AU2008201734B2 - Method of pretreatment of sample for quantitating cholesterol and method for quantitating cholesterol in specific lipoproteins by using the same - Google Patents

Abstract

A method of a pretreatment of a sample for quantitating cholesterol characterized by, before measuring cholesterol contained in specific lipoproteins, treating the sample 5 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 10 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.

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: 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: 827037 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 - DESCRIPTION METHOD OF PRETREATMENT OF SAMPLE FOR QUANTITATING CHOLESTEROL 5 AND METHOD FOR QUANTITATING CHOLESTEROL IN SPECIFIC LIPOPROTEINS BY USING THE SAME The present application is a divisional application from Australian patent application 2005202380, which is in turn a 10 divisional application from Australian Patent Application Number 54263/00, the entire disclosures of which are incorporated herein by reference. Technical Field 15 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 20 lipoprotein fraction by using the pretreatment method. Background Art Lipids such as cholesterol are complexed with apoproteins 25 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 30 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 in4 .9f 01 66.ncxqn t&6w an Inverse cdrrelatoi ith the f requency ofncnsls.f arteriosclerotic disease. These days. measurements of qboivstero1 ip, LPLorIWtL ate, therefore, wide ly conducted fdr thrvotion or Aid'gxjosis~p 'jf-imhnc heart A~seases. t s ~methocqk known ifor the in~uef~of cholesterol in tit erl!$.' there Ate. toIr exi piP4; a7* 9jo4 in which LV,)L or HDL i s separated -f Tomvtohbr lipprotin" -by ultraicentrifun 9 al z-,. ~ euMbecause they Wi: ;v-g - -p i-t md n:th a ei~ o, a eepdclr snard 4~ 'b&- . preciEs fAareon ncd is ittar Tis bfmeto4i, ?& opae wt utaimirfdiipo ltrpoess ~~ iJt, thec and vo',-erfor t%%t4l sexi t 1*47 re e e~±ach tsmpl is aI~ reai*l ag gtiy and in- esaPtnia rbe 2t1 pe of aus.±nsap aflJA~ji~ Ing ani 41 t uribrore, the entire analysis steps ,of, this thgcan itq be fll automated On the other nd.enzjytatipxne.tbiods have been studied U r thfra~cilonal quaztittion o? tbcijlesterolin HDL. Known *ahdsldue fobr~e*am to con0O','t an enzymatic reaction Mn Of.......... - an d :t a 14 noii sUrf~ctAnt -. j T~ ruetjhodso' ie$ qfe o'th ~tj tta -eriymaab r actoh ~~ ce s 1 pro it 9 t6 theconc~ntristIdi fb 0 1- ta'<qplbtdoei n iid .s_, e%.- n -.. af 'Conieiat~onnf choleptero I±xrHP!L. APtproblemnbhowek:texists j1h accuracy beo~use :the- reduction vIth :the chol-esterol in tint> a~d~h~~atip tl~hoaserd ih tb~~4poteins a~t V -k A of4a4 s to ractia tn*~ fi4rand L*disposalto the rgntse 3. 3 As still futOh~rrb ±s'tIo' (JP: 9-299 A-), which compk44,s crea-4ti' n; ah61estero1 oxidase and dfbes o te ct up.o1 9.iipoprqteins other than HD6L 14a-the presenee-qf a speci4. sprfactiant and to have Q~psta j S4l spch 9thet lijpoproteins, pv.e-" Il-. S X uring oho 1eserol i DL Thtlnitho )wss~;,s cpasiderably X2' iatic tftet'11t4L 4I#A Sv: 1401 t, *Iatb I 64xth TI, I~ sai-,acray i ~1 4 that) t3,05-320 which aia cosdreslto of th publshed ofater 4 flu be-~hd~-~e iMM~t~m~§iuttte d~es that, under toe t1 df HL-C in a se~i~r hyerip u icpant by tbe n4f ted enzyme due to~o~iv ere Itfhatis., to result Zita hig~r value compared w4~ttj~ ~tais~dd y e~1ita~vz~i~4d) xd4ceq When -thP 106"d, 01,14n1r. Acedaxnto i'Ceapi system,. -4 a.-t -,h ancdloj~ ,as -p .reol Itatd re#fe~ for' the -- b", , dra M *at4i P, s ac 4 ~~'retfltiw~~~~ i44&e jZ ywip b 1W -M ~~od& 4~e~' eh p~ecfr4A~at77 1!~&~ .......... qan~ 4akxw~3. £ tnge~4 asrfat~M 7",< 44, speqxil ,~~~~~ 1dp~~i.f~t~~ihutuigap~il t reaent a fied tant appicaion (JP 9-2A42). tis methd- an xtrmpl, 41h crreatin wth te cnveti~naa pr"Patonmehid;bu cmpre i masreet vlus with the~~~~~~~~~- rr"~iainmtti mto srcgie ohv .(~Q~5 a -0d~~3' WeS rgh*±a qed Xrolted I ~rI 1 Sif&Or U ET,~ *tMS'-ttiRLOGIAL SAAPIES)." prel~f~ti~ .e~bpd t.~b~&a1 ~sttutons- and the like, a itttpus ... ..... it ~ ~ ~ ~ ~ ~ -9I Psi~t~rr~ g~~nbb 1el~ nro£JI Yt ~ %WvN ~W ,_On all 2U'e~~a~t~b DIsoepfg"p r 'te nveton %q presen inv;B.........oceded.with..thoroug snvsti' Athqt for a aue hc a e epnil o h 6, znab.0

