U.S. patent application number 13/179928 was filed with the patent office on 2012-06-14 for method for quantitatively determining ldl cholesterols.
This patent application is currently assigned to SEKISUI MEDICAL CO., LTD.. Invention is credited to Koichi Hino, Mitsuhisa Manabe, Mitsuhiro Nakamura, Kazuo Nakanishi.
Application Number | 20120149047 13/179928 |
Document ID | / |
Family ID | 15135195 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120149047 |
Kind Code |
A1 |
Nakamura; Mitsuhiro ; et
al. |
June 14, 2012 |
METHOD FOR QUANTITATIVELY DETERMINING LDL CHOLESTEROLS
Abstract
A method for quantitatively determining LDL cholesterol,
including the steps of adding to serum a surfactant selected from
among polyoxyethylenealkylene phenyl ethers and
polyoxyethylenealkylene tribenzylphenyl ethers and a
cholesterol-assaying enzyme reagent so as to preferentially react
cholestrols in high density- and very low density-cholesterols
among lipoproteins, and subsequently determining the amount of
cholesterol that reacts thereafter. This method can eliminate the
necessity for pretreatments such as centrifugation and
electrophoresis, enables the quantitative determination to be
conducted in an efficient, simple manner, and can be applied to
various automatic analyzers.
Inventors: |
Nakamura; Mitsuhiro;
(Ibaraki, JP) ; Nakanishi; Kazuo; (Ibaraki,
JP) ; Hino; Koichi; (Ibaraki, JP) ; Manabe;
Mitsuhisa; (Ibaraki, JP) |
Assignee: |
SEKISUI MEDICAL CO., LTD.
Tokyo
JP
|
Family ID: |
15135195 |
Appl. No.: |
13/179928 |
Filed: |
July 11, 2011 |
Related U.S. Patent Documents
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Application
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Patent Number |
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12782447 |
May 18, 2010 |
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13179928 |
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12265202 |
Nov 5, 2008 |
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12782447 |
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12025369 |
Feb 4, 2008 |
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12265202 |
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11399447 |
Apr 7, 2006 |
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12025369 |
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10859999 |
Jun 4, 2004 |
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11399447 |
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09971673 |
Oct 9, 2001 |
6764828 |
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10859999 |
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09510170 |
Feb 22, 2000 |
6333166 |
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09971673 |
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09147296 |
Nov 23, 1998 |
6057118 |
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PCT/JP97/01232 |
Apr 10, 1997 |
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09510170 |
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Current U.S.
Class: |
435/11 |
Current CPC
Class: |
C12Q 1/60 20130101 |
Class at
Publication: |
435/11 |
International
Class: |
C12Q 1/60 20060101
C12Q001/60 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 1996 |
JP |
8-134727 |
Claims
1. A method for quantitatively determining low density lipoprotein
cholesterol, comprising the steps of adding to serum a surfactant
selected from among polyoxyethylenealkylene phenyl ethers and
polyoxyethylenealkylene tribenzylphenyl ethers and a
cholesterol-assaying enzyme reagent, to thereby induce preferential
reactions of cholesterols in high density- and very low
density-lipoproteins among lipoproteins, and subsequently
determining the amount of cholesterol which reacts thereafter.
2. A method for quantitatively determining low density lipoprotein
cholesterol, characterized by comprising the steps of adding to
serum a surfactant selected from among polyoxyethylenealkylene
phenyl ethers and polyoxyethylenealkylene tribenzylphenyl ethers, a
substance exhibiting stronger bonding affinity to very low density
lipoprotein than to low density lipoprotein, and a
cholesterol-assaying enzyme reagent, to thereby induce preferential
reactions cholesterols in high density- and very low
density-lipoproteins among lipoproteins, and subsequently,
determining the amount of cholesterol which reacts thereafter.
3. The method for quantitatively determining low "density
lipoprotein cholesterol according to claim 2, wherein the substance
exhibiting stronger bonding affinity to very `low density
lipoprotein than to low density lipoprotein is a`polyanion or a
substance forming a divalent metal salt.
