U.S. patent application number 10/898385 was filed with the patent office on 2005-01-13 for method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same.
This patent application is currently assigned to DAIICHI PURE CHEMICALS CO., LTD.. Invention is credited to Manabe, Mitsuhisa, Nakamura, Mitsuhiro, Taniguchi, Yuriko, Yamamoto, Mitsuaki.
Application Number | 20050009125 10/898385 |
Document ID | / |
Family ID | 26496172 |
Filed Date | 2005-01-13 |
United States Patent
Application |
20050009125 |
Kind Code |
A1 |
Nakamura, Mitsuhiro ; et
al. |
January 13, 2005 |
Method of pretreatment of sample for quantitating cholesterol and
method for quantitating cholesterol in specific lipoproteins by
using the same
Abstract
A method of a pretreatment of a sample for quantitating
cholesterol characterized by, before measuring cholesterol
contained in specific lipoproteins, treating the sample containing
lipoproteins with an enzyme, the substrate of which is free
cholesterol, optionally together with a reaction accelerator; a
method for quantitating cholesterol in specific lipoproteins by
using the above method; and a kit for quantitating cholesterol in
specific lipoproteins to be used in the above quantification
method. By using this quantification method, cholesterol in a
specific fraction can be conveniently, accurately and efficiently
quantitated fundamentally without resort to polyanion, etc. Thus,
this method is appropriately usable in various automatic
analyzers.
Inventors: |
Nakamura, Mitsuhiro;
(Ryugasaki-shi, JP) ; Taniguchi, Yuriko;
(Ryugasaki-shi, JP) ; Manabe, Mitsuhisa;
(Ryugasaki-shi, JP) ; Yamamoto, Mitsuaki;
(Ryugasaki-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
DAIICHI PURE CHEMICALS CO.,
LTD.
Tokyo
JP
|
Family ID: |
26496172 |
Appl. No.: |
10/898385 |
Filed: |
July 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10898385 |
Jul 26, 2004 |
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09926818 |
Dec 21, 2001 |
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6818414 |
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09926818 |
Dec 21, 2001 |
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PCT/JP00/03860 |
Jun 14, 2000 |
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Current U.S.
Class: |
435/11 |
Current CPC
Class: |
G01N 33/92 20130101;
C12Q 1/60 20130101; G01N 2800/044 20130101 |
Class at
Publication: |
435/011 |
International
Class: |
C12Q 001/60 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 1999 |
JP |
11-174624 |
Feb 3, 2000 |
JP |
2000-26737 |
Claims
1-37. (Canceled).
38. A method for quantitating free cholesterol and an esterified
cholesterol in a sample which contains various lipoproteins, which
comprises causing at least an enzyme, which acts upon free
cholesterol as a substrate, and a reaction accelerator, which is
selected from flufenamic acid, mefenamic acid,
2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone
acetate, monensin and mevinolin, to act upon said free
cholesterol.
39. A method for quantitating cholesterol existing in a specific
lipoprotein in a sample, which comprises causing cholesterol
oxidase, which acts upon free cholesterol as a substrate, to act
upon said sample with said lipoprotein contained therein to convert
only said free cholesterol under conditions that said lipoproteins
remain substantially unchanged; and then measuring said
cholesterol, which exists in said specific lipoprotein, by using a
substance which acts upon said specific lipoprotein only.
Description
TECHNICAL FIELD
[0001] This invention relates to a pretreatment method for
accurately and efficiently discriminating and quantitating
cholesterol, which exists in the specific lipoprotein fraction, by
simple procedures while using a small amount of a sample, and also
to a method for measuring cholesterol in the specific lipoprotein
fraction by using the pretreatment method.
BACKGROUND ART
[0002] Lipids such as cholesterol are complexed with apoproteins in
blood to form lipoproteins. Depending on differences in physical
properties, lipoproteins are classified into chylomicron, very low
density lipoprotein (VLDL), low density lipoprotein (LDL), high
density lipoprotein (HDL), and so on. Among these lipoproteins, LDL
is known to be one of causative substances which induce
arteriosclerosis, while HDL is known to show anti-arteriosclerotic
activity.
[0003] Epidemiologically, the level of cholesterol in LDL is known
to exhibit a positive correlation with the frequency of onset of
arteriosclerotic disease while the level of cholesterol in HDL is
known to show an inverse correlation with the frequency of onset of
arteriosclerotic disease. These days, measurements of cholesterol
in LDL or HDL are, therefore, widely conducted for the prevention
or diagnosis of ischemic heart diseases.
[0004] As methods known for the measurement of cholesterol in LDL
or HDL, there are, for example, a method in which LDL or HDL is
separated from other lipoproteins by ultracentrifugal separation
and is then subjected to a cholesterol measurement; and another
method in which subsequent to separation of LDL or HDL from other
lipoproteins by electrophoresis, its lipid is stained, and the
intensity of a developed color is measured. These methods are
however not used practically, because they involve one or more
problems in that procedures are intricate and many samples cannot
be handled.
[0005] A method for the measurement of cholesterol in HDL, which is
used at present in the field of clinical tests, is the
precipitation method in which a precipitation reagent is added to a
sample to agglutinate lipoproteins other than HDL, the resulting
agglutinate is removed by centrifugation, and cholesterol in
isolated supernatant which contains only HDL is then measured. This
method is simpler compared with ultracentrifugation or
electrophoresis, but due to the inclusion of the procedures to add
the precipitation reagent and to perform the separation, requires
each sample is a relatively large quantity, and involves a
potential problem of causing an analytical error. Furthermore, the
entire analysis steps of this method can not be fully
automated.
