Sample pretreatment method for detecting activity of CD38 enzyme in whole blood sample and method for detecting activity of CD38 enzyme in blood by high performance liquid chromatography

Abstract

The invention provides a sample pretreatment method for detecting the activity of a CD38 enzyme in a whole blood sample and a method for detecting the activity of the CD38 enzyme in the blood through high performance liquid chromatography, and belongs to the technical field of high performance liquid chromatography detection. According to the method, a whole blood sample is pre-incubated, endogenous beta-NAD is consumed, an exogenous substrate APAD is introduced for enzymatic reaction, then an exogenous product 3-acetylpyridine (3-AP) is generated, the generation of quantitative 3-AP is used for reflecting the enzymatic activity of CD38, and the interference of endogenous products is avoided. Meanwhile, 2-Me-5-AP is selected as an internal standard to be added into an enzymatic reaction system for quantification, high performance liquid chromatography quantitative detection after sample pretreatment is facilitated, and the detection accuracy is higher.

Description

Technical Field

[0001] The present invention belongs to the technical field of high performance liquid chromatography detection, and in particular relates to a sample pretreatment method for detecting CD38 enzyme activity in a whole blood sample and a method for detecting blood CD38 enzyme activity by high performance liquid chromatography. Background Art

[0002] CD38 is a multifunctional enzyme widely distributed in various tissues. Its main function is to consume nicotinamide adenine dinucleotide (NAD) in the body and plays an important role in NAD catabolism and maintaining NAD homeostasis in the body. Compared with quantitative methods such as measuring CD38 content, measuring CD38 enzyme activity can better reflect its biological function. Existing methods for measuring CD38 enzyme activity include enzyme cycling, thin layer chromatography, fluorescence spectrophotometry, etc., which have the disadvantages of cumbersome operation, need for radioactive isotopes, and low accuracy. High performance liquid chromatography (HPLC) to measure the reduction of enzyme reaction substrate or product generation to reflect CD38 enzyme activity is a better detection strategy. The principle of HPLC to measure CD38 enzyme activity is: β-NAD is an important coenzyme involved in redox reactions in the body, which can directly or indirectly affect many key cell functions, and its content is mainly regulated by CD38. Under the action of CD38, β-NAD can be degraded to generate adenosine diphosphate ribose (ADPR) and NAM, and all three have strong ultraviolet absorption. The product NAM is detected by HPLC, and the standard curve external standard method is used for quantification. The activity of CD38 enzyme is determined by the amount of NAM generated.

[0003] Recently, Zhao Xianghui et al. disclosed a method for determining CD38 enzyme activity by HPLC. The specific steps are as follows: 50 μl of whole blood is selected as the test sample, 150 μl of β-NAD with a concentration of 500 μmol / L is used as the enzyme reaction substrate, whole blood is pre-incubated to consume endogenous β-NAD, substrate β-NAD is added, incubated at 37°C for 40 minutes, perchloric acid (PCA) is used to terminate the reaction, and HPLC is used to determine the change in the product nicotinamide (NAM) before and after the enzymatic reaction and calculate the CD38 enzyme activity. The linearity, detection limit, quantification limit, precision, recovery rate and stability of the method are evaluated (see [Zhao Xianghui, Bu Xiaoxiao, Zhu Hanyu, et al. Establishment of a method for detecting blood CD38 enzyme activity by high performance liquid chromatography. Chinese Journal of Laboratory Medicine, 2023, 46(10): 1074-1082]). However, this HPLC method for determining CD38 enzyme activity also has some problems, such as the product NAM is an endogenous product, which interferes with the determination of the amount of enzymatic reaction products produced, and the quantitative accuracy needs to be improved. Summary of the invention

[0004] The purpose of the present invention is to provide a sample pretreatment method for detecting CD38 enzyme activity in whole blood samples and a method for detecting blood CD38 enzyme activity by high performance liquid chromatography. The method of the present invention can avoid the interference of endogenous products and has high quantitative accuracy.

[0005] The present invention provides a sample pretreatment method for detecting CD38 enzyme activity in a whole blood sample, comprising the following steps: pre-incubating the whole blood sample to consume endogenous β-NAD to obtain a pre-incubated whole blood sample; mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine to perform an enzymatic reaction, terminating the enzymatic reaction to obtain an enzymatic reaction product; centrifuging the enzymatic reaction product, collecting the supernatant to obtain a sample to be tested.

[0006] Preferably, the working concentration of acetyl adenine dinucleotide is 500 μmol / L; the working concentration of 2-methyl-5-acetyl pyridine is 15.04 μg / ml.

