Pyruvate oxidase stabilizer and application thereof in alanine detection kit

Abstract

The invention discloses a pyruvate oxidase stabilizer and application thereof in an alanine detection kit, and belongs to the technical field of medical analysis. According to the pyruvate oxidase stabilizer disclosed by the invention, specific substances in a proper mass percent range are selected and combined, so that the obtained pyruvate oxidase stabilizer can effectively stabilize pyruvate oxidase and also can effectively stabilize catalase; when the alanine fluorescent probe is applied to an alanine detection kit, interference of endogenous pyruvic acid can be effectively eliminated and alanine is converted into pyruvic acid by further selecting a reagent R1, a reagent R2 and a reagent R3 with proper components in the kit, so that the content of alanine is detected. The alanine detection kit provided by the invention is high in detection accuracy, high in sensitivity, simple and convenient to operate and beneficial to being widely applied to in-vitro alanine content detection.

Description

Technical Field

[0001] This application relates to the field of medical analysis and testing technology, and in particular to a pyruvate oxidase stabilizer and its application in an alanine detection kit. Background Technology

[0002] A physiological feedback system exists between liver and pancreatic α cells, known as the hepat-α cell axis, representing the interaction between amino acid-stimulated glucagon secretion and glucagon-stimulated amino acid metabolism. Glucagon regulates hepatic amino acid metabolism by stimulating amino acid conversion and urea production through both non-transcriptional and transcriptional methods; conversely, certain amino acids stimulate α cell growth and glucagon secretion, forming a classic feedback loop. Experiments have shown that this feedback loop may be disrupted by hepatic steatosis, which is associated with reduced glucagon-induced amino acid catabolism and urea production, leading to hyperaminoacidemia and subsequently hyperglucagonemia. Therefore, both clinical and experimental studies support the hepatic glucagon resistance hypothesis induced by steatosis. Numerous studies recommend using the glucagon-alanine index as a sensitive biomarker for glucagon resistance.

[0003] The glucagon-alanine index = fasting plasma glucagon (pmol / L) × fasting plasma alanine (mmol / L); the glucagon-alanine index is considered an indicator of the functional status of the hepat-α cell axis, with a higher index indicating dysfunction of the hepat-α cell axis. However, there is no good clinical method for detecting alanine. Currently, LC-MS / MS is commonly used, but LC-MS / MS is cumbersome and expensive, making it difficult to popularize in clinical practice. Furthermore, there is currently no LC-MS / MS or method specifically designed for alanine detection in clinical settings.

[0004] In in vitro diagnostic reagents, the stability of biological enzymes is crucial for ensuring reagent quality. Currently, there is no suitable stabilizer for alanine detection kits. Summary of the Invention

[0005] The purpose of this application is to address the lack of existing in vitro alanine content detection reagents and the technical problem of insufficient stability of related enzymes in in vitro alanine content detection. It provides a pyruvate oxidase stabilizer with good stability, and when added to an alanine detection kit, the resulting alanine detection kit exhibits good sensitivity and precision. The application of this pyruvate oxidase stabilizer in the alanine detection kit is also discussed.

[0006] To achieve the above objectives, a first aspect of this application provides a pyruvate oxidase stabilizer, said pyruvate oxidase stabilizer comprising the following components by mass percentage:

[0007] 3-20% ethylene glycol, 0.1-0.5% mannitol, 0.5-5% trehalose, 0.0002-0.002% flavin adenine dinucleotide, 0.0005-0.092% thiamine pyrophosphate, 0.01-0.24% water-soluble magnesium salt, 0.005-0.02% primary preservative, balance primary buffer.

[0008] This application combines the above-mentioned specific substances within a suitable mass percentage range to obtain a pyruvate oxidase stabilizer that can effectively stabilize pyruvate oxidase and also effectively stabilize catalase added for subsequent alanine detection.

[0009] Specifically, the addition of ethylene glycol within the aforementioned mass percentage range effectively prevents the degradation of pyruvate oxidase and improves its thermal stability. Simultaneously, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), as coenzymes for pyruvate oxidase, stabilize the enzyme. The addition of water-soluble magnesium salts releases magnesium ions, which, as activators of pyruvate oxidase, enhance its reactivity, thus improving the sensitivity and accuracy of the alanine assay kit. The addition of mannose and trehalose not only helps stabilize pyruvate oxidase but also improves the accuracy of alanine detection when applied to the kit.

[0010] Preferably, the pyruvate oxidase stabilizer comprises the following components by weight percentage:

[0011] 5-10% ethylene glycol, 0.1-0.5% mannitol, 0.5-5% trehalose, 0.0004-0.001% flavin adenine dinucleotide, 0.0023-0.05% thiamine pyrophosphate, 0.05-0.12% water-soluble magnesium salt, 0.005-0.02% primary preservative, balance primary buffer.

[0012] This study found that the mass percentage of the components in the pyruvate oxidase stabilizer affects its stabilizing effect on pyruvate. When the mass percentage of the components is further selected within the above range, the resulting pyruvate oxidase stabilizer has a better stabilizing effect on pyruvate oxidase, and when it is subsequently applied to an alanine detection kit, the results of alanine content detection are more reliable.

[0013] Preferably, the pH value of the first buffer solution is 7.0 to 7.4.

[0014] Preferably, the molar concentration of the solute in the first buffer solution is 10-50 mmol / L.