[0006] On the other hand, enzymatic methods have been studied for
the fractional quantitation of cholesterol in HDL. Known methods
include, for example, to conduct an enzymatic reaction in the
presence of a bile acid salt and a nonionic surfactant (JP
63-126498 A). This method makes use of the fact that an enzymatic
reaction proceeds in proportion to the concentration of cholesterol
in LDL in an initial stage of the reaction and the subsequent
reaction velocity is in proportion to the concentration of
cholesterol in HDL. A problem however exists in accuracy because
the reaction with the cholesterol in HDL and the reaction with
cholesterol in other lipoproteins cannot be fully
distinguished.
[0007] Also included in the known methods is to have lipoproteins
other than HDL agglutinated in advance, to cause cholesterol in HDL
alone to react enzymatically, and to inactivate the enzyme and at
the same time, to redissolve the agglutinate, followed by the
measurement of an absorbance (JP 6-242110 A). This method, however,
requires at least three procedures to add reagents so that it can
be applied only to particular automated analyzers, leading to a
problem in a wide applicability. Further, this method is not
satisfactory from the standpoint of damages to analytical equipment
and disposal of the reagents because of the use of a salt at a high
concentration upon redissolution of an agglutinate.
[0008] A still further method is also known (JP 9-299 A), which
comprises causing, in a first reaction, cholesterol oxidase and
cholesterol esterase to act upon lipoproteins other than HDL in the
presence of a special surfactant and to have cholesterol, which is
contained in such other lipoproteins, preferentially reacted, and
then measuring cholesterol in HDL while inhibiting any reaction to
cholesterol in lipoproteins other than HDL. This method, however,
is considerably different from the present invention inter alia in
that in the first reaction, the special surfactant, cholesterol
oxidase and cholesterol esterase are required at the same time to
put, outside the reaction system, both free cholesterol and
esterified cholesterol in the lipoproteins other than HDL.
[0009] Further, Japanese Patent No.2,600,065discloses a method
which makes combined use of a precipitation reagent, which is
adapted to cause precipitation of lipoproteins other than HDL, and
a cholesterol measuring reagent to measure cholesterol (HDL-C) in
unprecipitated HDL. This method has practical utility when a
modified enzyme is used as enzyme and .alpha.-cyclodextrin sulfate
is used as a precipitation reagent. This method, however, also
involves a problem in accuracy in that turbidity, which occurs as a
result of the use of the precipitation reagent, interferes with the
measurement system.
[0010] Concerning the measurement of HDL-C by a modified enzyme,
"SEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)", 19(5),
305-320, which is considered to be a published paper on the
above-described patented method, discloses that, under the
recognition of incapability of measurement of HDL-C in a serum of a
hyperlipidemic patient by the modified enzyme due to a positive
error (that is, to result in a higher value compared with that
obtained by the precipitation method) induced when the modified
enzyme is simply introduced into a reaction system, HDL-C was
measured by using cyclodextrin sulfate, a polyanion, and magnesium
chloride as a precipitation reagent for the avoidance of the
positive error.
[0011] To reduce the influence of turbidity caused by a
precipitation reagent in the above-described patented method,
certain techniques are also known, including to make a surfactant
exist concurrently (JP 8-116996 A), to use an antibody (JP 9-96637
A), and to employ a sugar compound (JP 7-301636 A). They, however,
all require as a premise the inclusion of a reagent which induces
formation of an agglutinate, so that it is fundamentally
indispensable for them to use a precipitation reagent such as a
polyanion.
[0012] The present inventors recently found that use of a
substance, which acts upon the specific lipoprotein only, makes it
possible to accurately quantitate cholesterol in the specific
lipoprotein fraction without using a precipitation reagent, and
filed patent applications (JP 9-244821). This method has an
extremely high correlation with the conventional precipitation
method, but compared in measurement values with the precipitation
method, this method is recognized to have a similar tendency as the
above-described method reported in "SEIBUTSU SHIRYO BUNSEKI
(ANALYSIS OF BIOLOGICAL SAMPLES)". To obtain data consistent with
those obtained by the conventional precipitation method at medical
institutions and the like, a polyanion or the like is added.
[0013] From the standpoint of the problem of a tarnish or the like
on a cuvette and scattering of measurement values, however, it is
not desired to add a polyanion or the like and to form a
precipitate in a measurement system. Accordingly, it has been
strongly desired to eliminate the precipitate from the system.
Further, it is also economically unreasonable to use a polyanion or
the like for making the resulting data consistent with those
obtained by the precipitation method although the polyanion or the
like is not needed from the standpoint of the principle of the
measurement. Hence, there is also an outstanding desire for its
solution.
[0014] An object of the present invention is, therefore, to provide
a method, which can accurately and efficiently quantitate
cholesterol in the specific lipoprotein fraction by simple
procedures fundamentally without needing a polyanion or the like
and is suitably applicable to various automated analyzers.
DISCLOSURE OF THE INVENTION
[0015] The present inventors proceeded with a thorough
investigation for a cause which may be responsible for the
above-described problem reported in "SEIBUTSU SHIRYO BUNSEKI
(ANALYSIS OF BIOLOGICAL SAMPLES)", that is, the problem that a
value of cholesterol in the specific lipoprotein fraction as
quantitated by using a substance which acts only upon a specific
lipoprotein such as HDL becomes higher than the corresponding value
as determined by the precipitation method; and came to a conclusion
that even from non-HDL lipoproteins (LDL, VLDL and the like) the
cholesterol of which is not supposed to be measured, a small amount
of free cholesterol existing on their surfaces or in the vicinity
of their surfaces is liberated to cause a positive error. Based on
this finding, it has been found that a cholesterol value obtained
by a quantitation method making use of a substance, which acts upon
a specific lipoprotein only, becomes consistent with the
corresponding value obtained by the precipitation method when the
cholesterol value is measured after consuming only free cholesterol
in advance under conditions that lipoproteins remain substantially
unchanged, leading to the completion of the present invention.
