GB1590736A - Chemical assay method - Google Patents

Description

(54) CHEMICAL ASSAY METHOD (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, 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 the analysis of aqueous liquids for triglyceride and/or glycerol content and more specifically to improved methods and compositions for the assay of blood serum triglycerides.

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. and Randle, P.J. Nature, 196 p. 987-988 (1962))

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

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

glyceol + NAD+ glycerol 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 of 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 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.

The present invention provides improved methods and compositions for the quantitative determination of glycerol and triglycerides, especially serum triglycerides, which methods and compositions are relatively free of any requirement for strict and narrow pH control and major concerns for reagent stability.

According to the present invention there is provided a method of detecting or assaying glycerol in aqueous liquid samples comprising treating the sample with a reagent composition whereby the glycerol is converted to L-a-glycerophosphate, the L-a-glycerophosphate is oxidised to liberate hydrogen peroxide and the hydrogen peroxide is allowed to react in a manner which provides a detectable change.

Free glycerol or glycerol formed by hydrolysis of fatty acid esters of glycerol, e.g., triglycerides is detected and assayed in aqueous liquids by the following steps: (I) contacting in the presence of an electron acceptor; 1 a sample of the aqueous liquid; and 2 enzymes and other reagents which effect an ordered sequence of reactions, pref erably quantitiative, wherein fatty acid esters of glycerol, if present, are enzymat ically hydrolyzed to glycerol, glycerol whether present in the free form initially or liberated by hydrolysis of the esters is converted to L-a-glycerophosphate which in turn is enzymatically oxidized, producing a detectable change; and (II) detecting the occurrence of the detectable change. According to a preferred embodiment, the electron acceptor is oxygen and an indicator composition which produces a detectable product on contact with hydrogen peroxide is included as a reagent. The detectable product is generally a colored material, which, according to a highly preferred embodiment, is quantifiable.

According to a further preferred embodiment, triglycerides present in the aqueous solution are first hydrolysed using a lipase to liberate glycerol.

According to yet another preferred embodiment glycerol is converted to L- a-glycerophosphate using glycerol kinase and the oxidation of L-a-glycerophosphate takes place in the presence of L-a-glycerophosphate oxidase.

A most preferred embodiment uses an indicator composition comprising substance having peroxidative activity and a dye precursor; the dye precursor comprising either (1) a compound which forms a dye in the presence of hydrogen peroxide and substance having peroxidative activity or (2) a compound or series of compounds which undergoes no detectable change in the visible range in the presence of hydrogen peroxide and substance having peroxidative activity but which interacts with another compound or series of compounds to produce a quantifiable product proportional to the glycerol or triglyceride content of the sample under analysis.

The method of this invention represents an improvement over prior art methods and compositions in that the present method and compositions do not rely on the production or disappearance of NADH with its attendant disadvantages which are well recognized and documented in the art.

In the combined reactions of the preferred composition, formation of the detectable species is proportional to glycerol and/or triglyceride concentration. This system has potential use in many clinical applications, in particular, the determination of blood serum triglycerides.

The procedure of this invention has many inherent advantages over conventional methods. First, any leuco dye that peroxidase will utilize as an electron donor 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 dye selection. Secondly, measurements made in the visible region are less subject to interferences than those taken at 340 nm. Third, in addition to dyes, substantially any means for detecting hydrogen peroxide can be used. Fourth, stability of NAD+ or NADH is not a concern since oxygen is the cofactor in the a-glycerophosphate oxidase reaction. Fifth, 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 instant procedure.

Sixth, any means which measures oxygen consumption can be used as a detection means when oxygen is used as the electron acceptor. Finally, the enzymes used in the proposed sequence are active over a relatively wide pH range; thus stringent pH control is not necessary.

Although the discussion hereinafter will centre primarily around solutions and solution methods for quantifying glycerol and triglycerides, it should be readily a parent to the skilled artisan that all of the reagents may be provided in dry of lyophilized form and reconstituted with water immediately prior to use. Compositions of this type are clearly contemplated hereby.

Hydrolysis: In its most sophisticated embodiment the method of the present invention is utilized to assay aqueous liquids, for example blood serum, for triglyceride content. According to this embodiment triglycerides are hydrolyzed to free glycerol by means of any of the well known techniques described in the art. Enzymatic techniques are preferred. These generally involve treatment of the serum sample with a lipase, either in combination with an effector such as a protease of 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 of lipase from Rhizopus arrhizus alone to achieve hydrolysis.

