JP5156911B2 - Method for adsorbing and separating phospholipids and glycolipids - Google Patents

Description

  The present invention relates to a method for adsorbing and separating phospholipids and glycolipids, which can separate phospholipids and glycolipids from a sample containing a plurality of types of lipids.

  Lipids constituting biological membranes are composed of simple lipids and complex lipids, and the latter include phospholipids and glycolipids. Phospholipids and glycolipids are composed of glycerolipids with a glycerol skeleton and sphingolipids with sphingoid bases. Phospholipids (glycerophospholipids) are included.

  Sphingolipids, on the other hand, have monoglycosylceramide, diglycosylceramide, triglycosylceramide (eg Gb3), tetraglycosylceramide (eg Gb4), or a sugar chain with 5 or more sugar chains. And sphingophospholipids such as sphingophospholipids such as gangliosides with sialic acid and sulfatides with sulfuric acid, and sphingomyelins with phosphorus such as sphingomyelin.

  These lipids have an important function as biologically active lipids. In particular, from recent studies, it is known that sphingolipids among these lipids form a microdomain on the cell membrane and play an important role in cell signal transduction. Therefore, there is an urgent need to establish a purification analysis method capable of handling a small amount, simple, and high throughput.

  However, these lipids are close to each other in their physicochemical characteristics, and thus comprehensive purification analysis of these lipids has been difficult.

Homogenize cells and biological tissues, extract total lipids by the Bligh-Dyer method and Folch method, fractionate them into neutral lipids and acidic lipids by anion exchange chromatography, etc., and phospholipids, glycolipids, and simple lipids. There is a process to obtain and analyze.
It can also be performed by normal phase chromatography. When analyzing glycolipids, it is necessary to weakly hydrolyze glycerophospholipids. When analyzing phospholipids, glycolipids are inhibitors.

  Since phospholipids and glycolipids have similar properties, they are often difficult to separate. Glycolipids are important compounds such as canceration of cells and apoptosis, and analysis of glycolipids has attracted attention in the clinical field. When a real sample is repeatedly injected into an HPLC or mass spectrometer, sample pretreatment is indispensable because it causes various adverse effects such as a decrease in resolution due to sample-derived contamination and column deterioration.

  For example, as a method for purifying sphingolipids, alkaline hydrolysis is often used. However, since most of the glycerolipids are broken, sphingolipids can be concentrated, but glycerolipids, particularly phosphatidylcholine, can be concentrated. It was difficult to recover major phospholipids such as phosphatidyl etanolamine and phosphatidylserine.

  In addition, although concentrated sphingolipids are concentrated, fatty acids or their derivatives generated from glycerolipids broken by alkaline hydrolysis remain, so further treatment such as column chromatography is required to remove them. It was.

  Furthermore, in the remaining sphingolipids, for example, sphingomyelin and tetraglycosylceramide are very similar in physicochemical properties. Conventionally, these lipids are acetylated, separated once by column chromatography, and deacetylated again. It was necessary to do work that said that

  Conventionally, when separating phospholipids and glycolipids in this way, chromatography using a normal phase column or an anion exchange column is used, but these columns are not specific to phospholipids or glycolipids. Therefore, there is a problem that many kinds of lipids are also mixed. There is also a problem that the sample is diluted. In order to solve this, a phospholipid-specific purification method using titanium dioxide has been attempted (Non-patent Document 1).

However, since the titania used here has almost no surface area, the recovery rate of phospholipid is low, and the removal rate of oleic acid and cholesterol other than phospholipid is low even in the purification method. In this method, adsorption / elution of glycolipid is not considered, and separation of phospholipid and glycolipid is not considered.
Further, although the target substance is different, there is a method using a titania column in the field of purification of a phosphorylated peptide in which a phosphate group is bound to a peptide. In Non-Patent Document 2, hydroxycarboxylic acid is also used for purification of phosphorylated peptide. However, the purpose of using the hydroxycarboxylic acid here is used to reduce the adsorption of non-specific substances other than phosphorylated peptides, and for the separation of substances containing sugar, which is the object of the present invention. This is not considered (Non-patent Document 2).

Yoshihiko IKEGUCHI et al. , ANALYTICAL SCIENCES 16, p541-543, 2000 Martin R. Larsen et al. , Molecular & Cellular Proteomics 4 (7), 873-86, 2005

  Therefore, in view of the above-described circumstances, the present invention provides a method capable of specifically separating and purifying phospholipids and glycolipids with high purity by a single operation without using various operation steps which are troublesome. The purpose is that.

In order to solve the above-mentioned problems and achieve the object, the present invention has been studied by the present inventor. As a result, a method using a metal oxide simultaneously adsorbs a phospholipid and a glycolipid having a trisaccharide or higher sugar, Only glycolipids with tri- or higher sugars can be eluted without inhibiting the adsorption of phosphate groups in phospholipids, and without inhibiting the adsorption of glycolipids with tri- or higher sugars It was discovered that only phospholipids can be eluted.
The sugars constituting the glycolipids mentioned here include hexose, (N-acetyl) glucosamine, or sialic acid having a cyclic structure in addition to sugars defined as “aldehyde or ketone having two or more hydroxyl groups”. It is a general term.

The present invention first proposes a method for adsorbing and separating phospholipids and glycolipids, wherein a sample containing phospholipids and glycolipids is brought into contact with and adsorbed to titanium oxide and / or zirconium oxide under acidic conditions. To do.