[0016] Described specifically, the present invention provides a
method for pretreating a sample, which contains various
lipoproteins, prior to measuring cholesterol existing in specific
one of the lipoproteins in the sample, which comprises causing an
enzyme, which acts upon free cholesterol as a substrate, to act
upon the sample.
[0017] The present invention also provides a method for
quantitating cholesterol existing in a specific lipoprotein in a
sample, which comprises causing an enzyme, which acts upon free
cholesterol as a substrate, to act upon the sample with the
lipoprotein contained therein; and then measuring the cholesterol,
which exists in the specific lipoprotein, by using a substance
which acts upon the specific lipoprotein only.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a diagram showing a correlation between the
present invention in Example 1 and the precipitation method;
[0019] FIG. 2 is a diagram showing a correlation between the
present invention in Example 2 and the precipitation method;
and
[0020] FIG. 3 is a diagram showing effects of a reaction
accelerator in Example 5.
BEST MODE FOR CARRYING OUT THE INVENTION
[0021] In the present invention, before measuring cholesterol
existing in a specific lipoprotein in a sample, an enzyme which
acts upon free cholesterol as a substrate is caused to act, as
pretreatment, upon the sample such that the free cholesterol is
consumed.
[0022] As the enzyme which acts upon free cholesterol as a
substrate, any enzyme can be used insofar as it acts upon free
cholesterol as a substrate. Illustrative are cholesterol
dehydrogenase and cholesterol oxidase. They can be of any origins
such as microorganism origins, animal origins or plant origins, and
can also be those prepared by genetic engineering. Further, they
can be either modified or unmodified chemically. The enzyme is
generally used at 0.001 to 100 U/mL, with 0.1 to 100 U/mL being
preferred.
[0023] No particular limitation is imposed on conditions under
which the above-described enzyme, which acts upon free cholesterol
as a substrate, is caused to act upon the sample, and conditions
recommended for the enzyme can be used. It is however necessary to
pay attention so that, during a stage in which the enzyme which
acts upon free cholesterol as a substate is caused to act upon the
sample, a reaction through which an esterified cholesterol is
converted into free cholesterol does not take place. Namely, it is
not important whether or not cholesterol esterase exists. What is
needed is to maintain conditions such that cholesterol esterase is
not allowed to act practically.
[0024] Along with the enzyme which acts upon free cholesterol as a
substrate, a coenzyme can be used as needed. As the coenzyme,
nicotinamide adenine dinucleotide or the like is usable. Such
coenzymes can be used either singly or in combination. The amount
to be used varies depending on the coenzyme. The coenzyme may be
used at 0.001 to 100 U/mL, preferably at 0.1 to 100 U/mL, although
no particular limitation is imposed thereon.
[0025] Concerning the enzyme which acts upon free cholesterol as a
substrate and is used in the present invention, no limitation is
imposed on its origin as described above. Its concentration and the
like can be chosen suitably to achieve desired performance and
handling ease. Accordingly, if it is desired to have the
pretreatment completed in a predetermined time, for example, it is
only necessary to use the enzyme in a greater amount, and if it is
conversely desired to save the enzyme, it is only necessary to make
the pretreatment time longer.
[0026] In the case of a diagnostic reagent for exclusive use in
measurements by automated analyzers, however, it is desired to meet
both of the requirements at the same time. Namely, it is required
to complete the pretreatment in a short time by using the enzyme in
a small amount. In such a case, concurrent existence of a reaction
accelerator selected from the below-described group in the
pretreatment, which uses an enzyme which acts upon free cholesterol
as a substrate, makes it possible to achieve desired performance
with a reduced amount of the enzyme without making the pretreatment
time longer.
[0027] Reaction accelerators usable for the above purpose can
include, for example, flufenamic acid, mefenamic acid,
2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone
acetate, monensin and mevinolin, including their salts and metal
derivatives (aluminum derivatives and the like) wherever such salts
and metal derivatives exist. Among these, flufenamic acid and
mefenamic acid are known as non-steroidal anti-inflammatory drugs,
and fusidic acid and monensin are known as antibiotics.
[0028] Upon using such a compound as a reaction accelerator, it is
necessary to suitably choose its concentration and the like by
taking into consideration its physical properties, pH and ionic
strength of the measurement system, and the kinds and
concentrations of substances existing together.
[0029] The concentration of the reaction accelerator can be
experimentally determined in accordance with conditions of a
measuring system. In general, however, flufenamic acid can may be
used at about 0.01 to 100 mM; fusidic acid at about 0.01 to 10 mM;
mefenamic acid, 2,2',6',2"-terpyridine and betamethasone acetate,
each, at about 0.01 to 5 mM; monensin and mevinolin, each, at about
0.01 to 1 mM; and tiglic acid at about 1 to 500 mM.
[0030] Use of the above-described reaction accelerator has made it
possible to reduce the amount of the enzyme, which acts upon free
cholesterol as a substrate, to one severalth or to one several
tenth. When the enzyme is used in the same amount, on the other
hand, the reaction accelerator can shorten the reaction time.