A further method involves the hydrolysis of serum triglycerides using 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. This process of hydrolysis is described and claimed in U.S. Patent application No. 34975/77 Serial No. 1590737. A compatible surfactant is one which stimulates triglyceride hydrolysis by the lipase as described in the test below. Thus, such a surfactant will not inhibit the activity of the lipase, but actually enhance it. The lipase is preferably from Candida cylindraces (Candida rugosa).

Useful lipases for triglyceride hydrolysis according to any of the foregoing techniques may be of plant or animal origin, but we prefer and find best, microbial lipases, such as the lipase from Candida cylindraces (Candida rugosa), when the lipase is used in combination with a surfactant as described below. Lipases from Chromobacterium viscosum, variant paralipolyticum crude or purified, the lipase from Rhizopus arrhizus (variant delmar), purified, for example as noted in Fukumoto et al, J. Gen. Appli. Microboil, 10, 257-265 (1964) and lipase preparations having similar activity are also useful.

Other useful lipases 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.

Since the lipases are readily available in lyophilized form, are easily incorporated into either dry mixtures for reconstitution with water or provided as stable solutions of reagent which can be combined with other such solutions to provide reaction mixtures for contact with samples for analysis.

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.

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.

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 1.3mM calcium chloride to aid precipitate formation), placed in a boiling water bath for 10 minutes and centrifuged to clarify (0 C 37,000 Xg, 10 minutes). Glycerol in a 0.4 ml aliquot of the clear supernatant is quantified in 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.

Examples of such preferred compositions are shown in Table II below.

When the protease-lipase combination is used for hydrolysis, proteases in general may be used. 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, be employed.

The useful concentrations of lipase and other effectors such as surfactants or protease, will vary broadly depending upon 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 nonlimiting examples of useful concentrations are described in the examples below.

Hydrolysis of triglycerides can also be achieved using any of the well known "nonenzymatic" techniques for obtaining the free glycerol prior to assay, including treatment with a strong base. Caution must be exercised, however, to insure that the glycerol is delivered to the enzymatic glycerol assay composition in a medium which does not contain materials which would inhibit the enzymes of the glycerol assay system or otherwise interfere with the reactions necessary to achieve an accurate glycerol determination.

Glycerol Assay Once triglyceride hydrolysis has been achieved, the enzymatic glycerol assay of the present invention can be implemented.

As shown in the reaction above, the first enzyme used in the glycerol assay is glycerol kinase 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 mycoderma 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, I. 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 provide, directly or indirectly, a radiometrically detectable, preferably coloured product. 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 hydrogen peroxide and measuring the consumption of oxygen 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 directly 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. Such an environment is described in Example 5 below.

Using this method certain indolphenols, potassium ferricyanide and certain tetrazolium salts have been found to be useful electron acceptors. Specifically, 2,6dichlorophenolindolphenol alone or in combination with phenazine methosulphate and 2-(p-iodophenyl)-3-(p- nitrophenyl)-5-phenyl-2H-tetrazolium chloride either alone or in combination with phenazine methosulphate have been found useful as electron acceptors in this reaction.

The detectable change may also be determined using potentiometric techniques, for example, by measuring oxygen consumption using an oxygen electrode.

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, F 24", Journal of Bacteriology, Vol. 98, No. 3, p. 10631068 (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.

Detection of glycerol in aqueous solutions containing glycerol and/or triglycerides, for example blood serum, is preferably achieved using an indicator composition which detects the level of hydrogen peroxide produced in the oxidation of L-a-glycerophosphate in the presence of oxygen. 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.

The hydrogen peroxide indicator composition generally comprises a substance having peroxidative activity, preferably peroxidase, and a dye precursor which undergoes a colour 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 substantial colour change upon oxidation in the presence of H202 and substance having peroxidative activity, but which in their oxidized form react with a colourforming or -changing substance (e.g., a coupler) to give visible 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, chromic salts (such as potassium chromic sulfate) absorbed in silica gel, etc. These substances are not satisfactory as peroxidase, per se, but are similarly useful.

