WO2014126207A1 - Method for detecting organic acid in sample - Google Patents

Abstract

Provided is a method whereby an organic acid, in particular an organic acid such as 3-HB, 3-HIB, etc. potentially usable as a biomarker, in a sample can be highly sensitively and highly accurately detected. The method for detecting an organic acid in a sample is characterized by comprising: ester-binding the organic acid in the sample to a derivatizing reagent represented by any of general formulae (1)-1 to (1)-3 [wherein one of R¹ and R² represents a C_1-6 hydroxyalkyl group and the other represents a hydrogen atom or a C_1-6 alkyl group] to synthesize a derivative; and detecting the derivative by the positive ion mode electrospray ionization LC-MS/MS (liquid chromatography-tandem mass spectrometry) method.

Description

Method for detecting organic acid in sample

The present invention relates to a method for detecting organic acids such as 3-hydroxybutyric acid and 3-hydroxyisobutyric acid in a sample with high sensitivity.
This application claims priority based on Japanese Patent Application No. 2013-28168 filed in Japan on February 15, 2013, the contents of which are incorporated herein by reference.

3-Hydroxybutyric acid (3-HB) is the most stable ketone body in serum and is synthesized in the liver during starvation and undernutrition. For this reason, 3-HB is useful as a biomarker for evaluating fatty acid catabolism in the liver. For quantification of 3-HB, an enzyme spectrophotometric method for detecting NADH after adding NAD and D-3-hydroxybutyrate dehydrogenase is used (see, for example, Non-Patent Document 1 or 2). However, since various enzymes exist in serum and NADH production can be inhibited, these methods have a problem of low detection sensitivity of 3-HB in serum. As another NADH determination method, 3HB is enzymatically converted to acetoacetic acid (AA), then derivatized with p-nitrobenzenediazonium fluoroboric acid, and this derivative is detected by high-sensitivity HPLC (high-performance liquid) that is detected by spectrophotometry or ultraviolet light. A method for quantification by a chromatography assay has been reported (for example, see Non-Patent Document 3 or 4). Further, as a method for directly detecting 3-HB, a method for detecting by derivatization after gas chromatography-mass spectrometry (GC-MS) (for example, see Non-Patent Document 5 or 6), or a method without derivatization. In addition, a method of detecting by LC-MS / MS (liquid chromatography-tandem mass spectrometry) (LC-P-ESI-MS / MS) by electrospray ionization (ESI) in positive ion mode (for example, Non-Patent Document 7). See also), but the detection sensitivity of these methods is as low as that of the enzyme spectrophotometry.

On the other hand, nutritional metabolism abnormalities occur in patients with gastrointestinal diseases due to decreased metabolism / digestion / absorption capacity or overnutrition. Many gastrointestinal diseases have abnormal sugar and lipid metabolism, so amino acids are highly important as an energy source. In particular, branched chain amino acids (BCAA: valine, leucine, isoleucine) are catabolized in the mitochondria of skeletal muscle instead of carbohydrates and lipids during starvation, undernutrition, long-term exercise, and metabolic diseases, and become energy sources. BCAA catabolized in skeletal muscle is metabolized to acetyl CoA and succinyl CoA. BCAA is bound to CoA that cannot pass through the mitochondrial membrane by a common enzymatic reaction in mitochondria. Specifically, BCAA is a branched chain α-keto acid (from valine to α-ketoisovaleric acid) by BCAT (BCAA aminotransferase) and BCKDH complex (branched α-keto acid dehydrogenase complex) in mitochondria. , Leucine to α-ketoisocaproic acid (KIC), isoleucine to α-keto-β-methylvaleric acid), and CoA form (α-ketoisovaleric acid to isobutyryl CoA, KIC to isovaleryl CoA, α-keto -Β-methylvaleric acid to α-methylbutyryl CoA). In the catabolism process of BCAA, CoA binding is essential, but in the middle of catabolism, CoA is once removed by a specific enzyme reaction in the middle of catabolism, and the intermediate metabolite 3-hydroxyisobutyric acid (3-HIB) Can do. Since 3-HIB to which CoA is not bound has a low molecular weight, it passes through the mitochondrial membrane, and a part of it leaks out of the cell. In fact, in patients with starvation or cirrhosis, serum 3-HIB levels increase with skeletal muscle atrophy.

From this, 3-HIB is expected to be used as an index reflecting the amino acid catabolism state in skeletal muscle, that is, as a BCAA catabolism marker in skeletal muscle. However, a method that can detect a trace amount of 3-HIB in a biological sample with sufficient sensitivity has not been reported so far. For example, as a method of directly detecting 3-HIB, it is detected by LC-MS / MS method (LC-N-ESI-MS / MS) by electrospray ionization (ESI) in negative ion mode without derivatization. Although a method (for example, see Non-Patent Document 7) has been reported, since the method has low sensitivity, for example, a large amount of serum is required to detect 3-HIB in serum. In addition, when 3-HB in blood is detected by the GC-MS method, it has been reported that 3-HIB could not be separated from 3-HB by chromatography (for example, non-patent literature). 8).

As a method for detecting organic acids other than 3-HB and 3-HIB, malonic acid is derivatized to di- (1-methyl-3-piperidinyl) malonic acid and then LC-P-ESI-MS / MS). A detection method (for example, see Non-Patent Document 9) has been reported. In this method, by introducing an amine residue into malonic acid to form a malonic ester, ionization was promoted and detection sensitivity was remarkably increased.

Also, as a metabolite of leucine, which is one of BCAAs, 3-hydroxyisovaleric acid (3-HMB) can be mentioned. Like other BCAAs, leucine is metabolized in the mitochondria via KIC to the CoA form (isovaleryl CoA), but about 5 to 10% of KIC passes through the mitochondrial membrane, and in the cytoplasm, 3-fold by KIC-dioxygenase. Metabolized to HMB. The effects of 3-HMB include protein synthesis promotion by mTOR pathway, protein degradation inhibition by inhibition of ubiquitin / proteasome pathway, and muscle synthesis by promotion of cholesterol synthesis by hydroxymethylglutaryl-CoA (HMG-CoA) converted from HMB-CoA. Sheath stabilization is known. Due to these effects, 3-HMB is expected to be used as a marker for synthesis of liver protein (albumin etc.) derived from BCAA and skeletal muscle hypertrophy. Actually, since 3-HMB is used as a supplement for the purpose of enhancing muscles, in order to study the blood dynamics when 3-HMB is ingested, the blood 3 It has been reported that -HMB concentration was measured by LC-N-ESI-MS / MS without derivatization (see, for example, Non-Patent Document 10). In addition, a method of simultaneously measuring 3-HB and 3-HMB without derivatization by HILIC (Hydrophilic Interaction Chromatography) -MS / MS has been reported. The detection limit is 0.4 μM, and 3-HMB in a biological sample (blood, saliva, etc.) is in a smaller amount than 3-HB or 3-HMB, so a method for detecting with higher sensitivity is required ( For example, refer nonpatent literature 11.).

Further, 2-hydroxybutyric acid (2-HB) is an organic acid similar in structure to 3-HB and the like. In 2-HB, α-ketobutyric acid, which is a metabolite of threonine and methionine, is produced as a by-product of the catabolism reaction by 2-DHB dehydrogenase (α-HBDH), which is an NADH-dependent LDH or LDH isozyme. Further, since α-ketobutyric acid is metabolized to propionyl CoA, which is a precursor of succinyl CoA, the increase in 2-HB is due to increased catabolism of α-ketobutyric acid by LDH or α-HBDH, or from α-ketobutyric acid to propionyl-CoA. Caused by metabolic failure. Furthermore, α-ketobutyric acid is also associated with the production of glutathione because cysteine, which is a precursor amino acid of glutathione, which is a powerful antioxidant, is also produced as a by-product when metabolized from cystathione. There is a report that 2-HB can be used as an early marker for predicting insulin resistance and glucose intolerance in non-diabetics (see, for example, Non-patent Document 12). This is because the development of insulin resistance and glucose intolerance involves lipid peroxidation and oxidative stress in the liver, and is due to increased glutathione production in the liver and an increase in NADH and NAD ⁺ associated with lipid peroxidation. Conceivable. In this study, 2-HB in serum and whole blood was measured with a UHPLC-MS or HILIC-MS / MS apparatus. From the serum concentration (1 μg / mL = about 9.6 μM or more) The measurement with a very small amount of biological sample (saliva etc.) has not been studied so far (for example, see Non-Patent Document 11 and Non-Patent Document 2).

The present invention is highly sensitive to organic acids, particularly organic acids such as 3-HB, 3-HIB, 3-HMB, and 2-HB that can be expected to be used as biomarkers contained in biological samples. The purpose is to provide a method of detecting well.

That is, the present invention provides a method for detecting an organic acid in the following samples [1] to [6], a method for evaluating [7], [8] and [10] below, a method for monitoring [11] below and The biomarker according to [9] is provided.
[1] A method for detecting an organic acid in a sample according to the present invention is a method for detecting an organic acid in a sample. The organic acid in a sample is represented by the following general formulas (1) -1 to (1) -3: (In the formula (1) -1 to 3, ^one of R ¹ and R ² represents a hydroxyalkyl group having 1 to 6 carbon atoms, and the other represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms). Derivatives are synthesized by ester linkage with a derivatization reagent represented by any of the above, and the derivatives are detected by an electrospray ionization LC-MS / MS (liquid chromatography-tandem mass spectrometry) method in positive ion mode. It is characterized by.

[2] In the method for detecting an organic acid in the sample of [1], the derivatization reagent is composed of 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl) -1-methylpyrrolidine. It is preferable that it is 1 or more types selected from a group.
[3] In the method for detecting an organic acid in the sample of [1] or [2], the organic acid is represented by the following general formula (2) (in the formula (2), R ³ is a group having 2 to 6 carbon atoms). It is preferably an organic acid represented by a hydroxyalkyl group.

