JP2012024014A - Method for measuring choline in whole blood - Google Patents

Abstract

A method and a composition for measuring choline in whole blood are provided.
A method for measuring choline in whole blood using choline oxidase, peroxidase and an oxidative coloring agent, comprising hemolyzed whole blood in solution, potassium iodate or sodium iodate, hydroxylamine or A method comprising a step of reacting the salt together.
[Selection figure] None

Description

  The present invention relates to a method for measuring choline in whole blood.

  In a biochemical test, blood cells are usually removed from collected whole blood and used as a sample as plasma or serum. The reasons are as follows: (1) Non-specific adsorption or aggregation of blood cell components and blood cell membrane components to the device to avoid the influence of blood cells and blood cell components, especially hemoglobin, on the optical system. (3) In order to avoid the reaction of the analysis reagent, for example, chemical reaction, enzyme reaction, immune reaction, etc., being inhibited by blood cell components, etc. That is, the more components in the sample, the more difficult the specific analysis of the measurement target substance in the sample due to their interference.

  Usually, blood cell removal is performed by centrifugation. Since this operation requires a centrifuge and is time consuming and inconvenient, several methods for measuring whole blood without removing blood cells as a sample have been proposed. Such measurement methods are divided into two methods: (I) devise that blood cells and blood cell components are not mixed into the analysis reagent, and (II) devise analysis reagents so that they are not affected by blood cells and blood cell components. Broadly divided.

  The method of (I) includes (i) a method of removing blood cells from whole blood using a dedicated device or membrane, and (ii) intervention for analysis of blood cell components by applying a device that prevents the destruction of blood cells to analysis reagents. There are ways to avoid it.

  The method (II) includes (i) a method using an antibody (Patent Document 1), (ii) a method using an inhibitor specific to endogenous alkaline phosphatase which is a blood cell component (Patent Document 2), and the like. is there.

  By the way, creatinine, uric acid, glucose, cholesterol, neutral fat, hemoglobin A1c, phospholipid, and the like, which are representative test items of biochemical tests, are obtained by using oxidase specific to each measurement target substance. It is often measured by a measurement method via hydrogen oxide, a so-called enzyme method. In this measurement method, it is known that when whole blood is directly measured as a sample, an error due to a reducing substance in the blood cell component occurs. Therefore, in the method (II), there is a great demand for a measurement method and a reagent for detecting hydrogen peroxide without being affected by blood cells or blood cell components. Patent Document 3 is a method for measuring blood components using hemolyzed whole blood and a kit thereof for detecting hydrogen peroxide including the use of a surfactant to avoid the influence of blood cell components ( A measurement method and a kit thereof are disclosed.

  The above measurement method via hydrogen peroxide is easily affected by the reducing substance because the reducing substance in the sample reduces hydrogen peroxide. Although it is a trace amount compared to whole blood, there are reducing substances in serum and plasma, and a method using formazan, imidazole, surfactant, sulfonic acid compound, nitro compound, etc. is proposed as a method to avoid this effect. (Patent Documents 4 to 6).

  On the other hand, choline in whole blood is expected as a biomarker such as acute coronary syndrome. Non-Patent Document 1 discloses a measurement method using LC / MS as a method for measuring choline in whole blood.

JP 2000-180439 A JP 2005-188987 WO2010 / 010881 WO2002 / 086151 JP2007-147630 WO2003 / 104815

Clinica Chimica Acta, 383, 2007, 103-109

  When measuring whole blood without removing blood cells as a sample, the measurement method of (I) above, where blood cells and blood cell components are not mixed into the analysis reagent, (a) When blood cells are destroyed even during blood collection Therefore, in order to obtain reliable measurement results, proper handling of samples and devices is essential. (B) Cannot handle specimens in which blood cells are destroyed (hemolyzed) during blood collection. (C) Blood cell components There is a problem that cannot be used as a measurement target substance. In addition, the measurement method (II) described above, which devises the analysis reagent so as not to be affected by blood cells and blood cell components, is not necessary because the method (i) requires antibodies only for the types of blood cell components of interest. The method (ii) is economical and is effective only when alkaline phosphatase is used as an analytical reagent.

  In the method of Patent Document 3 in which the measurement target substance in whole blood is measured by detecting (via) hydrogen peroxide, the influence of the blood cell component cannot be completely excluded, so the conversion of the measured value (correction by the calculation formula) is required. is necessary. For example, when choline oxidase (COD) is allowed to act on choline in whole blood and the generated hydrogen peroxide is detected (via) as a method for measuring choline in whole blood, reduction in blood cell components It has been found that measurement errors due to sexual substances (hemoglobin, reduced glutathione, etc.) occur and accurate measurement cannot be performed. In order to avoid the influence of reducing substances, each compound described in Patent Documents 4 to 6 for avoiding the influence of trace reducing substances in serum and plasma was examined. In some cases, it was insufficient to avoid the effects of high concentrations of reducing substances present. In addition, the method for measuring choline using LC / MS as described in Patent Document 1 is not suitable for simultaneously measuring choline in a large number of samples at low cost because the measuring instrument used is expensive. Met. The amount of choline in whole blood is very small (several μM to several tens μM), and a highly sensitive detection reagent is also required.

  Therefore, the problem to be solved by the present invention is to accurately measure whole blood by avoiding the influence of blood cells and blood cell components in a method of measuring the concentration of choline in whole blood via hydrogen peroxide using whole blood as a sample. It is to provide a measurement method and a composition for measurement capable of measuring the choline in it.

  The present inventors first studied the use of various oxidizing agents to avoid the influence of high concentration reducing substances present in whole blood, and found that potassium iodate was effective to some extent. Measurement was performed by adding choline as a standard substance to blood, and the recovery rate calculated was 52.8% at maximum.

  As a result of further investigations, the inventors of the present invention surprisingly more effectively reduced the concentration of reducing substances by using hydroxylamine hydrochloride known as a reducing agent simultaneously with potassium iodate. It was found that it could be avoided, and succeeded in increasing the addition recovery rate up to 80.3%. Furthermore, the present inventors succeeded in increasing the above addition recovery rate to 90% or more by using a cationic surfactant at the same time.

  Based on these findings, the present inventors have completed the present invention by creating a simple method for measuring choline in whole blood that can be applied to a general-purpose automatic analyzer, a composition for the measurement, and the like.