Second, a sample containing phospholipid and glycolipid is brought into contact with titanium oxide and / or zirconium oxide under acidic conditions to adsorb phospholipid and glycolipid at the same time. We propose a method for adsorbing and separating phospholipids and glycolipids, characterized by elution and separation of glycolipids with a hydroxycarboxylic acid-containing solvent.

  Thirdly, a method for adsorbing and separating phospholipids and glycolipids is proposed, wherein the hydroxycarboxylic acid is an aliphatic hydroxycarboxylic acid or an aromatic hydroxycarboxylic acid.

Further, the fourth, the titanium oxide, titanium dioxide, titanium monoxide, trioxide titanium, trititanium pentoxide, claims 1-3 wherein, characterized in that at least one selected from heptoxide titaniumtetrachloride A method for adsorbing and separating phospholipids and glycolipids according to any one of the above is proposed.

Fifth , the titanium oxide and / or zirconium oxide has a surface area of 3 m ² / g or more and a pore diameter in the range of 10 mm to 1000 mm and a surface area (unit: m ² / g) and a pore diameter (unit: mm). The product of) is 3000 or more, and the method for adsorbing and separating phospholipids and glycolipids according to any one of claims 1 to 4 is proposed.

Further, the sixth, the titanium oxide and / or zirconium oxide, phospholipids and glycolipids according to any one of claims 1-5, wherein characterized that it contained or attached to monoliths structure An adsorption separation method is proposed.

ADVANTAGE OF THE INVENTION According to this invention, the isolation | separation method of the novel phospholipid or glycolipid which can specifically refine | purify the phospholipid or glycolipid contained in a sample can be provided. The phospholipid or glycolipid according to the present invention can be purified with high selectivity.
Further, in the method for purifying phospholipids or glycolipids according to the present invention, it is possible to obtain phospholipids and glycolipids with less impurities, and the sample obtained after desalting operation is subjected to high performance liquid chromatography or mass It can be directly applied to the analyzer.

Hereinafter, the present invention will be described in detail.
The method for adsorbing and separating phospholipids and glycolipids according to the present invention is a method for separating and concentrating phospholipids and glycolipids contained in a sample from other components. Here, the sample is not particularly limited as long as it is a composition containing phospholipids or glycolipids. For example, it is a solution containing a plurality of types of lipids.

  In the method for separating phospholipids and glycolipids according to the present invention, the phospholipids and glycolipids are not limited at all, and any cell-derived phospholipids and glycolipids can be targeted for separation.

In the method for separating phospholipids and glycolipids according to the present invention, when a sample containing phospholipids and glycolipids is supplied to a separation means using metal oxide and adsorbed, the conditions are acidic.
Next, in order to selectively elute glycolipids having adsorbed sugars of three or more sugars, an organic solvent containing hydroxycarboxylic acids such as aromatic hydroxycarboxylic acid and aliphatic hydroxycarboxylic acid is added. Further, when the adsorbed phospholipid is selectively eluted, a basic organic solvent is added.
When it is desired to obtain only phospholipids, hydroxycarboxylic acids may be added to the sample in advance, or may be supplied alone in advance to the separation means before supplying the sample to the separation means. Thereby, adsorption | suction of the glycolipid to a isolation | separation means is inhibited, and phospholipid is selectively adsorbed and eluted. If glycolipids are required, hydroxycarboxylic acids should not be added to the sample or separation means in advance.

Here, the aliphatic hydroxycarboxylic acid refers to a hydroxycarboxylic acid having an aliphatic skeleton, and the aromatic hydroxycarboxylic acid refers to one having an aromatic ring in the skeleton. It may have both a group and an aromatic ring.
Specifically, examples of the hydroxycarboxylic acid include α-hydroxycarboxylic acids such as glycolic acid, lactic acid, malic acid, and citric acid, and aromatic carboxylic acids such as 2,5-DHB (dihydroxybenzoic acid). The specific compounds exemplified above may be used alone, or a plurality of types may be mixed and used.

Consider the reason for glycolipid elution with hydroxycarboxylic acid.
Metal oxides (such as titanium oxide and zirconia oxide) are known to work as Lewis acids. A Lewis acid refers to a substance that can accept an electron pair from a chemical species having OH or a lone electron pair, and this species has a ligand exchange ability.

  As a result, it is known that the hydroxyl group of a substance forms a complex with metal oxide ions (titanium ions and zirconium ions) and is adsorbed and retained. This adsorption / holding force depends on the three-dimensional position of the hydroxyl group in the molecule.

  From this, it is considered that a part of glycolipid having a hydroxyl group is adsorbed and held on the metal oxide surface by a three-dimensional structure. Hydroxycarboxylic acids also have a structure capable of forming a complex with metal oxides.

  For this reason, when hydroxycarboxylic acid is added to the state where glycolipids are adsorbed / held on metal oxide, hydroxycarboxylic acid forms a complex, so that glycolipids are released from the adsorption / retention on metal oxide and eluted. It is thought that such a phenomenon occurs.

The method for separating phospholipids and glycolipids according to the present invention will be described with reference to FIG.
This is an example, and the substance, the order of putting the substance, etc. are not limited to this. The lipids to be treated are extracted from cells and tissues with an organic solvent according to a conventional method, placed in a container, and the solvent is evaporated to dryness.

