Method for quantifying plasmalogens and kit for quantifying plasmalogens

Abstract

This method for quantifying plasmalogens comprises: a measurement sample preparation step for mixing a Schiff reagent with a specimen containing protein and plasmalogens to obtain a measurement sample; a control sample preparation step for mixing the specimen with a solvent of the Schiff reagent to obtain a control sample; a measurement step for measuring the extent of a first optical property of the measurement sample, the first optical property being obtained by reacting the Schiff reagent with an aldehyde generated from vinyl ether bonds of the plasmalogens, and measuring the extent of the optical property of the control sample; and a determination step for determining the concentration of plasmalogens in the specimen by comparing the correction extent, that is the extent to which the extent of the first optical property has been corrected by the extent of the optical property of the control sample, with the extent of a second optical property of a standard sample containing the Schiff reagent and a known concentration of plasmalogens, the second optical property being the optical property after the Schiff reagent has been reacted with the aldehyde generated from the vinyl ether bonds of the plasmalogens.

Description

Method for quantifying plasmalogen and kit for quantifying plasmalogen

[0001] The present invention relates to a method for quantifying plasmalogen and a kit for quantifying plasmalogen.

[0002] Plasmalogen is a type of phospholipid with antioxidant properties, a type of glycerophospholipid. Plasmalogen is present in all mammalian tissues, accounting for approximately 18% of the phospholipids in the human body. Because most plasmalogens are bound to polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid, they are involved in the storage of polyunsaturated fatty acids and the release of second messengers of intercellular signals, such as prostaglandins and leukotrienes, produced from these polyunsaturated fatty acids. Furthermore, plasmalogens are involved in cell fusion, ion transport, and other processes. Because the vinyl ether bond (alkenyl bond) of plasmalogens is particularly sensitive to oxidative stress, plasmalogens also fulfill a cellular antioxidant function. In vivo, plasmalogens exhibit protective effects against oxidative stress. For example, Non-Patent Documents 1 and 2 report that plasmalogen plasma concentrations serve as biomarkers for arteriosclerosis, cancer, and other conditions.

[0003] Plasmalogens are known to decrease in neurological diseases such as dementia, Parkinson's disease, depression, and schizophrenia. For example, Non-Patent Document 3 reports that ethanolamine-type plasmalogens are significantly decreased in the frontal lobe and hippocampus of Alzheimer's disease brains. Non-Patent Document 4 discloses that plasmalogen concentrations in the blood of patients with Alzheimer's dementia are reduced.

[0004] As the relationship between plasmalogens and diseases becomes clearer, a technology for easily measuring plasmalogens in vivo is needed. It is known that the vinyl ether bond of plasmalogens reacts specifically and quantitatively with iodine in methanol solution. As shown in Non-Patent Document 5, plasmalogens can be quantified by measuring the decrease in absorption of 355 nm wavelength light by iodine. The method using iodine disclosed in Non-Patent Document 5 is relatively simple, but has the disadvantage of low detection sensitivity.

[0005] The vinyl ether bond of plasmalogen is acid-labile and is decomposed by acid into aliphatic aldehydes and lysophospholipids. Aliphatic aldehydes can usually be measured as dimethyl acetals using gas chromatography-mass spectrometry. Lysophospholipids can be measured by two-dimensional thin-layer chromatography.

[0006] Patent Document 1 discloses a method for quantifying ethanolamine-type plasmalogen, which includes a step of treating ethanolamine-type lysoplasmalogen, which is produced by hydrolyzing ethanolamine-type plasmalogen in a sample with phospholipase A1, with phospholipase D.

[0007] Patent Document 2 discloses a method for quantifying choline-type plasmalogen, which utilizes the action of hydrolyzing the phosphate ester at the sn-3 position of choline-type plasmalogen to liberate choline using phospholipase D that acts on choline-type plasmalogen.

[0008] Furthermore, liquid chromatography-mass spectrometry (LC-MS) allows accurate identification of lipids in mixtures and identification of plasmalogens by detecting fragment ions characteristic of headgroup classes and fatty acids or alcohols esterified to the glycerol backbone.

