AU2005202380A1 - Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged:

O

O

0 00

(N

.rl Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Daiichi Pure Chemicals Co., Ltd.

Actual Inventor(s): Mitsuhiro Nakamura, Yuriko Taniguchi, Mitsuhisa Manabe, Mitsuaki Yamamoto Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: METHOD OF PRETREATMENT OF SAMPLE FOR QUANTITATING CHOLESTEROL AND METHOD FOR QUANTITATING CHOLESTEROL IN SPECIFIC LIPOPROTEINS BY USING THE

SAME

Our Ref: 746697 POF Code: 129840/154859 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1 T V

SDESCRIPTION

METHOD OF PRETREATMENT OF SAMPLE FOR QUANTITATING CHOLESTEROL AND METHOD FOR QUANTITATING CHOLESTEROL IN SPECIFIC SLIPOPROTEINS BY USING THE SAME 00 -q The present application is a divisional application from S 10 Australian patent application number 54263/00, the entire 0( disclosure of which is incorporated herein by reference.

Technical Field 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 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.

Epidemiologically, the level of cholesterol in LDL is known to exhibit a positive correlation with the frequency of X:1Files\659139\659 139 specadivisional.doc onset ofarteriosclerotic disease while:the level of cholesterol in HDL is? known to show an inverse correlation with the frequency Sof onset of arteriosclerotic disease. These days, measurements of cholesterol in LDL or HDL are, therefore, widely conducted 0 for the prevention or diagnosis of ischemic heart diseases.

00 As methods known for the measurement of cholesterol in Ci 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.

A method for the measurement of cholesterol in HDL, which is used at ,present in the field-of clinical tests, is the precipitation methodin 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 O of causing an analytical error. Furthermore, the entire analysis steps of this method can not be fully automated.

On the other hand, enzymatic methods have been studied for the fractional quantitation of cholesterol in HDL. Known O methods include, for example, to conduct an enzymatic reaction 00 ^i in thej presence of a bile acid salt and a nonionic surfactant CN JP 6 3-12 6498 A) This method makes use of the fact that an 0 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 ih other lipoproteins cannot be fully distinguished.

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 theisame time, to:redissolve the agglutinate, :-followed by the- measurement of an absorbance (JP 6-242110 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.

0 A still further method is also known (JP 9-299 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, 00 preferentially-reacted, and then measuring cholesterol in HDL Ci while inhibiting any reaction to cholesterol in lipoproteins 0 other than HDL This method, however, is considerably different from the present invention inter alla inmthat 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.

Further, JapanesefPatent No. 2,600,065 discloses amethod which mIiakes combined use of a precipitation reagent :which is adapted to cause precipitationof. lipoproteins other than1HDL, and a cholesterol measuring; reagent to measure cholesterol (HD -CG) in unprecipitated HDL. This method has practical utility when. a. modified enzyme is used as enzyme and G -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.

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 0 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 0 with that obtained by the precipitation method) induced when Mi the modif ied enzyme is simply introduced into a reaction system, 1 HDL-C was measured by using cyclodextrin sulfate, a polyanion, p and magnesium chloride as a precipitation reagent for the avoidance-of thepositive error. To reduce the influence of turbidity caused by a precipitation reagent .in the above-described patented method, certainhtechniques are alsoknown, including to make a surf actant exist concurrently (JP 8-116996 to use an antibody (JP 9-96637 and to employ a sugar compound (JP 7-301636 A).

They, .however., all-require as apremise the inclusionof 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.

The: present -nventors recently. found. that :use f 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 consistentwith those obtained by the conventional precipitation method at medical institutions and the like, a 0 polyanion or the like is added.

00 From the standpoint of the problem of a tarnish or the CN like on a cuvette-and scattering of measurement values, however it is not desired to add a polyanion or the like and to form0 a precipitate, in a measurement system. 'Accordingly, it-ahas been strongly desired to eliminate the precipitate fromithe 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 ,f the principle of the measurement. Hence, there is also- an outstanding desire for its solution.

-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.

Disolosure of the invention 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 lippprotein 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, V)DL and the like) the cholesterol of which is not supposed to, be-measured, a small amount of free cholesterol existingion 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 qonly free cholesterol in advance under conditidns that lipoproteins remain .substantially unchanged, leading to the completion of the preseit invention.

Sescribed 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.