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Abbvtf~tt~tts ~r~&qdrpoflO 4kt bt flMTffS kIRY BWJNSEKI a u va .6~ cbglestatol, jm- the specfi iiptoin traction as ~ gu$~e -y-sija I S s± c A4*ichr jcs only upon a s~Ti J±~poeamn- Pbu as- 7ipJ.b A#Ai 41$gbr than the (Sa $$seqtk"P*f Icu as dtpa onh t Qd - C Wp&' a ,7

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0 Th'~s ~ f d ~ i 1auim~~o~as h octsv uprt~on Thphoetro f~ana en *ne . su~stl~ate- to i-< b h fouxesn ievhdo as prvie a* meho forb 7T -nttttng cholest.roJ& eel 2 a lipoptotein Ia ~ae. -which acts upon free di 41stero1 as a s tle oat-uppn the sazhple with tha1tplotein, -con ained tbO7e1#: aad then measu~ring the eOh4jiet%660f,.whh'xists ln-iasec±itic lippt n,.by, using ~ g~t&jtwibh acts upnAp$64l i~ teiLn only. Brief TpPdofliptui bfe'l ~

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4,d 'c AT= -mt!o n aqt.# ,-sa to a a, Al 4 '. bkdse is Gdi fltd. Ahs the enzyme which acts upon free cholesterol as a s ubstite. any enzymne can be used Insof ar as it.'acts, upon free chole t e r~a as a substrate. Ifllistalv. are cholesterol dehydrogenase and cholesterol oxidase. They can be of any or%~kit~nbhan a4 -or plant otgfl:, And, can aen 1t43i6ee. £ifrthr. they..xb ~~ri6fe r nidfA hmcly The e' "he is serf~~s~ tCO1to 10OO!U/mT ., with 0.1 ~P 6 tovW -&OP WO;Uu bfl4 WW.&~j rP kt co - asu c - Qe -to1as a-p~ sngt,ho s tqul f to ct Pphbsl 4 4ienAp4i - <> 1*. A fl~*A e-s Is ~ ~ ~ ~ ~ ~ ~~c cisttoact1a yt±iXrog w4e alho4 n parlticuar4 limiatione ist ;R!Psc tero ~~J, - -crnn te - ehom whch ads o re hlseo as af susrt n sue ntepesn fvnin olmtto 9 t ~'-*-. * Isconcentration adfl~~The~s Qabp dr yt.achiqve desired petfnintad Axtfn medse.. '4pqrd!;ngly. if it is desired to i~etbep 6nt 'eixiste X. predptennined ti-me, toA saVeI etratttn±time 1on~rer. it ~ny ~A~s~,-ot "for< exciusxve 'use In t~a~pnf 4ip~rt~s6t.r 14~~~~ t nti~<.crf l in at shorttm by'Va .. eiymeAna sJI _!0 'It £ acse. cipurrnt wt tp~i g e -fo +.I. SIX _1 is 2.t.l4"'trwdl ae.v tdsP% Alaeidp.

