ganglioside preparation. GD1a represents ganglioside GD1a, GD1b represents ganglioside GD1b, GD3 represents ganglioside GD3, GM1 represents ganglioside GM1, GM2 represents ganglioside GM2, GQ1b represents ganglioside GQ1b, GT1G represents glioside G1 SM indicates sphingomyelin. FIG. 2 shows the result of separating and analyzing gangliosides of cultured cells by TLC. Lane 1 is a ganglioside preparation. Lanes 2, 3, 4, 5, and 6 are respectively HepG2 (human liver cancer-derived cell line), HuH (human liver cancer-derived cell line), HCT (human colon cancer-derived cell line), and HT-28 (human fibrosarcoma-derived). Cell line), DLD-1 cells (human colorectal cancer-derived cell line), and Colon 26 (mouse colorectal cancer-derived cell line). Lane 8 is a rat cerebrum-derived sample. GD1a represents ganglioside GD1a, GD1b represents ganglioside GD1b, GM1 represents ganglioside GM1, and GT1b represents ganglioside GT1b. FIG. 3 shows the results of separation analysis of neutral lipids of rat serum by TLC. Lanes 1-9 are serum samples of each rat. Lane 10 is a neutral lipid preparation. CE indicates cholesterol ester, TG indicates triglyceride, FA indicates fatty acid, C indicates cholesterol, DG indicates diglyceride, PE indicates phosphatidylethanolamine, PC indicates phosphatidylcholine, and SM indicates sphingomyelin Indicates. FIG. 4 shows the results of separation analysis of neutral lipids and phospholipids of rat serum by TLC. Lanes 1-9 are serum samples of each rat. Lane 10 is a sample of neutral lipid, phospholipid and phospholipid. CE indicates cholesterol ester, TG indicates triglyceride, FA indicates fatty acid, C indicates cholesterol, DG indicates diglyceride, PE indicates phosphatidylethanolamine, PC indicates phosphatidylcholine, and SM indicates sphingomyelin Indicates. FIG. 5 shows the results of separating and analyzing gangliosides (alkaline degradation treatment) of mouse cerebrum and cerebellum by TLC. Lanes 1 and 3 are samples (no treatment) derived from mouse cerebellum and cerebrum, respectively. Lanes 2 and 4 are samples (alkali decomposition treatment) derived from mouse cerebellum and cerebrum, respectively. Lane 5 is a ganglioside preparation. GD1a represents ganglioside GD1a, GD1b represents ganglioside GD1b, and GT1b represents ganglioside GT1b. FIG. 6 shows the results of separating and analyzing the lipids of hippocampal gray matter of Alzheimer's disease and Parkinson's disease patients and control patients by TLC. Lanes 1 to 4 are samples (no treatment) of hippocampal gray matter of Alzheimer's disease patients, respectively. Lanes 5 and 6 are samples of hippocampal gray matter (alkali decomposition treatment) of Parkinson's disease patients, respectively. Lanes 7 and 8 are samples of hippocampal gray matter of control patients (alkaline degradation treatment), and lane 9 is a sample of hippocampal gray matter of control patients (no treatment). FA indicates fatty acid, GalCer indicates galactosylceramide, Sulfatide indicates sulfatide (cerebrosid sulfate), SM indicates sphingomyelin, and Gangliosides indicates gangliosides. FIG. 7 shows the results of separation analysis of hippocampal white matter lipids of Alzheimer's disease and Parkinson's disease patients and control patients by TLC. Lanes 1 to 4 are samples of hippocampal white matter (untreated) from patients with Alzheimer's disease. Lanes 5 and 6 are samples of hippocampal white matter (alkali decomposition treatment) of Parkinson's disease patients, respectively. Lanes 7 and 8 are samples of hippocampal white matter from the control patients (alkaline degradation treatment), and lane 9 is a sample of hippocampal white matter from the control patients (no treatment). FA indicates fatty acid, GalCer indicates galactosylceramide, PE indicates phosphatidylethanolamine, PC indicates phosphatidylcholine, Sulfatide indicates sulfatide (cerebrosid sulfate), LysoPE indicates lysoethanolamine plasmarogen, and SM indicates sphingomyelin. FIG. 8 shows the results of quantitative analysis of plasmalogens in hippocampal white matter of Alzheimer's disease and Parkinson's disease patients and control patients. FIG. 9 shows the results of separating and analyzing gangliosides of neuroblastoma cells. Band a represents GD3 and band b represents GD2.

