CA1114269A - Integral element for the detection of glycerol or triglycerides - Google Patents

Abstract

Abstract of the Disclosure An integral element for the detection Or glycerol or triglyceride in liquids is described. The element is the type which comprises at least two superposed layers including a spreading layer and a reagent layer in fluid contact under conditions of use and, optionally, a support.
The element includes a novel assay composition comprising (1) optionally, a lipase preparation demonstrating triglycerlde hydrolysis capability and (2) a glycerol assay composition comprising enzymes and reagents which effect (a) the conversion of glycerol to L-.alpha.-glycerophosphate and (b) the production of a detectable change from L-.alpha.-glycerophosphate.
The lipase preparation is included when the element is intended for the detection/determination of triglycerides.

Description

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The present invention relates to e],emen-ts for the essentially dry analysis oE triglycerides or glycerol in aqueous solutions, such as blood serum.
Serum triglyceride levels are becoming increasingly important in the diagnosis of several -types of hyperlipemia and atherosclerotic heart disease (Kahlke, W. Med. Wscht. 91, p. 26 (1966), Kuo, P.T. and Basset, D. R., Amer.'In'tern. Med., 59, p.465 (1963)). Conventional procedures for serum triglyceride determina-tion involve hydrolyzing the triglyceride to liberate glycerol and treating the glycerol with various reagents -to produce a compound that can be quantitated spectrophotome-trically. Generally hydrolysis is achieved using a base. However, U.S. Patent Nos.
3,703,591 to ~ucolo et al and 3,759,793 to Stork et al. describe , en7ymatic techniques using a lipase alone ('793) or in combination ` ; with a pro-tease ('591) to achieve hydrolysis. Other non-enzymatic hydrolysis techniques are described in German Patent Nos. 2,229,849 , ' and 2,323,609.
~"~ Currently three enzymatic methods are conventionally used for the determination of glycerol from whatever source.

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, `' ~ ' , Z~9 Modifications o:E the method of (a) are also described in German Patent No. 2,665,556, British Patent No. 1,322,462 and U.S. Patent No. 3,759,793. In all cases NADH production or disappearance is measured at 3~0 nm in a U.V. spectrophotometer.
Method (a), utilized in many commercial "kits," is a three enzyme sequence and NADH disappearance is measured. Method (b) involves a two enzyme sequence in which NADH production is measured as is the case with the single enzyme glycerol dehydro-genase reaction (method (c)). The latter two procedures are extremely pH-sensitive and subject to error if strict pH control is not maintained. Also, in all three methods (especially ;;~ method (a)) stability of not only the diagnostic enzymes but also the cofactor, NAD(H), is a major concern. The short-comings of current enzymatic methods are discussed in greater . ~
;~ detail in Chen, H.P. and El-Mequid, S.S., Biochemical ~ Medicine, 7, p. 460 (1973).
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~-~ Another method for triglyceride analysis is described in German Patent No. 2,139,163. The method of this patent involves hydrolysis of the triglycerides, oxidation of the 20 resulting glycerol to formaldehyde and reaction of the formalde- -hyde with ammonia and a stable, water- and alcohol-soluble, colorless metal complex of acetylacetone to produce a colored compound.
Belgian Patent No. 801,742 and corresponding U. S.
Patent 3,992,158 describe integral elements for use in the qualitative and quantitative analysis of liquids such as blood serum and urine, which elements preferably comprise a porous spreading layer in fluid contact or communication with a re-agent layer which comprises at least one material interactive with a component or decomposition product of a component of the 2~

liquid. This patent lncludes no suggestion that any triglycer-ide/glycerol assay composition, much less one of the type described herein, could be incorporated into such a dry analy-tical element.
Koditschek et al in the Journal of Bacteriology, 98:3 pp 1063-1068 (1969) and Jacobs et al in Archives of Biochemistry and Biophysics 88, pp 250-255 (1960) describe the preparation and properties of ~-glycerophosphate oxidase, an enzyme which mediates electron transfer from ~-glycerophospha-te to 2 with the concomitant production of H2O2 and dihydroxyacetone phos-;` phate.

Figure I is a schematic cross-sec-tional view of an element according to the present invention.
Figure II shows calibration curves for typical elements ` of the type described herein.

The elements of the present invention simplify greatly the assay of liquids for triglyceride/glycerol content. Using these elements such an assay requires no reagent mixing and can be automated to permit rapid determination of triglyceride/
glycerol with a minimum of laboratory personnel participation.
According to the present invention there is provided an integral element for the detection of glycerol or triglycer-ides in aqueous liquids. The element comprises a spreading layer in fluid contact under conditions of use with a reagent ; layer and contains interactive materials comprising (a) enzymes which catalyze an ordered sequence of reactions wherein fatty acid esters of glycerol (e.g., triglycerides), if present, are hydrolyzed to glycerol, glycerol, whether present in the free .~i .

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form initially or libera-ted by hydrolysis of the esters, is converted to L-~-glycerophosphate, ~: which in turn is oxidized, producing a detectable .~ change preferably in proportion to the tri-~ glyceride and/or glycerol concentration o~ the i~. solution.

` According to a preferred embodiment, the element includes a further indicator composition comprising enzymes and/or re-,;
........... agents to facilitate determining the presence and/or extent of the detectable change.

The various interactive materials are disposed within ~ the element so that any triglycerides contained in a liquid . sample applied to the element are hydrolyzed and glycerol, , . . .
whether liberated by this hydrolysis or otherwise present in the liquid sample, is enzymatically oxidized to produce, in the element, a detectable change that is related, pre~erably quantitatively, to the triglyceride/glycerol content of the liquid sample. Optionally, the element may include a support.
The interactive materials which accomplish triglyceride hydrolysis and the glycerol detection are preferably incorporated `~
into the element as follows:
(a) all in the reagent layer; or (b) the triglyceride hydrolyzing enzyme in the spreading layer and the materials interactive :~

with glycerol and its oxidation products in `
one or more discrete reagent layers; or (c) the triglyceride hydrolysis composition in a .
- d:iscrete layer intervening the spreading and reagent layers and all other interactive materials in a reagent layer.

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The reagent layer preferably contains the indicator composition which reacts with hydrogen peroxidé generated in the enzymatically catalyzed oxidation of glycerol when oxygen is the electron accep-tor, to produce, in the element, a color change related to the triglyceride or glycerol content of liquid sample applied to the element.

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In a highly preferred embodiment, the triglyceride hydrolyzing enzyme and any associated material are contained in ~;; a spreading layer overlaying a reagent layer con-taining the interactive materials required for glycerol detection.
The analytical elements described herein will be re-ferred to primarily as elements for the determination of tri-- glycerides, however, it should be clear that they are similarly useful for the determination of glycerol, either with or without incorporated hydrolytic enzyme which hydrolyzes tri-glyceride, or for the detection of any single interactive material required for the production of a detectable product by inclusion of all of the other required interactive materials.
Integral analytical elements having a spreading layer and a reagent layer are described in U.S. Patent 3,992,158.
The elements described herein are of this type and comprise:
(1) a spreading layer which serves to deliver a uniform apparent concentration of analyte to (2) a reagent layer in fluid contact with the spreading layer under conditions of use; and

(3) optionally, a support.
Various enzymes and other interactive materials which serve to (1) hydrolyze triglycerides contained in a liquid sample applied to the spreading layer, .~ ~