Dye precursors which produce a colour formation 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 phenyleneidamine, 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 and hloroglucinol; 5 Aromatic acids, such as salicylic, pyroacetechuic and gallic acids; 6 Leuco 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- ethylbenzothiazoline-(6)-sulphonic acid) and 3,3'-diaminobenzidine.

Other indicator compositions that are oxidizable by peroxides in the presence of peroxidase and can provide a radiometrically detectable species include certain dye-providing compositions. In one aspect indicator compositions can include a compound that, when oxidized in the presence of peroxidase, can couple with itself or with its reduced from 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 salicyclic acid. 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, (1966), especially at Chapter 17. In another aspect, the detectabel change can be provided by oxidation of a leuco dye in the presence of peroxidase to provide the corresponding dyestuff form. Representative leuco dyes include such compounds as leucomalachite green and leucophenolophthalein. 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. Patent No. 3,880,658 are considered preferable in the practice of this invention. In yet another aspect, the detectable change can be 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 benzidine and its homologs, p-phenylenediamines, p-aminophenols, and 4-aminoantipyrine. 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-Sensitive Systems, 1965, pages 215-249.

The indicator composition of the present invention preferably comprises 4methoxy-l-naphthol which undergoes self coupling in its oxidized state or a combination of 1,7-dihydroxynaphthalene and 4-aminoantipyrine hydrochloride. In the latter composition the oxidized pyrine compound couples with the dihydroxynaphthalene. 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, 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.

Of course, other means for detecting hydrogen peroxide may also be used in the successful practice of the present invention. For example, enzymes and other reagents are described herein can be incorporated into membranes of oxygen sensitive polarographic electrodes as described in Rawls, Rebecca L., "Electrodes Hold Promise in Biomedical Uses," Chemical and Engineering News, January 5, 1976, p. 19.

As a further alternative, instead of measuring the hydrogen peroxide produced, it is also possible to measure oxygen consumption using an oxygen sensitive electrode and thereby determine the quantity of glycerol produced in above-described reaction (1) of Table I which would result in the consumption of that quantity of oxygen in reaction (3) in Table I.

The concentration of the other components of the assay compositions according to the invention described herein 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 II 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 II Generally useful Preferred Enzyme range U/ml level U/ml Lipase (when used) 20-160 80 Glycerol kinase 0.05-1 0.2 Glycerophosphate 1-10 4 oxidase Protease (when used) 300-2400 1200.0 Peroxidase 0.2-1.4 0.7 g/ml g/ml Surfactant (when used) 0.01-.05 .02 Of course, useful results may be obtained outside of these ranges.

In the foregoing Table II, 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 37"C and pH 7.

As is well recognized in the art, each of the enzymes possesses a activity profile, i.e., the activity of the enzyme varies with pH. These data are described in detail for a-glycerophosphate oxidase in the Examples. As demonstrated by that data, the pH activity profile of L-a-glycerophosphate oxidase peaks at between pH 5 and 8.5. The pH range over which each of the enzymes in the novel reaction sequence is most active is shown in Table III.

TABLE III 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 most desirable to buffer the assay compositions described herein at a pH of between 6.0 and 8.0 and most preferably between 7.0 and 8.0. Techniques for achieving this type of buffering are well known in the art and involve dissolving, dispersing, or otherwise distributing, suitable concentrations of buffer materials in the reagent composition or, alternatively, providing them in dry form when a reconstitutable mixture is provided. 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.

The concentration of detectable species produced can, of course, be detected using any of the well known methods. For example, by comparison to a standard colour chart and spectro photometrically.

The following enzyme preparation techniques and standardized procedures and composi tions were used in the examples which follow.

Standard Solutions - Exact concentrations of glycerol standard solutions were determined by the method of Garland and Randle (Nature, 196, 987-988 (1962)). Hydrogen peroxide solutions were standardized by measuring the A240 (optical absorbance at 240 nm) and using E240 =43.6 for pertinent calculations. Serum samples were analyzed for triglyceride concentration by the semi-automated fluorometric method of Kessler and Lederer (Fluorometric Measurement of Triglycerides, Automation in Analytical Chemistry, Techni can Symposia, L.T. Sheggs, Jr., Ed. Medical Inc., N.Y., N.Y. 341 (1966)).