[0015] Preferably, the first buffer comprises any one of phosphate buffer, Tris-HCl buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, and 2-morpholinoethanesulfonic acid (MES) buffer.

[0016] This study found that the first buffer in the pyruvate oxidase stabilizer can stabilize the pH of the system. When the pH, the molar concentration of the solute, and the type of the first buffer are further selected within the above range, the overall effect of the obtained pyruvate oxidase stabilizer is better.

[0017] Preferably, the water-soluble magnesium salt includes at least one of magnesium sulfate and magnesium chloride.

[0018] Choosing the above-mentioned types of water-soluble magnesium salts not only allows for faster dissociation of magnesium ions, but also ensures that the anions do not affect stability. Therefore, further selection of the above-mentioned types of water-soluble magnesium salts yields even better overall results.

[0019] Preferably, the first preservative includes at least one of gentamicin sulfate, Proclin 300, and KY100.

[0020] In a second aspect of this application, a method for preparing the pyruvate oxidase stabilizer is provided, the method comprising the following steps:

[0021] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well. Then let it stand at 35-40℃ for 20-24h to obtain mixture A.

[0022] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 20-30℃ for 30-60 min, then filter, collect the filtrate, and obtain pyruvate oxidase stabilizer.

[0023] This study found that the pyruvate oxidase stabilizer prepared by the above method can better stabilize pyruvate oxidase, and when it is subsequently applied to an alanine detection kit, the resulting alanine detection kit has better reliability.

[0024] In a third aspect of this application, the application of the pyruvate oxidase stabilizer in the preparation of an alanine detection kit is provided.

[0025] Pyruvate oxidase is generally added to alanine detection kits. The activity and stability of pyruvate oxidase affect the accuracy and sensitivity of alanine detection. The pyruvate oxidase stabilizer provided in this application can better stabilize pyruvate oxidase, and therefore can be widely used in alanine detection kits.

[0026] In a fourth aspect of this application, an alanine detection kit is provided, the alanine detection kit comprising reagent R1, reagent R2 and reagent R3;

[0027] The reagent R1 includes pyruvate oxidase, catalase, and the pyruvate oxidase stabilizer described in this application.

[0028] The reagent R2 includes a second buffer, alanine aminotransferase, α-ketoglutarate, and a second preservative.

[0029] The reagent R3 comprises dioxaneboronic acid (AMBPD) and carbonate buffer.

[0030] The alanine detection kit provided in this application can effectively eliminate the interference of endogenous pyruvate by selecting appropriate components of reagent R1, reagent R2 and reagent R3, converting alanine into pyruvate, thereby detecting the content of alanine.

[0031] Specifically, the pyruvate oxidase stabilizer added to reagent R1 effectively stabilizes pyruvate oxidase and also has a certain stabilizing effect on catalase, thus effectively eliminating the interference of pyruvate originally present in the test sample and improving the accuracy and precision of the detection results. The alanine aminotransferase, α-ketoglutarate, and the second preservative in reagent R2 can completely convert alanine into pyruvate and inhibit the activity of catalase in reagent R1, thereby improving the accuracy and precision of the detection results. The dioxaneboronic acid in reagent R3 reacts with hydrogen peroxide to decompose into AMP-D anions, which then decompose into 2-adamantanone and methyl 3-hydroxymethylbenzoate. Methyl 3-hydroxymethylbenzoate is a luminescent intermediate that releases a light signal after changing from the excited state to the ground state. The intensity of the light signal is proportional to the concentration of alanine, allowing the content to be generated in the form of a light signal, thus conveniently and quickly obtaining alanine content information.

[0032] Preferably, in reagent R1, the concentration of pyruvate oxidase is 2-50 U / mL and the concentration of catalase is 2-50 U / mL.

[0033] Preferably, in reagent R2, the concentration of alanine aminotransferase is 2-50 U / mL, the molar concentration of α-ketoglutarate is 0.2-2 mmol / L, and the mass percentage of the second preservative is 0.02-0.04%.

[0034] This study found that further selecting the concentrations or mass percentages of components in reagents R1 and R2 within the above-mentioned range can better ensure the reliability of alanine detection results.

[0035] Preferably, the second preservative includes sodium azide or thimerosal, and also includes at least one of gentamicin sulfate and Proclin 300.

[0036] Preferably, the second buffer solution comprises any one of phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 2-morpholine ethanesulfonic acid buffer.

[0037] Preferably, the pH value of the second buffer solution is 7.0-7.4.

[0038] Preferably, the molar concentration of the solute in the second buffer solution is 10-50 mmol / L.

[0039] Preferably, the pH value of the carbonate buffer solution in reagent R3 is 9.9-10.1.

[0040] Preferably, the alanine detection kit further includes calibrators and quality control products.

[0041] The calibrators and quality control samples are L-alanine solutions, prepared from L-alanine (commonly available product, purity ≥99%) and phosphate buffer.

[0042] The phosphate buffer solution used to prepare the calibrators and quality control samples has a pH of 7.0-7.4 and a solute molar concentration of 10-50 mmol / L.

[0043] In a fifth aspect of this application, the use of the alanine detection kit in the preparation of products for detecting alanine content is provided.

[0044] The alanine detection kit provided in this application can effectively detect alanine content, and therefore can be widely used in the detection of alanine content.

[0045] In a sixth aspect of this application, the use of the alanine assay kit in the preparation of products for assessing metabolic-associated fatty liver disease is provided.