[0007] Preferably, mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine comprises mixing the pre-incubated whole blood sample with a mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine; based on a volume of 50 μl of the whole blood sample, the amount of the mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine is 150 μl.

[0008] Preferably, the pre-incubation is carried out in a water bath; the pre-incubation temperature is 37° C.; and the pre-incubation time is 20 min.

[0009] Preferably, the enzymatic reaction is carried out in a water bath; the temperature of the enzymatic reaction is 37° C.; and the time of the enzymatic reaction is 40 min.

[0010] Preferably, the reagent used to terminate the enzymatic reaction includes acetonitrile; based on 50 μl of the whole blood sample, the amount of acetonitrile used is 200 μl.

[0011] Preferably, the centrifugal temperature is 4°C; the centrifugal speed is 8000 rpm; and the centrifugal time is 10 min.

[0012] The present invention also provides a method for detecting blood CD38 enzyme activity by high performance liquid chromatography, comprising the following steps: obtaining a sample to be detected by the sample pretreatment method described in the above scheme; performing high performance liquid chromatography detection on the sample to be detected to obtain the peak area of ​​the product 3-acetylpyridine and the peak area of ​​the internal standard 2-methyl-5-acetylpyridine, and performing quantitative analysis using the internal standard method to obtain the whole blood CD38 enzyme activity; calculating the blood CD38 enzyme activity according to the formula shown in the following formula 1; the CD38 enzyme activity is expressed in the international unit of enzyme activity Katal;

[0013] 1nKatal / L=[L1×(S1 / S2)×C1] / (L2×T) Equation 1;

[0014] In Formula 1, L1 is the volume of the mixed solution including acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine, unit: L; L2 is the volume of the whole blood sample, unit: L; S1 is the product peak area, unit: mAU; S2 is the internal standard peak area, unit: mAU; C1 is the internal standard concentration, unit: μmol / L; T is the time of the enzymatic reaction, unit: s.

[0015] Preferably, the chromatographic column used for the HPLC detection is a C18 reverse phase chromatographic column; the mobile phase for the HPLC detection consists of an aqueous NaH2PO4 solution and acetonitrile; the concentration of NaH2PO4 in the aqueous NaH2PO4 solution is 0.1 mol / L; the volume ratio of the aqueous NaH2PO4 solution to acetonitrile is 55:45; the elution mode for the HPLC detection is isocratic elution; the detection wavelength for the HPLC detection is 260 nm; the flow rate of the mobile phase is 1 ml / min; the injection volume for the HPLC detection is 20 μl; and the elution time for the HPLC detection is ≤5 min.

[0016] Preferably, after obtaining the whole blood CD38 enzyme activity, the method further comprises dividing the whole blood CD38 enzyme activity by the red blood cell count for correction.

[0017] The present invention provides a sample pretreatment method for detecting CD38 enzyme activity in a whole blood sample, comprising the following steps: pre-incubating the whole blood sample, consuming endogenous β-NAD, and obtaining a pre-incubated whole blood sample; mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide (APAD) and 2-methyl-5-acetyl pyridine (2-Me-5-AP), performing an enzymatic reaction, terminating the enzymatic reaction, and obtaining an enzymatic reaction product; centrifuging the enzymatic reaction product, collecting the supernatant, and obtaining a sample to be tested. The present invention pre-incubates the whole blood sample, consumes endogenous β-NAD, introduces exogenous substrate APAD for enzymatic reaction, and then generates an exogenous product 3-acetyl pyridine (3-AP), and quantifies the generation of 3-AP to reflect the enzyme activity of CD38, thereby avoiding the interference of endogenous products. At the same time, the present invention selects 2-Me-5-AP as an internal standard and adds it to the enzymatic reaction system for quantification, which is beneficial to the quantitative detection of high performance liquid chromatography after sample pre-treatment, and the detection accuracy is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 is the chemical formula of the enzyme reaction; wherein APAD is acetyl adenine dinucleotide; ADPR is adenosine diphosphate ribose; 3-AP is 3-acetyl pyridine;

[0020] Figure 2 The flowchart of CD38 enzyme activity assay is shown in the figure; wherein: RT, room temperature; Cocktail is a mixture of APAD, acetyl adenine dinucleotide and 2-Me-5-AP, 2-methyl-5-acetyl pyridine; ACN, acetonitrile; HPLC, high performance liquid chromatography; 3-AP, 3-acetyl pyridine; 2-Me-5-AP, 2-methyl-5-acetyl pyridine;

[0021] Figure 3 is the standard curve of 3-AP; 3-AP is 3-acetylpyridine;

