Preparation method of low molecular component derived from lipid on skin

Abstract

To provide a method for preparing a low molecular component which is a metabolite from an RNA extraction residue which is generated in a step for extracting nucleic acid using a phenol-chloroform method from lipid on a skin collected from a subject.SOLUTION: There is provided a preparation method for preparing a low molecular component derived from lipid on a skin other than a nucleic acid, which comprises following steps (1) and (2) for: (1) adding a strong base solution to an organic layer which is acquired from the lipid on a skin collected from a subject by a phenol-chloroform extraction method, for mixing them, then removing a separated water layer; and (2) adding water to the organic layer from which the water layer is removed, for mixing them, then collecting the organic layer.SELECTED DRAWING: None

Description

Technical Field

[0004] ,

[0003] , , ,

[0001] The present invention relates to a method for preparing low molecular weight components derived from epidermal lipids.

Background Art

[0002] In recent years, with the rapid development of analytical techniques, it has become possible to analyze molecules (nucleic acids, proteins, metabolites, etc.) in various biological samples in detail. Furthermore, the development of technologies for examining the current and even future physiological states of the human body through these molecular analyses is also progressing. For example, diagnostic and predictive technologies for diseases using nucleic acid molecules in minimally invasive or non-invasive biological samples are being developed by many research institutions around the world, and their application has advanced by leaps and bounds. Among them, the most widely used is the diagnostic technology using DNA or RNA. The diagnostic technology using DNA is generally a method of diagnosing future disease risks and one's own constitution by collecting saliva or cells in the oral cavity and analyzing single nucleotide polymorphisms in the genomic DNA contained therein. On the other hand, diagnostic technologies using RNA, proteins, and metabolites are methods of diagnosing the presence or absence of diseases in the current living body based on the expression information and existing concentrations contained in biological samples such as blood and urine.

[0003] Generally, a comprehensive analysis method has been established for RNA, and abundant information can be obtained by one analysis. On the other hand, in recent years, the analysis technology for metabolites has also advanced by leaps and bounds, and comprehensive analysis of metabolites has become possible. Metabolites have many stable low molecular weight components compared to RNA, which is easy to decompose, and are relatively easy to handle. In addition, they are closer to the phenotype than RNA and may be able to diagnose the current and future health status and disease risks with high accuracy. Furthermore, by using metabolite data as well as RNA data simultaneously, it may be possible to enhance the usefulness and expand the application range in the diagnosis of current and future health status and diseases.

[0004] Patent Document 1 has shown that RNA can be extracted from surface lipids (SSL) of the skin, and proposes its application to biomarker discovery and diagnostic techniques using RNA from SSL, which can be collected non-invasively and easily. There are several methods for RNA extraction, but the phenol-chloroform method is the most common because it is simple and yields high amounts of RNA. The phenol-chloroform method is particularly effective for lysing lipid-rich tissues and has been used for RNA extraction from SSL as described above. However, there are no descriptions of detecting metabolites simultaneously with RNA, nor are there any reports of techniques combining the phenol-chloroform method with metabolite extraction. A method has been reported for simultaneously measuring RNA and metabolites in which metabolites are first extracted with an organic solvent, and then RNA is extracted (Non-Patent Literature 1), but a method that extracts unstable RNA later is undesirable. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Public Gazette No. 2018 / 008319 [Non-patent literature]

[0006] [Non-Patent Document 1] Woodward, A. et al. (2021) 'Integrated metabolomics and transcriptomics using an optimized dual extraction process to study human brain cancer cells and tissues', Metabolites, 11(240). doi: 10.3390 / metabo11040240. [Overview of the project] [Problems that the invention aims to solve]

[0007] In one embodiment, the present invention provides a method for preparing low-molecular-weight components, which are metabolites, from RNA extraction residue generated during the process of nucleic acid extraction using the phenol-chloroform method from skin surface lipids collected from a subject. In another embodiment, the present invention provides a method for analyzing low molecular weight components, which includes analyzing the low molecular weight components prepared by the above method. [Means for solving the problem]

[0008] The inventors have discovered that by adding a strong basic aqueous solution to the organic layer (phenol-chloroform layer), which is the RNA extraction residue, phenol is converted to phenoxide, separating it into two layers: an aqueous layer containing phenoxide and an organic layer containing low-molecular-weight metabolites. After removing phenol from the organic layer, a washing operation with water is performed, which allows for efficient extraction of the low-molecular-weight components.

[0009] In other words, the present invention relates to the following 1) to 6). 1) A method for preparing low molecular weight components derived from skin surface lipids other than nucleic acids, comprising the following steps (1) and (2): (1) A process in which a strong basic aqueous solution is added to an organic layer obtained by phenol-chloroform extraction from skin surface lipids collected from the subject and mixed, and then the separated aqueous layer is removed. (2) After removing the aqueous layer, water is added to the organic layer and mixed, then the separated aqueous layer is removed and the organic layer is recovered. A preparation method including the following. 2) A method for analyzing low molecular weight components, comprising analyzing low molecular weight components derived from skin surface lipids other than nucleic acids prepared by the preparation method described in 1). 3) A method for selecting a low molecular weight component marker derived from skin surface lipids other than nucleic acids, comprising using a population having a specified disease or condition or a risk thereof as subjects, preparing a low molecular weight component derived from skin surface lipids other than nucleic acids by the preparation method described in 1), and comparing the amount of the prepared low molecular weight component with a control. 4) A method for detecting a low molecular weight component marker derived from skin surface lipids other than nucleic acids of a subject by the preparation method described in 1), comprising: detecting a low molecular weight component marker derived from skin surface lipids of a predetermined disease or condition from the prepared low molecular weight component. 5) A method for selecting a nucleic acid marker and a low molecular weight component marker derived from skin surface lipids other than nucleic acids from skin surface lipids of the same subject by the preparation method described in 1), and comparing the expression level of the prepared nucleic acid and the amount of the low molecular weight component with a control. 6) A method for detecting nucleic acid markers and low molecular weight component markers derived from skin surface lipids, comprising: preparing nucleic acids and low molecular weight component markers derived from skin surface lipids other than nucleic acids from skin surface lipids of the same subject by the preparation method described in 1); and detecting nucleic acid markers and low molecular weight component markers derived from skin surface lipids from the prepared nucleic acids and low molecular weight component markers of a predetermined disease or condition. [Effects of the Invention]