4. A kit for quantitatively determining low density lipoprotein
cholesterol, comprising a cholesterol-assaying enzyme reagent and a
surfactant selected from among polyoxyethylenealkylene phenyl
ethers and polyoxyethylenealkylene tribenzylphenyl ethers.
5. The kit for quantitative determination according to claim 4,
further comprising a substance which exhibits stronger bonding
affinity to very low density lipoprotein than to low density
lipoprotein.
Description
CONTINUATION DATA
[0001] This application is a Continuation of U.S. application Ser.
No. 10/859,999, filed Jun. 4, 2004, pending, which is a
Continuation of U.S. application Ser. No. 09/971,673, filed on Oct.
9, 2001, now allowed, which is a Continuation of U.S. application
Ser. No. 09/510,170, filed on Feb. 22, 2000, now U.S. Pat. No.
6,333,166, which is a Continuation of U.S. Ser. No. 09/147,296,
filed Nov. 23, 1998, now U.S. Pat. No. 6,057,118, which is a 371 of
PCT/JP97/01232, filed Apr. 10, 1997.
TECHNICAL FIELD
[0002] The present invention relates to a-method for quantitatively
and fractionally determining LDL (Low Density Lipoprotein)
cholesterol and cholesterol in lipoproteins other than LDL in an
efficient, simple manner which requires a small amount of samples
and requires no treatment for separation, such as centrifugation or
electrophoresis.
BACKGROUND ART
[0003] Lipids such as cholesterols bind to apoprotein in serum to
form lipoprotein. Lipoprotein is typically classified as
chylomicron, very low density lipoprotein (VLDL), low density
lipoprotein (LDL), high density lipoprotein (HDL), etc. according
to physical properties. Among them, LDL is known to be a causal
substance inducing arteriosclerosis.
[0004] Several epidemiological studies have clarified that the LDL
cholesterol level is strongly correlated to onset frequency of
arteriosclerotic disease. Therefore, realization of measurement of
LDL cholesterol through a simple routine method might be very
useful clinically.
[0005] With regard to conventional methods for measuring LDL
cholesterol, there have been known, for example, a method in' which
LDL is separated from other lipoproteins by ultracentrifugation to
measure cholesterol and a method in which lipid is stained after
separation through electrophoresis so as to measure the intensity
of developed color. However, most of these methods are not used
routinely, due to their intricate operations and limitations in
handling a number of specimens. There is also known a method in
which a carrier is sensitized with an antibody which binds a
lipoprotein other than LDL, then mixed with a sample, and a
fraction not bound to the carrier is fractionated to measure
cholesterols therein. Although this method is more suited I for
routine assay as compared with the previous two methods, the assay
procedure involves manual steps, which makes automation of the
assay procedures difficult. Thus, the method is still unsuited for
handling a large number of specimens.
[0006] Meanwhile, with regard to a method for quantitatively and
fractionally determining lipoproteins in a sample without using
means for separation such as ultracentrifugation or
electrophoresis, there has been known a method in which, upon
fractional determination of cholesterols in HDL and other
lipoproteins (i.e., chylomicron, VLDL, and LDL), reactivity of
enzymes employed (typically cholesterol oxidase and (cholesterol
esterase) is controlled to induce exclusively HDL cholesterol to
enzyme reaction. For example, Japanese Patent Application Laid-Open
(kokai) No. 7-301636 discloses a method for exclusively measuring
HDL cholesterol by use of a surfactant and a sugar compound, and
Japanese Patent Application Laid-Open (kokai) No. 6-242110
discloses a method for exclusively measuring cholesterol in a
target lipoprotein by agglutinating lipoproteins other than the
lipoprotein to be measured so as to control reactivity with an
enzyme. These methods are significantly useful in view of
applicability thereof to automatic analyzers which realize
automation of all steps. However, these methods have limitations in
that they can quantitatively determine only HDL fractionated from
lipoproteins other than HDL, and have no further ability to
determine LDL quantitatively and fractionally from a mixture of
VLDL and chylomicron. Therefore, these methods cannot meet an
objective to measure LDL cholesterol without using separation
means.