[4] In the method for detecting an organic acid in the sample of [1] or [2], the organic acid is 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, and 2-hydroxybutyric acid. It is preferable that it is 1 or more types selected from the group which consists of.
[5] In the method for detecting an organic acid in a sample according to any one of [1] to [4], the sample is preferably a biological sample.
[6] In the method for detecting an organic acid in a sample according to any one of [1] to [4], the sample is preferably serum, tears, saliva, or urine.
[7] The method for evaluating the likelihood of developing cirrhosis according to the present invention is selected from the group consisting of 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, and 2-hydroxybutyric acid in a biological sample collected from a subject. One or more organic acids detected by the method for detecting an organic acid in the sample of [1] or [2], and measuring the concentration of the organic acid in the biological sample; and the quantifying step Comparing the organic acid concentration obtained in step 1 with a preset threshold and evaluating the possibility of developing cirrhosis in the subject, wherein the biological sample is serum or saliva To do.
[8] The method for evaluating the possibility of developing hepatic encephalopathy according to the present invention includes the step of [1] or [2], wherein 3-hydroxyisobutyric acid in a biological sample collected from a subject who has developed cirrhosis is used. The biological sample collects the quantitative step of detecting the organic acid in the sample and measuring the 3-hydroxyisobutyric acid concentration in the biological sample, and the 3-hydroxyisobutyric acid concentration obtained in the quantitative step. Comparing with the 3-hydroxyisobutyric acid concentration in the biological sample collected from the subject before the determined time point, and evaluating the possibility of developing hepatic encephalopathy in the subject, It is serum or saliva.
[9] The biomarker of hepatic encephalopathy or the onset possibility thereof according to the present invention is characterized by comprising 3-hydroxyisobutyric acid.
[10] The method for evaluating an energy supply source according to the present invention comprises at least one organic substance selected from the group consisting of 3-hydroxyisobutyric acid and 3-hydroxybutyric acid in a biological sample collected from a subject during exercise. A quantitative step of detecting an acid by the method for detecting an organic acid in a sample of [1] or [2] and measuring the concentration of the organic acid in the biological sample, and the organic acid obtained in the quantitative step Comparing the concentration with a pre-set threshold or the organic acid concentration in a biological sample collected from the subject before exercise, and evaluating an energy source during or after exercise in the subject; and And the biological sample is serum or saliva.
[11] The method for monitoring an energy supply source according to the present invention includes 3-hydroxyisolation in a plurality of biological samples collected from a subject over time from immediately before the start of exercise to after a lapse of a certain period after the end of exercise. One or more organic acids selected from the group consisting of butyric acid and 3-hydroxybutyric acid are detected by the organic acid detection method in the sample of [1] or [2], respectively, and the organic acid in each biological sample is detected. Based on the monitoring process for examining the time-dependent change of the organic acid concentration by measuring the acid concentration, and the time-dependent change of the organic acid concentration obtained in the monitoring process, energy during or after exercise in the subject An evaluation step for evaluating a supply source, wherein the organic acid concentration in the biological sample is used as a marker of an energy supply source, and the biological sample is serum or saliva. It is characterized by that.

The method for detecting an organic acid in a sample according to the present invention is a method of derivatizing an organic acid such as 3-HIB, 3-HB, 3-HMB, 2-HB and the like and detecting it by LC-P-ESI-MS / MS It is possible to directly detect the organic acid to be detected in the sample. In addition, by using a specific derivatization reagent, the detection sensitivity is much higher than the detection method by conventional enzyme spectrophotometry and LC-MS / MS, so that not only serum but also organic acids such as tears and saliva are used. It can be expected to be useful for organic acid detection from a biological sample having a relatively low content.

FIG. 3 shows a typical P-ESI MS spectrum of 3-HB (2 PM-3-HB) derivatized with 2-pyridinemethanol. In Example 1, it is the figure which showed the total ion chromatogram (upper stage) and the MS chromatogram (lower stage) of the sample containing only 3-HB derivatized with 2-pyridinemethanol. It is the figure which showed the MS spectrum of FIG. 2A. In Example 1, it is the figure which showed the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 2-pyridinemethanol. It is the figure which showed the MS spectrum of FIG. 2C. In Example 1, it is the figure which showed the product ion chromatogram when using m / z 196 of the sample containing 3-HB and 3-HIB derivatized with 2-pyridinemethanol as a precursor ion. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 2E. In Example 2, it is the figure which showed the total ion chromatogram (upper part) and MS chromatogram (lower part) of the sample containing only 3-HB derivatized with 1-piperidine ethanol. It is the figure which showed the MS spectrum of FIG. 3A. In Example 2, it is the figure which showed the total ion chromatogram (upper part) and MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 1-piperidine ethanol. It is the figure which showed the MS spectrum of FIG. 3C. In Example 2, it is the figure which showed the product ion chromatogram when using m / z 216 of the sample containing 3-HB and 3-HIB derivatized with 1-piperidine ethanol as a precursor ion. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 3E. In Example 3, the figure which showed the total ion chromatogram (upper part) and MS chromatogram (lower part) of the sample containing only 3-HB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine. is there. It is the figure which showed the MS spectrum of FIG. 4A. In Example 3, the figure which shows the total ion chromatogram (upper part) and MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine. is there. It is the figure which showed the MS spectrum of FIG. 4C. In Example 3, a product ion chromatogram when m / z 216 of a sample containing 3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine is used as a precursor ion is shown. FIG. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 4E. In Comparative Example 1, the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only 3-HB derivatized with 3-pyridinemethanol are shown. It is the figure which showed MS spectrum of FIG. 5A. In comparative example 1, it is the figure which shows the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 3-pyridinemethanol. It is the figure which showed the MS spectrum of FIG. 5C. In Comparative Example 1, a product ion chromatogram of a sample containing 3-HB and 3-HIB derivatized with 3-pyridinemethanol when m / z 196 is used as a precursor ion is shown. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 5E. In comparative example 2, it is the figure which shows the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HB derivatized with 2-diethylaminoethanol. It is the figure which showed MS spectrum of FIG. 6A. In comparative example 2, it is the figure which shows the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 2-diethylaminoethanol. It is the figure which showed the MS spectrum of FIG. 6C. In Comparative Example 2, it is a diagram showing a product ion chromatogram when m / z 204 of a sample containing 3-HB and 3-HIB derivatized with 2-diethylaminoethanol is used as a precursor ion. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 6E. In Comparative Example 3, the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only 3-HB derivatized with 3-hydroxy-1-methylpiperidine are shown. It is the figure which showed MS spectrum of FIG. 7A. In Comparative Example 3, the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only 3-HIB derivatized with 3-hydroxy-1-methylpiperidine are shown. It is the figure which showed MS spectrum of FIG. 7C. In Comparative Example 3, it is a diagram showing a product ion chromatogram when m / z 202 of a sample containing 3-HB and 3-HIB derivatized with 3-hydroxy-1-methylpiperidine is used as a precursor ion. . It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 7E. In comparative example 4, it is the figure which showed the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HB derivatized with 4-pyridinemethanol. It is the figure which showed MS spectrum of FIG. 8A. In comparative example 4, it is the figure which showed the total ion chromatogram (upper part) and the MS chromatogram (lower part) of the sample containing only 3-HIB derivatized with 4-pyridinemethanol. It is the figure which showed the MS spectrum of FIG. 8C. In Comparative Example 4, it is a diagram showing a product ion chromatogram when m / z 196 of a sample containing 3-HB and 3-HIB derivatized with 4-pyridinemethanol is used as a precursor ion. It is the figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 8E. FIG. 6 shows a typical SRM chromatogram of 3-HB (2 PM-3-HB) derivatized with 2-pyridinemethanol and [ ¹³ C ₄ ] isotopes in Example 4. In Example 5, it is the figure which showed the result of having measured the 3-HB density | concentration in the serum extract | collected from a healthy male person every 2 hours. In Example 8, the total ion chromatogram (top) of a sample containing 3-HMB and 2-HB derivatized with 2-pyridinemethanol, and the product ion chromatogram when m / z 196 is used as a precursor ion ( FIG. 4 is a diagram showing a product ion chromatogram (lower stage) when m / z 210 is a precursor ion. FIG. 11B is a diagram showing an MS spectrum (2-HB 2PM ester derivative) in the middle product ion chromatogram of FIG. 11A. It is the figure which showed MS spectrum (2-PM ester derivative of 3-HMB) of the product ion chromatogram of the lower stage of FIG. 11A. In Example 9, 2-pyridine derivatized with methanol 3-HB and 3-HIB and 3-HMB and total ion chromatogram of a sample containing 2-HB and ^3-HB- 13 _{C 4} (1 stage) , Product ion chromatogram when m / z 196 is used as a precursor ion (second stage), product ion chromatogram when m / z 200 is used as a precursor ion (third stage), and m / z 210 as a precursor ion FIG. 6 is a diagram showing a product ion chromatogram (fourth stage). In Example 10, it is the figure which showed the measurement result of the 3-HB density | concentration and 3-HIB density | concentration in the serum left at room temperature after blood collection. In Example 10, it is the figure which showed the measurement result of 3-HB density | concentration and 3-HIB density | concentration in the saliva left at room temperature after collection | recovery. In Example 11, it is the figure which showed the result of having investigated the correlation of the serum concentration of 3-HB and the concentration in saliva (fluid). In Example 11, it is the figure which showed the result of having investigated the correlation of the serum concentration of 3-HIB, and the concentration in saliva (fluid). In Example 11, it is the figure which showed the result of having investigated the correlation of the density | concentration in the serum of 2-HB, and the density | concentration in saliva (fluid). In Example 12, it is the figure which showed the test subject's exercise | movement, the meal condition, and the collection time of blood, saliva, and urine. In Example 12, it is the figure which showed the time-dependent change of the density | concentration of 3-HIB and 3-HB in a test subject's serum. In Example 12, it is the figure which showed the time-dependent change of the amount of urine excretion per unit time (micromol / h) in each test | inspection time of a subject's 3-HIB and 3-HB. In Example 12, it is the figure which showed the time-dependent change of the density | concentration of 3-HIB and 3-HB in a test subject's saliva. In Example 13, it is the figure which showed the measurement result of the 3-HIB density | concentration and 3-HMB density | concentration in serum of a cirrhosis patient group and a healthy subject group. In Example 13, it is the figure which showed the measurement result of the 3-HIB density | concentration and 3-HMB density | concentration in the saliva of a cirrhosis patient group and a healthy subject group. In Example 16, change in serum 3-HIB concentration (upper), change in serum albumin concentration (middle), and change in serum ammonia concentration over time in cirrhosis patient A having relatively mild symptoms of cirrhosis It is the figure which showed (lower stage). In Example 16, change in serum 3-HIB concentration over time (upper stage), change in serum albumin concentration over time (middle stage), and change in serum ammonia concentration over time in cirrhosis patient B with relatively severe cirrhosis symptoms It is the figure which showed (lower stage).

The method for detecting an organic acid in a sample according to the present invention (hereinafter sometimes referred to as “the detection method according to the present invention”) is a method for detecting an organic acid in a sample, Derivatives are synthesized by ester linkage with a derivatizing reagent represented by any of the following general formulas (1) -1 to (1) -3, and the derivatives are detected by LC-P-ESI-MS / MS method It is characterized by doing. By esterifying with a derivatizing reagent represented by any one of the following general formulas (1) -1 to 3, the ionization efficiency of organic acids by P-ESI is improved, and the detection sensitivity by LC-MS / MS is remarkable. To improve.