[0022] Figure 4 The graphs are sample stability results; (a) is the stability result of the sample at 4°C; (b) is the stability result of the sample at room temperature; (c) is the stability result of the sample at room temperature after thawing; (d) is the stability result of the sample after repeated freezing and thawing for 3 times;

[0023] Figure 5The stability results of the standard products are shown in the figure; (a) is the stability results of the standard products APAD, 3-AP and IS at 4°C; (b) is the stability results of the standard products APAD, 3-AP and IS at room temperature; APAD is acetyl adenine dinucleotide; 3-AP is 3-acetyl pyridine; IS is 2-Me-5-AP, 2-methyl-5-acetyl pyridine;

[0024] Figure 6 The graphs are injection stability results; (a) is the stability result of the sample after pretreatment at 4°C; (b) is the stability result of the sample after pretreatment at room temperature;

[0025] Figure 7 This is a graph showing the effect of inhibitor 78c on CD38 enzyme activity assay;

[0026] Figure 8 Figure 1 is a comparison result diagram of CD38 activity in the tumor group and the healthy group before and after RBC count correction; A is a comparison result diagram of CD38 activity in the tumor group and the healthy group before RBC count correction; B is a comparison result diagram of CD38 activity in the tumor group and the healthy group after RBC count correction; RBC is red blood cell;

[0027] Fig. 9 The figure shows the changes of CD38 activity corrected by RBC count with age in the tumor group and the healthy group; RBC is red blood cell. DETAILED DESCRIPTION

[0028] The present invention provides a sample pretreatment method for detecting CD38 enzyme activity in a whole blood sample, comprising the following steps: pre-incubating the whole blood sample to consume endogenous β-NAD to obtain a pre-incubated whole blood sample; mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine to perform an enzymatic reaction, terminating the enzymatic reaction to obtain an enzymatic reaction product; centrifuging the enzymatic reaction product, collecting the supernatant to obtain a sample to be tested.

[0029] The sample pretreatment method of the present invention is based on Figure 1 The enzymatic reaction principle shown is to add substrate APAD to the matrix containing CD38 enzyme activity for enzymatic reaction, and quantify the generation of product 3-AP to reflect the activity of the enzyme, thus avoiding the interference of endogenous products. Therefore, the operation is simple, and only the level of enzyme reaction product at one time point after incubation needs to be measured, making the detection process more efficient and easy to operate.

[0030] The present invention first pre-incubates a whole blood sample to consume endogenous β-NAD to obtain a pre-incubated whole blood sample.

[0031] In the present invention, the whole blood sample is preferably a frozen-thawed whole blood sample. Compared with a fresh whole blood sample, a frozen-thawed whole blood sample is easier to obtain, and the present invention can still accurately measure the frozen-thawed whole blood sample, which is easier to implement at the operational level and does not have to be limited by the blood drawing time; the storage temperature of the frozen whole blood sample is preferably -80°C; the preparation method of the whole blood sample preferably includes: placing the frozen whole blood sample at room temperature for 15 minutes, until it is completely thawed. In the present invention, the pre-incubation is preferably carried out in a water bath; the pre-incubation temperature is preferably 37°C; the pre-incubation time is preferably 20 minutes. The present invention uses blood samples that are more widely used in clinical practice and easier to obtain, and will be easier to promote and apply in the future.

[0032] After obtaining the pre-incubated whole blood sample, the present invention mixes the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine to perform an enzymatic reaction, terminates the enzymatic reaction, and obtains an enzymatic reaction product.

[0033] In the present invention, the working concentration of acetyl adenine dinucleotide is preferably 500 μmol / L; the working concentration of 2-methyl-5-acetyl pyridine is preferably 15.04 μg / ml. In the present invention, the mixing of the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine preferably includes mixing the pre-incubated whole blood sample with a mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine; based on the volume of the whole blood sample of 50 μl, the amount of the mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine is preferably 150 μl. In the present invention, the enzymatic reaction is preferably carried out in a water bath; the temperature of the enzymatic reaction is preferably 37°C; the time of the enzymatic reaction is preferably 40 min. In the present invention, the temperature is stable and controllable under the 37°C water bath condition; the incubation time is 40 min, which greatly shortens the reaction time compared with the 2 to 18 h of the traditional method and improves the detection efficiency. In the present invention, the reagent used to terminate the enzymatic reaction preferably includes acetonitrile (ACN); based on 50 μl of the whole blood sample, the amount of ACN is preferably 200 μl.

[0034] After obtaining the enzymatic reaction product, the present invention centrifuges the enzymatic reaction product, collects the supernatant, and obtains the sample to be tested.