That is, according to the present invention, the following inventions are provided.
[1] A method for measuring choline in whole blood using choline oxidase, peroxidase, and oxidative color former, which comprises hemolyzed whole blood, potassium iodate or sodium iodate, hydroxylamine or The method comprising the step of allowing the salt to coexist and react.
[1-2] A method for measuring choline in whole blood,
In a solution, reacting hemolyzed whole blood together with potassium iodate or sodium iodate and hydroxylamine or a salt thereof;
Producing hydrogen peroxide from choline in whole blood using choline oxidase; and measuring the concentration of hydrogen peroxide;
Measuring method including
[1-3] The above-mentioned [1] or [1-2] is a step in which the step of reacting is a step of causing coexistence of hemolyzed whole blood, potassium iodate, and hydroxylamine hydrochloride in a solution. ] The measuring method of description.
[1-4] A method for measuring choline in whole blood,
[1] or [1], wherein the reacting step is a step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate or sodium iodate in a solution containing hydroxylamine or a salt thereof. -2].
Measuring method including
[1-5] The step of reacting is the step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate in a solution containing hydroxylamine hydrochloride. 2].
[1-6] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 33 μmol / L or more, and the concentration of potassium iodate or sodium iodate is 45 mmol / L or more, [1] to The measurement method according to any one of [1-5].
[1-7] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 33 to 142 μmol / L, and the concentration of potassium iodate or sodium iodate is 45 to 90 mmol / L. ] To [1-5].
[1-8] The concentration of hydroxylamine or a salt thereof in the solution in the reacting step is 63 to 92 μmol / L, and the concentration of potassium iodate or sodium iodate is 45 to 90 mmol / L. ] To [1-5].
[1-9] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 63 to 92 μmol / L, the concentration of potassium iodate or sodium iodate is 45 to 90 mmol / L, The measurement method according to any one of [1] to [1-5], wherein the blood dilution rate is 0.20 to 2.45%.
[2] The measuring method according to any one of [1] to [1-9], wherein the reacting step is a step of reacting in the presence of a cationic surfactant.
[2-2] In the reaction step, the reducing substance in the hemolyzed whole blood is oxidized with potassium iodate or sodium iodate in a solution containing hydroxylamine or a salt thereof and a cationic surfactant. The measuring method according to any one of [1] to [1-9], wherein
[2-3] The step of reacting is a step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate in a solution containing hydroxylamine hydrochloride and a cationic surfactant. 1] to [1-9].
[2-4] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 63 μmol / L or more, the concentration of potassium iodate or sodium iodate is 45 mmol / L or more, and a cationic surfactant. The measurement method according to any one of [2] to [2-3], in which the concentration of is 0.031 w / v% or more.
[2-5] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 63 to 92 μmol / L, the concentration of potassium iodate or sodium iodate is 45 to 90 mmol / L, and a cation interface The measuring method according to any one of [2] to [2-3] above, wherein the concentration of the activator is 0.031 to 0.045 w / v%.
[2-6] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 63 to 92 μmol / L, the concentration of potassium iodate or sodium iodate is 45 to 90 mmol / L, and a cation interface Any of [2] to [2-3] above, wherein the concentration of the active agent is 0.031 to 0.045 w / v%, and the dilution rate of whole blood to the solution is 0.20 to 2.45%. The measuring method as described in.
[3] The measuring method according to any one of [1] to [2-6], wherein the concentration of hydroxylamine or a salt thereof in the solution in the reacting step is 33 to 142 μmol / L.
[3-2] The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is 33 to 142 μmol / L, and the dilution ratio of whole blood to the solution is 0.20 to 2.45%. 1] to the measurement method according to any one of [2-6].
[4] The measuring method according to any one of [1] to [3-2] above, wherein the concentration of potassium iodate or sodium iodate in the solution in the reacting step is 45 mmol / L or more.
[4-2] The measuring method according to any one of [1] to [3-2] above, wherein the concentration of potassium iodate or sodium iodate in the solution in the reacting step is 45 to 90 mmol / L.
[4-3] The concentration of potassium iodate or sodium iodate in the solution in the reaction step is 45 to 90 mmol / L, and the dilution rate of whole blood to the solution is 0.20 to 2.45%. The measurement method according to any one of [1] to [3-2].
[5] The measurement method according to any one of [2] to [4-3], wherein the concentration of the cationic surfactant in the solution in the reacting step is 0.031 w / v% or more.
[5-2] The concentration of the cationic surfactant in the solution in the reacting step is 0.027 to 0.045 w / v%, according to any one of the above [2] to [4-3] Measuring method.
[5-3] The concentration of the cationic surfactant in the solution in the reaction step is 0.027 to 0.045 w / v%, and the dilution ratio of whole blood to the solution is 0.20 to 2.45%. The measurement method according to any one of [2] to [4-3].
[5-4] The measuring method according to any one of [2] to [5-3], wherein the cationic surfactant is hexadecyltrimethylammonium bromide.
[6] A method for measuring choline in whole blood, comprising the following steps (a) to (e):
(A) lysing whole blood;
(B) a step of reacting hemolyzed whole blood with potassium iodate or sodium iodate and hydroxylamine or a salt thereof in a solution;
(C) adjusting the pH of the solution of step (b) to near neutral;
(D) producing hydrogen peroxide from choline in whole blood using choline oxidase;
(E) A step of measuring the concentration of hydrogen peroxide generated in step (d).
[6-2] The method according to [6] above, wherein the step (b) is a step of allowing the hemolyzed whole blood, potassium iodate, and hydroxylamine hydrochloride to coexist in a solution.
[6-3] Step (b) is a step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate or sodium iodate in a solution containing hydroxylamine or a salt thereof. 6].
[6-4] The method according to [6], wherein step (b) is a step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate in a solution containing hydroxylamine hydrochloride. .
[6-5] The measurement method according to any one of [6] to [6-4] above, wherein the solution in the step (b) further contains a cationic surfactant.
[6-6] The process according to any one of [6] to [6-5], wherein the step (c) is a step of adjusting the pH of the solution in the step (b) to pH 6.5 to 8.5. Measuring method.
[7] Acute myocardial infarction, stable angina pectoris or non-Q wave (subendocardial) myocardial infarction using the method for measuring choline in pre-blood according to any one of [1] to [6-6] Inspection method.
[8] A kit for measuring choline in whole blood, comprising the composition according to the following (1) to (4);
(1) a composition containing hydroxylamine or a salt thereof,
(2) an acidic composition containing potassium iodate or sodium iodate,
(3) A composition near neutral containing an oxidative color former, and (4) a composition containing choline oxidase and peroxidase.
[8-2] A kit for measuring choline in whole blood, comprising the composition according to the following (1), (2) and (3 ′);
(1) a composition containing hydroxylamine or a salt thereof,
(2) An acidic composition containing potassium iodate or sodium iodate, and (3 ′) a composition having a pH of 6 to 8.7 containing an oxidative color former, choline oxidase and peroxidase.
[8-3] The above [8] or [8-2], wherein the composition (1) is a composition containing hydroxylamine hydrochloride and the composition (2) is an acidic composition containing potassium iodate. ] Kit.
[8-4] The kit according to any one of [8] to [8-3] above, wherein the composition (2) further contains a cationic surfactant.
[8-5] The kit according to any one of [8] to [8-4], wherein the composition (2) is a composition having a pH of 3 to 5.
[8-6] The kit according to any one of [8] and [8-3] to [8-5], wherein the composition (3) is a composition having a pH of 6.5 to 8.5.
In the above description, the item numbers cited as “[1] to [1-7]” are shown as ranges, and the items having branch numbers such as [1-2] are arranged within the ranges. In this case, it means that a term having a branch number such as [1-2] is also cited.

  The present invention can provide a method for more accurately measuring choline present in whole blood by an enzymatic method. Moreover, the composition for a measurement etc. for performing such a measurement can also be provided. These measurement methods and measurement compositions are also useful for diagnosis of various diseases.

It is a figure which shows an example of the measurement principle of the choline measuring method in whole blood of this invention. Average value obtained by measuring the absorbance of the reagent blank and 100 μmol / L choline solution in duplicate at the DA-67 concentrations of 0.033, 0.082, 0.163 and 0.326 mmol / L with the reagent composition of Example 3. Indicates. In the reagent composition of Example 4, each wavelength of the sub-wavelength 700, 750, or 800 nm is combined with the main wavelength of 660 nm, and the average value obtained by double measurement of the absorbance of the reagent blank and 100 μmol / L choline is shown. With the reagent composition of Example 5, 50 μmol / L choline addition recovery rate (%) when the potassium iodate concentration when mixed with hemolyzed whole blood is 0, 49, 98 and 196 mmol / L, respectively. Indicates. In the reagent composition of Example 6, when potassium iodate concentration when mixing hemolyzed whole blood and potassium iodate is 45 or 90 mmol / L, 8 different concentrations of hydroxylamine hydrochloride are used. The collection recovery rate (%) of 50 μmol / L choline is shown. 50 μmol / L when the hexadecyltrimethylammonium bromide concentration is 0, 0.023, 0.034 and 0.045% when mixing the hemolyzed whole blood and potassium iodate with the reagent composition of Example 7 The recovery rate of addition of choline (%) is shown. When mixing hemolyzed whole blood and potassium iodate with the reagent composition of Example 8, 50 μmol / L of 8 different concentrations of hydroxylamine hydrochloride were used for 90 mmol / L of potassium iodate concentration. The addition recovery rate (%) of L choline is shown. The dilution linearity obtained by measuring a 5.0 to 100.0 μmol / L choline solution with the reagent composition of Example 9 is shown. In Example 10, in order to obtain | require the minimum detection sensitivity (n = 3) by +/- 3SD method, the result of having measured the choline solution of 1.0-10.0 micromol / L is shown. In Example 14, the average value which measured each sample of ae shown in Table 4 twice is shown. In Example 15, the choline concentration measured in the presence of each interfering substance shown in Table 5 is shown as an error% when the measured value in the control physiological saline is 100%. The distribution of the choline concentration measurement result in the whole blood of a healthy person in Example 16 is shown.

Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be specifically described. The present invention is not limited to the following embodiment, and can be implemented with various modifications within the scope of the gist.
This embodiment is, in one aspect, a method for measuring choline in whole blood using choline oxidase, peroxidase, and an oxidative coloring agent, wherein the whole blood is hemolyzed in solution, and potassium iodate or iodate. It is a measuring method characterized by including the process of making sodium and hydroxylamine or its salt coexist, and making it react.
In one aspect, the present embodiment relates to a method for measuring choline in whole blood, including the following steps (a) to (e):
(A) a step of hemolyzing whole blood (hemolysis step);
(B) a step (reaction step) of reacting the hemolyzed whole blood together with potassium iodate or sodium iodate and hydroxylamine or a salt thereof in a solution;
(C) a step of adjusting the pH of the solution in step (b) to near neutrality (pH adjustment step);
(D) a step of generating hydrogen peroxide from choline in whole blood using choline oxidase (hydrogen peroxide generation step); and (e) a step of measuring the concentration of hydrogen peroxide generated in step (d). (Measurement process).

In this embodiment mode, choline is a compound represented by the chemical formula (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ OH, and includes known choline and salts thereof. When the salt is exemplified by choline chloride or the like, it is not limited by the counter ion, but a counter ion usually present in a living body such as whole blood is desirable. Choline is a component of the cell membrane and is expected to be normally contained in whole blood. The measurement target of the measurement method of the present embodiment is free choline in whole blood.

  In the present embodiment, “whole blood” is blood from which blood cells are not separated. The whole blood of the present embodiment may contain blood cell components that are intentionally hemolyzed or unintentionally hemolyzed. The whole blood of this embodiment may contain an anticoagulant or a glycolysis inhibitor such as ethylenediaminetetraacetic acid dipotassium or disodium salt, heparin, sodium fluoride, sodium citrate, monoiodoacetic acid, etc. contained in the blood collection tube. Well, whole blood collected with an ethylenediaminetetraacetic acid or heparin blood collection tube is preferable from the viewpoint of being widely used for blood collection. Whole blood collected using a puncture device or the like used for self blood glucose measurement or the like without using a blood collection tube may be used. The puncture site is not particularly limited as long as whole blood can be obtained, but is usually the fingertip, forearm, upper arm, abdominal wall, etc., and is usually the forearm in the case of a medical examination. The amount of blood collected is not limited as long as choline in whole blood can be measured, and may be adjusted to the amount of blood collected in the blood collection tube to be used (usually approximately 10 mL or less). When no blood collection tube is used, more blood can be collected, and when a puncture device or the like is used, the blood collection amount is usually 0.2 mL or less.

  Choline measured by the measurement method of the present embodiment is usually present in whole blood. When whole blood is distinguished by specific gravity, separation method, etc., or by function of components, for example, red blood cells, white blood cells, plasma, serum, etc., all blood can be present. Therefore, according to the measurement method of the present embodiment, choline in whole blood can be measured even in the presence of various components in blood, particularly components derived from red blood cells. May be a mixture of red blood cells or hemolyzed whole blood. By using hemolyzed whole blood for measurement, choline present in erythrocytes can also be measured.

  The whole blood in the method for measuring choline in whole blood according to the present embodiment may be whole blood expected to contain choline. Whether choline is contained in whole blood may be determined before the measurement method of the present embodiment is performed. In this case, for example, it is determined by a conventional method such as LC / MS / MS. Alternatively, it may be determined using the measurement method of the present embodiment.

