JPH0131880B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF INDUSTRIAL APPLICATION The present invention relates to the quantitative analysis of glycerin in serum samples. BACKGROUND OF THE INVENTION Three enzymatic methods are currently 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― Glycerophosphate + ADP ADP + phosphonoenolpyruvate pyruvate kinase ――――――――――――→ Pyruvate + ATP Pyruvate + NADH Lactate dehydrogenase ―――――――――――― ――――→ Lactate + NAD + (b) Weiland’s Method (Weiland, O. “Biochem Z” No. 329, p. 313 (1957)) Glycerin + ATP glycerol kinase ―――――――― ---→ L-α- Glycerophosphate + ADP L-α-Glycerophosphate + NAD+ α-Glycerophosphate dehydrogenase ---→ NADH + Dihydroxyacetone Phosphate + H ⁺ (c) Glycerol dehydrogenase method (Hagen, J.
H and Hagen, PB “Can J.Biochem and
Physiology, Vol. 40, p. 1129 (1962)) Glycerin + NAD + Glycerin dehydrogenase ――――――――――――――――→ Dihydroxyacetone + NADH + H ⁺ Modification of method (a) is described in German Patent No. 2665556 issue,
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. Triglyceride analysis method is German Patent No. 2139163
It is stated in the number. The patented process involves hydrolyzing triglycerides, oxidizing the resulting glycerin to formaldehyde, and reacting the formaldehyde with ammonia and a water-alcohol-soluble stable colorless acetylacetone metal complex to produce a colored compound. consists of doing. Problems to be Solved by the Invention The purpose of the present invention is to provide an improved analytical method for the quantitative analysis of glycerin in serum samples, in particular, which does not require strict and narrow pH control and in which the reagents used are stable. The object of the present invention is to provide a method for quantitatively analyzing glycerin components in serum. According to the invention: (a) in an aqueous medium, () a serum sample;
() Reactions in the presence of glycerin-containing serum in the following order: (A) reaction to convert glycerin to L-α-glycerophosphate, (B) reaction to convert L-α-glycerophosphate to α-glycerophosphate oxidase contacting an enzyme and a reagent in an aqueous medium that undergoes a reaction that oxidizes to produce a detectable change in the presence of (b) detecting the detectable change produced in (B). A method for quantitatively analyzing glycerin is provided. According to a preferred embodiment, the contact between the aqueous liquid test sample and the enzyme and reagent is carried out in the presence of oxygen as an electron acceptor. Said detectable product is, according to a highly preferred embodiment, a generally quantifiable colored substance. A highly preferred embodiment utilizes an indicator composition consisting of a peroxidizing agent and a dye precursor. The dye precursor is either (1) a compound that produces a dye in the presence of hydrogen peroxide and a substance that has a peroxidizing effect, or (2) a compound that is detectable to the naked eye in the presence of hydrogen peroxide and a substance that has a peroxidizing effect. does not carry out a reaction, but
It is characterized by consisting of either a compound or a series of compounds that react with another compound or series of compounds to produce a quantifiable product in proportion to the glycerin content in the analytical sample. The methods of the present invention differ from prior art methods and compositions in that they do not rely on the production or depletion of NADH, a disadvantage well recognized in the art and described in the literature. It shows an improvement. In the present invention, glycerin is suitably quantitatively measured by the following series of reactions (Table 1). Table 1 Glycerin + ATP Glycerol Kinase――――――――――→ L-α-Glycerophosphate + ADP 2 L-α-Glycerophosphate + Electron Acceptor α-Glycerophosphate Oxidase―――――― ――――――――――――→ Dihydroxyacetone phosphate + Species for analytical measurement When oxygen is the electron acceptor, H ₂ O ₂ is used for analytical measurement in (3) above. produced as a substance,
The measurement of H ₂ O ₂ is preferably carried out by the following reaction. 3 H ₂ O ₂ + H ₂ A (reduced type) peroxidase――――――――――→ A (oxidized type) + H ₂ O (detectable substance) However, H ₂ A (reducing type) is the reduction of dye It is a dye precursor that is in the form of a dye precursor. A (oxidized type) is a dye produced by oxidation of H ₂ A. In a combinatorial reaction of a preferred composition, a detectable substance is produced in proportion to the concentration of glycerin. This method can be used in many clinical applications, specifically for measuring 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, the stability of NAD ⁺ or NADH is not important since O ₂ is a cofactor for the α-glycerophosphate oxidase reaction. Fifth, NAD ⁺ or
Serum components that utilize NADH (e.g., lactate plus lactate dehydrogenase) are
Do not interfere with this process. Sixth, any means of measuring _O2 consumption when _O2 is used as an electron acceptor can be used as a detection means. Finally, 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 glycerin, 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. Glycerin Analysis The novel enzymatic glycerin analysis of the present invention can be performed after triglyceride hydrolysis if necessary. The first enzyme used in glycerin analysis is glycerin kinase, which catalyzes the conversion of glycerin to L-α-glycerophosphate in the presence of adenosine triphosphate (ATP). In general, any glycerol kinase can be used successfully to practice the invention, but 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 literature on their preparation and reactivity can be found in T.E.
"Enzyme Hand book I" by E. Barman (Springer 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. This detectable change is preferably a color change or the production of a color, which in preferred cases is quantitatively related 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 α-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, and 2-(p-iodophenyl)-3-(nitrophenyl)-5- as the electron acceptor in this reaction.
It has been found that phenyl-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-glycerate 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. Streptococcus faecalis
Enzymes from the American Type Culture Collection
Particularly preferred are the specialized strains available from the Microbial Collection. Particularly useful and preferred enzymes include bacterial species identified on the basis of the collection archives.
Obtained from ATCC 11700, ATCC 19634 and ATCC 12755. As described and illustrated in the examples below, the ATCC 12755 enzyme has activity over a slightly wider PH range than the enzymes from the other two bacterial species, and is therefore most preferred. The following two documents describe enzymes and useful techniques for their preparation and extraction.