[0007] Japanese Patent Application Laid-Open (kokai) No. 7-280812
discloses a method for determining LDL cholesterol comprising the
steps of agglutinating LDL; removing cholesterols in other
lipoproteins by a system which differs from a system for
determining LDL; dissolving the agglutination of LDL; and reacting
the LDL cholesterol. However, similar to the methods described in
the above two publications, Japanese Patent Application Laid-Open
(kokai)7-280812 proposes no resolution to quantitative and
fractional determination of LDL and VLDL and/or chylomicron, which
is absolutely essential for determining LDL cholesterol. There is
also a problem with this method; it cannot be applied to
commonly-used automatic analyzers due to a large !number of steps
required for the assay, making this method of very limited use.
[0008] Thus, with conventional techniques, LDL cholesterol can
never be assayed effectively without performance of an operation
for separation, and, moreover, there has been no information
indicating possibility of the above measurement.
[0009] Accordingly, an object of the present invention is to
provide a method for quantitatively and fractionally determining
LDL cholesterol efficiently in a simple manner while eliminating
necessity for pretreatments such as centrifugation or
electrophoresis and which can be applied to a variety of automatic
analyzers.
DISCLOSURE OF THE INVENTION
[0010] In view of the foregoing, the present inventors have
conducted earnest studies, and have found that reaction with a
cholesterol-assaying enzyme reagent performed in the presence of a
specific surfactant which dissolves lipoproteins accelerates
reaction of HDL cholesterol and VLDL cholesterol and remarkably
retards reaction of LDL cholesterol; that reaction of HDL
cholesterol and VLDL cholesterol are terminated prior to reaction
of LDL cholesterol; and that LDL cholesterol can be measured
quantitatively and fractionally by appropriate selection of a point
of measurement, allowing for application to automated analyzers.
The present invention was accomplished based on these findings.
[0011] Accordingly, the present invention provides a method for
quantitatively determining LDL cholesterol, comprising the steps of
adding to serum a surfactant selected from among
polyoxyethylenealkylene phenyl ethers and polyoxyethylenealkylene
tribenzylphenyl ethers and a cholesterol-assaying enzyme reagent,
to thereby induce preferential reactions of cholesterols in high
density- and very low density-lipoproteins among lipoproteins, and
subsequently determining the amount of cholesterol which reacts
thereafter.
[0012] The present invention also provides a method for
quantitatively determining LDL cholesterol, characterized by
comprising the steps of adding to serum a surfactant selected from
among polyoxyethylenealkylene phenyl ethers and
polyoxyethylenealkylene tribenzylphenyl ethers, a substance
exhibiting stronger bonding affinity to VLDL than to LDL, and a
cholesterol-assaying enzyme reagent, to thereby induce preferential
reactions of cholesterols in high density- and very low
density-lipoproteins among lipoproteins, and subsequently
determining the amount of cholesterol which reacts thereafter.
[0013] Furthermore, the present invention provides a kit for
quantitatively determining LDL cholesterol, comprising a
cholesterol-assaying enzyme reagent and a surfactant selected from
among polyoxyethylenealkylene phenyl ethers and
polyoxyethylenealkylene tribenzylphenyl ethers. Furthermore, the
present invention provides a kit for quantitatively determining LDL
cholesterol as described above, further comprising a substance
which exhibits stronger bonding affinity to VLDL than to LDL.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 shows correlation of measurements of LDL cholesterol
obtained in Example 1 through a method of the present invention and
measurements of LDL cholesterol obtained through
ultracentrifugation.
[0015] FIG. 2 shows correlation of measurements of LDL cholesterol
obtained in Example 2 through a method of the present invention and
measurements of LDL cholesterol obtained through
ultracentrifugation.
[0016] FIG. 3 shows correlation of measurements of LDL cholesterol
obtained in Example 3 through a method of the present invention and
measurements of LDL cholesterol obtained through
ultracentrifugation.
BEST MODES FOR CARRYING OUT THE INVENTION
[0017] The surfactants which are used in the present invention are
selected from among polyoxyethylenealkylene phenyl ethers and
polyoxyethylenealkylene tribenzylphenyl ethers and dissolve
lipoproteins. Examples of the former ethers include Emulgen A-60
(Product of Kao Corporation) and examples of the latter ethers
include Emulgen B66 (Product of Kao Corporation). The surfactants
may be used singly or in combination of two or more species.
