Method for quantitatively detecting pyrophosphate

Abstract

The invention discloses a method for quantitatively measuring pyrophosphoric acid (hereinafter referred to as PPi) in human plasma. According to the method, a sample and radioactive isotope labeled uridine diphosphate glucose are subjected to an enzymatic reaction in a reaction system, and the radioactive intensity before and after the enzymatic reaction is measured so as to calculate the content of pyrophosphate in the sample. According to the method, unreacted uridine diphosphate glucose is adsorbed by activated carbon before determination, the use concentration of the activated carbon is 3-3.5% (w / v), and the pH value is 7.2-8.5. The method can be used for detecting the content of PPi in plasma and is not influenced by the matrix effect.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and relates to a method for quantitatively detecting pyrophosphate, and specifically to a method for quantitatively detecting pyrophosphate in human plasma. Background Art

[0002] Hypophosphatasia (HPP) is a rare hereditary metabolic bone disease characterized by low activity of tissue nonspecific alkaline phosphatase (TNSALP), which leads to excess extracellular organic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP). Therefore, clinical diagnosis can be made by detecting the content of the biomarker PPi in plasma. Radioactive isotopes such as tritium-labeled UDP-G* and PPi generate glucose*-1-phosphate under the action of 2-phosphouridine-glucose pyrophosphorylase. Then, it reacts with D-glucose-1,6-diphosphate under the action of glucose phosphomutase to generate glucose*-6-phosphate. And it continues to generate 6-phospho-gluconolactone* with glucose-6-phosphate dehydrogenase under the action of NADP+ coenzyme. The UDP-G* that does not participate in the reaction is adsorbed with activated carbon, and the remaining radioactivity is detected to estimate the content of PPi. Summary of the invention

[0003] The technical problem to be solved by the present invention is that the existing technology lacks a detection method directly targeting plasma samples due to the significant matrix effect when detecting the content of organic pyrophosphate (PPi). A method for quantitatively detecting pyrophosphate is provided, which can be directly applied to the detection of PPi content in plasma samples. By optimizing the reaction conditions and eliminating the interference of plasma matrix, the accurate detection of plasma PPi content can be achieved.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0005] The first aspect of the present invention provides a method for quantitatively detecting pyrophosphate (PPi), characterized in that the method comprises subjecting a sample to an enzymatic reaction with radioactive isotope-labeled uridine diphosphate glucose in a reaction system, and measuring the radioactivity intensity before and after the enzymatic reaction to calculate the content of pyrophosphate in the sample;

[0006] In some embodiments of the present invention, the method comprises using activated carbon to absorb unreacted UDP-glucose before the determination, wherein the activated carbon is used at a concentration of 3-3.5% (w / v) and a pH of 7.2-8.5.

[0007] In some embodiments of the present invention, the activated carbon is used at a pH of 7.2-7.6, 7.6-8.0 or 8.0-8.5.

[0008] In some embodiments of the invention, the sample is a plasma sample.

[0009] In some embodiments of the present invention, the activated carbon is used at a concentration of 3% (w / v) and a pH of 7.2.

[0010] In some embodiments of the present invention, the activated carbon is used at a concentration of 3.5% (w / v) and a pH of 7.2.

[0011] In some embodiments of the present invention, the activated carbon is used at a concentration of 3.5% (w / v) and a pH of 7.6.

[0012] In some embodiments of the present invention, the activated carbon is used at a concentration of 3.5% (w / v) and a pH of 8.0.

[0013] In some embodiments of the present invention, the activated carbon is used at a concentration of 3.5% (w / v) and a pH of 8.5.

[0014] In some embodiments of the invention, the radioisotope is selected from phosphorus ( 32 P), carbon ( 14 C) and tritium ( 3 H).

[0015] In some embodiments of the present invention, the reaction system comprises D-glucose-1,6-diphosphate, phosphoglucomutase and glucose-6-phosphate dehydrogenase.

[0016] A second aspect of the present invention provides a use of a reagent for detecting pyrophosphate in the preparation of a reagent for diagnosing hypophosphatasia.

[0017] In some embodiments of the present invention, the reagent for detecting pyrophosphate detects pyrophosphate by the method described in the first aspect of the present invention.