The present invention is a method for identifying changes in lipid patterns comprising the following steps:
(A) directly adsorbing a small amount of biological sample to a TLC plate;
It relates to a method comprising (b) separating one or more lipids in a trace biological sample by TLC, and (c) detecting one or more lipids separated on TLC.

<Fat for analysis>
In the present invention, the lipid is not particularly limited as long as it is a lipid detected by a usual method.

In the present invention, the lipid is, for example, a simple lipid, a complex lipid, or a derived lipid, preferably a neutral lipid, a phospholipid (sphingophospholipid, glycerophospholipid), a glycolipid (sphingoglycolipid, glyceroglycolipid), and One or more lipids selected from the group consisting of sulfolipids, and more preferably, the glycerophospholipid is one or more lipids selected from the group consisting of plasmalogen, lysophosphatidylcholine, and lysophosphatidic acid Or the glycosphingolipid is ganglioside (GD1a, GD1b, GD2, GD3, GM1, GM2, GQ1b, GT1b) or galactosylceramide.

In the present invention, the neutral lipid includes, for example, cholesterol ester, cholesterol, steryl glucoside, ceramide, sphingosine, triglyceride, diglyceride, monoglyceride, fatty acid, preferably cholesterol ester, cholesterol, ceramide, triglyceride, diglyceride, monoglyceride, And one or more neutral lipids selected from the group consisting of fatty acids.

In the present invention, the phospholipid includes sphingophospholipid and glycerophospholipid, such as phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylcholine, choline plasmalogen, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid, lysophosphatidylethanol. Amine, lysophosphatidylcholine, lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidic acid, sphingomyelin, lysosphingomyelin (psychosin), lysoethanolamine plasmarogen, lysocholine plasmalogen, phosphatidylglycerol, phosphatidylglucose, lyso Bisphosphatidic acid Phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylcholine, choline plasmalogen, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidine, Sphingomyelin, lysosphingomyelin (psychosin), lysoethanolamine plasmalogen, lysocholine plasmalogen, phosphatidylglycerol, phosphatidylglucose, more preferably phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, cardiolipin Phosphatidic acid, lyso phosphatidyl ethanolamine, sphingomyelin, and lyso ethanolamine plus Malo Zhen, most preferably phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, a lyso ethanolamine plus Malo Gen.

In the present invention, the glycolipid includes a sphingoglycolipid and a glyceroglycolipid. Lactotetraosylceramide, i-type glycolipid, I-type glycolipid, Le ^a , Le ^b , Le ^x , Le ^y , sialylparagloboside, sialyl lactotetraosylceramide, sialyl i-type glycolipid, sialyl I-type glycolipid Lipid, sialyl Le ^a , sialyl Le ^b , sialyl Le ^x , sialyl Le ^y , seminolipid, O-type glycolipid, A-type glycolipid, B-type glycolipid, sulfated glycolipid, preferably ganglioside, glucosylceramide, Galactosylceramide, sulfatai (Cerebroside sulfate), lactosylceramide, globotriaosylceramide, globoside, paragloboside, lactotetraosylceramide, i-type glycolipid, I-type glycolipid, Le ^a , Le ^b , Le ^x , Le ^y , sialyl Paragloboside, sialyl lactotetraosylceramide, sialyl i type glycolipid, sialyl type I glycolipid, sialyl Le ^a , sialyl Le ^b , sialyl Le ^x , sialyl Le ^y , more preferably ganglioside, galactosylceramide, sulfa Tide (cerebroside sulfate).