[0018] The amount of use depends on the compound and is not
particularly limited. Under normal conditions, the surfactants are
preferably used at a concentration of 0.01-2 wt. % so as to obtain
a sensitivity that permits detection of LDL cholesterol within a
desired assay time, which differs in accordance with the
janalytical apparatus to which a reagent is applied.
[0019] The method for assaying cholesterol according to the present
invention is preferably practiced in the presence of a substance
exhibiting stronger bonding affinity to VLDL than to LDL.
Particularly, when the specimen is chylomicron-containing serum,
addition of the above substance provides excellent assay results.
Examples of such substances include polyanions and substances
forming a divalent metal salt. Specific examples of the polyanions
include phosphotungstic acid and salts thereof, dextran sulfate,
and heparin; and more specific examples of the above substances
include divalent metal chlorides such as MgCl2, CaCl.sub.2, MnCl2,
or NiCl2 or hydrates thereof. These substances may be used singly
or in combination of two or more species. The amount of use depends
on the compound and is not particularly limited. Preferably,
polyanions are used in an amount of 0.002-10 wt. % and the
substances forming divalent metal ions are used in an amount of
0.01-1 wt. %, both in terms of a terminal concentration in
reaction.
[0020] A surfactant and a substance exhibiting stronger bonding
affinity to VLDL than to LDL are added to serum serving as a
specimen and may be added separately or in the form of a mixture.
Briefly, the former, the latter, and a cholesterol-assaying enzyme
reagent may be added separately; either of the former and the
latter and a mixture of the counterpart and a cholesterol-assaying
enzyme reagent may be added separately; or a mixture of the three
components may be added as a reagent.
[0021] Any known enzymatic assay methods may be used for assaying
cholesterols. Examples of the methods include a method employing a
combination of cholesterol esterase and cholesterol oxidase as an
enzyme reagent, as well as a method employing a combination of
cholesterol esterase and cholesterol dehydrogenase as an enzyme
reagent. Of these, a method employing a combination of cholesterol
esterase and cholesterol oxidase is preferred. No particular
limitation is imposed on the method for finally detecting
cholesterols following addition of these cholesterol-assaying
enzyme reagents, and examples thereof include an absorptiometric
analysis employing a further combination of peroxidase and a
chromogen and direct detection of a coenzyme or hydrogen
peroxide.
[0022] In order to perform an LDL cholesterol assay, the amount of
relevant reaction is determined after termination of reactions of
cholesterols in lipoproteins other than LDL. There may be employed
a method in which reaction of cholesterols in lipoproteins other
than LDL is substantially completed after allowing the reaction to
proceed for a specific time, and a reaction which proceeds
thereafter is kinetically monitored. Alternatively, there may be
employed a method in which an additional reaction-accelerating
agent is further added so as to accelerate reaction of LDL; the
reaction that has caused therefrom is measured through a reaction
end-point method; and the value is adjusted by use of a blank value
(2-points method). With regard to the reaction-accelerating agents
which may be used in the 2-points method include the same
surfactants that are used in reaction of cholesterols in
lipoproteins other than LDL in a higher concentration and another
kind of surfactant. In the 2-points method, cholesterols may be
introduced into another reaction system isolated from a system for
determining LDL to exclusively detect reaction of LDL cholesterol
during reaction of cholesterols in lipoproteins other than LDL.
[0023] Examples of other lipoproteins contained in serum include
chylomicron, which typically appears exclusively after ingestion of
food. Chylomicron has approximately the same reactivity as that of
VLDL. Therefore, reactivity of chylomicron is also accelerated in a
manner similar to the case of VLDL by addition of polyanions, a
substance which forms divalent metal ions, etc. and reaction of
chylomicron is also completed when the reaction of VLDL is
completed. Thus, LDL cholesterol may be determined quantitatively
and fractionally through measurement of the reaction amount of
cholesterols thereafter.
EXAMPLES
[0024] The present invention will next be described by way of
examples, which should not be construed as limiting the invention
thereto.
Example 1
[0025] Normal-lipid serum specimens were assayed for LDL
cholesterol through a method of the present invention by use of a
Hitachi model 7070 automatic analyzer, and the measurements were
compared with those obtained through ultracentrifugation. The
results are shown in FIG. 1.