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(2) oxidize glycerol, free in the liquid or liberated by such hydrolysis, and (3) provide detectable changes (e.g., color production) related to the triglyceride/glycerol content of the liquid, are incorporated into one or more layers of the element.
Reference herein to fluicl contact between layers in an analytical element identifies the ability of a fluid, whe-ther liquid or gaseous, to pass in such element between superposed regions of a spreading layer and a reagent layer or other layers in fluid contact. Sta-ted in another manner, fluid contact refers to the ability to transport components of a fluid between the layers in fluid contact. Although such layer in fluid contact can be contiguous, they may also be separated by intervening layers as described in detail hereinafterO However, layers in the element that physically intervene layers in mutual fluid contact will not prevent the passage of fluid between the fluid contacting layers.
Interactive Materials: In elements of the present inven-tion, triglycerides and/or glycerol are determined quantitativelythe following series of reactions:

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;tion, the amount Or detectable species formed is proportional to glycerol and/or triglyceride concentration.
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is reagent system provides the element of this invention with many inherent advantages over conventional triglyceride assay techniques. First, any electron acceptor capable of reacting with the a-glycerophosphate in the presence ,: .
of the oxidase to produce a detectable change (preferably an intermediate which in turn reacts to produce a detectable chan~e is poter,tially useful in the indicator composition; thus one can measure the reaction at one of several wavelengths in the visible region of the spectrum;
depending upon electron donor selection. Measurements made in the visible region are less subject to interferences than those taken at 340 nm. Second, stability of NAD~ or NADH is not a concern since 2 or some other electron acceptor is the cofactor in the ~-glycerophosphate oxidase reaction. Serum components that utilize NAD or NADH (for example, lactate plus lactate dehydrogenase) which might interfere with prior art reaction sequences, do not interfere with the reactions utilized in the instant elements. Finally, the enzymes used in the reaction sequence are active over a relatively wide pH range; thus, stringent pH control is not necessary in the element.
The preferred element of the present invention includes a triglyceride hydrolyzing composition which hydrolyzes triglyceride,present in a sample applied to the element,to glycerol. According to this e~bodiment,triglycerides are hydrolyzed to free ~lycerol using any of the well known techniques described in the art which can be incorporated ~nto the multilayer element. Enzymatic techniques are preferred, These generally ~nvolve treatment Or the ~erum sample with a lipase preparation, either in combination 2~
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. 1, with a hydrolysis stimulator such as a protease or an effector that is a surfactant or alone depending upon the nature of the trigly-. ~ .
~eride. Detailed discussions of useful triglyceride hydrolysis compositions are contained in U.S. Patent No. 3,703,591 to Bucolo et al issued November 21, 1972 and U.S. Patent No.
3,7595793 to Stork et al issued September 18, 1973. Bucolo .
et al uses a lipase preferably from Rhizo~us arrhizus (var.
delemar) and similar materials in combination with a protease to achieve hydrolysis of serum triglycerides while Stork 10 et al discloses the use of lipase from Rhizopus arrhizus alone to achieve hydrolysis. Since the lipase preparations, proteases, surfactants and other interactive materials are readily available in lyophilized form or easily dried, they are readily incorporated into elements of the type described herein in the manner described hereinafter.

Preferred compositions for achieving hydrolysis of serum triglycerides in these elements, especially protein-bound triglycerides, use a compatible mixture (as defined below) of a lipase which may, of itself, not be capable of hydrolyzing protein associated 20 triglycerides as found in serum or only capable of performing such hydrolysis at an unacceptably slow rate and, as an effector of hydrolysis, a surfactant.
Useful lipases for triglyceride hydrolysis according to any of the foregoing techniques may be of plant or animal ' ~ ~

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origin, but we prefer and rind best for use in elements of the type described herein, microbial lipases such as the lipase from Candida rugosa, particularly when the lipase ~:; is used in combination wi-th a surfactant as described in detail below. Lipases from Chromobacterium visco um, variant paralipolyticum crude or purified, the lipase from Rhizopus arrhizus (variant delemar), purified, for example, as described in Fukumoto et al, J. Gen. A~li. Microbiol, 10, 257-265 (1954) and lipase preparations having simi]ar activity are also useful.
Other useful lipases and methods for their preparation are described in the following U.S. Patents:
; 2,888,385 to Grandel issued ~ay 26, 1959;
3,168,448 to Melcer et al issued February 2, 1965;
3,189,529 to Yamada et al issued June 15, 1965;
3,262,863 to Fukumoto et al issued July 26, 1~o6, and 3,513,~73 to Mauvernay et al issued May 19, 1970.
Specifically preferred commercial lipases include wheat germ lipase supplied by Miles Laboratories of Elkhart, - 20 Indiana, Lipase 3000 supplied by Wilson Laboratories~
Steapsin supplied by Sigma Chemical Company (both of the latter are pancreatic enzymes), and Lipase M (from Candida !~
cylindracea (Candida rugosa))supplied by Enzyms Development Company.
As mentioned above, nonionic and anionic surfactants have been found useful as effectors for lipase preparations which of themselves are incapable of hydrolyzing protein-bound triglycerides or only accomplish such hydrolysis at unacceptably slow rates. Useful combinations of lipase . . .

2~ 9 preparation and surfactant are referred to hereinafter as com-patible mixtures.
In theory, any surfactant is a suitale candidate for use in the successful practice of the present invention.
Certain surfactants, however, inhibit the hydrolase activity of certain lipase preparations. For example, micro-bial enzyme from Rhizopus arrhizus is inhibited by octyl phe-noxy polyethoxy ethanol surfactant materials. Consequently, it is important that, before any attempt is made to combine a lipase preparation and a surfactant, some determination of the compatibility of the two members of the composition be made.
Such a determination is peferably made by using the test described below. A lipase preparation and surfactant mixture which successfully meets this test is referred to herein as a compatible mixture and each member thereof is said to be com-patible with the other.
Compatible mixtures of lipase and surfactant are readily defined by the following test:
The proposed surfactant under evaluation is added to unbuffered reconstituted serum (specifically Validate'~, a serum standard available from General Diagnostics Division of ~;
Warner Lambert Company, Morris Plains, N J) at varyin~ concen-tratons of between about 1 and 10 percent by weight and the solution incubated for about 5 minutes at 37 C. At this time, a sample of the proposed lipase preparation is added and incubation continued for a period of about 20 minutes. Ali-quots (~0.2 ml) of this solution are then diluted to 1.6 ml with water (containing 1.3 mM CaC12 to aid precipitate forma-tion), placed in a boiling water bath for 10 minutes and cen-trifuged to clarify (0, 37,000 Xg, 10 minutes). Glycerol in a 0.4-ml aliquot of the clear supernatant is determined quanti-.

`~ -13-tatively in a total volume of 1.2 mL by the method described by Garland, P B, and Randle~ P J, Natule, 196, 987-988 (1962).
When performing the fore~oin~ test, it is most desirable to run a blank which contains all of the components of the mixture but the lipase preparation so that any reaction which may be due to free ~lycerol or other components of the serum can be sub-tracted. Any composition which stimulates release of at least about 50 percent of the theoretical concentration of available glycerol is considered useful. The pre~erred compositions accomplish at least 70 percent hydrolysis of the available tri-~lyceride in less than 10 minutes and most preferred are those which achieve substantially complete hydrolysis, i.e., above about 90 percent hydrolysis of the available triglyceride, in less than about 10 minutes. Examples of such preferred compo-sitions are shown in the tables and examp]es below.
Among the surfactants which have been found useful to stimulate the hydrolase activity of the fore~oin~ useful lipase materials are nonionic and anionic surfactants including many of the natural surfactants such as the bile salts including deoxy-cholate, chenodeoxycholate, cholate and crude bile salt mixturesand synthetic surfactants such as sodium salts of alkylaryl polyether sulfonates commercially available from Rohm and Haas Company as Triton X-200~ and Alkanol XC~ commercially available from E I duPont Corporation, and alkyl phenoxy poly-ethoxy ethanols such as those available commercially from Rohm and Haas Company under the trade names Triton X-114~, Triton X-100~, Triton X-102'~ and Triton n-101~. Preferred alkyl phenoxy polyethoxy ethanols comprise a polyoxyethylene chain r~

