CA1138312A

CA1138312A - Composition and method for determination of cholesterol

Info

Publication number: CA1138312A
Application number: CA000190165A
Authority: CA
Inventors: Peter N. Tarbutton
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1973-03-01
Filing date: 1974-01-15
Publication date: 1982-12-28
Also published as: AU467471B2; FR2220062B1; HU170194B; NO146558C; CS176267B2; NO740693L; ES423559A1; BE811728A; FR2220062A1; NL7401974A; DE2409696C2; JPS502594A; NO751299L; AR201939A1; IT1013052B; DE2409696A1; NL181368C; BG26959A3; US4186251A; GB1463133A

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention relates to compositions and methods for use in determining free and total cholesterol levels in fluids such as body fluids. A test for free cholesterol is disclosed based on the determination of hydrogen peroxide released through the action of a chemical system having cholesterol oxidase activity on free choles-terol. A preferred means for determining the released hydrogen peroxide involves the use of a substance having peroxidative activity and an oxidation-reduction indicator.
The addition of a chemical system having cholesterol ester hydrolase activity to the free cholesterol test composi-tion provides an integral test composition for the deter-mination of total cholesterol.

Description

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BACKGR~UND OF THE INVENTION
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Cholesterol is found in nearly all plant and animal cells, either in its free form or in an ester form. Free cholesterol refers to cholesterol in its unre~cted state (~5 - cholesterolp or cholest-5-en-3~-ol). Total choles-terol refers to the sum o~ free cholesterol and its ester derivatives such as ~he linoleate~ oleate, palmit:ate, 1 arachidonate, palmitoleate, linolenate, stearate, and myristate esters. Cholesterol is found in constant amounts in serum under normal conditions In general, 25~ of the total cholesterol level in serum is free cholesterol while the remaining 75~ is in the form of ester derivatives It is fairly well established that the total choles-terol content of whole blood is directly related to certain maladies in man and animals. Among the many maladies which have been found to be related to total cholesterol levels in blood are hepatocellular diseases, thyroid metabolism disorders, biliary obstruction and perhaps most importantly atherosclerosls and other vascular dif-ficulties. Until the last decade it was customary to determine th~ total cholesterol content of whole blood, but it is now known that the serum level is altered by factors whicll do not affect the red cell level. As a result 7 most clinical determinations of both free and total cholesterol are per-formed on serum rather than on whole blood.

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As the clinical significance of free and total choles-~erol levels became increasingly accepted, the need for rapid and reliable methods or de~ermining cholesterol in fluids resulted in research efforts which yielded numerous new procedures and modifications of basic conventional procedures. A general treatise of the vas$ methodology available in cholesterol determinations can be found in American Journal of Clinical Pathology Vol. 25:1(1955) at pp, 433-46 In general, the presen~ methods of determin-ing total cholestero:l in fluids, particularly in serum, 3~

l involve the four basic steps of extracting the free choles-terol and the cholesterol esters from the fluid, saponify-ing the cholesterol esters to free cholesterolp isolating the free cholesterol which results, and determining the isolated free cholesterol, The generally accepted or stan-dard method for determining total cholesterol is the method of Abell 9 et al. in the Journal of Biological Chemistry~ Vol. 195 ~1952) at p. 357, In this method the cholesterol esters are saponified by incubation with alcoholic potassium hydroxide, and the free cholesterol is then extracted with petroleum ether. The isolated free cholesterol is determined spectrophotometrically using a modified Liebermann-Burchard reagent (acetic anhydride, sulfuric acid and acetic acid). This standard method and the numerous cther available methods possess the clini-cally disadvantageous characteristic of being highly complicated procedures which use numerous reagents and which are extremely time consuming.