In the present invention, examples of the ganglioside include GM2, GM3, GM4, GM1, GM1b, GD1a, GD1b, GD1α, GD3, GD2, GT1a, GT1b, GQ1b, O-acetylGD3, GalNAc-GD1a, and preferably GM2. , GM1, GM1b, GD1a, GD1b, GD3, GD2, GT1b, GQ1b, and more preferably GM2, GM1, GM1b, GD1a, GD3, GT1b, GQ1b.

<Lipid pattern>
In the present invention, the lipid pattern is a pattern specified by one or more kinds of lipids, their amounts, Rf values, and / or masses, molecular weights, and the like.

<Change in lipid pattern>
In the present invention, the change in lipid pattern means, for example, gender, age, gene, eating habits, lifestyle, exercise habits, living environment, work environment, physical condition, illness, medical history, medical history, family history, etc. It means that there is a difference in the type and amount of lipid.

In the present invention, the change in the lipid pattern is, for example, a symptom or disease that changes the lipid pattern, such as a neurodegenerative disease (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis). (MS), lipid accumulation disease, etc., cancer (neuroblastoma, lung cancer, melanoma, liver cancer, brain tumor, pancreatic cancer, bone marrow tumor, lymphoma, colon cancer, stomach cancer, breast cancer, etc.), autoimmune disease (Guillan) Valley syndrome, ulcerative colitis, etc.).

<Process (a)>
In the present invention, step (a) is a step of directly adsorbing a small amount of biological sample to the TLC plate.

<Biological sample>
In the present invention, the biological sample is, for example, a tissue (for example, brain (cerebrum, cerebellum, hippocampus (hippocampal gray matter, hippocampal white matter))), heart, lung, liver, stomach, intestine, collected from a subject or subject. Kidney, pancreas, breast), body fluid (eg blood (whole blood, plasma, serum), cerebrospinal fluid, urine, colostrum, milk) and cells, preferably cerebrum, cerebellum, hippocampus, whole blood, plasma Serum and cells, more preferably cerebrum, cerebellum, hippocampal gray, hippocampal white matter, serum and cells.

In the present invention, the cells are, for example, normal cells, cancer cells, cancer tissue-derived cells, etc., for example, human liver cancer-derived cells (HepG2, HuH), human colon or colon cancer-derived cells (HCT, DLD-1 cells). Human fibrosarcoma-derived cells (HT-28), mouse colon cancer-derived cells (Colon 26), and neuroblastoma cells.

<Trace biological sample>
In the present invention, a small amount of biological sample is, for example, a small amount of tissue, body fluid, or cell. For example, body fluid 0.1 to 2000 μl, tissue 0.1 to 200 mg, or cell 1.0 × 10 ⁵ to 1.0 × 10 ⁸ , preferably 0.5 to 400 μl of body fluid, 5.0 × 10 ⁵ to 5.0 × 10 ⁷ cells, or 5.0 to 150 mg of tissue, more preferably 1.0 to 1.0 ˜100 μl, 1.0 × 10 ⁶ to 1.0 × 10 ⁷ cells, or 10 to 100 mg of tissue.

<TLC plate>
In this invention, TLC will not be restrict | limited especially if a lipid can be isolate | separated, For example, a HPTLC plate (The Merck company make, 10cmx10cm) can be used.

<Method of directly adsorbing to TLC>
In the present invention, a method for directly adsorbing a trace amount biological sample to a TLC plate includes, for example, applying the trace amount biological sample onto a TLC using a syringe or a glass capillary and drying it.

In the step (a) of the present invention, the trace biological sample may be a supernatant obtained by adding a trace amount of water and / or an organic solvent to the trace biological sample and allowing to stand.

<Organic solvent>
In the present invention, the organic solvent is not particularly limited as long as it can be added to a biological sample, and examples thereof include nonpolar and polar solvents. Nonpolar solvents include chloroform, hexane, toluene, benzene, diethyl ether and ethyl acetate. Examples of the polar solvent include methanol, ethanol, acetone, n-propanol, isopropanol, butanol, acetic acid, formic acid, dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran and 1,4-dioxane.