In this container, an adsorbing solvent (mild acidic organic solvent such as HIPWHCl) in which appropriately treated titania powder is suspended is added, and the lipids and titania powder are mixed well. Through this process, phospholipids and tri- or higher glycolipids (other than simple lipids and glycolipids up to disaccharides) are adsorbed to titania.
The solution containing titania particles is separated into titania particles and a supernatant by a method such as centrifugation, and the supernatant is collected. This supernatant contains simple lipids and glycolipids with sugars up to disaccharides. If necessary, a washing solvent (HIPW or HIPWHCl) is added to the separated titania particles, and washing is performed, and titania particles are recovered by a method such as centrifugation.
The solvent used as the adsorption solvent contains an organic solvent capable of dissolving lipids and needs to exhibit acidity. As an example of a solvent that satisfies this requirement, a solvent (HIPWHCl) appropriately mixed with hexane, isopropanol, water, and hydrochloric acid can be used. For example, a solvent adjusted with hexane: isopropanol: water: hydrochloric acid = 120: 80: 5: 1 can be used.
As the washing solvent, a mixed solvent having the same composition as the adsorption solvent or a solvent in which hexane, isopropanol and water are mixed can be used.

Add phospholipid elution solvent A (for example, 0.1 mol / L ammonium methanol) to the collected titania and mix well. By this process, only the phospholipid adsorbed on the titania particles is eluted in the solvent. Only phospholipid adsorbed on the titania particles is eluted in the solvent by a method such as centrifugation of this solution. This solution is separated from titania and the supernatant by a method such as centrifugation, and the supernatant is collected and removed. This supernatant contains phospholipids. If necessary, titania precipitated with a washing solvent (for example, HIPW or phospholipid elution solvent) is washed, and titania is recovered by a method such as centrifugation.
The solvent used as the phospholipid elution solvent A is not limited to ammonium methanol. If the solvent is an organic solvent that can dissolve lipids and has basic characteristics, phospholipids can be selectively eluted. Is possible.

Glycolipid elution solvent B (for example, 10 mg / ml DHB in MeOH) is added to the collected titania and mixed well. By this process, glycolipids of trisaccharide or higher adsorbed on the titania particles are eluted in the solvent. From this solution, titania and the supernatant are separated by a method such as centrifugation, and the supernatant is recovered. This supernatant contains glycolipids having a sugar of 3 or more sugars.
If the solvent used as the glycolipid elution solvent B contains a solvent capable of dissolving lipid and contains a hydroxycarboxylic acid, the glycolipid can be selectively eluted.

  Here, the metal oxide used for the separation means is meant to include all substances known to have affinity for one or both of phospholipid and glycolipid. Among these, examples of the metal oxide include titanium oxide and zirconium oxide.

As titanium oxide, in addition to titanium dioxide (titania, TiO ₂ ), titanium monoxide (TiO), nitric oxide (Ti ₂ O ₃ ), trititanium pentoxide (Ti ₃ O ₅ ), tetratitanium oxide ( Ti ₄ O ₇ ).

  In the method for separating phospholipids and glycolipids according to the present invention, these metal oxides may be used alone, or a plurality of types may be mixed and used. In particular, as the metal oxide, titanium oxide and zirconium oxide are preferably used alone or in combination because of their high affinity for phospholipids and glycolipids.

In the method of the present invention, the metal (such as titania or zirconia) on the surface of the carrier (separation medium) itself is involved in the separation of phospholipids and glycolipids. By the way, in the separation by the reverse phase method, the thing related to the separation is the “amount of functional group” bound on the separation medium.
That is, in the present method, “separation performance” = “the amount of functional groups bound on the carrier”, not “separation performance” = “surface area of a single substance”.

Hereinafter, the reason for the difference in recovery due to the difference in pore diameter and surface area will be considered.
As a premise, it is considered that the principle of adsorption / elution does not differ greatly depending on the type of metal oxide, so the pore diameter and surface area, which are other factors, are considered.

First, regarding the pore size, the molecular size of the substance to be separated is important.
While the molecular size of phospholipids and glycolipids (separation target substances) to be separated is about 40 to 60 mm, simple lipids that are not separation target substances and a large amount of oleic acid and cholesterol that exist in the living body (such as oleic acid and cholesterol) The non-separation target substance) has a molecular size of about 20 to 30 cm.

If the pore size is more than twice the molecular size of the substance to be separated (approximately 100cm), both the separation target substance and the non-separation target substance can enter and exit the pores without interfering with each other. If the substance enters the pore, it will leave the pore as it is, and if the substance to be adsorbed enters the pore, a reaction (exchange reaction) occurs that is adsorbed in the pore. Adsorption inhibition is unlikely to occur.
Therefore, a sufficient recovery rate (separability) can be obtained when the surface area is large and the pore diameter is large, such as the zirconia particles used in Example 1.

  On the other hand, when the pore diameter is about 60 to 70 mm, the separation target substance and the pore diameter are close to each other. Therefore, when a small non-separation target substance coexists, a competition occurs with respect to entering the pores. The substance to be separated tends to enter the pores first.

Furthermore, since the pore diameter is small, it is difficult for an exchange reaction to enter and exit the pore, and the probability that a substance to be separated having a large molecular size is adsorbed in the pore decreases.
Since such a phenomenon occurs, it is considered that titania particles having a surface area of 42 m ² / g particle diameter of 5 μm and a pore diameter of 70 mm did not provide sufficient recovery and separation (see Example 3).

According to this concept, it can be said that a larger pore size is more useful, but since the pore size and surface area are generally inversely proportional, an excessively large pore size will decrease the surface area and reduce the adsorption / separation performance. cause. In addition, when the pore diameter is increased, the strength of the particles is reduced, so that the particles are damaged. The broken particles are difficult to separate by a method such as centrifugation, and the operation becomes complicated.