[0046] This study found that the glucagon-alanine index is considered an indicator of the functional status of the liver-α cell axis. A higher index indicates dysfunction of the liver-α cell axis. The glucagon-alanine index can be obtained by detecting the alanine content in the sample, thereby assessing the functional status of the liver-α cell axis and further evaluating metabolic-related fatty liver disease.

[0047] In a seventh aspect of this application, a method for detecting alanine content using the alanine detection kit described in this application is provided, the method comprising the following steps:

[0048] The sample to be tested is mixed with reagent R1 for the first action, then reagent R2 is added for the second action, followed by reagent R3 for the third action. Finally, the light signal intensity is detected, and the alanine content in the sample is calculated based on the light signal intensity-concentration standard curve.

[0049] This application first mixes the sample to be tested with reagent R1, which converts pyruvate in the sample into hydrogen peroxide under the action of pyruvate oxidase. The hydrogen peroxide is then decomposed by catalase, thus eliminating the influence of endogenous pyruvate. Next, reagent R2 is added, where alanine in the sample is converted into pyruvate under the action of alanine aminotransferase. Pyruvate oxidase in reagent R1 oxidizes pyruvate to produce hydrogen peroxide, while the secondary preservative (such as sodium azide) in reagent R2 inhibits the catalase in reagent R1. Then, reagent R3 is added, where dioxaneboronic acid (AMBPD) in reagent R3 reacts with hydrogen peroxide, decomposing into AMP-D anions, which then decompose into 2-adamantanone and methyl 3-hydroxymethylbenzoate (reaction diagram shown). Figure 1 As shown in the figure, methyl 3-hydroxy-methylbenzoate is a luminescent intermediate that releases a light signal after changing from the excited state to the ground state. The intensity of the light signal is proportional to the concentration of alanine. Thus, the alanine content in the sample can be obtained by measuring the intensity of the light signal.

[0050] Preferably, the duration of the first action is 3-8 minutes, and the temperature of the first action is 35-40°C.

[0051] Preferably, the duration of the second action is 3-8 minutes, and the temperature of the second action is 35-40°C.

[0052] Preferably, the duration of the third action is 5-8 minutes, and the temperature of the third action is 35-40°C.

[0053] Preferably, the sample to be tested includes either serum or plasma.

[0054] Compared with existing technologies, the advantages of this application are:

[0055] This application combines specific substances within a suitable mass percentage range to obtain a pyruvate oxidase stabilizer that effectively stabilizes both pyruvate oxidase and catalase. When applied to an alanine assay kit, further selection of appropriate reagents R1, R2, and R3 effectively eliminates interference from endogenous pyruvate, converting alanine into pyruvate and thus detecting its content. The alanine assay kit provided by this application offers high accuracy and sensitivity, and is easy to operate, making it suitable for widespread application in in vitro alanine content detection. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the reaction process between reagent R3 and hydrogen peroxide.

[0057] Figure 2 The graph shows the correlation between the alanine detection kit prepared in Example 1 and the LC-MS / MS results in Example 3. Detailed Implementation Plan

[0058] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0059] Unless otherwise specified, the reagents, methods and equipment used in this application are all conventional reagents, methods and equipment in the field.

[0060] Example 1

[0061] This application provides a pyruvate oxidase stabilizer A, which comprises the following components by mass percentage:

[0062] 10% ethylene glycol, 0.3% mannitol, 2.5% trehalose, 0.001% flavin adenine dinucleotide, 0.0023% thiamine pyrophosphate, 0.12% water-soluble magnesium salt (magnesium sulfate), 0.01% primary preservative (gentamicin sulfate), balance primary buffer (phosphate buffer with pH 7.2, where the molar concentration of the solute in the buffer is 30 mmol / L);

[0063] The preparation method of the pyruvate oxidase stabilizer is as follows:

[0064] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence, stir well and let stand at 37°C for 22 hours to obtain mixture A.

[0065] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 25°C for 45 min, then filter, collect the filtrate, and obtain pyruvate oxidase stabilizer A.

[0066] Example 2

[0067] This application provides a pyruvate oxidase stabilizer B, which differs from the stabilizer in Example 1 in that the mass percentage of the component is different. The mass percentage of the component in this embodiment is as follows:

[0068] 3% ethylene glycol, 0.1% mannitol, 0.5% trehalose, 0.0002% flavin adenine dinucleotide, 0.0005% thiamine pyrophosphate, 0.01% water-soluble magnesium salt (magnesium sulfate), 0.005% primary preservative (gentamicin sulfate), balance primary buffer (pH 7.2 phosphate buffer, with a solute molar concentration of 30 mmol / L).

[0069] Example 3

[0070] This application provides a pyruvate oxidase stabilizer C. The difference between the pyruvate oxidase stabilizer C and that in Example 1 lies in the mass percentage of the components and the selection of the components. The mass percentage of the components in this embodiment is as follows:

[0071] 20% ethylene glycol, 0.5% mannitol, 5% trehalose, 0.002% flavin adenine dinucleotide, 0.092% thiamine pyrophosphate, 0.24% water-soluble magnesium salt (magnesium sulfate), 0.015% primary preservative (gentamicin sulfate), balance primary buffer (pH 7.2 phosphate buffer, with a solute molar concentration of 30 mmol / L).