The present invention also provides a method for 0 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 O cholesterol, which exists in the-sp.cific lipoprotein, by using p a substance which acts .upon the specific lipoprotein only.

Brief Description of the Figures S FIG. 1 is a diagram showing a "correlation between the present invention in Example 1 and the precipitation method; 'FIG. 2 is a diagram showing a correlation between the present invention in Example 2 and the precipitation method; and.

FIG. 3 is a diagram showing effects of a reaction accelerator in Example Best Modes for Carrying Out the Invention 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.

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.

00 The enzyme is generally used at 0.001 to 100 U/mL, with 0.1 0 to 100U/mL being preferred.

No particular limitation is imposedon conditions under ^C which the above-described enzyme, which acts pon free 0 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 t 't cholesterol esterase exists. What is neededis:to maintain conditions such that. cholesterol esterase is not allowed to act: practically. 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.

Concerning the enzyme which acts upon free cholesterol as a substrate and is used in the present invention, no limitation is imposedp n i its origin as described above. Its concentration and the lke can be chosen: suitably to achieve desired performance and handling ease. Accordingly, if it is desired to hae the pretreatment completed in a predetermined time, for example, it is onlynecessary 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.

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 suchacase, 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- as substrate, iakes it possible to achieve desired performance with a reduced amount: of the enzyme without making. the pretreatment time longer.

Reaction accelerators usable for the above purpose can include,- for example, flufenamic acid, mefenamic acid,.

2,2 2 -terpyridine, tiglic acid. fusidic acid, betamethasone acetate, monensin anidmevinolin, 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 andmonensin are known as antibiotics.

SUpn 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 0 and concentrations of substances existing together.

00 ¢I The concentration of the reaction accelerator can be CI experimentally determined i accordance with conditions of a p measuring system. In general, however, flufenamic acid can may be used at about 0.01 :to 100lmM; fusidic acid at about 0.01 to 10- mefenamic acid, 2,21',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..

Use of the above-described reaction acceleratorhas mnade it possible to reduce the aibUnt of the:enzyme, which Pcts upon free cholesterol as a substrate, to one severalth or itoone several tenth. When the enzyme is used inthe same amount, Son-the-other .hand, the reaction' accelerator,. can shorten -the reaction time.

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 forpH regulation, surfactants (with exclusion of those giving substantial influence to lipoproteins), preservatives, proteins such as 0 albumin, and agent s having af finity o specific lipoproteins, such asantibodies, 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.

00 N VIn the present invention,.those containing the following Cq ingredients -can, therefore, be used as pretreatment agents for measuring cholesterol existing in specific lipoproteins in (Essential ingredients) Enzymes which act upon free cholesterol as a substrate, for example, cholesterol dehydrogenase and cholesterol -oxidase.

(Optional ingredients) Reaction accelerators,7 for example, flufenamic acid, mefenamic acid, ,2"-terpyridine, tiglic acid.

fusidic acid, betamethasone acetate, monensin and (.Other. ingredients) 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, Trinder's -reagent, electron acceptors such as phenazine _methosulfate, and reductive color developers such as nitroblue tetrazolium.

In the present invention, cholesterol which exists in 00 ci a -specific lipoprotein in a sample is measured after having Ci free cholesterol in lipoproteins consumed by the 0 above-described pretreatmenrt.

An A 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.

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 poyoxyethylene alkyene 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.

Examples of commercial products of the former surfactants, polyoxyethylene alkylene phenyl ethers, can include "Emulgen (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).

As an alternative method, there is a method which makes use of hthe modified enzymes;, which are disclosed on pages 305-320 of "SEIBUTSU SHIRYOBUNSEKI(ANALYSISOFBIOLOGICAL

SAMPLES,

19 as substances which act only upon specificlipoproteins, respectively. Although a -cyclodextrin suifate and magnesium chloride are used in the methodof this paper to inhibitre actions 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.

Except for the use of the, substance which acts upon the specifi 3 c 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 f ingredients which may be contained in reagents to be used ca include enzymes such as cholesterol-esterase, icholesteroloxidase., cholesteroldehydrogenase, isocitrate dehydrogenase, diaphorase and peroxidase, color developers, coenzymes, electron acceptors, proteins (albumin, etc.), preservatives, surfactants, salts, acids, and buffers for pH regulation.

