JPH06105700A - Measurement of l-carnitine and apparatus therefor - Google Patents

Abstract

PURPOSE:To provide a method for sensitively and accurately measuring L- carnitine and the apparatus therefor. CONSTITUTION:The method for measurement of L-carnitine as one of this inventions involves characteristically four processes composed of the first stage for making carnitine transferase(CAT) act on an examination sample in the presence of acetyl coenzyme A, the second stage for making acyl coenzyme A synthetase(ACS) act on coenzyme A synthesized in the first stage in the presence of a fatty acid and ATP, the third stage for making acyl coenzyme A oxidase (ACO) act on acyl coenzyme A produced through the second stage so as to oxidize the acyl coenzyme A and the fourth stage for measuring hydrogen peroxide generated in the third stage. The apparatus therefor as another invention is an equipment for measurement of L-carnitine, characteristically equipped with a column supporting three kinds of enzymes, i.e., CAT, ACS and ACO and with a chemiluminescence detector for measuring the generated hydrogen peroxide.

Description

Detailed Description of the Invention

[0001]

The present invention relates to L-carnitine, particularly L-carnitine contained in biological samples and foods with high sensitivity,
The present invention relates to a method for measuring with high accuracy and an apparatus used for carrying out the measuring method.

[0002]

2. Description of the Related Art Fat taken up in a living body is decomposed into free fatty acids and undergoes β-oxidation in intracellular mitochondria to produce ATP. L-carnitine has an important function as a carrier that transports long-chain fatty acids to the mitochondrial matrix, and the lipid metabolism rate depends on the L-carnitine concentration. In addition, when L-carnitine is deficient, it is reported that not only abnormalities in lipid metabolism are caused, but also skeletal muscles and myocardium are damaged, resulting in diseases such as ischemic heart disease. For patients and athletes in
L-carnitine is being administered. Further, secondary L-carnitine metabolism abnormality in renal failure and dialysis patients is also a problem. As described above, L-carnitine has already been studied at the clinical level such as analysis of pathological conditions and application to therapeutic drugs, but as for its measuring method, an appropriate method at the clinical level has not yet been developed. Not not.

The conventional methods for measuring L-carnitine include, for example, the following methods. Method for quantifying microorganisms using Candida yeast (Japanese Patent Laid-Open No. 1-225500). L-carnitine and acetyl coenzyme A (acetyl Co
A method in which carnitine transferase (CAT) is allowed to act on A) and the thiophenolate ion produced by the reaction of free CoASH and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) is determined by colorimetry (DTNB method) ) [J. Biol. C
hem. , Vol. 238, 2509 (1963), J. Lipid Researc
h, vol. 5, 184-187 (1964), Methods Enzymol. , V
ol. 14, 612-622 (1969) and clinical pathology, Vol. 36, No. 11, 12
96-1302 (1988)]. Acetyl Co labeled with L-carnitine and ¹⁴ C or ³ H
Method for measuring radioactivity of radiolabeled acetylcarnitine by allowing CAT to act on A (RI method) [J. LipidResearc
h, vol. 17, 277-281 (1976) and the Journal of Japan Society of Nutrition and Food Science, vol. 41 (5), 389-395 (1988)]. A method in which L-carnitine is fluorescently labeled, and CoASH generated by the action of CAT and acetyl-CoA that decreases are measured by HPLC (HPLC method) [Anal. Biochem. , Vol. 158, 3
46-354, (1986), J. Chromatogr. , Vol. 420, 385
-393 (1987), J. Chromatogr. , Vol. 445, 175-182
(1988), J. Nutr. Sci. Vitaminol. , Vol. 35, 475
-479 (1989)].

In the reaction in which L-carnitine dehydrogenase is allowed to act on L-carnitine and NAD to produce 3-dehydrocarnitine and NADH, the carnitine dehydrogenase method [Europe] which measures an increase in NADH in the ultraviolet region
an J. Biochem. , Vol. 6, 196-201 (1968), Freseni
us Z. Anal. Chem., vol. 320 (3), 285-289 (198
Five)]. CAT was allowed to act on L-carnitine and acetyl CoA, and N- [p- (2-benzimidazolyl) -phenyl] -maleimide (BIPM) was allowed to act on the produced CoA to produce CoA.
-Fluorescence method for measuring the fluorescence intensity of BIPM [Ministry of Health, Labor and Welfare
61 research report, 315-318, (1986)]. A method for measuring formazan produced by reacting L-carnitine and NAD with L-carnitine dehydrogenase (JP-A-3-61499). A method of reacting L-carnitine and NAD with L-carnitine dehydrogenase, allowing a small amount of NADH to coexist in this reaction system, performing a reversible cycling reaction between L-carnitine and dehydrocarnitine, and measuring the amount of NADH produced ( JP-A-3-251196).