In general formulas (1) -1 to 3, R ¹ and R ² each represents a hydroxyalkyl group having 1 to 6 carbon atoms, and the other represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. That is, when R ¹ is a hydroxyalkyl group having 1 to 6 carbon atoms, R ² is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ² is a hydroxyalkyl group having 1 to 6 carbon atoms, ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. An organic acid can be efficiently ionized by using a compound having a 6- or 5-membered heterocycle containing a nitrogen atom and having a hydroxyl group linked with an alkyl group in the vicinity of the nitrogen atom as a derivatization reagent. Furthermore, since the retention time of LC of the obtained derivative (esterified product) is relatively long, it can be clearly separated from the reagent used for derivatization.

The alkyl group having 1 to 6 carbon atoms may be a linear alkyl group or a branched alkyl group. Specifically, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, s-butyl group, t-butyl group, n-pentyl group, n-hexyl Groups and the like. R ¹ or R ^{2 in the} general formulas (1) -1 to 3 is preferably a methyl group, an ethyl group, an n-propyl group, or an i-propyl group.

The hydroxyalkyl group having 1 to 6 carbon atoms may be a group in which one hydrogen atom in a linear alkyl group having 1 to 6 carbon atoms is substituted with a hydroxyl group. A group in which one hydrogen atom in the branched chain alkyl group is substituted with a hydroxyl group may be used. Specific examples include 2-hydroxyethyl group, 3-hydroxypropyl group, 4-hydroxybutyl group, 5-hydroxypentyl group, 6-hydroxyhexyl group and the like.

The derivatization reagent represented by the general formula (1) includes at least one selected from the group consisting of 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl) -1-methylpyrrolidine. Preferably there is. By esterifying the carboxyl group with these compounds, organic acids having similar structures such as 3-HIB, 3-HB, 3-HMB and 2-HB can be separated well by LC. As a result, both of them can be detected simultaneously (detection by one LC-P-ESI-MS / MS for one sample). Among the derivatization reagents represented by the general formula (1), the retention time is slow, and the influence of the peak by the derivatization reagent on the derivatized organic acid ester peak to be detected is small. Methanol is particularly preferred.

In the detection method according to the present invention, the LC-P-ESI-MS / MS method can be performed by a conventional method except that the compound represented by the general formula (1) is used as a derivatization reagent. Specifically, HPLC and MS in LC-MS / MS can be performed in the same manner as known conditions for detecting a carboxylic acid ester or under appropriately modified conditions.

In the detection method according to the present invention, only one type of organic acid may be detected in one LC-P-ESI-MS / MS, or two or more types of organic acid may be detected simultaneously. The organic acid to be detected in the detection method according to the present invention is not particularly limited as long as it is a compound having a carboxyl group, and the detection method is applicable to detection of many organic acids having a carboxyl group. it can. The detection method according to the present invention is preferably used for detection and quantification of organic acids which are intermediate metabolites such as glycolysis, TCA cycle, and fatty acid synthesis. Especially, it is preferable that the detection method concerning this invention is used for the detection of the organic acid represented by following General formula (2).

In the general formula (2), R ³ represents a hydroxyalkyl group having 2 to 6 carbon atoms. The hydroxyalkyl group having 2 to 6 carbon atoms may be a branched hydroxyalkyl group or a linear hydroxyalkyl group. Examples of the hydroxyalkyl group include 2-hydroxyethyl group, 3-hydroxypropyl group, 2-hydroxypropyl group, 4-hydroxybutyl group, 2-hydroxybutyl group, 2- (hydroxymethyl) propyl group, 2- Examples thereof include a hydroxypentyl group, 3-hydroxy-2, 2-dimethylpropyl group, 3-hydroxypentyl group, 5-hydroxypentyl group, 6-hydroxyhexyl group, 2-hydroxyhexyl group and the like.

In the organic acid represented by the general formula (2), R ³ is preferably a hydroxyalkyl group having 2 to 3 carbon atoms. In particular, the organic acid represented by the general formula (2) is preferably at least one selected from the group consisting of 3-HIB, 3-HB, 3-HMB, and 2-HB. More preferably, HB and 3-HIB or 3-HIB, 3-HB and 3-HMB are detected simultaneously. As described above, 3-HB is a marker for fatty acid catabolism in the liver, and 3-HIB and 3-HMB can be BCAA catabolism markers in skeletal muscle. That is, the detection method according to the present invention can simultaneously detect a fatty acid catabolism marker in the liver and a BCAA catabolism marker in skeletal muscle.

In the detection method according to the present invention, as in other detection methods using LC-MS / MS, the organic acid to be detected is quantitatively detected based on a calibration curve created using an organic acid with a known concentration. can do. When preparing a calibration curve, it is also preferable to add a certain amount of stable isotopes to all dilution series of organic acids with known concentrations in order to improve the quantitativeness.

The sample used in the detection method according to the present invention is not particularly limited as long as it is a sample containing an organic acid. Specifically, biological samples, lakes, seas, rivers, soils, etc. Examples include samples collected from nature, cultured cells, cultures of microorganisms such as yeast and bacteria, and the like. Since the detection method according to the present invention is very sensitive, it is generally preferable to perform on a sample that contains only a very small amount of organic acid or a sample that contains a very small amount of organic acid. More preferably. The biological sample may be a sample collected from a living body, and is preferably collected from an animal such as a human, a mouse, or a rat. It may also be blood, plasma, serum, tears, saliva, cerebrospinal fluid, ascites, pleural effusion, joint fluid, bone marrow, bile, urine, nasal discharge, milk, sweat, semen, etc., skeletal muscle, liver, etc. It may be an extract (tissue extract) such as a tissue piece collected from the tissue. An extract such as a tissue piece can be prepared by homogenizing a tissue piece or the like by a conventional method. The sample used in the detection method according to the present invention is preferably serum, tears, saliva, or urine.

Also, in the detection of organic acids in biological samples, conventional analytical methods often require more biological samples because of insufficient sensitivity, or are too low to be detected in the first place. On the other hand, since the detection method according to the present invention has very high detection sensitivity, the target organic acid can be detected from a smaller amount of biological sample than in the past. In addition, it is possible to detect an organic acid in a biological sample that could not be detected because the content is extremely small.

For example, the detection sensitivity when 3-HIB is detected by the detection method according to the present invention is 100 times or more compared with the case where detection is performed by LC-N-ESI-MS / MS without conventional derivatization. . Further, the sensitivity is 10 times or more higher than when a derivative obtained by derivatizing 3-HIB with HCl-butanol is detected by positive ESI-LC-MS / MS. For this reason, the detection method according to the present invention can be used for simultaneous determination of 3-HIB and 3-HB, and for 3-HIB and 3-HB in 5 μL or less of serum, 5 μL or less of saliva, tears, or cell culture solution. Simultaneous determination of HB, 3-HMB and 2-HB is possible.

Since the concentration of 3-HIB and 3-HB in saliva and tear fluid is highly correlated with the serum concentration, the detection method according to the present invention can be used to make non-invasive treatment using saliva and tear fluid as a measurement sample. In particular, it is possible to evaluate fatty acid catabolism in the liver and BCAA catabolism in skeletal muscle.

Until now, there is no serum marker to grasp the catabolism of BCAA in skeletal muscle, clinically quantified by the BCAA concentration in blood and the ratio with aromatic amino acid (Fischer ratio), or indirect method by serum albumin level I only guessed. On the other hand, BCAA catabolism in skeletal muscle can be more directly evaluated by detecting 3-HIB by the detection method according to the present invention. That is, the detection method according to the present invention is also useful as a new clinical test for evaluating BCAA catabolism in skeletal muscle. In addition, the 3-HIB concentration in serum, tears, saliva and the like can also be used as an index (biomarker) for predicting the effect by administering the BCAA preparation.

The degree of catabolism of BCAA in skeletal muscle is, for example, cirrhosis, hepatic encephalopathy, myositis, steroid myopathy, heart failure, bad epidemic due to use of anticancer agents, rhabdomyolysis, disuse muscle atrophy, Blood albumin levels in patients suffering from diseases such as hyperthyroidism, chronic fatigue syndrome, sarcopenia (aging muscle weakening phenomenon), mitochondrial fatty acid oxidation disorders, dialysis patients, surgically invasive patients, elderly people, etc. Or an indicator (biomarker) of malnutrition or muscle protein degradation for a person whose blood BCAA concentration is low or may be low. It can also be used as an indicator of undernutrition and muscle protein degradation for athletes who are prone to increase muscle protein catabolism such as marathons and triathlons. In addition, since 3-HIB concentrations in serum, tears, saliva, etc. can be used as an indicator of nutritional status, 3-HIB concentrations in biological samples can be used, for example, for drug discovery screening, particularly for the treatment of various diseases described above. It is expected to be used as a marker showing the pharmacological effect of drug candidates and as a marker for toxicity evaluation in the safety test of active ingredients.

As energy consumption increases due to exercise, fatty acid catabolism in the liver increases and the amount of 3-HB in body fluids (blood, saliva) increases. Further, in the state where BCAA catabolism of skeletal muscle is increased, the amount of 3-HIB and 3-HMB increase. Therefore, 3-HIB concentration, 3-HB concentration, and 3-HMB concentration in serum or saliva are the energy source balance of liver and skeletal muscle during or after exercise (dependency of energy metabolism tissue and nutrient supply) Can be an index (biomarker) for evaluating If the 3-HB concentration or 3-HIB concentration in serum or saliva has not changed much since resting during or after exercise, it can be evaluated that sugar is used as an energy supply source. During or after exercise, if 3-HB or 3-HIB concentration in serum or saliva is significantly higher than at rest, energy is produced by fatty acid catabolism in the liver or BCAA catabolism in skeletal muscle. Can be evaluated. Nutrient sources for increased energy production, including during exercise and starvation, are usually lipids, free amino acids, and amino acids derived from proteins, following sugar. Therefore, in the blood during exercise, an increase in 3-HIB concentration appears after an increase in 3-HB concentration. However, when the rise in blood 3-HIB concentration appears early, it indicates that energy production by lipids is reduced, which may be caused by insufficient lipid nutrition or decreased liver function. In addition, if the 3-HIB concentration continues to increase even after nutritional supplementation after exercise, there is a possibility that energy production is caused by catabolism of skeletal muscle protein. As a biomarker for evaluating the use of skeletal muscle BCAA as an energy source during or after exercise, 3-HIB concentration in serum or saliva is preferable to 3-HIB, and 3-HIB concentration in saliva is more preferable than 3-HMB . As a biomarker for evaluating the energy production state by different tissues and different nutrient sources in the liver and skeletal muscle during or after exercise, it is necessary to measure both 3-HIB concentration and 3-HB concentration.

Specifically, 3-HIB and / or 3-HB in saliva or serum from saliva or blood collected from a subject during exercise is detected by the detection method according to the present invention, and 3-HIB concentration and / or Alternatively, the energy source during exercise in the subject can be evaluated by measuring the 3-HB concentration and comparing the concentration with a predetermined threshold value set in advance. The threshold is preferably set for each subject, but is set based on the results of measuring 3-HIB concentration and the like after performing an exercise that is expected to have the same amount of exercise load for many subjects. Also good.