[0009] In Non-Patent Document 6, the presence or absence of plasmalogen in Clostridium perfringens is qualitatively analyzed by a color reaction between an aliphatic aldehyde produced by acid treatment of plasmalogen and a Schiff reagent (fuchsin aldehyde reagent).

[0010] JP 2016-111929 A JP 2014-082991 A

[0011] Megumi Nishimuki and two others, "Absorption characteristics of plasmalogens and their usefulness as a biomarker for blood plasmalogens," Oleoscience, 2015, Vol. 15, No. 2, pp. 53-60; Marcia Cristina Fernandez Messias and three others, "Plasmalogen lipids: functional mechanism and their involvement in gastrointestinal cancer," Lipids in Health and Disease, 2018, pp. 17, pp. 41-53; Zhizhong Guan and five others, "Decrease and Structural Modifications of Phosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease”, J Neuropathol Exp Neurol, 1999, 58(7), 740-747 Dayan B. Goodenowe, 17 others, “Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia” 2007, J Lipid Res, 48(11), 2485-2498Eugene L. Gottfried and Maurice M. Rapport, “The biochemistry of plasmalogens. I. Isolation and characterization of phosphotidal choline, a pure native "plasmalogen", 1962, J Biol Chem, 237, 329-333 David R. Jackson, and 7 others, “Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operan Responsible for Plasmalogen Production in Clostridium perfringens”, 2021, ACS Chem Biol, 16, 6-13

[0012] All of the methods disclosed in Non-Patent Document 5, Patent Documents 1 and 2 require an extraction process of lipid fractions using organic solvents. Furthermore, these methods require time-consuming and expensive analytical equipment for analysis, making it difficult to measure a large number of samples. Although the method using the Schiff reagent in Non-Patent Document 6 can qualitatively detect plasmalogens in cells, it cannot be used for quantitative analysis.

[0013] The present invention has been made in view of the above-mentioned circumstances, and aims to provide a method for quantifying plasmalogen and a kit for quantifying plasmalogen that can quantify plasmalogen simply and quickly without undergoing an extraction operation of a lipid fraction.

[0014] Even when a Schiff's reagent is used to quantify plasmalogens in samples containing proteins such as plasma, no proportional relationship is observed between the volume of the sample, i.e., the amount of plasmalogens, and the degree of coloration, making it impossible to quantify plasmalogens. The present inventors have conducted extensive research and found conditions for quantifying plasmalogens in cell-free samples using the color reaction of the Schiff's reagent, thereby completing the present invention.

[0015] A method for quantifying plasmalogen according to a first aspect of the present invention includes: a measurement sample preparation step of mixing a specimen containing a protein and plasmalogen with a Schiff reagent to obtain a measurement sample; a control sample preparation step of mixing the specimen with a solvent for the Schiff reagent to obtain a control sample; a measurement step of measuring the degree of a first optical property of the measurement specimen obtained by reacting an aldehyde generated from a vinyl ether bond of the plasmalogen with the Schiff reagent and the degree of the optical property of the control specimen; and a determination step of determining the concentration of plasmalogen in the specimen by comparing the degree of correction obtained by correcting the degree of the first optical property with the degree of the optical property of the control specimen with the degree of a second optical property obtained after reaction of the Schiff reagent with an aldehyde generated from a vinyl ether bond of the plasmalogen with the Schiff reagent, which is obtained by a standard sample containing a Schiff reagent and a plasmalogen of a known concentration.

[0016] The standard samples may be multiple samples containing different concentrations of the plasmalogen and the Schiff's reagent, and in the determination step, the concentration of the plasmalogen in the specimen may be determined based on a calibration curve created from the degree of the second optical property of the multiple standard samples after the reaction and the degree of correction.

[0017] The degree of the first optical property, the degree of the optical property of the control sample, and the degree of the second optical property may be measured as fluorescence intensity.