[0005]

However, the above-described method for quantifying microorganisms has the drawbacks that the measurement takes time and the measurement sensitivity is low. Further, the above DTNB method has a problem that unstable acetyl-CoA is decomposed because the reaction time of CoASH and DTNB is long.
Although the method is excellent in sensitivity and specificity, there is a problem that it requires special equipment because it requires work in a radiation controlled area. Further, the above-mentioned HPLC method is not preferable because it requires a very long time for measurement, and the above-mentioned fluorescence method is complicated in its operation. In addition, the above-mentioned method, and the measurement method using NAD and NADH in the reaction, there is a problem in that the effect of these coenzymes remaining in the sample cannot be ignored, the measurement accuracy, the sample amount, etc. there were. Therefore, it is not suitable for the analysis of a small amount of sample, such as when measuring the L-carnitine content in foods or biological components. On the other hand, a device for simply measuring L-carnitine has not yet been developed.

Therefore, it is desired to develop a method for accurately measuring a sample containing such a trace amount of L-carnitine and an apparatus used for carrying out such a method. The present invention solves the above-mentioned problems of the prior art,
It is an object of the present invention to provide a novel method for measuring L-carnitine and a measuring device used for carrying out such a method.

[0007]

Means for Solving the Problems As a result of repeated studies to achieve the above object, the present inventors have found that peroxidation produced after L-carnitine is subjected to a series of enzymatic reactions as described below. By measuring hydrogen, it is possible to solve the above-mentioned problems of the prior art, and even in a small amount of sample,
The inventors have found that L-carnitine can be measured with high accuracy and high sensitivity, and completed the present invention.

A series of enzymatic reactions consists of the following three steps. Acetylation of L-carnitine by carnitine acetyltransferase (CAT)

[Chemical 1] Acyl coenzyme A (acyl CoA) synthetase (A
Acylation of CoA by CS)

[Chemical 2] Oxidation of acyl CoA by acyl CoA oxidase (ACO)

[Chemical 3]

That is, the present invention provides acetyl coenzyme A
In the presence of a carnitine acetyl transferase (CAT) in the presence of the first step, coenzyme A produced in the first step, in the presence of fatty acid and ATP acyl coenzyme A synthetase (ACS ) Is acted on, the acyl coenzyme A oxidase (ACO) is acted on the acyl coenzyme A produced in the second step to oxidize the acyl coenzyme A, and the third step, A method for measuring L-carnitine, comprising a fourth step of measuring hydrogen peroxide produced in the third step. The present invention also provides an apparatus for carrying out the above-mentioned method for measuring L-carnitine, which comprises 3 of CAT, ACS and ACO.
It comprises an apparatus having an enzyme-immobilized column in which immobilized enzymes in which various types of enzymes are immobilized on a carrier are packed in a column, and a chemiluminescence detector for measuring the produced hydrogen peroxide. .

The CAT, ACS and ACO used in the present invention are all known enzymes and widely distributed in the living body. These enzymes are also commercially available, and in the present invention, commercially available products or crude products obtained from organisms may be used. Further, when these three types of enzymes are used as immobilized enzymes immobilized on an immobilization carrier, the enzymes can be reused or continuously used, which is also preferable from the viewpoint of operation. As the immobilized enzyme, it is preferable to use a carrier obtained by immobilizing the above-mentioned three kinds of enzymes by a conventional method in a single column.

Before carrying out the method for measuring L-carnitine of the present invention, it is necessary to prepare a measurement sample. The solution-type measurement sample can be used as it is, but if it is not in solution, it is preferable to once dissolve it in water or disperse it in water to form a solution. In addition, the measurement sample is preferably adjusted to pH 7.

