Method For Quantifying Plasmalogens Using PLA1 Processing

Abstract

The present invention provides methods for quantifying an amount of plasmalogens in samples with high accuracy in an easy, convenient and inexpensive manner by using a hydrolysis processing to samples and a lipid extraction followed by the measurement using High Performance Liquid Chromatography (HPLC) with Evaporative Light Scattering Detector (ELSD) or Mass Spectrometer (MS), a fluorescence plate reader or a plate reader. The present invention also relates to a method for examining a subject by using the above method, a biomarker for disease detection, a method for using the biomarker for the disease detection, as well as a kit for the disease detection.

1 . A method for examining a subject to determine a risk of developing a disease that is closely associated with a change in the amount of plasmalogens comprising:
 (1) a step of quantifying plasmalogens contained in a sample derived from a subject; and   (2) a step of comparing the amount of plasmalogens in the sample measured at the step (1) with the amount of plasmalogens in samples derived from a healthy subject,   wherein the step of quantifying plasmalogens comprises:   (A) a step of providing hydrolysis processing of the sample, after which lipid extraction is performed to obtain a lipid sample,   (B′) a step of reacting the lipid sample obtained by the step (A) with glycerophospholipid specific phospholipase D to get an ethanolamine or a choline; and   (C′) a step of reacting the ethanolamine or choline obtained by the step (B′) with an amine oxidase or a choline oxidase to produce hydrogen peroxide (H 2 O 2 ), respectively; and   (D′) a step of reacting the hydrogen peroxide (H 2 O 2 ) produced by the step (C′) with a fluorescent reagent in the presence of a peroxidase to produce a fluorescent compound, and measuring the fluorescent compound by a fluorescence plate reader; or   (D″) a step of reacting the hydrogen peroxide (H 2 O 2 ) produced by the step (C′) with coloring reagents in the presence of a peroxidase to produce a colored compound, and measuring the colored compound by a plate reader.   
     
     
         2 . The method for examining a subject according to  claim 1 , wherein the sample is serum or plasma. 
     
     
         3 . The method for examining a subject according to  claim 1 , wherein the plasmalogens are ethanolamine plasmalogens or choline plasmalogens. 
     
     
         4 . The method for examining a subject according to  claim 1 , wherein the hydrolysis processing is performed by phospholipase A1 (PLA1). 
     
     
         5 . The method for examining a subject according to  claim 1 , wherein the lipid extraction process is performed by a mixture of hexane/isopropanol (3:2, v/v) or chloroform-methanol (1:2, v/v). 
     
     
         6 . The method for examining a subject according to  claim 1 , wherein the fluorescent reagent is Amplex Red. 
     
     
         7 . The method for examining a subject according to  claim 1 , wherein the coloring reagents are 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-toluidine (TOOS). 
     
     
         8 . The method for examining a subject according to  claim 1 , wherein after the step (A) and before the step (B′), unnecessary water-soluble substances are removed by using alcohol, sodium sulfate, water or any combination thereof. 
     
     
         9 . The method for examining a subject according to  claim 1 , further comprising:
 (3) a step of determining that a risk of the subject developing a disease is high as a consequence of the step (2), in the case where the amount of plasmalogens contained in the sample derived from a subject measured at the step (1) is either less or greater than that of plasmalogens contained in the samples derived from a healthy subject.   
     
     
         10 . The method for examining a subject according to  claim 1 , wherein the disease is dementia, depression, brain fatigue, insomnia, Parkinson's disease, metabolic syndrome, diabetes or arteriosclerosis. 
     
     
         11 . A method for using plasmalogens contained in serum or plasma, as a biomarker for detecting a disease that is closely associated with a change in the amount of plasmalogens. 
     
     
         12 . The method for using plasmalogens contained in serum or plasma according to  claim 11 , wherein the plasmalogens are ethanolamine plasmalogens or choline plasmalogens. 
     
     
         13 . A test kit for examining a subject a risk of developing a disease that is closely associated with a change in the amount of plasmalogens comprising:
 a reagent used for hydrolysis processing of samples;   organic solvents for lipid extraction;   an amine oxidase or a choline oxidase, a peroxidase, and a fluorescent reagent or coloring reagent; and   glycerophospholipid specific phospholipase D.