<Mixed solvent>
In the present invention, the organic solvent includes a mixed solvent of chloroform and methanol, and preferably includes a mixed solvent having a volume ratio of chloroform to methanol of 75:25 to 25:75, for example, chloroform: methanol = 1: 1. Or chloroform: methanol = 1: 2.

<Amount of water and / or organic solvent added to trace biological sample>
In the step (a) of the present invention, the amount of water and / or organic solvent added to a trace biological sample is not particularly limited as long as a lipid pattern can be obtained, and is preferably a small amount, for example, 0 μl to 2000 μl, 1 μl to 500 μl, preferably 5 μl to 100 μl.

<Amount of water and / or organic solvent added to tissue>
In step (a) of the present invention, the amount of water and / or organic solvent added to the tissue is not particularly limited as long as a lipid pattern can be obtained. For example, 10 to 20 wt. Add the mixture.

<Amount of water and / or organic solvent added to body fluid>
In the step (a) of the present invention, the amount of water and / or organic solvent added to the body fluid is not particularly limited as long as a lipid pattern can be obtained, and water and / or organic solvent may not be added to blood. The blood may be subjected to hemolysis treatment, anticoagulation treatment, and the like, and includes, for example, whole blood, plasma, and serum, preferably serum.

<Amount of water and / or organic solvent added to cells>
In the step (a) of the present invention, the amount of water and / or organic solvent added to the cells is not particularly limited as long as a lipid pattern can be obtained, and is preferably a small amount, for example, 0.05 to 0.1 ml. is there.

<Time for adding water and / or organic solvent and allowing to stand>
In the step (a) of the present invention, the time for standing by adding water and / or an organic solvent to the biological sample is not particularly limited, and is 0 to 24 hours, preferably 0 to 12 hours, and more preferably 0 to 8 hours. It's time.

<Temperature by adding water and / or organic solvent>
In step (a) of the present invention, the temperature at which water and / or an organic solvent is added to the biological sample and allowed to stand is not particularly limited, and is, for example, 4 ° C. to room temperature, preferably 4 ° C. or room temperature.

In the present invention, a trace amount biological sample (0.1 to 100 mg) is obtained by adding a trace amount of water and / or an organic solvent to a trace amount of body fluid, tissue, or cell, and further adding a trace amount of inorganic base to the trace amount biological sample. The supernatant obtained by standing still is included.

<Inorganic base>
In the step (a) of the present invention, the inorganic base added to the trace biological sample is not particularly limited as long as one or more lipids in the biological sample can be alkalinely decomposed. For example, sodium hydroxide, potassium hydroxide, sodium Methylate can be used, preferably 0.1 to 1.0 N sodium hydroxide in methanol, more preferably 0.5 N sodium hydroxide in methanol.

<Amount of inorganic base>
In the present invention, the amount of the inorganic base used for alkali decomposition is not particularly limited as long as one or more lipids in a biological sample can be alkali decomposed, and contains lipid obtained by adding water and / or an organic solvent. The amount is, for example, 1/50 to 1/2 times, preferably 1/30 to 1/2 times, more preferably 1/20 to 1/5 times the volume of the liquid volume.

<Time for adding water and / or organic solvent and allowing to stand>
In the step (a) of the present invention, water and / or an organic solvent is added to the biological sample, an inorganic base is further added, and the time for standing still may cause one or more lipids in the biological sample to undergo alkaline decomposition. If possible, it is not particularly limited, and is 0 to 30 minutes, preferably 0 to 15 minutes, and more preferably 0 to 10 minutes.

<Step (b)>
In the present invention, step (b) is a step of separating one or more lipids in a trace biological sample by TLC.

In the step (b) of the present invention, the TLC used is not particularly limited as long as one or more lipids can be separated using at least one developing solution.

In the step (b) of the present invention, as long as one or more lipids can be separated using TLC, at least one developing solution to be used is not particularly limited.