[0035] In the present invention, the centrifugal temperature is preferably 4°C; the centrifugal speed is preferably 8000 rpm; and the centrifugal time is preferably 10 min.

[0036] The present invention also provides a method for detecting blood CD38 enzyme activity by high performance liquid chromatography, comprising the following steps: obtaining a sample to be detected by the sample pretreatment method described in the above scheme; performing high performance liquid chromatography detection on the sample to be detected to obtain the peak area of ​​the product 3-acetylpyridine and the peak area of ​​the internal standard 2-methyl-5-acetylpyridine, and performing quantitative analysis using the internal standard method to obtain the whole blood CD38 enzyme activity; calculating the blood CD38 enzyme activity according to the formula shown in the following formula 1; the CD38 enzyme activity is expressed in the international unit of enzyme activity Katal;

[0037] 1nKatal / L=[L1×(S1 / S2)×C1] / (L2×T) Equation 1;

[0038] In Formula 1, L1 is the substrate volume, unit: L; L2 is the volume of the whole blood sample, unit: L; S1 is the product peak area, unit: mAU; S2 is the internal standard peak area, unit: mAU; C1 is the internal standard concentration, unit: μmol / L; T is the time of the enzymatic reaction, unit: s; nKatal is nakatal, 1nKatal=1×10 -9 Katal; the substrate volume refers to the volume of the mixed solution including the substrate acetyl adenine dinucleotide and the internal standard 2-methyl-5-acetylpyridine.

[0039] In the present invention, the chromatographic column used for the HPLC detection is preferably a C18 reverse phase chromatographic column, more preferably a Waters Symmetry ShieldRP18; the mobile phase for the HPLC detection is preferably composed of a NaH2PO4 aqueous solution and acetonitrile; the concentration of NaH2PO4 in the NaH2PO4 aqueous solution is preferably 0.1 mol / L; the volume ratio of the NaH2PO4 aqueous solution to acetonitrile is preferably 55:45; the elution mode for the HPLC detection is preferably isocratic elution; the detection wavelength for the HPLC detection is preferably 260 nm; the flow rate of the mobile phase is preferably 1 ml / min; the injection volume for the HPLC detection is preferably 20 μl; and the elution time for the HPLC detection is preferably ≤5 min.

[0040] The present invention only needs to quantify the final exogenous product 3-AP, and introduces the internal standard 2-Me-5-AP during quantification. The internal standard method is used for quantification, which corrects the variation in sample processing and chromatographic analysis and significantly improves the accuracy and repeatability of the measurement results.

[0041] After obtaining the whole blood CD38 enzyme activity, the present invention preferably further comprises dividing the whole blood CD38 enzyme activity by the red blood cell (RBC) count for correction. The quantitative method of the present invention corrected by RBC count can effectively reflect the CD38 enzyme activity at the cellular level and has good clinical significance in clinical testing.

[0042] The method of detecting blood CD38 enzyme activity by high performance liquid chromatography of the present invention can rapidly and accurately determine CD38 enzyme activity in a blood sample, and provides powerful technical support for the diagnosis and treatment of related diseases.

[0043] In the present invention, the product 3-AP and the internal standard 2-Me-5-AP can be effectively separated within 5 minutes, which greatly shortens the analysis time and significantly improves the detection efficiency.

[0044] Terminology explanation:

[0045] HPLC: High Performance Liquid Chromatography, high performance liquid chromatography technology, is used to separate and analyze compounds in a mixture. It separates the sample solution by passing it through a column filled with tiny particles and using the differences in the interactions of different compounds on the column. HPLC has the characteristics of high resolution, high sensitivity and rapid analysis, and is widely used in the fields of chemistry, biology, medicine, etc. It is the main detection technology used in this invention.

[0046] β-NAD: β-Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, it participates in energy metabolism and redox reactions in the body. Its main hydrolase is CD38, and it is the main substrate that consumes CD38 in the body.

[0047] APAD: Acetylpyridine adenine dinucleotide, a derivative of β-NAD, has the same glycosidic bond as β-NAD, can be hydrolyzed by CD38, and is an exogenous substrate of CD38.

[0048] 3-AP: 3-acetylpyridine, 3-acetylpyridine, is the product of APAD hydrolysis by CD38.

[0049] 2-Me-5-AP: 2-methy-5-acetylpyridine, 2-methyl-5-acetylpyridine, is the structural analog of 3-AP and the internal standard.

[0050] To further illustrate the present invention, a sample pretreatment method for detecting CD38 enzyme activity in a whole blood sample and a method for detecting CD38 enzyme activity in blood by high performance liquid chromatography provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1 A method for detecting blood CD38 enzyme activity using high performance liquid chromatography (HPLC) technology

[0052] Flowchart as Figure 2 shown.

