A reagent for determining serum choline and its method of use

Abstract

This invention discloses a reagent for determining choline in serum and its usage method, relating to the field of in vitro diagnostic reagent technology. It comprises the following components: MOPS, polyether F-68, dimethyl sulfoxide, prolin 300, potassium chloride, lithium acetate, calcium acetate, sodium tetraborate, sodium chloride, and choline. This invention provides an electrode filling solution that can be directly used to measure serum choline content using an electrode method. This method requires no sample preparation, has a short detection time, and provides stable and reliable results, avoiding the instability of choline in serum. It is compatible with appropriate equipment for quantitative detection. It solves the problem of inaccurate results due to the easy decomposition of choline during storage and also provides a low-cost method for detecting choline with results equivalent to tandem mass spectrometry.

Description

Technical Field

[0001] This invention relates to the field of medical in vitro diagnostic reagent technology, specifically to a reagent for measuring choline in serum and its method of use. Background Technology

[0002] Choline is a water-soluble essential nutrient, often classified as a member of the B vitamins (vitamin B4). It plays an irreplaceable role in the human body: Cell membrane structure: It is a key component of phospholipids (such as phosphatidylacetylcholine, i.e., lecithin), constituting all cell membranes. Neurotransmitter synthesis: It is a precursor to the synthesis of acetylcholine, a key neurotransmitter for memory, mood, muscle control, and many other brain functions. Lipid transport: It promotes fat transport and metabolism in the liver, preventing fatty liver. Methyl donor: It participates in one-carbon unit metabolism, influencing gene expression and DNA synthesis. Differences across different life stages: Newborns and infants: Serum choline levels are typically much higher than in adults, reaching 20-40 µmol / L or higher. This is closely related to their rapid growth, nervous system development, and specific metabolic needs. Pregnant women: During pregnancy, the need for choline increases to support fetal brain development. Plasma choline levels in pregnant women may change, making adequate choline intake crucial. High-risk groups for choline deficiency include: patients on long-term total parenteral nutrition (TPN) without choline supplementation, severely malnourished individuals, and certain populations with increased choline requirements (such as pregnant women). Consequences of deficiency: Choline deficiency can lead to fatty liver, muscle damage, and nervous system dysfunction. Monitoring serum choline levels helps in early detection and intervention.

[0003] Existing technology uses liquid chromatography-tandem mass spectrometry for detection, but the instruments are very expensive, the operation is complex, it requires professional personnel, and the analysis time is relatively long. Summary of the Invention

[0004] The purpose of this invention is to provide a reagent for determining choline in serum and a method for using it, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a reagent for determining choline in serum, comprising the following components: MOPS 1.18±10% g / l Polyether F-68 0.9±10% g / l Dimethyl sulfoxide 300±10% ml / l Prolin 300 0.5±10% ml / l Potassium chloride 0.2917±5% g / l Lithium acetate 0.0927±3% g / l Calcium acetate 0.2345±5% g / l Sodium tetraborate 0.8182±10% g / l Sodium chloride 5.9±10% g / l Choline 0.038±3% g / l.

[0006] Preferably, weigh and tare a 1-liter beaker, add 900 ml of purified water, and then add MOPS, polyether F-68, dimethyl sulfoxide, prolin300, potassium chloride, lithium acetate, calcium acetate, sodium tetraborate, sodium chloride, and choline in sequence. Stir until homogeneous and bring to a final volume to obtain the reagent.

[0007] Preferably, a method of using a reagent for determining choline in serum includes the following steps: Step 1: Inject the prepared choline reagent into the corresponding channel or reagent position of the electrode; Step 2: Perform two-point calibration using low-concentration and high-concentration choline standard substances. The instrument automatically measures the potential value generated by the standard substances and establishes a standard curve between the potential and the logarithm of the choline concentration. Step 3: Measure the quality control serum to verify the effectiveness of the calibration curve; Step 4: Directly aspirate or drip the serum sample to be tested into the sample detection area and read the result.

[0008] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an electrode filling solution for directly measuring serum choline levels using an electrode method. This method requires no sample preparation, has a short detection time, and provides stable and reliable results, avoiding the instability of choline in serum. It can be used with appropriate equipment for quantitative detection. It solves the problem of inaccurate results due to the easy decomposition of choline during storage, and also provides a low-cost method for choline detection with results equivalent to tandem mass spectrometry. Detailed Implementation