[0031] In the above-described pretreatment by the enzyme which acts
upon free cholesterol as a substrate (and also by the reaction
accelerator, if needed), it is also possible to use other enzymes
(with exclusion of those giving substantial influence to
lipoproteins) and salts, buffers for pH regulation, surfactants
(with exclusion of those giving substantial influence to
lipoproteins), preservatives, proteins such as albumin, and agents
having affinity to specific lipoproteins, such as antibodies,
antibiotics, saponins, lectins and polyanions to extents not
causing agglutination of the specific lipoprotein, such that the
action of the enzyme is adjusted without impairing the specificity
of the measurement.
[0032] In the present invention, those containing the following
ingredients can, therefore, be used as pretreatment agents for
measuring cholesterol existing in specific lipoproteins in
samples.
[0033] (Essential Ingredients)
[0034] Enzymes which act upon free cholesterol as a substrate, for
example, cholesterol dehydrogenase and cholesterol oxidase.
[0035] (Optional Ingredients)
[0036] Reaction accelerators, for example, flufenamic acid,
mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid,
betamethasone acetate, monensin and mevinolin.
[0037] (Other Ingredients)
[0038] coenzymes such as NAD, other enzymes such as peroxidase,
catalase, diaphorase and ascorbate oxidase, acids such as pyruvic
acid, salts, buffers for pH regulation, surfactants giving no
substantial influence on lipoproteins, preservatives, proteins such
as albumin, antibodies, antibiotics, saponins, lectins, polyanions
and couplers such as 4-aminoantipyrine, oxidative color developers
such as hydrogen donors, e.g., Trinder's reagent, electron
acceptors such as phenazine methosulfate, and reductive color
developers such as nitroblue tetrazolium.
[0039] In the present invention, cholesterol which exists in a
specific lipoprotein in a sample is measured after having free
cholesterol in lipoproteins consumed by the above-described
pretreatment.
[0040] Any method can be used for the measurement of the
cholesterol existing in the specific lipoprotein in the sample
insofar as the method can measure the cholesterol existing in the
specific lipoprotein by using a substance which acts upon the
specific lipoprotein only.
[0041] An illustrative example of the method may comprise
providing, as the substance which acts upon the specific
lipoprotein, a surfactant selected from polyoxyethylene alkylene
phenyl ethers or polyoxyethylene alkylene tribenzylphenyl ethers
disclosed in JP 11-56395 A; adding a cholesterol measuring enzyme
reagent in the presence of the substance; and then measuring the
amount of cholesterol reacted in a time during which cholesterol in
high density lipoprotein out of lipoproteins preferentially reacts
with the cholesterol measuring enzyme reagent.
[0042] Examples of commercial products of the former surfactants,
polyoxyethylene alkylene phenyl ethers, can include "Emulgen A-60"
(trade name, product of Kao Corporation), while examples of
commercial products of the latter surfactants, polyoxyethylene
alkylene tribenzylphenyl ethers, can include "Emulgen B66" (trade
name, product of Kao Corporation).
[0043] As an alternative method, there is a method which makes use
of the modified enzymes, which are disclosed on pages 305-320 of
"SEIBUTSU SHIRYO BUNSEKI (ANALYSIS OF BIOLOGICAL SAMPLES)", 19(5),
as substances which act only upon specific lipoproteins,
respectively. Although .alpha.-cyclodextrin sulfate and magnesium
chloride are used in the method of this paper to inhibit reactions
with lipoprotein fractions other than HDL, the use of the
above-described pretreatment method of this invention makes it no
longer necessary to use such substances.
[0044] Except for the use of the substance which acts upon the
specific cholesterol, the method for the measurement of cholesterol
existing in the specific lipoprotein can be practiced by using
reagents employed in conventional cholesterol-measuring methods.
Examples of ingredients which may be contained in reagents to be
used can include enzymes such as cholesterol esterase, cholesterol
oxidase, cholesterol dehydrogenase, isocitrate dehydrogenase,
diaphorase and peroxidase, color developers, coenzymes, electron
acceptors, proteins (albumin, etc.), preservatives, surfactants,
salts, acids, and buffers for pH regulation.
[0045] As surfactants out of the above-described ingredients, both
ionic and nonionic surfactants are usable. Illustrative are
polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers,
polyoxyethylene-polyoxy- propylene condensate, polyoxyethylene
alkyl ether sulfates, alkylbenzenesulfonate salts, and bile acid
salts. The amount of the surfactant to be used varies depending on
the compound. The surfactant may however be used in 0.0001% to 5%,
preferably in 0.001% to 5%, although no particular limitation is
imposed thereon.
[0046] No particular limitation is imposed on the buffers.
Conventional buffers such as Good's buffer, phosphate buffer, Tris
buffer and phthalate buffer are usable. The buffer may be used at
0.005 M to 2 M, preferably 0.01 M to 1 M, although no particular
limitation is imposed thereon.
[0047] The method for quantitating cholesterol in a specific
lipoprotein fraction by the present invention typically comprises
firstly adding a pretreatment agent, which acts upon free
cholesterol only, into a measureing sample and causing the
pretreatment agent to act upon the sample, and then adding and
mixing a cholesterol measuring reagent (hereafter called a
"quantitation reagent"), which contains a substance capable of
acting upon the specific lipoprotein and a reagent employed for a
conventional cholesterol-measuring method, to measure the amount of
cholesterol in the specific lipoprotein fraction.
[0048] Specific examples can include, but are not limited to, a
method which comprises mixing cholesterol dehydrogenase and a
coenzyme (NAD) with a sample and then adding a
cholesterol-measuring reagent which comprises cholesterol esterase
and cholesterol oxidase; a method which comprises mixing
cholesterol dehydrogenase and NAD with a sample and then adding a
cholesterol-measuring reagent which comprises cholesterol esterase;
a method which comprises mixing a sample and cholesterol oxidase
together with peroxidase, 4-amino antipyrine or catalase and then
adding a cholesterol-measuring reagent which comprises cholesterol
esterase; and a method which comprises mixing a sample and
cholesterol oxidase together with peroxidase, 4-aminoantipyrine,
etc. and then adding a cholesterol-measuring reagent which
comprises cholesterol esterase, cholesterol dehydrogenase and
NAD.