As surfactants out of the above-described ingredients, both ionic and nonionic surfactants are usable. Illustrative are polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers;, polyoxyethylere-polyoxyprpyene condensate polyoxyethylene alkyl ether sulfates, alkylbenzenesulfonate salts, and bile acid salts. The amount of the surfactant to be used varies depending oni the compound. The surfactant may however be used in 0.0001% to preferably in 0.001% to ci although no particul-ar limitation is 'imposed 'thereon.

No particular limitation is imposed on the buffers,.

Conventional buffers such. as Good'-s buffer, phosphate .buffer, Tris buffer and phthalatebuffer are usable. The-buffer may be used at 0.005 M to 2 preferably 0.01 M to 1: M, although no particular limitation is iimposed thereon.

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 addingand.mixing a cholesterol measuring reagent (hereafter called.

a "quantitation-reagent"), which contains a .substance capable.

of acting ,upon' the specif ic lipoprotein and a reagent employed for a conventional cholesterol-measuring method, to measure; the amount of cholesterol in the specific lipoprotein fraction.

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 0 antipyrine or catalase and then adding a cholesterol-measuring C-q reagent which'comprises cholesterol esterase; andamethod which comprises mixing a sample and cholesterol oxidase together with 0 peroxidase., 4-aminoantipyrine, etc. and then adding a cholesterol-measuring reagent which comprises cholesterol esterase. cholesterol dehydr6genase and NAD.

Examples of the method for measuring cholesterol in a specific lipoprotein fraction. can include a method making combinedi 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.

In thepresent 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 O specific lipoprotein to be measured, so that most cholesterol (free cholesterol esterified cholesterol) forming the lipoprotein can be quantitated.

Further, no particular limitation is imposed on the method O for finally detecting cholesterol after the addition of such 00 M a cholesterol-measuring enzyme reagent. It is possible to use, (1 for ^example, absorptiometry in which detection is conducted 0 by combining peroxidase with a chromogen or diaphorase or an electron acceptor with a reductive color-developing reagent; or a metliod in which a coenzymeor hydrogen peroxide is directly detected. The coenzyme may be amplified by a coenzyme cycling system To practice the method of the present invention with ease, it ispreferred'to use a quantitation kit which is suited for measuring cholesterol in the specific lipoprotein.

Although such kits can be readily designed based on the above explanation,. their examples will be described nextby dividing them into those making use of cholesterol oxidas eand those making use of cholesterol dehydrogenase as typical example of enzymes which act 'upon freecholesterol as a substrate.

[Kits making use of cholesterol oxidase] A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents and a first reagent comprising cholesterol oxidase and a hydrogen peroxide consuming substance (and further comprising a reaction accelerator in some instances); and S(2) a second reagent comprising a substance which acts upon the specific lipoprotein only, cholesterol esterase, and a color developer.

A quantitation kit for cholesterol in a specific 0 lipoprotein, comprising the following reagents and 00 S(1) a first reagent :comprising cholesterol oxidase, Cq cholesterol esterase! .and a hydrogen peroxide consuming C substance, (and. further comprising a reaction accelerator in some- instances); and 2 a sscong a substance -whch acts upon the specific lipoprotein only, and a color developer.

(c)A quantitation kit for cholesterol in a specific lipoprotein, comprising the following, reagents and a firs% reagent comprising ;holesterol oxidase and ahydrogen-peroxideconsuming substance (and-further-eomprising a reaction, accelerator in some: instances); a second reagent comprising a substance which act uppn the.specific lipoprotein only; and a third reagent comprising cholesterol esterase and a color, developer.

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.

-Among these, a coupler such as 4-aminoantipyrine and a hydrogen donor such as Trinder's reagent develop a color when 0 reacted, Min combination, with hydrogen peroxide, and are usable O0 c- as the, color developer in the above-described reagent or (3 JAs the reagent for use in the pretreatment step 0 according to the present invention, i 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-deve-loping 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 thereagent from the intensity of a color developed by the, reagent or- the reagent [Kits making use of cholesterol dehydrogenase) A quantitation.kit for cholesterol in a :specific lipoprotein, comprising the-following reagents 1) and a first reagent comprising cholesterol dehydrogenase and a coenzyme(and further comprising a reaction accelerator in some instances); and a second reagent comprising a substance, which acts upon the specific lipoprotein only, and cholesterol esterase.