SUMMARY OF THE INVENTION
It has now been found that test compositions and methods for determining both free and total cholesterol may be provided based on the catalyzed degradation reactions of free cholesterol and cholesterol esters, The present -invention discloses a test composition for determining free cholesterol in a fluid comprising a chemical system having cholesterol oxidase activity and means for determin- -ing hydrogen peroxide, A particularly preerred means for determining hydrogen peroxide comprises a reagent system 3~2 1 which includes a substance having peroxidative activity and an indicator material which is oxidized in the presence of peroxide and the substance having peroxidative activity and which changes color thereupon. It is also preferred to include a buffering material which is capable of main-taining a p~l of between about 4 and 9 when contacted with the fluid sample. A surfactant capable of solubilizing substantially all of the free cholesterol present in the fluid sample is also preferably included in the test compo-sition for free cholesterol.
A ~est composition for cletermining total cholesterolin a fluid is also disclosed comprising a chemical system having cholesterol ester hydrolase activity and the test composition for determining free cholesterol described above. A preferred chemical system having cholesterol ester hydrolase activity comprises the enzyme cholesterol ester hydrolase and a biliary enzyme co-factor therefor.
Where the test composition is modified to determine total ~ cholesterol, the buffering material which is preferab:ly included is capable oi maintaining a pH of between about 5 and 8 when contacted with the fluid sample. Also 9 the test composition may include a bacterial inhibitor, such as sodium azide, to prevent interferring bacterial growth.
The disclosed method for determining free cholesterol in a fluid sample comprises contacting the fluid sample with a test composition described above for determiTling free cholesterol and observing the response which results, It is preferred that the response which results be allowed to develop for a predetermined period of time before observing such response, Also, it is preferred to contact 3~

l the mixture of the fluid sample and the test composition wi~h an acidic substance in order to enhance the develop-ment of the indicator response. Semi-quantitative results are obtained by observing the resulting colorimetric response visually and comparing such response to a standard color chart. Quantitative results are obtained by observ-ing the resulting colorimetric response with instrumental means. Similar method steps are followed for the deter-mination of total cholesterol in a fluid sample; however, the test composition used for this purpose is that which has been previously described for use in determining total cholesterol, A method for preparing an extract containing choles-terol ester hydrolase which is substantially free of proteolytic activity is also disclosed and includes the steps of combining a substance containing cholesterol ester hydrolase, such as pancreatin, and a first aqueous liquid which may comprise an inorganic salt in solution, separat-ing the liquid and solid phases which are formed thereby, passing the separated liquid phase through a column con-taining an adsorbent which comprises an adsorbent-active diethylaminoethyl portion, and passing a second aqueous liquid comprising an inorganic salt solution through the column, which inorganic salt solution is capable of elut-ing cholesterol ester hydrolase from the column. Thecolumn adsorbent preferably includes diethylaminoethyl cellulose. A substance capable of protecting cholesterol ester hydrolase from proteolytic activity is also prefer-ably included in both the first and second liquids.

1 BRIEF DESCRIPTION OF THE DRA~INGS
The sole :Eigure of the drawings i5 a graphical representation showing the composition of the fractional components of the column effluent using the cholesterol ester hydrolase purification process described in Example 4 DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the context of this disclosure, determination means either qualitative, semi-quantitative, or quantitative ana-lysis unless otherwise specified The present invention is based, in part, on the :Eind-ing that hydrogen peroxide is formed in the reaction of free cholesterol in the presence of molecular oxygen and certain chemical systems having cholesterol oxidase activity As used herein, cholesterol oxidase activity refers to the catalysis of free cholesterol through an oxidation reaction which forms hydrogen peroxide as a product. Tlle reaction between such chemical systems and . free cholesterol and the subsequent preEerred means for determining the hydrogen peroxide which is :Eormed using reagent means can be represented by the following chemical equations;
cholesterol free cholesterol ~2 acXt-iiv~ Q4-cholesten-3-one H 0 ~ indicator material ~ oxidized indicator

2 2 actlvlty (color change) It is contemplated that free cholesterol can also be deter-mined by determining the ~4-cholesten-3-one released as well, - - -

3~

1 It has also been found that when the above system is used in conjunction with a chemical system having cholesterol ester hydrolase activity wh:ich is effective to convert the cholesterol esters to -free cholesterol, a means for determining total cholesterol is provided, As used herein, cholesterol ester hydro:Lase activity refers to the catalysis of cholesterol esters to free cholesterol through hydrolysis. This catalysis can be represented by the following equation:

cholesterol ester5 c~lleStetroil ~ er hiydrolase~ free h ~ fatty acids A chemical system having cholesterol oxidase activity can be obtained by a variety of means and in numerous forms, Usually such a system is obtained through the extraction of naturally occurring materials such as microorganisms which may be exemplified by the following; Various Mycobacteria such as Mycobacterium rubrum, various Nocardia such as Nocardia erythropolis~ and various _ . ~ ~ ., , Streptomyces.
A particular chemical system having cholesterol oxi-dase activity is the enzyme choles~erol oxidase obtained through an extraction of Mycobacterium rubrum according to the method disclosed in Methods in Enzymology Vol, 1, ed, Colowick and Kaplan, Academic Press ~New York~1955) at pp, 678-81, The preparation may be briefly described as follows, Wet packed cells of Mycobac~erium rubrum are ruptured by a conventional method such as by grinding in sand, The ground material is suspended in a buffer such as a phosphate buffer and centrifuged to remove the 1 insoluble material. The buffer which contains the soluble protein, of which cholesterol oxidase is a part, is then treated to remove interferring low molecular weight materials such as by dialysis.
A chemical system having cholesterol ester hydrolase activity can be obtained by a variety of means and in numerous forms. Usually such a systam i5 obtained ~hrough the extraction o~ naturally occurring substances such as animal or human pancreas, liver, and inteStines, Commer-cial pancreatin is particularly useul; however, due to its high proteolytic activity content it must unclergo purification to remove such activity as will be discussed more fully hereinafter According to the present invention, the determination lS of free cholesterol using a chemical system having choles-terol oxidase activity is dependent upon a means for determining hydrogen peroxide formed by the catalyzed reaction Such means which can be used include all known techniques for determining hydrogen peroxide in fluids A particularly preferred means involves the use of a re-agent means comprising a substance having peroxidative activity and an indicator material which is oxidized in the presence of peroxide and the substance having peroxida-tive activity, and which yields a colorimetric response or color change.
Substances having peroxidative activity comprise such naturally occurring peroxidases as horseradish peroxidase and potato peroxidase, Other substances having peroxida-~ive activity include certain organic materials such as normal whole blood9 red blood c~lls alone, lyophilyzed ~ . . . .

3~2 1 whole blood, urohemin~ metalloporphyrins and so forth.
Certain inorganic compounds are also useful such as iodide salts and molybdate salts, iron sulfocyanate, iron tannate, :ferrous ferrocyanide and potassium chromic sulfate.
Indicator materials of the type disclosed herein include those which are oxidation-reduction indicators having an oxidation-reduction potential appropriate to detecting hydrogen peroxide in the presence of the substance having peroxidative activity. Such indicators include o-tolidine, syringaldazine, vanillinazine, the combination of phenol and 4-aminoantipyrine, 2,7-diaminofluorine, ben~idine and derivat.ives of benzidine such as o-dianisi-dine.
In the determination of free cholesterol, a buffering material is preferably included in the composition and is preferably capable of maintaining a pH of between about

4 and 9 upon contacting the fluid sample to be tested, Such a pH range assures ~he stability of the components of the test composition especially where the enzyme choles terol oxidase is used. The optimum pH range for the detection of ~ree cholesterol using cholesterol oxidase is from about 7 0 to about 7.2. Where a chemical system having cholesterol ester hydrolase activity is included in order to determine total cholesterol, the pH range is preferably between about 5 and 8 with an optimum at 6.6 where the enzyme cholesterol ester hydrolase is used. Any buffering materîal may be utilized so long as the appro-priate pH range criteria i5 met and the effectiveness of the test composition in detecting free cholesterol ar total cholesterol is not substantially impaired, -~ 3~ ~

1 A surfactant capable of solubilizing substantially all of the ~ree cholesterol present in the fluid sample is preferably included in the test composition for free cholesterol since free cholesterol is only slightly soluble in certain fluids such as aqueous solutions, Where cholesterol ester hydrolase and a biliary co-factor there-for is included to determine total cholesterol, a surfac-tant is not desirable since the biliary co-factor itself acts as a solu'oilizing agent, Thus, the use of cholesterol ester hydrolase and its biliary co-factor eliminates all need for a solubilizing agent for free cholesterol, thereby simplifying the test composition, Surfactants which may be used include both ionic and non-ionic surfactants and various other detergents which are well known as solubiliz-ing agents, Bile salts are paFtic~larly useful, Thosesurfactants which function both as solubilizing agents for free cholesterol and as co-factors or cholesterol ester hydrolase include cholic, glycocholic, taurocholic, and taurcdeoxycholic acids and their salt$. The sodium salts of these compounds are particularly useful.
The disclosed method for de~ecting free cholesterol or total cholesterol basically comprises con~acting the fluid sample to be tested with ~h~ appropriate test composi-tion described herein and observing the response which results, The response is pref~rably a colorimetric response or a color change which is obtained through the use of indicator materials described hereinbefore. The appropriate test compositio~ ~ay be contacted with the fluid sample in a variety of ways9 including mixing the : . :