[0010] According to the present invention, low-molecular-weight components, which are metabolites, can be easily and non-invasively collected from a subject. The low-molecular-weight components prepared by the present invention are useful as samples for analyzing the subject's skin, as well as other parts of the subject's body or the condition of the entire body (for example, for the diagnosis of various diseases). Furthermore, since the method of the present invention utilizes the residue after RNA extraction in the phenol-chloroform method, which is a common RNA extraction method, RNA and low-molecular-weight components can be obtained from the same sample. This allows for the combination and use of biomarkers that are optimal for the diagnosis of various diseases and conditions, and is expected to expand the range of applications. [Brief explanation of the drawing]

[0011] [Figure 1] The results of reverse-phase HPLC-mass spectrometry (MS) analysis (TIC, Total Ion Chromatogram) of the recovered samples are shown. [Figure 2]It shows the separation state of the aqueous layer and the organic layer after adding an aqueous sodium hydroxide solution. [Figure 3] It shows the results of reverse-phase HPLC-Mass Spectrometry (MS) analysis of the recovered sample. [Figure 4] It shows the recovery rate of testosterone derived from SSL. [Figure 5] It shows the recovery rates of four androgens. [Figure 6] It shows the recovery rates of four androgens. [Figure 7] It shows the recovery rates of four androgens dropped onto the oil-absorbing film. [Figure 8] It shows the results (TIC) of reverse-phase HPLC-Mass Spectrometry (MS) analysis of the recovered sample.

Mode for Carrying Out the Invention

[0012] The method for preparing low-molecular components derived from skin surface lipids other than nucleic acids of the present invention comprises the following steps (1) and (2): (1) A step of adding a strong base aqueous solution to the organic layer obtained by phenol-chloroform extraction from the skin surface lipids collected from a subject, mixing them, and then removing the separated aqueous layer (2) A step of adding water to the organic layer after removing the aqueous layer, mixing them, then removing the separated aqueous layer, and recovering the organic layer. Hereinafter, in this specification, the "low-molecular components derived from skin surface lipids other than nucleic acids" may be simply referred to as "low-molecular components".

[0013] 〔Step (1)〕 This step is a step of adding a strong base aqueous solution to the organic layer obtained by phenol-chloroform extraction from the skin surface lipids collected from a subject, mixing them, and then removing the separated aqueous layer. In this specification, "superficial lipids (SSL)" refers to the lipid-soluble fraction present on the surface of the skin, and is sometimes called sebum. Generally, SSL mainly consists of secretions from exocrine glands such as sebaceous glands in the skin, and is present on the skin surface in the form of a thin layer covering the skin surface. Furthermore, unless otherwise specified, "skin" is a general term for the region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands.

[0014] A "subject" can be any organism that has SSL on its skin. Examples of subjects include mammals, including humans and non-human mammals, with humans being preferred. Preferably, the subject is a human or non-human mammal that requires or desires analysis of its own nucleic acid. Also preferably, the subject is a human or non-human mammal that requires or desires gene expression analysis in the skin, or analysis of the condition of the skin or other parts of the body using nucleic acid.

[0015] SSL extracted from the subject contains nucleic acids, proteins, and low-molecular-weight components, which are metabolites, expressed in the subject's skin cells. "Nucleic acid" is not particularly limited to DNA, RNA, etc., but is preferably RNA. Examples of RNA include mRNA, tRNA, rRNA, small RNA (e.g., microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), etc.), long intergenic non-coding (linc)RNA, etc. Low molecular weight components in this specification will be described later.

[0016] This process may further include a step of collecting SSL from the subject. The skin from which SSL is collected may be any part of the body, such as the head, face, neck, trunk, hands, or feet; skin with diseases such as atopic dermatitis, acne, inflammation, or tumors; or skin with wounds; however, it is not particularly limited. Furthermore, the skin from which SSL is collected preferably does not include the skin of the palms, back, soles of the feet, or fingers.

[0017] Any means used for the recovery or removal of SSL from the skin can be employed to collect SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping SSL off the skin, as described later, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has an affinity for SSL, and examples include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include methods of absorbing SSL onto a sheet material such as oil-blotting paper or oil-blotting film, methods of adhering SSL to a glass plate or tape, and methods of scraping off and collecting SSL with a spatula or scraper. To improve the adsorption of SSL, an SSL absorbent material containing a highly lipid-soluble solvent beforehand may be used. On the other hand, since the adsorption of SSL is inhibited if the SSL absorbent material contains a highly water-soluble solvent or water, it is preferable that the content of highly water-soluble solvents or water is low. It is preferable to use the SSL absorbent material in a dry state.

[0018] SSL collected from a subject may be stored for a certain period of time. To minimize the degradation of the contained RNA, it is preferable to store the collected SSL under low temperature conditions as quickly as possible after collection. The storage temperature conditions for SSL in this invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The storage period for SSL under these low temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours to 12 months, more preferably 6 months or less, for example, 1 day to 6 months, and even more preferably 3 months or less, for example, 3 days to 3 months.

[0019] Phenol-chloroform extraction can be performed according to conventional methods. For example, phenol solution and then chloroform are added to the nucleic acid extraction sample and mixed, then centrifuged. Centrifugation separates the sample into an aqueous layer and an organic layer (phenol-chloroform layer). The upper layer is an aqueous layer containing RNA, the lower layer is an organic layer containing low molecular weight components (metabolites) and proteins, and an intermediate layer containing DNA is formed at the boundary between the aqueous and organic layers. In RNA extraction from SSL, the upper aqueous layer is recovered and the lower organic layer is discarded as RNA extraction residue. However, in this invention, the low molecular weight components contained in the organic layer are extracted.

[0020] Examples of strong base aqueous solutions used in this process include aqueous solutions of strong bases such as alkali metal or alkaline earth metal hydrides, hydroxides, carbonates, and alkoxides. Specifically, examples include aqueous solutions of sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium methoxide. Among these, aqueous sodium hydroxide solution is preferred from the viewpoint of availability and versatility. One or more strong bases can be used in combination. For the water used in the strong alkaline solution, tap water, purified water, distilled water, ion-exchanged water, pure water, ultrapure water, etc., can be used.