whetsixei such solts ane jnta1 dettVAtkP es exist - Among these, flufe'tle acid and, 4p nenri aid axe-khown as non-steroidal ant>T3J4flanJatory drugs, andfusid±&lird. and monensin are known as antWiotics.

Vj~zip~ng ~cb~4~11 j*ta apc7elerator. tt~ln reessary to s~itt bods&aen ativn and the 1i23te4' 4akin no nsd~t t 9 it b s >a l:propertjias. PH and4j6q16cistren-pth of -t sYs3temand the, kinds rcan be expAlrgrtfl .ttnnJi xW~ ~ tEis 'of a mne~rIfl systemu. lp s nqr a xo~t fbfiC can wqbtpsd-a&cetad in~~t 4 so iA t:i6vknikneeamc afd 2fr2N&.;0':t an it~t~ tli; acid. * )-,

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batet~ison tbfl$ . ix' aec%6tAtaro aso rnEsm fr1si radI4 rnitnol.each, pt at0.*tCI iia; , tgi ai a. it to u so surf~ ~ 4cfst (-ihixlsono -fitv~y usat i->t jev lic to lippoen-'rsra-tv~,poen uha suck 1 ~~~~ $tniois atbocssjpins, lectins ap d sin _91-nax polyanXOns to e*'tpntP not oqsn.-g",*"I tiof the specif ic fortlsuc4 htathe a64t0ii oqff enzyme is adjusted wi thquat -4a&jitt, 'ttf~y Off, Z asurement. At trtf) icont±e foi1i' ±~ge~~tkca rtbref~reagentsfo mda,'rhobesterp ixs+4.±lslci itoprqtemns in r.- -~ : - 4 W ]4@0oaenas cds-such as pruvc 40, JA; t. b* E~sf pr H rgulaion surfbtats gvin nosubsantal-------- on l~oorotinspreervaive, Prteis suh a albmin -atbdeatboic. . ~~s et ns, po&&actd. andCoplrszuQ s =#>~antip~in fl~afev........ *~, ~$E, * x~e12 ilopvns4$ rSobnTDD 4lrs. 6.9., Trihdprls =ta ent.- AIetro acpO bas pbienaztne, .m~thsia~ae~ an< eiluctive color 0ev6iopetrs such as ndltcblue tort A&44um 2n the preaelt.t incittn hoisestseat i~hick eitws in i..otin ale me a fre j -f~ellY 1csre ~he, abbe s, 3.l 64 em~itatte tl~e -P- de Mbi -acts upon s~c~~c 1popxte~r us~g in it the ~%t it~irtt Y. ; rXV fi o4~%4totai%#*I-, masu fltresea A-60" (trade nane. -product of Rao Corporation) , while eXanples 13 -14 Pxk %?4 od ft -efatns 1eay,% tm. ~hylehe. all..e~n oxiaZ bny ethers, can include B~nagn6t' (trad 'n~jne product. af ao Corpoation). As'an atern~ativemeh thl 'e. is amiehod whithrmakes ~$d Ia 2 ' «et' eapZ . qp eclo -p F;-. - - -t - - -' ,4 :Me, Atttof~ c~d~ri sjt~te'tn h~gefu Ze~~Per "M ItkTG. t -~~1 . h 't- .4 -4' V - ;&~-t 'j"~ ; ~ 2 it~bn*j'W''w fs aoge nec~kr' t4 sseW 'a.e proffni (41*u~dn' tol tPrse..gs. surf ' cas sats 41 at. p~oltyefle' 1D~~d eijir ~i~$~ g1eznsonate 6wait I~db~ Thed ; D16amo~3!t of-'T thA urfactanit to US usp ~e eedn Jx.ft S cogp~n& Thsurftetant may 4p ta'. - n bAtf ea AA*$;f, 9t2p,*$~V e~ ltp t " c~i!y. r(NAnD wit a, 4a-p, an ' a a. -hlieo mesirn ±4.'UUon IhltC 61*1esterolt~'~ -- trw C." -blsed aiae ite ~ihcrpi 1±p5 miiffolto4'uest er Z i AAA4it a:sa e and then achclesterre 1 ri~ tgft 1cb.,compriSes 9bo1s~I esterasg6 I &mtoWiltprises mixingasml and cjboe sterol l ds-teehrwt peroxidasie, 4-am-mo an'tip~zp -Or -bat dase,4ten-atd& ~a cholestetdl1-measuring 1, g~g 4 .1 TWlhcmks hA* vr~ itta.a~ja amethodwb ich ox heA pr07Qas~. 