Glycerol and Triglyceride Quanfltaflbn by the a- GP Oxidase Method - Incubation mix tures for glycerol detection contained in a total volume of 1.0 ml; 200 moles potassium phosphate buffer, pH 8.0, 4.2 purpurogallin units horseradish peroxidase. 2.5 moles magnesium sulphate 2.4 moles ATP, 10 mg "Triton' X-100 96 yg 4-aminoantlpyrene hydrochloride, 32 Zg 1,7-dihydroxynaphthalene (added as an 0.8% solution in ethanol), and 4 units of a-GP oxidase (excess glycerol kinase was present in the a-GP oxidase preparation). For triglyceride quantitation, incubation mixtures contained 10 mg (8 units/mg) lipase from Candida rugosa in addition to the above components. All components were equilibrated at 370C for five minutes and A490 (initial) was determined. Reactions were initiated by addition of either a glycerol standard (5-100 nmoles) or serum (20 ,ul) and allowed to proceed for 20 to 30 minutes. The A490 (final) was then measured.

Variations of this standard system are indicated where necessary.

Calculation of Triglyceride Concentrations - Triglyceride glycerol concentrations of unknown samples were determined in the following way: The he490 (A490 (final) minus A490 (initial)) for samples incubated in the presence of the standard glycerol detection system was subtracted from the AA490 of the same samples incubated in the presence of Lipase M and the standard glycerol detection system. Triglyceride concentrations were determined from this Lipase M dependent change in absorbance by use of a calibration curve with either glycerol or pre-analyzed serum samples as standards.

Growth of S. faecalis S. faecalis (species designated in Table IV below) was maintained on slants containing 0.1% glucose, 1% tryptone, 1% yeast extract, 0.65% potassium hydrogen phosphate and 1.5% agar. Water suspension of the slant colonies (0.2 ml of 1.0 ml suspension per flask) were used to inoculate flasks filled with 25 ml of media each. These were shaken at 120 rpm (2 inch throw) in a New Brunswich Psycrotherm Incubator Shaker at 300C for 22 hours.

Preparation of Cell-Free Extracts The cells from 100 ml of media were harvested by centrifugation (4"C, 10,000 X g, 10 min), washed with 40 ml of cold 0.05 M potassium phosphate buffer, pH 7.0, centrifuged again, and suspended in 10 ml of buffer. Cells then were disrupted by sonication (Branson J-17A sonifier operating at a setting of 40) in a Rosett cooling cell for 7 minutes; the temperature was maintained below 8"C. The supernatant from a centrifugation at 10,000 X g for 10 minutes was used as enzyme source. In all cases the amount of soluble protein had reached a maximum during the indicated sonication period. Using bovine serum albumin as standard, protein concentration was determined by the method of Lowry et al Lowry, D.H., Roseborough, N.S., Farr, A.L. and Randall, R.J., J. Biol. Chem. 193, 265 (1951).

Isolation of a-Glycerophosphate Oxidases from Streptococcus faecalis The results in Table IV compare a-glycerophosphate oxidases isolated from three strains of Streptococcus faecalis. In each case the organism was cultured aerobically at 300C for 22 hours in a glucose medium; cells were collected by centrifugation and then disrupted by sonication. Routinely, the supernatant from a 10,000 X g centrifugation was used as enzyme source (crude extract). However, the oxidases remained in solution even after centrifugation at 100,000 X g for 1 hour. In all cases the rate of decrease in dissolved oxygen was proportional to the amount of crude extract and was absolutely dependent on both D, L-a-glycerophosphate and the extract. As can be seen, all three strains displayed oxygen-linked activity. The enzyme from strain ATCC 11700 reportedly has a pH optimum of 5.8 and the oxidase from strain ATCC 19634 displays a maximum at pH 7.0. This trend is also seen in Table IV. By analogy the activity from strain 12755 was similar to the one from strain ATCC 19634.