  When creating a substance having pores, it is generally created by controlling the pore diameter. However, in a substance prepared by controlling the pore diameter, the relationship between the pore diameter and the surface area becomes inversely proportional. That is, when the pore diameter is increased, the surface is decreased, and when the pore diameter is decreased, the surface area is increased. In the present invention, if the pores are small, it affects the entry / exit of the substance to be separated, which makes it difficult to implement. If the pore diameter is increased, the surface area involved in adsorption / retention decreases, and sufficient adsorption / retention cannot be realized.

In other words, it is difficult to implement unless the pore diameter and the surface area are within a certain range. In setting this feasible range, a range in which the product of “surface area (unit: m ² / g)” and “pore diameter (unit: Å)” is 3000 or more is considered as the feasible range.
However, those having a pore diameter of 1000 mm or more are difficult to implement because the strength of the separation carrier is significantly reduced in actual use.

Surface area is related adsorption capacity of the target substance (the load), because they depend on the substance concentration in the sample, but can not be determined unconditionally, with respect to what is the surface area 3m 2 ^{/ g} or more, It can be carried out for measurement and surface area measurement.
In addition, those having a pore diameter of 10 mm or less are not included in the feasible range because the pore diameter is too small and the inner surface of the pore is hardly involved in adsorption / separation. As a result, the surface area must be 3 m ² / g or more.

Hereinafter, the surface area when the pore diameter is 60 to 70 mm is considered.
When the pore diameter is 60 to 70 mm, competition occurs in and out of the pores, and it is difficult to generate an exchange reaction. Therefore, not only phospholipids / glycolipids that are separation targets but also non-separation target substances are included in advance All lipids must have a surface area sufficient to contact the surface of the separation carrier.

  When the present invention is carried out on an actual sample such as blood, it is necessary to selectively adsorb and retain phospholipids and glycolipids from a sample containing lipids other than phospholipids and glycolipids. When selectively adsorbing, a separation carrier having an ability to adsorb and retain all lipids regardless of phospholipids and glycolipids is required.

  For example, in the case of a blood sample, the standard value for human total lipid is 400-800 mg / 100 mL. Since blood collection for collecting an analysis sample is a burden on the target animal or human, it should be 1 mL or less. Furthermore, since the collected sample is usually used for various studies other than phospholipids and glycolipids, the amount of blood that can actually be handled in this analysis is considered to be about 100 μL. In addition, it is preferable to collect a small amount of sample, but the amount of phospholipid / glycolipid to be separated is also small.

  In particular, in the case of glycolipids, in the present invention, glycolipids having a sugar chain of three or more sugars are separated, so the content is considered to be several percent of the total lipid, and the amount of each component is 1 μg or less. And detection is likely to be difficult. That is, about 100 μL is ideal as the total blood sample amount considered in the current detection method.

When 100 μL of blood sample is processed, the total amount of lipid contained in the sample is 0.4 to 0.8 mg as a standard value, but since this value is a standard value, approximately 1 mg of lipid is processed. It is hoped that it can be done. That is, a separation carrier capable of contacting all about 1 mg of lipid is effective in the present method.
In the present invention, since the phospholipid and glycolipid are separated by the interaction with a metal oxide such as a tyranol group present on the surface of the carrier (gel), the surface area that can hold at least 1 mg as a requirement for the pretreatment gel. It is recommended to have The molecular weight of lipids is widely distributed, but considering a lipid having a glycerol skeleton, the molecular weight is about 200. In order to hold 1 mg of this substance, it is necessary to hold 1 × 10 ⁻³ ÷ 250 = 4 × 10 ⁻⁶ mol = 4 μmol or more.

On the other hand, when a 100 μL sample is handled, the gel as the pretreatment solid phase is dispersed in the sample solution and mixed. Therefore, the amount of the gel needs to be 10 mg or less of 1/10 or less of the solution.
When more gel is added, the mixing of the solution and the gel becomes insufficient, and the contact efficiency decreases. In addition, there is a method of diluting the sample. In this method, however, the contact efficiency with the gel decreases due to the decrease in the concentration of the target component in the sample, resulting in a decrease in the separation efficiency.

  Therefore, in order to reliably hold and adsorb about 4 μmol (1 mg) of lipid contained in 100 μL of sample, a separation carrier having 4 μmol / 10 mg = 400 μmol / 1 g or more of tyranol groups (metal oxide groups) is required. Become.

Since the tyranol group on the surface of titania is considered to be 8 μmol (8 μmol / m ² ) per 1 m ² like silanol, the surface area is 400 μmol / 1 g ÷ 8 μmol / m ^{2 in} order to have a tyranol group of 400 μmol / 1 g or more. = 50 m ² / g is required.

From the above, when using a metal oxide having a pore diameter of 60 to 70 mm, it is considered that the metal oxide cannot be used unless the surface area is at least 50 m ² / g. In other words, unless the product of “surface area (unit: m ² / g)” and “pore diameter (unit: Å)” is 3000 or more, it is considered that the product cannot be used.

A monolith structure to which metal oxide is attached or contained will be described.
The monolith structure is mainly produced by a sol-gel method. That is, using metal alkoxide or reactive organic monomer alone or in combination, partially hydrolyzed and polycondensed to form a colloidal oligomer (formation of sol), and further hydrolyzed and polymerized. It is synthesized by promoting cross-linking and creating a three-dimensional network skeleton structure (generation of gel).