It is desirable that the measurement sample be subjected to deproteinization treatment in advance. In particular, biological samples such as urine, blood, lymph, and milk usually contain the enzyme catalase, which decomposes hydrogen peroxide produced in the third step and reduces chemiluminescence. It is desirable to remove catalase by performing deproteinization treatment in advance. Examples of such deproteinization treatment operations include a method of precipitating a protein with perchloric acid and an adsorption removal method using a protein adsorption resin, but any method that does not destroy or decompose L-carnitine Can even be used. In addition, when it is clear that hydrogen peroxide is contained in the sample from the beginning, it is necessary to remove the hydrogen peroxide by subjecting the measurement sample to catalase treatment in advance. A commercially available product can be used as this catalase.

The measurement sample of the present invention is subjected to an enzymatic reaction together with a reagent for the enzymatic reaction. As such an enzyme reaction reagent, acetyl CoA is used in the first step, and fatty acid and ATP, which serves as energy for the enzyme reaction, are used in the second step. The fatty acid is preferably, but not limited to, n-octanoyl acid having 8 carbon atoms, which has relatively high activity as a substrate for ACS and ACO and has good solubility in water. Acetyl CoA is preferably used in an amount of 1 to 10 μmol, particularly 2.4 μmol, based on 2 ml of the measurement sample. Further, the fatty acid is preferably used at a concentration of 1 to 3 mM, particularly 3 mM with respect to the enzyme reaction reagent, and ATP is preferably used at a concentration of 1 to 5 mM, particularly 2 mM with respect to the enzyme reaction reagent. It is preferably used. Other than these, EDTA, magnesium chloride, flavin adenine dinucleotide and the like can be used as the enzyme reaction reagent.

The enzyme reaction from the first to the third step in the measuring method of the present invention may be carried out by sequentially adding the enzyme necessary for each step and the reagent for the enzyme reaction, or CAT. Alternatively, the measurement sample and the enzyme reaction reagent may be passed through a column in which three types of enzymes, ACS, and ACO are immobilized on an immobilizing carrier and the immobilized enzyme is packed. After the enzymatic reaction, the hydrogen peroxide produced in the third step is measured. This measurement may be carried out by any known method, but an enzymatic method using peroxidase, luminol or the like is used. The chemiluminescence method is preferable because it can be measured with high sensitivity. The chemiluminescence method using luminol has been widely used in recent years in combination with peroxidase as a means of high-sensitivity analysis method, and has a feature that luminescence proceeds in a very short time.

Hereinafter, in the present specification, an example of measurement by a chemiluminescence method using luminol as a means for measuring hydrogen peroxide is illustrated, but the present invention is not limited to this method. If a reducing substance such as ascorbic acid is present in the sample, it may be difficult to perform accurate measurement. Therefore, it is preferable to use these reductants as oxidants before the measurement. . This oxidation treatment is preferably carried out by an apparatus having an electric field cell that allows a standardized electrolysis current to flow through the sample to oxidize the reductant by almost 100%. In order to carry out the oxidation simply, it is appropriate to use an electrochemical detector for HPLC, which is commercially available as a coulometric detector, but the device is assembled according to the principle of the coulometric device. You may implement. If no reducing substance is present in the sample, this operation is unnecessary.

The reagents used in the chemiluminescence method are 2 mg of luminol and 0.37 g of EDTA in 0.2 M borate buffer (pH 9.5) 2.
It consists of a solution dissolved in 00 ml. This chemiluminescent reagent can be mixed with the measurement reaction product obtained after the third step, and the amount of chemiluminescence having a maximum wavelength near 426 nm generated can be measured. The amount of L-carnitine in the sample can be determined by comparing and converting the amount of chemiluminescence with a calibration curve prepared by measuring standard L-carnitine. Since chemiluminescence is a reaction that proceeds in a very short time, a series of steps from the introduction of the measurement sample before entering the first step to the measurement of the chemiluminescence amount in the fourth step are measured using an automated device. It is preferable.

The measuring apparatus invented by the present inventors as an apparatus for automatically performing the above-mentioned measuring method is an enzyme-immobilized column in which three kinds of enzymes of CAT, ACS and ACO are immobilized, and hydrogen peroxide produced. And a chemiluminescence detector for measuring the amount of This measuring device
A device for supplying the measurement sample and the reagent for enzyme reaction such as acetyl CoA, fatty acid and ATP to an enzyme-immobilized column, the enzyme-immobilized column, and the chemiluminescence for measuring hydrogen peroxide produced. It is preferable to provide a detector and, if necessary, provide the oxidation treatment device immediately before the chemiluminescence detector.