[0072] Example 4

[0073] This application provides a pyruvate oxidase stabilizer D, which differs from Example 1 in that the buffer solution is a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with a pH of 7.0, wherein the molar concentration of the solute in the buffer solution is 10 mmol / L.

[0074] Comparative Example 1

[0075] This application provides a pyruvate oxidase stabilizer a in comparison, wherein the difference between pyruvate oxidase stabilizer a and Example 1 is that the mass percentage of ethylene glycol is 30%.

[0076] Comparative Example 2

[0077] This application provides a comparative example of a pyruvate oxidase stabilizer b, which differs from Example 1 in that glycerol is used instead of ethylene glycol.

[0078] Comparative Example 3

[0079] This application provides a comparative example of a pyruvate oxidase stabilizer c, which differs from Example 1 in that glucose is used instead of trehalose.

[0080] Comparative Example 4

[0081] This application provides a comparative example of a pyruvate oxidase stabilizer d, which differs from Example 1 in that it does not contain ethylene glycol, but is supplemented with mannitol.

[0082] Comparative Example 5

[0083] This application provides a comparative example of a pyruvate oxidase stabilizer e, which differs from Example 1 in that it does not contain flavin adenine dinucleotide, but is supplemented with thiamine pyrophosphate.

[0084] Comparative Example 6

[0085] This application provides a pyruvate oxidase stabilizer c as a comparative example. The difference between the pyruvate oxidase stabilizer c and that in Example 1 lies in the different preparation methods. The preparation method of this comparative example is as follows:

[0086] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well to obtain mixture A.

[0087] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 25°C for 45 min, then filter, collect the filtrate, and obtain pyruvate oxidase stabilizer e.

[0088] Comparative Example 7

[0089] This application provides a pyruvate oxidase stabilizer f as a comparative example. The difference between the pyruvate oxidase stabilizer f and that in Example 1 is the preparation method. The preparation method of this comparative example is as follows:

[0090] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well to obtain mixture A.

[0091] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 25°C for 45 min, then filter, collect the filtrate and place at 37°C for 22 h to obtain pyruvate oxidase stabilizer f.

[0092] Comparative Example 8

[0093] This application provides a pyruvate oxidase stabilizer g in a comparative example. The difference between the pyruvate oxidase stabilizer g and that in Example 1 is the preparation method. The preparation method of this comparative example is as follows:

[0094] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well. Then place at 25°C for 22 hours to obtain mixture A.

[0095] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 25°C for 45 min, then filter, collect the filtrate and place at 37°C for 22 h to obtain g of pyruvate oxidase stabilizer.

[0096] Comparative Example 9

[0097] This application provides a pyruvate oxidase stabilizer h in a comparative example. The difference between the pyruvate oxidase stabilizer h and that in Example 1 is the preparation method. The preparation method of this comparative example is as follows:

[0098] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt, first preservative, flavin adenine dinucleotide and thiamine pyrophosphate in sequence and stir at 25°C for 45 min. Then filter, collect the filtrate and place it at 25°C for 22 h to obtain pyruvate oxidase stabilizer h.

[0099] Comparative Example 10

[0100] This application provides a pyruvate oxidase stabilizer i in a comparative example. The difference between the pyruvate oxidase stabilizer i and that in Example 1 is the preparation method. The preparation method of this comparative example is as follows:

[0101] (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well. Then place at 25°C for 44 hours to obtain mixture A.

[0102] (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 25°C for 45 min, then filter, collect the filtrate and place at 37°C for 22 h to obtain pyruvate oxidase stabilizer i.

[0103] The following are the specifications for the calibrators (S0-S5), interference samples (PY300), and quality control samples (CT1, CT2) used in the effect example test: The pH value of the phosphate buffer is 7.2, and the molar concentration of the solute is 30 mmol / L.

[0104] S0, L-alanine-free, solvent is phosphate buffer;

[0105] S1 contains 50 μmol / L (0.00045%) L-alanine in phosphate buffer solution;

[0106] S2 contains 250 μmol / L (0.00225%) L-alanine in phosphate buffer solution;

[0107] S3 contains 500 μmol / L (0.0045%) L-alanine in phosphate buffer solution;

[0108] S4 contains 1000 μmol / L (0.009%) L-alanine in phosphate buffer solution;

[0109] S5 contains 2000 μmol / L (0.018%) L-alanine in phosphate buffer solution;

[0110] PY300: Contains 300 μmol / L (0.0027%) pyruvate, in phosphate buffer solution;

[0111] CT1: Contains 400 μmol / L-600 μmol / L L-alanine, in phosphate buffer solution;

[0112] CT2: Contains 1200 μmol / L-1400 μmol / L L-alanine, in phosphate buffer solution.

[0113] Example 1

[0114] The stabilizing effect of the pyruvate oxidase stabilizers prepared in the experimental and comparative examples of this application on pyruvate oxidase includes the following steps:

[0115] Reagent R1 was prepared by mixing the pyruvate oxidase stabilizer, pyruvate oxidase, and catalase obtained in the examples and comparative examples, wherein the concentration of pyruvate oxidase in reagent R1 was 25 U / mL and the concentration of catalase was 20 U / mL; the accelerated stability of reagent R1 was investigated for 7 days, specifically as follows:

[0116] S1. Prepare four bottles of each of the prepared reagent R1 corresponding to the pyruvate oxidase stabilizers obtained in the examples and comparative examples, and seal them.