[0049] Examples of the method for measuring cholesterol in a
specific lipoprotein fraction can include a method making combined
use of cholesterol esterase and cholesterol oxidase as an enzyme
reagent and a method making combined use of cholesterol esterase
and cholesterol dehydrogenase, although known enzyme assays are all
usable.
[0050] In the present invention, the enzyme for use in the first
reaction as the pretreatment reaction and the enzyme for use in the
measurement of cholesterol as the quantitation method through the
second reaction may be either the same or different. Further, the
enzyme may be used in an excess amount in the first reaction and
may also be used in the second reaction. In essence, it is only
necessary to consume free cholesterol, which exists in a small
amount on lipoprotein surfaces, (first reaction/pretreatment
reaction) and then to bring the reaction system into a state, in
which the enzyme acts only upon the specific lipoprotein to be
measured, so that most cholesterol (free cholesterol+esterified
cholesterol) forming the lipoprotein can be quantitated.
[0051] Further, no particular limitation is imposed on the method
for finally detecting cholesterol after the addition of such a
cholesterol-measuring enzyme reagent. It is possible to use, for
example, absorptiometry in which detection is conducted by
combining peroxidase with a chromogen or diaphorase or an electron
acceptor with a reductive color-developing reagent; or a method in
which a coenzyme or hydrogen peroxide is directly detected. The
coenzyme may be amplified by a coenzyme cycling system.
[0052] To practice the method of the present invention with ease,
it is preferred to use a quantitation kit which is suited for
measuring cholesterol in the specific lipoprotein.
[0053] Although such kits can be readily designed based on the
above explanation, their examples will be described next by
dividing them into those making use of cholesterol oxidase and
those making use of cholesterol dehydrogenase as typical example of
enzymes which act upon free cholesterol as a substrate.
[0054] [Kits Making Use of Cholesterol Oxidase]
[0055] (a) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0056] (1) a first reagent comprising cholesterol oxidase and a
hydrogen peroxide consuming substance (and further comprising a
reaction accelerator in some instances); and
[0057] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only, cholesterol esterase, and a color
developer.
[0058] (b) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0059] (1) a first reagent comprising cholesterol oxidase,
cholesterol esterase, and a hydrogen peroxide consuming substance
(and further comprising a reaction accelerator in some instances);
and
[0060] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only, and a color developer.
[0061] (c) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1), (2) and
(3):
[0062] (1) a first reagent comprising cholesterol oxidase and a
hydrogen peroxide consuming substance (and further comprising a
reaction accelerator in some instances);
[0063] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only; and
[0064] (3) a third reagent comprising cholesterol esterase and a
color developer.
[0065] In the above-described kits, the term "hydrogen peroxide
consuming substance" means a substance which consumes and
eliminates hydrogen peroxide produced by the reaction between
cholesterol oxidase and cholesterol. Illustrative are catalase,
couplers such as 4-aminoantipyrine, and oxidative-reductive color
developer agents including hydrogen donors such as Trinder's
reagent.
[0066] Among these, a coupler such as 4-aminoantipyrine and a
hydrogen donor such as Trinder's reagent develop a color when
reacted, in combination, with hydrogen peroxide, and are usable as
the color developer in the above-described reagent (2) or (3). As
the reagent (1) for use in the pretreatment step according to the
present invention, it is preferred to use only one of a coupler and
a hydrogen donor and to have hydrogen peroxide consumed through a
non-color developing reaction. Needless to say, it is also possible
to subject hydrogen peroxide to a color-developing reaction and
then to make an adjustment to a measured value [this adjustment can
be made by subtracting the intensity of a color, which is developed
by the reagent (1), from the intensity of a color developed by the
reagent (2) or the reagent (3)].
[0067] [Kits Making Use of Cholesterol Dehydrogenase]
[0068] (d) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0069] (1) a first reagent comprising cholesterol dehydrogenase and
a coenzyme(and further comprising a reaction accelerator in some
instances); and
[0070] (2) a second reagent comprising a substance, which acts upon
the specific lipoprotein only, and cholesterol esterase.
[0071] (e) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0072] (1) a first reagent comprising cholesterol dehydrogenase and
a coenzyme(and further comprising a reaction accelerator in some
instances); and
[0073] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only, cholesterol oxidase, cholesterol
esterase, peroxidase, and a color developer.
[0074] (f) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0075] (1) a first reagent comprising cholesterol dehydrogenase, a
coenzyme, and cholesterol esterase(and further comprising a
reaction accelerator in some instances); and
[0076] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only.
[0077] (g) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0078] (1) a first reagent comprising cholesterol dehydrogenase, a
coenzyme, and cholesterol esterase(and further comprising a
reaction accelerator in some instances); and
[0079] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only, cholesterol oxidase, peroxidase, and
a color developer.
[0080] (h) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1), (2) and
(3):
[0081] (1) a first reagent comprising cholesterol dehydrogenase and
a coenzyme(and further comprising a reaction accelerator in some
instances);
[0082] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only; and
[0083] (3) a third reagent comprising cholesterol esterase.
[0084] (i) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1), (2) and
(3):
[0085] (1) a first reagent comprising cholesterol dehydrogenase and
a coenzyme(and further comprising a reaction accelerator in some
instances);
[0086] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only; and
[0087] (3) a third reagent comprising cholesterol oxidase,
cholesterol esterase, peroxidase, and a color developer.
