JP4019463B2 - Method for analysis of lipid components in serum - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for analyzing the amount of lipid components contained in serum, and more particularly to a method for analyzing the concentration of each component such as glycerin fatty acid ester, cholesterol fatty acid ester, and phospholipid in serum.
[0002]
[Prior art]
In recent years, the number of people who have deviated from normal values in serum cholesterol, glycerin fatty acid ester, or phospholipid concentration tests has increased rapidly due to the westernization of food, increased drinking opportunities, lack of exercise, and stress. For each component, the cholesterol level is a risk factor for lifestyle-related diseases represented by diabetes, arteriosclerosis, hypothyroidism, etc., and the level of glycerin fatty acid ester, commonly called neutral fat, is the lipid metabolism. Abnormalities, cerebrovascular disorders, myocardial infarction, angina pectoris, diabetes risk factors, and phospholipid levels are attracting attention as risk factors for dyslipidemia, obstructive liver disorders, hyperthyroidism, and fulminant hepatitis Yes.
[0003]
Against this background, there is a need for a method for measuring the concentration of each component specifically, quickly, simply and with high sensitivity, and an inexpensive analyzer using the method.
[0004]
Conventional methods for measuring serum components are roughly classified into enzyme methods and chemical methods. For example, in the case of cholesterol fatty acid ester (ester cholesterol), the enzymatic measurement method mainly used at present is measured by three enzyme reactions.
[0005]
That is, (1) cholesterol fatty acid ester is hydrolyzed by cholesterol esterase into free cholesterol. (2) Free cholesterol is oxidized by cholesterol oxidase and produces hydrogen peroxide as a by-product. (3) This hydrogen peroxide is oxidatively condensed with 4-aminoantipyrine and a dimethylaniline derivative in the presence of peroxidase to produce a quinone dye. Cholesterol fatty acid esters are quantified by measuring the pigment produced here with a spectrophotometer or the like.
[0006]
In the case of glycerin fatty acid ester, measurement is performed through four reactions.
[0007]
(1) Glycerin fatty acid ester in serum is hydrolyzed into glycerol and fatty acid by lipoprotein lipase or lipase and protease. (2) The glycerol produced here is phosphorylated to glycerol-1-phosphate by glycerol kinase in the presence of adenosine triphosphate (ATP) and magnesium ions (Mg ++). (3) This glycerol-1-phosphate is oxidatively decomposed into hydrogen peroxide and dihydroxyacetone by glycerol phosphate oxidase. (4) This hydrogen peroxide is oxidatively condensed with 4-aminoantipyrine and a dimethylaniline derivative in the presence of peroxidase to produce a quinone dye. The pigment | dye produced here is measured with a spectrophotometer etc., and quantification of glycerol fatty acid ester is performed.
[0008]
In the case of phospholipids, measurement takes place via three reactions.
[0009]
(1) Phospholipids in serum are degraded by phospholipase D to produce choline. (2) Choline is oxidized and decomposed into hydrogen peroxide and betaine by choline oxidase. (3) This hydrogen peroxide is oxidatively condensed with 4-aminoantipyrine and a dimethylaniline derivative in the presence of peroxidase to produce a quinone dye. Phospholipids are quantified by measuring the dye produced here with a spectrophotometer or the like.
[0010]
Next, many chemical methods have been reported and put into practical use. However, after the enzymatic method has been generalized, it takes too much time to measure and the operation is complicated. Almost no longer used.
[0011]
For example, as an example of a chemical method of cholesterol, glycerol fatty acid ester is analyzed by extracting glycerol fatty acid ester with an organic solvent such as methanol / chloroform (1: 2 V / V), and then oxidizing with sodium periodate, Color is measured with acetylacetone and measured.
[0012]
[Problems to be solved by the invention]
By the way, each of these conventional measuring methods has the following problems.
[0013]
Problems in the enzymatic method are as follows: (1) Since four or three different enzymes and a large number of reagents are required, the work is complicated. (2) Correct values cannot be obtained unless all four-step or three-step reactions proceed completely. (3) If a problem occurs, it cannot be immediately determined which reaction process has the problem.