The measurement sample and the enzyme reaction reagent are supplied to the enzyme-immobilized column by an apparatus for supplying them to the enzyme-immobilized column, subjected to the enzyme reaction there, and then the sample discharged from the column is It is mixed with the luminol reagent and sent together to a chemiluminescent detector to measure the amount of hydrogen peroxide produced in the sample. Also, if necessary,
An oxidation treatment device (electrochemical detector) that electrochemically oxidizes a reducing substance may be installed in front of the enzyme-immobilized column. Furthermore, in the case of measuring a sample containing hydrogen peroxide from the beginning, a column packed with a carrier on which catalase is immobilized can be placed immediately before an enzyme-immobilized column on which the above-mentioned three types of enzymes have been immobilized. .

[0019]

EXAMPLES The present invention will be described in more detail with reference to Examples and Reference Examples. Example 1 Measuring Device of the Present Invention FIG. 1 is a diagram showing the configuration of the measuring device of L-carnitine of the present invention. In the figure, 1 is a reagent tank for enzyme reaction, 2 is a pump for moving the reagent for enzyme reaction and the measurement sample into the column, and 3 is an injector for injecting the measurement sample. The measurement sample is injected from the injector 3, and when a reductive substance such as ascorbic acid is present in the measurement sample, the action of the pump 2 causes the oxidation treatment device 4 to move into the oxidation treatment device 4 as the enzyme reaction reagent moves. Be flowed in. When a substance having a reducing property such as ascorbic acid and inhibiting chemiluminescence is present in the measurement sample, it is electrolyzed in this oxidation treatment device 4 and then flown into the enzyme immobilization column 5. When the substance having a reducing property such as ascorbic acid does not exist in the measurement sample, the measurement sample and the enzyme reaction reagent directly flow into the enzyme-immobilized column 5 without passing through the oxidation treatment device 4. This enzyme-immobilized column 5 has a structure in which two columns are connected in series, the first half column is filled with a catalase-immobilized carrier, and the second half column is CAT, ACS, ACO.
The carrier on which the three types of enzymes are immobilized is filled. The mobile phase containing the measurement sample flowing into the enzyme-immobilized column 5 is
First, the hydrogen peroxide contained in the sample is decomposed by catalase, and then the enzymatic reaction of L-carnitine proceeds in the latter half column to generate hydrogen peroxide in proportion to the amount of carnitine.

On the other hand, 7 indicates a chemiluminescent reagent tank, and 8 indicates a pump for sending it to the chemiluminescent detector 6. Hydrogen peroxide generated in the enzyme-immobilized column 5 reacts with a chemiluminescent reagent in the chemiluminescence detector 6 to emit chemiluminescence, and the emitted light is recorded by the recorder 9. By adopting the above-mentioned configuration, it is possible to automatically carry out the injection of the measurement sample to the measurement of L-carnitine contained therein in a short time.

Reference Example 1 Preparation of Immobilized Enzyme Column The immobilized enzyme column used in the apparatus of Example 1 was prepared as follows. Aminopropyl CPG (Co
ntrolled Pore Glass) 0.1 g (manufactured by CPG Inc. USA., 500 Å, 200/400 mesh) was added with 10 ml of 2.5% glutaraldehyde aqueous solution and gently stirred for 1 hour while degassing under reduced pressure. After filtering the resulting mixture through a glass filter, the residue is treated with 500 ml of water and 3 parts of phosphate buffer solution.
Washed with 0 ml. Next, carnitine acetyltransferase (CAT) 80Unit (manufactured by Sigma), acyl C
oA Synthetase (ACS) 30Unit (Toyobo) and Acyl C
It was mixed with oA oxidase (ACO) 33 Unit (manufactured by Wako Pure Chemical Industries) and reacted at 4 ° C. for 20 hours to obtain an immobilized enzyme. The immobilized enzyme was packed in a stainless steel column (60 mm x 2 mm id) to give an enzyme-immobilized column. In the same manner, 10 units of catalase (manufactured by Nagase Biochemicals) were also immobilized to obtain an enzyme-immobilized column.