[0018] The optical property may be coloration.

[0019] The degree of the first optical property, the degree of the optical property of the control sample, and the degree of the second optical property may be measured as absorbance.

[0020] The plasmalogen having a known concentration may be dissolved in ethanol.

[0021] The specimen may be cell-free.

[0022] The solvent for the Schiff's reagent may be aqueous sulfurous acid.

[0023] A kit for quantifying plasmalogen according to a second aspect of the present invention comprises: a Schiff reagent; and a solvent for the Schiff reagent.

[0024] According to the present invention, plasmalogen can be quantified simply and quickly without the need for extraction of lipid fractions.

[0025] FIG. 1 is a diagram showing the relationship between corrected absorbance and plasma volume in a sample according to Test Example 1. FIG. 2 is a diagram showing a separation method by high performance liquid chromatography (HPLC) of plasmalogen used to create a calibration curve according to Example 1. FIG. 3 is a diagram showing a chromatogram of a lipid fraction extracted by HPLC in Example 1. FIG. 4 is a diagram showing a chromatogram of ethanolamine-type plasmalogen (PL-PE) in Example 1. FIG. 5 is a diagram showing a calibration curve according to Example 1. FIG. 6 is a diagram showing plasmalogen concentrations by measuring fluorescence intensity in Example 2. FIG. 7 is a diagram showing plasmalogen concentrations by measuring absorbance in Example 2.

[0026] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0027] (Embodiment) A plasmalogen quantification method according to this embodiment includes a measurement sample preparation step, a control sample preparation step, a measurement step, and a determination step. In the measurement sample preparation step, a sample is obtained by mixing a sample with a Schiff's reagent. The quantification method according to this embodiment quantifies plasmalogen in a sample containing protein, so the sample contains protein and plasmalogen. Note that the sample may not contain plasmalogen, and in such cases, the quantification method determines that the sample does not contain plasmalogen or that the amount is below the detection limit.

[0028] More specifically, in the measurement sample preparation step, the Schiff reagent and the specimen are mixed under acidic conditions. For example, if the solvent of the Schiff reagent is acidic, the Schiff reagent and the specimen can be mixed under acidic conditions. More specifically, the pH of the mixture obtained by mixing the Schiff reagent and the specimen is less than 7. The pH is preferably less than 5, less than 4, less than 3, or less than 2.

[0029] The specimen is not particularly limited and may be a body fluid derived from a living organism, a cell culture medium, or the like. Examples of specimens include blood, plasma, serum, urine, sweat, cerebrospinal fluid, bile, pancreatic juice, amniotic fluid, and ascites. Preferably, the specimen does not contain cells. Suitably, the specimen is plasma. The living organism is a human or non-human animal, preferably a mammal. Examples of mammals include primates such as chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats.

[0030] Schiff's reagent is a reagent that specifically reacts with aldehydes. "Aldehyde-specific coloring" means that it reacts with aldehydes but not with ketones to produce a color. Because Schiff's reagent does not react with ketones, it specifically reacts with plasmalogens even if the sample contains protein. A commercially available Schiff's reagent may be used, or it may be prepared by dissolving basic fuchsin in hot water, cooling, adding anhydrous sodium sulfite and concentrated hydrochloric acid, and diluting appropriately with water.

[0031] The mixing ratio of the specimen and the Schiff reagent mixed in the measurement sample preparation step is not particularly limited, and for example, the volume ratio of the specimen to the Schiff reagent (specimen:Schiff reagent) is 1:5 to 1:50, 1:5 to 1:40, 1:5 to 1:30, or 1:5 to 1:20.

[0032] In the control sample preparation step, the specimen is mixed with the solvent of the Schiff's reagent to obtain a control sample. If the solvent of the Schiff's reagent used in the measurement sample preparation step is sulfite water, the specimen is mixed with sulfite water in the control sample preparation step. If the solvent of the Schiff's reagent used in the measurement sample preparation step is water containing anhydrous sodium sulfite and concentrated hydrochloric acid, the specimen is mixed with water containing anhydrous sodium sulfite and concentrated hydrochloric acid in the control sample preparation step. The mixing ratio of the specimen and solvent mixed in the control sample preparation step is preferably the same as the mixing ratio of the specimen and Schiff's reagent in the measurement sample preparation step.