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of less than about 20 oxyethylene units and have a hydrophile-lipophile balance (HLB) number below about 15. Other specifically useful surfactants are presented in the examples below.
Proteases in general are also useful as effectors for lipase preparations as described in prior patents described above. These include by way of example, chymotrypsin, Streptomyces griseus protease (commercially available under the registered traaemark "Pronase"), proteases from Aspergillus oryzae and Bacillus subtilis, elastase, papain and bromelain.
Mixtures of such enzymes may, of course, also be employed.
The useful concentrations in the element of lipase and effectors such as surfactants and protease? will vary ; broadly depending upon such variables as the time limitations imposed on the assay, the purity and activity of the enzyme preparations, the nature of the triglyceride, etc., and these are readily determined by the s'illed artisan. Typical non-limiting examples of useful concentrations are described in the examples below.

Glycerol Assay Once triglyceride hydrolysis has been achieved in - the element by any of the foregoing means, glycerol assay is achieved using the enzymes and interactive materials referred to above.
The first enzyme used in the glycerol assay is ~; glycerol kinase (2.7.1.30) which catalyzes the conversion of glycerol to L-~-glycerophosphate in the presence of adenosine triphosphate (ATP). Generally, any glycerol kinase is useful in the successful practice of the present invention although those obtained from ~. coli and Candida mycodermea are preferred.
Other glycerol kinase enzymes are well known in the art. A

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g complete discussion of such materials and further references to their preparation and reactivity may be found in T.E.
Barman, Enz~me Handhook, I, Springer-~erlag, N.Y. (1969) pgs. 401-402.
The next step in the reaction sequence involves the oxidation of L-~-glycerophosphate in the presence of L-a-glycerophosphate oxidase and an electron acceptor to produce a detectable change. The detectable change is preferably a color change or color formation which, in the preferred case, is quantitatively related to the glycerol contained in the liquid sample. Other detectable changes ; such as oxygen consumption may also be monitored to detect the analytical result.
Any electron acceptor which will permit oxidation of the a-glycerophosphate in the presence of the oxidase enzyme with the ; concomitant production of a detectable change is a suitable candidate for use in this reaction. Particularly preferred as electron acceptors are materials which result in the formation of a colored product or a chemical intermediate which, although not itself colored, can be detected via a reaction or reactions which result in color formation. The utility of any particular electron acceptor can be determined by experimentation with potentially useful electron acceptors.
; A highly preferred electron acceptor is oxygen which will oxidize the L-~-glycerophosphate in the presence of the oxidase to dihydroxyacetone phosphate and hydrogen peroxide.
Methods for deter~ining the presence or amount of hydrogen per-oxide in reactions Or this type are, of course, well known. An ~' .

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alternative preferred embodiment uses as electron acceptor material colored or uncolored which undergoes a change in or the production of color upon reduction in the presence of the enzyme and the substrate. As described above, such materials can be selected by testing in a specific use ; environment. Using this method certain indolphenols, potassium ferricyanide and certain tetrazolium salts have been found to be useful electron acceptors. Specifically, 2,6-dichloro-phenolindolphenol alone or in combination with phenazine methosulfate and 2-(p-indophenyl)-3-(p-nitropheny])-5-pnenyl-2H-tetrazolium chloride either alone or in combination with phenazine methosulfate have been found useful as electron acceptors in this reaction.

L-a-Glycerophosphate oxid~se is a microbial enz~e which can be derived from a variety of sources. The properties of enzyme from certain sources are more desirable than those from others as will be elaborated below. Generally, the enzyme may be obtained from Streptococcaceae, La^t~ e~-~; and Pediococcus. The enzyme from cultures of Stre~tococcus faecalis, specific strains of which are obtainable from theAmerican Type Culture Collection, are specifically preferred.
Particularly useful and preferred enzymes are obtained from strGins ATCC
11700, ATCC 19634 and ATCC 12755 identified on the basis of their deposit in that collection. As will be described and demonstrated by example below, the enzyme from ATCC 12755 demonstrates activity over a somewhat broader pll ranEe than enzymes derivecl from the other two strains and for this reason is most preferred.

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The ~ollowing two references describe both the enzyme and useful techniques ~or its preparation and extraction:
Koditschek, L.K. and Umbreit, W.W. ~-Glycerophosp~ate Oxidase in Streptococcus faecium, F 24, Journal of Bacteriology, Vol. 98, No. 3, p. 1063-1068 (196~) and Jacobs, N.J. and - Van Demark, P.J. "The Purification and Properties of the ~-Glycerophosphate Oxidizing Enzyme of strePtococcus faecalis, 10 Cl." Enzymes prepared according to the methods described in either of these publications are useful in the successful practice of the invention. When any enzyme preparation of unknown total composition is used, care should be exercised ; to extract any contaminants which may interfere w~th assay results. For example, certain preparations of L-~-glycero-phosphate oxidase, derived as described below, contained sufficiently high concentrations of impurities that the crude prepara~ion had to be purified using conventional fractionation and column separation techniques before ass~ys ; of blood serum triglycerides free from unwanted inter~erences -~ could be achieved.
The detection of glycerol in aqueous solutions containing glycerol and/or triglycerides, for example, blood serum, is preferably achieved using an indicator composition which quantifies the level of hydrogen peroxide generated in the oxidation of L-a-glycerophosphate. Indicator compositions for the detection of enzyma-tically generated hydrogen peroxide are well known in the art, particularly as indicator compositions in the enzymatic detection of glucose and uric acid. U.S.
Patent Nos. 3,092,465 and 2,981,606 among many others describe ~ such useful indicator compositions and such compositions are ; 3 readily incorporated into elements of the type described herein using the methods outlined hereinafter.
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The hydrogen peroxide indicator composltion generally comprlses a substance having peroxldative activlty, perrerably peroxidase, and a dye precursor which undergoes a color formation or change in the presence of hydrogen peroxide and the substance having peroxidative activity. Alternatively, the dye precursor may be one or more substances which undergo no substan-tial color change upon oxidation in the presence of H202 and peroxidase, but which in their oxidized form react with a color-forming or color-changing substance (e.g., a coupler) to give visible and preferably quantitative, evidence of chemical reaction. U.S. Patent No. 2,981,606 in particular provides a detailed description of such indicator compositions. The latter dye precursor, i.e., one which produces color by virtue of a coupling reaction, is preferred in the practice of the present invention.
A peroxidase is an enzyme which ~ill catalyze a reaction wherein hydrogen peroxide or other per~xide oxidizes another substance.
The peroxidases are generally conjugated proteins containing iron porphyrin. Peroxidase occurs in horseradish, p~tatoes, figtree sap and turnips (plant peroxidase)j in milk (lacto peroxidase); and in white blood corpuscles (verdo peroxidase);
also it occurs in microorganisms. Certain synthetic peroxidases, such as disclosed by Theorell and Maehly in Acta Chem. Scand., Vol. 4~ pages 422-434 (1950), are also satisfactory. Less satisfactory are such substances as hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivatives, and certain other substances which have peroxidative activity.

