A method for quantitatively detecting muscle inositol in blood

Abstract

This invention discloses a method for quantitatively detecting muscle inositol in blood. It employs an electrochemical method and specifically includes the following steps: a) preparation of a series of standard working solutions; b) preparation of a blank sample solution; c) plotting a standard curve using differential pulse voltammetry (DPV) with the blank sample solution and the series of standard working solutions; d) preparation of the test sample solution; e) determination of the muscle inositol content in the test sample. Experiments have demonstrated that the method of this invention yields accurate and reliable results and can be used for clinical blood testing.

Description

Technical Field

[0001] This invention provides a method for quantitatively detecting muscle inositol in blood. Background Technology

[0002] Myo-inositol, also known as hexahydroxycyclohexane, is a glucose-derived polyol. It plays a crucial role in regulating various cellular functions, serving as a structural basis for eukaryotic secondary messengers, particularly as a precursor to inositol phosphate, phosphatidylinositol, and phosphatidylinositol phosphate lipids. Therefore, it contributes to important functional and structural roles, including cell growth and survival, osteogenic processes, proliferation, and the development of the nervous system. Abnormalities in myo-inositol metabolism are associated with the development of several disease states, such as neural tube defects, Down syndrome, Alzheimer's disease, and diabetic glomerular disease. Previous studies have demonstrated chemopreventive and chemotherapeutic properties in human cancer cells and animal cancer models, including prostate, breast, colon, pancreatic, liver, and lung cancer. Simultaneously, myo-inositol has been tested as a pharmacological intervention for various conditions, including depression, panic disorder, and obsessive-compulsive disorder. Therefore, the potential applications of myo-inositol as a pharmacological agent and metabolic biomarker necessitate the establishment of methods for measuring serum myo-inositol.

[0003] Inositol in the blood is maintained at a certain concentration through incorporation into cells, excretion, reabsorption, and renal oxidation; normal serum inositol concentrations range from 18–41 μmol / L. Currently, various analytical methods have been developed for the detection of inositol, including gas chromatography-mass spectrometry (GC / MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (HPLC / MS), liquid chromatography pulsed amperometric detection, and enzyme cycling detection. Most of these detection techniques are generally complex and time-consuming, with expensive and bulky instruments, and require highly skilled operators. Furthermore, they require cumbersome sample pretreatment, thus limiting their application. Compared to these instrumental methods, electrochemical methods, due to their simplicity, small sample volume requirements, high sensitivity, and ease of miniaturization, show broad application prospects in the ultrasensitive detection of small molecule compounds, becoming a new hot topic in methodological research. However, there are currently no reports of applying electrochemistry to the detection of inositol levels in blood. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for quantitatively detecting muscle inositol in blood, which employs an electrochemical method to detect muscle inositol in blood.

[0005] Furthermore, it includes determining the response current values ​​of muscle inositol solution, blank serum solution, and blood sample solution to be tested using differential pulse voltammetry (DPV method), and calculating the muscle inositol content in the blood sample based on the muscle inositol standard curve established by the concentrations of muscle inositol solution and blank serum solution and the corresponding current values.

[0006] Furthermore, it specifically includes the following steps:

[0007] (1) Establishment of the muscle inositol standard curve

[0008] a. Preparation of a series of standard working solutions: Take muscle inositol, dissolve it in water, and dilute it to prepare a series of standard solutions. Take each series of standard solutions, add blank serum and mix well, then add acetonitrile and mix well. Centrifuge, take the supernatant and dry it. Dissolve the residue in glycine-NaOH buffer to obtain a series of standard working solutions.

[0009] b. Preparation of blank sample solution: Take blank serum, add acetonitrile, mix well, centrifuge, take the supernatant, dry it, and dissolve the residue in glycine-NaOH buffer to obtain the blank sample solution;

[0010] c. Take blank sample solution and series of standard working solutions of different concentrations respectively, add oxidized nicotinamide adenine dinucleotide solution (NAD+) and muscle inositol inositol dehydrogenase solution (IDH) and mix well. React for 10-30 min. Take the reaction mixture, attach it to the screen-printed carbon electrode, and then connect it to the electrochemical workstation. Use differential pulse voltammetry (DPV method) to measure the response current value. Plot the muscle inositol standard curve with muscle inositol concentration as the X-axis and the difference between the response current values ​​of the series of standard working solutions and the blank sample solution as the Y-axis.

[0011] (2) Determination of muscle inositol content in the sample to be tested:

[0012] d. Preparation of the sample solution to be tested

[0013] Take a serum sample and prepare it using the same method as in step b to obtain the sample solution to be tested;

[0014] e. Determination of muscle inositol content in the sample to be tested

[0015] Take the sample solution to be tested and measure the response current value using the same method as in step c. Calculate the muscle inositol content in the sample to be tested based on the standard curve in step (1).

[0016] Further, the volume ratio of the series of concentration standard solutions, blank serum and acetonitrile in step a is 1:9:40.

[0017] Further, the concentration of the series of standard solutions is 50 to 5000.0 μmmol / L, preferably 50.0 μmmol / L, 100.0 μmmol / L, 200.0 μmmol / L, 500.0 μmmol / L, 1000.0 μmmol / L, 2000.0 μmmol / L, 4000.0 μmmol / L and / or 5000.0 μmmol / L.

[0018] Further, the volume ratio of blank serum to acetonitrile in step b is 9-13:30-50, preferably 10:40.

[0019] Further, the mixing in steps a and b is vortex mixing; the centrifugation temperature is 2-8℃, the rotation speed is 10000-20000 rpm / min, and the time is 8-15 min, preferably the centrifugation temperature is 4℃, the rotation speed is 13300 rpm / min, and the time is 12 min.