[0033] In the measurement step, the degree of optical properties of a measurement sample in which an aldehyde generated from a vinyl ether bond of the plasmalogen has reacted with a Schiff reagent and the degree of optical properties of a control sample are measured. To react the aldehyde with the Schiff reagent, for example, the measurement sample may be incubated at room temperature, more preferably at 37°C. The reaction time is, for example, 5 to 60 minutes, 10 to 50 minutes, 20 to 40 minutes, or 25 to 35 minutes.

[0034] Optical properties include coloration, color development, light absorption, luminescence, fluorescence, etc. When measuring coloration as an optical property, the measurement step measures the degree of color A of a measurement sample in which an aldehyde generated from a vinyl ether bond of plasmalogen has reacted with a Schiff reagent, and the degree of color B of a control sample. As described above, since the vinyl ether bond of plasmalogen is unstable to acid, plasmalogen is decomposed under acidic conditions to generate aldehyde. In the measurement step, the degree of color A generated by the reaction of the generated aldehyde with the Schiff reagent is measured. In the measurement step, the degree of color A may be measured first, followed by the degree of color B, or the degree of color B may be measured after measuring the degree of color A, or the degree of color A and the degree of color B may be measured simultaneously.

[0035] The degree of color A and color B can be measured visually, but is preferably measured as absorbance using a spectrophotometer. Absorbance can be measured using known methods and devices. In measuring absorbance, for example, the mixed solution is irradiated with light of an absorption wavelength suitable for the Schiff reagent, and the absorbance is measured. The wavelength of the irradiated light is 500 to 600 nm, 520 to 580 nm, or 540 to 560 nm, preferably 550 nm.

[0036] Since the fuchsin contained in the Schiff's reagent has a structure with multiple benzene rings, the product of the reaction between the aldehyde and the Schiff's reagent is fluorescent. Therefore, fluorescence may be measured as an optical property. In this case, for example, the degree of optical properties of the measurement sample and the degree of optical properties of the control sample are measured as fluorescence intensity. Fluorescence intensity can be measured using known methods and instruments. When measuring fluorescence intensity as an optical property, the measurement step measures the fluorescence intensity of the measurement sample and the control sample in which the aldehyde generated from the vinyl ether bond of the plasmalogen has reacted with the Schiff's reagent. In fluorescence measurement, the fluorescence intensity of the measurement sample after the reaction may be measured followed by the fluorescence intensity of the control sample, or the fluorescence intensity of the measurement sample after the reaction may be measured after the control sample has been measured, or the fluorescence intensity of the measurement sample after the reaction and the fluorescence intensity of the control sample may be measured simultaneously.

[0037] In the determination step, the concentration of plasmalogen in the specimen is determined by comparing the degree of correction obtained by correcting the degree of optical properties of the measurement sample with the degree of optical properties of the control sample with the degree of optical properties of a standard sample containing Schiff reagent and a plasmalogen of known concentration after reaction between the Schiff reagent and the aldehyde produced from the vinyl ether bond of the plasmalogen.

[0038] The determination step will be described in more detail using an example in which coloration is measured as an optical property. In the determination step, the degree of coloration A is corrected by the degree of coloration B of the control sample, and the correction degree is compared with the degree of coloration C (second coloration) after the reaction of the standard sample, thereby determining the concentration of plasmalogen in the sample. When the degrees of coloration A and coloration B are measured by absorbance, the correction degree is, for example, the value (V １ The plasmalogen having a known concentration is, for example, plasmalogen dissolved in ethanol. The plasmalogen may be extracted from animal tissue or the like by a known method, or may be commercially available plasmalogen.

