JPH0246200B2 - TORIGURISERIDOKENSHUTSUYOSOSEIBUTSU - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a triglyceride detection composition suitable for use in the analysis of triglyceride-containing aqueous liquids, particularly serum triglycerides. Measurement of serum triglyceride concentrations has gained increasing importance in the diagnosis of several types of hyperlipidemia and atherosclerotic heart disease (see:
Kahlke, W. “Med.Wscht” Vol. 91, p. 26 (1966)
), Kuo, PT and Basset, DR, “Amer.
59, p. 465 (1963)). Conventional methods for measuring serum triglycerides consist of hydrolyzing triglycerides to liberate glycerin and treating this glycerin with various reagents to produce compounds that can be quantified spectroscopically. It is common to perform hydrolysis using a base, but US Pat. No. 3,703,591 to Bucolo et al. and US Pat.
No. 3759793 describes an enzymatic method in which hydrolysis is carried out using lipase alone (see above No. 3759793) or in combination with protease (see above No. 3703591). Other non-enzymatic hydrolysis methods are German Patent No. 2229849 and German Patent No. 2323609.
It is stated in the number. Currently, three enzymatic methods are routinely used to measure glycerin from any source. These are as follows. (a) Garland and Rundle method (Method of
Garland and Randle) (Garland, PB and Randle, RJ, Nature, Vol. 196, pp. 987-988 (1962)) Glycerin + ATP glycerol kinase --------→ L-α-glycerophosph ate + ADP ADP + phosphonoenolpyruvate pyruvate kinase――――――――――→ Pyruvate + ATP Pyruvate + NaDH Lactate dehydrogenase――――――――――――――→ Lactate +NAD+ (b) Weilands Method (Weiland, O. “Biochem Z” No. 329, p. 313 (1957)) Glycerin + ATP glycerol kinase――――――――――→ L-α-glycerophosph ate + ADP L-α-glycerophosphate + NAD + α-glycerate dehydrogenase――――――――――――――――――――→ NADH + dihydroxyacetone phosphate + H ⁺ (c) Glycerol dehydronase method (Hagen , J.H.
and Hagen, P.B. “Can J.Biochem and
Physiology, Vol. 40, p. 1129 (1962)) Glycerin + NAD + Glycerin Hydrogenase ――――――――――――――――→ Dihydroxyacetone + NADH + H ⁺ Modification of method (a) is described in German Patent No. 2665556 ,
It is also described in UK Patent No. 1,322,462 and US Pat. No. 3,759,793. In all cases, NADH production or loss is measured at 340 nm in UV spectroscopy. Method (a) is utilized in many commercial "kits". It is a three-enzyme step and the disappearance of NADH is measured. Method (b) consists of a two-enzyme step and the production of NADH is measured as in a single reaction with the enzyme glycerol dehydrogenase (method (c)). The latter two operations are extremely sensitive to pH and will produce erroneous values unless strict pH control is maintained. Also, in all three methods (especially method (a))
In this case, the stability of not only the diagnostic enzyme but also the cofactor, ie, NADH, is of major concern. Regarding errors in current enzymatic methods
Chen, HP and El-Mequid, SS, Biochemical Medicine, Vol. 7, p. 460 (1973)
(2013), which is discussed in detail. Another method for triglyceride analysis has been published in a German patent.
Described in No. 2139163. The method of this patent is
It consists of hydrolyzing triglycerides, oxidizing the resulting glycerin to formaldehyde, and reacting this formaldehyde with ammonia and a water-alcohol-soluble stable colorless acetylacetone metal complex to produce a colored compound. An object of the present invention is to provide a composition for quantitatively measuring triglycerides, particularly serum triglycerides, which does not require strict and narrow PH control and has excellent stability of reagents used. It's about doing. According to the invention, (a) lipase, (b) glycerol kinase, (c) adenosine triphosphate, (d) α-
A composition for detecting triglycerides in an aqueous liquid is provided, comprising glycerophosphate oxidase and (e) a substance exhibiting peroxidation activity. In the present invention, triglycerides are suitably quantitatively measured by the following reaction series (Table I). Table I 1 Triglyceride + HOH nopase――――→ Glycerin + Fatty acid 2 Glycerin + ATP glycerol kinase――――――――→ L-α-glycerophosphate + ADP 3 L-α-glycerophosphate + electron acceptor Body α-glycerophosphate oxidase――――――――――――――――――――――→ Dihydroxyacetone phosphate + substance for analytical measurement When oxygen is the electron acceptor , H ₂ O ₂ is produced as a substance for analysis and measurement in (3) above,
The measurement of H ₂ O ₂ is preferably carried out by the following reaction. 4 H ₂ O ₂ + H ₂ A (reduced type) peroxidase――――――――→ A (oxidized type, detectable substance) + H ₂ O However, H ₂ A (reduced type) is the reduced form of the dye. It is a dye precursor. A (oxidized type) is a dye produced by oxidation of H ₂ A. In a combinatorial reaction of preferred compositions, detectable substances are produced in proportion to the concentration of glycerin and/or triglycerides. This method is
It can be used in many clinical applications, in particular in the determination of serum triglycerides. The procedure of the present invention has many unique advantages over conventional methods. First, any leuco dye utilized by peroxidase as an electron donor can be effectively used in the indicator composition. The reaction can thus be measured at one of several wavelengths in the visible region of the spectrum, depending on the choice of dye. Second, measurements made in the visible range are less susceptible to interference than measurements made at 340 nm. Third, any means of detecting hydrogen peroxide besides dyes can be used. Fourth, since O ₂ is a cofactor for the α-glycerophosphate oxidase reaction,
The stability of NAD ⁺ or NADH is not important.