[0117] S2. Store one bottle of reagent R1 at 2-8℃, and place the remaining three bottles of reagent R1 in a constant temperature storage box at 37℃ on day 0, day 4, and day 6, respectively.

[0118] S3. Prepare reagents R2 and R3. Reagent R2 includes a second buffer (phosphate buffer with a pH of 7.2, wherein the molar concentration of the solute in the buffer is 30 mmol / L), alanine aminotransferase, α-ketoglutarate, and a second preservative (sodium azide and gentamicin sulfate). In reagent R2, the concentration of alanine aminotransferase is 25 U / mL, the molar concentration of α-ketoglutarate is 1 mmol / L, the mass percentage of sodium azide is 0.03%, and the mass percentage of gentamicin sulfate is 0.01%.

[0119] Reagent R3 consists of dioxaneboronic acid (AMBPD) and carbonate buffer, wherein the mass percentage of AMBPD in reagent R3 is 5% and the pH of the carbonate buffer is 10.0.

[0120] S4. On the 7th day, all reagent R1 was removed and tested using a fully automated chemiluminescence analyzer (model HomoG100) according to the following procedure: The test samples (calibrators S0, S2, S5 and PY300) were mixed with the removed reagent R1 and subjected to a first reaction at 37°C for 8 minutes. Then, reagent R2 was added and the mixture was subjected to a second reaction at 37°C for 6 minutes. Next, reagent R3 was added and the mixture was subjected to a third reaction at 37°C for 6 minutes. The fully automated chemiluminescence analyzer detected the light signal intensity of calibrators S0, S2, S5 and PY300 at 470 nm and recorded the luminescence value RLU. The measurement was repeated 3 times and the average value was used for statistical analysis.

[0121] S5. Using the results at 2-8℃ as the data for acceleration day 0, calculate the relative deviations between the results of acceleration day 1 (placed on day 6), day 3 (placed on day 4), and day 7 (placed on day 0) and day 0.

[0122] The results are shown in Tables 1-3.

[0123] Table 1

[0124]

[0125] Table 2

[0126]

[0127]

[0128] Table 3

[0129]

[0130] As can be seen from Tables 1-3, when the technical solution provided in this application is adopted, the changes in calibrators S0, S2, and S5 of reagent R1 during the 7-day acceleration process are small, and the absolute values ​​of the changes are all within 8%. The interfering sample PY300 is not affected by the acceleration. Reagent R1 can stably remove pyruvate, indicating that both pyruvate oxidase and catalase are stable.

[0131] As can be seen from Tables 1 and 2, when the type or amount of the component in the pyruvate oxidase stabilizer is not within the scope of this application, the changes in calibrators S0, S2, and S5 of the obtained reagent R1 during the 7-day acceleration process are all increased compared to Example 1, especially the changes in calibrators S2 and S5 are significantly increased, and the interfering samples are also affected by the acceleration to some extent.

[0132] As can be seen from Tables 1 and 3, when the preparation method of the pyruvate oxidase stabilizer is not provided in this application, the luminescence value of the calibrator increases or decreases significantly, and PY300 shows incomplete removal. The significant change in the calibrator indicates that the pyruvate oxidase is unstable, and the increase in PY300 indicates that the catalase is unstable. That is, the stabilizing ability of the pyruvate oxidase stabilizer for pyruvate oxidase is significantly reduced.

[0133] Example 2

[0134] The effect of the pyruvate oxidase stabilizer prepared in Example 1 of this application on pyruvate oxidase stabilization includes the following steps:

[0135] The pyruvate oxidase stabilizer, pyruvate oxidase, and catalase prepared in Example 1 were mixed to prepare reagent R1, wherein the concentration of pyruvate oxidase in reagent R1 was 25 U / mL and the concentration of catalase was 20 U / mL; the accelerated stability of reagent R1 was investigated at 37°C for 14 days, specifically as follows:

[0136] S1. Prepare 5 bottles of each of the reagents R1 prepared in the examples, and seal them;

[0137] S2. Store one bottle at 2-8℃, and place the remaining four bottles of reagent R1 in a constant temperature storage box at 37℃ on day 0, day 4, day 7, and day 11, respectively.

[0138] S3. Prepare reagents R2 and R3. Reagent R2 includes a second buffer (phosphate buffer with a pH of 7.2, wherein the molar concentration of the solute in the buffer is 30 mmol / L), alanine aminotransferase, α-ketoglutarate, and a second preservative (sodium azide and gentamicin sulfate). In reagent R2, the concentration of alanine aminotransferase is 25 U / mL, the molar concentration of α-ketoglutarate is 1 mmol / L, the mass percentage of sodium azide is 0.03%, and the mass percentage of gentamicin sulfate is 0.01%.

[0139] Reagent R3 consists of dioxaneboronic acid (AMBPD) and carbonate buffer, wherein the mass percentage of AMBPD in reagent R3 is 5% and the pH of the carbonate buffer is 10.0.

[0140] S4. On day 14, all reagent R1 was removed and tested using a fully automated chemiluminescence analyzer (model HomoG100) according to the following procedure: The test samples (calibrators S0-S5, PY300 plasma samples 1-2) were mixed with the removed reagent R1 and incubated at 37°C for 8 minutes. Then, reagent R2 was added and mixed at 37°C for 6 minutes. Next, reagent R3 was added and mixed at 37°C for 6 minutes. The fully automated chemiluminescence analyzer detected the light signal intensity of calibrators S0-S5, PY300, and plasma samples 1-2 at 470 nm and recorded the luminescence value RLU. The measurement was repeated 3 times and the results were statistically analyzed using the mean.