[0021] In this process, the amount of strong base aqueous solution added can be appropriately selected as long as it is enough to transfer phenol from the organic layer to the aqueous layer. However, from the viewpoint of facilitating the transfer of phenol from the organic layer to the aqueous layer, it is preferable that the value obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution [X(N) × Y(L)] is at least three times the volume (L) of the organic layer. The relationship between the normality X(N) of the strong base, the volume Y(L) of the strong base aqueous solution, and the volume (L) of the organic layer is shown in the following equation (1). X × Y ≥ Volume of the organic layer × 3 (1) From a similar viewpoint, the amount of strong base aqueous solution added is preferably such that the value obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution [X(N) × Y(L)] is 3 times or more the volume (L) of the organic layer. Furthermore, from the viewpoint of improving the extraction efficiency and recovery rate of low molecular weight components, it is preferable that the amount is 9.2 times or less, more preferably 6 times or less, more preferably 5 times or less, and more preferably 4 times or less the volume (L) of the organic layer. The amount of strong base aqueous solution added is preferably such that the value obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution [X(N) × Y(L)] is 3 to 9.2 times, more preferably 3 to 6 times, more preferably 3 to 5 times, and more preferably 3 to 4 times the volume (L) of the organic layer.

[0022] The normality (N) of the strong base can be appropriately adjusted according to the amount and volume of the strong base aqueous solution added. From the viewpoint of separating the organic layer from the aqueous layer and improving the recovery rate of low molecular weight components, the normality (N) of the strong base is preferably 0.2N or higher, more preferably 0.4N or higher, more preferably 0.5N or higher, more preferably 0.8N or higher, more preferably 1.0N or higher, more preferably 1.8N or higher, more preferably 3.0N or higher, even more preferably 4.0N or higher, even more preferably 4.3N or higher, and even more preferably 6.0N or higher. Furthermore, from the viewpoint of improving the extraction efficiency and recovery rate of low molecular weight components, the upper limit of the normality (N) of the strong base is preferably 12.0N or lower, more preferably 8.0N or lower. The normality (N) of the strong base is preferably 0.2 to 12.0 N, more preferably 0.4 to 12.0 N, more preferably 0.5 to 12.0 N, more preferably 0.8 to 12.0 N, more preferably 1.0 to 12.0 N, more preferably 1.8 to 8.0 N, even more preferably 3.0 to 8.0 N, even more preferably 4.0 to 8.0 N, even more preferably 4.3 to 8.0 N, and most still preferably 6.0 to 8.0 N.

[0023] The volume (L) of the strong base aqueous solution can be adjusted as appropriate depending on the amount of strong base aqueous solution added and the normality (N) of the strong base, but it is preferable that it be at least 0.9 times the volume (L) of the organic layer from the viewpoint of improving the recovery rate of low molecular weight components. From a similar viewpoint, the volume (L) of the strong base aqueous solution is preferably 0.92 times or more, more preferably 1.0 times or more, more preferably 2.0 times or more, more preferably 3.8 times or more, more preferably 5.5 times or more, and more preferably 7.0 times or more, relative to the volume (L) of the organic layer, and from the viewpoint of operability, it is preferably 20 times or less, more preferably 10 times or less.

[0024] In this process, it is preferable to further add an organic solvent to the organic layer obtained by phenol-chloroform extraction from skin surface lipids, from the viewpoint of improving operability. The organic solvent is not particularly limited, but examples include alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform, and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; and pyridines. These can be used individually or in combination of two or more. Among these, alcohols, halogenated hydrocarbons, and hydrocarbons are preferred, and chloroform and chloroform-methanol mixed solutions are more preferred. The ratio (by volume) of chloroform in the chloroform-methanol mixed solution is preferably 53% or more, more preferably 55% or more, from the viewpoint of facilitating phenol migration from the organic layer to the aqueous layer and improving recovery rate, and preferably 90% or less, from the viewpoint of extraction efficiency of low molecular weight components. The ratio (by volume) of chloroform in the chloroform-methanol mixed solution is preferably 53-90%, more preferably 55-90%.

[0025] The amount of organic solvent added is preferably 1:1 or more relative to the volume (L) of the organic layer.

[0026] Methods for separating the organic layer from the aqueous layer include static separation and centrifugation. The separation conditions can be adjusted as appropriate, but for centrifugation, it is preferable to adjust the conditions to atmospheric pressure, 5000 to 20000 r / min, for 5 to 30 minutes. The separation temperature is preferably 20°C or lower, more preferably 10°C or lower, for example, preferably 4°C. In this step, by adding a strong base aqueous solution to the organic layer and mixing it, the phenol in the organic layer becomes phenoxide and migrates to the upper aqueous layer. By separating and removing the aqueous layer containing phenoxide, phenol can be removed from the organic layer. The organic layer containing low molecular weight components after the removal of the aqueous layer is then subjected to the next step.

[0027] [Step (2)] This process involves adding water to the organic layer after removing the aqueous layer, mixing it, then removing the separated aqueous layer, and recovering the organic layer. The operation of adding water to the organic layer and mixing it, followed by removing the separated aqueous layer, may be repeated once or multiple times, for example, two or three times. From the viewpoint of efficiently removing strong bases remaining in the organic layer, it is preferable to repeat the process multiple times. Examples of water include tap water, purified water, distilled water, ion-exchanged water, pure water, and ultrapure water.

[0028] The amount of water added to the organic layer is preferably 1 or more times the volume (L) of the organic layer. The amount of water added is the value for a single addition.

[0029] Methods for separating the organic layer from the aqueous layer include static separation and centrifugation. The separation conditions can be adjusted as appropriate, but for centrifugation, it is preferable to adjust the conditions to atmospheric pressure, 5000 to 20000 r / min, for 5 to 30 minutes. The temperature at which separation is performed is preferably 20°C or lower, more preferably 10°C or lower, for example, preferably 4°C. By adding water to the organic layer and mixing it, any strong bases remaining in the organic layer migrate to the upper aqueous layer. By separating and removing the aqueous layer containing the strong bases, the organic layer containing low-molecular-weight components can be recovered. The recovered organic layer may be dried as needed. Examples of drying methods include vacuum drying, freeze-drying, spray drying, and heat drying.

[0030] Thus, low-molecular-weight components can be prepared from surface lipids of the skin collected from the subject. Low molecular weight components include bio-derived and non-biological components. Specifically, bio-derived components include fatty acids, acylglycerols, sphingolipids, phospholipids, sebum, hormones, and arachidonic acid cascades. Non-biological components include food-derived components, personal care product-derived components, chemical product-derived components, and pharmaceutical components. The low molecular weight components are preferably those whose precise mass (m / z), provided by mass spectrometry, is in the range of 107.0681 to 1,245.6860. The mass spectrometer conditions are as described in the examples below.