[0018] In the present invention, the term "organic pyrophosphate (PPi)" refers to an organic compound formed by two phosphate groups connected by an ester bond, which participates in various metabolic processes in the body. In the human body, PPi is a key product of phosphate metabolism, and its abnormal level is closely related to metabolic diseases such as hypophosphatasia.

[0019] "Radioactive isotope" refers to a radioactive atomic nucleus, the presence of which can be detected by emitting radiation. In the present invention, radioactive isotopes are used to label UDP-glucose, and quantitative detection of PPi is achieved by measuring the radioactivity intensity.

[0020] Enzymatic reaction: refers to a series of reactions carried out under the catalysis of a specific enzyme. The present invention includes three coupled enzymatic reactions: first, under the catalysis of UDP (uridine diphosphate)-glucose pyrophosphorylase, PPi reacts with UDP-G* to generate UDP and glucose-1-phosphate; then, under the catalysis of phosphoglucomutase, glucose*-1-phosphate is converted into D-glucose*-1,6-phosphate; finally, under the catalysis of glucose-6-phosphate dehydrogenase, D-glucose-1,6-phosphate reacts with NADP+ to generate 6-phospho-gluconolactone* and NADPH. The content of PPi in the sample is calculated by measuring the changes in the radioactive markers before and after the reaction.

[0021] w / v: Weight-to-volume concentration, which indicates the number of grams of solute contained in every 100 ml of solution. For example, 3.5% (w / v) activated carbon means that there are 3.5 grams of activated carbon in every 100 ml of solution.

[0022] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0023] The reagents and raw materials used in the present invention are commercially available.

[0024] The positive progressive effect of the present invention is that the present invention provides a method for detecting the content of organic pyrophosphate (PPi) in plasma using tritium-labeled UDP-G* and an enzymatic reaction chain, and the method has the following significant advantages: the detection limit can reach 750pmol / mL, which meets the clinical diagnosis needs; by optimizing the reaction conditions of 3.5% activated carbon concentration and pH 8.0, the accuracy and reproducibility of the measurement results are improved; the method adopts conventional enzymatic reaction conditions, and the operation is simple and controllable, which is convenient for long-term storage and batch testing of clinical samples; importantly, the method is not affected by the plasma matrix effect, can be directly applied to plasma sample detection, is accurate and reliable, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the enzymatic reaction chain for the detection of organic pyrophosphate (PPi).

[0026] Figure 2 Radioenzymatic standard curve for pyrophosphate (PPi) in human plasma (0.75-12 μM). DETAILED DESCRIPTION

[0027] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0028] 1. Applicability of the Method

[0029] Pyrophosphate (PPi) assay for quantitative determination of PPi concentration in human plasma (whole blood was treated with K2EDTA anticoagulant before collection, human plasma source: Hunan Women and Children's Hospital). The PPi enzymatic reaction pathway for the test is shown in Figure 1 shown.

[0030] 2. Main instruments

[0031] Tri-Carb® 4910 TR Liquid Scintillation Counter

[0032] Refrigerator (temperature controlled at 5 ± 3°C)

[0033] Biochemical incubator (temperature controlled at 37 ± 2°C)

[0034] Vortex mixer, micro refrigerated centrifuge, ultrapure water instrument, etc.

[0035] 3. Key Reagents

[0036]

[0037]

[0038] IV. Reagent Preparation

[0039] 1. Prepare 0.5M Trizma acetate (code: TrisA) at pH 8.0

[0040] 2. Prepare 0.1M magnesium acetate (code: MgAc)

[0041] 3. Prepare 10mM disodium NADP (code: NADP)

[0042] 4. Prepare 1.24mM α-D-glucose 1,6-diphosphate potassium salt hydrate (code: G1,6DP)

[0043] 5. Prepare 3.28mM uridine-5'-diphosphoglucose disodium salt hydrate (code: UDPG)

[0044] 6. Prepare 0.5U / μL of glucose-6-phosphate dehydrogenase (code: G6PD)

[0045] 7. Prepare 0.4U / μL phosphoglucomutase (code: PGM)

[0046] 8. Prepare 0.1U / μL uridine-5'-diphosphoglucose pyrophosphorylase (code: UDPGPP)

[0047] 9. Prepare 3.5% activated carbon with pH 8.0 (code: Char3.5%)

[0048] 10. Prepare 1 μCi / μL of tritiated uridine diphosphate glucose ammonium salt (code: 3H-UDPG)

[0049] 11. Prepare the reaction mixture according to the following ratio: (code: RxM)

[0050]

[0051] 5. Preparation of PPi Standards and QC Samples

[0052] 1. Prepare 10 mM PPi stock solution (PPi STD Stock 1 (Sigma-Aldrich, Cat#221368) or PPi QC Stock 1 (Sigma-Aldrich, Cat#221368)) with ultrapure water.