  Thus, the monolith structure has a structure having a continuous hole (through pore) that is continuous from one end to the other end between the skeleton structures. In addition, pores (mesopores) having an arbitrary pore diameter can be formed on the skeleton surface. For this reason, the monolith structure has a structure that also has pores for increasing the surface area involved in the separation while securing continuous pores that are passages of the liquid sample.

By using titanium alkoxide, zirconium alkoxide, or the like as a metal alkoxide that is a raw material used for producing the monolith structure, it is possible to create a monolith structure having titanium oxide or zirconium oxide as a skeleton.
In addition, a monolith structure having a metal oxide on the surface can be prepared by reacting titanium alkoxide, zirconium alkoxide, or the like with the surface of the monolith structure formed using silane alkoxide or the like.

Hereinafter, although the separation method of the phospholipid and glycolipid which concerns on this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
In addition to the method of dispersing and mixing the separation carrier in the liquid, the contact between the metal oxide and the liquid is carried out by placing the separation carrier across the diameter in a cylinder open at both ends, and the pressure difference (negative pressure due to suction, It is also possible to move the liquid by positive pressure due to discharge, etc., centrifugal force, gravity (spontaneous fall), and contact with the metal oxide. When a monolith structure is used, liquid can be passed through the structure by this method.

In Example 1, a sphingo, which is a phospholipid from a lipid mixed sample containing sphingomyelin (Sph), which is a phospholipid, galactocerebroside (GalCer), which is a glycolipid having a monosaccharide, and cholesterol (CHO), which is a simple lipid. Myelin was purified. First, 25 μg each of sphingomyelin (Sph), galactocerebroside (GalCer) and cholesterol (CHO) was dissolved in 300 μL of HIPWHCl (hexane / 2-propanol / water / HCl, 120/80/5/1).
In addition, 100 mg of titania powder (surface area 100 m ² / g, pore diameter 60 mm, particle size 5 μm) and zirconia powder (surface area 30 m ² / g, pore diameter 300 mm, particle diameter 5 μm) were added in methanol / water 4/6, 1 ml. Suspend, mix by inverting for 10 minutes, centrifuge, discard the supernatant, and add 1 ml of HIPWHCl (hexane / 2-propanol / water / HCl, 120/80/5/1) to each to suspend. Inverted for 10 minutes.

Next, the lipid mixed sample, the titania particle suspension and the zirconia particle suspension were mixed, mixed by inverting well for 10 minutes, and then centrifuged to collect the supernatant as a non-adsorbed fraction.
Furthermore, about 300 μL of HIPW (hexane / 2-propanol / water, 60/40/3) was added to the sediment (titania particles, zirconia particles) from which the non-adsorbed fraction was removed, and after mixing well, the supernatant was collected by centrifugation. Added to the previously collected non-adsorbed fraction.

  By adding 300 μL of 0.1 mol / L ammonium methanol to the sediment (titania particles, zirconia particles) from which the non-adsorbed fraction has been removed and mixing well by inverting for 20 minutes, the substances adsorbed on the titania particles and zirconia particles are removed. The supernatant collected after elution and centrifugation was used as an elution fraction.

  Each of the non-adsorbed fraction and the eluted fraction was evaporated to dryness, developed with thin layer chromatography (TLC) using developing solvent: chloroform: methanol: water = 65: 25: 4, and then carbonized with copper sulfate. Detected.

FIG. 2 is a developed image by TLC analysis of the non-adsorbed fraction, and FIG. 3 is a developed image by TLC analysis of the eluted fraction.
2 and 3, a standard mixed sample for identifying a development pattern was developed in “lane std”, and a lipid mixed sample (CHO, GalCer Sph 2 μg each) before treatment was developed in “lane 1”.
The “non-adsorbed fraction using titania particles” was developed in “lane 2” and “lane 3” in FIG. 2, and the non-adsorbed fraction using zirconia particles was developed in “lane 4” and “lane 5”.
Also in FIG. 3, the lipid mixed sample was developed in “lane 1” as in FIG. Elution fractions using titania particles were developed in “lane 2” and “lane 3”, and elution fractions using zirconia particles were developed in “lane 4” and “lane 5”.

  2 and 3, in Lane 1 where the lipid mixed sample was developed, a cholesterol (CHO) band was observed at the top, followed by two galactocerebroside (GalCer) bands, a position close to the origin (bottom). Shows two sphingomyelin (Sph) bands.

  In lanes 2 to 5 of FIG. 2 in which the non-adsorbed fraction using titania particles and zirconia particles was developed, bands corresponding to cholesterol and galactocerebroside were confirmed, but bands corresponding to sphingomyelin were not confirmed. .

  On the other hand, in lanes 2 to 5 of FIG. 3 where the elution fraction was developed, the bands corresponding to cholesterol and galactocerebroside seen in FIG. 2 were not confirmed, but only the bands corresponding to sphingomyelin were confirmed.

Therefore, when the above method is used, simple lipids (cholesterol) other than the phospholipid sphingomyelin and glycolipids having a monosaccharide (galactocerebroside) are not adsorbed to titania particles and zirconia particles. The sphingomyelin, which is a phospholipid, is adsorbed on titania particles and zirconia particles, so it does not appear in the non-adsorbed fraction and is eluted only in the eluted fraction by specific elution. I understand.
According to this method, it is possible to separate phospholipids and non-phospholipids by simple operations using titania particles and zirconia particles.