4 4in6nirn) An t ahig "piIa enind eito1 oxseas t'.hpX ,rr to- hetc e tel, whcoxiss it iss Apfeesa m e 1.e in~~ ~ a ~iaon n1,or~~nsr _fce, fis tec*l~rtrama recin n hntGbgterato system. Int a tti hchteezm ct nyuo h 16it l-a gpea-trIt 1ipoprotalnwt, be measr .6S that most cholesterol (.ifr e A :ceoatero-1 4 qpt ckfeied bVkro)forming the Jtip6']..vdtelh can bet qnatiatd. Furter, op~tcu~x flnit~to~imposed on the method tfS iI~ly ddtesting vh61esterot, ttt tbie addition of such fo 4>l-esj rlmaIsim en I m e rsrt Is Possiie touse, afottoar .4. I Atkfdtion is conducted by td a'bintbiomety -n 131 a tv-nmr pir I iiei t. 4 ;r diaphorase or ani ork 14 ;Pei,>i tdch P direcotly detett(~. he oefzremay-bf-a.V pi & :ba coenk'ina-.bbing a e. a ext~z with ease, ~% r issita& tw jff.h WnS 4P-' * ati A. i 6S inastpf 1±poprq-in, compr44in4. .tb4 fo0*iaX~u rea4gents <1) and (2): (- a first rpagent comTprisn "6b1esterol oxidase and ahydrvgenperoxide con~tingnsdhst-anc tanfldurtbgr comprising a reaction accelerator In some instances): and -17 upon qit 4-i~ ~ , Aiostgiol esterase, and LPqA uantittpiun it _f or. chqitrl in a sD6C~fic i±~pr~i. 6~pij thei~0q4 rea 1si). sand & 13 ~ Xis~rant dcinrn4i4 cho1-eptlra ggaas 14.. Fort-. -7< Its;i7 -SP, a qq34 iaosl' pe cholesterolod4as oant n ' at l hpl- rp uitatle te~ caaae oplr uha 4-a2bi.t-yie n ~t~ i w: r-edetle ~Includ-ing hydrogen juI&p StIxese. acrniler 4 snob'ft 4,-Tax-inoantipyrine and a bydrbqew sdvnorentbdeve is a colot-when reaced;in cmbiat'bff.~i~i~ide. n are usable -tr*a4t-nt t'p 1yent aIo 'tdsweiyfqrredit usp Ohyly 0- a - -Cn i21I9"qlUnl a ndA-c i;evbcl TMpto~ dldS' j~ -trX-t W- t an d. - i;eo an- i tt .(;2 a seon ne-get ofpism'. . a ,wihat upo th specif .c I ~ sk#~ 7%~ i nla~ih erietxa ,.A qun.Iit A-a:fb hbtotli pcf I~ ~~r , prrti.cmpiig hi oloil,2egis()ad() 4 '419 and a cq zzyie(n44 rt~rq.6dwp4 ing a reaction 4cc,61erator In scm& ntns ~ 2)a se~rorld raage&nt :coqpri~1ns a substance which acts li~0 t1e ~ qlfit ipOrotIn ho~ ch1eter3. X olAse, tflA ~ei'~n, -a spekofi lip~n. mtnpz au.dts an 2j.t~acaa an2 ip i a~ ie~sc Ain mq - ~ *~ ~.-~ 04 * uq "It fNI& tflkr yxn. idstI~teo1~terWpt (d1.a firsti reaentoprsli chl wq016hichgaa and~~~~~~~ ad conzm (adfrhr-piig erinacl -) -~ 20 A s3coa ieXgent conrsn ~substance which acts A,3) a thtra reagebt, coup 4alb.Qhpilesterb.l esterase. 