Reference Example 2 Effect of Carbon Number of Acyl CoA on Measured Value Acyl CoA oxidase (ACO) used in the reaction has different substrate specificity depending on the carbon number of acyl CoA as a substrate. Using acyl CoA having 10 and 16 carbon atoms, the effect on the measured value was confirmed, and the optimum carbon number of the fatty acid used for the enzyme reaction reagent was selected. In this embodiment,
Using the column of Reference Example 1 and the apparatus of Example 1, the amount of luminescence was measured for the acyl CoA prepared at the concentrations shown in FIG. Preparation of enzyme reaction reagent EDTA 0.74 g, ATP 0.48 g, MgCl ₂ 0.8 g and flavin adenine dinucleotide (FAD) 0.0334 g were dissolved in 50 mM phosphate buffer (pH 7.0) 400 ml to prepare an enzyme reaction reagent. did. Preparation of chemiluminescent reagent Luminol 2 mg and EDTA 0.37 g in 0.2 M borate buffer (pH 9.
5) It was dissolved in 200 ml and used as a chemiluminescent reagent. Effect of the difference in carbon number of acyl CoA on the measured values The measured results are shown in Fig. 2. No effect was observed due to the difference in the number of carbons, but due to the solubility in the reagents for enzyme reaction, C8
It was judged that it is preferable to use the fatty acid of. Therefore,
In the examples below, sodium n-octanoylate was used.

Example 2 Measurement of L-carnitine The enzyme-immobilized column prepared in Reference Example 1 and the enzyme reaction reagent prepared in Reference Example 2 (sodium n-octanoate)
0.2g) and chemiluminescent reagent
The carnitine content was measured. Preparation of calibration curve for L-carnitine Prepare a solution of L-carnitine at a concentration of 1 μM to 3 mM, and add 2.4 μmo to a standard solution of 2 ml at each concentration.
A solution was prepared with the addition of 1 acetyl CoA. This sample was measured using the apparatus of Example 1 to prepare a calibration curve. The calibration curve is shown in FIG. As is clear from the figure, the calibration curve is 1
It had linearity in the range of μM to 100 μM. The L-carnitine content can be determined by reading the measured value from this calibration curve and multiplying it by the dilution factor of the sample.

Measurement of L-carnitine content in milk and dairy products: 25 ml of 6% perchloric acid was used to deproteinize 25 ml of sterilized (UHT) milk and 40 ml of infant formula powdered milk. Then, centrifugation was performed and the supernatant was recovered. Adjust the pH of this supernatant to 7.0 with an aqueous potassium hydroxide solution, and then use a 0.45 μm-filtered solution as a sample to prepare 2 ml of this sample.
Then, acetyl-CoA was added to measure the amount of L-carnitine. Table 1 below shows the measurement results of the amount of L-carnitine in the measurement samples consisting of 5 sterilized milk samples and 5 infant formula powdered milk samples.

[0025]

[Table 1]

Measurement of standard L-carnitine added to milk and dairy products: 25 ml of 6% concentration of perchloric acid was used to deproteinize 25 ml of sterilized milk and 40 ml of infant formula powdered milk, and the mixture was centrifuged. Processing was performed and the supernatant was collected. The supernatant was adjusted to pH 7.0 with an aqueous solution of potassium hydroxide, then 0.45 μm filtered solution was used as a sample, and acetyl CoA was added to 2 ml of this sample. Carnitine was added, and the measurement was performed in the same manner as above. The calibration curve obtained by standard addition is shown in FIG. The calibration curve of L-carnitine added to the sample had linearity in the range of 10 to 100 μM, and it was confirmed that measurement was possible. Quantification of L-carnitine added to pasteurized milk and measurement of recovery rate Samples were prepared by adding L-carnitine to a concentration of 250 μM to pasteurized milk, and 5 samples of this sample were similarly analyzed. The measurement was performed. The measurement results are shown in Table 2 below.

[0027]

[Table 2] Recovery rate (369.4-145) /250×100=89.8% Therefore, it was confirmed that highly accurate measurement was possible.

Reference Example 3 Inhibitory Effect of Ascorbic Acid on Chemiluminescence The effect of ascorbic acid on inhibiting chemiluminescence in the measurement using the apparatus of Example 1, the column of Reference Example 1 and the measurement method of Example 2 was investigated. . Hydrogen peroxide 100 μM
On the other hand, a sample to which 0 to 200 μM of ascorbic acid was added was measured without operating the oxidation treatment apparatus 4 in the apparatus of Example 1. Results are shown in FIG. A remarkable decrease in the amount of luminescence was observed as the concentration of ascorbic acid increased. After that, when the oxidation treatment device 4 was operated, the phenomenon of chemiluminescence reduction disappeared, and the same amount of luminescence as in the case where ascorbic acid was not added was exhibited. It was confirmed that the oxidation treatment device is effective for the measurement of the sample containing the substance having the low redox potential.

