GB1590738A - Analytical element - Google Patents

Description

(54) ANALYTICAL ELEMENT (71) We, EASTMAN KODAK COMPANY, a Company organized under the Laws of the State of New Jersey, United States of America of 343 State Street, Rochester, New York 14650, United States of America do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement :- The present invention relates to elements for the essentially dry analysis of triglycerides or glycerol in aqueous solutions, such as blood serum.

The determination of serum triglyceride levels is 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. Intern. Med., 59, p. 465 (1963). Conventional procedures for serum triglyceride determination involve hydrolyzing the triglyceride to liberate glycerol and treating the glycerol with various reagents to produce a compound that can be quantitated spectrophotometrically. Generally hydrolysis is achieved using a base, however, U. S. Patent Nos. 3, 703, 591 and 3, 759, 793 describe enzymatic techniques using a lipase alone ('793) or in combination with a protease ('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. These are as follows : (a) Method of Garland and Randle (Garland, P.B.a dn Randle, P.J. Nature, 196 p.987 - 988 (1962))

glycerol kinase glycerol + ATP # L-α-glycerophosphate + ADP pyruvate kinase ADP + phosphoenolpyruvate # pyruvate + ATP lactate pyruvate + NADH # lactate + NAD+ dehy6drogenase (b) Weiland's Method (Weiland, O. Biochem Z., 329 p.313 (1957)

glycerol kinase glycerol + ATP # L-α-glycerophosphate + ADP α-glycerophosphate L-α-glycerophosphate + NAD+ # NADH + dihydroxyacetone phosphate + H+ dehydrogenase (c) Glycerol Dehydrogenase method (Hagen, J.H. and Hagen, P.B. Can. J. biochem. and Physiology, 40 p. 1129 (1962))

glycerol glycerol + NAD+ # dihydroxyacetone + NADH + H+ dehydrogenase Modifications of the method of (a) are also described in German Patent No. 2,665,556, U.K. Patent No. 1,322,462 and U.S. Patent No. 3,759,793. In all cases NADH production or disappearance is measured at 340 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 dehydrogenase 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, NADH, is a major concern. Errors in current enzymatic methods are discussed in greater detail in Chen, H.P. and El-Mequid, S.S., Biochemical Medicine, 7, p.460(1973).

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 resulting glycerol to formaldehyde and reaction of the formaldehyde with ammonia and a stable, water- and alcohol- soluble, colourless metal complex of acetylacetone to produce a coloured compound.

United Kingdom Patent No. 1,440,464 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 porour spreading layer in fluid contact or communication with a reagent layer which comprises at least one material interactive with a component or decomposition product of a component of the liquid.

Koditschek et al in the Journal of Bacteriololgy, 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 a-glycerophosphate oxidase, an enzyme which mediates electron transfer from a-glycerophosphate to oxygen with the concomitant production of hydrogen peroxide and dihydroxyacetone phosphate.

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 a multilayer analytical element comprising a spreading layer and a reagent-containing layer characterised in that the element contains (a) a glycerol kinase, (b) adenosine triphosphate, (c) an a-glycerophosphate oxidase and (d) an electron acceptor, the items (a) to (d) being disposed within the layers of the element such that any glycerol containing liquid applied to the spreading layer is converted to L-a-glycerophosphate by (a) with (b) and the L-a-glycerophosphate is oxidised by (c) with (d) to provide a detectable change. The reagent-containing layer composition is described and claimed in U.K. Patent Application 34974/77. (Serial No. 1590736) According to a preferred embodiment, the analytical element includes a further indicator composition comprising enzymes and/or reagents 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, preferably 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 discrete layer intervening the spread ing and reagent layers and all other interactive materials in a reagent layer.

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

In a highly preferred embodiment, the triglyceride hydrolyzing enzyme and any associated material are contained in a spreading layer overlyaing a reagent layer containing the interactive materials required for glycerol detection.

