METHODS OF DETERMINING A HIGH DENSITY LIPOPROTEIN PHOSPHOLIPID LEVEL IN A SAMPLE
The present disclosure provides economical and scalable (e.g., high-throughput) methods of determining an HDL-PL level in a sample from a subject, and methods of treating a subject comprising determining an HDL-PL level in a sample from the subject.
1 . A method of determining a level of HDL-PL associated with a subject, the method comprising:
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system.
2 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises contacting the sample with a precipitation reagent comprising dextran, a Mg 2+ salt and sodium azide.
3 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises contacting the sample with apolipoprotein B antisera to immunoprecipitate non-HDL particles.
4 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises ultracentrifuging the sample and thereafter removing any fractions having a density of at least 1.063 g/mL.
5 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises gel filtration of the sample.
6 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises gel filtering the sample.
7 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises filtering the sample through a column.
8 . The method of claim 1 , wherein the step of removing non-HDL lipoproteins comprises contacting the sample with cholesterol oxidase, peroxidase, phospholipidase D and N,N-bis-(4-sulfobutyl)-m-toulidine disodium.
9 . The method of claim 1 , wherein the indicator system comprises phospholipase D, choline oxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, 4-aminoantipyrine, and peroxidase.
10 . A method of determining a level of HDL-PL in a sample associated with a subject, the method comprising:
contacting the sample with a reagent system comprising cholesterol oxidase, peroxidase, phospholipase D and N,N-bis-(4-sulfobutyl)-m-toluidine disodium to solubilize free cholesterol and/or non-HDL phospholipids;
contacting the sample with a detergent to solubilize HDL-lipoproteins;
contacting the solubilized HDL-lipoproteins with phospholipase D, choline oxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline and 4-aminoantipyrine; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system.
11 . A method of treating a cardiovascular-related disease in a subject, the method comprising:
determining an HDL-PL level in a sample of the subject; and
administering to the subject a cardiovascular agent if the HDL-PL level is below a normal value.
12 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises contacting the sample with a precipitation reagent comprising dextran, a Mg 2+ salt and sodium azide.
13 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises contacting the sample with apolipoprotein B antisera to immunoprecipitate non-HDL particles.
14 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises ultracentrifuging the sample and thereafter removing any fractions having a density of at least 1.063 g/mL.
15 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises gel filtering the sample.
16 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises filtering the sample through a column.
17 . The method of claim 11 , wherein the step of determining an HDL-PL level in the sample comprises
removing non-HDL lipoproteins from a sample associated with the subject to produce a purified HDL-PL composition;
contacting the purified HDL-PL composition with an indicator system; and
measuring a concentration of the HDL-PL as a function of at least an absorbance of the indicator system,
wherein the step of removing non-HDL lipoproteins comprises contacting the sample with cholesterol oxidase, peroxidase, phospholipidase D and N,N-bis-(4-sulfobutyl)-m-toulidine disodium.
18 . The method of claim 11 , wherein the indicator system comprises phospholipase D, choline oxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, 4-aminoantipyrine, and peroxidase.