References: Kodicietsk, L. K.
(Koditschek, LK) and Ambright, D'Avrieux. D'Avrieux. (Umbreit, WW), Journal of Bacteriology.
Streptococcus faecium α-glycerophosphate oxidase (α-
glycerophosphate oxidase in Streptoccocus
faecium)” Jacob, N. J.A. (Jacob, NJ)
and Vandemark, P. J.A. (Van
Demark PJ) “The Purification and Properties of α-glycerophosphate oxidase of Strectococcus faecalis
the α-Glycerophosphate Oxidiying Enzyme
of Streptococeus Foecalis 10 cl.) Enzymes prepared according to the methods described in any of the above-mentioned publications are useful in the successful practice of the present invention. 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-
Because α-glycerophosphate oxidase preparations contain very high concentrations of impurities, conventional fractionation and column separation methods must be used to prepare the crude The formulation had to be purified. Detection of glycerin in an aqueous solution containing glycerin, such as serum, is preferably carried out using an indicator composition that detects the concentration of hydrogen peroxide produced by oxidation of Lα-glycerophosphate in the presence of oxygen. 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. Among many others, U.S. Pat.
No. 3,092,465 and No. 2,981,606 describe such useful indicator compositions. The hydrogen peroxide indicator composition comprises a substance having a peroxidizing effect, preferably peroxidase, and a dye precursor that undergoes coloration or discoloration in the presence of hydrogen peroxide and the substance having a peroxidizing effect. Alternatively, the dye precursor may be oxidized in the presence of H ₂ O ₂ and a substance with peroxidizing properties without undergoing a substantial color change, but in its oxidized form, with a color-producing or color-changing substance, e.g. , a color former) to visually prove 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 polyphyllin 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 peroxidative properties are somewhat inferior. Other substances that are not enzymes but have peroxidative effects 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 a peroxidizing effect include the following substances. A coloring agent is also included if necessary. (1) Monoamines such as aniline and its derivatives, orthotoluindipara-toluidine, etc. (2) ortho-phenylenediamine, N,N'-dimethyl-para-phenylenediamine, N,
Diamines such as N′-diethylphenylenediamine, benzidine, dianisidine, etc. (3) Phenol itself, phenols such as thymol, ortho-methani and para-cresol, α-naphthol, β-naphthol (4) Catechol, guaiacol, Ausinol,
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 the 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 a leuco dye in the presence of peroxidase to provide the corresponding dye form. Representative leuco dyes include compounds such as leucomalachite green and leucophenolphthalein. 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 chromogenic agent, such as having a phenol group or an activated methylene group. may be provided. Typical examples of such oxidizing compounds include benzidine and its similar substances, p-
Phenylene diamine, p-aminophenol, 4
- Compounds such as 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 drugs with aminoantipine (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 Promise in
Biomedical Uses” (Chemical and Biomedical Uses)
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 above. However, this consumes the above amount of oxygen in reaction (3) in Table 1. 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 Glycerin 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, 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 resulting concentration of detectable substance 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 was determined by the method of Garland and Randle (Nature, Vol. 196, pp. 987-988 (1962)). Hydrogen peroxide solution
Measure A ₂₄₀ (absorbance at 240 nm) and E ₂₄₀ =
It was set as a standard by appropriately calculating using 43.6. Serum samples were analyzed using the Kessler and Lederer 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 contained 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 In addition to the above ingredients, the incubate mixture for triglyceride determination contained 10 mg (8 units/mg) of Candida gosa lipase. All components were kept in equilibrium for 5 minutes and (initial) A ₄₉₀
was measured. Glycerin standard (5-
The reaction was started by adding either 100 nmol) or serum (20 μl) and 20
It lasted for about 30 minutes. Then (final) I measured A ₄₉₀ . Deviations for this standard system are indicated where appropriate. Calculation of Triglyceride Concentration The triglyceride glycerin concentration of the unknown specimen was determined as follows. The ΔA ₄₉₀ (A ₄₉₀ (final value) - A ₄₉₀ (initial value)) of the specimen incubated in the presence of the standard glycerin detection system is the ΔA ₄₉₀ of the same specimen incubated in the presence of Lipase M and the standard glycerin detection system. Subtracted from. Triglyceride concentrations were determined from this dependent change in lipase M absorbance by the 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) is grown in 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 extracts Cells from 100 ml of culture medium were obtained by centrifugation (4°C, 10,000xg, 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).
Sonication (operating with 40 sets)
Sonication) destroyed. The temperature was kept below 8°C. The supernatant obtained by centrifugation at 10,000×g for 10 minutes was used as an enzyme source. The amount of soluble protein reached its 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.Biol.Chan” No. 193.
265 (1951)). Isolation of α-glycerophosphate oxidase from Streptococcus faecalis The results in the table 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 10,000×g was used as the enzyme source (crude extract). However, the oxidase remained in solution even after centrifugation at 100,000 xg for 1 hour. In all cases the rate of reduction of dissolved enzyme was proportional to the amount of crude extract and was completely dependent on both the D,L-α-glycerophosphate and the extract. Apparently, all three bacterial species exhibited oxygen-linked activity. ATCC11700
The optimum pH of the enzyme from the bacterial species is 5.8, and
It has been reported that the oxidase of ATCC19634 strain shows the highest value at pH 7.0. This trend is also seen in the table. The activity of 12755 bacterial species is analogous to
It was similar to the bacterial species of ATCC19634.