  The origin of whole blood containing choline to be measured by the measurement method of the present embodiment is not limited. For example, in the case of an experimental animal, whole blood derived from monkeys, dogs, minipigs, rats, mice, guinea pigs, rabbits, etc. It is preferably whole blood derived from a disease model animal described later. When the measurement method of the present embodiment is used for testing various diseases in humans, human-derived whole blood is preferable as the measurement target.

  In the method for measuring choline in whole blood according to the present embodiment, whole blood may be diluted. Diluting whole blood refers to mixing whole blood with a solution (diluent) other than whole blood. For example, purified water, an aqueous solution containing sodium chloride, a composition (reagent (R-1) used in the examples described later) ), (R-2), (DIL)) may be used as a diluent, and usually purified water or sodium chloride is used. The dilution of whole blood may be performed before (after the step of (a) the hemolysis step and (b) the reaction step, or after any step), but (a) the hemolysis step and (b) the reaction. It is preferable to carry out before the process. In one embodiment, depending on the type of diluent, it is preferable to dilute whole blood simultaneously with (a) the hemolysis step.

  The whole blood (specimen amount) used in the method for measuring choline in whole blood according to the present embodiment varies depending on the equipment used. For example, when the measurement is performed according to the technique described in the examples below, 1 About 40 μL, preferably about 2-25 μL. When diluting whole blood, the whole blood is diluted 1.1 to 100 times, preferably about 2 to 50 times with a diluent. Even when whole blood is diluted, the diluted whole blood used is about 1 to 40 μL, preferably about 2 to 25 μL.

  In one aspect, the method for measuring choline in whole blood according to the present embodiment includes (a) a step of hemolyzing whole blood (hemolysis step). “Welding whole blood” refers to lysing red blood cells and white blood cells in whole blood, that is, destroying the cell membrane of blood cells, such as a solution containing a surfactant, a saponin solution or a hypotonic solution. You may carry out by the method of mixing with a hemolyzing agent, and you may carry out by the method including physical processes, such as freezing and thawing, ultrasonic treatment, and a pressurizing process. Preferably, the (a) hemolysis step of the present embodiment is performed by mixing with a solution containing a surfactant. Since the surfactant has a protein denaturing action, hemoglobin denaturation occurs simultaneously with the hemolytic effect, and when the choline concentration is measured using the absorptiometric method, an effect of stabilizing the absorbance can also be obtained. The presence or absence of hemolysis can be confirmed by observing whether there is a known shape of red blood cells in whole blood using a microscope. Preferably, in the (a) hemolysis step, hemolysis is performed to such an extent that almost all red blood cells are not observed with a microscope.

  (A) When a surfactant is used in the hemolysis step, the surfactant is not limited as long as it can lyse whole blood without affecting the measurement of choline in whole blood. Any of ionic surfactants, amphoteric surfactants, and nonionic surfactants may be used, but in the case of coexistence of enzymes, nonionic surfactants should be used from the viewpoint that the enzymes are difficult to destabilize. preferable. The nonionic surfactant is preferably used alone, but a plurality of types can be used simultaneously.

  (A) When a nonionic surfactant is used in the hemolysis step, examples of the nonionic surfactant include polyoxyethylene alkylphenyl ether and polyoxyethylene sorbitan fatty acid ester from the viewpoint of enhancing the hemolysis effect. Polyoxyethylene alkylphenyl ether is preferred. The alkyl group in the polyoxyethylene alkylphenyl ether is preferably a C7 to C10 alkyl group such as octyl or nonyl, such as nonion HS210 (Nippon Yushi Co., Ltd.), nonylphenyl ether (Wako Pure Chemical Industries, Ltd.), Triton X-100. (Wako Pure Chemical Industries, Ltd.), Emulgen 920 (Kao Corporation), etc. are mentioned, and Triton X-100 is particularly preferable. Other nonionic surfactants include octyl glucoside, heptyl thioglucoside, decanoyl-N-methylglucamide, polyoxyethylene dodecyl ether, polyoxyethylene heptamethylhexyl ether, polyoxyethylene isooctyl phenyl ether, polyoxyethylene Nonylphenyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitol ester and the like can be mentioned.

  (A) When a cationic surfactant is used in the hemolysis step, the same cationic surfactant that can be used in the (b) reaction step described later can be used as the cationic surfactant.

  (A) When an anionic surfactant is used in the hemolysis step, examples of the anionic surfactant include sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl-N-sarcosinate, sodium cholate, sodium deoxycholate, Examples include sodium taurodeoxycholate.

  (A) When an amphoteric surfactant is used in the hemolysis step, examples of the amphoteric surfactant include 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid, 3-[(3-chola Midpropyl) dimethylammonio] -2-hydroxy-1-propanesulfonic acid, palmitoyl lysolecithin, dodecyl-N-betaine, dodecyl-β-alanine and the like.

  (A) When a surfactant is used in the hemolysis step, the concentration thereof is not particularly limited as long as it does not affect the measurement of choline in whole blood and can lyse whole blood. The lower limit of the concentration is 0.0001 (w / v)% or more, preferably 0.001 (w / v)% or more, and more preferably 0.01 (w / v)% or more. The upper limit is 10 (w / v)% or less, preferably 5 (w / v)% or less, and more preferably 2 (w / v)% or less. When using a plurality of surfactants, the preferred surfactant concentration may vary, but it is usually in the above range for each.

  (A) A pH buffering agent can be used in the hemolysis step as desired. When a pH buffer is used, the pH buffer is not limited as long as it does not affect the measurement of choline in whole blood and can lyse whole blood. When the (a) hemolysis step and the later-described (b) reaction step are performed at the same time, it is preferable to use a pH buffer having the same conditions as the later-described (b) reaction step.

  In the method for measuring choline in whole blood according to the present embodiment, (b) in solution, hemolyzed whole blood, potassium iodate or sodium iodate, and hydroxylamine or a salt thereof are allowed to coexist and react. Including a step (reaction step). This reaction step is performed prior to measurement in the method for measuring choline in whole blood according to the present embodiment in order to avoid an error in measuring hydrogen peroxide from choline in whole blood. It is a process.

  In a preferred embodiment, the reaction step (b) includes a step of oxidizing a reducing substance in hemolyzed whole blood with potassium iodate or sodium iodate in a solution containing hydroxylamine or a salt thereof (oxidation). Process). Various reducing substances exist in blood cells, and the reducing substance has an action of reducing hydrogen peroxide. Therefore, in the method for measuring choline in whole blood according to this embodiment, when hydrogen peroxide is generated from choline and the concentration of hydrogen peroxide is measured, a negative error occurs in the measured value. In order to avoid this error, the reducing substance in the hemolyzed whole blood can be oxidized with potassium iodate or sodium iodate prior to measurement.

(B) The potassium iodate or sodium iodate used in the reaction step is preferably potassium iodate. Potassium iodate includes known potassium iodate and is represented by the chemical formula KIO ₃ . Further, sodium iodate includes known sodium iodate and is represented by the chemical formula NaIO ₃ . (B) The concentration of potassium iodate or sodium iodate in the solution in the reaction step is not particularly limited as long as the influence of the reducing substance in the whole blood on the measured value of choline can be substantially avoided. Is exemplified by 10 mmol / L or more, preferably 30 mmol / L or more, more preferably 45 mmol / L or more, and further preferably 50 mmol / L or more. As an upper limit, 200 mmol / L or less is illustrated, 150 mmol / L or less is preferable, 90 mmol / L or less is more preferable, and 80 mmol / L or less is more preferable.

  In the present embodiment, “substantially avoiding the influence of the reducing substance” refers to avoiding the influence of the reducing substance to such an extent that the measurement value of choline in whole blood is not substantially affected. Means that. As a specific example, for example, when choline is added to whole blood and the choline is measured using the measurement method of the present embodiment, choline is added to the whole blood calculated according to the method described in the examples described later. The addition recovery rate in the case of adding may be 50% or more, preferably 80% or more, and more preferably 90% or more. Further, when the method for measuring choline in whole blood according to the present embodiment is used for disease testing, it is preferable to avoid the influence of reducing substances to the extent that there is no problem in diagnosing the disease based on the measured values. Specifically, although it may vary depending on the disease, the recovery of addition when choline is added to whole blood calculated according to the method described in the examples below is preferably 70% or more, more preferably 80% or more. Preferably, 90% or more is more preferable, and 95% or more is particularly preferable.

(B) The hydroxylamine or salt thereof used in the reaction step is exemplified by hydroxylamine, hydroxylamine hydrochloride, hydroxylamine sulfate or hydroxylamine nitrate, preferably hydroxylamine hydrochloride. Hydroxylamine hydrochloride is also called hydroxylammonium chloride and is represented by the chemical formula NH ₂ OH · HCl. The above hydroxylamine or a salt thereof is generally used as a reducing agent. The present inventors surprisingly compared with the case of using only potassium iodate or sodium iodate due to the presence of hydroxylamine or a salt thereof in the solution in which (b) the reaction step is performed, It has been found that the effects of reducing substances present in the blood can be avoided more efficiently. (B) The concentration of hydroxylamine or a salt thereof in the solution in the reaction step is not particularly limited as long as the influence of the reducing substance present in the whole blood can be substantially avoided, but the lower limit is 10 μmol / L. Examples are given above, preferably 33 μmol / L or more, more preferably 63 μmol / L or more, and even more preferably 70 μmol / L or more. The upper limit is exemplified by 200 μmol / L or less, preferably 142 μmol / L or less, more preferably 92 μmol / L or less, and still more preferably 85 μmol / L or less.

  In one embodiment, the solution in the (b) reaction step further contains a cationic surfactant. Without being bound by theory, this cationic surfactant is obtained by oxidizing (b) a reducing substance in whole blood oxidized in the reaction step (for example, methemoglobin which is a reducing substance). It is considered that this has the function of more efficiently avoiding the influence of reducing substances present in whole blood.