[0141] S5. Using the results at 2-8℃ as the data for accelerated day 0, calculate the relative deviations between the results for accelerated days 3 (placed on day 11), 7 (placed on day 7), 10 (placed on day 4), and 14 (placed on day 0) and day 0; wherein the plasma 1-2 was sourced from employee volunteers of the applicant's company;

[0142] The results are shown in Table 4.

[0143] Table 4

[0144]

[0145] As shown in Table 4, after 14 days of accelerated treatment at 37℃ using the pyruvate oxidase stabilizer prepared in this application, the relative deviation of the luminescence values ​​of the calibrators (S0-S5) and plasma samples compared with the control group stored at 2-8℃ was within 10%, indicating that the pyruvate oxidase activity did not decrease. The luminescence value of the interfering substance (PY300, pyruvate concentration of 300μM) was close to that of S0, indicating that the clearance of the interfering substance pyruvate was not affected, and the catalase activity remained stable.

[0146] Application Example 1

[0147] This application provides an alanine detection kit, including reagent R1, reagent R2, reagent R3, quality control materials (CT1, CT2), and calibrators (S0-S5);

[0148] Reagent R1 includes the pyruvate oxidase stabilizer, pyruvate oxidase and catalase prepared in Example 1, wherein the concentration of pyruvate oxidase in reagent R1 is 25 U / mL and the concentration of catalase is 20 U / mL.

[0149] Reagent R2 comprises a second buffer (phosphate buffer with a pH of 7.2, wherein the molar concentration of the solute in the buffer is 30 mmol / L), alanine aminotransferase, α-ketoglutarate, and a second preservative (sodium azide and gentamicin sulfate). In reagent R2, the concentration of alanine aminotransferase is 25 U / mL, the molar concentration of α-ketoglutarate is 1 mmol / L, the mass percentage of sodium azide is 0.03%, and the mass percentage of gentamicin sulfate is 0.01%.

[0150] Reagent R3 consists of dioxaneboronic acid (AMBPD) and carbonate buffer, wherein the mass percentage of AMBPD in reagent R3 is 5% and the pH of the carbonate buffer is 10.0.

[0151] Application Example 2

[0152] This application provides an alanine detection kit, which differs from Application Example 1 in that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in Example 2.

[0153] Application Example 3

[0154] This application provides an alanine detection kit, which differs from Application Example 1 in that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in Example 3.

[0155] Application Example 4

[0156] This application provides an alanine detection kit, which differs from Application Example 1 in that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in Example 4.

[0157] Application Example 5

[0158] This application provides an alanine detection kit, which differs from Application Example 1 in that the concentration of pyruvate oxidase in reagent R1 is 50 U / mL and the concentration of catalase is 2 U / mL.

[0159] Application Example 6

[0160] This application provides an alanine detection kit, which differs from Application Example 1 in that the concentration of pyruvate oxidase in reagent R1 is 2 U / mL and the concentration of catalase is 50 U / mL.

[0161] Application Example 7

[0162] This application provides an alanine detection kit, which differs from Application Example 1 in that the concentration of alanine aminotransferase in reagent R2 is 2 U / mL, the molar concentration of α-ketoglutarate is 2 mmol / L, the mass percentage of sodium azide is 0.03%, and the mass percentage of gentamicin sulfate is 0.01%.

[0163] Application Example 8

[0164] This application provides an alanine detection kit, which differs from Application Example 1 in that the concentration of alanine aminotransferase in reagent R2 is 50 U / mL, the molar concentration of α-ketoglutarate is 0.2 mmol / L, the mass percentage of sodium azide is 0.03%, and the mass percentage of gentamicin sulfate is 0.01%.

[0165] Comparative Application Example 1

[0166] This application provides an alanine detection kit in a comparative application example. The difference between the alanine detection kit and application example 1 is that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in comparative example 1.

[0167] Comparative Application Example 2

[0168] This application provides an alanine detection kit in a comparative application example. The difference between the alanine detection kit and application example 1 is that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in comparative example 2.

[0169] Comparative Application Example 3

[0170] This application provides an alanine detection kit in a comparative application example. The difference between the alanine detection kit and application example 1 is that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in comparative example 3.

[0171] Comparative Application Example 4

[0172] This application provides an alanine detection kit in a comparative application example. The difference between the alanine detection kit and application example 1 is that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in comparative example 4.

[0173] Comparative Application Example 5

[0174] This application provides an alanine detection kit in a comparative application example. The difference between the alanine detection kit and application example 1 is that the pyruvate oxidase stabilizer is the pyruvate oxidase stabilizer prepared in comparative example 5.

[0175] Example 3

[0176] This application's effect examples explore the performance of the alanine detection kits prepared in the application examples and comparative application examples, including the following tests:

[0177] (1) Correlation test

[0178] The alanine concentration was determined using the alanine detection kit and LC-MS / MS prepared in this application. The concentrations were measured in 20 fresh human plasma samples (derived from volunteer employees of the applicant company), and correlation regression analysis was performed on the measured values.