Fifth, serum components that utilize NAD ⁺ or NADH (eg, lactate plus lactate dehydrogenase), which interfere with prior art reaction steps, do not interfere with the process. Sixth, any means of measuring O ₂ consumption when O ₂ is used as an electron acceptor can be used as a detection means. lastly,
The enzymes used in the present process are active over a relatively wide PH range, so strict PH control is not required. Although the discussion below will mainly focus on solution and solution methods for quantifying triglycerides, it will be appreciated by those skilled in the art that all reagents can be provided in dry or lyophilized form and reconstituted with water immediately before use. It should be easy to understand. Compositions of this type are clearly expected here. Hydrolysis In its most complex embodiment, the method of the invention is utilized to analyze aqueous fluids, such as serum, for triglyceride content. According to this embodiment, triglycerides are hydrolyzed to free glycerin by any of the well-known techniques described in the art. Enzymatic techniques are preferred. These usually consist of treating the serum sample with a lipase alone or in combination with effectors such as proteases or detergents, depending on the nature of the triglycerides. Detailed discussions of such techniques and compositions useful for their implementation are provided in U.S. Pat. Published in US Pat. No. 3,759,793. Hydrolyze serum triglycerides using a lipase, preferably Rhizopus arrhizus (var. delemar) lipase and similar substances, in combination with a protease, whereas Stork et al. It discloses that hydrolysis can be carried out using An alternative method is the hydrolysis of serum triglycerides using a compatible mixture of a lipase, which by itself cannot hydrolyze triglycerides complexed with proteins such as those normally found in serum, and a compatible surfactant as an effector. Consisting of Compatible surfactants are those that promote hydrolysis of triglycerides by lipases as described in the tests below. Therefore, the surfactant substantially enhances the activity of lipase without inhibiting it. Preferably, the lipase is from Candida cylindracea (Candidarugosa). Lipases useful for triglyceride hydrolysis according to any of the foregoing techniques may be derived from plants or animals, but when the lipase is used in combination with a surfactant, as described below, microbiol lipases such as Candida cylindracea can be used. things are the best. For example, as described by Fukumoto et al. in "J. Gen. Appli. Microbiol." Vol. 10, pp. 257-265 (1964), Chromobacterium viscosum
Also useful are the crude or purified lipases of various Paralipolyticum, the purified lipases of Rhizopus arizus (various Rhizopus delmar), and lipase concentrates with similar activity. Other useful lipases and methods for their preparation are described in the following US patents: U.S. Patent No. 2,888,385 issued to Grandel on May 26, 1959 U.S. Patent No. 3,168,448 issued to Melcer et al. on February 2, 1965 Issued on June 15, 1969 to Yamada et al. U.S. Patent No. 3,189,529 issued to Fukumoto et al. on July 26, 1966 U.S. Patent No. 3,513,073 issued to Mauvernay et al. on May 19, 1970 The lipase was lyophilized. As a stable reagent solution, the analytical sample can be easily incorporated into a dry mixture for reconstitution with water or combined with other such solutions to produce a reaction mixture for contacting. Either provide. Particularly preferred commercially available lipases include wheat germ lipase supplied by Miles Laboratories, Elkhart, Indiana, lipase 3000 supplied by Wilson Laboratories, and stearpsin supplied by Sigma Chemical Co. (the last two enzymes are both pancreatic Enzyme) and supplied by Enzyme Development Company (from Casogida Lugosa)
Contains lipase M. Nonionic and anionic surfactants cannot hydrolyze serum triglycerides by themselves;
It was found to be useful in combination with lipase preparations. Among these substances is one sold by Rohm and Haas under the trademark Triton.