[0039] When the degree of color A and the degree of color B are measured as absorbance, the degree of color C is also measured as absorbance. For example, the degree of color C can be calculated by the ratio of the Schiff's reagent and the concentration C １ is the absorbance of the standard sample prepared by mixing the plasmalogen. １ If the absorbance of the standard sample is the same as that of the plasmalogen in the sample, the concentration of plasmalogen in the sample is C １ is determined.

[0040] When measuring absorbance as the degree of optical property, the optical property of the standard sample after the reaction is also measured as fluorescence intensity. For example, the degree of correction is the value obtained by subtracting the fluorescence intensity value of the control sample from the fluorescence intensity value of the measurement sample (V ２ ) is the concentration of Schiff's reagent and ２ If the fluorescence intensity of the standard sample is the same as that of the plasmalogen mixed with the plasmalogen in the sample, the concentration of plasmalogen in the sample is C ２ is determined.

[0041] In the determination step, the plasmalogen concentration in the specimen may be determined based on the calibration curve and the degree of correction. The calibration curve is created using multiple standard samples containing different concentrations of plasmalogen and Schiff's reagent, and based on the degree of optical properties of the multiple standard samples after the reaction. For example, the plasmalogen concentration in the standard samples may be set within a range that encompasses the plasmalogen concentration expected in the specimen. The different plasmalogen concentrations in the standard samples may be, for example, three, four, five, six, seven, or ten or more. The plasmalogen concentration range of the standard samples is, for example, 0.005 to 0.1 μg / μL.

[0042] When the degree of optical property is absorbance, specifically, the value V obtained as the correction degree is added to the regression equation of the obtained calibration curve. １ In addition, when the degree of optical property is fluorescence intensity, the value V obtained as the correction degree is added to the regression equation of the obtained calibration curve. ２ By substituting the above, the concentration of plasmalogen in the sample can be determined.

[0043] According to the plasmalogen quantification method of this embodiment, the concentration of plasmalogen in a sample can be determined based on the degree of optical properties after reaction between a standard sample Schiff's reagent and a plasmalogen of known concentration. Therefore, plasmalogen can be quantified simply and quickly without extraction procedures such as lipid fractions. Furthermore, in the plasmalogen quantification method of this embodiment, the plasmalogen concentration in a sample may be determined based on a calibration curve and the degree of correction. As shown in Example 1 below, the correlation coefficient of the calibration curve obtained by this quantification method is good, so the plasmalogen concentration in a sample can be determined with high accuracy.

[0044] Furthermore, in the plasmalogen quantification method according to the present embodiment, the degree of coloration A, coloration B, and coloration C as optical properties may be measured as absorbance. Absorbance can be easily measured using a relatively inexpensive device, and a large number of samples can be easily measured.

[0045] In addition, in the plasmalogen quantification method according to the present embodiment, the optical properties of the measurement sample, control sample, and standard sample may be measured as absorbance. Fluorescence is preferable because it can be measured easily, has higher detection sensitivity than absorbance, and can quantify plasmalogens at lower concentrations.

[0046] In another aspect of the present embodiment, a kit for quantifying plasmalogen is provided. The kit for quantifying plasmalogen includes a Schiff reagent and a solvent for the Schiff reagent. The Schiff reagent is used to prepare the measurement sample and the standard sample as described above. The solvent is used to prepare the control sample as described above. The kit for quantifying plasmalogen may also include a plasmalogen that can be used to prepare the control sample. In another aspect of the present embodiment, a reagent for quantifying plasmalogen is provided that includes the solvent for the Schiff reagent.

[0047] In another aspect of the present embodiment, there is provided use of the above-mentioned solvent for the Schiff reagent for the manufacture of a kit for quantifying plasmalogen or a reagent for quantifying plasmalogen. In another aspect of the present embodiment, there is provided a solvent for the Schiff reagent for use in quantifying plasmalogen.