Other substances which are not enzymes but which have peroxidative activity are: iron sulfocyanate, iron tannate, ferrous ferrocyanide, chromic salts (such as potassium chromic sulfate) absorbed in silica gel, etc. These substances are not as satisfactory as peroxidase per se but are similarly useful.
Dye precursors which produce color in the presence of hydrogen peroxide and a substance having peroxidative activity include the following substances, with a coupler where necessary:
; (1) Monoamines, such as aniline and its derivatives, ortho-toluidine, para-toluidine, etc.;
(2) Diamines, such as ortho-phenylenediamine, N, N ' di-methyl-para-phenylenediamine, N, N ' -diethyl phenylenediamine, benzidine, dianisidine, etc.;
(3) Phenols, such as phenol per se, thymol, ortho-, meta-and para-cresols, alpha-naphthol, beta-naphthol~ etc.;

(4) Polyphenols, such as catechol, guaiacol, orcinol, pyrogallol, p,p-dihydroxydiphenyl and phloroglucinol;

(5) Aromatic acids, such as salicyclic, pyrocatechuic and gallic acids;

(6) Leuco dyes, such as leucomalachite green and leucophenolphthalein;

(7) Colored dyes, such as 2,6-dichlorophenolindophenol;

(8) Various biological substances, such as epinephrine, the flavones, tyrosine, dihydroxyphenylalanine and tryptophane;

(9) Other substances, such as gum guaiac, guaiaconic acid, potassium, sodium, and other water soluble iodides; and bilirubin; and

(10) Such particular dyes as 2,2'-azine-di(3-ethylbenzo-thiazoline-(6)-sulfonic acid) and 3,3'-diaminobenzidine.

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Other ind~cator compositions that are oxidizable by peroxides in the presence o~ peroxidase and can provide a detectable species include a compound that,when oxidized ~n the presence of peroxidase, can couple with itself or with its reduced form to provide a dye. Such autocoupling compounds include a variety of hydroxylated compounds such as orthoamincphenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, cresols, pyrogallol, guaiacol3 orcinol, catechol phloroglucinol, p,p-dihydroxydiphenyl, gallic acid, pyrocatechuic acid, salicylic acid, etc. Compounds of this type are well known and described in the literature, such as in The Theory of the Photographic Process, Mees and James Ed, (1956), especially at : Chapter 17. Other leuco dyes, termed oxichromic compounds, are described in U.S. Patent No. 3,880,658 and it is further described that such compounds can be diffusible ~' ~

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with appropriate substituent groups thereon. The non-stabilized oxichromic compounds described in U.S. Patent No. 3,880,658 are considered preferable in the practice of this invention.
In a preferred aspect, the detectable change is provided by indicator compositions that include a compound oxidizable in the presence of peroxidase and capable of undergoing oxidative condensation with couplers, such as those containing phenolic groups or activated methylene groups.
Representative such oxidizable compounds include such compounds as benzidene and its homologs, p-phenylenediamines, p-aminophenols, 4-aminoantipyrine, etc. A wide range of such couplers, including a number of autocoupling compounds, is described in the literature, such as in Mees and James (supra) and in Kosar, Light-Sensitiv~
Systems, 1955, pages 215-249.
Preferred dye precursors are 4-methoxy-1-naphthol, ~ 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl) ; imidazole; a combination of 1,7-dihydroxynaphthalene and 4-a~inoantipyrine (HCl);and 4-isopropoxy-1-naphthol. The concentrations of the components of the various indicator compositions useful in the elements described herein are dependent to a large extent upon the concentration of glycerol in the sample under test, the sophistication of the detection apparatus, the dye produced, etc., and are readily determinable by the skilled artisan. Typical values are shown in the examples below.
The concentration of the other components of the assay compositions may also vary broadly depending upon the - solution under assay (i.e., blood serum, diluted or undiluted, .
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or other complex aqueous solution o~ glycerol and/or triglycerides). Table I below provides a ready reference for the generally useful and preferred concentration ranges of the various components of the novel assay compositions described herein.

T e_ Generally us~ful Preferred Enzyme ranges U/m~ _ ranges U/m2 Lipase (when used) 9,000-27,000 13,000-26,000 Glycerol kinase 100-1,500 250-800 Glycerophosphate 800~5,500 1,500-3,300 oxidase ' ~ Protease (when used) 36,000-105,000 72,000-go,ooo Peroxidase 3,000-11,000 6,000-7,500 i~ g/m g/m ; Surfactant (when used) 1.5-10 4-6 Of course useful results may be obtained outside of these ranges .

In the foregoing Table I, one irternational unit of enzyme is defined as that quantity of enzyme which results in the conversion of one micromole of substrate in one minute at 37C and pH 7.
As is well recognized in the art~ each of the enzymes possesses a pH-activity profile, i.e., a graphic representation of variations in the activity of the enzyme with varying pH. The pH activity profile of L-a-glycerophos-phate oxidase peaks at between about pH 5 and 8.~. The optimum pH range over wh~ch each of the enzymes in the novel reaction sequence is active is shown in Table IIo ~ -23-.

.

4Z~t Table II

pH-value Lipase 5-9 Glycerol kinase 7-9 L-~-glycerophosphate oxidase 6.3-~.0 Peroxidase 6-8 From the foregoing table, it is readily apparent that it is generally desirable to buffer the layer(s) of the elemen~s described herein which contain the respective reagents at pH levels of between about 6.o and about 8.o and most preferably between about 7.0 and about 8.o. Specific layers containing only certain of the enzymes may be buffered to take advantage of the pH optimum of the particular enzyme(s). Techniques for achieving this type of buffering are well known in th~ art and involve dissolving or dispersing suitable concentrations of buffer in the compositions which are subsequently dried to form the layered element. ~uitable buffers for buffering to the aforementioned pH levels are described in detail by Good in Biochemistry 5, 467 (1966).
Particularly preferred buffers are the pho~phates such as potassium phosphate.

The Spreading Layer: As used herein, the term spreading layer refers to a layer, isotropically porous or otherwise, that can accept a liquid sample, whether applied ; directly to the spreading layer or provided to it from a layer or layers in fluid contact with the spreading layer, and within the layer distribute the solvent or dispersion medium of the sample and at least one dissolved or dispersed com-ponent such that a uniform apparent concentratlon o~ such component ~s provided at the sur~ace of the spreadlng layer racing the .

, `Z~

reagent layer(s) of the element. It should be understood that the unirormity Or such concentration is a perceived uniformity as measured by techniques like those described hereinarter.

In the context o~ this invention~
spread sample components will, Or course, include one or more Or triglycerides, glycerol or oxidation products of glycerol as present in the applied sample. It will be appreciated that such an apparent concentration can be achieved with concentration gradients present through the ~0 thickness o~, or otherwise in, the spreading layer. Such gradients do not present any dir~iculty to obtaining quanti-tative test results i~ they are not detectable during result measurement or can be accommodated using known calibration techniques.
The spreading layer can be an isotropically porous layer. Reference herein to iso+ropic porosity identifies the fact of substantial porosity in all directions within the spreading layer. It will be understood that the degree of such porosity may be variable, if necessary or desirable, for example, regarding pore size, percentage of void volume or otherwise. It shall be understood that the term isotropic porosity (or isotropically porous) as used herein should not be confused with the terms isoporous or ionotropic often used with reference to filter membranes to signify those membranes having pores that are continuous between membrane surfaces. Likewise, isotropic porosity should not be confused with the term isotropic, used in contradistinction to the term anisotropic, which signifies filter membranes having a thin "skin" along at least one surface of the membrane. See, for example, Membrane Science and Technolo~, James Flinn Ed, Plenum Press, New York (1970).

""~' .