[0053] 1. Sample preparation: Take out the whole blood sample from the -80℃ refrigerator and place it at room temperature for 15 minutes to completely thaw. Then, take 50μl of the whole blood sample and place it in a 37℃ water bath for 20 minutes to pre-incubate to consume endogenous β-NAD.

[0054] 2. Substrate and internal standard addition: Add 150 μl of a mixed solution containing 15.04 μg / ml 2-Me-5-AP internal standard and 500 μmol / L APAD standard to the pre-incubated whole blood sample. Incubate the mixture in a 37°C water bath for 40 min to perform the enzymatic reaction.

[0055] 3. Reaction termination and sample treatment: After incubation, add 200 μl of acetonitrile (ACN) to terminate the enzymatic reaction. Centrifuge the terminated sample at 8000 rpm for 10 min at 4°C. Pipette 200 μl of the supernatant and transfer it to a sample injection bottle for HPLC analysis.

[0056] 4.HPLC analysis conditions: C18 reverse phase column Waters Symmetry Shield RP18 was used for analysis. The mobile phase consisted of 0.1 mol / L NaH2PO4 aqueous solution and acetonitrile in a ratio of 55:45 (v / v), and isocratic elution was performed. The detection wavelength was set at 260 nm, the flow rate was 1 ml / min, and the injection volume was 20 μl. Under these conditions, the product 3-AP and the internal standard 2-Me-5-AP could be effectively separated within 5 min.

[0057] 5. Quantitative calculation: Use the internal standard method for quantitative analysis. CD38 enzyme activity is expressed in Katal, an international unit of enzyme activity, and the calculation formula is:

[0058] 1nKatal / L=

L1×(S1 / S2)×C1

[0059] Wherein, L1 is the substrate volume (unit: L), L2 is the whole blood volume (unit: L), S1 is the product peak area (unit: mAU), S2 is the internal standard peak area (unit: mAU), C1 is the internal standard concentration (unit: μmol / L), and T is the enzymatic reaction time (unit: S).

[0060] Test Example 1 Evaluation of the method of Example 1

[0061] 1. Linearity: The 3-AP standard solution (80 μg / mL) was diluted step by step by the two-fold dilution method to prepare a series of standard solutions with concentrations of 1, 5, 10, 20, 40, and 80 μg / mL. According to the above HPLC method, the concentration of 3-AP was used as the horizontal axis (X) and the peak area was used as the vertical axis (Y) to draw a standard curve, and a linear regression equation was obtained to calculate the 3-AP in the enzyme reaction system. In the concentration range of 1 to 80 μg / mL, the concentration and peak area of ​​3-AP had a good linear relationship. The regression equation for calculating the standard curve with the concentration of 3-AP as the horizontal axis (X) and the peak area as the vertical axis (Y) was Y=11.902X+1.874, and the linear coefficient R of multiple experiments was 2 =0.9999, see Figure 3 .

[0062] 2. Precision: The coefficient of variation was used to evaluate precision. The exogenous substrate APAD was used to measure the CV of the intra-batch (3 consecutive tests in a single day) and inter-batch (3 consecutive days) test results of quality control whole blood at three levels: low, medium, and high. Three parallel tubes were set up for each test, and the intra-batch CV and total CV were calculated separately. The average intra-batch CV of the three different levels of quality control whole blood determinations was 1.41% to 4.08%, and the total CV was 1.87% to 3.57%. The precision of the established method is good, see Table 1.

[0063] Table 1 Precision results of HPLC method for determining CD38 enzyme activity

[0064] Whole blood CD38(nKat / L) Intra-assay CV (%) Total CV (%) Low concentration 82.16±2.78 3.86 3.39 Medium concentration 115.07±2.15 1.41 1.87 High concentration 184.69±6.59 4.08 3.57

[0065] 3. Recovery rate: 5, 10, 20, and 40 μg / mL of 3-AP standards were added to whole blood for recovery experiments to evaluate the recovery rates at different concentrations. Low, medium, and high levels of 3-AP standards were added to whole blood samples, and the actual concentration was determined using the established HPLC method. The theoretical concentration was the actual concentration of added 3-AP. The relative recovery rate was calculated as: relative recovery rate = (measured concentration - endogenous concentration) / theoretical concentration × 100%. The recovery rate was in the range of 99.82% to 102.01%, close to 100%, and the method had good accuracy. The results are shown in Table 2.