A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents and S(1) a first reagent comprising cholesterol dehydrogenase and a coenzyme(and further comprising a reaction accelerator in some instances); and a second reagent comprising a substance which acts 0 upon the, specific lipoprotein only, cholesterol oxidase, 00 cholesterol esterase, peroxidase, and ;a color developer.

S(f) A quantitation kit for cholesterol in a specific lipoprotein, comprising the f6llowing reagents (.l)and (1)afirstreagentcomprisingcholesterol dehydrgenase, a coenzyme, and cholesterol esterase(and further cobprising a reaction accelerator in some instances); and a second reagent comprising a substance which acts upon the specific lipoprotein only.

A quantitation kit for cholesterol in. a specific." .lipoprotein, comprising the following reagents and af-irst reagent comprising:cholesterol dehydrogenase, a coenzyme, and cholesterol esterase(and further comprising a: reaction accelerator in some instances):; and:-.: a second.reagent comprising a substance which acts upon the specific lipoprotein only, cholesterol oxidase, peroxidase, and a color developer.

A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents and a first reagent comprising cholesterol dehydrogenase and a coenzyme(and further comprising a reaction accelerator inr some instances)l a second reagent comprising a substance which acts upon the specific li-poprotein only; and a third reagent comprising cholesterol esterase.

A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents and a first reagent comprising cholesterol dehydrogenase and a coenzyme(and further comprising a reaction acc.lerator in- some instances); a second reagent comprising ia, substance which acts upon the specific lipoprotein only and a third reagent comprising cholesterol oxidase, cholesterol esterase, peroxidase, and a color deyveioper quantitation- kit for cholesterol in a-specific lipoproteinh comprising the fol wi ng reagents and a first reagent comprising cholesteroldehydrogenase, a coenzyme-, and a coenzyme reaction -product consuming subs t an ce andfurther comprising a reaction accelerator in some instances):; and a second reagent comprising a substance, which acts upon the specific lipoprotein only, and cholesterol esterase.

A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents and a first reagent comprising cholesterol dehydrogenase, a coenzyme, and a coenzyme reaction product consuming 0 substance(and further comprising areaction accelerator in some instances); and a second reagent comprising a substance which acts upon the specific lipoprotein only, cholesterol esterase, and a color .developer..

00 In the above-described kits making use of cholesterol C- 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. Illiustrative is a combination of lactate dehydrogenase and pyruvic acid (substrate). In each of the above-described kits, the reaction productbof t-hecoenzyme is produced by the addition of the reagent S 1. In each of the kits and out of, the: above-described kits, light of the same, wavelength as' a color developed. bythe addition of the reagent 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 isdeveloped in: the pretreatment stage in which the reagent is added, from the intensity of a color developed by the reagent or the reagent As an alternative, it may also be possible to add beforehand the substance, which consumes the reaction product, to the reagent and subsequent to consumption of the reaction •product, to add the reagent or the reagent for the development of a color. In this case, addition Sof a substance, which reduces the action of the substance which consumes the reaction product, to the reagent or the reagent is preferred. In each of the kits I) and 00 C< ,on the iother hand, it isinot :absolutely necessary to subtract the intensity of the color, which: is developed in the 0 pretreatment stage, from the color intensity^ measured i the measurement stage, because in the measurement stage, adeveloped color of a wavelength different from the color developed 1h the pretreatment stage is measured.

It is to be noted. that the application of the above-mentioned reaction accelerators. such as flufenamic acid, -mefenamic acid. 2"-terpyridine, tiglic acid7, fusidic acid, betamethasone acetate, monensinandmevinolin, is limitd 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 lipoproteih, said quantitat ionmethod or kit-making use of the pretreatmernt ethod or agent.

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 maklng combined use of cholesterolloxidase, 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.

Further, reference.to the ,disclosure of, this. specification on: the cholesterol q.uantitation :m.:eth d (for.: example, selection of a surfactant to limit a target of aspecific lipoprotein to be measured) makes. it possible to more specifically design a quantitation method :as desired.

ndustrially Appliabi. ty.

The present invertion has made itpossible t efficiently quantitate cholesterol in a specific fraction: by simple procedureswi-thout using a polyanion or the like. to6s ay nothing of a mechanical pretreatment such as centrlfugation. As the methods of the present invention do not forma 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.