[0031] The low molecular weight component can be used for various analyses or diagnoses. Therefore, the present invention also provides a method for analyzing a low molecular weight component, which includes analyzing the low molecular weight component prepared by the method for preparing the low molecular weight component according to the present invention. Examples of analyses and diagnoses that can be performed using the low molecular weight component include: (i) Analysis of the subject's skin condition, for example, evaluation or future prediction of skin health, diagnosis or prognosis of skin diseases, evaluation of the efficacy of topical skin agents, diagnosis or prognosis of skin cancer, evaluation of subtle skin changes, etc. Specifically, for example, it has been shown that skin condition can be predicted by analyzing steroid hormones in SSL (Japanese Patent Publication No. 2020-143915). Furthermore, since it correlates with blood concentration, it is possible to analyze the amount of blood steroid hormones non-invasively without the need for blood sampling. (ii) Analysis of the subject's condition in areas other than the skin or the entire body, such as evaluation or prediction of overall health status, and diagnosis or prognosis of various diseases including neurological diseases, cardiovascular diseases, metabolic diseases, and cancer. Specifically, for example, it has been shown that Parkinson's disease may be diagnosed based on differences in the composition of lipid-related substances in sebum (Sinclair, Eleanor et al. 2021. “Metabolomics of sebum reveals lipid dysregulation in Parkinson's disease.” Nature Communications 12: 1592. http: / / dx.doi.org / 10.1038 / s41467-021-21669-4.)

[0032] The present invention also provides a method for selecting low-molecular-weight component markers derived from skin surface lipids for a disease or condition. In this method, a population with a predetermined disease or condition or at risk is used as a subject, and low-molecular-weight components other than nucleic acids are prepared from skin surface lipids according to the preparation method of the present invention. In the present invention, nucleic acids may also be prepared from the skin surface lipids of the same subject. The amount and expression (expression level, etc.) of a low molecular weight component, or nucleic acid and low molecular weight component, prepared from the population is compared with the amount and expression of a control. The control may include a population that does not have the specified disease or condition or its risk, and statistical data based on this control. Low molecular weight components that show different amounts or expressions compared to the control can be selected as markers or candidates for markers of the specified disease or condition.

[0033] The present invention also provides a method for detecting low-molecular-weight component markers derived from skin surface lipids of a disease or condition, or a method for determining a disease or condition, or its risk, based on the detection of said markers. In this method, low-molecular-weight components derived from skin surface lipids other than nucleic acids are prepared from a subject who desires or needs to determine a predetermined disease or condition, or its risk, according to the preparation method of the present invention. In the present invention, nucleic acids may also be prepared from the skin surface lipids of the same subject. Next, a low-molecular-weight component marker, or a nucleic acid marker and low-molecular-weight component marker, for a predetermined disease or condition is detected from the prepared low-molecular-weight component, or from the nucleic acid and low-molecular-weight component. Based on the presence or absence and expression level of the marker, the subject's disease or condition, or its risk, is determined.

[0034] (Preparation kit) In a further embodiment, the present invention provides a kit for preparing low molecular weight components derived from skin surface lipids other than nucleic acids. The kit includes equipment and reagents necessary for collecting and preserving SSL from a subject, and reagents for extracting low molecular weight components derived from skin surface lipids other than nucleic acids. Tools and reagents necessary for collecting and preserving SSL include, for example, tools for collecting SSL (e.g., oil-absorbing film), reagents for preserving the collected SSL, and storage containers. Reagents for extracting low-molecular-weight components derived from skin surface lipids other than nucleic acids include, for example, reagents for extracting and purifying low-molecular-weight components from collected SSL (e.g., phenol-chloroform extraction reagents), equipment necessary for testing, and guidance materials.

[0035] With regard to the embodiments described above, the present invention further discloses the following aspects.

[0036] <1> A method for preparing low-molecular-weight components derived from skin surface lipids other than nucleic acids, comprising the following steps (1) and (2): (1) A process in which a strong basic aqueous solution is added to an organic layer obtained by phenol-chloroform extraction from skin surface lipids collected from the subject and mixed, and then the separated aqueous layer is removed. (2) A preparation method comprising the steps of adding water to the organic layer after removing the aqueous layer and mixing it, then removing the separated aqueous layer and recovering the organic layer.

[0037] <2> In step (1) described above, the amount of strong base aqueous solution added is such that the value obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution [X(N) × Y(L)] is preferably 3 times or more the volume (L) of the organic layer, and more preferably 9.2 times or less, more preferably 6 times or less, even more preferably 5 times or less, and even more preferably 4 times or less, and also preferably 3 to 9.2 times, more preferably 3 to 6 times, even more preferably 3 to 5 times, and even more preferably 3 to 4 times. <1> The preparation method described. <3> The strong base aqueous solution is preferably an aqueous solution of alkali metal or alkaline earth metal hydrides, hydroxides, carbonates, or alkoxides, more preferably one or more selected from aqueous solutions of sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium methoxide, and even more preferably an aqueous solution of sodium hydroxide. <1> or <2> The preparation method described. <4> The normality (N) of the strong base is preferably 0.2N or higher, more preferably 0.4N or higher, more preferably 0.5N or higher, more preferably 0.8N or higher, more preferably 1.0N or higher, more preferably 1.8N or higher, more preferably 3.0N or higher, even more preferably 4.0N or higher, even more preferably 4.3N or higher, and even more preferably 6.0N or higher. It is also preferably 12.0N or lower, more preferably 8.0N or lower. Furthermore, it is preferably 0.2~12.0N, more preferably 0.4~12.0N, more preferably 0.5~12.0N, more preferably 0.8~12.0N, more preferably 1.0~12.0N, more preferably 1.8~8.0N, even more preferably 3.0~8.0N, even more preferably 4.0~8.0N, even more preferably 4.3~8.0N, and most preferably 6.0~8.0N. <1> ~ <3> The preparation method described in any one of the following. <5> The volume (L) of the strong base aqueous solution added to the organic layer is preferably 0.9 times or more, more preferably 0.92 times or more, more preferably 1.0 times or more, more preferably 2.0 times or more, more preferably 3.8 times or more, even more preferably 5.5 times or more, and even more preferably 7.0 times or more, and also preferably 20 times or less, more preferably 10 times or less, and also preferably 0.9 to 20 times, more preferably 0.92 to 20 times, more preferably 1.0 to 20 times, more preferably 2.0 to 20 times, more preferably 3.8 to 10 times, even more preferably 5.5 to 10 times, and even more preferably 7.0 to 10 times. <1> ~ <4> The preparation method described in any one of the following. <6> The above step (1) includes further adding an organic solvent to the organic layer, <1> ~ <5> The preparation method described in any one of the following. <7> The organic solvent is preferably one or more selected from alcohols, halogenated hydrocarbons, and hydrocarbons, and more preferably chloroform or a chloroform-methanol mixed solvent. <6> The preparation method described. <8> The ratio (by volume) of chloroform in the chloroform-methanol mixed solution is preferably 53% or more, more preferably 55% or more, and also preferably 90% or less, and also preferably 53-90%, more preferably 55-90%. <7> The preparation method described. <9> In step (2) above, after adding water to the organic layer and mixing, the separated aqueous layer is removed, and this operation is repeated at least twice. <1> ~ <8> The preparation method described in any one of the following. <10> Low molecular weight components derived from skin surface lipids are those whose precise mass (m / z), as provided by mass spectrometry, is between 107.0681 and 1,245.6860. <1> ~ <9> The preparation method described in any one of the following. <11> <1> ~ <10> A method for analyzing low molecular weight components, comprising analyzing low molecular weight components derived from skin surface lipids other than nucleic acids, prepared by the preparation method described in any one of the above. <12> Using a group of people with a specified disease or condition or at risk as subjects, <1> ~ <10> A method for selecting a low molecular weight component marker derived from skin surface lipids other than nucleic acids, comprising: preparing a low molecular weight component derived from skin surface lipids other than nucleic acids by the preparation method described in any one of the above; and comparing the amount of the prepared low molecular weight component with a control. <13> <1> ~ <10> A method for detecting a low molecular weight component marker derived from skin surface lipids other than nucleic acids of a subject by the preparation method described in any one of the above, comprising: detecting a low molecular weight component marker derived from skin surface lipids of a predetermined disease or condition from the prepared low molecular weight component. <14> From the skin surface lipids of the same subject, nucleic acids and <1> ~ <10> A method for selecting a nucleic acid marker and a low molecular weight component marker derived from skin surface lipids other than nucleic acids, comprising: preparing a low molecular weight component derived from skin surface lipids other than nucleic acids by the preparation method described in any one of the above; and comparing the expression level of the prepared nucleic acid and the amount of the low molecular weight component with a control. <15> From the skin surface lipids of the same subject, nucleic acids and <1> ~ <10> A method for detecting nucleic acid markers and low molecular weight component markers derived from skin surface lipids other than nucleic acids, comprising: preparing low molecular weight components derived from skin surface lipids other than nucleic acids by the preparation method described in any one of the above; and detecting nucleic acid markers and low molecular weight component markers derived from skin surface lipids from the prepared nucleic acids and low molecular weight components. [Examples]