Most preferred are octyl and nonyl phenoxy polyethoxy ethanol. The HLB (hydrophile lupophile balance) value is approximately
Best results are obtained when using surfactants with less than 15 and less than about 20 polyoxyethylene units in the polyoxyethylene chain. Compatible compositions of lipase and surfactant are easily defined by the tests described below. The surfactant of the composition to be evaluated was added to an unbuffered reconstituted serum (specifically, Theral Diagnostics Davey, Warner Lambert Company, Morris Plays, New Jersey). Serum standard Validate available from Jiyoung
is added at various concentrations between about 0 and 10% by weight, and the solution is incubated at 37° C. for about 5 minutes. At this point, add a sample of the desired lipase formulation and continue incubating for approximately 20 minutes.
A portion (~0.2 ml) of this solution was diluted to 1.6 ml with water (containing 1.3 mM _CaCl2 to encourage precipitate formation), placed on a boiling water bath for 10 min, and centrifuged. (4℃, 37000×g, 10 minutes) Clarify. Garland and peas with 0.4 ml of clear supernatant glycerin. quantitatively in a total volume of 1.2 ml by the method described by Garland, PB and Randle, PJ in Nature, vol. 196, pp. 987-988 (1962). Measure. Any composition that liberates at least about 50% of the theoretical concentration of effective glycerin is considered useful and is within the scope of this invention. When performing the above test, it is highly desirable to perform a blank test that includes all components of the mixture other than the lipase, so that any reactions attributable to free glycerin or other serum components can be subtracted. Preferred compositions achieve 75% hydrolysis of available triglycerides to glycerin in less than about 10 minutes, and more preferred compositions achieve substantially complete hydrolysis, i.e., about 75%, in less than about 10 minutes.
It effectively hydrolyzes triglycerides to glycerin by more than 90%. Examples of such preferred compositions are shown in the table below. For some reason, when using prior art punitease-lipase combinations for hydrolysis, more or less proteases can be used. these are,
For example, chymotrypsin, Streptomyces griseus protease (sold under the registered trademark Pronase), Aspergillus oryzae
oryzae) and Bacillus subtilis
subtilis protease, elastase, papain and bromelain. Of course, mixtures of such enzymes can also be used. Effective concentrations of lipase and other effectors such as detergents, proteases, etc. will vary widely depending on the time constraints imposed on the assay, etc., but these are readily determined by one skilled in the art. Typical, non-limiting examples of effective concentrations are described in the examples below. Triglycerides can be hydrolyzed by any of the known conventional "non-enzymatic" methods, including treatment with strong base, to obtain free glycerin prior to analysis. However, distributing the glycerin to the enzymatic glycerin assay composition in a medium that does not contain substances that inhibit the enzymes of the glycerin assay system or otherwise interfere with the reactions necessary to make accurate glycerin measurements. Care must be taken to ensure that. Glycerin Analysis Once triglyceride hydrolysis has been achieved, glycerin analysis can be performed with the novel enzymes of the present invention. As evidenced by the reaction above, the first enzyme used for glycerol analysis is glycerol kinase, which catalyzes the conversion of glycerol to L-α-glycerophosphate in the presence of adenosine triphosphate (ATP). shows. In general, any glycerol kinase can be used to successfully practice the invention, but those obtained from E. coli and Candidamycoderma are preferred. Other glycerol kinase enzymes are well known in the art. A complete discussion of such materials and further literature on their preparation and reactivity can be found in T.E.
"Enzyme Hand book I" by Springer Barman
Verlag, NY (1969), pp. 401-402). Glycerol Kinase from Worthington Biochemical Company provides a satisfactory commercially available enzyme. The next reaction step involves the oxidation of L-α-glycerophosphate in the presence of L-α-glycerophosphate oxidase and an electron acceptor to produce a detectable reaction. The detectable change is preferably a color change or color production;
In a preferred case, it relates quantitatively to the glycerin contained in the liquid sample. Other detectable changes, such as oxygen consumption, can also be monitored to confirm the analytical results. Any electron acceptor that oxidizes alpha-glycerophosphate with a detectable change in the presence of an oxidase enzyme is a suitable candidate for use in this reaction. Particularly preferred as electron acceptors are substances which give preferably colored products which can be detected radiometrically, either directly or indirectly. The utility of any individual electron acceptor can be determined by experimenting with potentially useful electron acceptors. A highly preferred electron acceptor is oxygen, which oxidizes L-glycerophosphate to dihydroxyacetone phosphate and hydrogen peroxide in the presence of an oxidase. It goes without saying that methods for measuring hydrogen peroxide and checking the amount of oxygen consumed in this type of reaction are known. In another preferred embodiment, a colored or colorless substance is used as the electron acceptor, which undergoes immediate discoloration or development upon reduction in the presence of the enzyme and substrate.