  (B) The cationic surfactant used in the reaction step is not particularly limited as long as the influence of a reducing substance present in the hemolyzed whole blood can be substantially avoided, but hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium chloride, didecyldimethylammonium chloride, didecyldimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, tetradecylammonium bromide, dodecylpyridinium chloride, hexadecylpyridinium Chloride, hexadecylpyridinium bromide, 1-laurylpyridinium chloride, tetradecyltrimethyl It includes Nmo bromide or the like, from the viewpoint of avoiding more efficiently the effects of reducing substances in whole blood, hexadecyltrimethylammonium bromide is preferred.

  (B) The concentration of the cationic surfactant in the solution in the reaction step is not limited as long as the influence of the reducing substance present in the hemolyzed whole blood can be substantially avoided, but the lower limit is 0. 0.027% or more is preferable, and 0.031% or more is preferable. The upper limit is not particularly limited as long as the surfactant is dissolved, but 0.045% or less is preferable.

  The amount of hemolyzed whole blood used in the (b) reaction step of the present embodiment varies depending on the equipment used and the like. For example, when measurement is performed according to the technique described in the Examples below, 1 to 40 μL The degree, preferably about 2 to 25 μL. The whole blood may be diluted 1.1 to 100 times, preferably about 2 to 50 times with a hemolytic agent. In this case, the diluted hemolyzed whole blood used is also about 1 to 40 μL, preferably Is about 2 to 25 μL.

  (B) The amount of the reagent in the reaction solution in which the reaction step is carried out varies depending on the equipment used for the measurement, but for example, when the measurement is carried out according to the method shown in the Examples described later, 50 to 250 μL, preferably Is about 100 to 200 μL. Accordingly, the lower limit of the dilution rate of whole blood with respect to the solution in which the (b) reaction step is performed is exemplified by 0.004% or more as the lower limit, preferably 0.04% or more, more preferably 0.2% or more, and 4% or more is more preferable. The upper limit is exemplified by 80% or less, preferably 25% or less, more preferably 2.45% or less, and further preferably 2.0% or less. (B) As the quantity of the whole solution in which a reaction process is performed, 50-500 microliters is illustrated, Preferably it is 200-400 microliters.

  In the method for measuring choline in whole blood according to the present embodiment, (b) reaction step may be performed after (a) hemolysis step, or (a) hemolysis step and (b) reaction step are performed simultaneously. You may go. From the viewpoint of simplicity, it is preferable to simultaneously perform the (a) hemolysis step and the (b) reaction step. Depending on the reagent used, there is another embodiment in which (b) the reaction step is preferably performed after the (a) hemolysis step.

  For example, when (b) the solution in the reaction step further contains a cationic surfactant and (a) the hemolysis step and (b) the reaction step are performed simultaneously, the cationic surfactant used in (b) the reaction step is used. It is preferable from the viewpoint of simplicity to hemolyze whole blood and simultaneously perform the (a) hemolysis step.

  (B) The reaction step is preferably performed in a solution containing a pH buffer as appropriate from the viewpoint of enhancing the effect of avoiding the influence of reducing substances present in whole blood. The use of a buffering agent can also be expected to stabilize the hemoglobin. The pH buffer is not limited as long as the target pH can be maintained, but Good's pH buffer (MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO , EPPS, Tricine, Bicine, TAPS, CHES, CAPS, etc.), Tris buffer, diethanolamine buffer, carbonate buffer, glycine buffer, borate buffer, phosphate buffer, glycylglycine buffer, acetate buffer, Examples include citrate buffer, succinate buffer, maleate buffer, trisethanolamine buffer, imidazole buffer, and the like. These buffers are used after adjusting to a pH range that can be used as a buffer with a strong acid such as hydrochloric acid or a strong alkali such as NaOH. Two or more buffering agents may be used in combination. From the viewpoints of avoiding the influence of reducing substances present in whole blood, stabilizing hemoglobin, and facilitating (c) pH adjustment step described later, (b) pH in the solution in which the reaction step is performed The lower limit is exemplified by pH 2.5 or more, preferably pH 3.0 or more, more preferably pH 3.5 or more, and the upper limit is pH 5.5 or less, preferably pH 5.0 or less, more preferably pH 4.5 or less. Is exemplified. Further, the concentration of the pH buffer is preferably low, and the lower limit is 3 mmol / L or more, preferably 5 mmol / L or more, more preferably 10 mmol / L or more, and the upper limit is 200 mmol / L or less, preferably 100 mmol. / L or less, more preferably 50 mmol / L or less.

  In one aspect, the method for measuring whole blood choline according to the present embodiment includes (b) a reaction step, (c) a step of adjusting the pH of the solution in step (b) to near neutrality (pH adjustment step). Including. (C) The pH adjustment step is a step performed for the purpose of facilitating the production of hydrogen peroxide from choline using choline oxidase in the subsequent (d) hydrogen peroxide production step. In the present embodiment, near neutral means a state that is neither strongly acidic nor strongly alkaline, and includes weak acidity and weak alkalinity, and the subsequent (d) hydrogen peroxide generation step and (d) measurement step are performed. It is not limited as long as it can be done. For example, the pH near neutral is exemplified by a pH of 6.0 or more, preferably 6.2 or more, more preferably 6.5 or more, and even more preferably 6.8 or more. The upper limit is exemplified by pH 8.7 or less, preferably pH 8.5 or less, more preferably pH 8.2 or less, further preferably pH 7.8 or less, and particularly preferably pH 7.5 or less. In the present embodiment, “adjusting the pH of the solution to near neutral” includes bringing the pH of the solution closer to pH 7 than the pH before adjustment.

  (C) The pH adjustment step can be performed by adjusting the pH using a pH buffer. The pH buffer that can be used is the same as that described in connection with the reaction step (b). (C) The pH of the solution after the pH adjustment step is adjusted to pH 6 or higher, preferably pH 6.2 or higher, more preferably pH 6.5 or higher, and the upper limit is pH 8.7 or lower, preferably pH 8 0.5 or less, more preferably pH 8.2 or less. (C) The concentration of the pH buffer in the solution after the adjustment in the pH adjusting step is 20 mmol / L or more, preferably 50 mmol / L or more, more preferably 100 mmol / L or more as the lower limit, and the upper limit is 1M. Hereinafter, it is preferably 500 mmol / L or less, more preferably 300 mmol / L or less.

  In one aspect, the method for measuring whole blood choline according to the present embodiment includes (d) a step of generating hydrogen peroxide from choline in whole blood (hydrogen peroxide generation step) using choline oxidase. (D) In the hydrogen peroxide generation step, choline oxidase is allowed to act to decompose choline in whole blood into betaine and / or betaine aldehyde and hydrogen peroxide.

  (D) Choline oxidase (sometimes abbreviated as choline oxidase, COD) used in the hydrogen peroxide production step contains an enzyme classified as EC 1.1.3.17, and produces hydrogen peroxide from choline. It is not particularly limited as long as it has an action to act, and those having an action to oxidize choline to betaine aldehyde and / or betaine and hydrogen peroxide are preferable. The optimum pH of COD is 7.5-8.0, the pH stability is between 7.5-9.0, and the optimum temperature is around 37 ° C. The secondary structure, tertiary structure and quaternary structure, properties, purity, origin, trade name and price of COD are not limited. A preferred example of COD is Arthrobacter globebiformis-derived COD (Asahi Kasei Pharma Corporation (T-05)). (D) The concentration of COD in the solution during the hydrogen peroxide generation step is not particularly limited as long as choline in whole blood is converted to hydrogen peroxide and the subsequent measurement step (e) can be performed. However, the lower limit is 1 U / mL or more, preferably 5 U / mL or more, more preferably 10 U / mL or more, and there is no particular upper limit, but 200 U / mL or less, preferably 100 U / mL or less, more preferably 80 U / mL or less is illustrated. The amount of the enzyme to be used is preferably large from the viewpoint of the stability of the reagent, and is preferably small from the viewpoint of economy. When performing the rate assay, the concentration of the enzyme used is preferably low, and the lower limit is exemplified by 0.01 U / mL. The enzyme activity value may vary depending on the enzyme activity measurement method.

  (D) It is preferable to implement a hydrogen peroxide production | generation process using a pH buffer suitably from a viewpoint of improving the reactivity of COD. The pH buffer that can be used is the same as that described in connection with the reaction step (b). (D) The pH in the solution when carrying out the hydrogen peroxide generation step is, as a lower limit, pH 6 or more, preferably pH 6.5 or more, more preferably pH 7 or more, and the upper limit is pH 9.0 or less, preferably Is pH 8.5 or less, more preferably pH 8 or less. The concentration of the pH buffering agent is not particularly limited as long as the target pH can be maintained, but the lower limit is 3 mmol / L or more, preferably 5 mmol / L or more, more preferably 10 mmol / L or more, and the upper limit is 500 mmol. / L or less, preferably 200 mmol / L or less, more preferably 100 mmol / L or less.

  In one aspect, the method for measuring whole blood choline according to the present embodiment includes (e) a step (measurement step) of measuring the concentration of hydrogen peroxide generated in step (d). The measurement of the concentration of hydrogen peroxide can be preferably performed by quantitative analysis from the viewpoint of simplicity, accuracy, etc., and more preferably, the quantitative analysis of hydrogen peroxide is performed using an oxidizing color former to produce a dye. The absorbance can be measured and colorimetric analysis can be performed. For example, whole blood that may contain choline (measurement target) and a sample (standard solution) containing choline at a known concentration are individually processed in the same process, and then choline is converted to hydrogen peroxide. And the amount of dye produced (change amount) is detected using an oxidative color former. The choline concentration in the measurement object can be quantified by comparing the production amount of the dye in the measurement object and the production amount of the dye in the standard solution.