[0038] Example 1. Extraction of metabolites from RNA extraction residue Skin surface lipids (SSL) were used as the sample. SSL was collected from the entire face of one subject using an oil-absorbing film (3M Japan). The oil-absorbing film was then cut to an appropriate size, and RNA extraction was performed using QIAzol (Qiagen) reagent (containing 50 v / v% phenol) according to the provided protocol. Specifically, QIAzol was added to the cut film, SSL was extracted, chloroform was added and mixed to the extract, and centrifugation was performed. Of the two layers that formed, the upper layer was an aqueous layer containing RNA, and the lower layer was a pale red RNA extraction residue containing metabolites. This lower layer was used for metabolite extraction. The pale red residue is rich in phenol, which inhibits metabolite analysis, and therefore it is preferable to remove it. The following procedure was performed to remove the large amount of phenol.

[0039] To 300 μL of RNA extraction residue, 1,350 μL of a chloroform:methanol = 3:1 (volume ratio) mixture was added and mixed. Then, 150 μL of 8N sodium hydroxide aqueous solution was added. After thoroughly mixing with a vortex mixer, the mixture was centrifuged at 15,000 r / min for 5 minutes. The solution separated into two layers, with a pale red layer separating to the top, which was carefully removed. 300 μL of ultrapure water (milliQ water, Merck Millipore) was added to the remaining lower layer and mixed by vortexing, then centrifuged at 15,000 r / min for 5 minutes. The resulting upper layer was carefully removed. This process of adding pure water, mixing, centrifugation, and removing the upper layer was repeated a total of three times. All centrifugation operations were performed at 4°C.

[0040] The remaining lower layer was concentrated to dryness under reduced pressure, and any remaining aqueous solution was dried by freeze-drying. 200 μL of 80% methanol (by volume) was added to the dried sample for redissolution, and the supernatant was used for reverse-phase HPLC-mass spectrometry (MS) analysis after centrifugation at 15,000 r / min for 5 minutes. Samples were prepared using the above RNA extraction procedure from films without SSL (silica spectroscopy) and samples performed using only the extraction procedure without film.

[0041] As a result, it was revealed that the RNA extraction residue contained numerous peaks derived from metabolites present in SSL (Figure 1). Based on the mass spectrometry data, the structures of the detected peaks were estimated to be components of biological origin, including fatty acids ((2E,18R)-18-hydroxynonadec-2-enoic acid, N-palmitoyl methionine, CAR 18:1, CAR 20:2, CAR 17:0, CAR 18:0; O,CAR 16:0, Octadecanamide, NAE 20:2, C16:0, FA16:1, FA18:1, FA14:1), acylglycerols (MG(14:0~20:0), DG(i-13:0 / 8:0 / 0:0)), and sphingolipids (Sphingosines, Sphinganines, N-acetylsphinganine, N,N-Dimethylsphingosine, Halaminol A,2-amino-3-methoxyoctadec-4-en-1-ol,Phytosphingosines,Dehydrosphingosine,Cer(d18:2 / 14:0),Cer(d18:2 / 15:0),Cer(d16:1 / 17:0)),phospholipids (PA,PC,PE,PS,LysoPC,LysoPE),sebum (7a-Hydroxy-cholestene-3-one,3-Oxo-4,6-choladienoic acid (bile acid),Sulfolithocholic acid (bile acid),7-Dehydrodesmosterol),hormones (cortisol 17-valerate,18-Hydroxycortisol,3b,17b-Dihydroxyetiocholane),arachidonic acid cascade (Arachidonic acid,Eicosenoic acid,Eicosapentaenoic acid) Acid, leukotriene-D4, etc., were suspected to be present. In addition, numerous other components were detected, including those suspected to be derived from food (caffeine), personal care products (Laureth-5), and chemical products (Octyl 4-methoxycinnamic acid). Fatty acids and other substances are known to be components found in sebum, and this extraction method has been shown to be a valid method for extracting metabolites from RNA extraction residue. Furthermore, caffeine was identified as one of the other components using a standard sample. Caffeine has been reported to be detectable on the skin after ingestion of caffeinated beverages in non-patent literature (Liou, Y., Chang, K. and Lin, C. (2017) 'Sampling and profiling caffeine and its metabolites from an eyelid using a watercolor pen based on electrospray ionization / mass spectrometry', International Journal of Mass Spectrometry, 422, pp. 51-55. doi: 10.1016 / j.ijms.2017.08.010.), and this method was shown to be a valid method for extracting metabolites from RNA extraction residue.