The analytical elements described herein will be referred to primarily as elements for the determination of triglycerides, however, it should be clear that they are similarly useful for the determination of glycerol, either with or without incorporated hydrolytic enzyme which hydrolyzes triglyceride, 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 United Kingdom Patent No. 1,440,464.

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, (2) oxidize glycerol, free in the liquid or liberated by such hydrolysis, and (3) provide detectable changes (e.g., colour production) related to the trig lyceride/glycerol content of the liquid, are incorporated into one or more layers of the element.

Reference herein to fluid contact between layers in an analytical element identifies the ability of a fluid, whether 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. Stated in another manner, fluid contact refers to the ability to transport components of a fluid between the layers in fluid contact. Although such layers in fluid contact can be contiguous, they may also be separated by intervening layers as described in detail hereinafter. 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 invention, triglycerides and/or glycerol are determined quantitatively by the series of reactions set out earlier.

In the combined reactions of the preferred composition, the amount of detectable species formed is proportional to glycerol and/or triglyceride concentration.

This 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 prescence of the oxidase to produce a detectable change (preferably an intermediate which in turn reacts to produce a detectable change) is potentially 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 oxygen or some other electron acceptor is the cofactor in the a-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 embodiment, triglycerides are hydrolyzed to free glycerol using any of the well known techniques described in the art which can be incorporated into the multilayer element. Enzymatic techniques are preferred. These generally involve treatment of the serum sample with a lipase preparation, either in combination with an effector such as protease or a surfactant or alone depending upon the nature of the triglyceride. Detailed discussions of such techniques and useful compositions for their performance are contained in U.S. Patent No. 3,703,591 and U.S. Patent No. 3,759,793. U.S. 3,703,591 uses a lipase preferably from Rhizopus arrhizus (var. delemar) and similar materials in combination with a protease to achieve hydrolysis of serum triglycerides while U.S. 3,759,793 discloses the use lipase from Rhizopus arrhizus alone to achieve hydrolysis. Since the lipase preparations, protease, 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 herein defined, of a lipase which may, of itself, not be capable of hydrolyzing protein associated triglycerides as found in serum or only capable of performing such hydrolysis at an unacceptably slow rate and, as an effector, a surfactant. Such compatible mixtures of a lipase and a surfactant are described and claimed in U.K. Patent Application 34975/77 (Serial No. 1590737).

Useful lipases preparations for triglyceride hydrolysis may be derived from plant or animal sources but we prefer preparations from microbial sources such as from Candida rugosa, Chromobacterium viscosum, variantparalipolyticum, crude or purified. Other useful enzyme preparations and methods for their preparation are described in the following U.S. Patents: 2,888,385; 3,168,448; 3,189,529; 3,262,863; and 3,513,073.

Specifically preferred commercial enzyme preparations include wheat germ lipase from Miles Laboratories of Elkhart, Indiana, Lipase 3000 from Wilson Laboratories, Stepsin from Sigma Chemical Company (both of the latter are pancreatic enzymes), and Lipase M (from Candida rugosa) from Enzyme 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.

Certain surfactants, however, inhibit the hydrolase activity of certain enzyme preparations.

For example, microbial enzyme from Rhizopus arrhizus is inhibited by octyl phenoxy polyethoxy ethanol surfactants. Consequently, it is important that before any attempt is made to combine an enzyme preparation and a surfactant for use as described herein some determination of the compatibility of the two members of the composition be made. Such a determination is preferably made by using the test described below. An enzyme preparation and surfactant mixture which successfully meet this test are referred to herein as a compatible mixture and each member thereof is said to be compatible with the other.