As will be appreciated, the extent of spreading isdependent in part on the volume of liquid to be spread.
However, it should be emphasized that the uniform apparent concentration obtained with spreading is substantially independent of liquid sample volume and will occur irres~ective of the extent of spreading. As a result~ elements of this invention generally do not require precise sample application ; techniques. However, a particular liquid sample volume may be desirable for reasons o~ preferred spread times or the like. Because the elernents of this invention are able to produce quantitative results using very small sample volumes that can be entirely taken up within a conveniently sized region of the spreading layer (e.g., one square centimeter), there is no need to remove excess moisture from the element after application of a liquid sample. Further, because spreading occurs in the spreadir:g layer and the spread component is provided to the fluid contacting reagent layer without apparent substantial lateral hydrostatic pressure, there is not the "ringing" problem often seen with prior analytical elements when soluble reagents were used.
The spreading layer need only produce a uniform apparent concentration of spread component per unit area at its surface facing a reagent layer with which the spreading layer is in fluid contact, and it is very convenient to determine whether a particular layer can be suitable for spreading purposes by means of the simple test described in the aforementioned U.S. Patent 3,992,158.

:- :
Isotropically porous layers can be prepared using a variety of components~ In one aspect, particulate material . .:

. ~ ;
... .

can be used to form such layers, wherein the isotropic porosity is created by interconnected spaces between the particles.
Various types of particulate matter, all desirably chemically inert to sample components under analysis, are useful.
Pigments, such as titaniu~ dioxicle, barium sulfate, zinc oxide, lead oxide, etc., are desirable. Other desirable particles are diatomaceous earth and microcrystalline colloidal materials derived from natural or synthetic polymers. Such microcrystalline materials are described in an article entitled "Colloidal ~acromolecular Phenomena, Part II, Novel Microcrystals of Polymers" by 0. A. Battista et al published in the Journal of Applied Pol~mer Science, Vol. II, pages 481-498 (1967). Microcrysvalline cellulose, which is comercially available from FMC Corporation under the name Avice ~ , is an example of such a colloidal material which is satisfactory for use in the present invention. Spherical particles of uniform size or sizes, such as resinous or glass beads, can also be used and may be particularly desirable where uniform pores are advantageous, such as for selective filtration purposes. If a particulate material of choice is not adherent, as in the case of glass beads or the like, it can be treated to obtain particles that can adhere to each other at points of contact and thereby facilitate formation of an isotropically porous layer. As an example of suitable treatment, non adherent particles can be coated ~ith a thin adherent layer, such as a solution of hydrophilic colloid like gelatin or polyvinyl alcohol, and brought into mutual contact in a layer. When the colloid (iOe., binder) coating dries, the layer integrity is maintained and open spaces remain between its component particles.

,' ~

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As ~n alternative or in addition to ~uch particulate materials, the spreading layer can be prepared using an isotropically porous continuous polymer phase. It is possible to prepare such polymers using techniques useful in forming "blush" polymers. "Blush" polymer layers can be formed on a substrate by dissolving a polymer in a mixture of two liquids, one of which is a lower boiling, good solvent for the polymer and the other of which is of a higher boiling point and is a non solvent or at least a poor solvent for the polymer. Such a polymer solution is then ccated on the substrate, and dried under controlled conditions. The lower boiling solvent evaporates more readily and the coating can become enriched in the liquid which is a poor solvent or non-solvent. As evaporation proceeds, under proper conditions, the pol~er formed is an isotropically porous layer. Many different polymers can be used, singly or in combination, for preparing isotropically porous "blusht' polymer spreading layers for use in this invention, typical examples being polycarbonates, ; polyamides, polyurethanes and cellulose esters such as cellulose acetate.
A wide range of materials are useful as the spreading . layer. Usually, however, materials that are resistant to, i.e., substantially non-swellable upon contact with, the -;~
liquid under analysis are desired. Swelling of about 10-40%
of the layer's dry thickness may be normal.
Furthermore, although it may be possible to obtain ` useful spreading layers having isotropic porosity, etc., it -~ is preferred th~t the material of the spreading layer be substantially non-fibrous to avoid wicking effects which tend ¦ 30 to produce apparent non-uniform dlstributions of analyte between , ~ ~ -28-, ~ , .

~ 4;~
.

and along fibers and mottle when spreading layers o~ fibrous material are used as the background or milieu Or spectro-photometric measurements.

The Reagent Layer~sl: Reagent layer(s) in the elements of this invention are desirably permeable, preferably uniformly permeable, and optionally porous if appropriate, to components spreadable within the spreading layer. As used herein the term permeability includes permeability arising from porosity, ability to swell or any other characteristic. Such layers generally include a matrix in which is distributed, i.e., dissolved or dispersed, the enz3~es and other reagents interactive with triglycerides, glycerol or decomposition products of glycerol. Interactive materials are discussed hereinafter. Layers which serve merely as a sump for receiving a detectable species are referred to herein as registra ion layers.
The distribution of interactive materials (i.e., enzymes and other reagents) can be obtained by dissolving or dispersing them in the matrix material. Although uniform distributions of interactive materials are often preferred, they may not be necessary if the interactive material is, for example, an enzyme such as lipase, glycerol kinase, a-glycerophosphate oxidase, etc. which is not consvmed in : , any reaction but serves as a catalyst which is continuously ' ~- reused.
: .

`~- Desirably~ reagent layers are uniformly permeable ~..
to spread components. Uniform permeability of a layer refers to permeability such that, when a homogeneous fluid is provided , ..
~ uniformly to a surface of the layer, measurements of the ."'`,' ~
~; -29-:, 2~ .

concentration of such fluid within the layer~ made with identical equipment and under identical conditions but through different regions of a surface of the layer, will yield (i.e., be capable of yielding) substantially equal results. By virtue of unirorm permeability, undesirable con-centration gradients within, for example, a reagent layer as ` described herein, are avoided.
The choice of a matrix material for the reagent or registration layers described herein is, of course, variable and dependent on the intended method of use of the ele;;lent as well as the particular interactive materials ~Jhich are incorporated therein as described hereinafter. Desirable matrix materials can include hydrophilic materials including both naturally occurring substances like gelatin~ gelatin derivatives, hydrophilic cellulose derivatives~ polysaccharides such as dextran, gum arabic, agarose and the like, and also synthetic substances such as water-soluble polyvinyl compounds like poly(vinyl alcohol) and poly(vinyl pyrrolidone), acrylamide polymers, etc. Organophilic materials such as -; 20 cellulose esters and the like can also be useful, and the choice of materials in any instance will reflect the use `~ parameters ~or any particular element. For example, if a protease is used to assist in hydrolysis o~ triglycerides as described below, gelatin is not a particularly suitable reagent ~- matrix. To enhance permeability of the reagent layer, if not .~:
lnitially porous, it is often useful to use a matrix material that --is moderately swellable in the solvent or dispersion medium o~
liquid under analysis.
In addition to its permeability5 the reagent layer 3 is desirably substant~ally free ~rom any characteristic that might appear as or contribute to mottle or other noise in the . .
30- ~

:

- ,,. , . ., - . . - .

detection of an analytical result produced in an integral element of the invention. For example, variations in color or in texture within the reagent layer, as may occur when fibrous ~aterials such as papers are used as a permeable medium, may be disadvantageous due to non-uni~orm reflectance ; or transmittance of detecting energy, e.g., when the detectable change has occurred in and is detected in the reagent layer.
Also, although fibrous materials like filter and other papers are highly permeable overall, they typically eYhibit widely ran~ing degrees of permeability between regions of the paper, for example, based on structural vari2tions such as fiber dimensions and spacing. As a result, such materi21s are not considered uniformly permeable and, as such, although useful, are not preferred in either the spreading or reagent layers of the present invention. In various preferred embodiments the spreading and reasent layers of elements absorbed herein are prepared using non-fibrous ~aterials. It should be appreciated that the use of fibrous constituents, such as in appropriate combination with the non-fibrous materials, may . .
`~ 20 be desirable.