In the present invention, the at least one developing solution is not particularly limited as long as one or more lipids can be separated. For example, CHCl ₃ : MeOH: 0.2% CaCl ₂ (60 to 75:25 to 40: 4-9, v / v / v), MeOAc: nPrOH: CHCl ₃ : MeOH: 0.25% KCl (20-30: 20-30: 20-30: 5-15: 5-10, v / v / v / v / v), Hexane: Ether: AcOH (60-80: 20-40: 0.5-1.5, v / v / v), Petroleum ether: Ether: AcOH (60-80: 20-40) : 0.5 to 1.5, v / v / v), preferably CHCl ₃ : MeOH: 0.2% CaCl ₂ (60: 40: 9, v / v / v), MeOAc: nPrOH: CHCl ₃ : MeOH: 0.25% KCl (25: 25: 25: 10: 9, v / v / v / v / v), Hexane: Ether: AcOH (70: 30: 1, v / v / v) may be used. it can.

<Step (c)>
In the present invention, step (c) is a step of detecting one or more lipids separated on TLC.

<Detection of lipid>
The means for detecting one or more lipids separated on TLC is not particularly limited as long as the lipid can be detected. For example, UV, Rf value (comparison with standard), detection reagent, immunostaining, binding to lectin Tests and mass spectrometry can be used.

<Detection with detection reagent>
In the present invention, the detection reagent is not particularly limited as long as it can detect lipids. For example, a primulin reagent, a molybdenum reagent, an orcinol reagent, a resorcinol reagent, a ninhydrin reagent, and the like can be used.

<Detection by mass spectrometry>

The method for identifying a change in the lipid pattern of the present invention comprises the following steps following the steps (a) to (c):
(D) transferring one or more separated lipids to a membrane; and (e) attaching the membrane to which one or more lipids have been transferred to an analysis plate, and subjecting the analysis plate to mass spectrometry. Including that.

<Step (d)>
In the present invention, the membrane is not particularly limited as long as it can transfer one or more separated lipids. For example, a PVDF membrane can be used.

<Process (e)>
In the present invention, mass spectrometry is not particularly limited as long as the molecular weight of one or more separated lipids can be measured. For example, a MALDI method and a time-of-flight mass spectrometer can be used.

The method for identifying a change in lipid pattern of the present invention further comprises the following steps:
(F) comparing a predetermined lipid pattern standard with one or more lipid patterns in a trace biological sample.

<Step (f)>
In the present invention, the predetermined lipid pattern standard means one or more corresponding lipid patterns of a living body having no cause for changing the lipid pattern.

The present invention also relates to a method for preparing a lipid-containing liquid for analysis by TLC.

The method for preparing the lipid-containing liquid of the present invention comprises the following steps:
(X) adding a trace amount of water and / or an organic solvent to a trace amount biological sample;
Further, if necessary, it may include alkali decomposition by adding a trace amount of an inorganic base.

<Process (x)>
The description of the step (a) can be applied to the step (x).

One embodiment of the present invention is a kit for identifying changes in lipid patterns.

The kit of the present invention includes, for example, an organic solvent, TLC, a developing solution, and a predetermined lipid pattern standard.

The kit of the present invention can further include a device that can collect a small amount of biological sample by an individual or a group, for example, a device for self-collecting blood.

Hereinafter, the present invention will be described in more detail with reference examples and examples, but the scope of the present invention is not limited thereto.