[0009] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0010] 1. Reagent preparation (electrode filling solution / stabilizing reagent) Prepare the core reagents of this patent according to the following formula and steps: Formula (for preparing 1L): Material Name Theoretical dosage Function Description MOPS 1.18 g Buffer system to maintain pH stability Polyether F-68 0.9 g Buffer system, stabilizes pH Dimethyl sulfoxide 300 mL Key: Ensure the stability of choline storage Prolin300 0.5 mL antibacterial agent Potassium chloride 0.2917 g To maintain a specific ion concentration, the necessary equipment is required. Lithium acetate 0.0927 g To maintain a specific ion concentration, the necessary equipment is required. Calcium acetate 0.2345 g To maintain a specific ion concentration, the necessary equipment is required. Sodium tetraborate 0.8182 g To maintain a specific ion concentration, the necessary equipment is required. Sodium chloride 5.9 g To maintain a specific ion concentration, the necessary equipment is required. choline 0.038 g The object being measured provides a calibration benchmark. Preparation steps: Take a 1L beaker, tare it, and zero it.

[0011] Add approximately 900 mL of purified water.

[0012] Weigh and add MOPS, polyether F-68, dimethyl sulfoxide, Prolin300, potassium chloride, lithium acetate, calcium acetate, sodium tetraborate, sodium chloride, and choline in sequence.

[0013] Stir until completely dissolved and mixed evenly.

[0014] Make up to 1L with purified water and mix thoroughly.

[0015] 2. Preparation of Instruments and Materials Instruments: A matching electrolyte analyzer or an ion analyzer with a choline detection module, and a dedicated choline ion selective electrode.

[0016] Calibrator: Choline standard solutions of known concentrations (e.g., 40 μmol / L and 10 μmol / L quality control standards).

[0017] Sample: Fresh or properly preserved serum sample (no complicated pretreatment required).

[0018] 3. Testing Steps Step 1: Instrument Start-up and Preparation Turn on the electrolyte analyzer and install the choline ion selective electrode and reference electrode.

[0019] Inject the prepared choline reagent (electrode filling solution) into the corresponding channel or reagent position of the electrode.

[0020] Step 2: System Calibration Two-point calibration was performed using choline standards at low concentrations (e.g., 10 μmol / L) and high concentrations (e.g., 40 μmol / L).

[0021] The instrument automatically measures the potential value generated by the standard substance and establishes a standard curve between the potential (E) and the logarithm of choline concentration (log C).

[0022] Example of calibration parameters (from the manual): CHO = 40 K correction y=kc+dk=1.00 d=-0.15, indicating that the system has good linearity.

[0023] Step 3: Quality control sample testing Determine the quality control serum (with known concentration) to verify the effectiveness of the calibration curve.

[0024] The quality control results must be within the allowable range of the theoretical concentration (e.g., ±10%).

[0025] Step 4: Sample Testing The serum sample to be tested is directly aspirated or dripped into the sample detection site.

[0026] The instrument automatically introduces the sample into the measuring cell, and the choline electrode detects the change in its potential.

[0027] The instrument's internal processor automatically converts the potential value into choline concentration (μmol / L) based on the calibration curve.

[0028] After the test is completed, the instrument automatically cleans the electrodes and prepares for the next sample.

[0029] Step 5: Result Reading and Reporting The choline concentration results of the serum sample can be read directly on the instrument display screen or the connected computer software. II. The results of detection using liquid chromatography-tandem mass spectrometry are compared with those of this application. 1. Main Instruments Liquid Chromatography System: Agilent Technologies 1200 series Mass spectrometry system: Agilent 6410A Triple Quad mass spectrometer (equipped with an electrospray ionization source) Centrifuge: Eppendorf-5415R High-Speed ​​Refrigerated Centrifuge Pure water system: Milli-Pore pH meter: Denver UB-10 2. Main reagents Standard: Choline (Sigma-Aldrich) Isotope internal standard: d3-choline (Isotec) Chromatographic reagents: Acetonitrile (Merck, chromatographic grade), ammonium formate (AlfaAesar) Other: Phosphate buffer 3. Testing Steps Step 1: Sample Collection and Processing Collect 3 mL of fasting venous whole blood in the morning.

[0030] Centrifuge at 4℃ and 3000×g for 15 minutes within 30 minutes to separate the serum.

[0031] Dispense into EP tubes and store as required.

[0032] Step 2: Solution preparation Standard stock solution: Prepare 20 mmol / L choline stock solution with PBS and store at -20°C.

[0033] Standard application solution: Dilute the stock solution to a series of concentrations of 0.62–400 μmol / L with mixed serum diluted 10 times.

[0034] Internal standard solution (also for protein removal): Prepare 10 mmol / L d3-choline stock solution with PBS, and dilute to 10 μmol / L with acetonitrile before use.

[0035] Mobile phase: 15 mmol / L ammonium formate solution (pH 3.5), filtered and degassed before use.