In addition, blood or saliva is sampled from a subject over time between immediately before the start of exercise and after a lapse of a certain period after the end of exercise, and the 3-HIB concentration and / or 3-HB concentration in each sample is recorded. By measuring with the detection method according to the invention, the 3-HIB concentration and / or the 3-HB concentration in blood or saliva can be monitored (change over time). Based on the 3-HIB concentration and / or 3-HB concentration change over time obtained as a result of monitoring, the energy source during exercise in the subject can be evaluated. In addition, when it is not at rest (normal state) immediately before the start of exercise, blood or saliva is collected as a sample from the subject at rest in advance, and the 3-HIB concentration and / or 3-HB concentration in each sample is determined. It is preferable to measure by the detection method according to the present invention and use the obtained measurement value as a reference for evaluation of the energy supply source during exercise.

In addition to excessive exercise, inadequate rest and nutrition results in a so-called overwork state where the relative exercise load becomes excessive. In an undernutrition state such as starvation, energy production by activation of ketone bodies obtained by fatty acid catabolism in the liver and BCAA catabolism obtained by proteolysis in skeletal muscle is activated. -HB and 3-HIB concentrations increase. In particular, endurance athletes and athletes and enthusiasts who are losing weight or restricting their diets are less dependent on the use of amino acids in their skeletal muscles as energy sources because they have less lipid storage as an energy source after sugar consumption. Get higher. Increased and sustained utilization of such skeletal muscle amino acids increases muscle protein degradation, resulting in a systemic energy metabolism deficiency state accompanied by symptoms such as skeletal muscle atrophy. Many of these cases are in a so-called overwork state. In such an overwork state, by measuring the concentration of 3-HB and 3-HIB in serum or saliva, the energy supply balance by catabolism derived from fatty acid catabolism of liver and BCAA catabolism derived from skeletal muscle protein, Since the degree of dependence can be evaluated, it can be an indicator of overwork status. For example, 3-HIB concentration and 3-HB concentration at rest (normal state) when not exercising, and 3-HIB concentration and 3-HB in saliva or serum after exercise that does not cause overwork The concentration is measured by the detection method according to the present invention, and from these measured values, the 3-HIB concentration and 3-HB concentration in the overwork state and the 3-HIB concentration and 3-HB in the non-overwork state are determined. A threshold for dividing the density (overwork threshold) is set in advance. By detecting the 3-HIB and 3-HB concentrations in saliva or serum from saliva or blood collected from a subject after excessive repetitive exercise that causes overwork, the detection method according to the present invention 3 -Assess the energy source balance in the subject by measuring the HIB and 3-HB concentrations and comparing the concentrations to a preset overwork threshold. Specifically, if the subject's 3-HIB concentration and 3-HB concentration are lower than the overwork threshold, the subject's exercise load is appropriate, and the subject is likely not overworked, When the 3-HIB concentration of the subject is equal to or higher than the overwork threshold, it can be evaluated that the subject's exercise load is excessive and the subject is likely to be overworked. Further, when only the 3-HB concentration of the subject is equal to or higher than the threshold value, it can be evaluated that there is a high possibility that the subject has not yet overworked even if the subject is in an energy-deficient state. The overwork threshold is preferably set for each subject, but it is set from the results of measuring 3-HIB concentration or 3-HB concentration after performing exercise to such an extent that many healthy subjects do not overwork. Also good. Collecting saliva or blood over time from an exercising subject and measuring and monitoring 3-HIB and 3-HB concentrations over time, thereby ending exercise before a dangerous overwork condition You can also. In this case, the sample collection from the subject does not necessarily have to be during exercise, and the results obtained from the sample taken at rest of the subject who is performing a long-term exercise program or training are the same as those before the exercise program or before training. By comparing with the result obtained from the sample collected from the subject, it can also be used for evaluation of the overwork state. That is, by monitoring the 3-HIB concentration and 3-HB concentration during exercise or long-term by the method according to the present invention, an appropriate amount of exercise can be evaluated and an overwork state can be prevented.

Further, for example, by measuring the concentration of 3-HIB or 3-HB in a biological sample (serum, tears, saliva, etc.) collected from one individual over time by the detection method according to the present invention, The state of muscle BCAA catabolism and fatty acid catabolism in the liver can also be monitored. 3-HIB concentration monitoring and 3-HB concentration monitoring are, for example, the treatment of BCAA preparations and the start of nutrition therapy for the prevention of skeletal muscle atrophy and the improvement of undernutrition for patients with cirrhosis and undernutrition or bedridden conditions. It can be used to determine whether or not there is a need and to determine their treatment. For example, if only 3-HIB concentration is increasing despite the fact that 3-HB concentration does not change, the ability to produce low energy in the liver due to the decrease in liver function is reduced by skeletal muscle protein catabolism. It can be evaluated that there is a high possibility of undernutrition that compensates for energy production derived from amino acids.

In addition, by measuring the concentration of 3-HIB or 3-HB by the detection method according to the present invention, the energy source-dependent balance (energy metabolism state) of lipids and amino acids in the liver and skeletal muscles is measured in the field of sports medicine. It is also possible to monitor using saliva or tears as well as blood. Furthermore, in the area of weight management and beauty by anti-aging and dieting, saliva or tears are analyzed by the detection method according to the present invention, and the concentration of 3-HIB or 3-HB is measured, so that sugar, amino acid, fatty acid It is also possible to effectively manage nutrition, exercise, and physical condition while evaluating the metabolic state. Furthermore, the detection method according to the present invention can also be used for evaluation of nutrition and intake effects when supplements and nutrient drinks containing nutrients such as BCAA are ingested.

In addition, the concentration of 3-HIB, 3-HMB, and 2-HB in the body fluid tends to be higher in the cirrhosis patient group than in the healthy group. Therefore, 3-HIB, 3-HMB, and 2-HB can be used as biomarkers for cirrhosis. Specifically, a threshold value for separating a cirrhosis patient from a healthy person is set in advance, and the concentrations of 3-HIB, 3-HMB, and 2-HB in the body fluid of the subject are measured by the detection method according to the present invention. The possibility of developing cirrhosis can be evaluated by comparing the concentration with a predetermined threshold value. Specifically, when the concentration of 3-HIB or the like in the body fluid of the subject, particularly serum or saliva, is equal to or higher than a predetermined threshold, the subject is highly likely to develop cirrhosis. Can be evaluated. The threshold value for separating a cirrhosis patient from a healthy person can be set experimentally. For example, the detection method according to the present invention is applied to a body fluid collected from a population known not to develop cirrhosis and a body fluid collected from a population known to develop cirrhosis. The concentration can be set as appropriate by measuring the concentration of 3-HIB and the like in these body fluids and comparing the measured values of both groups. For example, in the case of 3-HIB concentration in serum, the threshold value for separating the cirrhosis patient group from the healthy subject group can be 18 μM, preferably 20 μM, more preferably 24 μM. As a biomarker for cirrhosis, 3-HIB or 3-HMB is preferable, and since the difference between the cirrhosis patient group and the healthy group is clearer, 3-HIB in saliva or 3-HMB in saliva is more preferable.

When liver cirrhosis progresses and the urea cycle, which is one of the liver functions, decreases, ammonia is metabolized to glutamine and alanine in a compensatory manner in the skeletal muscle and detoxified. At that time, glutamic acid generated as a by-product in the process of BCAA being catabolized in skeletal muscle to be branched chain α-keto acid by BCAT is used for ammonia detoxification. Therefore, it is necessary to increase catabolism of skeletal muscle BCAA during ammonia detoxification that compensates for skeletal muscle at the time of cirrhosis, resulting in an increase in 3-HIB concentration in body fluids. It is thought that encephalopathy is likely to be induced if ammonia detoxification is insufficient and the concentration of ammonia in body fluids increases. Therefore, in addition to the decrease in the ammonia detoxification ability in the liver of cirrhotic patients, a decrease in the ability to compensate for ammonia detoxification through skeletal muscle BCAA catabolism is considered to cause hepatic encephalopathy. For these reasons, 3-HIB can be used as a biomarker for hepatic encephalopathy or its onset, and can provide useful information for its prevention and early treatment. For example, for cirrhosis patients who are evaluated to have a high risk of developing hepatic encephalopathy from 3-HIB levels, a protein-restricted diet is recommended, or BCAA intake is recommended even if encephalopathy has not been manifested. It may be possible to prevent the development of hepatic encephalopathy by making recommendations.

For example, when monitoring the 3-HIB concentration in body fluid (serum, tears, saliva, etc.) collected from a cirrhotic patient over time, and the 3-HIB concentration tends to decrease, The possibility of developing encephalopathy is high. Therefore, a biological sample collected from a subject who has developed cirrhosis, in particular, 3-HIB concentration in serum or saliva is measured by the detection method according to the present invention, and the obtained 3-HIB concentration is measured with the biological sample. By comparing with the 3-HIB concentration in the biological sample collected from the subject before the time when was collected, the possibility of developing hepatic encephalopathy can be evaluated. Specifically, when the 3-HIB concentration obtained is significantly lower than the 3-HIB concentration of a previously collected biological sample, or as a result of monitoring, the 3-HIB concentration of a previously collected biological sample When the tendency to become lower than this continues for a certain degree, it is evaluated that the subject is highly likely to develop hepatic encephalopathy.

Whether the 3-HIB concentration in cirrhosis patients is lowered can also be evaluated using a threshold value that separates cirrhosis patients from healthy individuals. Specifically, the 3-HIB concentration in a biological sample collected from a subject who has developed cirrhosis, particularly serum or saliva, is measured by the detection method according to the present invention, and the obtained 3-HIB concentration is The possibility of developing hepatic encephalopathy can be evaluated by comparing with a predetermined threshold value (for example, the threshold value that separates the cirrhosis patient from the healthy person). Specifically, the subject develops hepatic encephalopathy when the 3-HIB concentration of the subject is below a predetermined threshold or when the state of being below the predetermined threshold continues to some extent. It can be evaluated that there is a high possibility of doing.

By using the detection method according to the present invention, in addition to a biological sample such as blood having a relatively high content of 3-HIB and the like, a living body having a very small content of 3-HIB such as saliva or tears It is also possible to monitor using a sample. In particular, by using non-invasively collected tears, saliva, etc. as samples, sample collection such as blood collection and special collection devices (needle, syringe, blood collection tube, noncoagulant) Samples can be collected regardless of location, time, and sampler. In this case, there is no need to go to hospital or hospitalization, and it is possible to collect samples by the patient at home. In addition, measurement with a saliva sample is also effective for children who do not like blood collection, elderly people, and patients who cannot collect blood for religious reasons.