<Analysis of mouse cerebellar and cerebellar gangliosides>
Mouse cerebrum or cerebellum 50 mg was weighed and cut finely with surgical scissors on aluminum foil placed on ice. The treated sample was placed in a glass test tube, an organic solvent (1 ml of CHCl ₃ : MeOH (1: 1, v / v) was added and shaken, and stored overnight at 4 ° C. to prepare a lipid-containing solution.
Using a 10 μl syringe, 5 μl of each lipid-containing solution (sample) was applied to a band of about 4 mm at a position 1.5 cm from the lower end of an HPTLC plate (Merck, 10 cm × 10 cm). The ganglioside preparation was purchased from Sigma and used.
Using the first developing solution (CHCl ₃ : MeOH: 0.2% CaCl ₂ (60: 35: 8.5, v / v / v), the HPTLC plate was developed from the lower end to A (see FIG. 1). ).
Subsequently, using a second developing solution (MeOAc: PrOH: CHCl ₃ : MeOH: 0.25% KCl (25: 25: 25: 10: 9, v / v / v / v / v)), Deployed once from the lower end to B (see FIG. 1).
After removing the developing solvent with a drier, the primulin reagent was sprayed and detected at UV 315 nm.

<Separation analysis of gangliosides in cultured cells>
Each cell 5.0 × 10 ⁶ cultured under normal culture conditions was collected, transferred to a glass centrifuge tube, and centrifuged. After washing with PBS and further centrifuging, 200 μl of an organic solvent (CHCl ₃ : MeOH (1: 2, v / v)) was added and stored at 4 ° C. overnight to prepare a lipid-containing solution.
Using a 10 μl syringe, 10 μl of each lipid-containing solution (sample) was applied to a band of about 4 mm at a position 1.5 cm from the lower end of an HPTLC plate (Merck, 10 cm × 10 cm). The ganglioside preparation was purchased from Sigma and used.
Using the first developing solution (CHCl ₃ : MeOH: 0.2% CaCl ₂ (60: 40: 9, v / v / v)), the plate was developed once from the lower end of the HPTLC plate to A (FIG. 2). reference).
Subsequently, using the second developing solution (MeOAc: nPrOH: CHCl ₃ : MeOH: 0.25% KCl (25: 25: 25: 10: 9, v / v / v / v) from the lower end of the HPTLC plate Development was performed once up to B (see FIG. 2).
After removing the developing solvent with a drier, the primulin reagent was sprayed and detected at UV 315 nm.

<Separation analysis of neutral lipids in rat serum>
10 μl of rat serum derived from 9 rats was diluted 5-fold with water.
Using a 10 μl Eppendorf micropipette tip, 2 μl of each lipid-containing solution (sample) was applied to a band of about 4 mm at a position 1.5 cm from the lower end of an HPTLC plate (Merck, 10 cm × 10 cm). The neutral lipid preparation was purchased from Sigma and used.
Development was performed for 15 to 20 minutes using a developing solution (Hexane: Ether: AcOH (70: 30: 1, v / v / v) (see FIG. 3).
After removing the developing solvent with a drier, the primulin reagent was sprayed and detected at UV 315 nm.

<Separation analysis of neutral lipid and complex fat in rat serum>
10 μl of rat serum derived from 9 rats was diluted 5-fold with water.
Using a 10 μl Eppendorf micropipette tip, 2 μl of each lipid-containing solution (sample) was applied to a band of about 4 mm at a position 1.5 cm from the lower end of an HPTLC plate (Merck, 10 cm × 10 cm). Neutral lipid and complex fat preparations were purchased from Sigma and used.
Using the first developing solution MeOAc: nPrOH: CHCl ₃ : MeOH: 0.25% KCl (25: 25: 25: 10: 9, v / v / v / v) from the lower end of the HPTLC plate to A Deployed once (see FIG. 4).
Subsequently, using the second developing solution Hexane: Ether: AcOH (70: 30: 1, v / v / v), the plate was developed once from the lower end of the HPTLC plate to B (see FIG. 4).
After removing the developing solvent with a drier, the primulin reagent was sprayed and detected at UV 315 nm.

<Separation and analysis of gangliosides in mouse cerebrum and cerebellum (alkaline degradation)>
Mouse cerebrum or cerebellum 50 mg was weighed and cut finely with surgical scissors on aluminum foil placed on ice. A mouse cerebrum or cerebellum sample was placed in a glass test tube, an organic solvent (1 ml of CHCl ₃ : MeOH (1: 1, v / v) was added, and stored at 4 ° C. overnight to prepare a lipid-containing solution.
20 μl of the lipid-containing solution was placed in a 10 ml glass tube, 2 μl of 0.5N NaOH (MeOH solution) was added, and the mixture was allowed to stand at room temperature for about 5 minutes.
Separation analysis using HPTLC was performed in the same manner as in Example 1 using a lipid-containing liquid, an alkali-treated lipid-containing liquid, and a ganglioside preparation.