[0066] Table 2 Recovery results of the HPLC method for determining CD38 enzyme activity

[0067]

[0068] 4. Stability: In order to investigate the stability of whole blood at different temperatures and for different periods of time, whole blood was immediately packaged after blood collection and placed at 4°C for 0, 1, 2, 4, 8, 24, and 48 hours, and at room temperature for 0, 0.5, 1, 2, 4, and 8 hours. After the frozen whole blood was thawed, it was placed at room temperature for 0, 0.5, 1, and 2 hours. After reaching the storage time, it was immediately stored in a -80°C refrigerator to detect the activity of the whole blood CD38 enzyme. In addition, the effect of repeated freezing and thawing of whole blood 1 to 3 times on the determination of CD38 enzyme activity was investigated. In order to investigate the stability of the standard, the substrate APAD, product 3-AP, and internal standard 2-methyl-5-AP standards were placed at 4°C for 0, 1, 2, 4, 8, 24, and 48 hours, and then placed at room temperature for 0, 0.5, 1, 2, 4, and 8 hours and then stored in a -80°C refrigerator. The peak area changes were detected by HPLC. In order to investigate the injection stability, after pretreatment, the samples were placed at 4°C for 0, 1, 2, 4, 8, 24, and 48 hours, and at room temperature for 0, 0.5, 1, 2, 4, and 8 hours before HPLC was used to detect the 3-AP peak area and enzyme activity. The CD38 enzyme activity of whole blood samples was stable at 4°C for at least 48 hours, and at room temperature for at least 8 hours. Placing frozen blood at room temperature for 2 hours after thawing did not affect the enzyme activity, and there was no significant change in CD38 enzyme activity after three repeated freeze-thaw cycles. The substrate APAD, product 3-AP, and internal standard 2-methyl-5-AP standards were stable at 4°C for at least 48 hours, and at room temperature for at least 8 hours. Placing the pretreated samples at 4°C for 48 hours and at room temperature for 8 hours did not affect the enzyme activity determination results, and the CD38 enzyme activity determination had good stability. Results are shown in Figures 4 to 6 .

[0069] 5. Effect of inhibitors on enzyme activity determination: In order to explore the effect of enzyme activity inhibitors on CD38 enzyme activity determination, CD38 inhibitor 78c solutions with concentrations of 0, 0.73, 7.3, 73, 365, 730, 3650, 7300, 21900, and 45900 nmol / L were prepared and added to the reaction system to participate in the reaction. The activity of CD38 enzyme in the reaction system containing different concentrations of 78c was measured, and a dose-response curve was established. After the CD38 specific inhibitor 4-aminoquinoline derivative 78c was increased to 7.3 nmol / L, the CD38 enzyme activity began to gradually decrease with the increase of 78c concentration. When the 78c concentration increased to 7300 nmol / L, the CD38 enzyme activity was almost completely inhibited. The enzyme activity basically did not change as the 78c concentration continued to increase. The method of this example can effectively reflect the changes in CD38 enzyme activity. See the results. Figure 7 .

[0070] Example 2 A quantitative method corrected by RBC counting

[0071] 1. Collect whole blood samples from 259 apparently healthy people who underwent physical examinations in Beijing hospitals in 2022 and 234 newly diagnosed tumor patients who did not receive treatment (including 43 people with lung cancer, 54 people with colorectal cancer, 56 people with gynecological tumors, 29 people with blood tumors, 19 people with benign tumors, and 33 people with other tumors such as thymomas and stromal tumors). Collect 2 ml of fasting venous blood in the early morning and store it at -80°C. Collect RBC count information from 493 people, and perform HPLC analysis to determine the CD38 enzyme activity in whole blood as described in Example 1.

[0072] 2. Statistical methods: SPSS26.0 software was used to analyze the data of 259 healthy people and 234 tumor cancer patients. Spearman correlation analysis was used to compare the relationship between CD38 activity before and after RBC correction and age. One-way analysis of variance was used to compare the CD38 activity of each tumor group with that of the healthy group before and after RBC correction. Independent sample t-test was used to compare the inter-group differences in CD38 activity between the healthy group and the tumor group before and after RBC correction.

[0073] 3. Quantitative results: In both healthy and tumor populations, whole blood enzyme activity was present and had no significant correlation with age. However, after correction for red blood cell count, enzyme activity was significantly positively correlated with age (see Figure 8 ), and the correlation coefficients are shown in Table 3. The CD38 activity of the tumor group (including all tumors and each tumor group) was also significantly higher than that of the healthy group (such as Fig. 9 As shown in Table 4). In summary, it can be seen that the clinical significance of measuring enzyme activity in whole blood is different from that after calibration of red blood cell count. Comparison between the two shows that enzyme activity after calibration of red blood cell count can better reflect clinical significance such as aging and tumors.