3-HMB can be used as a marker for protein synthesis and skeletal muscle hypertrophy in BCAA-derived liver and skeletal muscle, and 2-HB is a marker for early insulin resistance and glucose intolerance. is there. By using the detection method according to the present invention, these biomarkers can be detected with high sensitivity using blood, saliva, or the like.

As shown in Example 10 below, the concentrations of 3-HIB and 3-HB in biological samples such as saliva and blood are unlikely to decrease even when left at room temperature for a long time. Therefore, even when it is difficult to immediately refrigerate / freeze a biological sample collected from a subject, 3-HIB and 3-HB can function as stable and excellent biomarkers. For this reason, even when the location for collecting the biological sample from the subject and the test location for detecting an organic acid such as 3-HIB in the biological sample are separated, such as in a health checkup, for example. By using the detection method according to the present invention, the concentrations of 3-HIB and 3-HB can be accurately measured.

Next, the present invention will be described in more detail with reference to examples and the like, but the present invention is not limited thereto.

<Reagents>
The reagents used in the following examples and the like are as follows. D-3-HB, L-3-HB, and 3-HIB sodium salts were manufactured by Sigma-Aldrich Chemical. DL-3-HB- ¹³ C ₄ sodium salt was manufactured by Taiyo Nippon Sanso Co., Ltd. 2-pyridinemethanol, 2-methyl-6-nitrobenzoic anhydride, 1-piperidineethanol, 2- (2-hydroxyethyl) -1-methylpyrrolidine, 3-pyridinemethanol, 2-diethylaminoethanol, 3-hydroxy- 1-methylpiperidine and 4-pyridinemethanol were manufactured by Tokyo Chemical Industry Co., Ltd., and 4-dimethylaminopyridine was manufactured by Wako Pure Chemical Industries. A 3-HB dehydrogenase manufactured by Roche Diagnostics was used.

<Preparation of serum sample>
Serum was collected from blood collected from healthy individuals and stored at −20 ° C. until the time of use for measurement. Serum (10 [mu] L), placed in a 1.5mL centrifuge plastic tube, further acetonitrile / water (1/19 containing ^DL-3-HB- 13 _{C 4} sodium salt of 769pmol (100ng) as an internal standard, volume ratio ) After the solution (100 μL) was added and stirred for 1 minute, it was centrifuged at 2000 × g for 1 minute. The supernatant was put in a water / acetonitrile (1/19, volume ratio) solution and subjected to deproteinization treatment, and then the liquid phase was recovered and evaporated to dryness at 80 ° C. under nitrogen gas blowing.

<Preparation of 3-HB-free serum>
3-HB-free serum was prepared by adding normal serum (10 μL) to 150 mM Tris-HCl (pH 8.6) supplemented with 1.8 mM NAD and 0.075 U 3-hydroxybutyrate dehydrogenase at 37 ° C. For 30 minutes.

<Esterification reaction of organic acid using 2-pyridinemethanol as derivatization reagent>
The esterification reaction of an organic acid using 2-pyridinemethanol as a derivatization reagent was performed by improving the method for synthesizing carboxyesters by Siina et al. (See Non-Patent Document 13). Specifically, a mixed reagent for 2PM derivatization comprising 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and 2-pyridinemethanol (100 μL) Was used. First, the reaction solution prepared by adding the 2PM derivatization mixed reagent (50 μL) prepared immediately before to a dried sample containing an organic acid was allowed to stand at room temperature for 30 minutes. Subsequently, n-hexane (1 mL) was added to the reaction solution and stirred for 30 seconds, followed by centrifugation at 700 × g for 1 minute, and the collected supernatant was evaporated at 80 ° C. under nitrogen gas blowing. The residue was redissolved in a 1% by volume formic acid aqueous solution (150 μL), centrifuged again at 7000 × g for 1 minute, and the collected supernatant was subjected to ESI-LC-MS / MS as a measurement sample.

<LC-P-ESI-MS / MS>
The LC-MS / MS system is a triple quadrupole mass spectrometer TSQ Vantage (manufactured by Thermo Fisher Scientific) equipped with a HESI-II probe and Prominence Ultra Fast Liquid Chromatography (UFLC) system (manufactured by Shimadzu Corporation). . Separation in chromatography was performed at 40 ° C. using a Hypersil GOLD aQ column (2.1 × 150 mm, 3 μm, manufactured by Thermo Fisher Scientific). First, the mobile phase was allowed to flow for 5 minutes at a flow rate of 300 μL / min as acetonitrile / water (1/19, volume ratio) containing 0.2% by volume of formic acid. After 5 minutes, the mobile phase was changed to acetonitrile containing 0.2% by volume of formic acid and allowed to flow for 7 minutes at a flow rate of 300 μL / min. General MS / MS conditions are as follows:
Spray voltage: 3000V
Nebulizer temperature: 450 ° C,
Sheath gas (nitrogen) pressure: 50 psi,
Auxiliary gas (nitrogen) flow rate: 15 arbitrary units,
Ion transfer capillary temperature: 220 ° C.
Impact gas (argon) pressure: 1.0 mTorr,
Collision energy: 15V
Ion polarity: positive mode.

FIG. 1B shows a typical P-ESI MS spectrum of 3-HB (2 PM-3-HB) derivatized with 2-pyridinemethanol. In 2 PM-3-HB, [M + H] ⁺ ions are found at m / z 196 as the base peak. In the product ion MS spectrum (FIG. 1A) when m / z 196 is used as a precursor ion, a fragment ion of [C ₅ H ₄ NCH ₂ OH + H] ⁺ is seen at m / z 110 as the most prominent peak. SRM is performed using m / z 196 → m / z 110 for detection of 2 PM-3-HB, and m / z 200 → m / z 110 for detection of [ ¹³ C ₄ ] isotope (internal standard). It was performed using.

<Create calibration curve>
For the preparation of the calibration curve, a sample stock solution (200 ng / μL) in which D-3-HB sodium salt was dissolved in acetonitrile / water (1/19, volume ratio) was further added to acetonitrile / water (1/19). D-3-HB sodium standard solution (1 to 200 ng / 100 μL) having a known concentration prepared by appropriately diluting using a volume ratio). Each standard solution as an internal standard, were added ^DL-3-HB- 13 _{C 4} sodium salt (100 ng), after the mixture was evaporated to dryness and the obtained by the derivatization (esterification with derivatives) , Measured by ESI-LC-MS / MS. To examine the matrix effect of the calibration curve, 3-HB-free serum was added as a blank matrix to each standard solution.

<LC-N-ESI-MS / MS>
LC-N-ESI-MS / MS of 3-HB was performed using the same LC-MS / MS system used in LC-P-ESI-MS / MS without derivatization. Separation in chromatography was performed at 40 ° C. using a Hypersil GOLD aQ column (2.1 × 150 mm, 3 μm, manufactured by Thermo Fisher Scientific). As the mobile phase, methanol / water (1/9, volume ratio) containing 0.1% by volume of formic acid was allowed to flow at a flow rate of 200 μL / min. General MS / MS conditions are as follows:
Spray voltage: 2500V
Nebulizer temperature: 450 ° C,
Sheath gas (nitrogen) pressure: 50 psi,
Auxiliary gas (nitrogen) flow rate: 15 arbitrary units,
Ion transfer capillary temperature: 220 ° C.
Impact gas (argon) pressure: 1.0 mTorr,
Collision energy: 15V
Ion polarity: negative mode,
3-HB SRM (selective reaction monitoring): m / z 103 → m / z 59,
[ ¹³ C ₄ ] SRM: m / z 107 → m / z 61.

<Statistical processing>
The numerical values in the following examples and the like are mean ± SD (standard deviation). The linearity of the calibration curve was analyzed by single regression analysis. Reproducibility was analyzed by one-way analysis of variance (one-way ANOVA) (JMP software, manufactured by SAS Institute). An estimated value ± 95% confidence limit was obtained as an accuracy index (see Non-Patent Document 14). In order to calculate the values, orthogonal regression analysis was performed in a reproducibility test using JMP software. Statistical significance of differences between different groups was assessed by Student's two-tailed t-test. In all analyses, P <0.05 was considered significant.

[Example 1]
3-HB and 3-HIB derivatized with 2-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, a sample solution in which only D-3-HB sodium salt (standard, 1 μg) was dissolved in acetonitrile / water (1/19, volume ratio) (100 μL), D-3-HIB sodium salt (standard) Sample solution in which only 1 μg) is dissolved, and a sample solution in which D-3-HB sodium salt (standard, 1 μg) and D-3-HIB sodium salt (standard, 1 μg) are dissolved, Each was evaporated to dryness and then derivatized (esterified with a derivative) with the mixed reagent for 2PM derivatization. Finally, it was redissolved in a 1% by volume aqueous formic acid solution (1 mL), and 1 μL (corresponding to 1 ng each of D-3-HB sodium salt and D-3-HIB sodium salt) was added to LC-P-ESI- Measured by MS / MS.

FIG. 2 shows the results of detection of 3-HB and 3-HIB derivatized with 2-pyridinemethanol by LC-P-ESI-MS / MS. 2A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only derivatized 3-HB, FIG. 2B shows the MS spectrum of FIG. 2A, and FIG. 2C shows the derivatized 3 -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 2D contains the MS spectrum of Fig. 2C, and Fig. 2E contains derivatized 3-HB and 3-HIB. A product ion chromatogram when m / z 196 of the sample is used as a precursor ion is shown in FIG. 2F, and an MS / MS spectrum of the 3-HIB peak in FIG. 2E is shown. *

As a result, even though the sample used for measurement contained only 1 ng, both 3-HB and 3-HIB derivatives could be spectrally analyzed in the scan mode with low sensitivity and high information content. It was. In the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, since the LC retention time is slow, the effect of the derivatization reagent (eg, 4-dimethylaminopyridine, etc.) that emerges first from the column on the peaks of 3-HB derivatives and 3-HIB derivatives is very high. It was small. That is, from these results, it was found that organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity by derivatization with 2-pyridinemethanol.

[Example 2]
3-HB and 3-HIB derivatized with 1-piperidineethanol were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for 1PE derivatization composed of 1-piperidineethanol (100 μL) was used, and measurement was performed by LC-P-ESI-MS / MS.

FIG. 3 shows the results of detection of 3-HB and 3-HIB derivatized with 1-piperidineethanol by LC-P-ESI-MS / MS. 3A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only derivatized 3-HB, FIG. 3B shows the MS spectrum of FIG. 3A, and FIG. 3C shows the derivatized 3 -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 3D contains the MS spectrum of Fig. 3C, and Fig. 3E contains derivatized 3-HB and 3-HIB. FIG. 3F shows a product ion chromatogram when m / z 216 of the sample is used as a precursor ion, and FIG. 3F shows an MS / MS spectrum of the 3-HIB peak in FIG. 3E. *

As a result, even though the sample used for measurement contained only 1 ng, both 3-HB and 3-HIB derivatives could be spectrally analyzed in the scan mode with low sensitivity and high information content. It was. In the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, each peak of the 3-HB derivative and the 3-HIB derivative had a good S / N in the product ion chromatogram and a particularly good sensitivity. That is, from these results, it was found that organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity by derivatization with 1-pyridineethanol.