Table IV Isolation of a-Glycerophosphate Oxidases from Three Strains of Streptococcus faecalis Reactions were carried out at 210 in 0.05 M potassium phosphate buffer at the pH indicated with 0.13 M DL- a-glycerophosphate as substrate pH of S. faecalis Incubation Decrease in % culture Mixture Dissolved 2 A%/ min/ mg ATCC 11700 6.0 4.52 7.3 1.49 ATCC 19634 6.1 3.94 7.5 6.90 ATCC 12755 5.9 4.93 6.8 7.10 Oxygen Electrode Assay of a-Glycerophosphate Oxidase L-a-glycerophosphate oxidase was assayed by measuring the decrease in dissolved oxygen with a New Brunswick D.O. Analyzer. The oxygen'electrode was calibrated against both nitrogen and air saturated water with constant agitation provided by a magnetic stirrer.

Incubation mixtures containing buffer and D, L-a-glycerophosphate in a total volume of 7.5 ml were allowed to equilibrate at 210C. Then reaction was initiated by enzyme addition and the rate of decrease in dissolved oxygen was calculated from the linear portion of the curve. Exact conditions and concentrations for each experiment are given where appropriate.

Spectrophotometric Assay of a-Glycerophosphate Oxidase a-GP oxidase was assayed with a reagent containing in a total volume of 1.0 ml: 100 ,moles potassium phosphate buffer, pH 7.0, 66,ag o-dianisidine, 25 ,ug horseradish peroxidase (4.6 purpurogallin units) and 200 moles D,L-a-glycerophosphate (at pH 7.0). The reagent was equilibrated at 37"C, and the reaction was initiated by the addition of an aliquot of enzyme. Activity was calculated from the initial linear slope of the reaction trace at 430 nm, with e = 1.08 x 104.

Properties of the oe-Glycerophosphate Oxidase in Crude Extracts A detailed pH-activity profile of the a-glycerophosphate oxidase from strain 12755 is shown in Figure 1 in which the percentage change in dissolved oxygen per minute is plotted aganist pH. Optimum activity was observed over the broad pH range of 6.3 to 7.5; below pH 6.0 and above pH 8.0 activity decreased rapidly. Also shown in Figure 1 is the apparent inhibition of the enzyme by either tris-HC1 (A-A) or glycine-KOH (X) buffers. At pH 7.7 the activity in 0.1 M potassium phosphate buffer was 4- times that observed in 0.1 M glycine-KOH. However, when an incubation was carried out in the presence of both 0.1 M glycine-KOH and 0.07 M potassium phosphate buffer, pH 7.7, 82% of the original activity (in presence of 0.1 M potassium phosphate buffer) was restored. This suggests that tris-HC1 and glycine-KOH were not inhibitors, but rather that potassium phosphate buffer activated the enzyme. Sodium acetate buffer also must stimulate the enzyme (Figure 1), since at pH 6.5 activity in sodium acetate buffer was at least 90% that observed in potassium phosphate buffer.

L- < x-glycerophosphate Oxidase Purification Preliminary investigations of the a-glycerophosphate oxidase-glycerol detection system indicated that the crude a-glycerophosate (a-GP) oxidase preparation contained impurities. Some of these impurities apparently prevented the use of the crude enzyme in serum studies since substrates for these enzymes were apparently present in the serum at concentrations compaparable to normal triglyceride levels. Certain of these impurities also acted on substrates present in serum to produce hydrogen peroxide which, of course, interfered with the preferred detection technique. The results of the purification using protein fractionation techniques are shown in Table V.

TABLE V PURIFICATION OF &alpha;-GLYCEROPHOSPHATE OXIDASE FROM S. FAECALIS ATCC 12755 TOTAL UNITS* Total Units &alpha;-GP OX PROCEDURE &alpha;-GP OX protein, mg mg protein Purification Yield Crude cell-free 6300 9850 0.64 1 100 extract Protamine . SO4 fractionation 6280 9540 0.66 1.03 100 (0.05%) Ammonium . SO4 fractionation 5590 4700 1.2 1.9 89 (50-80%) DEAE-cellulose 3600 1007 3.6 5.6 57 fractionation Dialysis and 4250 1007 4.22 6.6 68 Concentration * 1 unit = amount of enzyme required to convert 1 mole substrate to 1 mole product in 1 minute at 37 C.

Stability of a-Glycerophosphate Oxidase The enzyme solution was completely stable for at least four months when stored frozen at -20 C. Repeated freezing and thawing did not denature the enzyme. Also the enzyme was not inhibited by 'Triton' X-100 even at surfactant concentrations as high as 2%.

The following examples serve to illustrate particular embodiments of the present invention.