[0048] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0049] Test Example 1: Study of the color reaction between human plasma and Schiff's reagent. Venous blood from a subject was collected in a heparin-containing blood collection tube (Terumo Corporation) and centrifuged at 1,000 × g for 5 minutes. The supernatant plasma was collected and frozen. 190, 185, and 180 μL of Schiff's reagent (pH < 2.0, Sigma, 84655) was added to 10, 15, and 20 μL of plasma, respectively, and the mixture was incubated at 37°C for 30 minutes to obtain measurement samples. The absorbance at 550 nm of the measurement samples was measured using an iMark plate reader (BioRad).

[0050] Separately, 190, 185, and 180 μL of sulfite water (Fujifilm Wako Pure Chemical Industries, Ltd., 195-11955) were added to 10, 15, and 20 μL of plasma from each subject, respectively, and the mixture was allowed to react at 37°C for 30 minutes. After that, the absorbance was measured as background in the same manner as above.

[0051] The relationship between the absorbance of the measurement sample (corrected absorbance) after subtracting the background value and the plasma volume in the sample is shown in Figure 1. An increase in the corrected absorbance was observed in proportion to the plasma volume.

[0052] Comparative Example: Measurement of Plasmalogen in Human Plasma by LC-MS 1. Preparation of Stock Standard Solution The reagents used were phosphatidylethanolamine (PE, Funakoshi Co., Ltd., A-34, dissolved in chloroform at 10 mg / mL), phosphatidylcholine (PC, Funakoshi Co., Ltd., A-29B, dissolved in chloroform at 5 mg / mL), and sphingomyelin (SM, Funakoshi Co., Ltd., A-401). 25 mg of SM was diluted with chloroform to a concentration of 5 mg / mL.

[0053] 100 μL of PE, 200 μL each of PC and SM were placed in separate screw-top tubes. ２ After drying with gas, 1 mL each of isopropanol / chloroform (2:1) was added to PE, PC, and SM to adjust the concentration to 1 mg / mL. After mixing, the mixture was sonicated and stored at -30°C as a stock standard solution.

[0054] 2. Preparation of measurement standard solutions The stock standard solutions were diluted with HIP (3:2) on ice (5, 10, 15, 20, 25, 50 μg / mL for PE and PC, and 20, 40, 60, 80, 100, 200 μg / mL for SM), and filtered to prepare 0.45 μL standard solutions.

[0055] 3. Lipid Extraction from Blood Venous blood from the same subject as in the above test example was collected in a heparin-containing blood collection tube (Terumo Corporation), centrifuged at 1,000 × g for 5 minutes, and the supernatant plasma was collected and frozen. The preserved plasma was thawed by immersion in room temperature water and centrifuged at 13,000 rpm for 5 minutes. 280 μL of the plasma was pipetted into an Eppendorf tube using a 1 mL pipette. 70 μL of PLA1 (×2) solution, prepared fresh by mixing Phospholipase A1 (Sigma) and 0.1 M citrate buffer (HCl pH 4.5) at a 1:1 ratio, was added to the Eppendorf tube and mixed. The mixture was allowed to react at 45°C for 1 hour, and then immediately immersed in ice water to stop the reaction. After mixing, the PLA1-treated plasma was pipetted and dispensed into Eppendorf tubes in 100 μL portions.

[0056] Lipids were extracted by the hexane-isopropanol (HIP) method as follows: 800 μL of HIP (3:2) was added to the plasma and mixed. The mixture was treated with ultrasound for 5 minutes to remove Na. ２ SO ４ 400 μL of water (1 g / 15 mL) was added and mixed. After standing for 5 minutes, 400 μL of the upper layer was taken without pipetting and transferred to another Eppendorf tube. 400 μL of HIP (7:2) was then added and mixed. After standing for 5 minutes, 300 μL of the upper layer was taken without pipetting and combined with the upper layer previously collected in the Eppendorf tube to form a sample. The sample was stored at -30°C until measurement.

[0057] 4. Before LC-MS measurement, the sample was ２ The sample was redissolved in 200 μL of HIP (3:2) and filtered through a 0.45 μL filter.