<Separation analysis of plasmalogens in hippocampal gray matter of patients with Alzheimer's disease and Parkinson's disease>
100 mg of hippocampal gray matter from patients with Alzheimer's disease and Parkinson's disease from whom informed consent was obtained was weighed and cut finely with surgical scissors on aluminum foil placed on ice. The treated sample was placed in a glass test tube, an organic solvent (2 ml of CHCl ₃ : MeOH (1: 1, v / v) was added, and stored at 4 ° C. overnight to prepare a lipid-containing solution.
20 μl of the lipid-containing solution was placed in a 10 ml glass tube, 2 μl of 0.5N NaOH (MeOH solution) was added, and the mixture was allowed to stand at room temperature for about 5 minutes.
Separation analysis using HPTLC was performed in the same manner as in Example 1 using each of the lipid-containing liquid and the lipid-treated liquid subjected to alkali treatment.

<Separation analysis of plasmalogens in hippocampal white matter of Alzheimer's and Parkinson's disease patients and control patients>
100 mg of hippocampal white matter from patients with Alzheimer's disease and Parkinson's disease from which informed consent was obtained was weighed and cut finely with surgical scissors on aluminum foil placed on ice. The treated sample was placed in a glass test tube, an organic solvent (2 ml of CHCl ₃ : MeOH (1: 1, v / v) was added, and stored at 4 ° C. overnight to prepare a lipid-containing solution.
20 μl of the lipid-containing solution was placed in a 10 ml glass tube, 2 μl of 0.5N NaOH (MeOH solution) was added, and the mixture was allowed to stand at room temperature for about 5 minutes.
Separation analysis using HPTLC was performed in the same manner as in Example 1 using each of the lipid-containing liquid and the lipid-treated liquid subjected to alkali treatment.

<Quantitative analysis of plasmalogens in hippocampal white matter in Alzheimer's and Parkinson's disease patients and control patients>
-Transfer of lipid on HPTLC plate to PVDF membrane Band on HPTLC plate obtained in Example 7 (a band detected with primulin reagent and UV 315 nm) was marked with a red pencil, and transfer solvent (isopropanol: 0.2 It was immersed in% CaCl ₂ : MeOH (40: 20: 7, v / v / v) for 10 seconds.
Immediately after that, a PVDF membrane (Clear Blot-P, ATTO Co. Ltd.) was placed, and further a Teflon membrane (PTEE membrane AC5973, ATTO Co. Ltd), a glass filter membrane, (AC5972 (100 mm × 100 mm), ATTO Co. Ltd.). .) In order. Furthermore, a glass plate (100 mm × 100 mm) was placed thereon, and lipid was transferred by applying pressure at 180 ° C. for 30 seconds using TLC Blotter (manufactured by Bio-X).
Mass spectrometry Fix PVDF membrane to MALDI target plate (P / N: 241-08727-91, Shimadzu, Kyoto, Japan) with conductive double-sided tape (P / N: 241-08727-92, Shimadzu, Kyoto, Japan) did.
DHB (prepared to 50 mg / ml using methanol: 0.1% TFA = 1: 1) as a matrix was applied in two portions so as to be 5 μl / band.
Using a mass spectrometer (MALDI-TOF MS: AXIMA-QIT, Shimadzu, Japan), mass analysis of lipid molecules contained in each band was performed, and m / z: 508.3 was integrated.