As mentioned above, such materials can be tested and selected for specific use situations. Such a situation is described in Example 5 below. Using this method, certain indophenols, potassium ferricyanide, and certain tetrazolium salts have been found to be useful electron acceptors. Specifically, 2,6-dichlorophenol indophenol alone or in combination with phenazine methosulfate was used as the electron acceptor in this reaction, and 2-(p-iodophenyl)-3-(nitrophenyl)-5-phenyl- It has been found that 2H-tetrazolium chloride, either alone or in combination with phenazine methosulfate, is useful. Detectable changes can also be measured using potentiometric techniques, for example by measuring oxygen consumption using an oxygen electrode. L-α-glycerophosphate oxidase is a microbial enzyme that can be obtained from a variety of sources. As detailed below, the properties of enzymes from certain sources are more favorable than those from others. Generally, the enzymes can be obtained from the Streptococcus family, the Lactobacillus family and Pediococcus. Particularly preferred are enzymes from Streptococcus faecalis cultures available from the American Type Culture Collection. Particularly useful and preferred enzymes are strains ATCC 11700, ATCC 19634 and
Obtained from ATCC12755. As described and illustrated in the examples below, the ATCC 12755 enzyme is compatible with other two
It has activity over a slightly broader pH range than enzymes obtained from bacterial strains, and is therefore the most preferred. The following two documents describe enzymes and useful techniques for their preparation and extraction. References: Kodicietsk, L. K. (Kodits
-chek, LK) and Ambright, D'Avrieux.
D'Avrieux. (Umbreit, WW), “Journal
Obacteriology (Journal of
"α-glycerophosphate oxidase of Streptoccocus faecium", ``Streptoccocus faecium'', published in ``Bacteriology'' Vol. 98, No. 3, pp. 1063-1068 (1969); Jacob, N.; J.A. (Jacob, NJ)
and Vandemark, P. J.A. (Van
"Purification and properties of α-glycerophosphate oxidase of Streptococcus faecalis" by Demark P.J) Enzymes prepared according to the methods described in any of the above-mentioned publications may be used to successfully carry out the present invention. It is useful for When using any enzyme preparation of unconventional composition, care must be taken to filter out any contaminants that would interfere with the analytical results. For example, L derived as below
- Because α-glycerophosphate oxidase preparations contain very high concentrations of impurities, conventional fractionation and column separation methods have to be used before achieving analysis of serum triglycerides free of undesired interfering substances. , the crude formulation had to be purified. Detection of glycerin in aqueous solutions containing glycerin and/or triglycerides, such as serum, is suitable using an indicator composition that detects the concentration of hydrogen peroxide produced by the oxidation of L-α-glycerophosphate in the presence of oxygen. will be implemented. Indicator compositions for detecting hydrogen peroxide produced by enzymes are well known in the art as indicator compositions for detecting enzymes, particularly glucose and uric acid. US Pat. No. 3,092,465 and US Pat. No. 2,981,606, among many others, describe such useful indicator compositions. The hydrogen peroxide indicator composition comprises a substance having peroxidation activity, preferably peroxidase, and a dye precursor that undergoes coloration or discoloration in the presence of hydrogen peroxide and the substance having peroxidation activity. Alternatively, the dye precursor may be oxidized in the presence of H ₂ O ₂ and a substance with peroxidation activity without undergoing a substantial color change, but in its oxidized form, the dye precursor may be oxidized in the presence of H 2 O 2 and a substance with peroxidation activity, but in its oxidized form, a color-producing or color-changing substance (e.g., a color former) to visually demonstrate a chemical reaction,
It can be one or more substances. In particular, US Pat. No. 2,981,606 provides a detailed description of such indicator compositions. The latter dye precursor, that is, one that develops color through a coupling reaction, is preferred for carrying out the present invention. Peroxidases are enzymes that catalyze reactions in which hydrogen peroxide or other peroxides oxidize other substances. The peroxidase is generally a complex protein containing porphyrin iron. Peroxidase is derived from horseradish, yam, fig sap, turnip (vegetable peroxidase), and milk (lactoperoxidase).