[Table] Oxygen electrode analysis of α-glycerophosphate oxidase New Brunswick Day. Oh. By measuring the decrease in dissolved oxygen with an analyzer (New Brunswick DO Analyzer), L-α
- Glycerophosphate oxidase was analyzed. To water saturated with both _N2 and air, with constant stirring with a magnetic stirrer,
The scale of this oxygen electrode was measured. Add an 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 100μml potassium phosphate buffer (PH7.0), 66μ
g 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 (PH7.0) 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. Activity was calculated from the initial linear slope of the reaction trace at 430 nm with ε=1.08×10 ⁴ . Properties of α-glycerophosphate oxidase in crude extract The detailed PH activity of α-glycerophosphate oxidase of 12755 bacterial species is shown in Figure 1. The optimal activity is
It was observed in a wide PH range from 6.3 to 7.5, and the activity decreased sharply at the end of PH 6.0 and above PH 8.0. Also shown in FIG. 1 is the apparent inhibition of the enzyme by either tris-HCl (△-△) or glycine-KOH (X) buffers. Activity in 0.1M potassium phosphate buffer at PH7.7 is 0.1M glycine-KOH
The activity was four times higher than that observed in the cells. 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 means that tris-HCl and glycine-KOH
This suggests that the potassium phosphate buffer is not an inhibitor, but rather activates the enzyme. Sodium acetate buffer also appears to activate this enzyme (Figure 1) since the activity at pH 6.5 in sodium acetate buffer is at least 90% of the activity observed in 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 1.