Supports: The integral analytical elements of the present invention can be self-supporting or the spreading layer, reagent layer and any other asso^iated layers can be coated on a support. Useful support materials, when such are used, include paper and polyolefin coated paper, as well as ~';
a variety of polymeric materials such as cellulose acetate, . .. ....
~ poly(ethylene terephthalate), polycarbonates and polyvinyl , ~ compounds s~ch as polystyrenes, etc. The support can be `~ -31-': ~

~L$~L~2~

opaque or it can transmit lighJ or other energy depending, o~ course, on the mode of detection used. A support Or choice in any case will be compatible with the intended mode of result detection. Pre~erred supportsinclude transparent support materials capable o~ transmitting electromagnetic radiation of a wavelength within the region between about 200 nm and about 900 nm. Transparent supports need not, o~ course, transmit over the entire 200-900 nm region but may transmit only in the reglon of the indicating radiation. When an element incl~des a support, the reagent layer is interposed in the element between the support and the spreading layer.
Speci~ically preferred transmission ranges ~or elements of ;
the present invention will be apparent from the discussion of the various preferred indicator compositions discussed above.
When used~ supporcs ~aving thicknesses of between about 1 and about lO mils have been found satisfactory, although the ~ ~ ,,;.
thickness can vary broadly depending on such factors, for example~ as the intensity of the detecting radiation and the : .
sensitivity of the detecting apparatus.

. ` , Other Layers: In a preferred embodiment, analytical ;.~., elements of the present invention are adapted for use in analytical procedures employing reflection techniques of spectrophotometric analysis. In accordance with this "
embodiment, such elements will generally include a reflecting layer to provide a suitable background for spectrophotometric measurement of colorimetric or other indicator reactions.

I~ a support is used, measurement will usually be ; made through the support. The reflecting layer : ~ , permits the passage o~ triglycerides, glycerol and/or decomposition products o~ glycerol to the reagent or ., ~ . ~. . ; ~ :

2~

indicator layer ti.e., a layer underlying a reagent layer containing enzymes which catalyze the decomposition o~
triglycerides or glycerol and only contains the means for detecting hydrogen peroxide) and should provide an effective background for reflection spectrophotometry. A white background is generally preferred for this purpose. In view of its function as a background for indicator formed in the reagent or indicator layer, any re~lective layer will normally intervene the spreading and reagent or registration layers.

Such a layer may, however, intervene 2 reagent and indicator layer where such structure is appropriate. Reflectance can be provided by a layer also serving, for example, as a ; spreading layer or it can be provided by an additional layer ~; that may not have an additional function within the element.
Pigments, such as titanium dioxide and barium sulfate, are reflective and can be used to advantage in a reflecting layer.
Blush polymers can also constitute a suitaDle reflecting . ,~, material. As can be appreciated, pigment spreading layers may be useful for this purpose as can blush polymer layers that may also be spreading layers. In one preferred aspect, blush pol~er layers can also incorporate a pigment to enhance spreading and/or reflectivity. The amount of pigment that ., can be included in a layer together with blush polymer is highly variable, and amounts of from about 1 to about 10 parts by weight of pigment per part by weight of blush polymer ~ are preferred, with from about 3 to about 6 parts pigment per ,~ part of blush polymer being most preferred.

- Filtering layers may also be present in the element.
The composition and preparation of such layers are well known in the art an~, when present, they serve to remove - . .. . .. ..

~rom the sample components which could interfere with the indicating reaction or otherwise hinder the determination.
Thus, in the use o~ the multilayer analytical element for analysis of triglycerides in whole blOodg a separate filtering layer could serve to remove red blood cells while transmi~ting the serum to the layer below. In the analysis of blood serum or other fluids, the filtering layer may serve to remove unwanted components which could hinder or confuse the primary indicating reaction. The aforementioned blush polymer layers may also, under certain circumstances, serve as filtering layers. If the element is to be used for analysis of whole blood, it is desirable that any riltering layer ha-~e a pore size of from about 0.5 to about 5 microns.
` The incorporation of a protease into a reagent layer whose matrix is composed primarily of, for example, gelatin or some other natural or synthetic material which is attacked by protease will result in the normal proteolytic reactions when such reagent layer is wetted, such as by sample ` application to the element. Although some measurements can be made in an element which includes the protease and conse-quently the hydrolyzing composition in a gelatin or similar ` reagent matriY~, it is most desirable that the hydrolyzing composition be incorporated into a layer, which is resistant ^~ to the action of the protease and that, as a further measure to protect the gelatin (or similar) matrix of the reagent or other layer from the protease, that an analyte permeable barrier layer be incorporated into the element to selectively prevent protease rrom contacting gelatin or other proteinaceous matrix materials in the element. In this configuration, it may also be desirable to place the glycerol degrading enzymes in a layer with the components Or the hydrolyzing composition so that indication requires ~nly that the relatively small hydrogen peroxide molecules ~, . . .

z~

permeate the barrier layer to an underlying layer while the larger protease enzyme molecules are prohibited from migrating into the protease susceptible layer. Optionally, the glycerol determining reagents may be incorporated into a reagent layer while the hydrolysis reagents are incorporated - into the spreading layer.
If used to inhibit protease migration, the barrier layer may be comprised of any of a large variety of materials compatible with the various components of the element. Pre-ferred materials include hydlophilic polymeric materialswhich permit migration of the hydrogen peroxide or glycerol as just described in the desired ~ashion while excluding the protease enzyme and demonstrate no inhibitory effect on any of the other components of the system. Particularly preferred as the protective barrier layer is a coating of agarose or a poly(acrylamide) resin, e.g., poly(isopropylacrylamide). ~-Element Preparation: In preparing integral analytical elements of this invention3 the layers can be preformed separately and laminated to form the overall element. Layers prepared in such a manner are typically coated from solution or dispersion on a surface from which the dried layer can be physically stripped. However, a -convenient method which can avoid the necessity for multiple stripping and lamination steps is to coat an initial layer on a stripping surface or a support, as desired, and thereafter to coat successive layers directly on those coated previously.
Such coating can be accomplished by hand, using a blade coating device or by machine, using techniques such as dip or bead coating. I~ machine coating techniques are used, it is often possible to coat adjacent layers simultaneously, ~4~

using hopper coating techniques well known in the preparation of light-sensitive photographic films and papers. Interlayer adhesion problems can be overcome without harmful effect ~y means of surface treatments including extremely thin application(s) of subbing materia:Ls such as are used in photographic films.
According to one embodiment of the present invention, wherein the spreading layer performs the functions `
of filtering and spreadinga this layer is advantageously prepared by simultaneously coating two strata of a binder such as cellulose acetate dissolved in a mixed organic solvent to provide "blush" polymer layers as described below. Such a technique simplifies the manufacturing operation by reducing the multiple coating of multiple layers to a : .
single multiple coating operation while providing a highly useful spreading and/or filtering layer. Optionally, if .
desired, either or bcth of the discrete layers may contain dispersed therein a reflective pigment such as TiO2-The physical structure of layers prepared in this `~ 20 fashion generally consists of an isotropically porous upperlayer which functions primarily as a spreading layer to provide a uniform apparent concentration of analyte to an underlying layer in spite of variations in volume of sample applied (as described above), and a porous underlayer which functions primarily as a filter layer. The porosity of these two strata is controlled during manufacture by the use of different ratios of mixed organic solvents as described in British Patent No. 134,228 or in the discussion of "blush"
polymer layers hereinabove. A particularly useful combination ; -36-z~