and white blood cells (Verdoperoxidase). It also occurs in microorganisms. Theorell and Maehly
"Actachem.Scand." Volume 4, pp. 422-434 (1950
Synthetic peroxidases such as those published in 2010 are also satisfactory. Substances such as hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemoglobin, alkaline hematin, hemin derivatives and other compounds with peroxidation activity are somewhat inferior. Other substances that are not enzymes but have peroxidation activity are: iron thiocyanate, iron tannate,
Ferrous ferrocyanide, dichromate (potassium chromium sulfate) absorbed in silica gel, etc., these substances are used in a similar manner, although not as fully as peroxidase itself. Dye precursors that exhibit coloration in the presence of hydrogen peroxide and a substance having peroxidation activity include the following substances. A coloring agent is also included if necessary. (1) Monoamines such as aniline and its derivatives, ortho-toluindipara-toluidine, (2) ortho-phenylenediamine, N,N'-dimethyl-para-phenylenediamine, N,
Diamines such as N'-diethylphenylenediamine, benzidine, dianisidine, etc. (3) Phenol itself, thymol, ortho-,
Phenols such as meta- and para-cresol, α-naphthol, β-naphthol (4) Catechol, guaiacol, osinol,
Polyphenols such as pyrogallol, p,p-dihydroxydiphenyl and phloroglucinol (5) Aromatic acids such as salicylic acid, pyrocatechinic acid and gallic acid (6) Leucomalachite green and leucophenolphthalein Leuco dyes such as (7) Colored dyes such as 2,6-dichlorophenol indophenol (8) Various biological substances such as epinephrine, flavones, tyrosine, dihydroxyphenylalanine and tryptophan (9) Others, Substances such as guaya gum, guaiaconic acid, potassium iodide, sodium iodide and other water-soluble iodides, and bilirubin, and (10) 2,2'-azine-di(3-ethylbenzothiazoline-(6)-sulfone) other indicator compositions that can be oxidized by peroxidase in the presence of peroxidase and provide a substance that can be detected by radiometric techniques. includes certain paint-donor compositions. In this embodiment, the indicator composition can include a compound that, when oxidized in the presence of peroxidase, self-couples as such or with its reduced form to provide a paint. Such self-coupling compounds include orthoaminophenol, 4-alkoxynaphthol, 4-amino-5-pyrazolone, cresol, pyrogallol, guaiacol, oscinol, catechol, phloroglucinol, p,p-
Mention may be made of various hydrolyzed compounds such as dihydroxydiphenyl, gallic acid, pyrocatechinic acid, salicylic acid, etc. Compounds of this type are well known and are described in Mees and James Ed.'s The Theory of Photographic Process.
In particular, it is described in the literature such as Chapter 17. In another embodiment, a detectable change is obtained by oxidizing the leuco dye in the presence of peroxidase to provide the corresponding dye form. Representative leuco dyes include compounds such as leucomalachite green and leucophenolphthalein. They are called oxichromic compounds.
Other leuco dyes are described in US Pat. No. 3,880,658, which further states that such compounds can be disseminated with appropriate substituents thereof. U.S. Patent No. 380658
The non-stabilized oxychromic compounds described in this issue are considered preferred for the practice of this invention. In another embodiment, the detectable change is caused by an indicator composition comprising a compound that can be oxidized in the presence of peroxidase and oxidatively condensed with a color former, such as having a phenol group or an activated methylene group. may be provided. Typical examples of such oxidized compounds include benzidine and its analogues, p-
Phenylene diamine, p-aminophenol, 4
-Aminoantipyrine, etc. may be mentioned. A wide variety of such color formers, including many self-coupling compounds, are described in Mace and James, supra, and Kosar, Light Sensitive System, pp. 215-249 (1965).
year). Indicator compositions of the present invention include 4-methoxy-1-naphthol or 1-7-dihydroxynaphthalene and 4-
Contains concomitant medicines with aminoantipyrine (HCl). In the latter composition, an oxidized pirine compound is coupled with dihydroxynaphthalene. The component concentrations of the various indicator compositions useful in the elements described herein are highly dependent on the glycerin concentration in the sample, the accuracy of the detection equipment, the dye produced, etc.
It can be easily determined by one skilled in the art. Typical values are shown in the examples below. Other means of detecting hydrogen peroxide may also be used in the successful practice of this invention. For example, the enzymes and other reagents described herein are described in Rawls, Rebetzka, L.; (Rebecca,
L.) says “Electrode Hold Promie in
Biomedical Uses” (Chemical and Biomedical Uses)
Oxygen can be incorporated into the membrane of a sensitive polarographic electrode as described in Engineering News, January 5, 1976, p. 19). Alternatively, instead of measuring the hydrogen peroxide produced, an oxygen sensitive electrode is used to measure the oxygen consumption and thus determine the amount of glycerin produced in reaction (1) of Table I above. However, this consumes the above amount of oxygen in reaction (3) of Table I. The concentrations of other components of the novel analytical compositions described herein vary widely depending on the analyte solution (i.e., diluted or undiluted serum or other complex aqueous solutions of glycerin and/or triglycerides). You can also. The table below will serve as a handy reference for generally useful and preferred concentration ranges for the various components of the novel analytical compositions described herein.