[0058] The prepared standard solution and sample were each placed in a glass insert with stems, and 10 μL was injected into the LC-MS. The LC-MS conditions are as follows: Column: CHEMCOBOND 5OH (Diol) 3.0 x 30, 30 x 250 (w) (Chemco Plus) Column temperature: 50°C Ionization mode: API-ES Polarity: Negative Scan mass range low: 680 Scan mass range high: 950 Gas temperature: 300°C Drying gas flow: 10.0 L / min Nebulizer pressure: 45 psig Vcap (negative): 3500 V Solvent A: 53 mM formic acid - acetonitrile Solvent B: 60 mM ammonium formate and 53 mM formic acid - water

[0059] The LC-MS time schedule is shown in Table 1.

[0060]

[0061] 5. Results In the comparative example, the concentration of PL-PE in plasma quantified by LC-MS was 0.053 μg / μL.

[0062] Example 1: Measurement of human plasma plasmalogen by absorbance using Schiff's reagent 1. Preparation of sPls Plasmalogen (sPls) consisting only of PL-PE was prepared from scallops (Mizuhopecten yessoensis) using the following method. sPls was extracted from raw scallops using hexane, as described in Japanese Patent No. 7304643. The steps for extracting plasmalogen from scallops are as follows: (1) Cocurase P (manufactured by Mitsubishi Chemical Foods Corporation) and phospholipase A1 (PLA1, manufactured by Mitsubishi Chemical Foods Corporation) were added to the scallops and digested for 1 hour. (2) Hexane / isopropanol was added and stirred. After allowing to stand, the supernatant was filtered under suction. (3) Aqueous sodium sulfate solution was added to the filtrate and mixed thoroughly. (4) The upper layer was evaporated to dryness using a rotary evaporator. (5) 40 mL of acetone cooled to 4°C was added to this and mixed. (6) Separation was performed using a centrifuge at 3,000 rpm for 10 minutes at 4°C. (7) The supernatant was discarded and the precipitate was collected. (8) Drying was performed overnight in a desiccator. Following the specification of Japanese Patent No. 5,430,566, separation was performed by HPLC using the mobile phase solvent shown in Figure 2A. Figure 2B shows the lipid composition of the fractions extracted in steps (1) to (8) above. As shown in Figure 2C, only PL-PE was separated and designated sPls.

[0063] 2. Preparation of a calibration curve sPls was dissolved in ethanol to give concentrations of 0.005, 0.01, 0.05, and 0.1 μg / μL to obtain sPls solutions. 185 μL of Schiff solution (pH < 2.0, Sigma, 84655) was added to 15 μL of each sPls solution, and the mixture was incubated at 37°C for 30 minutes. The absorbance at 550 nm was measured in the same manner as in Test Example 1 above.

[0064] 3. Measurement of plasmalogens in human plasma 185 μL of Schiff's reagent was added to 15 μL of plasma prepared from the venous blood of the same subject as in Example 1, and the mixture was allowed to react at 37° C. for 30 minutes to obtain a measurement sample. The absorbance of the measurement sample at 550 nm was measured in the same manner as in Test Example 1 above. Meanwhile, 185 μL of sulfite water was added to 15 μL of the plasma from the same subject, and the mixture was allowed to react at 37° C. for 30 minutes, after which the absorbance was measured as background in the same manner as in Test Example 1 above.

[0065] 4. Results The calibration curve prepared is shown in Figure 3. A good positive correlation was observed between the sPls concentration and absorbance (correlation coefficient = 0.9997).

[0066] The absorbance (corrected absorbance) of the measurement sample after subtracting the background value was 0.038, and the plasmalogen concentration in plasma calculated from the calibration curve was 0.058 μg / μL. Compared to the results of the comparative example, the value was higher than the PL-PE concentration of 0.053 μg / μL because the plasmalogen concentration calculated from the calibration curve also includes the concentration of choline-type plasmalogen in plasma.