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1 fluid sample with a solution, such as an aqueous solution, which contains the test composition, Also, the test composi-tion may be in a dried state, such as a lyophilized state, and added directly to the fluid sample or rehydrated prior to mixing, It is contemplated that the test composit.ion may be incorporated with a carriar member, such as by impregnation of adsorbent pads, to form a test device.
Such a device could be dipped into the fluid sample in order to bring the test composition into contact with the fluid sample.
It is preferred to allow the reaction between ~he components of the test composition and the fluid sample to proceed for a sufficient period of time to promote the development of a sufficient colorimetric response for reliable and sensitive results~ This incubation may vary from as short a time as about one minute to a longer period such as overnight. Using the preferred means of detecting hydrogen peroxide, the incubation is usually between about 0.5 hour and 1.0 hour at between about O~C.
and 50C, and preferably at about 37C, Also~ the addition of an acidic substance such as sulfuric acid to the test composition and fluid sample mixture has been found to enhance the color development using the preferred test composition.
Semi-quantitative results are provided where ~he response is observed visually and compared to a standard color chart which illustrates the responses developed using known concentrations of free or total cholesterol, 3~

l Quantititative results are provided using spectrophoto-metric or reflectance means for determining the response which is then compared to a standard curve, In the present invention the phrase "chemical system having 'certain' enzymatic activity" implies all chemical compounds singularly or in combination which accomplish the stated enzymatic activity, It is not known whether discrete molecules or enzymes are involved in the activity desired, since the composition is prepared from extracts of naturally occurring substances7 which extracts exhibit the desired activity, For example, cholesterol oxidase activity may be provided from an extract of the microorgan-ism Mycobacterium rubrum and it is not known whether a ,, . . . ~ . . _ single moleçule or enzyme is invnlved, The activity may involve certain enzyme co^factors or more complicated enzyme systems, It is not necessary that enzymes be involved, since the system having cholesterol oxidase activity provides only catalytic activity and does not provide a reagent which is either used up in the reaction or ac~ed upon to produce a product, Thus, the signifi-cance is only in resultant catalytic activity and the present invention is therefore described in terms of a chemical system having cholesterol oxidase activity, The same situation applies ~o cholesterol ester hydrolase activity; however, more is known concerning cer~ain chemical systems having this activity, For instance, it is known that the enzyme cholesterol ester hydrolase requires an enzyme co-factor derived from bile, Those biliary enzyme co-factors include cholic, glycocholic, taurocholic, and taurodeoxycholic acids and their salts, 1 It is necessary that where the test composition includes proteins such as enzymes in the catalytic chemi-cal system~ the test composition be substantially free of proteolytic activity; that is, free o~ substances capable of promoting the degradation of proteins. If proteolytic activity is present the proteins are destroyed, thereby impairing their participation in the catalytic activ:ity.
The enzyme co-factor for cholesterol ester hydrolase inherently protects this enzyme from proteolytic destruction in addition to its function in the catalytic activity of the chemical system. Cholesterol oxidase and peroxidase are no~ so protected. Thus, where the test composition includes these substances, proteolytic activity must be absent In the present invention the combination of extracted cholesterol ester hydrolase with such proteins as cholesterol oxidase and peroxidase requires a new method of preparing an extract containing cholesterol ester hydrolase which is substantially free of proteolytic activity Such a method is provided comprising the steps of combining a substance containing cholesterol ester hydro-lase activity and a first aqueous liquid which may comprise an inorganic salt in solution, separating the resultant liquid and solid phases, passing the liquid phase through a column containing an adsor~ent which has an adsorbent -active diethylaminoethyl portion, and passing a second aqueous liquid comprising an inorganic salt solution through the column, which inorganic salt solution is capable of eluting cholesterol ester hydrolase -from the column It has been found that, at an appropriate'inorganic ~3~ 2 1 salt concen~ration, cholesterol ester hydrolase has a greater affinity for adsorbents ha~ing diethylamino-ethyl portions than do those substances having proteolytic activity, A preferred adsorbent is diethylaminoethyl cellulose, althowgh the diethylaminoethyl portion may be attached to a skeletal or supportive portion other than cellulose, Other supportive portions may include the various derivatives of cellulose and so forth.
The first aqueous liquid may or may not include an inorganic salt in solution. Both the first and second aqueous liquids preferab]y include a buffer in order to maintain a pH environment for cholesterol ester hydrolase which is not deleterious to its catalytic activity, The pH of the buffer is preferably between about 5 and 8 with an optimum at a pH of 6.6. It is further pre-ferred that the inorganic salt itself perform such a buffering function, Particularly useful first and second liquids are potassium phosphate solutions having a concentration of up to 0.05M
and betweerl 0,15M and l.OM respectively. Inorganic salt solutions having ionic strengths equivalent to potassium phosphate solutions within the ranges recited above may also comprise the first and second aqueous liquids, Examples of inorganic salt buffers which may be used include acetate~
citrate, tartrate, and phthalate buffers. A particularly useful procedure is described in Example 4 which follows~
It is also preferred that both the first and second liquids also comprise a substance capable of protecting cholesterol ester hydrolase from proteolytic activity9 such as cholic, glycocholic, ~aurocholic, and taurodeoxycholic acids and ii ~ 3 ~ 3 ~ ~