  In one embodiment, the measurement step (e) can be performed by causing hydrogen peroxide to develop color using a leuco dye as an oxidizing color former in the presence of peroxidase (POD). Specific examples of leuco dyes include O-dianisidine, O-tolidine, 3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine (above Doujin Chemical Laboratory), N- (carboxymethylamino) Carbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67), and the like. In one embodiment, it may be preferable to use an oxidative color former having a wavelength of 600 nm or more that does not overlap with the absorption wavelength of the hemoglobin, particularly in order to avoid influence on the measurement of hemoglobin in whole blood. An example of such an oxidative color former is DA67 (maximum absorption 666 nm).

  (E) When a leuco dye such as DA64 or DA67 is used in the measurement step, a known method of coexisting a dye having an effect of absorbing ultraviolet rays or visible light (see Japanese Patent Application Laid-Open No. 2008-201968) is applied. The leuco dye can be stabilized. Such stabilizing dyes include Orange G, Orange II, Food Red No. 2, Food Red No. 3, Food Red No. 40, Food Red No. 102, Food Red No. 104, Food Red No. 106, Food Yellow No. 4, Food Yellow Yellow No. 5 is mentioned, and orange G is preferable from the viewpoint that DA67 has a high stabilizing effect. The concentration of the stabilizing dye is not limited as long as it does not affect the measured value of choline in whole blood and can stabilize the leuco dye, and the lower limit is 0.01 mmol / L or more, preferably 0.05 mmol / L or more, more preferably 0.1 mmol / L or more. The upper limit is 1 mmol / L or less, preferably 0.5 mmol / L or less, and more preferably 0.2 mmol / L or less. When using a plurality of stabilizing dyes, the preferred concentration may vary but is usually in the above range for each.

  (E) When a leuco reagent such as DA64 or DA67 is used in the measurement step, a known method in which a reducing agent coexists (see Japanese Patent Application Laid-Open No. 2008-201968) may be applied to stabilize the leuco dye. it can. Examples of such a reducing agent include cysteine, cysteamine, N-acetylcysteine, thioglycerol, sodium thiosulfate, sodium sulfite, and sodium disulfite, and sodium thiosulfate is preferable from the viewpoint that DA67 has a high stabilizing effect. . The concentration of the reducing agent does not affect the measured value of choline in the whole blood and is not limited as long as the leuco dye can be stabilized. The lower limit is 0.1 mmol / L or more, preferably 0.5 mmol / L or more. More preferably, it is 1 mmol / L or more. The upper limit is 200 mmol / L or less, preferably 100 mmol / L or less, and more preferably 50 mmol / L or less. When using a plurality of reducing agents, the preferred concentration may vary, but is usually in the above range for each.

  (E) When an oxidation color former is used in the measurement step, for example, referring to the examples described later, in addition to the dominant wavelength corresponding to the oxidation color former using a technique known to those skilled in the art, appropriate for each measurement as appropriate. By selecting the sub-wavelength and correcting the measurement value, more accurate measurement can be performed.

  (E) When peroxidase (may be abbreviated as peroxidase, POD) is used in the measurement step, POD is not limited as long as it can develop an oxidative color former with hydrogen peroxide, but EC 1.11.1 Enzymes classified as .7 are preferred. The optimum pH of POD is 6-7, the pH stability is between 5-10, and the optimum temperature is around 45 ° C. The secondary structure, tertiary structure and quaternary structure of POD, properties, purity, origin, trade name and price are not limited. Suitable examples of POD include general-purpose enzymes derived from plants such as horseradish (SIGMA (P8375, etc.), Amano Enzyme Co., Ltd.). The concentration of POD to be used can be determined with reference to the description regarding (d) the concentration of COD in the hydrogen peroxide production step.

  (E) The measurement step is performed by producing and quantifying hydrogen peroxide in the presence of peroxidase (POD) by generating a dye by oxidative condensation between a chromogen such as a oxidative color former such as a Trinder reagent and a coupler. You can also. In this case, a phenol derivative, an aniline derivative, a toluidine derivative or the like can be used as the oxidizing color former, and specific examples include N, N-dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N- Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N -Sulfopropyl-m-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-s Hopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -3-m-anisidine (ADPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3-sulfo) Propyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS) (above Doujin Chemical Laboratory Co., Ltd.) and the like. Examples of couplers include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazone (MBTH), and the like.

  In the method for measuring choline in whole blood of the present embodiment, each of the above steps can be performed discontinuously or simultaneously, and the origin of whole blood, the concentration of choline in whole blood, the purpose of measurement, and use The order in which the steps are performed can be determined so as to obtain a preferable result depending on the apparatus or the like. That is, each process may be performed in arbitrary order and two or more may be performed simultaneously. For example, possible embodiments of the (a) hemolysis step and (b) reaction step are as described above, and (b) the pH adjustment step and (d) the hydrogen peroxide generation step are simultaneously performed after the (b) reaction step. Embodiment to perform; (b) After the reaction step, (c) pH adjustment step, (d) Hydrogen peroxide generation step and (e) Measurement step are simultaneously performed; (b) After the reaction step, (c) pH adjustment step (D) A mode in which (d) a hydrogen peroxide generation step and (e) a measurement step are simultaneously performed; (b) a (c) pH adjustment step is performed after the reaction step, and then (d) a hydrogen peroxide generation step is performed. A mode in which (e) a measurement step is performed, and then a combination of these modes is also possible.

  As a preferred embodiment, (a) the hemolysis step and (b) the reaction step are performed simultaneously, then (c) the pH adjustment step is performed, then (d) the hydrogen peroxide generation step is performed, and then (e) the measurement step The mode which performs is mentioned.

  In the method for measuring choline in whole blood of the present embodiment, each step may be performed in a different reaction tank (phase) or in the same reaction tank (phase). In one aspect, when whole blood is diluted and used for measurement, it is preferable to carry out only dilution of whole blood in a separate reaction vessel (phase).

  In the present embodiment, the reaction time of each step is not limited as long as choline in whole blood can be measured, but the lower limit is 15 seconds or more, preferably 1 minute or more, more preferably 3 minutes or more, respectively. is there. Although there is no particular upper limit, it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less. The reaction time of each step may be uneven. The temperature at the time of performing each step is not limited as long as choline in whole blood can be measured, and the temperature in each step may be uneven. When an enzyme is used in each step, it is preferable to carry out each step within the range of the working temperature of the enzyme used, and the lower limit is 15 ° C or higher, preferably 20 ° C or higher, more preferably 25 ° C or higher. The upper limit is 70 ° C. or lower, preferably 50 ° C. or lower, more preferably 40 ° C. or lower, and each step is preferably performed at around 37 ° C.

  In one aspect, a suitable example of the method for measuring choline in whole blood of this embodiment is mixing 2 to 30 μL of whole blood with 50 to 250 μL of a composition containing at least hydroxylamine hydrochloride. Next, 2 to 30 μL of the mixture and 50 to 250 μL of a composition having a pH of 3 to 5 containing at least potassium iodate and a cationic surfactant are mixed in a different reaction tank and hemolyzed whole blood, iodine Potassium acid and hydroxylamine hydrochloride coexist. Next, 50 to 250 μL of a composition having a pH of 6.5 to 8.5 containing at least an oxidizing color developer is added to the reaction vessel, and finally 50 to 250 μL of a composition containing at least POD and COD is added to the reaction vessel. Hydrogen peroxide is produced from choline, and this hydrogen peroxide is developed with POD and an oxidizing color former.

  The method for measuring choline in whole blood of the present embodiment is preferably performed in the liquid phase, but may be performed in the gas phase, the solid phase, or each critical plane. Examples of the liquid phase include sol and gel in addition to the solution. In order to obtain a sol-gel, for example, a polysaccharide such as agar may be used. When distinguishing between sol-gel and emulsion, it may be carried out in emulsion. In order to obtain an emulsion, for example, an organic solvent or the like may be used, and if an amphiphilic substance is used, micelles can be used. In any case, when using a pH buffer, the above description can be referred to. From the viewpoint of convenience, the method for measuring choline in whole blood of the present embodiment is preferably performed in an aqueous solution.

  An example of the measurement principle of the method for measuring choline in whole blood according to the present embodiment is shown in FIG. By the action of COD, two molecules of hydrogen peroxide are generated from one molecule of choline. This hydrogen peroxide oxidizes DA-67 by the action of POD, and the coloration of the generated methylene blue is colorimetrically determined at a measurement wavelength of 666 nm.

In one aspect, this embodiment includes a method for examining acute myocardial infarction, stable angina pectoris, and non-Q wave (subendocardial) myocardial infarction using the method for measuring choline in whole blood. Non-Patent Document 1 shows the relationship between adverse heart events including acute myocardial infarction, stable angina and non-Q wave (subendocardial) myocardial infarction and choline concentration in whole blood. According to Non-Patent Document 1, choline in whole blood and plasma is a predictive marker for major adverse cardiac events independent of age, sex, history of myocardial infarction, coronary risk factors, and electrocardiogram. In addition, choline in whole blood reflects an increase in phospholipase D (PLD) activity, increased platelet responsiveness, and the vulnerability of coronary unstable plaque, and many important processes in cell invasion and activation around unstable plaque. Appears as a change in choline concentration in blood cells. Thus, choline in whole blood and plasma is involved in tissue ischemia, but whole blood choline is particularly useful for detecting the risk of coronary vulnerable plaque. For the detection of early major adverse cardiac events due to vulnerable plaque, the measurement of choline concentration in whole blood is more significant than choline in plasma (Oliver Danne et al., Clin. Chem, 51, 7, 1315). -1317, 2005). In Non-Patent Document 1, the cut-off value for major adverse cardiac events is 28.2 μmol / L for choline in whole blood. The cut-off value of choline in plasma is not determined, but the 99th percentile has a cut-off value of 25.0 μmol / L and a low cut-off value of 18.5 μmol / L. The sensitivity and specificity for high-risk unstable angina was reported to be 86%. Therefore, by performing the whole blood choline measurement method of the present embodiment using whole blood derived from a subject as a measurement target, it can be examined whether the subject is suffering from the above-mentioned disease. Also, Martin Mockel et al., Clinica. Chimica. Acta, 393, 103-109, 2008, reported that the combination of NT-proBNP, choline in whole blood and lipoprotein-related phospholipase A ₂ (Lp-PLA ₂ ) is optimal for early risk stratification. Therefore, early risk stratification can be performed by measuring Lp-PLA ₂ using a known technique in combination with the method for measuring whole blood choline according to the present embodiment.