[Example 3]
3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for 1PE derivatization consisting of 2- (2-hydroxyethyl) -1-methylpyrrolidine (100 μL) was used, and LC-P-ESI-MS / MS It was measured.

FIG. 4 shows the results of detecting 3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine by LC-P-ESI-MS / MS. 4A shows the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only derivatized 3-HB, FIG. 4B shows the MS spectrum of FIG. 4A, and FIG. 4C shows the derivatized 3 -The total ion chromatogram (top) and MS chromatogram (bottom) of the sample containing only HIB, Fig. 4D contains the MS spectrum of Fig. 4C, and Fig. 4E contains derivatized 3-HB and 3-HIB. FIG. 4F shows a product ion chromatogram when m / z 216 of the sample is used as a precursor ion, and FIG. 4F shows an MS / MS spectrum of the 3-HIB peak in FIG. 4E. *

As a result, even though the sample used for measurement contained only 1 ng, both 3-HB and 3-HIB derivatives could be spectrally analyzed in the scan mode with low sensitivity and high information content. It was. In the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, each peak of the 3-HB derivative and the 3-HIB derivative had a good S / N in the product ion chromatogram and a particularly good sensitivity. In other words, these results indicate that organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity by derivatization with 2- (2-hydroxyethyl) -1-methylpyrrolidine. .

[Comparative Example 1]
3-HB and 3-HIB derivatized with 3-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for derivatization of 3PM consisting of 3-pyridinemethanol (100 μL) was used, and measurement was performed by LC-P-ESI-MS / MS.

FIG. 5 shows the results of detection of 3-HB and 3-HIB derivatized with 3-pyridinemethanol by LC-P-ESI-MS / MS. 5A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only derivatized 3-HB, FIG. 5B shows the MS spectrum of FIG. 5A, and FIG. 5C shows the derivatized 3 -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 5D contains the MS spectrum of Fig. 5C, and Fig. 5E contains derivatized 3-HB and 3-HIB. A product ion chromatogram when m / z 196 of the sample is used as a precursor ion is shown in FIG. 5F, and an MS / MS spectrum of the 3-HIB peak in FIG. 5E is shown. *

As a result, both 3-HB and 3-HIB derivatives could be detected, but the separation of 3-HB derivative and 3-HIB derivative was poor in the LC chromatogram. The LC retention time overlapped with 4-dimethylaminopyridine in the mixed reagent for 3PM derivatization. In particular, the 3-pyridine methanol derivative of 3-HB was extremely insensitive due to the matrix effect of 4-dimethylaminopyridine.

[Comparative Example 2]
3-HB and 3-HIB derivatized with 2-diethylaminoethanol were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for 2DEAE derivatization consisting of 2-diethylaminoethanol (100 μL) was used, and measurement was performed by LC-P-ESI-MS / MS.

FIG. 6 shows the results of detecting 3-HB and 3-HIB derivatized with 2-diethylaminoethanol by LC-P-ESI-MS / MS. 6A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only derivatized 3-HB, FIG. 6B shows the MS spectrum of FIG. 6A, and FIG. 6C shows the derivatized 3 -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 6D shows the MS spectrum of Fig. 6C, and Fig. 6E contains derivatized 3-HB and 3-HIB. A product ion chromatogram when m / z 204 of the sample is used as a precursor ion is shown in FIG. 6F, and an MS / MS spectrum of the 3-HIB peak in FIG. 6E is shown. *

As a result, both 3-HB and 3-HIB derivatives could be detected, but the separation of 3-HB derivative and 3-HIB derivative was poor in the LC chromatogram. The LC retention time overlapped with 4-dimethylaminopyridine in the mixed reagent for 2DEAE derivatization. Particularly, 3-HIB 2-diethylaminoethanol derivative was extremely insensitive due to the matrix effect of 4-dimethylaminopyridine. Further, a huge broad peak that appeared to be derived from the reagent of 2-diethylaminoethanol appeared around 6.5 minutes.

[Comparative Example 3]
3-HB and 3-HIB derivatized with 3-hydroxy-1-methylpiperidine were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for 3H1MP derivatization composed of 3-hydroxy-1-methylpiperidine (100 μL) was used, and measurement was performed by LC-P-ESI-MS / MS.

FIG. 7 shows the results of detection of 3-HB and 3-HIB derivatized with 3-hydroxy-1-methylpiperidine by LC-P-ESI-MS / MS. FIG. 7A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only derivatized 3-HB, FIG. 7B shows the MS spectrum of FIG. 7A, and FIG. -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 7D includes the MS spectrum of Fig. 7C, and Fig. 7E includes derivatized 3-HB and 3-HIB. A product ion chromatogram when m / z 202 of the sample is used as a precursor ion is shown in FIG. 7F, and an MS / MS spectrum of the 3-HIB peak in FIG. 7E is shown. *

As a result, three peaks were detected for each of the 3-HB and 3-HIB derivatives. Further, in the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was poor. The LC retention time overlapped with 4-dimethylaminopyridine in the mixed reagent for 3H1MP derivatization. Further, a huge broad peak that appeared to be derived from the 3-hydroxy-1-methylpiperidine reagent appeared around 7 minutes.

[Comparative Example 4]
3-HB and 3-HIB derivatized with 4-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, as a derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was performed in the same manner as in Example 1 except that a mixed reagent for derivatization of 3H1MP consisting of 4-pyridinemethanol (100 μL) was used, and measurement was performed by LC-P-ESI-MS / MS.

FIG. 8 shows the results of detection of 3-HB and 3-HIB derivatized with 4-pyridinemethanol by LC-P-ESI-MS / MS. 8A shows the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only derivatized 3-HB, FIG. 8B shows the MS spectrum of FIG. 8A, and FIG. 8C shows the derivatized 3 -Total ion chromatogram (top) and MS chromatogram (bottom) of a sample containing only HIB, Fig. 8D contains the MS spectrum of Fig. 8C, and Fig. 8E contains derivatized 3-HB and 3-HIB. A product ion chromatogram when m / z 196 of the sample is used as a precursor ion is shown in FIG. 8F, and an MS / MS spectrum of the 3-HIB peak in FIG. 8E is shown.

As a result, both 3-HB and 3-HIB derivatives could be detected, but the separation of 3-HB derivative and 3-HIB derivative was poor in the LC chromatogram. The LC retention time overlapped with 4-dimethylaminopyridine in the mixed reagent for 3H1MP derivatization.

[Example 4]
A sample obtained by adding DL-3-HB- ¹³ C ₄ sodium salt as an internal standard to 10 μL of serum collected from a healthy person was derivatized with 2-pyridinemethanol in the same manner as in Example 1, and LC-P-ESI -Measured by MS / MS. FIG. 9 shows a typical SRM chromatogram of 3-HB (2 PM-3-HB) and [ ¹³ C ₄ ] isotopes derivatized with 2-pyridinemethanol. From the chromatogram, the peak area ratio of 2 PM-3-HB to the [ ¹³ C ₄ ] isotope was calculated, and the ratio was applied to a calibration curve to determine the 3-HB concentration in serum. The peak of 2 PM-3-HB in the chromatogram corresponded to 5.1 pmol (76 μM). The peak seen at 4.53 minutes in chromatogram A was identified as a 2PM ester derivative of 3-HIB by comparison with 2 PM-3HIB preparation.

[Example 5]
In the same manner as in Example 4, serum 3-HB concentration collected from healthy male subjects every 2-3 hours was measured. The measurement results are shown in FIG. In the figure, the black arrow indicates the time spent eating, and the shaded portion indicates sleep time. As a result, it was found that 3-HB in 10 μL of serum can be detected by the detection method according to the present invention. It was also found that 3-HB in serum tended to decrease with food intake, and that 3-HB concentration in serum was controlled by food intake.

[Example 6]
The accuracy and accuracy of the detection method according to the present invention were examined using serum collected from healthy individuals as a sample. Specifically, a sample obtained by adding DL-3-HB- ¹³ C ₄ sodium salt as an internal standard to 10 μL of serum collected from a healthy person was derivatized with 2-pyridinemethanol in the same manner as in Example 1, Measured by LC-P-ESI-MS / MS.
The reproducibility was measured by LC-P-ESI-MS / MS for each of four types of samples (samples A to D). The measurement results are shown in Table 1. The measurement results were analyzed by one-way ANOVA. At that time, the analysis error was divided into two causes: sample preparation and SRM measurement. As shown in Table 2, the error during sample preparation was not significantly greater than the error between measurements, so the variance was considered not due to sample preparation. The inter-test variation counts of dispersion between or within samples were 2.2% or 1.2%, respectively. In Table 2, “S” is the sum of deviation squares, “f” is the degree of freedom, “V” is unbiased variance, and “F ₀ ” is the dispersion ratio ([V _{sample preparation} ] / [V _error ]). “F (3, 8, α)” means the F value when the degree of freedom is 3 and 8, and the significance level is α. As a result, since a trace amount of 3-HB of 200 pmol could be detected, it was found that the detection method was excellent in sensitivity. Further, in any of the four types of samples, the variation between measurements was very small, and a very small amount of 3-HB of 200 pmol could be detected, indicating that the detection method was excellent in accuracy.

In the addition recovery test, a known amount of 3-HB (158.7, or 317.5, or 476.2 pmol) was placed in a 10 μL serum sample (n = 2). After deproteinization treatment and derivatization reaction, each sample was measured three times by SRM. As shown in Table 3, the recovery rate of the known added amount of 3-HB was in the range of 93.7 to 98.2%, and the average was 96.7%. In addition, the amount of endogenous 3-HB when 10 μL of serum was not added was within the 95% confidence limit of the estimated amount of 3-HB calculated by orthogonal regression analysis. In Table 3, numerical values with “a” are values obtained from Table 1, “recovery rate (%) ^b ” is a value represented by the following formula, and “estimated amount ± 95% The “confidence limit ^c ” is a value calculated by orthogonal regression analysis.

[Example 7]
The sensitivity of 2 PM-3-HB measured by LC-P-ESI-MS / MS was compared with the sensitivity measured by LC-N-ESI-MS / MS.
Specifically, a dilution series with a known concentration of 2 PM-3-HB solution was measured by LC-P-ESI-MS / MS (SRM), and the minimum concentration at which 2 PM-3-HB was detected (minimum detection sensitivity) ). Similarly, a dilution series with a known concentration of the 3-HB solution was measured by LC-N-ESI-MS / MS (SRM) to examine the minimum detection sensitivity. Each measurement sample was added with 3-HB-free serum as a blank matrix.
As a result, LC-P-ESI-MS / MS was able to detect 2 PM-3-HB with an on-column injection amount of 6.0 fmol (S / N = 3). In contrast, LC-N-ESI-MS / MS was able to detect only up to 3-HB with an on-column injection amount of 481 fmol (S / N = 3).