[0036] Step 3: Sample pretreatment Take 30 μL of serum or standard working solution.

[0037] Add 90 μL of internal standard solution (acetonitrile containing internal standard) and vortex to mix.

[0038] Centrifuge at 4℃, 13,000×g for 10 minutes.

[0039] The supernatant was filtered through a centrifugal filter column (3000×g, 2 minutes).

[0040] Collect approximately 90 μL of filtrate, transfer it to a sample vial, and prepare it for instrumentation.

[0041] Step 4: Chromatographic and Mass Spectrometric Conditions Chromatographic column: Ultimate SiO2 column (2.1 mm × 100 mm, 5 μm) Mobile phase: Acetonitrile : 15 mmol / L ammonium formate buffer (pH 3.5) = 60 : 40 (isocratic elution) Flow rate: 0.2 mL / min Column temperature: 25℃ Injection volume: 5 μL Ion source: Electrospray ionization source (positive ion mode) Monitoring mode: Multiple response monitoring Major MRM ion pairs: Step 5: Quantitative Analysis The series of standard application solutions and samples are processed and loaded onto the machine simultaneously.

[0042] Plot a standard curve with the concentration of the standard on the x-axis and the ratio of the peak areas of the standard to the internal standard on the y-axis.

[0043] The concentration is calculated by substituting the peak area ratio of the sample into the standard curve.

[0044] The following is a comparison of this application with existing mass spectrometry detection methods: Calibration parameters CHO = 40 K-correction y = kc + dk = 1.00 d = -0.15 Theoretical concentration of quality control standard 1: 40 μmol / L (1SD±10%), theoretical concentration of quality control standard 2: 10 μmol / L (1SD±10%) The test results show that both tandem mass spectrometry and the results of this invention are within the quality control range. The correlation coefficient between the two methods for testing serum samples is 0.9996, indicating that the test results of the two methods are equivalent, and the results of the enzyme method and the tandem mass spectrometry method are consistent.

[0045] The quality control test results were obtained after one year of reagent storage: Quality Control 1 concentration: 39.2, Quality Control 2 concentration: 10.3. Conclusion: The serum choline (CHO) assay kit (enzymatic method) and tandem mass spectrometry (MS / MS) yielded equivalent results, and the enzymatic method can replace MS / MS. The quality control results remained unchanged after one year of storage.

[0046] This method is based on the ion-selective electrode (ISE) method. A choline ion-selective electrode has an electrode membrane containing a sensing substance that selectively responds to choline ions. When the electrode is immersed in a solution containing choline ions (serum sample or standard solution), selective ion exchange occurs between the choline ions and the electrode membrane, generating a potential difference (membrane potential) across the membrane. This membrane potential is proportional to the logarithm of the choline ion activity in the solution (approximately equal to the concentration in dilute solutions), conforming to the Nernst equation. By measuring the potential difference between the electrode and the reference electrode and comparing it with a standard solution of known concentration (calibration), the concentration of choline in the sample can be calculated. This application provides a long-term stable choline electrode filling solution / reagent, in which key components (such as dimethyl sulfoxide) effectively maintain the stability of choline in solution, solving the problem of inaccurate results caused by the easy decomposition of choline.

Claims

1. A reagent for determining choline in serum, characterized in that, Includes the following components: MOPS 1.18±10% g / l Polyether F-68 0.9±10% g / l Dimethyl sulfoxide 300±10% ml / l Prolin 300 0.5±10% ml / l Potassium chloride 0.2917±5% g / l Lithium acetate 0.0927±3% g / l Calcium acetate 0.2345±5% g / l Sodium tetraborate 0.8182±10% g / l Sodium chloride 5.9±10% g / l Choline 0.038±3% g / l.

2. The method for preparing a reagent for determining choline in serum according to claim 1, characterized in that, Weigh and tare a 1-liter beaker, add 900 ml of purified water, and then add MOPS, polyether F-68, dimethyl sulfoxide, prolin300, potassium chloride, lithium acetate, calcium acetate, sodium tetraborate, sodium chloride, and choline in sequence. Stir until the mixture is homogeneous and the volume is adjusted to obtain the reagent described above.

3. The method of using the reagent for determining choline in serum according to claim 1, characterized in that, Includes the following steps: Step 1: Inject the prepared choline reagent into the corresponding channel or reagent position of the electrode; Step 2: Perform two-point calibration using low-concentration and high-concentration choline standard substances. The instrument automatically measures the potential value generated by the standard substances and establishes a standard curve between the potential and the logarithm of the choline concentration. Step 3: Measure the quality control serum to verify the effectiveness of the calibration curve; Step 4: Directly aspirate or drip the serum sample to be tested into the sample detection area and read the result.