  According to the method for measuring choline in whole blood according to the present embodiment as described in detail above, even choline in whole blood mixed with various reducing substances derived from blood cells can be detected with high accuracy. Sensitivity can be measured. For example, even a very small amount of choline of 2 μmol / L can be measured accurately. Therefore, it is very useful for the examination of the above diseases.

  The composition used in the method for measuring choline in whole blood of this embodiment is a composition for measuring choline in whole blood or a measurement kit (also referred to as “measuring composition etc.” in this specification). ).

  The composition for measuring choline in whole blood of the present embodiment is preferably at least hydroxylamine or a salt thereof, potassium iodate or sodium iodate, choline oxidase, a cationic surfactant, an oxidative coloring agent, and a peroxidase More preferably, it contains at least hydroxylamine hydrochloride, potassium iodate, choline oxidase, a cationic surfactant, an oxidative coloring agent and peroxidase.

  In one aspect, the present embodiment includes (1) a composition containing hydroxylamine or a salt thereof, (2) an acidic composition containing potassium iodate or sodium iodate, and (3) an oxidative color former. The present invention relates to a kit for measuring choline in whole blood, comprising a composition near neutrality and (4) a composition containing choline oxidase and peroxidase.

  Among these, (2) the acidic composition containing potassium iodate or sodium iodate may further contain a cationic surfactant. Examples of the cationic surfactant include the cationic surfactants described above in connection with the (b) reaction step.

  Hydroxylamine or a salt thereof, potassium iodate or sodium iodate, choline oxidase, positive, contained in the composition of the above (1) to (4) constituting the kit for measuring choline in whole blood of the present embodiment The types and effective amounts of ionic surfactant, oxidative color former and peroxidase, that is, the conditions effective for the composition to be a composition that can be used as a kit for measuring choline in whole blood, etc. The method for measuring choline in whole blood can be referred to. In addition, regarding the pH of the composition of said (2), the description regarding the pH of the reaction solution in which the said (b) reaction process is performed can be referred. Moreover, regarding the pH of the composition of said (3), the description regarding the solution pH after adjustment by the said (c) pH adjustment process can be referred. Specifically, as the pH near the neutrality of the composition (3), the lower limit is exemplified by pH 6.0 or more, preferably pH 6.2 or more, more preferably pH 6.5 or more, and pH 6.8 or more. More preferably, pH 7.0 or higher is particularly preferable, and the upper limit is exemplified by pH 8.7 or lower, pH 8.5 or lower is preferable, pH 8.2 or lower is more preferable, and pH 8.0 or lower is further preferable.

  The composition of the above (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment is for the purpose of improving the quality of the reagent such as sensitivity, accuracy, reproducibility, and stability. The composition may contain various salts, various sugars and / or preservatives such as sodium azide and antibiotics.

When the composition of (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment contains a salt such as NaCl, KCl, NH ₃ Cl, etc. The lower limit of the amount of the salt is 0.1 mmol / L or more, preferably 5 mmol / L or more, more preferably 50 mmol / L or more. The upper limit is not particularly limited, but is preferably 200 mmol / L or less, Preferably, it is 150 mmol / L or less, and particularly preferably 120 mmol / L or less. These salts can function as enzyme stabilizers.

  When the composition of the above (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment contains sugar, the concentration of the sugar in each composition is in a soluble range. For example, when sucrose is contained, the lower limit is 0.05 (w / v)% or more of the total composition, preferably 0.1 (w / v)% or more, more preferably 0.3 (w / v)% or more, and the upper limit is 30 (w / v)% or less, preferably 10 (w / v)% or less, more preferably 5 (w / v)% of the total composition. It is as follows. For example, when mannitol is contained, the lower limit is 0.05 (w / v)% or more of the total composition, preferably 0.1 (w / v)% or more, more preferably 0.3 (w / v)%. The upper limit is 3 (w / v)% or less of the total composition, preferably 2 (w / v)% or less, and more preferably 1 (w / v)% or less. Examples of other sugars include trehalose and cyclodextrin. These sugars can function as enzyme and composition stabilizers, or as freeze-drying excipients when the composition is freeze-dried.

  When the composition of the above (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment contains a preservative, the type and concentration of the preservative are not limited. In the case of sodium fluoride, the lower limit is 0.005 (w / v)% or more, preferably 0.01 (w / v)% or more, more preferably 0.03 (w / v) as the concentration in each composition. )% Or more, and the upper limit is 1 (w / v)% or less of the total composition, preferably 0.5 (w / v)% or less, more preferably 0.1 (w / v)% or less. . For example, in the case of antibiotics, the lower limit is 5 μg / mL or more, preferably 10 μg / mL or more, more preferably 30 μg / mL or more, and the upper limit is 100 μg / mL or less, preferably 75 μg / mL or less, more preferably 60 μg. / ML or less.

  When the composition of (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment contains a chelating agent such as EDTA, the type and concentration are not limited. For example, EDTA When it contains, it is 0.05 to 10 mmol / L normally as a density | concentration in each composition. Chelating agents such as EDTA, EGTA, and NAT may inhibit the activity when a protease that utilizes a metal for activity expression is present in the composition.

  When the composition of (1) to (4) constituting the kit for measuring choline in whole blood according to the present embodiment contains a protein such as bovine albumin, ovalbumin, human albumin, or crystallin, the protein The type and concentration are not limited. For example, when bovine albumin is contained, the concentration in each composition is usually in the range of 0.01 (w / v)% to 5 (w / v)%. Since these proteins serve as protease substrates, they may be enzyme stabilizers. Moreover, it can become a freeze-drying excipient during freeze-drying.

  The kit for measuring choline in whole blood according to the present embodiment may further contain a calibration reagent containing at least a known amount of choline as a composition constituting the kit.

  The calibration reagent of the present invention may be a reagent containing at least a known amount of choline, but is preferably a reagent containing a pH buffering agent, a preservative such as sodium azide or an antioxidant, and a stabilizer such as sugar. The conditions such as the type and concentration are the same as in the case of the above composition. As the calibration method, a single inspection quantity, a multi-inspection quantity (a broken line or a spline), a linear regression of the multi-inspection quantity, and the like can be selected.

  The known amount of choline in the calibration reagent is not particularly limited, and may be selected in order to accurately measure choline in whole blood. A calibration reagent containing a plurality of different known amounts of choline may also be used. In the case of choline, the lower limit is 1 μmol / L or more, preferably 3 μmol / L or more, more preferably 5 μmol / L or more, and the upper limit is 80 μmol / L or less, preferably 50 μmol / L or less, more preferably 30 μmol / L or less. This concentration can vary depending on the purpose of the measurement using the kit for measuring choline in whole blood (target disease, etc.). The approximate concentration is the same in the case of a multi-calibration reagent that also functions as a calibration reagent other than choline and choline.

  The composition for measuring choline in whole blood and the calibration reagent of the present embodiment are liquid products, frozen products of liquid products, freeze-dried products of liquid products, or dried products of liquid products (heat-dried, air-dried and / or Or by drying under reduced pressure or the like). In consideration of measurement accuracy, liquid products, frozen products of liquid products, and freeze-dried products of liquid products are preferable, and each of these can be selected as a preferable shape according to the purpose of use, storage method, measurement accuracy, and the like. The composition for measuring choline in whole blood of the present embodiment may be a composition of one reagent, but usually it is preferably separated into two or more reagents, for example, as in the above-described measurement kit, It can be separated into 4 or more reagents. In addition, for example, in the case of use for a capillary of a point-of-care device, or use as an enzyme sensor, the concentration of each component is preferably higher than usual, for example, it is fixed or soaked into paper or a film, It is preferable to use it as a gel-sol composition.

EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, the scope of the present invention is limited to a following example etc. and is not interpreted.
In this example, any reagent that is commercially available and can be easily obtained can be used. The reagent manufacturer, purity, price and the like are not particularly limited, and those skilled in the art can appropriately select and use them as necessary. The measurement values shown below may vary depending on various conditions such as the purity of the reagent, the measurement conditions, the accuracy of the equipment used, and the atmosphere such as the temperature and pressure of the equipment used. Those skilled in the art can easily understand that the results showing the same tendency as those obtained in the following examples and the like can be obtained when measured under conditions. In the examples,% means w / v% unless otherwise specified.
In this example, N-ethylmaleimide, L-alanine, L-serine, L-carnitine, reduced glutathione, L (+) ascorbic acid, uric acid, hydroxylamine hydrochloride (product name: hydroxylammonium chloride), iodic acid Potassium, hexadecyltrimethylammonium bromide, DA-67, choline chloride, and sodium thiosulfate are from Wako Pure Chemical Industries, creatinine monohydrate is from Hanai Chemicals, Triton X-100, and bovine albumin is from Sigma-Aldrich. (St. Louis, USA) Orange G was purchased from Kanto Chemical. Other reagents purchased from Wako Pure Chemical Industries, Sigma-Aldrich, etc. were used unless otherwise specified. POD was manufactured by Oriental Yeast Co., Ltd. (derived from wasabi, POD-03), and COD was manufactured by Asahi Kasei Pharma Corporation (derived from Arthrobacter globiformis, T-05).