[0074] Table 3 Comparison of whole blood CD38 activity between tumor groups and healthy groups before and after RBC count correction

[0075]

[0076] Table 4 Comparison of whole blood CD38 activity between tumor groups and healthy groups before and after RBC count correction

[0077]

[0078] When the method of detecting blood CD38 enzyme activity by high performance liquid chromatography of the present invention is used, it is found that after the enzyme activity is calibrated with the number of red blood cells, it is more closely related to aging or disease status.

[0079] Comparative Example 1

[0080] Prepare cell samples, quantitative dilution → add 50μmol / L NAD or cADPR → take a portion as blank value, and incubate the rest at room temperature for 2-18h → stop the reaction in ice bath / ice salt bath → cool on ice for 5-10min → centrifuge to remove cells → centrifuge for 10min using a 10KD cutoff centrifugal concentrator to remove proteins or cell particles → incubate cells without NAD to remove background signals → C18 reverse phase column, gradient elution, a total of 27min, detection at 270nm → NAD, cADPR, ADPR, AMP, adenosine as standards → 24μl of each standard and 96μl of mobile phase → use the peak area of ​​the standard nucleotide to calculate the amount of ADPR product. (For details, see [Kirchberger T, Guse AH. Measuring CD38 (ADP-ribosylcyclase / cyclic ADP-ribosehydrolase) activity by reverse-phase HPLC. Cold Spring Harb Protoc. 2013 Jun; 2013 (6): 569-73].)

[0081] The sample matrix of the method in Comparative Example 1 is cells; secondly, the enzymatic reaction condition is room temperature, which has a large variable; because the amount of ADPR product measured reflects the enzyme activity, and cADPR can also produce ADPR, and NAD can also produce cADPR, the incubation time is 2 to 18 hours, which is too long and the detection efficiency is low.

[0082] Comparative Example 2

[0083] 100 μl whole blood, bovine lung tissue + 1 mmol / L β-NAD or cADPR, respectively, containing 70 mmol / L, pH = 7.5 potassium phosphate buffer → incubate at 38°C → add HClO4 at 0, 5, 10, and 15 min, respectively, to terminate the reaction → place on ice for 10 min → centrifuge → add K2CO3, centrifuge again → C18 reverse phase chromatography column, the mobile phase is 100 mmol / L potassium phosphate (pH = 6.0) - methanol (20%), gradient elution, a total of 26 min, monitor the absorbance at 254 nm → detect the amount of product ADPR synthesized. (For details, see [Polzonetti V, Carpi FM, Micozzi D, et al. Population variability in CD38 activity: correlation with age and significant effect of TNF-α-308G>A and CD38184C> GSNPs. Mol Genet Metab. 2012 Mar; 105(3):502-7.] and [Polzonetti V, Pucciarelli S, VitaA, et al. CD38 inbovine lung:Amulticatalytic NADase.JMembrBiol.2009Feb;227(3):105-110.】)

[0084] The sample matrix of the method of comparative example 2 is whole blood, and the substrate is endogenous β-NAD. The amount of ADPR synthesized from NAD per minute is calculated to reflect the enzyme activity. The enzyme activity needs to be calculated through the product generation at multiple time points, so the operation is cumbersome (such as multiple termination of the reaction, multiple injections, and complex calculations); secondly, the liquid phase requires a long time for gradient elution separation. In addition, the results lack methodological evaluations such as precision and recovery rate to prove that the method can be clinically applied.

[0085] Comparative Example 3

[0086] 50μl frozen whole blood was thawed at room temperature → pre-incubated at 37℃ for 20min → a portion + 150μl 500μmol / L β-NAD was taken and immediately added with perchloric acid to terminate the reaction, a portion + 150μl 500μmol / L β-NAD was taken and incubated in a 37℃ water bath for 40min and then perchloric acid was added to terminate the reaction → centrifugation → 150μl supernatant was taken and added with 600μl Na2HPO4 → the difference between NAM after incubation for 40min and NAM before incubation for 40min was detected. (For details, see [Zhao Xianghui, Bu Xiaoxiao, Zhu Hanyu, et al. Establishment of a method for detecting blood CD38 enzyme activity by high performance liquid chromatography. Chinese Journal of Laboratory Medicine, 2023, 46(10): 1074-1082]).