[Table] It goes without saying that useful results can be obtained outside these ranges. In the above table, one international unit of enzyme is 37
It is defined as the amount of enzyme that causes the conversion of 1 μmol of substrate in 1 minute at ℃ and pH 7. As is well recognized in the art, each enzyme has the property of PH activity. That is, the activity of the enzyme is
Varies depending on pH. These data are detailed for α-glycerophosphate oxidase in the example below. As shown in the data, the nature of the PH activity of L-α-glycerophosphate oxidase peaks between about PH 5 and 8.5. The pH range at which each enzyme becomes most active in the new reaction process is shown in the table below. Table PH value Lipase 5-9 Glycerol kinase 7-9 L-α-glycerophosphate oxidase
6.3-8.0 Peroxidase 6-8 From the table above, it can be seen that the analytical compositions described herein are between about 6.0 and about 8.0, most preferably about
It will be readily appreciated that buffering to a pH between 7.0 and about 8.0 is most desirable. Methods for obtaining this type of buffering effect are known in the art and may include dissolving, dispersing, or otherwise distributing, or alternatively reconstitution of, appropriate concentrations of buffer materials in the reagent composition. When the mixture is provided, it is provided in dry form. Buffers suitable for buffering the above pH value are described in detail by Good in "Biochemistry", Vol. 5, p. 467 (1966).
A particularly preferred buffering agent is a phosphate salt such as calcium phosphate. It goes without saying that the concentration of the detectable substance produced can be detected using any known method, such as comparison with a standard color diagram, spectroscopic analysis, and the like. In the examples described below, the following enzyme preparation method and standardized procedures and compositions were used. Standard solution The exact concentration of the glycerin standard solution is determined by the method of Garland and Randle (Nature, Vol. 196, 987-988).
(1962)). The hydrogen peroxide solution was measured at A ₂₄₀ (absorbance at 240 nm),
A standard was obtained by appropriately calculating using ε ₂₄₀ =43.6 (ε: molecular extinction coefficient at 240 nm). Kessler serum sample
and Lederer's Semi-Automated Fluorometric Measurement of
Triglycerides，Automation in Analytical
"Chemistry", Technican Symposia, LT
Sheggs, Jr., Ed.Medical Inc., NY, NY341
(1966)) for the concentration of triglycerides. Determination of glycerin and triglycerides by α-glycerophosphate oxidase method Mixture to be incubated to detect glycerin in a total volume of 1.0 ml Potassium phosphate buffer (PH 8) 200 μmol Horseradish peroxidase 4.2 purpurogallin units MgSO ₄ 2.5 μmol ATP 2.4 μmol Triton was present in the α-glycerophosphate oxidase preparation.) 4 units The incubate mixture for triglyceride determination contained 10 mg (8 units/mg) of Candidal goza lipase in addition to the above components. . All components were kept in equilibrium for 5 minutes and (initial) A ₄₉₀
was measured. Glycerin standard (5-
Start the reaction by adding either 100 nmol) or serum (20 μl), and
It lasted for about 30 minutes. Then (final) I measured A ₄₉₀ . Variations of this standard system are shown where appropriate. Calculation of Triglyceride Concentration The triglyceride glycerin concentration of the unknown specimen was determined as follows. △A ₄₉₀ (A ₄₉₀ (final value) - A ₄₉₀ (initial value)) of the sample incubated in the presence of the standard glycerin detection system, and △ of the same sample incubated in the presence of Lipase M and the standard glycerin detection system. Deducted from _A490 . Triglyceride concentrations were determined from this dependent change in lipase M absorbance by use of a calibration curve using either glycerin or previously analyzed serum samples as standards. Cultivation of Streptococcus faecalis (types shown in the table below): 0.1% glucose, 1% tryptone, 1% yeast extract, 0.65% K ₂ HPO ₄ and 1.5
% agar was kept on a slant containing agar. Suspension water of the slope colony (per flask)
0.2 ml of 1.0 ml suspension water was used to inoculate flasks filled with 25 ml of each medium. These are New Brunswick cyclotherm incubator shakers (New Brunswick
22 at 30°C in a Psycrotherm Incubator Shaker)
Shaking was performed at 120 rpm (2 inch throw) for the duration. Preparation of cell-free extract Cells from 100 ml of medium were obtained by centrifugation (4°C, 10000xg, 10 min) and cooled.
Washed with 40 ml of 0.05M potassium phosphate buffer (PH7.0), centrifuged again and suspended in 10 ml of buffer. The cells were then placed in a Rosett cooling cell for 7 minutes (Branson J-17A Sonifier).
Destroyed the Sonication (by driving with a set of 40). The temperature was kept below 8°C. The supernatant obtained by centrifugation at 10000xg for 10 minutes was used as an enzyme source. The amount of soluble protein reached a maximum during the indicated period of sonication in all cases. Using bovine serum albumin as a standard, protein concentrations were determined by the method of Lowry et al. (Lowry, DH, Roseborovgh, NS, Farr, A.