Example 1 Calibration Curve for Glycerol and Hydrogen Peroxide A glycerol response curve is shown in Figure 2 in which the dye formation in fifteen minutes, measured as a change in optical absorbance he490, is plotted against glycerol concentration in micromoles. Mixtures were prepared as described above under Glycerol and Triglyceride Quantitation by the a-GP Oxidase Method. Reactions were initiated by substrate addition and were essentially complete in 15 minutes at 370C. A good relationship between glycerol concentrations (.) and dye formation (x) was observed for coupled reactions 2, 3 and 4.

Example 2 Quantitative Determination of a Triglyceride Substrate A triglyceride emulsion was prepared by sonicating olive oil (3.6 ,amoles/ml) in 0.4% 'Triton' X-100 (octyl phenoxy polyethoxyethanol -- available from Rohm and Haas Company) in an ice bath for 10 minutes.

Quantitative determination of the triglyceride substrate by coupling reactions 1, 2, 3 and 4 was compared to quantitative determination by the method of Garland and Randle.

Sufficient lipase from Candida rugosa was added to catalyze rapid (less than one minute) and complete hydrolysis of the triglyceride. Triglyceride glycerol was determined by comparing the he430 after a 30 minute incubation to the glycerol concentration response relationship similar to Figure 2. The results, shown in Table VI, demonstrate good agreement between the two methods. Triglyceride values determined with the a-glycerophosphate oxidase method were slightly higher in all cases but the difference was greater than 10% only in sample 2.

Table Vl Triglyceride Concentration Garland and a-Glycerophosphate Oxidase Sample Randle Method 1 18.4 19.0 2 36.8 42.0 3 73.6 78.0 4 110.0 114.0 Example 3 Quantitative Determination of Serum Triglycerides by the a-Glycerophosphate Oxidase Method By means of the preferred buffer system of 0.2 M potassium phosphate buffer at pH 8.0, ten serum samples were assayed for triglyceride glycerol in concentrations ranging from 0.50 to 6.50 mM.

Control mixtures contained only the standard components for glycerol detection; sample mixtures contained lipase from Candida rugosa plus the standard components for glycerol detection. All mixtures were equilibrated at 370C for 5 minutes and the initial A490 was determined. Reactions were initiated by addition of 20 ,ttl of each serum sample and after 20 minutes incubation, the final A490 was measured. Triglyceride glycerol concentrations were determined from an aqueous glycerol calibration curve such as in Figure 2 after the he490 Of the controls were subtracted from the he490 of the samples. Results of comparison to the reference method of Kessler and Lederer are shown in Table VII. Good agreement was observed between the two methods.

TABLE Vll Comparison of Serum Triglyceride Quantitative Determination by the a-Glycerophosphate Oxidase System to a Semi-Automated Chemical Method Triglyceride Concentration ablycerophosphate Reference Method Oxidase Method Sample mM mg/dl mM mg/dl 1 6.59 560.15 6.48 550.80 2 4.82 409.70 4.90 416.50 3 3.76 319.60 3.97 337.45 4 3.30 280.50 3.70 314.50 5 2.00 170.00 1.90 161.50 6 1.71 145.35 1.10 93.50 7 1.06 90.10 0.50 42.50 8 0.59 50.15 0.63 53.55 9 1.25 106.25 1.35 114.75 10 1.00 85.00 0.95 80.75 Example 4 The precision of the method described herein was tested by repetitive assay of two different pooled serum samples; one contained a normal and one contained a high triglyceride level.

The results are shown in Table VIII. Coefficients of variation of 5.1% and 2.6% were calculated for the normal and abnormal sera respectively.

TABLE VHI Reproducibility of the a-GP Oxidase System for Triglyceride Quantitative Determination Triglyceride Concentration mM Normal Serum Level High Serum Level 1.60 4.90 1.66 4.82 1.61 4.80 1.42 5.25 1.62 4.76 1.55 5.00 1.54 4.78 1.70 4.92 1.66 4.96 1.54 4.83 4.91 4.79 4.90 4.80 mean 1.59 4.89 S.D. + 0.081 0.13 COV 5.10 2.60 Example 5 Alternate Electron Acceptors To illustrate electron acceptors other than oxygen, reaction mixtures containing the followirig ingredients were prepared: 0.1 M potassium phosphate buffer to pH 7; 0.2 M D,L-a-glycerophosphate; electron acceptor as and at the level specified in Table IX.