l their salts A bacterial inhibitor may also be included in the first and second aqueous liquids to prevent inter-ferring bacterial growth. A particularly useful bacterial inhibitor is sodium azide.
Fluid samples which can be assayed using the present invention preferably are aqueous solutions where the pre-ferred reagent means for d0tecting hydrogen peroxide is used. Such fluid samples include body fluids such as serum, urine, plasma, and so forth, as well as other solu-tions containing free or total cholesterol such as those prepared in laboratory or manufacturing processes, The test composition may be in the form of a solution such as an aqueous solution, a dried or lyophylized powder, or may be incorporated with a carrier member in a dried state. The components may also be stored separately and combined at the time of use Suitable carrier membeTs are capable of absorbing the fluid sample being tested and include bibulous paper, cotton, polymeric pads, porous plastics, and so forth. The dry test composition may be impregnated in the carrier member, coated on the carrier membeT~ chemically bonded to the carrier member, physically entrapped within the carrier member or any combination thereof, and so forth. The carrier member may also be attached or o*herwise associated with a holder or support The present invention provides a means of determining both free and total cholesterol which is simple and rapid Wit}l respect to the prior art means. It is eminen~ly suit-able for çlinical use by personnel having a minimum amount of technical skill and provides an inducement to increase ~he number of cholesterol determinations requested by :~3~3~

l physicians. It is particularly surprising to find a work-able assay system as highly complex as is presented by the preferred means for detecting total cholesterol, In its preferred embodiment this means comprises the enæymes cholesterol oxidase, cholesterol ester hydrolase, and peroxidase~ an enzyme co-factor, and an oxidation-reduction indicator material, yet does not require elaborate control of assay conditions in order to give quantitative results, The present invention will now be illustrated by the following examples~ but is not intended to be limited thereby, ...1.___~.. _A . ..
The formation of hydrogen peroxide from the contact~
ing of a fluid sample containing free cholesterol with a test composition of the present invention is described herein, A cell-free extract of cholesterol oxidase from Mycobacterium rubrum was obtained using the procedure described by Stadtman in Methods in En~ymology Vol, I, ~ . _ . ~
ed, Colowick and Kaplan, Academic Press (New York71955~ at ppr 678-81, Three solutions were prepared having the following ingredients:

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Bolled Enzyme Enzyme Cholesterol Control Blank solutlon solution solution ml, _ ml, ml, ~l Potassium Phos-phate buffer (pH 7,5) 0,2 0.2 0,2 cholesterol oxidase extract (0,13 units/ml) 0,8 0.8 cholesterol oxidase extract boiled for 15 min, - - 0.8 acetone - 0,1 1% cholesterol in acetone 0,1 - 0,1 peroxidase/o-di-anlsidine 1.0 1.0 1.0 A unit of cholesterol oxidase enzyme activity is defined herein as the amount capable of catalyzing the oxidation of one micromole cholesterol to one micromole ~4-choles-ten-3-one and one micromole hydrogen peroxide per hour. A
unLt of cholesterol ester hydrolase enzyme activity is defined herein as the amount capable of catalyzing the hydrolysis of one micromole of cholesteryl oleate per hour.
The peroxidase/o-dianisidine solutions contained 1 mg peroxidasa/ml and was saturated with respect to o-dianisi-dine 2 HCl, These solutions were incubated at 37C. -for one hour after being purged with molecular oxygen. After incubation, 1 ml of 65% H2S~4 was added to each solution.
The solutlons were filtered and the absorbance at 540 nm determined. The control solution and the boiled enzyme blank gave optical densities of 0,080 and 0,020 respec-tively. The en7yme-cholesterol solution gave an cjptical ~ . .

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1 density of 0,540 confirming the procluction o:E hydrogen peroxide as detected through the oxidation of o dianisidine.