4&U-A. .banti- .t ifl;l ki - n ai j)ecif ic ±7, 9C 6 (),afls reagent- -ohkil - a sbfac-W.-c b K.'a Upon th spepf c lp~protlr Orl IdJjrasi ~~fl~~4jfl bholl.asttr4>o's0da a f<- irf dphy-'* 0"-e caent ~ ~ ~ ~ 4 joe, an -onyh edi a&~c o~~n a -4* acceier~tor in some insta4+1; aid a eod ~~ ht cop~4W, -substa ce. which acts '4~9n te svcitn 1pprqtgip only,. qhQes-re1 estet4pse. and aj&6 th4djve]pe.te ±rntb .... t stibsa~S nn A sutIsanoe zh~vs c ecd~6n I L' $, tbtreqffibOtb Chofe1 -1 a VIC~e f 1,141,, te n a IM& WI In eab ~4~~$&teet41 .. ....... * ~the 0 6 b eef&b ~sbfret0teintens "at c4Ireap1 in ttiepr tre'atmen stagenWhh th req ptil)' sadde4., from the Iiensity of a colo aatnd- he rleaget(-)ote reagent* (3). As an alierjpatlve. i-t nit4o, be posble: to add befoehad te abkane. which p thi4 reacti-oh product, to the reagent (f) afidsubseqnen to con~uiuptioi -of 22 & ThistasA.a4dition ot a Suvt*'abce. -wlq iae b~btnee which consiamas tbox rt404 - 40- t'b s vtAZ) &he Traageht (IgJ tsS4ferteAd IfY~% 9,. 1Ja t)CC n'_T nl2A. or tbIL' ,,% ~ ~ ..... ..... ~&) l h 44. to A:!'t cqp k)tT 4, lp er4 1,- .. peseo as a§-. $ -B~t~t atlnthqda te eroafr if tb: peroodas~~~e, a-oo o~opj n h ~otloo§o ;2 choeseroestsVo$e, p&rOxi&4se, a c]rd ~e and the lik , j~t is qbvou 1Y bosi~ie oAbtocut- ad Vantazgeous effecot &11cA-r tvat the4 aplqvnt . of~ tha e1et be uesi act~g ~ . - 4tol as a rop, ttn fbEthe fnzyif44 3 .reactaoh -t on te 4hfti_' 0110 ~4*~~ta set.P - -v y-<. .+.Atisn 4-~ -- b ~~~-v S. -tp -- *w xw Mb% c Ih I A S. over jnoaured vaue also.2~ Or, UPX~t, t~he atlnvdeftaewb 1fleige-o m*easiaring nafts tseify.uet~adtelk 24 msui&' va 44g 'a vt~#oIAakn-ith, 'tfiose dbtatnA oyth qo jnOn c4q~t~tb miithad Can be obq iel?!Tl~S1~r~~pe q npAw thgh-trigiy.*erlde - a 1~acw-Sf lie~ethoa o te prtesen-teq tn& -1 n Ko .t;q -; Re*~1n1ItattIy~ ai b t * "*&rw y --. q v~4%it~ia~eh 4 xx a 7 -trbctot~~- - rgt Iy.5r Jn method)n~a * k~ ~ 25 PH68) 30. r Sr vo to lsap aeut;; C&ut.t2Sdg.lU ad03 Ch-i s. 24-A) W..1- m"i-~ tesml '0 AL)AiGd b rei~nt&AaSuvf ,O-gefoh4mit' all ~ ~ ~ .'a-i .4 aTJwaclsto 40ktb h &" x. *~b e~tl qawrt~atom ea2 t (.3 mlVtL coop e - owf 16as C~fl4D~b26 ib if-&L Q4 ~!4~'~$~(AiM$AA~ I /27 4oil I~ s readxul .' as~ 4tjAt the invention _,ep te iA50sii6ii~ dnucv or te like, showed 'an' eatreI4 good cOr-rnt-tn 4~f '~~ conventional _'Atia e& tb ±iwrmtton met-bod -~~0u0±~U~h) EXU±* ret that lWm"t -P fkrs Teaget W~ML Ici 4 bct '" Of'opb0oco. - A - -S A~ SS. -s C A - - - ;t A -~ 4. 4L ~j 42~ I & * . *.~; ,s ~ n C Cl ~LK;i.