using hopper coating techniques well known in the preparation of light-sensitive photographic films and papers. Interlayer adhesion problems can be overcome without harmful effect by means of surface treatments including extremely thin application(s) of subbing materials such as are used in photographic films.
According to one embodiment of the present invention, wherein the spreading layer performs the functions of filtering and spreadin~g this layer is advantageously prepared by simultaneously coating two strata of a binder such as cellulose acetate dissolved in a mixed organic solvent to provide "blush" polymer layers as described below. Such a technique simplifies the manufacturing operation by reducing the multiple coating of multiple layers to a single multiple coating operation while providing a highly useful spreading and/or filtering layer. Optionally, if .
desired, either or bcth of the discrete layers may contain dispersed therein a reflective pigment such as TiO2-The physical structure of layers prepared in this - 20 fashion generally consists of an isotropically porous upper layer which functions primarily as a spreading layer to provide a uniform apparent concentration of analyte to an underlying layer in spite of variations in volume of sample applied (as described above), and a porous underlayer which functions primarily as a filter layer. The porosity of these two strata is controlled during manufacture by the use Or different ratios of mixed organic solvents as described in British Patent No. 134,228 or in the discussion of 1'blush"
polymer layers hereinabove. A particularly useful combination ~ '~
; 36 z~
described in Chalkley, Journal oE the National Cancer Institute, 4, 47 (1943) and by direct welghing and determining the ratio of actual weight of the layer to the weight of solid material equal in volume to that of the layer, comparably composed of constituents from the layer. It will be appreciated that the pore size in any case should be sufficient to permit spreading of triglycerides and decomposition products of triglycerides may be appropriate in view of the location of the various inter-active materials in the element.
For reagent layers, a coating solution or dispersion including the matrix and incorporated interactive materials can be prepar`ed, coated as discussed herein and dried to form a dimensionally stable layer. The thickness of any reagent layer and its degree of permeability can be varied widely and depend on actual usage. Dry thicknesses in the range of from about 10 microns to about 100 microns have been found useful.
The hydrolyzing composition may be incorporated into the reagent layer. However, according to a highly preferred embodi-ment of the present invention, the hydrolysis composition is ' ! `
incorporated into the spreading layer, for example, by dis-persing the enzymes in a lyophilized state in the coating medium used to form the spreading layer, and then coating this mixture over the reagent layer. According to this embodiment, spreading of the sample and hydrolysis of any triglycerides are accom-plished substantially simultaneously and glycerol in the sample, whether initially present as a fatty acid ester or free glycerol, is transmitted to the reagent layer. Such a configuration uti-lizes the time needed to spread the sample to prepare it for immediate reaction with the glycerol assay reagents in the reagent layer. As ' $~

another alternative, a distinct layer which includes the hydrolyzing composition may be incorporated between the spreading layer and the glycerol assay reagent_containing layer to accomplish hydrolysis before the sample reaches the glycerol detection reagents but af-ter spreading is complete.
~;Wherever the enzymatic hydrolyzing composition is incorporated, optimum results are achieved when the composition is buffered to a pH of between about 6 and 8.o and preferably between about 7.0 and 8.o. Thus, when the hydrolyzing 10 composition is incorporated into the reagent layer, or into I
another layer with the glycerol detecting reagents, a pH of about 7.0 produces optimum results. Similar pH values are used when the hydrol~zing composition is present in a second reagent layer or in the spreading layer.
,~ :.
The concentration of lipase in the layer containing ~, ~ .
the hydrolysis composition may vary over a broad range.

Generally, however, concentrations of lipase ranging from ;about 9,000 to about 27,000 U/m2 have been found useful.
. ~ ~
Below this level substantially complete hydrolysis may not occur. Concentrations above these levels, although perhaps still useful, are not usually necessary. According to a preferred embodiment of the present invention, lipase levels on the order of from about 13,000 to about 26,000 U/m2 are used. When protease is used as the hydrolysis stimulator in combination with the lipase it is used at a level of between about 36,000 and about 105,000 U/m2. According to a preferred embodiment,protease concentrations of between about 72,000 and about 90,000 U/m2 are used. As with the concentrations of the other components of the elements of this in~ention, these levels may, of course~ vary broadly depending on factors listed hereinabove.

_39_ , z~

As all of the layers described herein are preferably formed by coating from solutions or dispersions as described in the aforementioned, US Patent 3,992,158, it is often necessary to include coating aids which impart uniform coating properties -~
to the layers.
Whatever coating aids are used for this purpose, or those described above in connection with hydrolysis stimulation, it is important that they do not inhibit nor interfere with the ~ lipase or any of the other enzymes or reagents present in any of ;~ 10 the various layers. Particularly useful coating aids include nonionic surfactants such as the octyl phenoxy polyethoxy etha-nols commercially available from Rohm and Haas Company under the Triton trade name (X-lOO'n, X-102'~, X-165'~, X_305in and X-405ii~ being particularly useful), p-nonylphenoxy)glycerol com-mercially available from Olin Mathieson Corporation under the trade name Surfactant lOG'~, and polyethylene glycols such as the Carbowax'~ materials available from Union Carbide. Of course, surfactants which are useful as hydrolysis stimulators may also serve as coating aids which improve the coating charac-20 teristics of the materials in manufacture. When used as coatingaids, concentrations of surfactant on the order of between about 0.1 g/m2 and about l.0 g/m2 have been useful. Preferred concentration ranges for surfactants as coating aids range between about 0.3 g/m2 and about 0.6 g/m2.
Use of the Element: In use, as demonstrated by the following examples, a sample usually on the order of from about 5 to about 50 ~1 :is applied to the spreading or other outer-most layer of the element. It is usually applied as a contact spot or free drop. In passage through the spreading layer the F~ O--d~
sample drop is spread and is then delivered to the underlying reagent layer. Also during passaye through the spreading layer or the reagent layer, depending upon the embodiment used, -tri-glycerides contained in the applied sample are hydrolyzed to glycerol, and glycerol thus formed or otherwise contained in the sample contacts glycerol kinase to form L-~-glycerophosphate ~` which in turn contacts ~-glycerophosphate oxidase in the presence of oxygen to produce H2O2. The detectable change pro-duced by the intervention of the indicator composition which reacts with the H2O2 can then be determined quantitatively using known techniques and the concentration of triglycerides/
. glycerol present in the applied sample determined ~ The following examples are included to illustrate :~. further the present invention.
-- Example 1 ~ A coated element for the assay of total glycerol i- `~
including that present as triglyceride in blood serum was pre~ ~
pared by coating a gelatin subbed .18 mm.poly(ethylene tere- ;
- phthalate) film with a reagent layer comprising 21.5 g/m2 of : 20 deionized gelatin, 4-isopropoxy-1-naphthol (.54 g/m2), 5,5-dimethyl-1,3-cyclohexanedione (0.1 g/m2), alkylphenoxypolyethoxy ethanol (0.4 g/m2), peroxidase (6994 U/m2), adenosine 5'-: triphosphate disodium salt (1.3 g/m2, ~-glycerophosphate oxidase (1506 U/m2) and glycerol kinase (645 U/m2) (glycerol kinase from Worthington Biochemical Company provides a satis-factory commercial source of the enzyme) in 0.1 M potassium phosphate buffer at pH 7. A layer comprising 0.3 g/m of poly-n-isopropylacrylamide is applied over the reagent layer, and spreading/reflecting layer comprising 45.2 g/m , :, ,, :.: :, 2~

TiO2, 6.5 g/m2 cellulose acetate, 900 U/m2 Or lipase and 5,4 g/m2 Or octylphenoxy polyethoxy ethanol is coated over the subbing layer.
With components at these concentrations, reactions with serum applied to the spreading layer were essentially completed in 5-7 minutes at 37~C and reactions with glycerol or L-a-glycerophosphate were completed in less than 5 minutes at 37 DC.
~`~ Serum samples (10 ~1 aliquots) were applied to the above coating and the reflection densities DR at 660 nm were measured after 8 minutes at 37~C.
~; A calibration curve (Figure 2) was prepared from H202, aqueous glycerol and L-~-glycerophosphate standards.
Serum concentrations of total ~lycerol were determined from ; that curve. The DR resulting from application of a 10 ~
H20 sample to the coating was subtracted in each case. A
comparison of the results of serum triglyceride quantitation by this coated system and the semiautomated chemical method of Xessler and Lederer are shown in Table III. There was good ` 20 correlation between the two methods indicating that the coated element responded quantitatively to serum triglyceride : samples.