Compatible compositions of lipase and surfactant according to the present invention are defined by the following test. The 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., U.S.A.) at varying concentrations of between 0 and 10% by weight and the solution incubated for 5 minutes at 37"C. At this time, a sample of the proposed lipase preparation is added and incubation continued for a period of 20 minutes. Aliquots (NO.2 ml) of this solution are then diluted to 1.6 ml with water (containing 1.3 mM calcium chloride to aid precipitate formation), placed in a boiling water bath for 10 minutes and spun in a refrigerated centrifuge to clarify (0 C, 37,000 Xg, 10 minutes). Glycerol in a 0.4 ml aliquot of the clear supernatant is quantified in a total volume of 1.2 ml by the method described by Garland, P.B. and Randle, P.J., Nature, 196, 987-988 (1962). When performing the foregoing test it is most desirable to run a blank which contains all of the components of the mixture but the enzyme preparation so that any reaction which may be due to free glycerol or other components of the serum can be subtracted. Any composition which effects release of amounts of glycerol greater than those released by the control is considered useful, preferably at least 50% of the theoretical concentration of available glycerol is released. The preferred compositions accomplish hydrolysis of at least 70%, preferably 75%, of the available triglyceride in less than 10 minutes and most preferred are those which achieve substantially complete hydrolysis, i.e., above 90% hydrolysis, of the available triglyceride to glycerol in less than 10 minutes.

Among the surfactants which have been found useful to stimulate the hydrolase activity of the foregoing useful enzyme preparations are nonionic and anionic surfactants including many of the natural surfactants such as the bile salts including deoxycholate, chenodeoxycholate, cholate and crude bile salt mixtures and synthetic surfactants such as sodium salts of alkylaryl polyether sulphonates commercially available from Rohm and Haas Company under the Trade Mark 'Triton' X-200 and E.I. duPont deNemours and Company under the Trade Mark 'Alkanol' XC alkyl phenoxy polyethoxy ethanols such as those available commercially from Rohm and Haas Company under the Trade Marks 'Triton' X-114, 100, 102 and 'Triton' n-101. Synthetic surfactants are preferred due to the large selection of such materials which are available and the ability to tailor them to meet specific needs and requirements. Preferred alkyl phenoxy polyethoxy ethanols comprise a polyoxyethylene chain of less than about 20 oxyethylene units and have a hydrophile-lipophile balance number below 15. Other 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 Trade Mark "Pronase"), proteases from Aspergillus oryzae and Bacillus subtilis, elastase, papain and bromelain. Mixtures of such enzymes may, of course, also be used.

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 and the nature of the triglyceride, and these are readily determined by the skilled 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-a-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 E. coli and Candida mycodermea are preferred.

Other glycerol kinase enzymes are well known in the art. A complete discussion of such materials and further references to their preparation and reactivity may be found in T.E.

Barman, Enzyme Handbook, 1, Springer-Verlag, N.Y. (1969) pgs. 401-402. Glycerol kinase from Worthington Biochemical Company provides a satisfactory commercial source of the enzyme.

The next step in the reaction sequence involves the oxidation of L-a-glycerophosphate in the presence of L-a-glycerophosphate oxidase and an electron acceptor to produce a detectable change. The detectable change is preferably a colour change or colour 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 coloured product or a chemical intermediate which, although not itself coloured, can be detected via a reaction or reactions which result in colour 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 La-glycerophosphate in the presence of the oxidase to dihydroxyacetone phosphate and hydrogen peroxide. Methods for determining the presence or amount of hydrogen peroxide in reactions of this type are, of course, well known. An alternative preferred embodiment uses as electron acceptor material coloured or uncoloured which undergoes a change in or the production of colour 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-dichlorophenolindolphenol alone or in combination with phenazine methosulphate and 2 (p-indophenyl)-3-(p-nitrophenyl)-5-phenyl 2H-tetrazolium chloride either alone or in combination with phenzine methosulphate have been found useful as electron acceptors in this reaction.

L-a-glycerophosphate oxidase is a microbial enzyme 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, Lactobacillaceae and Pediococcus. The enzyme from cultures of Streptococcus faecalis, specific strains of which are obtainable from the American Type Culture Collection, are specifically preferred. Particularly useful and preferred enzymes are obtained from strains 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 pH range than enzymes derived from the other two strains and for this reason is most preferred.