ro .. showed - '-4.';-- V )at "ute,% th4ufi- a *itt a s~tt rtb 1*~ sowe ito ;Ot -s-L t h -#la Im i-V;. .e 2 4sI oIt~gn~bgth peflt ~ietton$ihrepet bth s33e 419 344 I ' k

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L ~ ~>t~ t! > 's s-t t -~~~~~t CS f1tStStgA~ 'QcP1T4.U wAs d- hpwpf, 0 44-" ............. rrhaszi eA4316 Rlyieft 21'Ct~-~Ve '~lsefloxidase 2 F .Z~o cetq A.&4emc o, ~ ~os~44~ftY Iq' atreatv absor!bAncas of the four stzj~Ies. are presented In Fl-G. 3, in 37 which "COD" stands for cholesterol oxidase. 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 5 confirmed that the use of the reaction accelerator makes it possible to reduce the amount of cholesterol oxidase to be used. 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 10 upon free cholesterol as a substrate. 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. 15 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 20 general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

Claims (11)

1. A method for pretreating a sample, which contains various lipoproteins, prior to measuring cholesterol existing in 5 specific one of said lipoproteins in said sample, which comprises causing an enzyme, which acts upon free cholesterol as a substrate, to act upon said sample.

2. A method for pretreating a sample, which contains 10 various lipoproteins, prior to measuring cholesterol existing in 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 15 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 20 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 25 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. 30

5. 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, and a reaction accelerator, which is selected from flufenamic acid, mefenamic acid, 2,2',6',2"-terpyridine, tiglic acid, fusidic 39 acid, betamethasone acetate, monensin or mevinolin, 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 5 lipoprotein only.

6. A quantitation method according to claim 4 or 5, wherein said enzyme is cholesterol oxidase and/or cholesterol dehydrogenase. 10

7. A quantitation method according to any one of claims 4-6, wherein said specific lipoprotein is high density lipoprotein. 15

8. 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, 20 to act.

9. A method for quantitating total cholesterol, which comprises causing at least an enzyme, which acts upon free cholesterol as a substatrate, and a reaction accelerator, which 25 is selected from flufenamic acid, mefenamic acid, 2,2',6',2" terpyridine, tiglic acid, fusidic acid, betamethasone acetate, monensin or mevinolin, to act.

10. A method according to any one of claims 1, 2, 4 and 5, 30 substantially as herein described with reference to any of the Examples.

11. A method according to claims 8 or 9, substantially as herein described with reference to any of the Examples. 35 40