L. and Randall, R.J., “J.Boil.Chan” No. 193.
265 (1951)). Isolation of α-glycerophosphate oxidase from Streptococcus faecalis The results in Table 1 compare α-glycerophosphate oxidase isolated from three species of Streptococcus faecalis. In each case, this bacterium was cultured aerobically in glucose medium at 30°C for 22 hours. Cells were collected by centrifugation and then sonicated. Usually, the supernatant obtained by centrifugation at 10000xg was used as the enzyme source (crude extract). However, the oxidase
It remained in solution even after centrifugation at 100,000xg for 1 hour. In all cases, the rate of reduction of dissolved enzymes was proportional to the amount of crude extract, and D,L
It was completely dependent on both -α-glycerophosphate and the extract. Apparently, all three bacterial species exhibited oxygen-linked activity. Optimization of enzymes from ATCC11700 strain
The pH is 5.8, and it has been reported that the oxidase of ATCC19634 strain shows the highest value at PH7.0. This trend is also seen in the table. 12755
The activity of the strain was analogously similar to that of strain ATCC19634.

[Table] Oxygen electrode analysis of α-glycerophosphate oxidase L-α-
Glycerophosphate oxidase was analyzed. The oxygen electrode was calibrated against water saturated with both N ₂ and air while being constantly stirred with an electrode stirrer. Incubate mixture containing buffer and D,L-α-glycerophosphate to a total volume of 7.5 ml at 21°C.
It was kept in equilibrium at. The reaction was then started by addition of enzyme and the percent reduction in dissolved oxygen was calculated from the linear portion of the curve. The exact conditions and concentrations for each experiment were established accordingly. Spectroscopic analysis of α-glycerophosphate oxidase 100 μml potassium phosphate buffer (PH7.0), 66 μg
of O-dianisidine, 25 μg of horseradish peroxidase (4.6 purpurogalin units) and
α-glycerophosphate oxidase was analyzed using a reagent containing 200 μmol of D,L-α-glycerophosphate (PH 7.0) in a total volume of 1.0 ml.
The reagents were equilibrated at 37°C and the reaction was initiated by adding a portion of the enzyme. From the initial linear slope of the spectrophotometric curve tracing the reaction, ε = 1.08 × 10 ⁴ (ε:
The activity was determined using the molecular extinction coefficient (at a wavelength of 430 nm) of the dye produced in the reaction. Properties of α-glycerophosphate oxidase in crude extract The detailed PH activity of α-glycerophosphate oxidase of strain 12755 is shown in FIG. Optimal activity is observed over a wide PH range of 6.3 to 7.5;
The activity rapidly decreased below 6.0 and above 8.0. Also shown in FIG. 1 is the apparent inhibition of the enzyme by either tris-HCl (Δ-Δ) or glycine-KOH (X) buffers. 0.1M at PH7.7
The activity in potassium phosphate buffer was four times that observed in 0.1M glycine-KOH. However, when incubated in the presence of both 0.1M glycine-KOH and 0.07M potassium phosphate buffer (PH7.7), 82% of the initial activity (in the presence of 0.1M potassium phosphate buffer) was I went back. This suggests that tris-HCl and glycine-KOH are not inhibitors, but rather that the potassium phosphate buffer activates the enzyme. PH in sodium acetate buffer
Sodium acetate buffer also appears to activate this enzyme (Figure 1) since the activity at 6.5 is at least 90% of that observed with potassium phosphate buffer. L-α-Glycerophosphate Oxidase Purification Preliminary testing of the α-glycerophosphate oxidase-glycerin detection system indicated that the crude α-glycerophosphate (α-GP) oxidase preparation contained impurities. . Some of these impurities clearly interfered with the use of the crude oxidase enzyme in serum tests. This is because it is clear that the oxidase enzyme substrate is present in serum at a concentration comparable to normal triglyceride concentrations. Some of these impurities acted on substrates present in the serum to produce hydrogen peroxide, which of course interfered with the preferred detection technique. Purification results using protein fractionation techniques are shown in Table V.

Table: Stability of α-glycerophosphate oxidase The enzyme solution was completely stable for at least 4 months when stored frozen at -20°C. The enzyme did not denature even after repeated freezing and thawing. Also, this enzyme was not inhibited by Toridone X-100 at a concentration of 2%. The following examples will serve to explain detailed embodiments of the invention. Example 1 Calibration curve for glycerin and hydrogen peroxide The glycerin reaction curve is shown in FIG. For the determination of glycerin and triglycerides by the α-glycerophosphate oxidase method, mixtures were prepared as described above. The reaction was initiated by addition of substrate and substantially completed for 15 minutes at 37°C.
FIG. 2 shows the series of reactions 2, 3 and 4 (Part I) described above.