The following two references describe both the enzyme and useful techniques for its preparation and extraction: Koditschek, L.K. and Umbreit, W.W. a-glycerophosphate Oxidase in Streptococcus faecium, F24, Journal of Bacteriology, Vol. 98, No. 3, p.1063-1068 (1969) and Jacobs, N.J. and Van Demark, P.J. "The Purification and Properties of the a-glycerophosphate Oxidizing Enzyme of Streptococcus faecalis, 10 C1." 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 with assay results. For example, certain preparations of L-a-glycerophosphate oxidase, derived as described below, contained sufficiently high concentrations of impurities that the crude preparation had to be purified using conventional fractionation and column separation techniques before assays of blood serum triglycerides free from unwanted interferences could be achieved.

The detection of glycerol in acqueous 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 La-glycerophosphate. Indicator compositions for the detection of enzymatically 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 readily incorporated into elements of the type described herein using the methods outlined hereinafter.

The hydrogen peroxide indicator composition generally comprises a substance having peroxidative activity, preferably peroxidase, and an indicator material (i.e., a chromogen) which undergoes a colour formation or change in the presence of hydrogen peroxide, such as a dye precursor, and the substance having peroxidative activity. Alternatively, the dye precursor may be one or more substances which undergo no substantial colour change upon oxidation in the presence of hydrogen peroxide and peroxidase, but which in their oxidized form react with a colour-forming or colour-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 colour by virtue of a coupling reaction, is preferred in the practice of the present invention.

A peroxidase is an enzyme which will catalyze a reaction wherein hydrogen peroxide or other peroxide oxidizes another substance. The peroxidases are generally conjugated proteins containing iron porphyrin. Peroxidase occurs in horseradish, potatoes, figtree sap and turnips (plant peroxidase ; 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 haemin, methaemoglobin, oxyhaemoglobin, haemoglobin, haemochromogen, alkaline haematin, haemin derivatives, and certain other substances which have peroxidative activity.

Other substances which are not enzymes but which have peroxidative activity are: iron sulphocyanate, iron tannate, ferrous ferrocyanide and chromic salts (such as potassium chromic sulphate) absorbed in silica gel. These substances are not satisfactory as peroxidase per se but are similarly useful.

Dye precursors which produce colour 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 and para-toluidine; 2 Diamines, such as ortho-phenylenediamine, N,N'-dimethyl-para-phenylenediamine, N,N'-diethyl phenylenediamine, benzidine and dianisidine; (3) Phenols, such as phenol per se, thymol, ortho-meta-and para-cresols, alpha-naphthol and beta-naphthol; (4) Polyphenols, such as catechol, guaiacol, orcinol, pyrogallol, p,p-dihydroxydiphenyl andhloroglucinol; Aromatic acids, such as salicylic, pyrocatechuic and gallic acids; 6Leuco dyes, such as leucomalachite green and leucophenolphthalein; (7) Coloured 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-ethyl-benzothiazoline-(6)-sulphonic acid) and 3,3 '-diaminobenzidine.

Other indicator compositions that are oxidizable by peroxides in the presence of peroxidase and can provide a detectable species include a compound that, when oxidized in 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 orthoaminophenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, cresols, pyrogallol, guaiacol, orcinol, catechol phloroglucinol, p,p-dihydroxydiphenyl, gallic acid, pyrocatechuic acid and salicylic acid. Compounds of this type are well known and described in the literature, such as in The Theory ofthe Photographic Process, Mees and James Ed, (1966), 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 with appropriate substituent groups thereon. The non-stabilized oxichromic compounds described in U.S. Pa such couplers, including a number of autocoupling compounds, is described in the literature, such as in Mees and James (supra) and in Kosar, Light-Sensitive Systems, 1965, 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-aminoantipyrine (HC1); 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, 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, or other complex aqueous solution of 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.