Further, as wilbe demonrated n subs equet Exampes, measurement values showing a high correlat ion with those obtained by the conventional precipitation method can be obtained even with respect to samples with high triglyceride 0 levels. Therefore, the methods according .to the present c- invention are also excellent in that they are applicable: to r various (samples without limitaton..

0 In. addition, the use of the reaction accelerator makes it possible to use the enzyme, whlch acts upon-freecholesterol as a substrate, in a smaller amount in the pretreatment stage.

As, has been described above, the methods according to the present invention permit accurate and specificmeasurements 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. The present- invention will next be described :in further detail by the Examples. It should however be borne in mind that the present invention isby no means limited to the Examples.

Example 1 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.

(invention method) lmMphosphate b fer (First Reagent; pH 8.5) (300 I L) which containedO. 1U/mLicholesteroldehydrogenase (product Pof Amano Pharmaceutical Co., Ltd.), 2.5 mM NAD and 0.03%

O

4-aminoantipyrine, was added to each sample (3 9 L) (pretreatment) About 5 minutes later, cholesterol 0O ci quantitation reagent (Second Reagent) (100 which was .composedof 100 mMMESbuffer (pH6) containin-g :EmulgnB-66" 1.3 U/mL cholesterol esterase (product of: Asahi Chemical 3.Um'coe teol eo u in ersa.l-.

Industry Ltd.), 2 U/mLcholesteroloxidase.(product-of As ahi Chemical Industry co., Ltd 5: U/mL:, peroxidase (product of Toyobo Co., Ltd. andd 0'.04% disulifobutylmetatoluidine was added.

Just before the addition of the Second Reagent and upon ani elapsed time of five minutes after_. the addition,. the.

absorbance .was measured at 600 nm. From a differehce 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. wa: us ed. The above-procedures were conducted -using "Hitachi 7150 automated. analyzer Precipitation method) HDLC 2 'Daiichi' Precipitant" (product of Dailchi Pure Chemicals Co., Ltd.) (200 IL) was mixed with the sample (200 9L), followed by centrifugation at 3,000 rpm for 10 minutes.

The supernatant (50 was collected, followed by the mixing with a ,cholesterol quantitation reagent (3 mL) composed of 100 MW MES buffer containing 1 iton X-10), 1 U/mL cholesterol esterase. 1 U/mL cholesterol oxidase, 5 UimL peroxidas 0. 0 4% -disulf obutylmetatoluidine and 0.04% 4-aminoantipyrine. After the resulting mixture as incubated at 37- for 10 inutes, its absorba c at 600 nm was measured c to determine- the concentration of the cholesterol in HDL.

(Results,).

The results are shown in Table 1 and, FIG. 1.

2005202380 01 Jun 2005 Table 1 Pre]e o.icition: Inhv ent ion Si Inhvepnt io No. meho ~g/L)method,. (mg Jwe o. mt d(m/L ehod. m/L 4 45'19, 50.4 75771 83 8 52 .223353 951 434t482,3 1249 51' 27 -3 3 13443 46 28 5231 147 5-29.6 63 159 356 738 30474 SAs. is readily envisaged frolmtheresults, the invention method despite the omission of a poyanion or the like, showed an extremely good correlation with the conventional precipitation method.

C Example, 2 Measurements were conducted by another method of the present invention, which was similar to the invention method 0 conducted in Example 1 except that in the first reagent.

cholesterol dehydrogenase, NAD and the phosphate buffer were replaced byi 5 U/mL cholesterol oxidase (product of Toyobo C. Ltd. 5 U/mL peroxidase (product of Toyobo Co., Ltd.) and 100 mM MES buffer (pH The measurement values were compared with those obtained by the precipitation method in Example 1.

S Results) The results are shown in Table 2 and' FIG. 2.

2005202380 01 Jun 2005 Table 2 Sa ple No. Pr c p t t on'vn i nSample No. m eho mg dL method., ,nve t- dL) metho (m/L)Pecipiatio Invntio 173 73 ~1 2 39 38:174 54 3 3 5.18. 6061 6 756r 74, 21~3 7 7 51 522 656- 525 3' 353 9-4 44-6 24~ 92 -58. 58! -25 63 61 1.2 495527333 13 44 28525 14 70' 69 2 56 337 3 4 4 As Iisreadily envisaged from the results, the invention method, idespite the omission of a poyanion or the lik showed an extremely good correlation with the conventional precipitation method.