[Example 8]
3-HMB and 2-HB derivatized with 2-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, D-3-HMB sodium salt (standard, 1 μg) and D-2-HB sodium salt (standard, 1 μg) were dissolved in acetonitrile / water (1/19, volume ratio) (100 μL). The prepared sample solution was prepared, evaporated and dried, and then derivatized (esterified with a derivative) using the mixed reagent for 2PM derivatization. Finally, it was redissolved in 1% by volume aqueous formic acid solution (20 μL), and 1 μL (corresponding to 50 ng each of D-3-HMB sodium salt and D-2-HB sodium salt) was added as described above to LC-P-ESI- Measured by MS / MS.

FIG. 11 shows the results of detection of 3-HMB and 2-HB derivatized with 2-pyridinemethanol by LC-P-ESI-MS / MS. FIG. 11A shows a total ion chromatogram of the sample containing derivatized 3-HMB and 2-HB (upper stage), a product ion chromatogram when using m / z 196 as a precursor ion (middle stage), and m / z. Product ion chromatograms (lower row) when 210 is used as a precursor ion are respectively shown. 11B shows the MS spectrum of the middle product ion chromatogram of FIG. 11A, and FIG. 11C shows the MS spectrum of the lower product ion chromatogram of FIG. 11A.

As a result, even though the sample used for measurement contained only 50 ng, both 3-HMB and 2-HB derivatives could be simultaneously analyzed in the low-sensitivity high-information scan mode. did it.
In addition, when a sample containing 100 pg or 10 pg each of 2-HM ester derivatives of 3-HMB and 2-HB was measured in the same manner by LC-P-ESI-MS / MS, either sample was measured. , 3-HMB and 2-HB derivatives could all be analyzed simultaneously (not shown).
From these results, by derivatizing with 2-pyridinemethanol, it is possible to simultaneously detect highly structurally similar 3-HMB and 2-HB with high sensitivity even in the case where only a trace amount of 10 pg is contained. I understood.

[Example 9]
3-HB, 3-HIB, 3-HMB, and 2-HB derivatized with 2-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, acetonitrile / water (1/19, volume ratio) (100 μL), D-3-HB sodium salt (standard, 30 ng), D-3-HIB sodium salt (standard, 30 ng) and D Sample solution in which -3-HMB sodium salt (standard, 30 ng), D-2-HB sodium salt (standard, 30 ng) and DL-3-HB- ¹³ C ₄ sodium salt (standard, 80 ng) were dissolved After evaporating and drying, derivatization (esterification with a derivative) was performed using the mixed reagent for 2PM derivatization. Finally, redissolved in 1% by volume aqueous formic acid solution (1 mL), and 1 μL (D-3-HB sodium salt, D-3-HIB sodium salt, D-3-HMB sodium salt, D-2-HB sodium salt) Each corresponding to 3 ng and DL-3-HB- ¹³ C ₄ sodium salt corresponding to 8 ng) was measured by LC-P-ESI-MS / MS as described above.

FIG. 12 shows the results of detection of 3-HB, 3-HIB, 3-HMB, and 2-HB derivatized with 2-pyridinemethanol by LC-P-ESI-MS / MS. 12 shows the total ion chromatogram of the sample, the product ion chromatogram when m / z 196 is used as the precursor ion in the second stage from the top, and m / z 200 as the precursor ion in the third stage from the top. The product ion chromatogram when m / z 210 is used as the precursor ion is shown at the bottom, respectively.

As a result, even though the sample used for the measurement contained only 3 ng, in the scan mode with low sensitivity and high information content, 2 PM of 3-HB, 3-HIB, 3-HMB, and 2-HB was obtained. All the ester derivatives could be spectrally analyzed. In the LC chromatogram, the separation of the 3-HB derivative, the 3-HIB derivative, the 3-HMB derivative, and the 2-HB derivative was good. In other words, these results indicate that structurally similar 3-HB, 3-HIB, 3-HMB, and 2-HB can be detected simultaneously with high accuracy and high sensitivity by derivatization with 2-pyridinemethanol. .

[Example 10]
The stability of 3-HIB and 3-HB in serum and saliva when serum and saliva collected from a subject were allowed to stand at room temperature for a certain time was examined.

<Serum>
Blood was collected from 6 healthy blood collection tubes. One of the six blood collection tubes was centrifuged immediately after blood collection, and the collected serum was stored frozen at -20 ° C. The remaining 5 bottles were allowed to stand at room temperature for 1, 2, 4, 6, or 24 hours, respectively, centrifuged, and the collected serum was stored at -20 ° C.
The concentration of 3-HIB and 3-HB in each serum after storage at -20 ° C., using a ^DL-3-HB- 13 _{C 4} sodium salt as an internal standard, using 2-pyridinemethanol as a derivatizing reagent In the same manner as in Example 4, the measurement was performed by LC-P-ESI-MS / MS. From the chromatogram, calculate the peak area ratio of 2 PM-3-HB to the [ ¹³ C ₄ ] isotope and apply the ratio to the calibration curve to determine the 3-HB and 3-HIB concentrations in the serum. did. The measurement results are shown in FIG. As a result, the 3-HB concentration and 3-HIB concentration in the serum decreased until the time allowed to stand at room temperature after blood collection reached 6 hours, but did not change until 24 hours thereafter.

<Saliva>
Saliva collected (fluid) from one healthy person into one tube was dispensed into six tubes. One of the 6 tubes was stored frozen at −20 ° C. immediately after collection. The remaining five were each allowed to stand at room temperature for 1, 2, 4, 6, or 24 hours and then stored at −20 ° C. The frozen saliva sample was naturally thawed at room temperature during analysis. After mixing using a vortex mixer, centrifugation (3000 rpm, 15 minutes) was performed to decompose the viscous protein (mucin), which was precipitated together with meals contained in saliva, and the supernatant was used for analysis.
The concentration of 3-HIB and 3-HB in the saliva after storage at -20 ° C., using a ^DL-3-HB- 13 _{C 4} sodium salt as an internal standard, using 2-pyridinemethanol as a derivatizing reagent In the same manner as in Example 4, the measurement was performed by LC-P-ESI-MS / MS. From the chromatogram, calculate the peak area ratio of 2 PM-3-HB to the [ ¹³ C ₄ ] isotope and apply the ratio to the calibration curve to determine the 3-HB concentration and 3-HIB concentration in saliva. did. The measurement results are shown in FIG. As a result, the 3-HB concentration and 3-HIB concentration in saliva were almost stable regardless of the time of standing at room temperature after collection.

[Example 11]
The correlation between the concentrations of 3-HIB, 3-HB, and 2-HB in serum and saliva (fluid) collected from the subjects was examined. Specifically, serum and saliva were collected from 3 healthy individuals (subjects α, β, and γ), 5 samples each, and the concentrations of 3-HIB, 3-HB, and 2-HB were determined. Measured and examined the correlation between serum concentration and saliva concentration. The concentrations of 3-HIB, 3-HB, and 2-HB in serum and saliva were measured in the same manner as in Example 10. The measurement result of 3-HB concentration is shown in FIG. 15A, the measurement result of 3-HIB concentration is shown in FIG. 15B, and the measurement result of 2-HB concentration is shown in FIG. 15C. As a result, it was confirmed that the saliva concentration correlated with the serum concentration in any of 3-HIB, 3-HB, and 2-HB.

[Example 12]
The 3-HIB and 3-HB concentrations in serum, saliva, and urine before and after running exercise were measured, and changes over time were observed.
Specifically, a subject (a healthy male person) collected urine for 24 hours from 11:00 am on the previous day to 11:00 am on the day of running exercise, and then jogged for 60 minutes (traveling distance: about 9 km, speed: about 6.7) Min / km), and the time until the lapse of 24 hours (12:00 noon on the day following the running day) was set as the experiment period. The subjects took lunch and dinner the previous day, lunch and dinner on the day of running exercise, and breakfast the next day from 11:00 am the previous day until the end of the experiment period. FIG. 16 shows the exercise and meal status of the subject, and blood, saliva, and urine collection time points. The 60-minute jogging performed was an exercise with a slightly higher load than the exercise performed on a daily basis for the subject.

Serum, saliva, and urine 3-HIB and 3-HB concentrations were measured in the same manner as in Example 10. The measurement results of 3-HIB and 3-HB in each sample collected are shown in FIGS. FIG. 17 shows changes over time in the concentrations of 3-HIB and 3-HB in serum. FIG. 18 shows the change over time of urinary excretion per unit time ([urinary excretion] / [elapsed time from previous collection]) (μmol / h) at each collection time of 3-HIB and 3-HB. Each is shown. FIG. 19 shows changes over time in the concentrations of 3-HIB and 3-HB in saliva. The lower diagram in FIG. 19 is a diagram showing the measurement results during the upper running exercise with the vertical axis scale enlarged.
As a result, it was confirmed that the detection method according to the present invention can detect both 3-HIB and 3-HB in serum, saliva, and urine collected from a subject.

The concentration of 3-HB increased in both serum and saliva due to running exercise, but the serum 3-HB concentration also increased after running exercise, and the concentration of 3-HB in saliva was Although it decreased immediately after the running exercise, it increased again after that (FIG. 17 and FIG. 19 upper stage). From these results, it was confirmed that the 3-HB concentration in saliva increased by exercise as well as the 3-HB concentration in serum increased by exercise. After exercise, the 3-HB concentration in both serum and saliva continues to rise until the meal is consumed, so liver fatty acid catabolism (burning of fat) after exercise is applied to both blood and saliva samples. It was also confirmed that it can be monitored.

On the other hand, the concentration of 3-HIB increases in both serum and saliva due to running exercise, and after a certain amount of time has elapsed since the end of exercise, the concentration decreases rapidly and returns to the normal state before exercise. Was confirmed (FIG. 17 and the upper stage of FIG. 19). Skeletal muscle BCAA catabolism does not persist after exercise compared to during exercise, and normal individuals may not undergo catabolism of BCAA or free BCAA derived from skeletal muscle protein degradation only by performing transient exercise. confirmed. In particular, it was found that the 3-HIB concentration in saliva hardly fluctuated from 30 minutes after the end of exercise to the end of the experiment period, and was hardly affected by meals and the like. On the other hand, the serum 3-HIB concentration decreased to a normal state at 1 hour after the end of exercise, but slightly increased at 4 hours after the end of exercise. Since 2.25 hours after the end of exercise, food was consumed, the increase in serum 3-HIB concentration at the end of 4 hours after the end of exercise may have been slightly affected by the diet. It was.