In each instance the mixture was equilibrated at 370C and the reaction was initiated by enzyme addition. The activity of the enzyme was calculated as described hereinabove using E600 = 16 x 103 for 2,6-dichlorophenolindophenol, E400 = 1 x 103 for potassium ferricyanide and E505 = 18.5 x 103 for 2-(p-iodophenyl)-3-(p- nitrophenyl)- 5-phenyl- 2M-tetrazolium chloride (INT).

The results are shown in Table IX.

TABLE IX Alternate Electron Acceptors for &alpha;GP Oxidase Reaction Electron Acceptor moles &alpha;GP oxidized/min/mg Relative Rate &num; Oxygen 80 1 2,6-Dichlorophenolindolphenol (70 m) 4 0.05 2,6-Dichlorophenolindolphenol (70 m) 70 0.88 Phenazine Methosulphate (60 m) K3Fe(CN)6 (1 mM) 21 0.26 K3Fe(CN)6 (1mM) 35 0.44 Phenazine Methosulphate (60 m) INT* (0.16 mM) 0.8 0.01 INT (0.16 mM) 12 0.15 Phenazine Methosulphate (60 m) &num; Rate compared to that with oxygen as electron acceptor * INT = 2-(p-iodophenyl)- 3-(p-nitrophenyl)- 5-phenyl-2H-tetrazolium chloride The method described herein can of course be used to quantify any one of the various reagents and enzymes used in the total reagent system. For example, ATP can be determined with a composition which includes all of the reagents except ATP which would be introduced by the sample for assay. Similarly, glycerol kinase, lipase and a-glycerophosphate can be determined using compositions which include all of the other required materials but that under assay.

The assay compositions described herein may, of course, be incorporated into a matrix of absorbent material of the type well known in the art by impregnation or otherwise to yield test compositions suitable for qualitative or semi-quantitative assay of glycerol or triglycerides. Typical such materials and elements produced therewith which can be adapted for the assay of glycerol or triglycerides are those described, for example, in the following U.S. Patents: 3,092,465, 3,418,099, 3,418,083, 2,893,843, 2,893,844, 2,912,309, 3,008,879, 3,802,842, 3,798,064, 3,298,739, 3,915,647, 3,917,453, 3,933,594, 3,936,357, etc.

Claims (40)

WHAT WE CLAIM IS:

1. A method of detecting or assaying glycerol in aqueous liquid samples comprising treating the sample with a reagent composition whereby the glycerol is converted to La-glycerophosphate, the L-a-glycerophosphate is oxidised to liberate hydrogen peroxide and the hydrogen peroxide is allowed to react in a manner which provides a detectable change.

2. The method as claimed in Claim 1 wherein the L-a-glycerophosphate is oxidised by an a-glycerophosphate oxidase and an electron acceptor to provide a detectable change.

3. The method as claimed in Claim 1 or 2 wherein the a-glycerophosphate oxidase is derived from a microbial source belonging to the genus Streptococcaceae, Lactobacillaceae or Pediococcus.

4. The method as claimed in Claim 3 wherein the a-glycerophosphate oxidase is derived from the species Streptococcus faecalis.

5. The method as claimed in any of the Claims 2, 3 or 4 wherein the electron acceptor is 2,6-dichlorophenolindolphenol, 2-(p-indophenyl)-3-(p-nitrophenyl)- 5phenyl-2H-tetrazolium chloride or oxygen.

6. The method as claimed in any of the preceding Claims wherein the hydrogen peroxide is allowed to react with an indicator composition which provides a colour change in response to the presence of hydrogen peroxide.

7. The method as claimed in Claim 6 wherein the indicator composition includes a substance having peroxidative activity and a dye precursor composition.

8. The method as claimed in Claim 7 wherein the substance having peroxidative activity is a peroxidase.

9. The method as claimed in Claim 7 or 8 wherein the dye precursor composition is a leuco dye oxidisable to a coloured dye in the presence of hydrogen peroxide and a peroxidase.

10. The method as claimed in Claim 9 in which the dye precursor composition is 2-(3,5-dimethoxy-4-hydroxyphenyl)- 4,5-bis(4-dimethylaminophenyl)imidazole or 4-isopropoxy- 1 -naphthol.