[0014]
In addition, problems in the chemical method are as follows: (1) The operation is complicated and the measurement takes too much time. (2) Reactions with chemical substances have low specificity, and it is difficult to obtain accurate values due to many impurities other than the target component contained in serum. (3) Since a large amount of organic solvent is used, the pain of the analyzer is fast, and it takes time to dispose of the used organic solvent.
[0015]
In any of the analysis methods, a very large and expensive analyzer is required to analyze serum lipid components.
[0016]
Therefore, the present invention solves the problems of conventional measuring methods and analyzers, and lipid components contained in serum, specifically cholesterol fatty acid esters, glycerin fatty acid esters and phospholipids, etc. In the analytical method, for example, hydrogen peroxide is measured through four or three stages of reaction using four or three different enzymes by the most commonly used enzyme method. Focusing on the separation of fatty acids from each lipid component and analysis of the amount of fatty acids, the free fatty acid produced by the enzyme reaction is directly measured, so that the concentration of serum lipid components is specific, simple, rapid, and highly sensitive It is an object of the present invention to provide an analytical method that can be quantitatively determined.
[0017]
[Means for Solving the Problems]
The method for analyzing lipid components in serum of the present invention produces fatty acids using enzymes that specifically act on various lipid components in serum, and measures the total acid concentration in the serum to measure the total acid concentration in the serum. In the method for analyzing lipid components in serum for calculating the lipid component concentration of the serum, the first step of measuring the total acid concentration in the serum before the fatty acid production, and the specific action on each lipid component in the serum A second step of producing a fatty acid using an enzyme, a third step of measuring the total acid concentration in the serum after the fatty acid is produced in the second step, the measured value in the first step, and the And a fourth step of calculating a lipid component concentration in the serum based on the measurement value in the third step.
[0018]
The method for analyzing lipid components in serum of the present invention produces fatty acids and organic acids using enzymes that specifically act on various lipid components in serum, and measures the total acid concentration in the serum. In the method for analyzing a lipid component in serum to calculate the lipid component concentration in the serum, a first step of measuring the total acid concentration in the serum before the production of the fatty acid and the organic acid, and in the serum A second step of producing fatty acids and organic acids using enzymes that specifically act on each of the various lipid components, and the total acid concentration in the serum after the fatty acids and organic acids are produced in the second step. A third step of measuring, and a fourth step of calculating a lipid component concentration in the serum based on the measured value in the first step and the measured value in the third step.
[0019]
[Embodiments of the Invention]
(Embodiment 1)
First, the method for analyzing lipid components in serum in the present invention will be described. The first step is to use free fatty acids or organic acids using enzymes that act specifically on various components as described below. Is generated. The chemical formula shown in (Chemical Formula 1), which is mainly known as a method for generating free acids from various components, generates cholesterol and free fatty acids by hydrolyzing cholesterol fatty acid esters in serum with cholesterol esterase. It is a reaction formula.
[0020]
[Chemical 1]
[0021]
The chemical formula shown in (Chemical Formula 2) is a reaction formula in which glycerol fatty acid ester in serum is hydrolyzed by lipoprotein lipase to produce glycerol and free fatty acid. In this case, the number of fatty acids bound to glycerol of the glycerin fatty acid ester is considered to be 1 to 3, but in the case of serum, it is found that 90 to 95% of the glycerin fatty acid ester is triglyceride of 3 fatty acids. Therefore, glycerin fatty acid ester and triglyceride are almost synonymous. Therefore, when glycerin fatty acid ester in serum is an analysis target, it can be considered that three free fatty acids are generated from one molecule of glycerin fatty acid ester.
[0022]
[Chemical 2]
[0023]
Furthermore, the chemical formula shown in (Chemical Formula 3) shows a reaction for producing free fatty acid from phospholipid in serum. However, in the case of phospholipids, the term “phospholipid” does not refer to a single compound, but is a general term for compounds having an organic phosphate group such as lecithin, sphingomyelin, lysolecithin, phosphatidyl, ethanolamine and the like. Most phospholipids, including the compounds listed here, produce free fatty acids with phospholipase A.