[0088] (j) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0089] (1) a first reagent comprising cholesterol dehydrogenase, a
coenzyme, and a coenzyme reaction product consuming substance (and
further comprising a reaction accelerator in some instances);
and
[0090] (2) a second reagent comprising a substance, which acts upon
the specific lipoprotein only, and cholesterol esterase.
[0091] (k) A quantitation kit for cholesterol in a specific
lipoprotein, comprising the following reagents (1) and (2):
[0092] (1) a first reagent comprising cholesterol dehydrogenase, a
coenzyme, and a coenzyme reaction product consuming substance (and
further comprising a reaction accelerator in some instances);
and
[0093] (2) a second reagent comprising a substance which acts upon
the specific lipoprotein only, cholesterol esterase, and a color
developer.
[0094] In the above-described kits making use of cholesterol
dehydrogenase, the term "coenzyme reaction product consuming
substance" means a substance which converts a reduced coenzyme (for
example, NADH), which occurs through the reaction among
cholesterol, cholesterol dehydrogenase and a coenzyme (for example,
NAD), back into the original coenzyme. Illustrative is a
combination of lactate dehydrogenase and pyruvic acid (substrate).
In each of the above-described kits, the reaction product of the
coenzyme is produced by the addition of the reagent (1). In each of
the kits (d), (f), (h) and (j) out of the above-described kits,
light of the same wavelength as a color developed by the addition
of the reagent (1) may be measured in the measurement stage without
advance consumption of the reaction product. In this case, however,
it is necessary to quantitate the cholesterol in the specific
lipoprotein by subtracting the intensity of a color, which is
developed in the pretreatment stage in which the reagent (1) is
added, from the intensity of a color developed by the reagent (2)
or the reagent (3). As an alternative, it may also be possible to
add beforehand the substance, which consumes the reaction product,
to the reagent (1) and subsequent to consumption of the reaction
product, to add the reagent (2) or the reagent (3) for the
development of a color. In this case, addition of a substance,
which reduces the action of the substance which consumes the
reaction product, to the reagent (2) or the reagent (3) is
preferred. In each of the kits (e), (g), (I) and (k), on the other
hand, it is not absolutely necessary to subtract the intensity of
the color, which is developed in the pretreatment stage, from the
color intensity measured in the measurement stage, because in the
measurement stage, a developed color of a wavelength different from
the color developed in the pretreatment stage is measured.
[0095] It is to be noted that the application of the
above-mentioned reaction accelerators, such as flufenamic acid,
mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid,
betamethasone acetate, monensin and mevinolin, is limited neither
to the pretreatment method or agent of the present invention nor
the quantitation method or kit of the present invention for
cholesterol in a specific lipoprotein, said quantitation method or
kit making use of the pretreatment method or agent.
[0096] If a reaction accelerator such as fulfenamic acid is allowed
to exist concurrently upon conducting a cholesterol quantitation
method making use of an enzyme which acts upon free cholesterol as
a substrate, for example, a free cholesterol quantitation method
making combined use of cholesterol oxidase, peroxidase, a color
developer and the like or a total cholesterol quantitation method
making combined use of cholesterol oxidase, cholesterol esterase,
peroxidase, a color developer and the like, it is obviously
possible to bring about advantageous effects such that the amount
of the enzyme to be used, said enzyme being capable of acting upon
free cholesterol as a substrate and being cholesterol oxidase in
the above-exemplified method, can be reduced and the time of the
enzymatic reaction can be shortened.
[0097] Further, reference to the disclosure of this specification
on the cholesterol quantitation method (for example, selection of a
surfactant to limit a target of a specific lipoprotein to be
measured) makes it possible to more specifically design a
quantitation method as desired.
INDUSTRIALLY APPLICABILITY
[0098] The present invention has made it possible to efficiently
quantitate cholesterol in a specific fraction by simple procedures
without using a polyanion or the like, to say nothing of a
mechanical pretreatment such as centrifugation. As the methods of
the present invention do not form a precipitate which would
otherwise occur by the addition of the polyanion or the like,
measuring apparatus (especially, cuvettes) and the like remain free
of a tarnish and moreover, measured values also remain free of
scattering. The methods according to the present invention are,
therefore, superior to the conventional cholesterol measuring
methods.
[0099] Further, as will be demonstrated in subsequent Examples,
measurement values showing a high correlation with those obtained
by the conventional precipitation method can be obtained even with
respect to samples with high triglyceride levels. Therefore, the
methods according to the present invention are also excellent in
that they are applicable to various samples without limitation.
[0100] In addition, the use of the reaction accelerator makes it
possible to use the enzyme, which acts upon free cholesterol as a
substrate, in a smaller amount in the pretreatment stage.
[0101] As has been described above, the methods according to the
present invention permit accurate and specific measurements of a
variety of samples by simple procedures while using the samples in
small quantities. Accordingly, they can be applied to various
automated analyzers and are also extremely useful in the field of
clinical tests.
[0102] The present invention will next be described in further
detail by the Examples. It should however be borne in mind that the
present invention is by no means limited to the Examples.
EXAMPLE 1
[0103] With respect to each of 30 serum samples containing
lipoproteins, the cholesterol in HDL was quantitated by the
below-described method according to the present invention and the
precipitation method, and the measurement values were compared.