[0042] Example 2. Investigation of the amount of base required for the separation and removal of phenol. RNA extraction was performed using QIAzol without using biological samples such as sebum, and RNA extraction residue was prepared. 300 μL of the extraction residue was mixed with 300 μL of chloroform, and then 150 μL of sodium hydroxide aqueous solutions of different concentrations were added. After thorough mixing by vortexing, the mixture was centrifuged at 15,000 r / min for 5 minutes. Sodium hydroxide solutions of concentrations 2, 4, 5, 6, 7, and 8 mol / L (N) were added, respectively. The centrifugation was performed at 4°C.

[0043] As a result, with 2, 4, and 5N sodium hydroxide, some or all of the pale red layer remained in the lower layer, while with 6N and above, the pale red layer completely moved to the upper layer (Figure 2). This indicates that a certain amount of sodium hydroxide relative to the extraction residue is necessary to remove phenol contained in the extraction residue. Furthermore, it was shown that the condition for the separation and removal of phenol is that the ratio of sodium hydroxide concentration (N) × volume of sodium hydroxide solution (μL) to the volume of residue (μL) must be 3 or more. Incidentally, the 300 μL of residue prepared above contained approximately 2.77 mmol of phenol, but 150 μL of 6N sodium hydroxide, which was able to separate and remove phenol, contained approximately 0.90 mmol, indicating that separation is possible with a significantly smaller amount than the theoretical amount required for neutralization.

[0044] Example 3. Effect of different base addition amounts on analytical results We investigated whether adding more sodium hydroxide than necessary for phenol removal would adversely affect the yield of extracted metabolites. As in Example 1, SSL was collected from the entire face of the subject and RNA extraction was performed. RNA extraction was performed from two oil-blotting films, and the obtained RNA extraction residues were combined. Four tubes of 300 μL each of RNA extraction residue were prepared, and 1,350 μL of a chloroform:methanol = 3:1 (volume ratio) mixture was added and mixed. Then, 150 μL of 6, 8, 12N, and 18.4N sodium hydroxide aqueous solutions were added. Note that 18.4N is the theoretical amount required to neutralize the phenol present in the residue. After thoroughly mixing with a vortex mixer, the mixture was centrifuged at 15,000 r / min for 5 minutes. In all cases, the solution separated into two layers, with a pale red layer separating to the upper layer, which was carefully removed. 300 μL of ultrapure water (milliQ water, Merck Millipore) was added to the remaining lower layer, mixed with a vortex mixer, and then centrifuged at 15,000 r / min for 5 minutes. The resulting upper layer was carefully removed. This process of adding pure water, mixing, centrifugation, and removing the upper layer was repeated a total of four times. All centrifugation operations were performed at 4°C.

[0045] The remaining lower layer was concentrated to dryness under reduced pressure, and any remaining aqueous solution was dried by freeze-drying. 200 μL of 80% methanol (by volume) was added to the dried sample to redissolve it, and the supernatant was centrifuged at 15,000 r / min for 5 minutes. Reverse-phase HPLC-Mass Spectrometry (MS) analysis (positive mode) was performed using the supernatant.

[0046] As a result, the peak intensity derived from metabolites decreased overall as the sodium hydroxide concentration increased, especially at 12 and 18.4 N (Figure 3). This was thought to be because higher concentrations of sodium hydroxide reduced the extraction efficiency and thus the recovery rate. From this, it was shown that in order to detect and measure metabolites as efficiently as possible, it is desirable that the ratio of sodium hydroxide concentration (N) × volume of sodium hydroxide aqueous solution (μL) to the volume of residue (μL) be less than 6 times.

[0047] Example 4. Effect of chloroform-methanol ratio added to residue on phenol separation and removal Even if sodium hydroxide is added directly to the RNA extraction residue, the pale red layer containing phenol will migrate to the upper layer, but the volume of the lower layer containing metabolites will become extremely small, and there is a possibility of losing the lower layer when removing the upper layer, which has a significant impact. Therefore, to improve operability, it is desirable to add an organic solvent such as a chloroform-methanol mixture to make the volume of the lower layer roughly equivalent to that of the upper layer.

[0048] RNA extraction was performed using QIAzol without using biological samples such as sebum, and RNA extraction residues were prepared. 300 μL of the extraction residue was mixed with 450 μL of mixed solutions of different chloroform / methanol ratios (volume ratio), and then 150 μL of 8N sodium hydroxide aqueous solution was added. After thorough mixing by vortexing, the mixture was centrifuged at 15,000 r / min for 5 minutes. Solutions with chloroform ratios (%) of 50, 55, 60, 66.70, 75, 80, 85.7, 90, 95.2, and 100% were added, respectively. The centrifugation was performed at 4°C.

[0049] As a result, as shown in Table 1, when a mixed solvent with a chloroform ratio of 55% or more was used, the pale red layer containing phenol migrated to the upper layer and could be removed. This indicates that when adding a chloroform-methanol mixed solvent to the residue, it is desirable that the chloroform ratio be 55% or more.

[0050] [Table 1]

[0051] Example 5. Effect of chloroform-methanol ratio added to residue on testosterone yield Regarding testosterone, one of the male hormones expected to be detected in SSL, we investigated the effect of the chloroform-methanol ratio added to the RNA extraction residue on the recovery rate of metabolites. RNA extraction residue was prepared by performing RNA extraction using QIAzol without using biological samples such as sebum. 300 μL of the extraction residue was mixed with 400 pg of testosterone standard (4 μL of 100 ppb methanol solution), then 450 μL of mixed solutions with different chloroform / methanol ratios (volume ratio) were added and mixed. Finally, 150 μL of 8N sodium hydroxide aqueous solution was added. After thorough mixing by vortexing, the mixture was centrifuged at 15,000 r / min for 5 minutes. Chloroform solutions with ratios (%) of 75%, 80%, 85.7%, 90%, 95.2%, and 100% were added, respectively. Under all conditions, a pale red layer separated to the upper layer, which was carefully removed. 300 μL of ultrapure water (milliQ water, Merck Millipore) was added to the remaining lower layer and mixed by vortexing, followed by centrifugation at 15,000 r / min for 5 minutes. The resulting upper layer was carefully removed. The process of adding pure water, mixing, centrifugation, and removing the upper layer was repeated a total of four times. All centrifugation operations were performed at 4°C.