[0024]
[Chemical Formula 3]
[0025]
The method for producing free fatty acids from various components using enzymes is not intended only for the above reaction, and any method capable of specifically producing free fatty acids from various components can be used. However, it is preferable to select an enzyme reaction that generates free fatty acids from various components in one step in consideration of simplicity. The above-mentioned various enzymes are not particularly specified for their origin, and may be derived from any origin such as animal organs, microorganisms, and DNAs artificially prepared and modified by genetic manipulation. One thing to note is that the reaction produced by the substance produced by the enzyme reaction cannot interfere with the next measurement, ie the analysis of free fatty acids. In the above-mentioned enzyme reaction given as an example, no problem was found.
[0026]
Commercially available enzymes can be used as they are as the examples of enzymes used in the present invention. For the conditions such as the amount of enzyme used, reaction temperature, reaction time, etc., it is possible to use the numbers presented by the seller as desirable, but the analyst can freely set them according to the purpose. It is also possible to change.
[0027]
The second stage measurement for quantifying the free fatty acid concentration produced by the first stage enzyme reaction is a direct measurement of the free fatty acid. The method or measuring apparatus for measuring acid will be specifically described below.
[0028]
The method of measuring the acid concentration electrochemically using voltammetry is to mix an organic solvent, a quinone derivative and an electrolyte with serum that has been freed of fatty acids by enzymatic treatment, and gradually charge the coexisting electrolyte. In this method, the concentration of acid is analyzed by measuring the peak value of the voltammogram reduction pre-wave that is characteristic only when the acid is present in the solution when the generated current is measured. In this coexisting electrolyte, the fatty acid acts as a strong proton donor and gives protons to the quinone derivative present in the coexisting electrolyte. Adducts of proton donors and quinones such as fatty acids are more easily reducible than quinone alone, so that in the voltammogram, a reduction wave (a voltammogram reduction pre-wave) before the reduction wave normally exhibited by quinones in organic solvents. Produce. It has been found that the current value of the pre-reduction wave is proportional to the acid concentration in the solution. The acid concentration in the sample is accurately measured by comparing with a calibration curve obtained in advance with a known acid concentration solution. Since the position of the pre-reduction wave hardly changes depending on the type of fatty acid, the total amount of acid can be accurately measured even if there are many types of fatty acid produced from serum by the first stage enzyme reaction.
[0029]
By measuring the total acid amount before the enzymatic reaction in the same manner and taking the difference from the total acid amount after the reaction, various lipid components in the sample, specifically cholesterol fatty acid ester, glycerin fatty acid ester, and phospholipid are individually measured. Content can be quantified. The kind of various quinone derivatives, solvents or electrolytes used in the electrochemical measurement method described above is not particularly limited as long as it shows a reduction pre-wave proportional to the acid concentration.
[0030]
An embodiment of an acid concentration measuring apparatus applying this principle will be described below.
[0031]
FIG. 1 is an apparatus for analyzing lipid components in serum using an electrochemical acid concentration measurement method according to an embodiment of the present invention.
[0032]
In FIG. 1, 1 is an enzyme reaction part, 2 is an enzyme reaction container, 3 is a temperature holding device, 4 is a stirring device, and 5 is serum and enzyme solution. In addition, 6 is an acidity measurement unit, 7 is a coexisting electrolyte container, 8 is a coexisting electrolyte, 9 is a working electrode, 10 is a counter electrode, 11 is a comparison electrode, 12 is a control unit, and 13 is a display unit.
[0033]
The enzyme reaction unit 1 includes an enzyme reaction container 2 containing an enzyme that specifically acts on each lipid component in serum, and a temperature at which this enzyme reaction can be stably maintained (many 30 ° C. to 40 ° C.) The temperature holding device 3 that can maintain the temperature is provided. By introducing the serum into the enzyme reaction vessel 2, fatty acids can be released from the serum.
[0034]
The stirrer 4 advances the enzyme reaction quickly.