Table I Generally useful Preferred Enzyme ranges U/m2 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 72,000-90,000 Peroxidase 3,000-11,000 6,000-7,500 g/m2 g/m2 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 international unit of enzyme is defined as that quantity of enzyme which results in the conversion of one micromole of substrate in one minute at 370C 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-glycerophosphate oxidase peaks at between about pH 5 and 8.5. The optimum pH range over which each of the enzymes in the novel reaction sequence is active is shown in Table II.

Table II pH-value Lipase 5-9 Glycerol kinase 7-9 L-a-glycerophosphate oxidase 6.3-8.0 Peroxidase 6-8 From the foregoing table, it is readily apparent that it is generally desirable to buffer the layer(s) of the elements described herein which contain the respective reagents at pH levels of between 6.0 and 8.0 and most preferably between 7.0 and 8.0. 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 the art and involve dissolving or dispersing suitable concentrations of buffer in the compositions which are subsequently dried to form the layered element. Suitable buffers for buffering to the aforementioned pH levels are described in detail by Good in Biochemistry 5, 467 (1966).

Particularly preferred buffers are the phosphates such as potassium phosphate.

One form of the multilayer analytical element according to the invention is shown in Figure 1. In this figure a support 10 carries a reagent layer 11. This layer may be a composite of two or more layers. The reagent layer 10 carries a subbing layer 12 over which is coated a porous spreading layer 13.

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 component such that a uniform apparent concentration of such component is provided at the surface of the spreading layer facing the ragent layer(s) of the element. It should be understood that the uniformity of such concentration is a perceived uniformity as measured by techniques like those described hereinafter.

In the context of this invention, spread sample components will, of course, include one or more of 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 thickness of, or otherwise in, the spreading layer.

Such gradients do not present any difficulty to obtaining quantitative test results if 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 isotropic 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 Technology, James Flinn Ed, Plenum Press, New York (1970).

As will be appreciated, the extent of spreading is dependent 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 irrespective 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 of preferred spread times. Because the elements of this invention are able to produce quantitative results using very small sample volumes that can be entirely taken up with a conveniently sized region of the spreading layer (e.g., one square centimetre), there is no need to remove excess moisture from the element after application of a liquid sample. Further, because spreading occurs in the spreading 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 and U.K. Patent 1,440,464.

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 titanium dioxide, barium sulphate, zinc oxide and lead oxide, 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 Macromolecular Phenomena, Part II, Novel Microcrystals of Polymers" by O.A. Battista et al published in the Journal of applied Polymer Science, Vol. II, pages 481-498 (1967). Microcrystalline cellulose, which is commercially available from FMC Corporation under the trade mark 'Avicel', 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 with 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 (i.e., binder) coating dries, the layer integrity is maintained and open spaces remain between its component particles.

As an alternative or in addition to such 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 coated 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 polymer formed is an isotropically porous layer. Many different polymers can be used, singly or in combination, for preparing isotropically porous "blush" 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, it is preferred that the material of the spreading layer be substantially non-fibrous to avoid wicking effects which tend to produce apparent non-uniform distributions of analyte between and along fibres and mottle when spreading layers of fibrous material are used as the background or milieu of spectrophotometric measurements.

The Reagent Layer(s): 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 permeablility 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 enzymes 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 registration 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 and a-glycerophosphate oxidase, which is not consumed 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 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 uniform permeability, undesirable concentration 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 element as well as the particular interactive materials which 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 dectran, gum arabic and agarose, and also synthetic substances such as water-soluble polyvinyl compounds like polyvinyl alcohol) and polyvinyl pyrrolidone) and acrylamide polymers. Organophilic materials such as cellulose esters can also be useful, and the choice of materials in any instance will reflect the use parameters for any particular element. For example, if a protease is used to assist in hydrolysis of triglycerides as described below, gelatin is not a particularly suitable reagent matrix. To enhance permeability of the reagent layer, if not initially porous, it is often useful to use a matrix material that is moderately swellable in the solvent or dispersion medium of liquid under analysis.