This example illustrates the use of the test cornposi-tion and method of the present invention for the deter-mination of both free and total chol.esterol, The following reagents were prepared:
Cholesterol ester hydrolase - 32 units/ml, 9,4 mg protein /ml in 0,lM potassium phosphate buffer ~ph 6.6), 0.28% sodium taurocholate and 0,01% NaN3 Cholesterol oxidase - 4 units/ml 3 16 mg protein/ml in 0,lM potassium phosphate buffer (ph 7,0) and 0,01% NaN3 Peroxidas~ solution - 0.1 mg/ml, in O.lM potassium phos-phate buffer (pH 7.0) and 0.01% NaN3 o-dianisidine - 1 mg/ml in 90% ethylene glycol monomethul ether Buffer - 0,lM potassium phosphate buffer (pH 6.6), 0,28%
sodium taurocholate and 0,01% NaN3 Serum - Serachol* standardized serum lot #1560031 *Trade Mark 3~3~ 1 1 These reagen*s were mixed as follows:
Total Total Free Free Choles- Choles- Choles- Choles-terol -terol terol terol Blank Sample Blank Sample ml. ml. ml. ml.
._ Cholesterol ester hydrolase 1.0 1,0 cholesterol oxidase 1.0 1.0 1.0 1.0 11 ~, peroxidase 0,8 0~8 0,8 0.8 o-dianisidine0,2 0.2 0.2 0.2 buffer - - 1.0 1,0 serum - 0,05 0.05 I, *The serum used was a standardized serum containing 346 mg /100 ml total cholesterol and 74 mg/100 ml free cholesterol prepared by Warner-Chilcott Laboratories, Morris Plains, New Jersey under the tradename*Serachol. The four solutions were then incubated at 37C. for 1 hour and at the end of that hour the optical densities (OD) at 450 nm were found to be as follows:

total cholesterol blank 0.245 total cholesterol sample 0.845 free cholesterol blank 0~140 free cholesterol sample 0~280 Analysis of the data shows that upon subtracting the blanks from both the free and total c~holesterol a ratio *Trade Mark ~l~L3~3~

l of 0.233 is obtained between free cholesterol and total cllolesterol, This closely matches the known ratio of 0,214 in the standardized serum, This example illustrates that increasing levels of free cholesterol yield increasing colorimetric responses using the preferred detection means for free cholesterol.
0,5 mg/ml and 1,0 mg/ml free cholesterol standard acetone solutions were prepared, 0~5 ml of an enzyme solution containing cholesterol oxidase 0,13 units/ml and 0,5 ml of a peroxidase/o-dianisidine solution containing 1 mg peroxidase/ml and saturated with o-dianisidine 2 HCl, were each added to 0,05 ml of the standard solu-tions and 0,05 ml of a control solution comprising 0,05 ml acetone. The three solutions were purged with molecular oxygen and incubated at 37C, for 1,0 hour at which time l ml of 65% H2SO4 was added, Optical density (OD) was measured at 540 nm to give the following results:
OD
control 0,07 0.5 mg/100 ml 0,12 l,0 mg/100 ml 0,21 This example relates to the preparation of extracted cholesterol ester hydrolase which is substantially free of proteolytic activity, A solution of 10 g, commercial pancreatin 4XNF ~pre-pared by the method of National Formula y XIII Amer:lcan 3~:

1 Pharmaceutical Associat.ion3 Washington, D.C., 1970) in 100 ml of 0 05M potassium phosphate bu:ffer (pH 6.6), 0.28%
sodium taurocholate, and 0 01% NaN3 was stirred at room ~emperature for about 30 minutes. 1 m:l of this pancreatin S suspension was mixed with 2 ml of 0.05M potassium phosphate bufer (pH 6.6) 0.28% sodium taurocholate, and 0 01% NaN3 and assayed for enzymatic activity using the procedure of Example 5. The remaining volume of suspension was divided into four equal volumes and centrifuged for 30 minutes at lS,000 rpm and 4C. The supernatants were combined and 0 1 ml of this buffer extract was mixed wi.th 9.9 ml of 0,lM potassium phosphate bu:Efer (pH 6.6), 0.28% sodium taurocholate, and 0.01% NaN3. This diluted extract was assayed for protein using the method described in Methods lS in Enzymology Vol. III, ed, Colowick and Kaplan, Academic Press (New York, 1957) at pp. 451-4 Also, Osl ml of the buffer extract was mixed with 0.2 ml of 0 lM potassium phosphate buffer (pH 6.6), 0.28% sodium taurocholate, and . 0 01% NaN3 and assayed for enzymatic activity using the ~0 procedure of Example 5 DE-52 (DEAE-cellulose) manufactured by Whatman Bio-chemicals Ltd., Maidstone, Kent~ England~ was washed in 0 O5M potassium phosphate buffer (pH 6.6), 0.28~ sodium taurocholate, and 0.01% NaN3 and added as a slurry to a