In Example 2, the separation of glycolipids and phospholipids by titania particles was examined. The following mixtures were used as samples for separation studies.
Monohexosylceramide (CMH): glycolipid with monosaccharide Dihexosylceramide (CDH): glycolipid with disaccharide Trihexosylceramide (Gb3): glycolipid with trisaccharide Globoside (Gb4) : Glycolipid with tetrasaccharide Sphingomyelin (Sph): Phospholipid

Titania particles (surface area 100 m ² / g, pore size 60 mm, particle size 5 μm, 100 mg) used for separation are first suspended in methanol / water 4/6, 1 ml, mixed by inverting for 10 minutes, centrifuged, and the supernatant And 1 ml of HIPWHCl (hexane / 2-propanol / water / HCl, 120/80/5/1) was added to resuspend the titania powder and mixed by inverting for 10 minutes.

  About 300 μL of this titania powder suspension was added to the above sample and mixed well by inverting for 10 minutes, and then the supernatant was removed by centrifugation. Furthermore, about 300 μL of HIPW (hexane / 2-propanol / water, 60/40/3) was added to the sediment (titania powder) after centrifugation, and after mixing well, the supernatant was removed by centrifugation.

Next, 300 μL of 0.1 mol / L ammonium methanol was added to the sediment (titania) after centrifugation, mixed well by inverting for 20 minutes, and the centrifuged supernatant was collected as elution fraction 1.
Again, the above process was performed and elution fraction 2 was collected.

  Next, 300 μL of DHB / methanol (10 mg / mL) was added, mixed, and centrifuged, and the supernatant was collected as elution fraction 3. Each of elution fraction 1, elution fraction 2, and elution fraction 3 was evaporated to dryness, and then developed by thin layer chromatography (TLC) using a developing solvent: chloroform: methanol: water = 65: 25: 4. Detection was performed by carbonization with copper sulfate and sugar with orcinol.

  In carbonization coloring with copper sulfate, all the substances in which hydrocarbons exist are colored, so all the mixtures used as samples are colored. On the other hand, in the case of sugar coloring with orcinol, only those containing sugar are colored, and therefore, substances other than sphingomyelin (Sph): phospholipid are colored in the sample mixture.

  In the separation under the above TLC development conditions, it is difficult to distinguish sphingomyelin and Gb4 because they overlap and are detected. Therefore, it was determined whether or not Gb4 is a glycolipid by using sugar coloring.

FIG. 4 uses carbonization coloring, and FIG. 5 uses sugar coloring.
4 and 5, “lane std” is a standard mixed sample for identifying a development pattern, “lane 1” is a mixture of globoside (Gb4) and sphingomyelin (Sph) before processing, and “lane 2” is Elution fraction 1, which is the first elution fraction with 0.1 mol / L ammonium methanol, “lane 3” is elution fraction 2, which is the second elution fraction with 0.1 mol / L ammonium methanol, “ Lane 4 "developed elution fraction 3, which is the third elution fraction with DHB / methanol.

  In FIG. 4 using carbonized color development, in lanes 2, 3, and 4, bands are observed at positions corresponding to globoside (Gb4) or sphingomyelin (Sph).

  On the other hand, in FIG. 5 using sugar coloring, a band is detected only in lane 4 at the same position as in FIG. In elution fraction 1 (lane 2) and elution fraction 2 (lane 3), which are elution fractions with ammonium methanol, a band is detected in carbonized color development, but no band is detected in sugar color development. It can be seen that the lipid not contained, that is, sphingomyelin (Sph): phospholipid was specifically eluted.

  Moreover, since a band is detected for the first time in the elution fraction 3 (lane 4) due to sugar coloration, it is understood that a lipid (globoside (Gb4)) containing a sugar is eluted for the first time by elution with DHB / methanol.

  From this, phospholipids are eluted with 0.1 mol / L ammonium methanol, but not with DHB, and glycolipids are not eluted with 0.1 mol / L ammonium methanol, but are selectively eluted from titania particles with DHB. It was shown that it can be done.

  Lane std: Standard, Lane 1: Sphingomyelin + Gb4, Lane 2: 0.5 M ammonia methanol (first time), Lane 3: Ammonium methanol (second time), Lane 4: 10 mg / mL DHB / methanol

Lane std: Standard Lane 1: Sphingomyelin + Gb4
Lane 2: elution with 0.1 mol / L ammonium methanol (first time): elution fraction 1
Lane 3: elution with 0.1 mol / L ammonium methanol (second time): elution fraction 2
Lane 4: DHB / methanol: elution fraction 3

With the same operation as in Example 1, the recovery rate of phospholipid sphingomyelin (Sph) was examined using titania particles having different surface areas. The titania particles used have a surface area of 42 m ² / g particle size of 5 μm and a pore size of 70 μm and a surface area of 100 m ² / g particle size of 5 μm and a pore size of 60 μm.

FIG. 6 shows TLC developed images of the non-adsorbed fraction and the eluted fraction.
Lane 1: lipid mixed sample before treatment (CHO, GalCer, Sph)
Lanes 2 and 3: titania particles surface area 42 m ² / g particle size 5 μm pore size 70 mm
Lanes 4 and 5: titania particles surface area 100 m ² / g particle size 5 μm pore size 60 mm

In Example 1, the results of using zirconia particles (surface area 30 m ² / g particle size 5 μm, pore size 300 mm) in FIG. 2 lanes 4 and 5 and FIG. .