In addition to its permeability, the reagent layer is desirably substantially free from any characteristic that might appear as or contribute to mottle or other noise in the detection of an analytical result produced in an integral element of the invention. For example, variations in colour or in texture within the reagent layer, as may occur when fibrous materials such as papers are used as a permeable medium, may be disadvantageous due to non-uniform 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 exhibit widely ranging degrees of permeability between regions of the paper, for example, based on structure variations such as fibre dimensions and spacing. As a result, such materials 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 reagent layers of elements absorbed herein are prepared using non-fibrous materials. It should be appreciated that the use of fibrous constituents, such as in appropriate combination with the non-fibrous materials, may be desirable.

Supports: The integral analytical elements of the present invention can be selfsupporting or the spreading layer, reagent layer and any other associated 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 such as polystyrenes.

The support can be opaque or it can transmit light or other energy depending, of course, on the mode of detection used. A support of choice in any case will be compatible with the intended mode of result detection. Preferred supports include transparent support materials capable of transmitting electromagnetic radiation of a wavelength within the region between 200 nm and 900 nm. Transparent supports need not, of course, transmit over the entire 200-900 nm region but may transmit only in the region of the indicating radiation. When an element includes a support, the reagent layer is interposed in the element between the support and the spreading layer. Specifically preferred transmission ranges for elements of the present invention will be apparent from the discussion of the various preferred indicator compositions discussed above. When used, supports having thicknesses of between about 1 and about 10 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. If a support is used, measurement will usually be made through the support. The reflecting layer permits the passage of triglycerides, glycerol and/or decomposition products of glycerol to the reagent or indicator layer (i.e., a layer underlying a reagent layer containing enzymes which catalyze the decomposition of 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 reflective layer will normally intervene the spreading and reagent or registration layers. Such a layer may, however, intervene a 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 suitable 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 polymer 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 1 to 10 parts by weight of pigment per part by weight of blush polymer are preferred, with from 3 to 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 and, when present, they serve to remove from the sample components which could interfere with the indicating reaction or otherwise hinder the determination. Thus, in the use of the multilayer analytical element for analysis of triglycerides in whole blood, a separate filtering layer could serve to remove red blood cells while transmitting 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 filtering layer have a pore size of from 0.5 to 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 consequently the hydrolyzing composition in a gelatin or similar reagent matrix, 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 from 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 of the hydrolyzing composition so that indication requires only that the relatively small hydrogen peroxide molecules 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 incorpo rated 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. Preferred materials include hydrophilic polymeric materials which permit migration of the hydrogen peroxide or glycerol as just described in the desired fashion 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 invention, 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. If machine coating techniques are used, it is often possible to coat adjacent layers simultaneously, 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 spreading, 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 both of the discrete layers may contain dispersed therein a reflective pigment such as titanium dioxide.

The physical structure of layers prepared in this 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 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 of solvents when cellulose acetate is used as the binder comprises acetone, xylene and dichloroethane in ratios of from about 3.5:2:1.1 to 4.5:1:0.

The thickness of the spreading layer can be varied and will depend in part on the intended sample volume, which for convenience and cleanliness the spreading layer should be able to absorb, and on the layer's void volume, which also affects the amount of sample that can be absorbed into the layer. Spreading layers having thicknesses in the range of from 50 microns to 300 microns have been particularly useful, although other thicknesses are acceptable and may be desirable for particular elements.