S. Example 00 C: Using the reagents of Example and Example 2, five serum samples of different triglyceride levels were measured. The 0 measurement values were -then compared with those obtained by the precipitation method. The results are -shown inrrTable 3.

2005202380 01 Jun 2005 Table 3 pecpitto Iveninmto n Invenition method in Tr -g Lycid .meh~o (igiL.): -exam'1 1 rdL) Exalm ee m/L SAmplA 47 49~9198 Sample. B 4,9 5049301.

Sample C. 26724: 742, Sple D 606 1517 Sample E 37. 40 36 428 0 .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 0 Example 4 00 Measurements were conducted in a similar manner as in iN Example 2 except that in the first reagent, 511UmL cholesterol oxidase was. changed to give reagent compositions of the, ingredient concentrations and combinations shown be'low inTable 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 Example1 iwas used. The results are shown in Table ~(ClfOmosit-ions of -testing.:reaenets.) Test-, sytmContents of composition Stanard .Cholste o xidase.

U m4% A Cholesterol',oxidase Flufenamicacd+coetrloxde Meenai -acid, -cholesterol1 oxidase: 2,2' ,2-terpyidin +,5~etrj choeserloxidase Tig2lic:--2 acpyidin choleste o l. xidase E~O. (5,mM) (U/i F uii cd+ cholesterol.: oXIldase (0.1 mM):(1.U/niL) Betamethasonel'aceta-te .cholestero6l oids H Monensin choles tero oxIdas J.Moll-oin, cholesterol oxiJdase:, (Results) 2005202380 01 Jun 2005 Table Precipitati-on:____ Ts ytm(rga) Sample Method

T

(ga) Standard A B CD E G H 3 270 72 66 71 707401 7 4t 71773'0 '6 9 6 71 69 71 7 1 070 691 61: 6, 67 6,8 70, 6 9 6 71 70 67.70 63; 68 68 6 9 7' 6 79666:3 6 611 65 65 65 64; 65 66: 867 6'70V 68 i6 68 i67 6-6 69 68 68 9. 665- "65 5 9 65. 64 63 65 65 651 '6 64 6 65.4,26 65' 63 157556 .57 54' 5 56 57 8 125.56 5 55 49 54 53 55 13 54. 5. 54 5 50'. .54. 53 533 544 14 535-45' 4.'3 5 5 4 52 534 1552'"352 51 4 7' t 512 4-5245 52 16 51, 53 511 .50. 46' 0' 51 451 5 1 17 49 50 48- 4 8 44' 47'48 47 48 4 8 49: 18' 47 48: 4-8 46 I41. 46 44547- 47: 47 19 45 64 44 3-8" 4-6 43' 4 47.. 47 46 47, 47 49.' 45 4 4 6 4 5, 45 48: 47' 47 21 42 4 4' 44; 43'' 39, 43* 4Z2 4,1 43 44 43.

2 .,39 4 42.. 4' 37 41.4 4' 41i1 4 233 5 ~3 1: 36 34- '3:2 34. 34-3 2418, 202 9 '7 2' 9 1' 20 19 254 4 4. 1 45 4 1: 's 40- 41 42, 41 Corrlalition coef. -0 96 0.90 0.199.' 0.99 095' 0..909, 0.995::0;:.97 slop "0.-90,5'.0.83j8 0.4 0'.832 ~0.456 0.915 .877, 0.891 0.91 7 intercept 5. 8.7 2.6 6' 6. 3 57!' 4.1 S,-When the amount of cholesterol oxidase was reduced to one fifth (the test system A) compared with the standard test system (Example the correlation coefficient slightly declined and the value of intercept slightly increased. When the reaction accelerator was used, however, result s 00 c- substantially comparable with those of the standard test system were obtained even when the amount of cholesterol oxidase was 0 one fifth. It has hence become evident from these results that the use of a reaction accelerator makes it possible: to, reduce Sthe amount of cholesterol oxidase to be used.

Example 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;l the kind andpH of buffer and the concentrations of cholesterol oxidase (productiof Toyobo Co. Ltd.) andful-fenamic acid (product of, Sigma. Chemical Co. b Table 6 B' uffer Reagent Concentration of cholesterol oxidase S:Concentration of fulfenamic acid 50 mM Bis-Tris (pH J A 0.5, 1.0, 2.5, 5.0 U/mL T 0, 0.01, 0.05, 0.1 mM mM PIPES (pH K 0.5, 1.0, 2.5, 5.0 U/mL mM MOPS (pH L 0.5, 1.0 2.5, 5.0 U/mL.