In the TLC developed image of the non-adsorbed fraction, when zirconia particles (surface area 30 m ² / g particle diameter 3 to 5 μm, pore diameter 300 mm) are used (FIG. 2, lanes 4 and 5), titania particles (surface area 100 m ² / g particle diameter) In the case of using 5 μm pore diameter (60 mm) (FIG. 6, non-adsorbed fraction lanes 4 and 5), sphingomyelin was not detected in the non-adsorbed fraction.
On the other hand, when titania particles (surface area 42 m ² / g particle diameter 5 μm, pore diameter 70 mm) are used (FIG. 6, lanes 1 and 2), sphingomyelin is detected. This indicates that unadsorbed sphingomyelin remained in the non-adsorbed fraction due to lack of sufficient adsorption performance.

In the TLC developed image of the eluted fraction, when zirconia particles (surface area 30 m ² / g particle size 3 to 5 μm, pore size 300 mm) are used (FIG. 3, lanes 4 and 5), titania particles (surface area 100 m ² / g particle size) When using titania particles (surface area 42 m ² / g particle size 5 μm pore size 70 mm) compared to the case using 5 μm pore diameter 60 mm (FIG. 6 elution fraction lanes 4 and 5) (FIG. 6 elution fraction) In the lanes 2 and 3), the detection amount of the eluted fraction was small.
This means that with titania particles (surface area 42 m ² / g particle size 5 μm, pore size 70 mm), sufficient phospholipid adsorption performance cannot be obtained, and phospholipids remaining without being adsorbed are detected in the non-adsorbed fraction. Therefore, it shows that the detection amount of the eluted fraction is also reduced .

Example 4 uses Titansphere® TiO, a column (10 × 4.0 mm ID) packed with titania (surface area 100 m ² / g, particle size 5 μm) to specifically select phospholipids Separation was considered.

  The sample used for analysis is a mixture of non-phospholipid D-erythro-Sphingosine (1.8 mg / 500 μl MeOH) and phospholipid Sphingomycin (1.8 mg / 500 μl MeOH) in equal amounts. did.

The analysis was performed by the following method.
First, the sample mixed sample was injected in a state where a mixed solution of H ₂ O (0.1% TFA): CH ₃ CN: MeOH = 8: 82: 10 was allowed to flow through the column. Collect as a pass-through fraction. FIG. 7 is a chromatogram showing the position of the preparative fraction, and the pass-through fraction is the portions 1 and 2 in FIG.

Next, the solvent was switched to H ₂ O (0.1% TFA): CH ₃ CN: MeOH: H ₃ PO ₄ = 8: 82: 10: 0.6, and the elution fraction from the column was changed to 3 in FIG. 10 to 10 fractions.
The above fractions were analyzed by D-erythro-Sphingosine and Sphingomyelin using a separable column Inertsil (registered trademark) NH ₂ (150 × 4.6 mm ID), and the presence or absence of each substance. (FIG. 8 shows the analysis results of the fractions collected, and the chromatogram comparison diagram of each fraction).

The measurement conditions are as follows.
Column: Titansphere (registered trademark) TiO (10 × 4.0 mm ID)
Eluent: A: H ₂ O (0.1% TFA): CH ₃ CN: MeOH = 8: 82: 10
B: H ₂ O: CH ₃ CN: MeOH: H ₃ PO ₄ = 8: 82: 10: 0.6
0 to 2.9 min A / B = 100/0
3-20min A / B = 0/100
Flow rate: 1.0 ml / min
Column temperature: Room temperature Detection wavelength: UV210nm
Sample: 50 μl of prepared lipid mixture

The chromatogram shown in preparative fraction number 0 (top) in FIG. 8 is the result of confirming the presence or absence of each substance without using a column packed with titania particles. Since no processing is performed, both D-erythro-Sphingosine and Sphingomyelin are detected.
The chromatogram shown in the middle of FIG. 8 is an analysis of fraction 1, which is a fraction passed through the column under the conditions of H ₂ O (0.1% TFA): CH ₃ CN: MeOH = 8: 82: 10. It is a result. A peak corresponding to D-erythro-Sphingosine was detected, but a peak corresponding to Sphingomyelin was not detected.

The chromatogram shown in the lower part of FIG. 8 shows the analysis result of fraction 8 eluted from the column under the conditions of H ₂ O: CH ₃ CN: MeOH: H ₃ PO ₄ = 8: 82: 10: 0.6. It is. Contrary to fraction 1, which is a normal fraction, no peak corresponding to D-erythro-Sphingosine was detected, but a peak corresponding to Sphingomyelin was detected.

The analysis conditions are as follows.
Column: InertSil® NH ₂ (150 × 4.6 mm ID)
Eluent: H ₂ O: CH ₃ CN: MeOH = 8: 87: 5
Flow rate: 1.0 ml / min
Column temperature: room temperature Detection wavelength: UV 205 nm
Sample: 0. 50 μl of the prepared lipid mixture diluted 66-fold with eluent
1-10. 50 μl of each fraction

  In addition, in the method in which the column is pretreated with the column using the metal oxide particles and the monolith structure and then directly introduced into the analytical column, the so-called column switching method, the present invention can be used to selectively select phospholipids. It can be analyzed.

An embodiment using a monolith structure will be described.
Phospholipids and glycolipids were separated using titania monolith 1 prepared by coating titania on the surface of the silica monolith structure. The titania monolith 1 is fixed in a general-purpose pipette tip 2 in a disc-like shape, and is attached to the pipetter 3 so that the sample solution / solvent and the titania monolith 1 are brought into contact with each other by suction / discharge operation of the pipetter 3. I can do it.