11. The method as claimed in Claims 6 or 7 wherein 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.

12. The method as claimed in Claim 11 wherein the dye precursor composition is 4-aminoantipyrine and 1,7-dihydroxynaphthalene.

13. The method as claimed in any of the preceding Claims wherein the glycerol is converted to L-a-glycerophosphate by a combination of a glycerol kinase and adenosine triphosphate.

14. The method as claimed in Claim 13 wherein the glycerol kinase is derived from E. coli or Candida mycoderma.

15. The modification of the method as claimed in any of the preceding Claims wherein the sample contains free or protein-bound triglycerides and is first treated or simultaneously treated with a lipase having triglyceride hydrolysis capability to liberate free glycerol.

16. The method as claimed in Claim 15 wherein the triglyceride is protein-bound and the sample is treated with a lipase and a protease.

17. The method as claimed in Claim 16 wherein the protease is chymotrypsin, elastase, papain, bromelain or a protease from Stepotomyces griseus, Aspergillus oryzae or Bacillus subtilis.

18. The method as claimed in Claim 15 wherein the triglyceride is protein-bound and the sample is treated with a mixture of a lipase and a compatible surfactant, as herein defined.

19. The method as claimed in Claim 18 wherein the compatible surfactant is an octyl or nonyl phenoxy polyethoxy ethanol.

20. The method as claimed in Claim 19 and wherein the compatible surfactant has less than 20 carbon atoms in the polyoxyethylene chain and the hydrophilelipophile balance number is less than 15.

21. The method as claimed in any of the Claims 15 to 20 in which the lipase is derived from Rhizopus arrhizus, Candida rugosa or Chromobacterium viscosum.

22. The method as claimed in any of the preceding Claims in which the reagent composition is buffered to a pH from 6 to 8.

23. Methods of detecting or assaying glycerol as claimed in Claim 1 and as herein described.

24. The modification of the method as claimed in Claims 13 or 14 wherein the glycerol is present in the reagent composition and the adenosine triphosphate is present in the aqueous liquid sample.

25. Methods of detecting or assaying triglycerides as claimed in Claim 15 and as herein described.

26. A reagent composition comprising (a) a glycerol kinase, (b) adenosine triphosphate, (c) an a-glycerophosphate oxidase and (d) an electron acceptor.

27. The reagent composition as claimed in Claim 26 which includes an indicator composition comprising a substance having peroxidative activity and a dye percursor which undergoes a detectable change in the presence of peroxide and the substance having peroxidative activity.

28. The reagent composition as claimed in Claim 27 in which the substance having a peroxidative activity is a peroxidase.

29. The reagent composition as claimed in any of the Claims 26 to 28 in which the glycerol kinase is derived from E. coli or Candida mycoderma.

30. The reagent composition as claimed in any of the Claims 26 to 29 in which the a-glycerophophate oxidase is derived from a microbial source belonging to the genus Streptococcaceae, Lactobacillaceae or Pediococcus.

31. The reagent composition as claimed in Claim 30 in which the a-glycerophosphate oxidase is derived from the species Strepocccus faecalis.

32. The reagent composition as claimed in any of the Claims 26 to 31 which includes a lipase.

33. The reagent composition as claimed in Claim 32 in which the lipase is derived from Rhizopus arrhizus, Candida rugosa or Chromobacterium viscosum.

34. The reagent composition as claimed in Claim 33 which includes a protease.

35. The reagent composition as claimed in Claim 34 in which the protease is chrymotrypsin, elastase, papain, bromelain or a protease derived from Streptomyces griseus, Aspergillus oryzae or Bacillus subtilis.

36. The reagent composition as claimed in Claim 33 which includes a compatible surfactant as herein defined.

37. The reagent composition as claimed in Claim 35 in which the surfactant is an octyl or nonyl phenoxy polyethoxy ethanol.

38. The reagent composition as claimed in Claim 36 in which the surfactant has less than 20 carbon atoms in the polyethoxy chain and the hydrophil-lipophile balance number is less than 15.

39. The reagent composition as claimed in any of the Claims 26 to 37 which is buffered to a pH from 6 to 8.

40. Reagent compositions as claimed in Claim 26 and as herein described.