When preparing an isotropically porous spreading layer, it is useful to have void volume comprise at least 25% of the total layer volume, and void volumes of from 50-95 % may be desirable. Variations in void volume of porous spreading layers can be used advantageously to modify element characteristics such as total permeability of the spreading layer or the time needed for sample spreading to occur. As can be appreciated, void volume within the layer can be controlled, for example, by selecting particulate materials of appropriate size, or by varying the solvents or drying conditions when isotropically porous blush" polymers are used in the spreading layer. The void volume of any such layer can be calculated with reasonable accuracy by a variety of techniques, such as the statistical method described in Chalkley, Journal of the National Cancer Institute, 4, 47 (1943) and by direct weighing 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 tht 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 interactive materials in the element.

For reagent layers, a coating solution or dispersion including the matrix and incorporated interactive materials can be prepared, 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 10 microns to 100 microns have been found useful.

The hydrolyzing composition may be incorporated into the reagent layer. However, according to a highly preferred embodiment of the present invention, the hydrolysis composition is incorporated into the spreading layer, for example, by dispersing 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 accomplished 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 utilizes 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 after spreading is complete.

Wherever the enzymatic hydrolyzing composition is incorporated, optimum results are achieved when the composition is buffered to a pH of between 6 and 8.0 and preferably between 7.0 and 8.0. Thus, when the hydrolyzing composition is incorporated into the reagent layer, or into another layer with the glycerol detecting reagents, a pH of 7.0 produces optimum results. Similar pH values are used when the hydrolyzing 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, concentr ethanol (0. 4 g/m2), peroxidase (6994 U/m2), adenosine 5'-triphosphate disodium salt (1.3 g/m2), a-glycerophosphate oxidase (1506 U/m2) and glycerol kinase (645 U/m2) (glycerol kinase from Worthington Biochemical Company provides a satisfactory commercial source of the enzyme) in 0.1 M potassium phosphate buffer at pH 7. A layer comprising 0.3 g/m2 of poly-n-isopropylacrylamide is applied over the titanium dioxide 6.5 g/m cellulose acetate, 900 U/m of lipase and 5.4 g/m2 of 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 370C. Atmospheric oxygen acted as the electron acceptor.

Serum samples (10 Zl aliquots) were applied to the above coating and the reflection densities DR at 660 nm were measured after 8 minutes at 370C.

A calibration curve, (see Figure 2), qas prepared from hydrogen peroxide, aqueous glycerol and L-a-glycerophosphate standards. Serum concentrations of total glycerol were determined from that curve. The curve shows Dr. measured at 660 nm plotted against the triglyceride content of the samples in milligrams per decilitre. The Dr resulting from application of a 10 il water 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 Kessler and Lederer are shown in Table III. There was good correlation between the two methods indicating that the coated element responded quantitatively to serum triglyceride samples.

Table III Comparison of Serum Triglyceride Quantitation by the Coated Element and a Semi-Automated Chemical Method Triglyceride Concentration mg/dl Kessler and Coated a-glycerophosphate Serum Sample Lederer System 1 115 105 2 260 275 3 490 450 Example 2 An element prepared as described in Example 1 was tested in replicate following the procedure described in Example 1. Two levels of serum triglyceride were used; a normal level containing 90 mg/dl and a high level containing 560 mg/dl. The results of this data are shown in Table IV.

Table IV Triglyceride level 90 mg/dl 560 mg/dl DR DR Sample 1 0.519 1.146 2 0.519 1.169 3 0.532 1.123 4 0.572 1.130 Mean 0.5355 1.142 Coefficient of Variation % 4.65 1.47 DR = Reflection density at 540 nm.

The results of these tests demonstrate the quantitative 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 quantitate L-a-glycerophosphate, ATP or any compound which can be coupled to the production of one of these materials such as glycero-phospholipids, phosphoenolpyruvate, lipase or glycerol kinase.