[0026] Briefly, to a specimen (4 [1), a reagent (300.about.L1)
containing sodium phosphotungstate (0.02 wt. %) and MgCl2-6H2O (0.2
wt. %) was added. Approximately five minutes later, there was added
a cholesterol-assaying reagent (100 .mu.l) containing Emulgen A-60
(product of Kao Corporation) (0.5 wt. %), cholesterol esterase (1
U/ml), cholesterol oxidase (1 U/ml), peroxidase (1 U/ml),
4-aminoantipyrine (0.005 wt. %), and N,N-dimethyl-m-toluidine (0.04
wt. %), and the changes in absorbance at 545 nm during the period
of one minute to five minutes after the addition of the second
reagent were measured.
[0027] For ultracentrifugation, the serum was subjected to
centrifugation at 100,0008 for two hours by use of an
ultracentrifuge, to thereby remove the upper layer. To an aliquot
(1 ml) collected from the resultant lower layer, a heparin solution
(40 .mu.l; heparin=5000 usp units/ml) and a 1M MgCl2 solution (50
.mu.l) were added, and the mixture was `centrifuged at 5000 rpm for
30 minutes, to thereby obtain a supernatant. The solution
(containing LDL and HDL) of the lower layer obtained through
ultracentrifugation and the`; fractionated supernatant (containing
HDL) obtained through addition of a solution of heparin and a
solution of MgCl2 were subjected to cholesterol assay, and the
value obtained, by subtracting the latter from the former
represents the LDL cholesterol level (Reference; Paul S. Bachorik
et al., Clin. Chem. 41/10, 1414-1420, 1955).
[0028] As shown in FIG. 1, the present invention provides
measurements having excellent correlation to those obtained through
conventional centrifugation, even though the method of the present
invention requires a small amount of sample and can be carried out
in a simple manner.
Example 2
[0029] A specimen that contains chylomicron-containg serum having a
high triglyceride level was assayed for LDL cholesterol through a
method of the present invention by use of a Hitachi model 7070
automatic analyzer, and the measurements were compared with those
obtained through ultracentrifugation. The results are shown in FIG.
2.
[0030] Briefly, to a specimen (4 .mu.l), a reagent (300 .mu.l)
containing Emulgen B66 (product of Kao Corporation) (0.5 wt. %),
cholesterol esterase (0.3 U/ml), cholesterol oxidase (0.3 U/ml),
peroxidase (0.3 U/ml), and 4-aminoantipyrine (0.002 wt. %) was
added. Approximately five minutes later, there was added a reagent
(100 .mu.l) containing Triton X-100 (1 wt. %) and
N,N-dimethyl-m-toluidine (0.04 wt. %), and the changes in
absorbance were measured by subtracting the absorbance measured at
545 nm before the addition of the second reagent from that measured
five minutes after the addition thereof (correction in
consideration of the change in amount of the reagents).
[0031] In the ultracentrifugation step, the procedure of Example 1
was repeated.
[0032] As shown in FIG. 2, similar to the case of Example 1, in
Example 2 measurements of LDL cholesterol having excellent
correlation to those obtained through conventional centrifugation
were obtained.
Example 3
[0033] The procedure of Example 2 was repeated by use of the same
specimen and reagents except that phosphotungstic acid (0.3 wt. %)
was further incorporated in the first reagent, and the measurements
were compared with those obtained through ultracentrifugation. The
results are shown in FIG. 3.
[0034] As shown in FIG. 3, similar to the case of Example 1, in
Example 3 measurements of LDL cholesterol having excellent
correlation to those obtained through conventional centrifugation
were obtained, even though a serum specimen containing
chylomicron-containing serum was used.
INDUSTRIAL APPLICABILITY
[0035] The present invention eliminates the necessity for
pretreatment such as centrifugation and electrophoresis, and
enables quantitative determination of LDL cholesterol, fractional
to cholesterols contained in other lipoproteins, to be performed in
an efficient, simple manner, and thus can be applied to various
automatic analyzers used in clinical examinations. Thus, the
invention is remarkably useful in the clinical field.
* * * * *