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An element as in Example 1 was tested in replicate following the procedure described ln Example 1. Two ' levels of serum triglyceride were used; a normal level con-taining 90 mg/dl and a high level containing 560 mg/dl. The results of this data are shown in Table IV.

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The results of these tests demonstrate the quan-titative response of the analytical elements of the present : invention when used in the analysis of liquids for their -; triglycerides content.
Although the elements of this invention have been - described herein in connection with their use in the assay of aqueous fluids for glycerol or triglyceride content it ~" should be apparent that elements can be prepared to quan-titate L-a-glycerophosphate, ATP or any compound which can be colpled to the production of one of these materials such as glycero-phospholipids, Phosphoenolpyruvate, liDase or glycerol kinase.
While the invention has been described in detail with particular reference to preferred embodiments thereof, it will be understood that variations and modifications can be effected within the spirit ar.d scope of the invention.

.

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-46- :
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Claims (29)

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

1. An integral multilayer element for the detection of triglycerides in a liquid sample, the element characterized by a spreading layer and a reagent layer, and containing (a) lipase having triglyceride hydrolysis capability;
(b) glycerol kinase;
(c) adenosine triphosphate;
(d) .alpha.-glycerophosphate oxidase; and (e) an electron acceptor all disposed within the element such that triglyceride contained in liquid applied to the spreading layer is hydrolyzed to glycerol, the resultant glycerol is converted to L-.alpha.-glycero-phosphate, and the resultant L-.alpha.-glycerophosphate is oxidized with concurrent reduction of the electron acceptor to provide a detectable change.

2. The element of claim 1 wherein the lipase has associated with it a protease or an effector which is a compatible surfactant.

3. The element of claim 2 wherein the electron acceptor is selected from the group consisting of 2,6-dichloro-phenolindolphenol, 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and oxygen,.

4. The element of claim 1 further including a support upon which the spreading layer and the reagent layer are super-posed, the reagent layer being interposed between the support and the spreading layer.

5. The element of claim 4 wherein the spreading layer contains the lipase and either a protease or an effector which is a compatible surfactant.

6. The element of claim 1 wherein the .alpha.-glycerophos-phate oxidase is derived from a microbial source selected from the group consisting of Streptococcaceae, Lactobacillaceae, and Pediococcus.

7. The element of claim 6 wherein the .alpha.-glycerophos-phate oxidase is from Streptococcus faecalis.

8. The element of claim 4 further including an indicator composition comprising a substance having peroxidative activity and a dye precursor which undergoes a detectable change in the presence of hydrogen peroxide and said substance having peroxidative activity.

9. The element of claim 8 wherein the substance having peroxidative activity is a peroxidase.

10. The element of claim 9 wherein the dye precursor comprises a leuco dye which is oxidized to form a colored dye in the presence of hydrogen peroxide and peroxidase.

11. The element of claim 9 wherein the dye precursor comprises material which is oxidized in the presence of hydrogen peroxide and peroxidase yielding a colorless product which in turn reacts with a coupler to yield a colored product in pro-portion to the amount of hydrogen peroxide present.

12. The element of claim 11 wherein the dye precursor comprises 4-aminoantipyrine and 1,7-dihydroxynaphthalene.

13. An integral multilayer element for analysis of triglycerides in a liquid sample, the element characterized by a spreading layer and a reagent layer, and wherein the element contains (a) lipase having triglyceride hydrolysis capability, (b) glycerol kinase, (c) adenosine triphosphate, (d) .alpha.-glycerophosphate oxidase, and (e) a hydrogen peroxide indicator composition all disposed within the element such that triglyceride contained in an applied liquid is hydrolyzed to glycerol, glycerol is converted to L-.alpha.-glycerophosphate, L-.alpha.-glycerophosphate is oxidized in the presence of an electron acceptor and hydrogen peroxide is formed which contacts the indicator composition and produces a detectable change.

14. The element of claim 13 wherein the lipase has associated with it a protease or an effector which is a sur-factant.

15. The element of claim 13 further including a support upon which the spreading layer and the reagent layer are superposed, the reagent layer being interposed between the support and the spreading layer.

16. The element of claim 15 wherein the spreading layer contains the lipase and either a protease or an effector which is a compatible surfactant.

17. The element of claim 16 wherein the lipase is a microbial lipase from a source selected from the group con-sisting of Rhizopus arrhizus, Candida rugosa and Chromo-bacterium viscosum.

18. The element of claim 16 wherein said protease is selected from the group consisting of chymotrypsin, elastase, papain, bromelain and the proteases from Streptomyces griseus, Aspergillus oryzae and Bacillus subtilis.

19. The element of claim 16 wherein the compatible surfactant is selected from the group consisting of octyl and nonyl phenoxy polyethoxy ethanols.

20. The element of claim 19 wherein the compatible surfactant has the hydrophile-lipophile balance number below about 15 and the number of carbon atoms in the polyoxyethylene chain is less than 20.

21. The element of claim 13 wherein the glycerol kinase is from E. coli or Candida mycodermea.

22. The element of claim 13 wherein the .alpha.-glycerophos-phase oxidase is derived from a microbial source selected from the group consisting of Streptococcaceae, Lactobacillaceae, and Pediococcus.

23. The element of claim 22 wherein the .alpha.-glycerophos-phate oxidase is from Streptococcus faecalis.

24. The element of claim 15 wherein the indicator composition comprises a substance having peroxidative activity and a dye precursor which undergoes a detectable change in the presence of hydrogen peroxide and said substance having peroxi-dative activity.

25. The element of claim 24 wherein the substance having peroxidative activity is a peroxidase.

26. The element of claim 25 wherein the dye precursor comprises a leuco dye which is oxidized to form a colored dye in the presence of hydrogen peroxide and peroxidase.

27. The element of claim 25 wherein the dye precursor comprises material which is oxidized in the presence of hydrogen peroxide and peroxidase to yield a product which in turn reacts with other material to yield a colored product.

28. The element of claim 27 wherein the dye precursor is selected from the group consisting of 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl)imidazole; 4-iso-proproxy-1-naphthol; and a combination of 4-amino-antipyrine and 1,7-dihydroxynaphthalene.

29. A multilayer element for the detection of glycerol in a liquid sample, the element characterized by a spreading layer and a reagent layer, and wherein the element contains (a) glycerol kinase, (b) adenosine triphosphate, (c) 4-glycerophosphate oxidase, and (d) a hydrogen peroxide indicator composition all disposed within the element such that glycerol contained in liquid applied to the element is converted to L-.alpha.-glycerophos-phate, the resultant L-.alpha.-glycerophosphate is oxidized in the presence of oxygen to form peroxide which contacts the indicator composition and produces a detectable change.