Claims (24)

WHAT WE CLAIM IS:-

1. A multilayer analytical element comprising a spreading layer and a reagent-containing layer characterised in that the element contains (a) a glycerol kinase, (b) adenosine triphosphate, (c) an o-glycerophosphate oxidase and (d) an electron acceptor, the items (a) to (d) beind disposed within the layers of the element such that any glycerol containing liquid applied to the spreading layer is converted to L-a-glycerophosphate by (a) with (b) and the L-a-glycerophosphate is oxidised by (c) with (d) to provide a detectable change.

2. The element as claimed in Claim 1 in which the spreading layer and the reagent layer are superposed and the layer combination is carried by a support with the spreading layer outermost.

3. The element as claimed in Claim 1 or 2 which contains a lipase having triglyceride hydrolysis capability disposed within the layers of the element such that any triglyceride containing liquid applied to the spreading layer is hydrolysed to glycerol.

4. The element as claimed in Claim 3 in which the spreading layer contains the lipase and a protease.

5. The element as claimed in Claim 4 in which the protease is chymotrypsin, elastase, papain, bromelain or a protease from Streptomyces griseus, Aspergillus oryzae or Bacillus subtilis.

6. The element as claimed in Claim 3 in which the spreading layer contains a mixture of the lipase and a compatible surfactant as herein defined.

7. The element as claimed in Claim 6 in which the compatible surfactant is an octyl or nonyl phenoxy polyethoxy ethanol.

8. The element as claimed in Claim 7 in which the compatible surfactant has less than 20 carbon atoms in the polyoxyethylene chain and the hydrophile-lipophile balance number is less than 15.

9. The element as claimed in any of the preceding Claims in which the lipase is derived from Rhizopus arrhizus, Candida rugosa or Chromobacterium viscosum.

10. The element as claimed in any of the prceding Claims in which the a-glycerophosphate oxidase is derived from a microbial source belonging to the genus Streptococcaceae, Lactobacillaceae, or Pediococcus.

11. The element as claimed in Claim 10 in which the a-glycerophosphate oxidase is derived from the species Streptococcus faecalis.

12. The element as claimed in any of the preceding Claims in which the electron acceptor is 2,6-dichlorophenolindolphenol, 2-(p-indophenol)-3-(p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride or oxygen.

13. The element as claimed in any of the preceding Claims which contains an indicator composition which provides a colour change in response to the presence of hydrogen peroxide.

14. The element as claimed in Claim 13 in which the indicator composition includes a substance having peroxidative activity and dye precursor composition.

15. The element as claimed in Claim 14 in which the substance having peroxidative activity is a peroxidase.

16. The element as claimed in claims 14 or 15 in which the dye precursor composition is a leuco dye oxidisable to a coloured dye in the presence of hydrogen peroxide and peroxidase.

17. The element as claimed in Claim 16 in which the dye precursor composition is 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl)imidazole or 4-isopropoxy- 1 -naphthol.

18. The element as claimed in Claims 14 or 15 in which the dye precursor composition is a material which is oxidisable in the presence of hydrogen peroxide or peroxidase to yield a colourless compound together with a coupler with which it reacts to form a coloured compound.

19. The element as claimed in Claim 18 in which the dye precursor composition is 4-amino-antipyrine and 1,7-dihydroxynaphthalene.

20. The element as claimed in any of the preceding Claims 2 to 19 in which the support is cellulose acetate or poly(ethylene terephthalate).

21. The element as claimed in any of the preceding Claims in which the spreading layer is a porous blush polymer layer.

22. Multilayer analytical elements as claimed in Claim 1 and as herein described.

23. The method of determining the amount of glycerol in an aqueous liquid sample comprising placing some of the liquid on the spreading layer of an element as claimed in any of the Claims 1 to 22 and measuring the detectable change caused by the oxidation of the a-glycerophosphate formed therein.

24. The method of determining the amount of glycerol and triglycerides in an aqueous liquid sample comprising placing some of the liquid on the spreading layer of an element as claimed in any of the Claims 3 to 22 and measuring the detectable change caused by the oxidation of the a-glycerophosphate formed therein.