[0104] (Invention Method)
[0105] 10 mM phosphate buffer (First Reagent; pH 8.5) (300 .mu.L),
which contained 0.1 U/mL cholesterol dehydrogenase (product of
Amano Pharmaceutical Co., Ltd.), 2.5 mM NAD and 0.03%
4-aminoantipyrine, was added to each sample (3 .mu.L)
(pretreatment). About 5 minutes later, a cholesterol quantitation
reagent (Second Reagent) (100 .mu.L)--which was composed of 100 mM
MES buffer (pH6) containing 1% "EmulgenB-66", 1.3 U/mL cholesterol
esterase (product of Asahi Chemical Industry Co., Ltd.), 2 U/mL
cholesterol oxidase (product of Asahi Chemical Industry Co., Ltd.),
5 U/mL peroxidase (product of Toyobo Co., Ltd.) and 0.04%
disulfobutylmetatoluidine--was added.
[0106] Just before the addition of the Second Reagent and upon an
elapsed time of five minutes after the addition, the absorbance was
measured at 600 nm. From a difference in absorbance, the
concentration of HDL cholesterol in the serum sample was determined
(2-point method). As a calibration substance, a control serum
sample with a known concentration of HDL cholesterol was used. The
above procedures were conducted using "Hitachi 7150 automated
analyzer".
[0107] (Precipitation Method)
[0108] "HDLC 2 `Daiichi` Precipitant" (product of Daiichi Pure
Chemicals Co., Ltd.) (200 .mu.L) was mixed with the sample (200
.mu.L), followed by centrifugation at 3,000 rpm for 10 minutes. The
supernatant (50 .mu.L) was collected, followed by the mixing with a
cholesterol quantitation reagent (3 mL) composed of 100 mM MES
buffer (pH6.5) containing 1% Triton X-100, 1 U/mL cholesterol
esterase, 1 U/mL cholesterol oxidase, 5 U/mL peroxidase, 0.04%
disulfobutylmetatoluidine and 0.04% 4-aminoantipyrine. After the
resulting mixture was incubated at 37.degree. C. for 10 minutes,
its absorbance at 600 nm was measured to determine the
concentration of the cholesterol in HDL.
[0109] (Results)
[0110] The results are shown in Table 1 and FIG. 1.
1TABLE 1 Precipitation Invention Sample No. method (mg/dL) method
(mg/dL) 1 73 72 2 39 39 3 53 52 4 54 54 5 57 58 6 75 71 7 51 51 8
52 50 9 43 43 10 58 58 11 59 59 12 49 51 13 44 46 14 70 65 15 35 38
16 54 54 17 45 47 18 60 59 19 50 52 20 58 56 21 38 39 22 56 55 23
35 37 24 29 31 25 63 60 26 51 50 27 33 36 28 52 51 29 65 63 30 47
49
[0111] As is readily envisaged from the results, the invention
method, despite the omission of a polyanion or the like, showed an
extremely good correlation with the conventional precipitation
method.
EXAMPLE 2
[0112] Measurements were conducted by another method of the present
invention, which was similar to the invention method conducted in
Example 1 except that in the first reagent, cholesterol
dehydrogenase, NAD and the phosphate buffer were replaced by 5 U/mL
cholesterol oxidase (product of Toyobo Co., Ltd.), 5 U/mL
peroxidase (product of Toyobo Co., Ltd.) and 100 mM MES buffer (pH
6). The measurement values were compared with those obtained by the
precipitation method in Example 1.
[0113] (Results)
[0114] The results are shown in Table 2 and FIG. 2.
2TABLE 2 Precipitation Invention Sample No. method (mg/dL) method
(mg/dL) 1 73 73 2 39 38 3 53 52 4 54 56 5 57 57 6 75 74 7 51 52 8
52 50 9 43 44 10 58 58 11 59 57 12 49 51 13 44 45 14 70 69 15 35 37
16 54 54 17 45 47 18 60 61 19 50 50 20 58 55 21 38 37 22 56 56 23
35 35 24 29 29 25 63 61 26 51 52 27 33 33 28 52 52 29 65 66 30 47
46
[0115] As is readily envisaged from the results, the invention
method, despite the omission of a polyanion or the like, showed an
extremely good correlation with the conventional precipitation
method.
EXAMPLE 3
[0116] Using the reagents of Example 1 and Example 2, five serum
samples of different triglyceride levels were measured. The
measurement values were then compared with those obtained by the
precipitation method. The results are shown in Table 3.
3TABLE 3 Invention Invention method in method in Precipitation
Example 1 Example 2 Triglyceride method (mg/dL) (mg/dL) (mg/dL)
level(mg/dL) Sample A 47 49 49 198 Sample B 49 50 49 301 Sample C
26 27 24 742 Sample D 60 61 61 517 Sample E 37 40 36 428
[0117] As is shown in Table 3, measurement values of comparable
levels with those obtained by the conventional method were also
obtained by the present invention with respect to the samples of
the high triglyceride levels.
EXAMPLE 4
[0118] Measurements were conducted in a similar manner as in
Example 2 except that in the first reagent, 5 U/mL cholesterol
oxidase was changed to give reagent compositions of the ingredient
concentrations and combinations shown below in Table 4. The
measurement values were compared with those obtained by the
precipitation method and also with those obtained by the invention
method (standard test system) of Example 2. Incidentally, as a
second reagent, the same reagent as the second reagent employed in
Example 1 was used. The results are shown in Table 5.
