HDL Cholesterol Sensor Using Selective Surfactant
A method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, said method comprising reacting the sample with a surfactant which preferentially reacts with high density lipoproteins in the sample, said surfactant being selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives, and measuring the amount of cholesterol in the high density lipoproteins, for example using an electrochemical technique.
1 . A method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, said method comprising reacting the sample with (a) a surfactant which preferentially breaks down high density lipoproteins, said surfactant being selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives, and measuring the amount of cholesterol in the high density lipoproteins.
2 . A method according to claim 1 , wherein the surfactant is for improving the reproducibility of the determination of the amount of cholesterol in high density lipoproteins.
3 . A method according to claim 1 , wherein the surfactant (a) has a differentiation for HDL over LDL given by the equation (i) below of at least 50%:
Differentiation
(
%
)
=
G
HDL
-
G
LDL
G
HDL
×
100
wherein
G
HDL
is
measured
(
HDLcholesterol
)
known
[
HDLcholesterol
]
,
and
G
LDL
is
measured
(
LDLcholesterol
)
known
[
LDLcholesterol
]
.
(
i
)
wherein measured (HDL cholesterol) or measured (LDL cholesterol) is a measured value which relates to the concentration of HDL or LDL respectively.
4 . A method according to claim 1 , wherein the surfactant (a) is selected from HEGA-8, HEGA-9, HEGA-10, C-HEGA-9, C-HEGA-10, MEGA-8, MEGA-9, MEGA-10 and MEGA-12.
5 . A method according to claim 1 , wherein the method comprises repeating the measurement one or more times for a given sample to provide a data set of a plurality of measurements relating to the HDL concentration of the sample, and wherein R 2 for the data set is at least 0.6.
6 . A method according to claim 1 , wherein the amount of cholesterol in the high density lipoproteins is measured by reacting the sample with (b) a cholesterol ester hydrolysing reagent and (c) cholesterol oxidase or cholesterol dehydrogenase and determining the amount of cholesterol which has reacted with the cholesterol oxidase or cholesterol dehydrogenase.
7 . A method according to claim 1 , wherein the amount of cholesterol in the high density lipoproteins is measured by an electrochemical technique.
8 . A method according to claim 7 , wherein the method comprises reacting the sample with
(a) a surfactant which preferentially breaks down high density lipoproteins;
(b) a cholesterol ester hydrolysing reagent;
(c) cholesterol oxidase or cholesterol dehydrogenase;
(d) a coenzyme; and
(e) a redox agent capable of being oxidised or reduced to form a product;
and electrochemically detecting the amount of product formed.
9 . A method according to claim 8 , wherein the sample is additionally reacted with (f) a reductase.
10 . A method according to claim 1 , wherein the sample is reacted simultaneously with the surfactant (a), the cholesterol ester hydrolysing reagent (b) and the cholesterol oxidase or cholesterol dehydrogenase (c).
11 . A method according to claim 1 , wherein the high density lipoprotein containing sample is whole blood and wherein the method additionally comprises the step of filtering the sample to remove red blood cells.
12 . A method according to claim 1 , wherein the measurement of the amount of cholesterol in the high density lipoproteins is completed within a period of no more than 3 minutes from reaction of the sample with the surfactant.
13 . A reagent mixture for use in a method for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the reagent mixture comprising
(a) a surfactant which preferentially breaks down high density lipoproteins, said surfactant being selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives;
(b) a cholesterol ester hydrolysing reagent; and
(c) cholesterol oxidase or cholesterol dehydrogenase.
14 . A reagent mixture according to claim 13 , which additionally comprises (d)
a coenzyme, (e) a redox agent capable of being oxidised or reduced to form a product; and optionally (f) a reductase.
15 . A kit for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the kit comprising (a) a surfactant which preferentially breaks down high density lipoproteins, said surfactant being selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives, (b) a cholesterol ester hydrolysing reagent, and (c) cholesterol oxidase or cholesterol dehydrogenase, and optionally one or more of (d) a coenzyme, (e) a redox agent capable of being oxidised or reduced to form a product, and (f) a reductase, and means for measuring the amount of cholesterol which reacts with the cholesterol oxidase or cholesterol dehydrogenase.
16 . A kit according to claim 15 wherein the means for measuring the amount of cholesterol which reacts with the cholesterol oxidase or cholesterol dehydrogenase comprises
an electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
a power supply for applying a potential across the cell; and
a measuring instrument for measuring the resulting electrochemical response.
17 . A kit according to claim 15 , wherein the reagents (a), (b) and (c) and optionally one or more of (d), (e) and (f) are present in the form of a single reagent mixture.
18 . A kit according to claim 17 , wherein the reagent mixture is in dried form.
19 . A method of operating a kit for the determination of the amount of cholesterol in high density lipoproteins in a high density lipoprotein containing sample, the kit comprising:
(a) a surfactant which preferentially breaks down high density lipoproteins, said surfactant being selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives, (b) a cholesterol ester hydrolysing reagent, and (c) cholesterol oxidase or cholesterol dehydrogenase, and optionally one or more of (d) a coenzyme, (e) a redox agent capable of being oxidised or reduced to form a product and (f) a reductase;
and means for measuring the amount of cholesterol which reacts with the cholesterol oxidase or cholesterol dehydrogenase comprising
an electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
a power supply for applying a potential across the cell; and
a measuring instrument for measuring the resulting electrochemical response; said method comprising
(i) contacting (1) the reagents (a), (b) and (c), and optionally one or more of (d), (e) and (f), and (2) a high density lipoprotein containing sample, with each other and with the electrodes;
(ii) applying a potential across the electrochemical cell; and
(iii) electrochemically detecting the amount of product formed by measuring the resulting electrochemical response.
20 . A method according to claim 19 , wherein step (iii) is completed within a period of up to 3 minutes from the time at which the sample is contacted with the reagents (a), (b) and (c).
21 . A method of improving the reproducibility of a determination of the amount of cholesterol in high density lipoproteins, comprising reacting a high density lipoprotein containing sample with a surfactant selected from hydroxyethyl glucamide derivatives and N-acyl-N-methyl glucamine derivatives.