<Separation analysis of gangliosides in neuroblastoma cells>
300 μl of organic solvent (CHCl ₃ : MeOH (1: 2, v / v) 300 μl) was added to neuroblastoma tissue (wet weight 30 mg) cultured under normal culture conditions, shaken and stored overnight at 4 ° C. A containing liquid was prepared.
A 20 μl portion of the lipid-containing solution was placed in a 10 ml glass tube, 2 μl of 0.5N NaOH (MeOH solution) was added, and the mixture was allowed to stand at room temperature for 10 minutes.
As a control, 50 mg of mouse cerebrum was weighed and cut finely with surgical scissors on an aluminum foil placed on ice. The treated sample was placed in a glass test tube, an organic solvent (1 ml of CHCl ₃ : MeOH (1: 1, v / v) was added and shaken, and stored at 4 ° C. overnight to prepare a lipid-containing solution.
Using a 10 μl syringe, 5 μl of each lipid-containing solution (sample) was applied to a band of about 4 mm at a position 1.5 cm from the lower end of an HPTLC plate (Merck, 10 cm × 10 cm).
Using a developing solution (CHCl ₃ : MeOH: 0.2% CaCl ₂ (60: 40: 9, v / v / v)), the plate was developed once from the lower end of the HPTLC plate to A.
In accordance with the method described in Example 8, lipid transfer and mass spectrometry were performed.
As a result, band a was identified as GD3 and band b was identified as GD2. These two gangliosides were gangliosides characteristic of neuroblastoma tissue.

The present invention provides a method for identifying changes in lipid patterns in which (a) a microbiological sample is adsorbed directly to a TLC plate, (b) one or more lipids in the microbiological sample are separated by TLC, and (c ) Complex processes traditionally required for analysis of lipids in biological samples, including extraction, separation, purification and concentration of lipids from biological samples by detecting one or more lipids separated on TLC Therefore, the present invention has industrial applicability.

Claims (10)

1. A method for identifying changes in lipid patterns comprising the following steps:
   (A) directly adsorbing a small amount of biological sample to a TLC plate;
   (B) separating one or more lipids in a trace biological sample by TLC, and (c) detecting one or more lipids separated on TLC.
2. The method according to claim 1, wherein the trace biological sample is a supernatant obtained by adding a trace amount of an organic solvent and / or water to a trace amount of body fluid, tissue, or cell and allowing to stand.
3. The trace amount biological sample is a supernatant obtained by adding a trace amount of an organic solvent and / or water to a trace amount of body fluid, tissue, or cell, and further adding a trace amount of an inorganic base, and allowing to stand. the method of.
4. The trace amount biological sample is 0.1 to 2000 μl of body fluid, 0.1 to 200 mg of tissue, or 1.0 × 10 ⁵ to 1.0 × 10 ⁸ cells, according to any one of claims 1 to 3. Method.
5. The trace biological sample is a trace amount of body fluid, tissue or cell, 0.5 to 400 μl of body fluid, 5.0 × 10 ⁵ to 5.0 × 10 ⁷ cells, or 5.0 to 150 mg of tissue. The method according to any one of 1 to 4.
6. Said (c) is the following process:
   (D) transferring one or more lipids separated on the TLC to a membrane; and (e) attaching the membrane to which one or more lipids have been transferred to an analysis plate, The method according to any one of claims 1 to 5, comprising subjecting to analysis.
7. 7. The lipid according to claim 1, wherein the lipid is one or more lipids selected from the group consisting of neutral lipids, sphingophospholipids, glycerophospholipids, glycosphingolipids, glyceroglycolipids, and sulfolipids. The method described.
8. The lipid according to any one of claims 1 to 6, wherein the lipid is one or more lipids selected from the group consisting of neutral fat, plasmalogen, lysophosphatidylcholine, lysophosphatidic acid, ganglioside, and galactosylceramide. Method.
9. The method according to any one of claims 1 to 8, wherein the trace biological sample is cerebrum, cerebellum, hippocampus, whole blood, plasma, serum, or cells.
10. In addition, the following steps:
    The method according to any one of claims 1 to 9, comprising comparing (f) a predetermined lipid pattern standard with one or more lipid patterns in a trace biological sample.

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