<Devices used>
For the analysis, Hitachi spectrophotometer U-2810 and Hitachi 7170 type automatic analyzer (Hitachi High-Technologies) were used as an automatic analyzer. As the pH meter, a glass electrode type hydrogen ion concentration indicator (Iwaki Glass) was used at 25 ° C.

<Measurement reagent>
Diluent (DIL), first reagent (R-1), second reagent (R-2), and third reagent (R-3) were prepared as measurement reagents in the automatic analyzer. The compositions of (DIL), (R-1), (R-2), and (R-3) are shown in each example as necessary.

<Automatic analyzer usage conditions>
The reaction in the automatic analyzer was performed at 37 ° C.
When performing pre-dilution (when using DIL): 85 μL of (DIL) was added to 17 μL of a measurement sample (whole blood, standard solution, etc.), and this was used as an analysis sample. Dispense 20 μL from the sample for analysis into the reaction vessel, mix with (R-1) 180 μL, mix 1 minute 25 seconds with (R-2) 90 μL, and add 3 minutes 35 seconds (R-3) 20 μL. After 1 minute and 2 seconds, colorimetric determination was performed by a two-point end method with a main wavelength of 660 nm and a sub wavelength of 800 nm.
When pre-dilution is not performed (when DIL is not used): 3.5 μL is dispensed from the sample for measurement (whole blood, standard solution, etc.) into the reaction container, and other conditions are the same as “when pre-dilution is performed” The measurement method was used.

<Standard solution>
147 mg of choline chloride was dissolved in 100 mL of purified water, and this was used as a standard stock solution of choline. When used as a measurement material, it was diluted 100 times with purified water to obtain a 100 μmol / L choline standard solution.

<Whole blood sample>
As the whole blood sample, a sample obtained by collecting blood with an EDTA-2Na-containing blood collection tube (Terumo Corporation, Benedict II vacuum blood collection tube) was used. In addition, all the whole blood samples used were samples for which consent for blood collection was obtained according to the consent form.

[Example 1] Examination of hemolysis method of whole blood sample Using 0.01 mol / L phosphate buffer, 0.05%, 0.1%, 0.2% and 0.3% Triton X-100 solution This was designated as “hemolyzed blood”. When 180 μL of each “hemolyzed blood” and 3.5 μL of whole blood were mixed and the presence or absence of hemolysis was confirmed under a microscope, complete hemolysis was confirmed at a Triton X-100 concentration of 0.1% or more. In addition, since the surfactant has a protein denaturing action, the use of Triton X-100 as hemolyzed also has the effect of stabilizing the absorbance due to hemoglobin denaturation as well as the hemolytic effect.

[Example 2] Examination of enzyme concentration The amount of POD, which is a conjugated enzyme, was determined by Kumada, et al., "Reagents used in clinical tests-Contents of general-purpose automatic analysis and contents of commercially available reagents, performance list" The amount of enzyme to be added was set to 5.0 U / mL with reference to the Department of Clinical Chemistry Laboratory / Institutional Difference Correction Study Group, pages 60-63, 1996).

  The COD amount is calculated from the COD Km value (ASAHI KASEI Diagnostic Enzymes, July 2008, page 16) and Michaelis-Menten's formula so that the COD enzyme reaction reaches the end point within 1.3 minutes. did. It was calculated that the enzyme reaction reached the end point within 1.3 minutes when COD was added at 4.0 U / mL.

[Example 3] Examination of concentration of oxidation color former (DA-67) The composition of each reagent is shown below. (DIL) was not used. The absorbance of purified water and 100 μmol / L choline standard solution was measured using an automatic analyzer. For DA-67 of (R-2), four concentrations of 0.033, 0.082, 0.163 and 0.326 mmol / L were examined.

  The results are shown in FIG. The horizontal axis of FIG. 2 is the concentration of DA-67 in (R-2). The absorbance of the reagent blank was stable at all DA-67 concentrations. At a DA-67 concentration of 0.082 mmol / L or more (in (R-2)), the absorbance of 100 μmol / L choline became stable, so that the DA-67 concentration was 0.082 mmol / L (in (R-2)) ) Was selected.

(R-1)
0.07 mol / L phosphate buffer pH 7.5
(R-2)
0.07 mol / L phosphate buffer pH 7.5
0.033-0.326mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 4] Examination of sub-wavelength Each reagent composition is shown below. (DIL) was not used. The absorbance of purified water and 100 μmol / L choline standard solution was measured using an automatic analyzer. The main wavelength was 660 nm, and the sub wavelengths were 700 nm, 750 nm, and 800 nm.

  The results are shown in FIG. When the subwavelength was 800 nm, the absorbance of 100 μmol / L choline was maximum and the sensitivity was high, so the subwavelength was set to 800 nm.

(R-1)
0.07 mol / L phosphate buffer pH 7.5
(R-2)
0.07 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 5] Examination of potassium iodate concentration Each reagent composition is shown below. (DIL) was not used. As the potassium iodate concentration in (R-1), four concentrations of 0, 50, 100 and 200 mmol / L were examined. By measuring the addition recovery rate (%) when 50 μmol / L choline standard solution was added to whole blood, evaluation of avoidance of the influence of a high concentration reducing substance present in whole blood was performed. Using <whole blood sample> and 500 μmol / L choline solution, A (choline solution 1 volume + healthy subject sample 9 volumes), B (physiological saline 1 volume + healthy subject sample 9 volumes), C (choline solution 1 Volume + physiological saline 9 volume) and D (physiological saline volume 1 volume + physiological saline volume 9 volume) were prepared as samples, and the choline concentration in each sample was measured. The addition recovery rate (%) was calculated using the following formula.
Addition recovery rate (%) = (A−B) / (C−D) × 100

  The results are shown in FIG. By increasing the concentration of potassium iodate, a maximum addition recovery of 52.8% was obtained. In this example, when mixing hemolyzed whole blood and potassium iodate, 3.5 μL of specimen prepared with whole blood 9 + choline solution (or physiological saline) 1 is mixed with (R-1) 180 μL. Therefore, the dilution ratio of whole blood to the solution is 0.9 × {3.5 / (3.5 + 180)} × 100≈1.72%, and the concentration of potassium iodate is 180 / (3.5 + 180). ) ≒ 0.98 times. The horizontal axis of FIG. 4 shows the potassium iodate concentration when mixed with hemolyzed whole blood. In FIG. 4, the potassium iodate concentrations are 0, 49, 98 and 196 mmol / L, respectively.

(R-1)
0.01 mol / L acetate buffer pH 4.0
0-200 mmol / L Potassium iodate 0.1% Triton X-100
(R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 6] Examination of potassium iodate and hydroxylamine hydrochloride concentrations when mixing hemolyzed whole blood and potassium iodate Each reagent composition is shown below. (DIL) was used. The concentration of hydroxylamine hydrochloride in (DIL) was examined at 8 concentrations of 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50 and 1.75 mmol / L, The potassium iodate density | concentration in (R-1) examined 2 density | concentration of 50 and 100 mmol / L. By measuring the addition recovery rate when 50 μmol / L choline standard solution was added to whole blood, the effect of avoiding the influence of a high concentration reducing substance present in whole blood was evaluated. Using the whole blood sample and a 500 μmol / L choline solution, the addition recovery rate (%) was calculated in the same manner as in [Example 5].

  When mixing the hemolyzed whole blood and potassium iodate, first, 17 μL of the sample prepared with whole blood 9 + choline solution (or physiological saline) 1 is used as an analysis sample by adding 85 μL of (DIL). Dispense 20 μL into a reaction container and mix with 180 μL of (R-1), so the dilution ratio of whole blood to the solution when mixed with potassium iodate is 0.9 × {17 / (17 + 85)} × {20 /(20+180)}×100=1.50%, and the concentration of hydroxylamine hydrochloride in the solution when mixed with potassium iodate is {85 / (17 + 85)} × {20 / (20 + 180)} ≈0 0.083 times, the concentration of potassium iodate is 180 / (20 + 180) = 0.9 times. The horizontal axis of FIG. 5 shows the concentration of hydroxylamine hydrochloride when mixing hemolyzed whole blood and potassium iodate. In FIG. 5, the hydroxylamine hydrochloride concentrations are 0, 20.83, 41.67, 62.50, 83.33, 104.17, 125.00 and 145.83 μmol / L, respectively. When the recovery rate of addition of hydroxylamine hydrochloride 83.3 μmol / L with respect to 45 mmol / L of potassium iodate is 83.5% and when hydroxylamine hydrochloride 62.5 μmol / L is used with respect to 90 mmol / L of potassium iodate The recovery of addition was 80.3%, and the recovery of addition was greatly improved by the use of hydroxylamine hydrochloride.

  The addition recovery was similarly calculated using thiourea or sodium thiosulfate instead of hydroxylamine hydrochloride. When thiourea was used instead of hydroxylamine hydrochloride, the addition recovery was up to 33.6%. When sodium thiosulfate was used instead of hydroxylamine hydrochloride, the addition recovery was up to 30.5%.

(DIL)
0 to 1.75 mmol / L Hydroxylamine hydrochloride (R-1)
0.01 mol / L acetate buffer pH 4.0
50 or 100 mmol / L potassium iodate 0.1% Triton X-100
(R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 7] Concentration study of cationic surfactant in mixing hemolyzed whole blood and potassium iodate Each reagent composition is shown below. (DIL) was used. Hexadecyltrimethylammonium bromide generally used as a cationic surfactant was used. As the concentration of hexadecyltrimethylammonium bromide in (R-1), four concentrations of 0, 0.025, 0.038 and 0.05% were examined. By measuring the addition recovery rate when 50 μmol / L choline standard solution was added to whole blood, the effect of avoiding the influence of a high concentration reducing substance present in whole blood was evaluated. Using the whole blood sample and a 500 μmol / L choline solution, the addition recovery rate (%) was calculated in the same manner as in [Example 5].