[0052] The remaining lower layer was concentrated to dryness under reduced pressure, and any remaining aqueous solution was dried by freeze-drying. 200 μL of 50% methanol (by volume) was added to the dried sample to redissolve it, and the supernatant was centrifuged at 15,000 r / min for 5 minutes. LC-MS / MS analysis was performed on the supernatant to quantify testosterone and calculate the recovery rate.

[0053] As a result, the recovery rate tended to increase as the proportion of chloroform increased (Figure 4). This indicates that when a chloroform-methanol mixed solvent is added to the residue, a higher chloroform ratio leads to a higher recovery rate.

[0054] Example 6. Effect of water addition in step (1) on androgen yield (1) For dihydrotestosterone (DHT), testosterone (T), androstenedione (A-dione), and dehydroepiandrosterone (DHEA), which are male hormones expected to be detected in SSL, the effect of water addition in step (1) on the recovery rate of metabolites was investigated. RNA extraction residue was prepared by performing RNA extraction using QIAzol without using biological samples such as sebum. 200 pg each of DHT, T, A-dione, and DHEA standards were added (10 μL of a 20 ppb mixed solution (solvent: methanol)), and 600 μL of the extraction residue was added to a concentrated, dry tube. 900 μL of chloroform was then added and mixed. 0, 1, 5, or 10 mL of ultrapure water (milliQ water, Merck Millipore) was then added, followed by 300 μL of 8N sodium hydroxide aqueous solution (the combined volume of ultrapure water and sodium hydroxide aqueous solution was 0.3 mL, 1.3 mL, 5.3 mL, and 10.3 mL). After thorough mixing by vortexing, centrifugation was performed at 15,000 r / min for 5 minutes. Under all conditions, a pale red layer separated to the upper layer, which was carefully removed. 600 μL of ultrapure water was added to the remaining lower layer and mixed by vortexing, then centrifuged at 15,000 r / min for 5 minutes. The resulting upper layer was carefully removed. This process of adding pure water, mixing, centrifugation, and removing the upper layer was repeated a total of three times. All centrifugation operations were performed at 4°C.

[0055] The final lower layer was concentrated to dryness under reduced pressure, and any remaining aqueous solution was dried by freeze-drying. 90 μL of 50 mM methoxyamine hydrochloride methanol solution was added to the dried sample to redissolve it, and the mixture was reacted at 60°C for 30 minutes to derivatize the androgens. After 30 minutes, the mixture was cooled on ice for approximately 5 minutes, then 110 μL of 50% acetonitrile was added, and the mixture was filtered through a 0.45 μm syringe filter (DISMIC 13HP045AN, ADVANTEC). The filtrate was analyzed by LC-MS / MS to obtain the peak area values ​​for each male hormone, and the recovery rate was calculated. The recovery rate (%) was calculated as "peak area value of each male hormone under each extraction condition / peak area value of each male hormone that was only derivatized × 100".

[0056] As a result, the recovery rate significantly increased when water was added in step (1) in addition to the organic solvent and strong base aqueous solution (Figure 5). This indicates that increasing the volume of strong base aqueous solution added to the organic layer relative to the residue volume improves the recovery rate of metabolites.

[0057] Example 7. Effect of water addition in step (1) on androgen yield (2) The same test as in Example 6 was performed, but with a different amount of ultrapure water added to the extraction residue. Specifically, tests were conducted under each condition with 0, 0.25, 0.5, 1, 2, or 4 mL of ultrapure water added. The operation of adding pure water, mixing, centrifugation, and removing the upper layer in step (2) was repeated a total of two times. All other conditions were the same.

[0058] As a result, similar to Example 6, the recovery rate increased significantly as the volume of the strong base aqueous solution increased (Figure 6). A significant improvement in the recovery rate was observed even with an added amount of 0.25 mL of water, and the recovery rate further improved as the amount of water added increased. Under the condition of adding 0.25 mL of water, the volume of the aqueous solution, including the added sodium hydroxide aqueous solution, was 0.55 mL, which corresponds to 0.92 times the volume of the residue used (600 μL). This indicates that the recovery rate of metabolites is improved by adding sodium hydroxide aqueous solution to a volume of 0.92 times or more the volume of the residue.

[0059] Example 8. Effect of water addition in step (1) on androgen yield (3) DHT, T, A-dione, and DHEA, which are male hormones expected to be detected in SSL, were dropped onto an oil-absorbing film, and then an RNA extraction procedure was performed. Using the RNA extraction residue obtained therefrom, the effect of adding water in step (1) on the recovery rate of metabolites was investigated. Without using biological samples such as sebum, 250 pg each of DHT, T, A-dione, and DHEA standards were added to an oil-absorbing film (5 μL of a 50 ppb mixed solution (solvent: methanol)), and the solvent was air-dried. The oil-absorbing film was cut into 16 pieces with scissors and placed in 5 mL tubes. 1,450 μL of QIAzol Lysis Reagent (Qiagen) was added, vortexed for 10 seconds, and then allowed to stand for 1 minute. After repeating the vortexing-standing procedure a total of three times, as much of the extract as possible from the tube was transferred to a 2 mL tube. 260 μL of chloroform was added, vortexed for 10 seconds, and then centrifuged at 15,000 r / min for 15 minutes. The supernatant after centrifugation was removed, and the remaining lower organic layer, i.e., the RNA extraction residue, was obtained. 600 μL of the extraction residue was added to a new 5 mL tube, and then 900 μL of chloroform was added. Next, 0 mL or 3 mL of ultrapure water and 350 μL of 8N sodium hydroxide aqueous solution were added (the combined volumes of ultrapure water and sodium hydroxide aqueous solution were 0.35 mL and 3.35 mL, respectively). After thoroughly mixing with a vortex mixer, the mixture was centrifuged at 3,000 r / min for 5 minutes. After carefully removing the upper layer, 600 μL of ultrapure water was added to the remaining lower layer and mixed with a vortex mixer, then centrifuged at 15,000 r / min for 5 minutes. The resulting upper layer was carefully removed. This procedure of adding pure water → mixing → centrifugation → upper layer removal was repeated once more (a total of two times). All centrifugation operations were performed at 4°C.

[0060] The remaining lower layer was concentrated to dryness under reduced pressure. 90 μL of 50 mM methoxyamine hydrochloride methanol solution was added to the dry sample to redissolve it, and the mixture was reacted at 60°C for 30 minutes to derivatize the androgens. After 30 minutes, the mixture was cooled on ice for about 5 minutes, then 110 μL of 40% acetonitrile was added, and the mixture was filtered through a 0.45 μm syringe filter (DISMIC 13HP045AN, ADVANTEC). The filtrate was analyzed by LC-MS / MS to obtain the peak area values ​​for each male hormone, and the recovery rate was calculated. The recovery rate (%) was calculated as "peak area value of each male hormone under each extraction condition / peak area value of each male hormone that was only derivatized × 100".