5 cm. X 90 cm. column to form a 1 liter bed volume with a 200 ml volume of the washing buffer remaining on top of the bed. The entire 4 liter volume of the phosphate buffer extract was then added to the column, followed by 50 ml of a first eluting buffer of 0 05M potassium phos-3~ phate buffer (pH 6 6), 0.28% sodium taurocholate, 0.01%

1 NaN3, and O.OlM mercaptoethanolO A second eluting buffer was then continuously added consisting of 0.2M potassium phosphate buf-fer (pH 6.6)~ 0 28% sodium taurocholate, 0.01%
NaN3~ and 0 OlM mercaptoe~hanol The column effluen~ was collected in 22 ml fractions which were assayed for enzyma-tic activity using the procedure that follows this Example and for protein as above. The results are found in the following table and in the graph illustrated in the Figure of the drawing.
Specific Re-Enzyme Activity cov-Vol. Protein Activity Units/ ery ml mg. Units mg. _%
pancreatin 105 10,000 3150 0.3 100 1~ suspension buffer 915,9601840 0 3 58 extract DEAE- 103260 850 3.3 27 cellulose The cholesterol ester hydrolase containing effluent fractions were combined and the volume concentrated to about 100 ml on a ~iaflo UM-10 ultrafiltration membrane manufactured by Amicon Corporation, Lexington, Massachusetts and then dialyzed against 100 volumes of 0 05M potassium phosphate buffer (pH 6.6), 0.28% sodium taurocholate, and 0.01% NaN3 at 4C~ overnight with one buffer change.
The 0 2M potassium phosphate buffer (pH 6.6), 0.28%
sodium taurocholate, 0.01% NaN3 and O.OlM mercaptoethanol effluent fractions were found to contain no pro~eolytic ac~ivity from either trypsin or~chymotrypsin The following relates to the preparation of the choles-teryl oleate substrate for use in determining cholesterol *Trade Mark 3~æ

l ester hydrolase activity and to the assay procedure itself, a, Substrate preparation Ten mg cholesteryl oleate and 18 mg DL-lecithin were dissolved in 1 ml of ethyl ether in a homogeni~ing tube fitted with a Teflon plunger, Six ml of 0.lM potassium phosphate buffer (pH 6,6), 0,28% sodium taurocholate~ and 0.01% NaN3 were then added and the mixtuTe was homogenized using the plunger for 5 minutes, The ether was removed by holding the tube under hot running water with occasional activation of the plunger over a 15 minute per;od. The solution was cooled in an ice bath and subjected to sonica-tion for 15 minutes using a Sonifier cell disrupter Model W140 and a Sonifier converter Model L, manufactured by Branson Sonic Power Company, Plainview, New York. The solution was then centrifuged at 2500 rpm for 15 minutes and the insoluble material discarded~
b. Assay procedure Twenty-five ~1 of the enzyme solution was added to one ml of the substrate solution preheated to 37C, and incubated at 37C. for 10 minutes, Four ml of Bloor~s solvent ~75% ethyl alcohol:25% ethyl ether) were added and the solution was centrifuged at 2000 rpm for 10 minutes.
One ml of the supernatant was mixed with 1 ml of a 1.0% digitonin solution in 50% ethyl alcohol:SO% water.
The precipitate was allowed to coagulate for twelve hours and the solution was then cen'trifuged at 2500 rpm for 15 minutes, The precipitate wa$ washed with 4 ml acetone and centrifuged again. This precipitàte was dried in a *Trade Mark . . .

1 gentle stream of air at room temperature and 3 ml of 0,1% F_C13 6H2O in glacial acetic acid was added to dissolve the precipitate. Two ml of conc. H2SO4 was added slowly forming two layers which were quickly mixed and the optical density measured at 560 nm. These values were then divided by the optical density of a sample contain-ing a known amount of free cholesterol subjected to the same assay procedure. In order to obtain results in terms of units/ml as defined herein, the quotients were then multiplied by time and dilution factors to yield results in terms of ~moles cholesterol per ml per hour. Specifi-cally, 0,1 ~mole free cholesterol was subjected to the above assay procedure using 2.5 ml of the supernatant yielding an optical density of 1.248. Since the assay time was 10 minutes and the volume used was 25 ~1, the time and dilution factors were 6 and 40 respectively and cholesterol ester hydrolase activity was calculated as follows:
; units/ml = (12546~-) X 240 -24- -~

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A test device for determining cholesterol in a biological fluid sample comprising an absorbent carrier and incorporated therewith, a test composition comprising a chemical system having cholesterol ester hydrolase activity, a chemical system having cholesterol oxidase activity and reagent means for determining hydrogen peroxide or .DELTA.4-cholesten-3-one.