The titania monolith 1 used is as follows.
Continuous pore diameter (through pore) 10 μm, pore diameter (mesopore) 200 mm, surface area 200 m ² / g

  The sample used for the separation is a lipid mixed solution in which sphingomyelin (Sph), galactocerebroside (GalCer), cholesterol (CHO), and clobozide (Gb4) are dissolved in HIPWHCl.

The separation operation was performed according to the following procedure.
1. Aspirate and discharge 300 μL of PBS (phosphate buffer) and pass through titania monolith 1. Repeat this operation three times.
2. Pass 300 μL of methanol: water = 4: 6 through titania monolith 1 by suction and discharge.
3. Pass 300 μL of HIPWHCl (hexane: 2-propanol: water: HCl = 120: 80: 5: 1) through a titania monolith by suction and discharge. Repeat this operation twice.
4). The suction / discharge of 300 μL of the lipid mixture (cholesterol, galactocerebroside, sphingomyelin, clobozide) is repeated 9 times to bring the lipid mixture into contact with the titania monolith 1.
Repeat step 5.4 once more, but collect the discharge liquid in a new tube. This is referred to as “non-adsorbed fraction”.
6). 300 μL of HIPW (hexane: 2-propanol: water = 60: 40: 3) is aspirated and discharged, and the discharged liquid is collected and used as a washing fraction.
7. Aspirate and discharge 300 μL of 0.1 mol / L ammonium methanol, collect the discharged liquid, and set it as “elution fraction 1”.
8). Aspirate and discharge 300 μL of DHB / methanol, collect the discharged liquid, and set it as “elution fraction 2”.

  The obtained “non-adsorbed fraction”, “washed fraction”, “eluted fraction 1”, and “eluted fraction 2” were developed by TLC to perform carbonization coloring and sugar coloring. In FIG. 9, lane 1 has a lipid mixture solution before the separation operation, lane 2 has a “non-adsorbed fraction”, lane 3 has a “washed fraction”, lane 4 has “elution fraction 1”, lane 5 "Elution fraction 2" was developed.

In carbonized color development, in lane 2 (non-adsorbed fraction), galactocerebroside, which is a glycolipid having cholesterol and a monosaccharide, was detected, but sphingomyelin, which is a phospholipid, was not detected. In lanes 4 (elution fraction 1) and 5 (elution fraction 2), globozide, a glycolipid having sphingomyelin or tetrasaccharide, was detected.
In saccharide color development, only galactocerebroside and globozide develop color in the lipid mixed sample, so that galactocerebroside was detected in lane 1 and globozide was detected in lane 5.

  As a result, galactocerebroside, which is a glycolipid having simple cholesterol and monosaccharide, is not adsorbed to titania monolith under the conditions of HIPWHCl, and is a glycolipid having sphingomyelin and tetrasaccharide as phospholipids. It can be seen that a certain globozide is adsorbed. Furthermore, it can be seen that sphingomyelin is eluted under ammonium methanol conditions, and globozide is eluted under DHB / methanol conditions.

This result is equivalent to Example 2 using particles.
According to this method, even when a pipette tip fitted with a monolith structure is used, it is possible to selectively adsorb and separate phospholipids and glycolipids.

The present invention--the present invention separation method schematic explanatory diagram based on examples TLC image of non-phospholipid based on the present invention-Example TLC image of phospholipid based on the present invention-Example TLC developed image showing phospholipid elution state based on the present invention-Example TLC development image showing glycolipid elution state based on the present invention-Example TLC developed image showing difference in recovery due to difference in surface area of titania gel Chromatogram of preparative phospholipid according to the present invention-Example Chromatogram of preparative fraction with and without titania according to the present invention-Example TLC development image showing elution state of glycolipid based on one embodiment of the present invention TLC developed image showing elution state of phospholipid based on one embodiment of the present invention Explanatory drawing of the monolith chip | tip used for one Example of this invention

Claims (6)

A method for adsorbing and separating phospholipids and glycolipids, wherein a sample containing phospholipids and glycolipids is brought into contact with and adsorbed to titanium oxide and / or zirconium oxide under acidic conditions. A sample containing phospholipids and glycolipids is brought into contact with titanium oxide and / or zirconium oxide under acidic conditions to adsorb phospholipids and glycolipids at the same time, and then the phospholipids are eluted with a basic solvent. A method for adsorbing and separating phospholipids and glycolipids, wherein the glycolipids are eluted with a solvent to separate them separately.   The method for adsorbing and separating phospholipids and glycolipids according to claim 2, wherein the hydroxycarboxylic acid is an aliphatic hydroxycarboxylic acid or an aromatic hydroxycarboxylic acid. The titanium oxide, titanium dioxide, titanium monoxide, trioxide titanium, trititanium pentoxide, to any one of claims 1 to 3, wherein, characterized in that at least one selected from heptoxide titaniumtetrachloride The method for adsorbing and separating phospholipids and glycolipids as described. The titanium oxide and / or zirconium oxide has a surface area of 3 m ² / g or more and a pore diameter (unit: m ² / g) and a pore diameter (unit: Å) in the range of 10 to 1000 pores. It is 3000 or more, The adsorption separation method of the phospholipid and glycolipid as described in any one of Claims 1-4 characterized by the above-mentioned. The method for adsorbing and separating phospholipids and glycolipids according to any one of claims 1 to 5, wherein the titanium oxide and / or zirconium oxide is contained in or attached to a monolith structure.

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