[0067] [Test Example 2: Examination of the fluorescence of Schiff's reagent in reaction with plasmalogen-derived aldehyde] Since fuchsin contained in the Schiff's reagent has a structure with multiple benzene rings, the possibility of fluorescence was examined. HeLa cells that synthesize plasmalogen were fixed with paraformaldehyde solution and then reacted with Schiff's reagent at 37°C for 30 minutes, and the fluorescence of the obtained sample was detected using the white laser of a microscope SP8 (manufactured by Leica).

[0068] (Results) The maximum excitation wavelength of the sample was found to be 580 nm, and the maximum fluorescence wavelength was found to be 620 nm. This fluorescence was not detected in plasmalogen synthesis-deficient cells treated in the same way, and it was determined that fuchsin reacted with plasmalogen-derived aldehydes to be fluorescent.

[0069] Example 2: Measurement of plasmalogens in human plasma by fluorescence using Schiff's reagent Generally, measurements using fluorescence intensity are more sensitive than measurements using absorbance. Therefore, plasmalogens in plasma were measured by fluorescence using the same method as in Example 1 above, except that fluorescence intensity was measured instead of absorbance. A fluorometer DTX880 (manufactured by Beckman Coulter) was used to measure fluorescence intensity. An excitation filter was used to set the wavelength of excitation light to 535 nm, and a fluorescence filter was used to detect fluorescence at 595 nm. In this example, plasmalogens in plasma were measured using absorbance, as in Example 1 above.

[0070] (Results) Figure 4 shows the plasmalogen concentrations of measurement samples A to E measured using fluorescence intensity or LC-MS. Figure 5 shows the plasmalogen concentrations of measurement samples F to I measured using absorbance or LC-MS. Plasma plasma could be quantified even when fluorescence intensity was used.

[0071] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to illustrate the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0072] This application is based on Japanese Patent Application No. 2024-000431, filed on January 5, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-000431 are incorporated herein by reference.

[0073] The present invention is useful for quantifying plasmalogens.

Claims

1. A measurement sample preparation step of obtaining a measurement sample by mixing a sample containing a protein and a plasmalogen with a Schiff reagent; a control sample preparation step of obtaining a control sample by mixing the sample and a solvent of the Schiff reagent; a measurement step of measuring the degree of the first optical property of the measurement sample and the degree of the optical property of the control sample, in which an aldehyde generated from a vinyl ether bond of the plasmalogen reacts with the Schiff reagent; and a determination step of determining the concentration of the plasmalogen in the sample by comparing the corrected degree, obtained by correcting the degree of the first optical property with the degree of the optical property of the control sample, with the degree of the second optical property after the reaction of the Schiff reagent and an aldehyde generated from a vinyl ether bond of a plasmalogen of known concentration in a standard sample containing the Schiff reagent and the plasmalogen of known concentration. A method for quantifying a plasmalogen, comprising the above steps.

2. The standard sample is a plurality containing the plasmalogen and the Schiff reagent at different concentrations. In the determination step, the concentration of the plasmalogen in the sample is determined based on a calibration curve created from the degrees of the second optical property of the plurality of standard samples after the reaction and the corrected degree. The method for quantifying a plasmalogen according to claim 1.

3. The degree of the first optical property, the degree of the optical property of the control sample, and the degree of the second optical property are measured as fluorescence intensity. The method for quantifying a plasmalogen according to claim 1 or 2.

4. The optical property is color development. The method for quantifying a plasmalogen according to claim 1 or 2.

5. The degree of the first optical property, the degree of the optical property of the control sample, and the degree of the second optical property are measured as absorbance. The method for quantifying a plasmalogen according to claim 1 or 2.

6. The plasmalogen of known concentration is dissolved in ethanol. The method for quantifying a plasmalogen according to claim 1 or 2.

7. The sample does not contain cells. The method for quantifying a plasmalogen according to claim 1 or 2.

8. The solvent of the Schiff reagent is sulfurous acid water. The method for quantifying a plasmalogen according to claim 1 or 2.

9. A kit for quantifying a plasmalogen, comprising a Schiff reagent and a solvent of the Schiff reagent.

Images