2. A test device as claimed in claim 1 wherein the said chemical system having cholesterol ester hydrolase activity comprises cholesterol ester hydrolase and a bili-ary enzyme co-factor therefor.

3. A test device as claimed in claim 2 wherein said biliary enzyme co-factor is cholic, glycocholic, tauro-cholic or taurodeoxycholic acid or a salt thereof.

4. A test device as claimed in claim 1 which addi-tionally comprises a buffering material.

5. A test device as claimed in claim 1 wherein the said reagent means for determining hydrogen peroxide com-prises a substance having peroxidative activity and an in-dicator material which is oxidized in the presence of hy-drogen peroxide and said substance having peroxidative activity and which changes color thereupon.

6. A test device as claimed in claim 1 wherein the said carrier is absorbent relative to said sample.

7. A method for determining cholesterol in a bio-logical fluid sample comprising the steps of:

(a) contacting said fluid sample with the test device as claimed in claim 1, and (b) observing the response which results.

8. A method for determining total cholesterol in a biological fluid sample which comprises the steps of:
(a) contacting substantially simultaneously in the presence of molecular oxygen the said fluid sample, a chemical system having cholesterol ester hydrolase activity, and a chemical system having cholesterol oxidase activity, (b) contacting the hydrogen peroxide of .DELTA.4-cholesten-3-one produced with reagent means for determining hydrogen peroxide or .DELTA.4-cholesten-3-one, and (c) observing the response which results.

9. A method as claimed in claim 8 wherein at least one of the said chemical system having cholesterol ester hydrolase activity and the said chemical system having cholesterol oxidase activity are incorporated with an ab-sorbent carrier.

10. A method as claimed in claim 8 which comprises the additional step of allowing the resulting response to develop for a predetermined period of time between steps (b) and (c).

11. A method as claimed in claim 8 wherein the said response is observed visually and which comprises the additional steps of comparing the observed colorimetric response with a standard color chart.

12. An enzymatic method for the determination of total cholesterol in a serum sample, said method compris-ing:

hydrolyzing the cholesterol esters in said sample in the presence of a bile acid or salt thereof to free choles-terol by reaction with cholesterol esterase;
oxidizing the free cholesterol to .DELTA.4-cholestenone and hydrogen peroxide in the presence of cholesterol oxidase;
reacting the hydrogen peroxide produced with a hydro-gen donor which can be oxidized by hydrogen peroxide, in the presence of horseradish peroxidase to produce a color, said color being directly proportional to the cholesterol content of the sample; and measuring the color formed to determine the choles-terol content of the sample.

13. A single reagent for conducting an enzymatic assay for total cholesterol content of a serum sample com-prising a water soluble, solid, substantially anhydrous, storage stable mixture of:
the enzymes cholesterol esterase and cholesterol oxidase;
the enzyme horseradish peroxidase;
a hydrogen donor capable of being oxidized by hydro-gen peroxide; and a bile acid ox salt thereof.

14. The single reagent of claim 13 wherein the hy-drogen donor capable of being oxidized by hydrogen per-oxide is selected from the class consisting of O-dianisi-dine and 4-aminoantipyrine plus phenol.

15. The single reagent of claim 14 including a bile acid or salt thereof selected from the class consist-ing of cholic acid, taurocholic acid, glycocholic acid and salts thereof.

16. The single reagent of claim 15 including a buffer capable of maintaining the pH between 6.4 and 7.

17. A test composition for determining total choles-terol in a biological fluid sample comprising (1) a mixture of a chemical system having cholesterol ester hydrolase activity and a chemical system having cholesterol oxidase activity and (2) reagent means for determining hydrogen peroxide or .DELTA.4-cholesten-3-one, wherein said chemical sy-stem having cholesterol ester hydrolase activity comprises cholesterol ester hydrolase and a biliary enzyme co-factor therefor.

18. A test composition as claimed in claim 17 where-in said biliary enzyme co-factor is cholic glycocholic, taurocholic, or taurodeoxycholic acid or a salt thereof.