From these results, the 3-HB and 3-HIB concentrations in serum and saliva are increased by exercise, and these are simultaneously evaluated as liver fatty acid catabolism (fat burning) markers and skeletal muscle BCAA catabolism markers by exercise. Be useful as a marker for assessing the energy metabolism state (and hence the balance of energy supply sources) of tissues (liver and skeletal muscle) during or after exercise, and being less susceptible to meals, etc. From the results, it was found that for 3-HIB, the concentration in saliva is more preferable as a marker than the concentration in serum.

As shown in the lower part of FIG. 19, the concentration of 3-HIB in saliva during running exercise does not change much from the start of exercise (normal state) until 15 minutes after the start of exercise, but then tends to increase gradually. Was observed. The salivary 3-HB concentration during running exercise does not change much from the exercise start point (normal state) until 35 minutes after the start of exercise, but then increases more rapidly than the 3-HIB concentration A trend was observed. Sugar is preferentially used as an energy source during exercise, but fat and amino acids are used as energy sources as exercise time and intensity increase. From these results, increases in 3-HB concentration and 3-HIB concentration indicate that fats and amino acids were used as energy sources instead of sugar, and when both increased, the dependence on energy production Is considered to indicate the point of transition from sugar metabolism to lipid and amino acid metabolism. In recent years, it has been suggested that in addition to sugar and lipids, amino acids are also used as an energy source from the early days of exercise, but 3-HIB starts to rise earlier than 3-HB. It is thought that metabolism is also activated from the early stage of exercise. In this example, the concentration of 3-HIB in saliva decreased to a steady state immediately after the end of running, but when the exercise load becomes excessive, the so-called overwork state is a steady state even after the end of exercise. It is estimated that it will be difficult to return to

FIG. 18 shows urinary 3-HB excretion and 3-HIB excretion per unit time before and after exercise. Unlike serum and saliva, urinary 3-HB excretion and 3-HIB excretion were not increased by exercise, and urinary 3-HIB excretion tended to decrease (1 hour after exercise) ). The urinary 3-HB excretion thereafter tended to increase until 3 hours of exercise and then once decreased, but then tended to increase again until 12.5 hours of exercise. Urinary 3-HIB excretion increased up to 9.5 hours of exercise while repeating slight increases and decreases. Neither urinary 3-HB or 3-HIB excretion was associated with changes in serum or saliva concentrations, but it was presumed that reabsorption in the kidney tissue had an effect. When using urine as a sample, considering the necessity of correction with urine or animal urine as needed, or creatinine As a result, it was found that evaluation with serum or saliva is preferable to urinary excretion.

[Example 13]
Blood and saliva were collected from 15 healthy subjects and 20 cirrhosis patients, and 3-HIB and 3-HMB concentrations in serum and saliva were measured and compared. The 3-HIB concentration and 3-HMB concentration in serum and saliva were measured in the same manner as in Example 10.
The measurement results of 3-HIB and 3-HMB concentrations in serum are shown in FIG. 20A, and the measurement results of 3-HIB and 3-HMB concentrations in saliva are shown in FIG. 20B. As a result, in both saliva and serum, the 3-HIB and 3-HMB concentrations in the cirrhosis patient group tended to be higher than those in the healthy group, indicating that these are useful as cirrhosis markers. It was done. In particular, 3-HIB and 3-HMB concentrations in saliva were significantly higher in the cirrhosis patient group than in the healthy group.

[Example 14]
Blood was collected from 48 healthy subjects and 32 cirrhosis patients, and the 3-HIB concentration in serum was measured in the same manner as in Example 10. As a result, the average value of serum 3-HIB was 12.6 ± 0.7 μM for healthy subjects and 27.7 ± 3.6 μM for cirrhosis patients.

[Example 15]
Blood was collected twice from a patient with hepatic encephalopathy (male) over time, and the 3-HIB concentration in serum was measured in the same manner as in Example 10. Table 4 shows the measurement results. As a result, the 3-HIB concentration in the serum of patients with hepatic encephalopathy was much lower than the average value (12.6 μM) of healthy subjects.

[Example 16]
Blood was collected over time from cirrhosis patient A (female) with relatively mild cirrhosis and cirrhosis patient B (female) with relatively severe cirrhosis. Serum 3-HIB (μM) and albumin ( g / dL) and ammonia concentration (μg / dL) were measured. Albumin and ammonia were usually measured by a standard method commonly used in clinical examinations. The albumin concentration was measured by BCG method using the obtained patient serum using albumin HR-II (manufactured by Wako Pure Chemical Industries, Ltd.). The ammonia concentration was measured using Fuji Dry Chem slide NH3-WII (Fuji Film Co., Ltd.) and using Fuji Dry Chem biochemical analyzer. The 3-HIB concentration in serum was measured in the same manner as in Example 10. The albumin concentration in serum was measured as one of the liver function (liver protein synthesis ability) markers. Serum ammonia concentration was measured as a hepatic encephalopathy marker or to know the ammonia clearance status. Both patients were administered the BCAA agent for a certain period during the measurement period. FIG. 21 shows the measurement results of cirrhosis patient A, and FIG. 22 shows the measurement results of cirrhosis patient B. 21 and 22, the upper graph is a graph showing the change in serum 3-HIB concentration over time, the middle graph is the graph showing the change in serum albumin concentration over time, and the lower graph is the serum ammonia concentration. It is the graph which showed change with time. In the upper part of FIG. 21, “BCAA” indicates the BCAA administration period.

As shown in FIG. 21, although the serum albumin concentration is higher than the reference value (3.8 g / dL) and the cirrhosis patient A has a relatively mild symptom of cirrhosis, the serum 3-HIB concentration varies, but cirrhosis. Although it tended to be higher than the average value of patients, especially during the BCAA drug administration period, the serum ammonia concentration was lower than the upper limit of the reference value (86 μg / dL), and the risk of developing hepatic encephalopathy was I was able to evaluate that it was not so high.
On the other hand, in the case of cirrhosis patient B in which the serum albumin concentration tends to be lower than the reference value and the symptoms of cirrhosis are relatively severe, as shown in FIG. 22, the serum 3-HIB concentration is higher than the average value of cirrhosis patients. There was a tendency that the ammonia concentration in the serum exceeded the upper limit of the reference value. Further, when BCAA agent was administered thereafter, the serum 3-HIB concentration increased, and at the same time, the ammonia concentration in the serum tended to decrease (not shown). Thus, in patients with cirrhosis, when the serum 3-HIB concentration decreases, the serum ammonia concentration tends to increase, and when the serum 3-HIB concentration increases, the serum ammonia concentration tends to decrease. From these results, it was shown that 3-HIB concentration in body fluid can be a marker of hepatic encephalopathy in cirrhosis patients.

The detection method according to the present invention can be used in fields such as clinical tests because organic acids useful as biomarkers such as 3-HIB and 3-HB can be detected with high sensitivity and quantitative.

Claims (11)

1. A method for detecting an organic acid in a sample, comprising:
   The organic acid in the sample is represented by the following general formulas (1) -1 to (1) -3
   

   

   (In the formula (1) -1 to 3, ^one of R ¹ and R ² represents a hydroxyalkyl group having 1 to 6 carbon atoms, and the other represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)
   A derivative is synthesized by ester linkage with a derivatization reagent represented by any of the above, and the derivative is detected by an electrospray ionization LC-MS / MS (liquid chromatography-tandem mass spectrometry) method in positive ion mode A method for detecting an organic acid in a sample.
2. 2. The sample according to claim 1, wherein the derivatization reagent is at least one selected from the group consisting of 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl) -1-methylpyrrolidine. Method for detecting organic acids.
3. The organic acid is represented by the following general formula (2)
   

   

   (In Formula (2), R ³ represents a hydroxyalkyl group having 2 to 6 carbon atoms.)
   The method for detecting an organic acid in a sample according to claim 1, wherein the organic acid is represented by the formula:
4. The sample according to claim 1 or 2, wherein the organic acid is at least one selected from the group consisting of 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, and 2-hydroxybutyric acid. Method for detecting organic acids.
5. The method for detecting an organic acid in a sample according to any one of claims 1 to 4, wherein the sample is a biological sample.
6. The method for detecting an organic acid in a sample according to any one of claims 1 to 4, wherein the sample is serum, tears, saliva, or urine.
7. 3. One or more organic acids selected from the group consisting of 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, and 2-hydroxybutyric acid in a biological sample collected from a subject according to claim 1 or 2. A quantitative step of detecting the organic acid in the sample by measuring the concentration of the organic acid in the biological sample;
   Compare the organic acid concentration obtained in the quantification step with a preset threshold value or the organic acid concentration in the biological sample collected from the subject before the biological sample was collected, An evaluation process for evaluating the likelihood of developing cirrhosis,
   Have
   The method for evaluating the possibility of developing cirrhosis, wherein the biological sample is serum or saliva.
8. 3-hydroxyisobutyric acid in a biological sample collected from a subject who has developed cirrhosis is detected by the method for detecting an organic acid in a sample according to claim 1 or 2, and 3-hydroxyisobutyric acid in the biological sample is detected. A quantitative process for measuring hydroxyisobutyric acid concentration;
   The 3-hydroxyisobutyric acid concentration obtained in the quantification step is compared with a preset threshold value or a 3-hydroxyisobutyric acid concentration in a biological sample collected from the subject before the biological sample was collected. And an evaluation step for evaluating the possibility of developing hepatic encephalopathy in the subject,
   Have
   A method for evaluating the possibility of developing hepatic encephalopathy, wherein the biological sample is serum or saliva.
9. A biomarker of hepatic encephalopathy or its onset possibility, characterized by comprising 3-hydroxyisobutyric acid.
10. The organic acid in the sample according to claim 1 or 2, wherein one or more organic acids selected from the group consisting of 3-hydroxyisobutyric acid and 3-hydroxybutyric acid in a biological sample collected from a subject in motion are used. A quantitative step of detecting by the acid detection method and measuring the concentration of the organic acid in the biological sample;
    The organic acid concentration obtained in the quantification step is compared with a preset threshold value or the organic acid concentration in a biological sample collected before exercise from the subject, and during or after exercise in the subject An evaluation process for evaluating energy sources;
    Have
    The method for evaluating an energy source, wherein the biological sample is serum or saliva.
11. One selected from the group consisting of 3-hydroxyisobutyric acid and 3-hydroxybutyric acid in a plurality of biological samples collected over time from immediately before the start of exercise to after a lapse of a certain period after the end of exercise The above-mentioned organic acid is detected by the method for detecting an organic acid in a sample according to claim 1 or 2, and the organic acid concentration in each biological sample is measured. Monitoring process to examine
    An evaluation step for evaluating an energy supply source during or after exercise in the subject based on the change over time of the organic acid concentration obtained in the monitoring step;
    Have
    An energy supply source monitoring method, wherein the organic acid concentration in the biological sample is used as a marker of an energy supply source, and the biological sample is serum or saliva.

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