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 selectively reacts with high density lipoproteins in the sample, said surfactant being selected from sucrose esters, and maltosides, 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 selectively breaks down high density lipoproteins and which optionally attenuates the reaction of low density lipoproteins, said surfactant being selected from sucrose esters and maltosides, and measuring the amount of cholesterol in the high density lipoproteins.
2 . 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 HDL or LDL cholesterol concentration respectively.
3 . A method according to claim 1 , wherein the surfactant is (i) a sucrose ester comprising a sucrose moiety in which one or more HO— groups is independently replaced with a group RCOO—, wherein R is an alkyl or alkenyl group having up to 18 carbon atoms, or (ii) a β-maltoside of formula (II)
wherein R is an alkylene or alkenylene group having up to 18 carbon atoms and A is methyl or a cycloalkyl group having from 4 to 7 carbon atoms, or a corresponding α-maltoside.
4 . A method according to claim 1 to 3 , wherein the surfactant is sucrose monocaprate, cyclohexylbutyl-β-D-maltoside (Cymal-4) or cyclohexylpentyl-β-D-maltoside (Cymal-5).
5 . 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.
6 . A method according to claim 1 , wherein the amount of cholesterol in the high density lipoproteins is measured by an electrochemical technique.
7 . A method according to claim 6 , wherein the method comprises reacting the sample with
(a) a surfactant which selectively breaks down high density lipoproteins and which optionally attenuates the reaction of low 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.
8 . A method according to claim 7 , wherein the cholesterol ester hydrolysing reagent is a lipase.
9 . A method according to claim 7 , wherein the sample is additionally reacted with (f) a reductase.
10 . A method according to claim 7 , 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 selectively breaks down high density lipoproteins and which optionally attenuates the reaction of low density lipoproteins said surfactant being selected from sucrose esters and maltosides;
(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 selectively breaks down high density lipoproteins and which optionally attenuates the reaction of low density lipoproteins, said surfactant being selected from sucrose esters and maltosides, (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 selectively breaks down high density lipoproteins and which optionally attenuates the reaction of low density lipoproteins said surfactant being selected from sucrose esters and maltosides, (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, 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).