[0061] As a result, the recovery rate of each male hormone from the film increased significantly under the condition with 3 mL of water added (after modification) compared to the condition without water added (before modification) (Figure 7). This indicates that even when extracting metabolites from oil-absorbing films to which standards have been dropped using RNA extraction residue prepared by the phenol-chloroform method, the recovery rate improves as the volume of the strong base aqueous solution increases in step (1).

[0062] Example 9. Effect of water addition in step (1) on the yield of low molecular weight components SSL was collected from the entire face of one subject in two separate samples (at different times). The oil-blotting film was then cut to an appropriate size, and RNA extraction was performed using QIAzol (Qiagen) reagent (containing 50 v / v% phenol) according to the provided protocol. Specifically, 1,450 μL of QIAzol was added to the cut film to extract SSL. The extract was collected in a new tube, 260 μL of chloroform was added, and the mixture was vigorously mixed and centrifuged. Of the two resulting layers, the lower layer (RNA extraction residue) was used for metabolite extraction. The obtained RNA extraction residues (from both samples) were combined, and four 300 μL tubes of RNA extraction residue were prepared.

[0063] To 300 μL of RNA extraction residue, 450 μL of a chloroform:methanol = 3:1 (volume ratio) mixture was added and mixed. Then, 150 μL of 8N sodium hydroxide aqueous solution was added. Subsequently, 0 mL, 0.5 mL, 1.0 mL, or 2.0 mL of ultrapure water was added (the total ratio of sodium hydroxide aqueous solution and ultrapure water to the residue was 0.5, 2.17, 3.83, and 7.17 times, respectively). After thorough mixing by vortexing, the mixture was centrifuged at 15,000 r / min for 5 minutes, and the pale red upper layer was carefully removed. 300 μL of ultrapure water was added to the remaining lower layer and mixed by vortexing. Then, the mixture was centrifuged at 15,000 r / min for 3 minutes, and the resulting upper layer was carefully removed. This process of adding pure water, mixing, centrifugation, and removing the upper layer was repeated twice (a total of three times). All centrifugation operations were performed at 4°C.

[0064] The remaining lower layer was concentrated to dryness under reduced pressure. 300 μL of 80% methanol (by volume) was added to this sample for redissolution, and the mixture was filtered through a 0.45 μm syringe filter. The filtrate was then analyzed by reverse-phase HPLC-Mass Spectrometry (MS) (positive mode).

[0065] As a result, the peak intensity derived from metabolites increased significantly and overall as the volume of the sodium hydroxide aqueous solution increased, especially under conditions where 0.5 mL or more of ultrapure water was added (the volume of the strong base aqueous solution was 2.17 times or more relative to the volume of the residue (μL)) (Figure 8). Therefore, similar to the male hormone shown in Example 8, it was shown that the recovery rate of metabolites extracted from SSL improved as the volume of the strong base aqueous solution increased in step (1).

Claims

1. A method for preparing low molecular weight components derived from lipids on the skin surface other than nucleic acids, comprising the following steps (1) and (2): (1) A step of adding a strong base aqueous solution to an organic layer obtained by phenol-chloroform extraction of lipids on the skin surface collected from a subject, mixing the organic layer, and then removing the separated aqueous layer. (2) A preparation method comprising the steps of adding water to the organic layer after removing the aqueous layer, mixing the mixture, removing the separated aqueous layer, and recovering the organic layer.

2. 2. The method according to claim 1, wherein the amount of the strong base aqueous solution added in step (1) is such that the product of the normality X(N) of the strong base and the volume Y(L) of the strong base aqueous solution [X(N) x Y(L)] is at least three times the volume (L) of the organic layer.

3. 2. The method according to claim 1, wherein the strong base aqueous solution is an aqueous sodium hydroxide solution.

4. The method according to claim 1, wherein step (1) further comprises adding an organic solvent to the organic layer.

5. 5. The method according to claim 4, wherein the organic solvent is chloroform or a chloroform-methanol mixed solvent.

6. 2. The method according to claim 1, wherein in step (2), the operation of adding water to the organic layer, mixing, and then removing the separated aqueous layer is repeated two or more times.

7. 2. The method according to claim 1, wherein the low molecular weight components derived from lipids on the skin surface are components having an exact mass (m / z) of 107.0681 to 1,245.6860 as determined by mass spectrometry.

8. 2. The method according to claim 1, wherein in step (1), the volume (L) of the strong base aqueous solution added to the organic layer is at least 0.9 times the volume (L) of the organic layer.

9. A method for analyzing low molecular weight components, comprising analyzing low molecular weight components derived from lipids on the skin surface other than nucleic acids, prepared by the preparation method according to any one of claims 1 to 8.

10. A method for selecting markers of low molecular weight components derived from lipids on the skin surface for a predetermined disease or condition, comprising preparing low molecular weight components derived from lipids on the skin surface other than nucleic acids by the preparation method described in any one of claims 1 to 8, using a population having a predetermined disease or condition or a risk thereof as a subject, and comparing the amount of the prepared low molecular weight components with that of a control.

11. A method for detecting a low molecular weight component marker derived from lipids on the skin surface for a predetermined disease or condition, comprising: preparing low molecular weight components derived from lipids on the skin surface other than nucleic acids of a subject by the preparation method according to any one of claims 1 to 8; and detecting a low molecular weight component marker derived from lipids on the skin surface for the predetermined disease or condition from the prepared low molecular weight components.

12. A method for selecting a nucleic acid marker and a marker for a low molecular weight component derived from lipids on the skin surface for a predetermined disease or condition, the method comprising: preparing nucleic acids and low molecular weight components derived from lipids on the skin surface other than nucleic acids from lipids on the skin surface of the same subject by the preparation method described in any one of claims 1 to 8; and comparing the expression levels of the prepared nucleic acids and the amounts of the low molecular weight components with those of a control.

13. A method for detecting nucleic acid markers for a predetermined disease or condition and markers for low molecular weight components derived from lipids on the skin surface, comprising: preparing nucleic acids and low molecular weight components derived from lipids on the skin surface other than nucleic acids from lipids on the skin surface of the same subject by the preparation method of any one of claims 1 to 8; and detecting nucleic acid markers for the predetermined disease or condition and markers for low molecular weight components derived from lipids on the skin surface from the prepared nucleic acids and low molecular weight components.

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