[0035]
The acid-containing serum enzyme-treated in the enzyme reaction unit 1 is mixed with a quinone derivative, an organic solvent, and an electrolyte and led to the coexisting electrolyte container 7. As the quinone derivative, an orthobenzoquinone derivative or a parabenzoquinone derivative is desirable. This is because, with these quinone derivatives, the position of the pre-reduction wave of voltammogram described above appears shifted from the position of the reduction waveform of dissolved oxygen in the liquid, so it is accurate even without deoxygenation. This is because the acid concentration can be analyzed. The organic solvent is preferably an ethanol solution containing 35% or more of isooctane. At this time, the fatty acid can be sufficiently dissolved. As the electrolyte, lithium peroxide is suitable.
[0036]
A coexisting electrolyte solution container 7 into which acid-containing serum is introduced is filled with a coexisting electrolyte solution 8, and a working electrode 9, a counter electrode 10, and a reference electrode 11 are immersed therein. The control unit 12 composed of a microcomputer or the like sweeps the potential of the working electrode 9 within a predetermined range from the electrode potential of the comparison electrode 11 and simultaneously measures the value of the current flowing between the working electrode 9 and the counter electrode 10. Such a measuring method is usually called voltammetry. Furthermore, the control part 12 detects the electric current value of the reduction | restoration front wave which exists in the front position of the reduction | restoration waveform which a quinone derivative shows among this measured electric current value. This current value is proportional to the total fatty acid concentration in serum, or the total fatty acid concentration and the total organic acid concentration. By the way, in most enzyme reactions, fatty acids are produced. However, some enzymes that act on phospholipids produce organic acids in a slight but specific manner. Therefore, the acid concentration including this organic acid can be measured.
[0037]
Next, the control part 12 calculates the density | concentration of the lipid component in serum from the measured electric current value by collating with a calibration curve. When the density is calculated, the control unit 12 displays the density on the display unit 13 including an LCD or the like. In the conventional analysis by the enzymatic method, since the concentration analysis of hydrogen peroxide is performed after the enzyme reaction of 3 to 4 steps, there remains a problem in accuracy and it takes time, but the voltammetry of the first embodiment According to the serum lipid component analyzer according to the above, since the free fatty acid generated from the serum by a one-step enzymatic reaction is directly measured, the concentration of the serum lipid component can be quantified specifically, simply, quickly and with high sensitivity.
[0038]
(Embodiment 2)
FIG. 2 is a schematic diagram of an apparatus for analyzing lipid components in serum using high performance liquid chromatography according to another embodiment of the present invention.
[0039]
In FIG. 2, 1 is an enzyme reaction unit, 2 is an enzyme reaction vessel, 3 is a temperature holding device, 4 is a stirring device, 5 is serum and enzyme solution, 60 is an acidity measurement unit, 12 is a control unit, 13 is a display unit, 14 is an injection part, 15 is a metering high-pressure pump, 16 is a separation column, and 17 is a detection part.
[0040]
The operation of the enzyme reaction unit 1 provided with the enzyme reaction vessel 2 and the temperature holding device 3 is the same as that of the first embodiment.
[0041]
In the second embodiment, after the sample treated with the enzyme in the enzyme reaction vessel 2 is deaerated as necessary, a certain amount is weighed by an injection unit 14 such as an autosampler and injected into the acidity measurement unit 6. It may be a thing.
[0042]
The acidity measuring unit 60 in the second embodiment constitutes high performance liquid chromatography, and includes a quantitative high-pressure pump 15, a separation column 16, and a detecting unit 17. This fixed high-pressure pump 15 is capable of delivering a small amount of sample at a high pressure and a constant flow rate. The separation column 16 is uniformly packed with a filler adjusted for fatty acid measurement, and the sample fed by the metering high-pressure pump 15 is separated for each substance constituting the sample by the separation column 16. . The detection part 17 detects the density | concentration of a fatty acid among this substance. As the detection unit 17, a differential refractometer, an ultraviolet-visible spectrophotometer, a laser beam excitation fluorescence detector, or the like is appropriate. By calculating the total amount of fatty acid from the concentration of the fatty acid thus obtained, and further determining the difference from the total amount of acid before enzyme treatment, various lipid components in the sample, specifically cholesterol fatty acid ester, glycerin Quantification of the individual contents of fatty acid esters and phospholipids becomes possible.