[0119] (Compositions of Testing Reagents)
4 TABLE 4 Test system Contents of composition Standard Cholesterol
oxidase (5 U/mL) A Cholesterol oxidase (1 U/mL) B Flufenamic acid +
cholesterol oxidase (0.15 mM) (1 U/mL) C Mefenamic acid +
cholesterol oxidase (0.1 mM) (1 U/mL) D 2,2',6',2"-terpyridine +
cholesterol oxidase (0.5 mM) (1 U/mL) E Tiglic acid + cholesterol
oxidase (50 mM) (1 U/mL) F Fusidic acid + cholesterol oxidase (0.1
mM) (1 U/mL) G Betamethasone acetate + cholesterol oxidase (0.2 mM)
(1 U/mL) H Monensin + cholesterol oxidase (0.2 mM) (1 U/mL) I
Mevinolin + cholesterol oxidase (0.05 mM) (1 U/mL)
[0120] (Results)
5 TABLE 5 Precipitation method Test system (mg/dL) Sample (mg/dL)
Standard A B C D E F G H I 1 80 77 72 77 68 76 74 74 74 74 76 2 76
74 72 74 64 73 71 73 74 74 73 3 75 72 70 72 66 71 70 70 70 71 71 4
71 72 71 70 66 69 69 71 69 71 72 5 71 70 70 69 61 68 67 68 70 69 70
6 71 70 67 70 63 68 68 67 69 70 68 7 69 66 63 66 61 65 65 65 64 65
66 8 67 69 70 68 60 68 67 66 69 68 68 9 66 65 65 65 59 65 64 63 65
65 65 10 65 65 64 65 58 65 64 62 64 65 63 11 57 58 56 57 54 57 56
57 57 58 57 12 56 56 55 55 49 55 54 53 55 55 55 13 54 55 54 55 50
54 53 53 53 54 54 14 53 54 54 52 46 53 52 52 54 52 53 15 52 53 52
51 47 52 51 49 52 51 52 16 51 53 51 50 46 50 51 49 51 51 51 17 49
50 48 48 44 47 48 47 48 48 49 18 47 48 48 46 41 46 46 45 47 47 47
19 45 46 48 44 38 46 43 45 47 47 46 20 47 47 49 45 40 46 45 45 48
47 47 21 42 44 44 43 39 43 42 41 43 44 43 22 39 42 43 41 37 41 41
39 41 41 41 23 32 35 36 33 31 36 34 32 34 34 33 24 18 20 22 19 17
23 19 18 21 20 19 25 40 42 42 41 38 45 41 40 41 42 41 Correlation
coef. -- 0.996 0.990 0.998 0.992 0.995 0.997 0.997 0.994 0.995
0.997 Slope 0.905 0.838 0.941 0.832 0.856 0.888 0.915 0.877 0.891
0.917 Intercept 5.6 8.7 2.6 3.4 7.5 4.6 2.8 6.3 5.7 4.1
[0121] When the amount of cholesterol oxidase was reduced to one
fifth (the test system A) compared with the standard test system
(Example 2), the correlation coefficient slightly declined and the
value of intercept slightly increased. When the reaction
accelerator was used, however, results substantially comparable
with those of the standard test system were obtained even when the
amount of cholesterol oxidase was one fifth. It has hence become
evident from these results that the use of a reaction accelerator
makes it possible to reduce the amount of cholesterol oxidase to be
used.
EXAMPLE 5
[0122] Reagents J to L shown below in Table 6 were prepared, which
commonly contained 1.25 U/mL peroxidase (product of Toyobo Co.,
Ltd.), 0.01% 4-aminoantipyrine, 0.02% disulfo butyl-m-toluidine and
50 mM NaCl and were different from each other in the kind and pH of
buffer and the concentrations of cholesterol oxidase (product of
Toyobo Co., Ltd.) and fulfenamic acid (product of Sigma Chemical
Co.).
6 TABLE 6 {circle over (1)} Buffer (pH) {circle over (2)}
Concentration of cholesterol oxidase Reagent {circle over (3)}
Concentration of fulfenamic acid J {circle over (1)} 50 mM Bis-Tris
(pH 6.0) {circle over (2)} 0.5, 1.0, 2.5, 5.0 U/mL {circle over
(3)} 0, 0.01, 0.05, 0.1 mM K {circle over (1)} 50 mM PIPES (pH 7.0)
{circle over (2)} 0.5, 1.0, 2.5, 5.0 U/mL {circle over (3)} 0, 0.1,
0.5, 1.0 mM L {circle over (1)} 50 mM MOPS (pH 8.0) {circle over
(2)} 0.5, 1.0, 2.5, 5.0 U/mL {circle over (3)} 0, 1.0, 5.0, 10.0
mM
[0123] Reagents J to L (300 .mu.L) were separately added to
aliquots (3 .mu.L) of each serum sample. After the resultant
mixtures were incubated at 37.degree. C. for 5 minutes, their
absorbances were measured at 600 nm. The above procedures were
conducted using the Hitachi 7150 automated analyzer.
[0124] Four serum samples were measured with Reagents J to L.
[0125] With respect to each of Reagents J to L, relative
absorbances were calculated for the individual concentrations of
cholesterol oxidase and fulfenamic acid by assuming that the
absorbance obtained with a reagent containing 5.0 U/mL cholesterol
oxidase and 0 mM fulfenamic acid was 100.
[0126] (Results)
[0127] Results, which had been obtained by averaging the relative
absorbances of the four samples, are presented in FIG. 3, in which
"COD" stands for cholesterol oxidase.
[0128] As is readily appreciated from the results, the relative
absorbance increased depending upon the concentration of fulfenamic
acid irrespective of the pH. It has, therefore, been confirmed that
the use of the reaction accelerator makes it possible to reduce the
amount of cholesterol oxidase to be used.
[0129] It has also become clear that the reaction accelerator is
also usable in a method for the measurement of free cholesterol or
total cholesterol, which makes use of an enzyme which acts upon
free cholesterol as a substrate.
* * * * *