  The dilution rate of whole blood in the solution when mixing hemolyzed whole blood and potassium iodate was the same as in [Example 6], 1.50%, and the concentration of hydroxylamine hydrochloride was 0.75 × {85 / ( 17 + 85)} × {20 / (20 + 180)} = 62.5 μmol / L, and the concentration of potassium iodate is 100 × 180 / (20 + 180) = 90 mmol / L. The horizontal axis of FIG. 6 shows the concentration of hexadecyltrimethylammonium bromide when hemolyzed whole blood and potassium iodate are mixed. In FIG. 6, the concentrations of hexadecyltrimethylammonium bromide are 0.000, 0.023, 0.034 and 0.045%, respectively. When the amount of hexadecyltrimethylammonium bromide was 0.045%, the addition recovery rate was 97.6%, and the addition recovery rate was greatly improved.

(DIL)
0.75 mmol / L Hydroxylamine hydrochloride (R-1)
0.01 mol / L acetate buffer pH 4.0
100 mmol / L potassium iodate 0.1% Triton X-100
0-0.05% hexadecyltrimethylammonium bromide (R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 8] Examination of hydroxylamine hydrochloride concentration 2 when mixing hemolyzed whole blood and potassium iodate 2
(DIL) was used. As the concentration of hydroxylamine hydrochloride in (DIL), 8 concentrations of 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50 and 1.75 mmol / L were examined. The addition recovery rate (%) was calculated in the same manner as in [Example 5].
When the hemolyzed whole blood and potassium iodate were mixed, the dilution ratio of the whole blood with respect to the solution was 1.50% as in [Example 6], and the concentration of hydroxylamine hydrochloride was 0 as in [Example 6]. 0.083 times, the concentration of potassium iodate is 90 mmol / L as in [Example 7]. The horizontal axis of FIG. 7 shows the concentration of hydroxylamine hydrochloride when mixing hemolyzed whole blood and potassium iodate. When 62.5 μmol / L hydroxylamine hydrochloride was used with respect to 90 mmol / L potassium iodate, the maximum addition recovery rate of 91.9% was obtained. In FIG. 7, the concentrations of hydroxylamine hydrochloride are 0.00, 20.83, 41.67, 62.50, 83.33, 104.17, 125.00, and 145.83 μmol / L, respectively.

(DIL)
0 to 1.75 mmol / L Hydroxylamine hydrochloride (R-1)
0.01 mol / L acetate buffer pH 4.0
100 mmol / L potassium iodate 0.1% Triton X-100
0.05% hexadecyltrimethylammonium bromide (R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 9] Dilution linearity Each reagent composition is shown below. (DIL) was not used. The linearity of the measured choline concentration was examined using 7 concentrations of choline solutions of 5.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 μmol / L.
The results are shown in FIG. The horizontal axis of the graph indicates the measured concentration of the choline solution, and the vertical axis indicates the actual measured value of the choline concentration in each choline solution measured by the automatic analyzer after calibration with the standard choline solution. Linearity through the origin was obtained up to a choline concentration of 100 μmol / L.

(R-1)
0.01 mol / L acetate buffer pH 4.0
100 mmol / L potassium iodate 0.1% Triton X-100
0.05% hexadecyltrimethylammonium bromide (R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 10] Minimum detection sensitivity The same reagent as used in [Example 9] was used. (DIL) was not used. The minimum detection sensitivity (n = 3) was determined by the ± 3SD method using 5 concentrations of choline solutions of 1.0, 2.0, 2.5, 5.0 and 10.0 μmol / L.
The results are shown in FIG. The horizontal axis of the graph represents the concentration of the choline solution measured, and the vertical axis represents the average value of the choline concentration in each choline solution actually measured by the automatic analyzer. From FIG. 9, the minimum detection sensitivity was 2 μmol / L. The cut-off value of choline in whole blood for a major adverse cardiac event that can be diagnosed by measuring choline in whole blood was 28.2 μmol / L, which was considered to be sufficient detection sensitivity.

[Example 11] Simultaneous reproducibility test and daily difference reproducibility test The same reagents as in [Example 9] were used. (DIL) was not used. A simultaneous reproducibility test (n = 20) was performed using 10 μmol / L and 100 μmol / L choline solutions.
Further, using each reagent shown below and (DIL), a daily difference reproducibility test (n = 20) was performed using <whole blood sample>.
The results are shown in Table 1. It was possible to measure choline in whole blood with good reproducibility.

(DIL)
0.75 mmol / L Hydroxylamine hydrochloride (R-1)
0.01 mol / L acetate buffer pH 4.0
100 mmol / L potassium iodate 0.1% Triton X-100
0.05% hexadecyltrimethylammonium bromide (R-2)
0.2 mol / L phosphate buffer pH 7.5
0.082 mmol / L DA-67
10mmol / L Sodium thiosulfate 0.16mmol / L Orange G
(R-3)
0.07 mol / L phosphate buffer pH 7.5
77.5U / mL POD
60U / mL COD

[Example 12] Specificity test The same reagent as used in [Example 9] was used. (DIL) was not used. Ethanolamine, creatinine, carnitine, alanine and serine whose chemical structure is similar to choline were selected as structurally similar substances from among nitrogen-containing components, sugars and amino acids present in whole blood. Table 2 shows the reaction ratio (%) of each structurally similar substance of 50 μmol / L when the measured value (absorbance) of 50 μmol / L choline is 100%. Almost no reactivity was observed with any of the substances, and it was revealed that the method for measuring choline in whole blood of the present invention has high specificity for choline.

[Example 13] Addition / recovery test Each reagent and (DIL) shown in [Example 11] were used. Using the <whole blood sample> and 100 μmol / L and 500 μmol / L choline solutions, the addition recovery rate (%) was calculated in the same manner as in [Example 5].
The results are shown in Table 3. A high addition recovery rate was obtained by the method for measuring choline in whole blood of the present invention.

[Example 14] Specimen dilution linearity test Each reagent and (DIL) shown in [Example 11] were used. Using a <whole blood sample> and a 500 μmol / L choline solution, a (saline 1 volume + <whole blood sample> 9 volumes) and e (choline solution 1 volume + <whole blood samples> 9 volumes) are prepared. Then, a concentration series as shown in Table 4 was prepared. The choline concentration of each specimen a to e was measured twice.
The results are shown in FIG. Good dilution linearity was obtained by the method for measuring choline in whole blood of the present invention.

[Example 15] Influence of interfering substances in whole blood The reagent of [Example 9] was used. (DIL) was not used. As reducing substances present in whole blood, ascorbic acid, albumin, reduced glutathione, and uric acid were selected as interference substances, and solutions having concentrations shown in Table 5 were prepared. Nine volumes of each interfering substance was mixed with one volume of a 500 μmol / L choline solution, and the error (%) in the presence of the interfering substance was measured when the concentration of the control (saline) was 100%.

The results are shown in FIG. None of the interfering substances affected the method for measuring choline in whole blood of the present invention.

[Example 16] Measurement of healthy subject samples Using each reagent and (DIL) shown in [Example 11], <whole blood sample> 20 cases (20 to 25 years old, average 21 years old, male 13 cases, female 16 cases) were measured. The distribution of choline measurements in whole blood is shown in FIG. In three cases with measured values of 25 μmol / L or more, hemolysis was confirmed with the naked eye before hemolysis for measurement. In 26 cases where hemolysis before measurement was not observed, the average value of choline concentration in whole blood was 8.23 μmol / L, and the standard deviation was 4.06 μmol / L. When the reference standard range of this sample was an average ± 2SD, it was 0.1 to 16.3 μmol / L.

  According to the present invention, a method for measuring choline in whole blood and a composition for measurement are provided. Thereby, for example, it has industrial applicability that examination of diseases such as acute myocardial infarction, stable angina pectoris, non-Q wave (subendocardial) myocardial infarction becomes possible.

Claims (9)

  A method for measuring choline in whole blood using choline oxidase, peroxidase and oxidative color former, comprising: whole blood that has been hemolyzed in solution; potassium iodate or sodium iodate; and hydroxylamine or a salt thereof. The measuring method characterized by including the process made to coexist and react. A method for measuring choline in whole blood,
In a solution, reacting hemolyzed whole blood together with potassium iodate or sodium iodate and hydroxylamine or a salt thereof;
Producing hydrogen peroxide from choline in whole blood using choline oxidase; and measuring the concentration of hydrogen peroxide;
Measuring method including   The measuring method according to claim 1 or 2, wherein the step of reacting is a step of further reacting in the presence of a cationic surfactant.   The measuring method according to any one of claims 1 to 3, wherein the concentration of hydroxylamine or a salt thereof in the solution in the reacting step is 33 to 142 µmol / L.   The measuring method according to claim 1, wherein the concentration of potassium iodate or sodium iodate in the solution in the reacting step is 45 mmol / L or more. The measuring method according to claim 3, wherein the concentration of the cationic surfactant in the solution in the reacting step is 0.031 w / v% or more. A method for measuring choline in whole blood, comprising the following steps (a) to (e);
(A) lysing whole blood;
(B) a step of reacting hemolyzed whole blood with potassium iodate or sodium iodate and hydroxylamine or a salt thereof in a solution;
(C) adjusting the pH of the solution of step (b) to near neutral;
(D) a step of producing hydrogen peroxide from choline in whole blood using choline oxidase; and (e) a step of measuring the concentration of hydrogen peroxide produced in step (d).   A method for examining acute myocardial infarction, stable angina pectoris or non-Q wave (subendocardial) myocardial infarction using the method for measuring choline in whole blood according to any one of claims 1 to 7. A kit for measuring choline in whole blood containing the composition according to the following (1) to (4):
(1) a composition containing hydroxylamine or a salt thereof,
(2) an acidic composition containing potassium iodate or sodium iodate,
(3) A composition near neutral containing an oxidative color former, and (4) a composition containing choline oxidase and peroxidase.