Example 3 Measurement of L-carnitine in human serum According to the method for measuring L-carnitine in foods shown in Example 2, L-carnitine in human serum was measured. Blood was collected from a patient taking an enteral nutrient, and serum was separated by centrifugation according to a conventional method. The measurement samples were 5 samples shown in Table 3 below. Of these, sera 1 and 2 were derived from hypertensive patients, and sera 3, 4 and 5 were derived from patients who underwent gastrectomy. Resin having a site that specifically binds to an amino group (CN-Br activated Sepharose 4B:
A solution prepared by dispersing 0.2 g of Pharmacia (made by Pharmacia) in 1 ml of 1 mM hydrochloric acid was added to 100 μl of each of the above serum samples, and the stirring was repeated to adsorb the protein in the serum sample to the resin. Then, after centrifugation, the supernatant was collected and used as a measurement sample. To 200 μl of this measurement sample,
Example 2 after addition of 0.24 μmol of acetyl CoA
L-carnitine was measured in the same manner as in. The measurement results are shown in Table 3 below. All the measurement samples could be measured without interference phenomenon. That is, it was confirmed that the method and apparatus of the present invention can be used for clinical examination.

[0030]

[Table 3]

[0031]

INDUSTRIAL APPLICABILITY The present invention provides a method for measuring L-carnitine with high sensitivity and accuracy, and an apparatus used for carrying out the method. According to the measuring method of the present invention,
Compared with the conventional method, it is possible to measure a trace amount of components with high sensitivity and accuracy by a simple method. Further, since it is not necessary to provide a dedicated facility and the measurement time is short, it is possible to measure a large amount of samples at one time. Furthermore, the measuring device of the present invention has a simple structure and is inexpensive, and the above measuring method can be automatically carried out by a simple operation. Further, since the immobilized enzyme column is used, the enzyme can be reused or continuously used, and since the chemiluminescence detector is used, the measurement can be performed with high sensitivity and high accuracy.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a measuring apparatus according to the present invention.

FIG. 2 is a graph showing the influence of the carbon number of acyl CoA on the measured value.

FIG. 3 is a graph showing a measurement calibration curve of L-carnitine.

FIG. 4 is a graph showing a calibration curve of L-carnitine added to milk and infant milk powder.

FIG. 5 is a graph showing the inhibitory effect of ascorbic acid on chemiluminescence.

[Explanation of symbols]

 1 Reagent tank for enzyme reaction 2 Pump 3 Injector 4 Oxidation treatment device 5 Enzyme-immobilized column 6 Chemiluminescence detector 7 Chemiluminescence reagent tank 8 Pump 9 Recorder

Claims (7)

[Claims] 1. A carnitine acetyltransferase (CA) is added to a measurement sample in the presence of acetylcoenzyme A.
T), a second step of reacting the coenzyme A produced in the first step with an acyl coenzyme A synthetase (ACS) in the presence of a fatty acid and ATP, The acylcoenzyme A oxidase (ACO) is allowed to act on the acylcoenzyme A produced in the second step to oxidize the acylcoenzyme A, and the hydrogen peroxide produced in the third step is measured. A method for measuring L-carnitine, comprising the fourth step of: 2. The method for measuring L-carnitine according to claim 1, wherein the measurement of hydrogen peroxide is performed by a chemiluminescence method. 3. The reaction in the first to third steps is CA
The method for measuring L-carnitine according to claim 1 or 2, which is carried out by using an immobilized enzyme in which three kinds of enzymes of T, ACS and ACO are immobilized on a carrier. 4. The method according to any one of claims 1 to 3, wherein the measurement sample is preliminarily deproteinized.
Method for measuring L-carnitine. 5. The method for measuring L-carnitine according to claim 3, wherein the immobilized enzyme is packed in one column. 6. An apparatus for carrying out the method for measuring L-carnitine according to claim 1, which comprises CAT, ACS and ACO.
L is characterized by having an enzyme-immobilized column in which immobilized enzymes in which the three types of enzymes are immobilized on a carrier are packed in a column, and a chemiluminescence detector for measuring the produced hydrogen peroxide. -Carnitine measuring device. 7. The L according to claim 6, wherein an oxidation treatment device for electrochemically oxidizing a reducing substance contained in the sample is provided immediately before the enzyme-immobilized column. -Carnitine measuring device.