(see table) versus glycerin concentration (substrate concentration)
and dye production concentration (plotted as △A ₄₉₀ /15 min)
This shows that there is a linear relationship between Example 2 Quantitative determination of triglyceride substrates by sonicating olive oil (3.6 μmol/ml) in 0.4% Triton X-100 (octylphenoxypolyethoxyethanol available from Rohm and Haas) in an ice bath. A triglyceride emulsion was prepared. The quantitative determination of triglyceride substrates by reaction series 1, 2, 3 and 4 (see Table I) above was compared with the quantitative determination by the method of Garland and Randle. A sufficient amount of lipase from Candidal goza was added to act as a catalyst to quickly (less than 1 minute) and completely hydrolyze the triglycerides. Triglyceride glycerol was determined by comparing the ΔA ₄₃₀ after 30 minutes of incubation to a glycerol concentration response curve similar to FIG. These results, presented in Table 1, show good agreement between the two methods. The triglyceride values measured by the α-glycerophosphate oxidase method were somewhat high in all cases, but the difference was only greater than 10% in sample 2.

[Table] Example 3 Quantitative determination of serum triglycerides by the α-glycerophosphate oxidase method For triglyceride glycerol at concentrations ranging from 0.50 to 6.50 mM, depending on the preferred buffer system of 0.2 M potassium phosphate buffer at pH 8.0: Ten serum samples were analyzed. The control mixture contained only the standard component for glycerin detection. The sample mixture contained Candidal-Goza lipase and other standard components for glycerin detection. The entire mixture was allowed to equilibrate for 5 minutes at 37°C, and 20μ of each serum sample for which initial _A490 was measured was added.
Start the reaction by adding and
After incubating for 20 minutes, the final A ₄₉₀ was measured. After subtracting △A ₄₉₀ of the control from △A ₄₉₀ of the specimen,
The triglyceride glycerin concentration was determined from an aqueous glycerin calibration curve as shown in FIG. The results compared to the reference method of Kessler and Lederer are shown in Table 1. Good agreement was observed between these two methods.

EXAMPLE 4 The method described herein was accurately tested by repeated analysis of two separately pooled serum samples. One had normal triglyceride levels and one had elevated triglyceride levels. The results are shown in Table 1. Coefficients of variation (cov) of 5.1% and 2.6% were calculated for normal and abnormal serum, respectively.

Table Example 5 Alternative Electron Acceptors To demonstrate electron acceptors other than oxygen, a reaction mixture containing the following components was prepared. 0.1 M potassium phosphate buffer (to pH 7) 0.2 MD, L-α-glycerophosphate Electron acceptors and their concentrations as specified in the table In each case, the mixture was kept in equilibrium at 37°C and the enzyme The reaction was started by the addition of . The activity of this enzyme is ε ₆₀₀ = 16 × 10 ³ (molecular extinction coefficient of the dye 2,6-dichlorophenol indophenol at 300 nm), ε ₄₀₀ = 1 × 10 ³ (molecular extinction coefficient of the dye K ₃ Fe (CN) _{6 at 400} nm). extinction coefficient) and ε ₅₀₅
= 18.5×10 ³ (dye 2-(p-iodophenyl)-3-(p-nitrophenyl)-5- at 505 nm
Using the molecular extinction coefficient of phenyl-2M-tetrazolium chloride (INT), it was determined from the initial linear slope of the spectral curve according to Beer's law. These results are shown in Table 1.

Table Lazolium Chloride The method described herein can of course be used to quantify any of the various reagents and enzymes used in the overall reagent system. For example, ATP can be measured using a composition consisting of all reagents except the ATP introduced by the analytical sample. Similarly, glycerol kinase, lipase and α
- Glycerophosphate can be measured using a composition containing all other required substances besides the analyte. In order to obtain test compositions suitable for qualitative or semi-quantitative analysis of glycerin or triglycerides,
The analytical compositions described herein may be incorporated into matrices of absorbent materials of the type known in the art by impregnation or other methods. Typical products and elements that can be adapted for analysis of glycerin or triglycerides are described, for example, in U.S. Pat.
3092465, 3418099, 3418083, 2893843, 2893844, 2912309,
Same No. 30018879, Same No. 3802842, Same No. 3798064
No. 3298739, No. 3915647, No. 3915647, No.
It is described in No. 3917453, No. 3933594, No. 3936357, etc. Although the present invention has been described in particular detail with respect to its preferred embodiments, it will be understood that various changes and modifications can be made within the spirit and scope of the invention.

[Brief explanation of the drawing]

Figure 1 shows Streptococcus faecalis
This figure shows the PH activity of α-glycerophosphate oxidase from the ATCC12755 strain. In the figure, marks (○) are based on phosphate buffer, and (□), (△-△), and (×) are based on sodium acetate, tris-HCl, and glycine-KOH buffers, respectively. FIG. 2 is a graph showing the relationship between glycerin concentration (substrate concentration) and dye production concentration in the analysis according to the present invention.

Claims (1)

[Claims] 1. An aqueous product characterized by comprising: 1 (a) lipase (b) glycerol kinase (c) adenosine triphosphate (d) α-glycerophosphate oxidase (e) a substance exhibiting peroxidation activity A composition for detecting triglycerides in a liquid.