[0179] The specific testing process of the alanine detection kit prepared in this application is as follows: The detection is performed using a fully automated chemiluminescence analyzer (model: HomoG 100). The sample to be tested is mixed with reagent R1 and reacted at 37°C for 8 min. Then, reagent R2 is added and mixed at 37°C for 6 min. Next, reagent R3 is added and mixed at 37°C for 6 min. Finally, the light signal intensity is detected at 470 nm. Based on the light signal intensity-concentration standard curve, the fully automated chemiluminescence analyzer automatically calculates the alanine content in the sample to be tested.

[0180] The specific test procedure for determining alanine concentration using LC-MS / MS is as follows:

[0181] S1. Pretreatment: Add 5 μL of internal standard solution (prepared from L-alanine-3,3,3-d3) and 100 μL of methanol:acetonitrile (1:1) solution to 20 μL of sample plasma. Vortex for 2 min, let stand on ice for 30 min, and then centrifuge at 12000 rpm for 15 min at 4℃. Collect the supernatant as the sample solution.

[0182] S2. Perform LC-MS / MS detection:

[0183] Instrument model: AB SCIEX 3200+Shimadzu LC-20AT; Column: InfinityLab Poroshell 120 HILIC, 4.6×250mm, 4μm; Mobile phase A: 20mmol / L ammonium formate, pH 3.0; Mobile phase B: 20mmol / L ammonium formate in acetonitrile:water (90:10) solution, pH 3.0; Injection volume: 2μL; Quantification method: internal standard method.

[0184] The correlation coefficient between each alanine assay kit and the results obtained by LC-MS / MS was calculated and recorded in Table 5.

[0185] (2) Accuracy testing

[0186] Using the alanine assay kit prepared in this application, the national purity standard reference material for alanine (purchased from the National Institute of Metrology, China) was prepared with purified water to three concentrations (350 μmol / L, 750 μmol / L, and 1500 μmol / L) for determination. Three measurements were performed, and the relative deviation was calculated for each determination. The formula for the relative deviation is:

[0187]

[0188] Among them, B i Indicates relative deviation, x i T represents the measured concentration, and T represents the labeled concentration.

[0189] The results are shown in Table 5.

[0190] (3) Recovery rate detection

[0191] Alanine national purity standard material was prepared into high-concentration stock solutions (25mM and 50mM) using purified water. Three fresh plasma samples (from volunteer employees of the applicant company) were taken, and each sample was divided into three equal parts. Two plasma samples were added with equal volumes of the 25mM and 50mM stock solutions, respectively, while the third plasma sample was added with an equal volume of purified water. To ensure that the added solvent did not cause matrix interference, the amount added did not exceed 5% (specifically, 3 wt% of the sample mass). The three plasma samples were tested using the alanine detection kits of the application example and the control application example, and the average recovery rate was calculated. The results are shown in Table 5 below.

[0192] Table 5

[0193]

[0194]

[0195] As shown in Table 5, the alanine detection kit prepared using the technical solution of this application exhibits good correlation with the LC-MS / MS detection results, with a correlation coefficient above 0.979; among them, the alanine detection kit prepared in Application Example 1 shows a good correlation with the LC-MS / MS results. Figure 2 As shown, the alanine detection kit provided in this application has good accuracy in determining alanine content; while in contrast, when the pyruvate oxidase stabilizer in Examples 1-5 was not provided by the technical solution of this application, the correlation coefficients were significantly reduced; in addition, the accuracy test results show that the alanine detection kit prepared using the technical solution of this application has high detection accuracy, with relative deviations all within 10%; and the recovery rate is between 90-110%, which means that the alanine detection kit provided in this application has good accuracy in determining alanine content.

[0196] Example of effect 4

[0197] This application explores the application of the alanine detection kit prepared in Example 1 in assessing metabolic-associated fatty liver disease (MAFLD), with individual data sourced from the Third Hospital of Hebei Medical University.

[0198] The specific testing procedure was as follows: Fasting plasma samples were tested on a fully automated chemiluminescence analyzer (model: HomoG100) using a glucagon (GCG) assay kit (provided by the applicant) and an alanine (Ala) assay kit prepared in Example 1, respectively. The fully automated chemiluminescence analyzer calculated the glucagon content (pmol / L) and alanine content (μmol / L) in the samples. The glucagon-alanine index was calculated using the following formula:

[0199] Glucagon-alanine index = fasting plasma glucagon (pmol / L) × fasting plasma alanine (μmol / L) / 1000;

[0200] Grouped as follows:

[0201] Group A (27 cases): Normal reference individuals,

[0202] Group B (9 cases): Patients clinically diagnosed with hepatitis B,

[0203] Group C (14 cases): Patients clinically diagnosed with hepatitis B complicated with fatty liver.

[0204] Group D (39 cases): Patients clinically diagnosed with fatty liver;

[0205] The data were statistically analyzed using SPSS 20 statistical analysis software, and the results for different groups are summarized in Table 6.

[0206] Table 6

[0207]

[0208] The t-test was performed between the disease group and the normal group, and the results are shown in Table 7.

[0209] Table 7

[0210]

[0211] A t-test between the hepatitis B patient group and the normal group showed no significant difference in glucagon-alanine index. However, comparisons between the hepatitis B patient group with fatty liver, the fatty liver patient group, and the normal group showed significant differences (P < 0.05), indicating that the glucagon-alanine index in patients with fatty liver differs from that in the normal group.