0, 1.0, 5.0, 10.0 mM Reagents J to L (300 were separately-added to aliquots (3 of each serum sample. After the resultant mixtures were incubated at 37t. for 5 minutes, their absorbances weremeasured at,600 nin. The above procedures were conducted usingtheHitachi 7150 automated analyzer.

Four serum samples were measured with Reagents J to L.

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.

(Results) Results, which had been obtained by averaging the relative absorbances of the four samples, are presented in FIG. 3, in 0 which "COD" stands for cholesterol oxidase.

(N

As is readily appreciated from the results, the relative absorbance increased depending upon the concentration of flufenamic 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.

00 0 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.

(Ni Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

X:\Files\6591 39\659139_specidiisional.doc

Claims (31)

1. A method for pretreating a sample, which contains various lipoproteins, prior to measuring cholesterol existing in specific one of said lipoproteins in said sample, which Scomprises causing an enzyme, which acts upon free cholesterol as 00 Mg a substrate, to act upon said sample. (N q 2. A method for pretreating a sample, which contains various lipoproteins, prior to measuring cholesterol existing in C( specific one of said lipoproteins in said sample, which comprises causing 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 or mevinolin, to act upon said sample.

3. A pretreatment method according to claim 1 or 2, wherein said enzyme is cholesterol oxidase or cholesterol dehydrogenase.

4. 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 said sample with said lipoprotein contained therein; and then measuring said cholesterol, which exists in said specific lipoprotein, by using a substance which acts upon said specific lipoprotein only.

5. A method for quantitating cholesterol existing in a specific lipoprotein in a sample, which comprises causing an X:\Files\659139\659139 specidivisional.doc Senzyme, 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 or mevinolin, to act upon said sample with said lipoprotein contained therein; and then Smeasuring said cholesterol, which exists in said specific 00 M lipoprotein, by using a substance which acts upon said specific lipoprotein only. (N

6. A quantitation method according to claim 4 or C( wherein said enzyme is cholesterol oxidase and/or cholesterol dehydrogenase.

7. A quantitation method according to any one of claims 4-6, wherein said specific lipoprotein is high density lipoprotein.

8. A pretreatment agent for a sample to be measured for cholesterol, comprising an enzyme, which acts upon free cholesterol as a substrate, and substantially no substance which acts upon lipoprotein.

9. A pretreatment agent for a sample to be measured for cholesterol, comprising an enzyme, which acts upon free cholesterol as a substrate, a reaction accelerator which is selected from flufenamic acid, mefenamic acid, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and substantially no substance which acts upon lipoprotein. A pretreatment agent for a sample to be measured for X:\Files%659139\659139_sp _aivisional.doc cholesterol, comprising an enzyme, which acts upon free cholesterol as a substrate, and substantially no cholesterol esterase.

11. A pretreatment agent for a sample to be measured for cholesterol, comprising an enzyme, which acts upon free 00 cholesterol as a substrate, a reaction accelerator which is (Ni selected from flufenamic acid, mefenamic acid, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, S 10 monensin or mevinolin, and substantially no cholesterol (CN esterase.

12. A pretreatment agent according to any one of claims 8-10, wherein said enzyme is cholesterol dehydrogenase or cholesterol oxidase.

13. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol oxidase and a hydrogen peroxide consuming substance; and a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol esterase, and a color developer.

14. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase and a coenzyme; and a second reagent comprising a substance' which acts upon said specific lipoprotein only, and cholesterol esterase. X;U ies6593965939_specioajvsona.doc A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase and a coenzyme; and a second reagent comprising a substance which acts Supon said specific lipoprotein only, cholesterol oxidase, 00 00 cholesterol esterase, peroxidase, and a color developer.

16. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: 0( a first reagent comprising cholesterol oxidase, cholesterol esterase, and a hydrogen peroxide consuming substance; and a second reagent comprising a substance which acts upon said specific lipoprotein only, and a color developer.

17. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, and cholesterol esterase; and a second reagent comprising a substance which acts upon said specific lipoprotein only.

18. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, and cholesterol esterase; and a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol oxidase, X.\Fies\693965939speci-divisionaldoC peroxidase, and a color developer. -a 19. A quantitative kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol oxidase ad a Shydrogen peroxide consuming substance; 00 00 a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol esterase and a color developer. (N A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase and a coenzyme; a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol esterase.

21. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase and a coenzyme; a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol oxidase, cholesterol esterase, peroxidase, and a color developer.

22. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: X\Filesk65gl39\659l39 specidjvisona.doc S(1) a first reagent comprising cholesterol dehydrogenase, a coenzyme, and a coenzyme reaction product consuming substance; and a second reagent comprising a substance which acts upon said specific lipoprotein only, and cholesterol esterase. 00 M 23. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme and a coenzyme reaction product consuming substance; (CN and a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol esterase, and a color developer.

24. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol oxidase, a reaction accelerator selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and a hydrogen peroxide consuming substance; and a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol esterase, and a color developer. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol X:\Files\659139\659139_speci divisional.doc Sdehydrogenase, a reaction accelerator selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and a coenzyme; and a second reagent comprising a substance which acts Supon said specific lipoprotein only, and cholesterol esterase. 00

26. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol oxidase, C( a reaction accelerator selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin,(c) cholesterol esterase, and a hydrogen peroxide consuming substance; and a second reagent comprising a substance, which acts upon said specific lipoprotein only, and a color developer.

27. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, a reaction accelerator selected from flufenamic acid, mefenamic acid, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and cholesterol esterase; and a second reagent comprising a substance which acts upon said specific lipoprotein only. X;\Files\ 59139\ 59139 _specd djvsjonaI.doc

28. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, a reaction accelerator selected from flufenamic acid, mefenamic acid, Sterpyridine, tiglic acid, fusidic acid, betamethasone acetate, 00 M monensin or mevinolin, and cholesterol esterase; and a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol oxidase, peroxidase, and a color developer.

29. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol oxidase, a reaction accelerator selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and a hydrogen peroxide consuming substance; a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol esterase and a color developer. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a reaction accelerator selected from X:\Filesl659139\659139_specidivisional.doc 4' flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and a coenzyme; a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol esterase. 00

31. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol (CN dehydrogenase, a reaction accelerator selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and a coenzyme; a second reagent comprising a substance which acts upon said specific lipoprotein only; and a third reagent comprising cholesterol oxidase, cholesterol esterase, peroxidase, and a color developer.

32. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, a reaction accelerator selected from flufenamic acid, mefenamic acid, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and coenzyme reaction product consuming substance; and a second reagent comprising a substance which acts X:UTies659139\6513Ospeci-divisionaldoc upon said specific lipoprotein only, and cholesterol esterase.

33. A quantitation kit for cholesterol in a specific lipoprotein, comprising the following reagents: a first reagent comprising cholesterol dehydrogenase, a coenzyme, a reaction accelerator 00 selected from flufenamic acid, mefenamic acid, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, and coenzyme reaction product consuming substance; and (CN a second reagent comprising a substance which acts upon said specific lipoprotein only, cholesterol esterase, and a color developer.

34. A reaction accelerator for an enzyme capable of acting upon free cholesterol as a substrate, which is selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin. A reaction accelerator according to claim 34, wherein said enzyme is cholesterol oxidase or cholesterol dehydrogenase.

36. A method for quantitating free cholesterol, which comprises causing 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 or mevinolin, to act. X:\Files\659139\659139_speci_oivional.doc 'C W ,%t

37. A method for quantitating total cholesterol, 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, terpyridine, tiglic acid, fusidic acid, betamethasone acetate, Smonensin or mevinolin, to act. 00

38. A method according to any one of claims 1, 2, 4 and substantially as hereinbefore described with reference to any S 10 of the Examples. (N

39. A pretreatment agent according to any one of claims 8 to 11, substantially as hereinbefore described with reference to any of the Examples. A quantitation kit according to any one of claims 13 to 33, substantially as hereinbefore described with reference to any of the Examples.

41. A method according to claims 36 or 37, substantially as hereinbefore described with reference to any of the Examples. DATED: 31 May 2005 PHILLIPS ORMONDE FITZPATRICK Attorneys for: DAIICHI PURE CHEMICALS CO., LTD X: Fies659I39659139_specidivisiona.doc