[0053] 2. Dilute PPi STD Stock 1 with ultrapure water to seven standard curve concentration points: 12.00, 10.00, 8.00, 4.00, 1.60, 1.00, and 0.75 μM

[0054] 3. Dilute PPi QC Stock 1 into three quality control concentration points of 9.00, 5.00, and 1.25 μM using blank plasma matrix and ultrapure water.

[0055] 6. Experimental process

[0056] 1. Add 50 μL of standard, QC sample or plasma sample to a 1.5 mL centrifuge tube.

[0057] 2. Add 230 μL of reaction mixture to all centrifuge tubes. Vortex all the centrifuge tubes at low speed to mix well.

[0058] 3. Incubate at 37°C with low-speed shaking for 30 minutes.

[0059] 4. Add 400 μL of pre-cooled 3.5% activated carbon.

[0060] 5. Vortex at low speed for 2 minutes.

[0061] 6. Incubate at 4°C for 30 minutes. Vortex again for 2 minutes at the midpoint of the incubation (around 15 minutes).

[0062] 7. Centrifuge at 12,000 rpm for 12 minutes at 4°C.

[0063] 8. Transfer 200 μL of supernatant to a scintillation vial.

[0064] 9. Add 4 mL of scintillation fluid (Optiphase Hisafe 3, Revvity, Cat#1200.437).

[0065] 10. Count for 1 minute using a liquid scintillation counter.

[0066] VII. Experimental Results

[0067] The data were collected and analyzed, and the results are shown in Tables 1 to 3.

[0068] Table 1 PPi STD test results

[0069]

[0070] Table 2 Detection results of plasma sample KB143

[0071]

[0072] Table 3 Summary of repeatability test results of plasma sample KB143

[0073]

[0074] The standard curve is as follows Figure 2 shown.

[0075] The above results indicate that this method can quantitatively determine the concentration of PPi in human plasma in the range of 0.75 μM-12 μM, and the precision, accuracy and specificity of the method are in line with the requirements of the ICH M10 Bioanalytical Method Validation and Sample Analysis guidelines.

Claims

1. A method for quantitatively detecting pyrophosphate (PPi), characterized in that: The method comprises carrying out an enzymatic reaction between a sample and radioactive isotope-labeled uridine diphosphate glucose in a reaction system, and measuring the radioactivity intensity before and after the enzymatic reaction to calculate the content of pyrophosphate in the sample; The method comprises the following steps: using activated carbon to absorb unreacted UDP-glucose before measuring, wherein the activated carbon is used at a concentration of 3-3.5% (w / v) and a pH of 7.2-8.

5.

2. The method according to claim 1, characterized in that The use pH of the activated carbon is 7.2-7.6, 7.6-8.0 or 8.0-8.5; And / or, the sample is a plasma sample.

3. The method according to claim 1, characterized in that The activated carbon was used at a concentration of 3% (w / v) and a pH of 7.

2.

4. The method according to claim 1, characterized in that The activated carbon was used at a concentration of 3.5% (w / v) and a pH of 7.

2.

5. The method according to claim 1, characterized in that The activated carbon was used at a concentration of 3.5% (w / v) and a pH of 7.

6.

6. The method according to claim 1, characterized in that The activated carbon was used at a concentration of 3.5% (w / v) and a pH of 8.

0.

7. The method according to claim 1, characterized in that The activated carbon was used at a concentration of 3.5% (w / v) and a pH of 8.

5.

8. The method according to any one of claims 1 to 7, characterized in that: The radioactive isotope is selected from phosphorus ( 32 P), carbon ( 14 C) and tritium ( 3 H).

9. The method according to claim 1, characterized in that The reaction system comprises D-glucose-1,6-diphosphate, phosphoglucomutase and glucose-6-phosphate dehydrogenase.

10. Use of a reagent for detecting pyrophosphate in the preparation of a reagent for diagnosing hypophosphatasia; The reagent for detecting pyrophosphate detects pyrophosphate by the method according to any one of claims 1-9.

Images