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 Triton X-100 at a concentration of 2%. EXAMPLES The following examples will serve to illustrate detailed embodiments of the invention. Example 1 Standard curve for glycerin and hydrogen peroxide The glycerin reaction curve is shown in FIG. A mixture was prepared as described above with determination of glycerin and triglycerides by the α-glycerophosphate oxidase method. The reaction was initiated by addition of substrate and substantially completed in 15 minutes at 37°C. In FIG. 2, a favorable relationship between the glycerin concentration (◯) and the amount of dye produced (x) was observed for coupling reactions 2, 3, and 4. Example 2 Quantitative determination of triglyceride substrates 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 by: The quantitative determination of triglyceride substrates by coupling reactions 1, 2, 3 and 4 was compared with the quantitative determination by the method of Garland and Randle. A sufficient amount of lipase from Candida Gosa was added to act as a catalyst to quickly (in less than 1 minute) and completely hydrolyze 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, shown 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
It was only larger than 10% in .

[Table] Example 3 Quantitative determination of serum triglycerides by the α-glycerophosphate oxidase method With the preferred buffer system of 0.2M potassium phosphate buffer at pH 8.0, 10 Serum samples were analyzed. The control mixture contained only the standard component for glycerin detection. The sample mixture contained Candidal-Gosa lipase and other standard components for glycerin detection. The entire mixture was equilibrated for 5 minutes at 37°C and the initial A ₄₉₀ was measured. Each serum sample
The reaction was started by adding 20 μl and the final A ₄₉₀ was measured after 20 minutes of incubation. After subtracting the ΔA ₄₉₀ of the control from the ΔA ₄₉₀ of the specimen,
Triglyceride glycerol concentrations were determined from an aqueous glycerol standard 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.

【table】

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 E ₆₀₀ = 16×10 ³ for 2,6-dichlorophenol indophenol;
For K ₃ Fe (CN ₆ ) E ₄₀₀ = 1×10 ³ and 2−
Calculated as above using E ₅₀₅ =18.5×10 ³ for (p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2M-tetrazolium chloride (INT). These results are shown in Table 1.

[Table] 〓 Ratio compared to the ratio of oxygen as an electron acceptor *=2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazoliu
Muchlorides The methods 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.

[Brief explanation of drawings]

Figure 1 shows Streptococcus faecalis.
This figure shows the PH activity of α-glycerophosphate oxidase from the ATCC12755 strain. In the figure, the marks (○) are due to phosphate buffer, and the marks (□), (△-△), and (×) are due to sodium acetate, tris-HCl, and glycine-KOH buffers, respectively. FIG. 2 is a glycerin reaction curve. In the figure, (○) indicates glycerin concentration, and × indicates dye production.

Claims (1)

[Scope of Claims] 1 (a) In an aqueous medium, () a serum sample;
() Reactions in the presence of glycerin-containing serum in the following order: (A) reaction to convert glycerin to L-α-glycerophosphate, (B) reaction to convert L-α-glycerophosphate to α-glycerophosphate oxidase contacting an enzyme and a reagent in an aqueous medium that undergoes a reaction that oxidizes to produce a detectable change in the presence of (b) detecting the detectable change produced in (B). A method for quantitative analysis of glycerin.