[0087] After evaluation, the linear R 2 =0.999; the intra-batch CV of precision was 3.22%-4.03%, and the total CV was 2.91%-4.70%; the recovery rate was in the range of 94.82%-96.81%; the CD38 enzyme activity in whole blood was stable for at least 48 hours at 4°C and for at least 8 hours at room temperature; the enzyme activity would not be affected if the frozen blood was thawed and placed at room temperature for 2 hours, and there was no significant change in the CD38 enzyme activity after repeated freezing and thawing for 3 times; the NAM and β-NAD standards were stable for at least 48 hours at 4°C and for at least 8 hours at room temperature; the enzyme activity determination results were not affected if the treated samples were placed at 4°C for 48 hours or at room temperature for 8 hours.

[0088] The product NAM of the method in Comparative Example 3 is an endogenous product, which interferes with the determination of the amount of the enzymatic reaction product; secondly, it is necessary to simultaneously determine the amount of NAM at two time points and then calculate the enzyme activity, and the detection efficiency is low.

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A sample pretreatment method for detecting CD38 enzyme activity in whole blood samples, characterized in that: The following steps are involved: Pre-incubating the whole blood sample to consume endogenous β-NAD to obtain a pre-incubated whole blood sample; mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine to perform an enzymatic reaction, terminating the enzymatic reaction, and obtaining an enzymatic reaction product; The enzymatic reaction product is centrifuged, and the supernatant is collected to obtain a sample to be tested.

2. The sample pretreatment method according to claim 1, characterized in that: The working concentration of the acetyl adenine dinucleotide is 500 μmol / L; the working concentration of the 2-methyl-5-acetyl pyridine is 15.04 μg / ml.

3. The sample pretreatment method according to claim 1, characterized in that: Mixing the pre-incubated whole blood sample with acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine includes mixing the pre-incubated whole blood sample with a mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine; based on a volume of 50 μl of the whole blood sample, the amount of the mixed solution containing acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine is 150 μl.

4. The sample pretreatment method according to claim 1, characterized in that: The pre-incubation was carried out in a water bath; the pre-incubation temperature was 37° C.; and the pre-incubation time was 20 min.

5. The sample pretreatment method according to claim 1, characterized in that: The enzymatic reaction was carried out in a water bath; the temperature of the enzymatic reaction was 37° C.; and the time of the enzymatic reaction was 40 min.

6. The sample pretreatment method according to claim 1, characterized in that: The reagent used to terminate the enzymatic reaction includes acetonitrile; based on 50 μl of the whole blood sample, the amount of acetonitrile used is 200 μl.

7. The sample pretreatment method according to claim 1, characterized in that: The centrifugal temperature is 4°C; the centrifugal speed is 8000 rpm; and the centrifugal time is 10 min.

8. A method for detecting blood CD38 enzyme activity by high performance liquid chromatography, characterized in that: The following steps are involved: Obtaining a sample to be tested by using the sample pretreatment method according to any one of claims 1 to 7; Performing high performance liquid chromatography on the sample to be tested to obtain the peak area of ​​the product 3-acetylpyridine and the peak area of ​​the internal standard 2-methyl-5-acetylpyridine, and performing quantitative analysis using the internal standard method to obtain the whole blood CD38 enzyme activity; The blood CD38 enzyme activity was calculated according to the formula shown in the following formula 1; the CD38 enzyme activity was expressed in Katal, an international unit of enzyme activity; 1nKatal / L=[L1×(S1 / S2)×C1] / (L2×T) Equation 1; In Formula 1, L1 is the volume of the mixed solution including acetyl adenine dinucleotide and 2-methyl-5-acetyl pyridine, unit: L; L2 is the volume of the whole blood sample, unit: L; S1 is the product peak area, unit: mAU; S2 is the internal standard peak area, unit: mAU; C1 is the internal standard concentration, unit: μmol / L; T is the time of enzymatic reaction, unit: s.

9. The method according to claim 8, characterized in that The chromatographic column used for the high performance liquid chromatography detection is a C18 reverse phase chromatographic column; the mobile phase of the high performance liquid chromatography detection is composed of a NaH2PO4 aqueous solution and acetonitrile; the concentration of NaH2PO4 in the NaH2PO4 aqueous solution is 0.1 mol / L; the volume ratio of the NaH2PO4 aqueous solution to acetonitrile is 55:45; the elution mode of the high performance liquid chromatography detection is isocratic elution; the detection wavelength of the high performance liquid chromatography detection is 260 nm; the flow rate of the mobile phase is 1 ml / min; the injection volume of the high performance liquid chromatography detection is 20 μl; and the elution time of the high performance liquid chromatography detection is ≤5 min.

10. The method according to claim 8, characterized in that After obtaining the whole blood CD38 enzyme activity, the whole blood CD38 enzyme activity is divided by the red blood cell count for correction.

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