[0212] Furthermore, the samples in group D were divided into two groups according to the degree of fatty liver degeneration. Among them, 16 samples were diagnosed with mild fatty liver (group 1) and 11 samples were diagnosed with moderate fatty liver (group 2). The statistical analysis software SPSS20 was used to perform t-tests on the fatty liver samples of different degrees in the two groups. The results are shown in Tables 8 and 9, where Table 8 is the group statistical data and Table 9 is the independent samples test data.

[0213] Table 8

[0214]

[0215]

[0216] Table 9

[0217]

[0218] The mean alanine levels in the mild fatty liver group (Group 1) and the moderate fatty liver group (Group 2) were 488.31 μmol / L (±82.36 μmol / L) and 590.09 μmol / L (±50.07 μmol / L), respectively. The alanine levels in the moderate fatty liver group were significantly higher than those in the mild fatty liver group. Statistical analysis showed a significant difference in alanine levels between the two groups (P = 0.001). The glucagon levels in the two groups were 17.52 pmol / L (±7.03 pmol / L). The glucagon-alanine levels were 20.52 pmol / L (±5.34 pmol / L) and 20.52 pmol / L (±5.34 pmol / L), respectively. The t-test showed no significant difference (P>0.05). The glucagon-alanine index was 8.59 pmol / L*mmol / L (±4.02 pmol / L*mmol / L) and 12.04 pmol / L*mmol / L (±2.93 pmol / L*mmol / L), respectively, showing a significant difference (P=0.023). Statistical analysis shows that alanine levels differ significantly among patients with different degrees of fatty liver, and the glucagon-alanine index also demonstrates statistical significance in assessing the degree of steatosis. This indicates that alanine has a good application in assessing metabolic-associated fatty liver disease (MAFLD).

[0219] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of this application and not to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A pyruvate oxidase stabilizer, characterized in that, The pyruvate oxidase stabilizer comprises the following components by mass percentage: 3-20% ethylene glycol, 0.1-0.5% mannitol, 0.5-5% trehalose, 0.0002-0.002% flavin adenine dinucleotide, 0.0005-0.092% thiamine pyrophosphate, 0.01-0.24% water-soluble magnesium salt, 0.005-0.02% primary preservative, balance primary buffer.

2. The pyruvate oxidase stabilizer according to claim 1, characterized in that, The pyruvate oxidase stabilizer comprises the following components by mass percentage: 5-10% ethylene glycol, 0.1-0.5% mannitol, 0.5-5% trehalose, 0.0004-0.001% flavin adenine dinucleotide, 0.0023-0.05% thiamine pyrophosphate, 0.05-0.12% water-soluble magnesium salt, 0.005-0.02% primary preservative, balance primary buffer.

3. The pyruvate oxidase stabilizer according to claim 1, characterized in that, The pH value of the first buffer solution is 7.0-7.4; And / or, the molar concentration of the solute in the first buffer solution is 10-50 mmol / L; And / or, the first buffer comprises any one of phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 2-morpholine ethanesulfonic acid buffer; And / or, the water-soluble magnesium salt includes at least one of magnesium sulfate and magnesium chloride; And / or, the first preservative includes at least one of gentamicin sulfate, Proclin 300, and KY100.

4. The method for preparing the pyruvate oxidase stabilizer according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) Add ethylene glycol to the first buffer solution and mix well. Then add mannitol, trehalose, water-soluble magnesium salt and the first preservative in sequence and stir well. Then let it stand at 35-40℃ for 20-24h to obtain mixture A. (2) Add flavin adenine dinucleotide and thiamine pyrophosphate to mixture A in sequence, stir at 20-30℃ for 30-60 min, then filter, collect the filtrate, and obtain pyruvate oxidase stabilizer.

5. The use of the pyruvate oxidase stabilizer as described in any one of claims 1-3 in the preparation of an alanine detection kit.

6. An alanine detection kit, characterized in that, The alanine detection kit includes reagent R1, reagent R2 and reagent R3; The reagent R1 includes pyruvate oxidase, catalase, and the pyruvate oxidase stabilizer as described in any one of claims 1-3; The reagent R2 includes a second buffer, alanine aminotransferase, α-ketoglutarate, and a second preservative. The reagent R3 comprises dioxaneboronic acid and carbonate buffer.

7. The alanine detection kit according to claim 6, characterized in that, In reagent R1, the concentration of pyruvate oxidase is 2-50 U / mL, and the concentration of catalase is 2-50 U / mL. And / or, in reagent R2, the concentration of alanine aminotransferase is 2-50 U / mL, the molar concentration of α-ketoglutarate is 0.2-2 mmol / L, and the mass percentage of the second preservative is 0.02-0.04%. And / or, the second preservative includes sodium azide or thimerosal, and also includes at least one of gentamicin sulfate and Proclin 300; And / or, the second buffer comprises any one of phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and 2-morpholine ethanesulfonic acid buffer.

8. The use of the alanine detection kit as described in any one of claims 6-7 in the preparation of products for detecting alanine content.

9. The use of the alanine assay kit according to any one of claims 6-7 in the preparation of products for assessing metabolic-associated fatty liver disease.

10. A method for detecting alanine content using the alanine detection kit according to claim 6 or 7, characterized in that, The method includes the following steps: The sample to be tested is mixed with reagent R1 for the first action, then reagent R2 is added for the second action, followed by reagent R3 for the third action. Finally, the light signal intensity is detected, and the alanine content in the sample is calculated based on the light signal intensity-concentration standard curve.

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