[0043]
The control unit 12 measures the operation time of the metering high-pressure pump 15 and the time when the separation of the substance in the separation column 16 is completed, and then operates the detection unit 17 to calculate the total amount from the detected fatty acid concentration, The difference from the total amount of acid before the enzyme treatment is determined to determine the content of various lipid components in the sample.
[0044]
As described above, according to the serum component analyzer using the high performance liquid chromatography of the second embodiment, the serum component is optically analyzed, so that the sample can be measured without giving electrochemical stimulation, and the existing high speed liquid chromatography can be performed. The lipid component in serum can be measured specifically, simply, rapidly and with high sensitivity using liquid chromatography.
[0045]
In addition to the examples in the first and second embodiments described above, various apparatuses for measuring the total acid concentration can be applied. For example, an automatic or semi-automatic neutralization titration apparatus, a potentiometric titration apparatus, a photoelectric photometry A total can be considered.
[0046]
【The invention's effect】
In the present invention, the concentration of each component such as cholesterol fatty acid ester, glycerin fatty acid ester and phospholipid in serum can be measured specifically, highly sensitively, simply and rapidly, and lipid component analysis in serum for performing such analysis Equipment can also be provided at low cost.
[0047]
In addition, the application of the analysis method and analysis apparatus of the present invention makes it easy to obtain reliable and accurate serum lipid component concentrations, and not only clinical tests at medical sites but also personal health at home. Information useful for management can also be provided. In addition, it is possible to improve research speed and research accuracy in basic research and application / development research on each component.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a serum lipid component analyzer using an electrochemical acid concentration measurement method in one embodiment of the present invention. FIG. 2 utilizes high performance liquid chromatography in another embodiment of the present invention. Schematic diagram of the analyzed serum lipid component analyzer [Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Enzyme reaction part 2 Enzyme reaction container 3 Temperature holding device 4 Stirrer 5 Serum and enzyme solution 6 Acidity measurement part 7 Coexisting electrolyte container 8 Coexisting electrolyte 9 Working electrode 10 Counter electrode 11 Comparison electrode 12 Control part 13 Display part 14 Injection 15 Quantitative high-pressure pump 16 Separation column 17 Detection unit 60 Acidity measurement unit

Claims (5)

  Fatty acid is produced using enzymes that specifically act on various lipid components in serum, and the concentration of lipid components in serum is calculated by measuring the total acid concentration in the serum. In the analysis method, a first step of measuring the total acid concentration in the serum before the fatty acid production, a second step of producing fatty acids using enzymes that specifically act on various lipid components in the serum, The third step of measuring the total acid concentration in the serum after the fatty acid is generated in the second step, and the serum based on the measured value in the first step and the measured value in the third step And a fourth step of calculating the lipid component concentration of the serum.   A fatty acid and an organic acid are produced using enzymes that specifically act on various lipid components in the serum, and the concentration of the lipid component in the serum is calculated by measuring the total acid concentration in the serum. In the lipid component analysis method, using the first step of measuring the total acid concentration in the serum before the production of the fatty acid and the organic acid, and an enzyme that specifically acts on each of the various lipid components in the serum A second step for producing a fatty acid and an organic acid, a third step for measuring the total acid concentration in the serum after the fatty acid and the organic acid are produced in the second step, and a measurement value in the first step. And a fourth step of calculating a lipid component concentration in the serum based on the measurement value in the third step, and a method for analyzing a lipid component in serum. 3. The method for analyzing a lipid component in serum according to claim 1, wherein the lipid component in the serum is a cholesterol fatty acid ester. 3. The method for analyzing a lipid component in serum according to claim 1, wherein the lipid component in the serum is a glycerin fatty acid ester. 3. The method for analyzing a lipid component in serum according to claim 1, wherein the lipid component in the serum is a phospholipid.