[0020] Further, the drying in steps a and b is nitrogen gas drying; the temperature of the nitrogen gas drying is 60-100°C, preferably 80°C.

[0021] Further, the amount of glycine-NaOH buffer solution added in steps a and b is 1 / 10 to 1 / 15 of the volume of acetonitrile, preferably 1 / 12.

[0022] Furthermore, the pH value of the glycine-NaOH buffer solution is 9-11, and the glycine content is 500-600 mmol / L, preferably pH value is 10.3 and glycine content is 550 mmol / L.

[0023] Further, the blank sample solution or series of concentration standard working solutions described in step c, with NAD... + The volume ratio of the solution to the IDH solution is 25-45:3-7:3-7, preferably 40:5:5.

[0024] Furthermore, the NAD + The concentration of the solution is 100–200 mmol / L, and the concentration of the IDH solution is 4000–5000 U / L, preferably NAD. + The concentration of the solution was 160 mmol / L, and the concentration of the IDH solution was 4500 U / L.

[0025] Furthermore, the reaction temperature in step c is 20–30°C, and the reaction time is 20 min.

[0026] Further, in step c, the volume of the reaction mixture attached to the screen-printed carbon electrode is 20–60 μL, preferably 40 μL.

[0027] Further, the parameters of the DPV method in step c are voltage 0.1-0.73V, increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s.

[0028] This invention provides a method for quantitatively detecting muscle inositol in blood. Compared with existing analytical methods for muscle inositol, this invention has advantages such as simple and rapid preparation, good stability, low detection cost, and suitability for on-site detection. It can be used for the detection of muscle inositol in serum.

[0029] The method of this invention has been verified to have high sensitivity, fast analysis speed, wide linear range, good reproducibility, and strong anti-interference ability. The detection limit is as low as 1.0 μM (S / N = 3), the quantitation limit is 2.5 μM (S / N = 10), and the analytical detection range is 5.0–500.0 μmol / L. Three working solutions (low (10.0 μmol / L), medium (100.0 μmol / L), and high (200.0 μmol / L)) were prepared for different concentrations of muscle inositol standard solution. Five consecutive measurements were performed on the same day for five consecutive days. The intra-day precision was 3.2%–6.2%, and the inter-day precision was 7.1%–9.0%. The intra-day recovery rate was 96.6%–106%, and the inter-day recovery rate was 90.3%–103%.

[0030] The method of this invention measures the concentration of muscle inositol in serum samples without bilirubin and hemoglobin, as well as in serum samples with different concentrations of bilirubin (0.0–200.0) μmol / L and hemoglobin (0.0–16.0) mg / mL. The results show that hemoglobin and bilirubin in serum at concentrations of (0.0–8.0) mg / mL and (0.0–160.0) μmol / L, respectively, do not significantly interfere with the determination of serum muscle inositol. The results are accurate and reliable and can be used for clinical blood testing.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 Response current change △ Working curve of I versus serum muscle inositol concentration Detailed Implementation

[0034] Example 1: Determination of muscle inositol content in blood

[0035] (1) Establishment of the muscle inositol standard curve

[0036] a. Preparation of a series of standard working solutions: Muscle inositol solid powder was dissolved in ultrapure water to prepare a 200 mmol / L muscle inositol stock solution. This stock solution was then diluted with ultrapure water to prepare a series of muscle inositol standard solutions (50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, 4000.0, 5000.0) μmmol / L. 100 μL of each muscle inositol standard solution was mixed with 900 μL of blank serum by vortexing to prepare muscle inositol concentrations of (5.0, 10.0) μmmol / L. Spiked mixed serum solutions of 20.0, 50.0, 100.0, 200.0, 400.0, and 500.0 μmol / L were prepared. 120 μL of the spiked mixed serum solution was taken and then 480 μL of acetonitrile was slowly added and vortexed for 30 s. The mixture was centrifuged at 13300 rpm / min for 12 min at 4 °C. The supernatant was then dried under nitrogen at 80 °C. The residue was dissolved in 40 μL of glycine-NaOH buffer (550 mmol / L, pH = 10.3) to obtain a series of standard working solutions.

[0037] b. Preparation of blank sample solution: Take 120 μL of blank serum, slowly add 480 μL of acetonitrile and vortex for 30 s. Centrifuge the mixture at 13300 rpm / min at 4℃ for 12 min. Then, take the supernatant and dry it under nitrogen at 80℃. Dissolve the residue in 40 μL of glycine-NaOH buffer (550 mmol / L, pH=10.3) to obtain the blank sample solution.

[0038] c. Take 40 μL of blank sample solution and a series of standard working solutions, add 5 μL of oxidized nicotinamide adenine dinucleotide solution (NAD+, 160 mmol / L) and 5 μL of muscle inositol dehydrogenase solution (IDH, 4500 U / L), mix well, and carry out the enzyme reaction at room temperature for 20 min. Take 40 μL of the reaction mixture, attach it to a screen-printed carbon electrode, and then connect it to an electrochemical workstation (the reference electrode of the electrochemical workstation is AgCl ink, and the working electrode and counter electrode are carbon ink). The response current value is measured by differential pulse voltammetry (DPV method), where the parameters are voltage (0.1V to 0.73V), increment 0.005V, amplitude 0.10V, pulse width 0.05s, and pulse period 0.2s. Plot the muscle inositol standard curve with muscle inositol concentration as the X-axis and the difference between the response current values ​​of the series of standard working solutions and the blank sample solution as the Y-axis.

[0039] (2) Determination of muscle inositol content in the sample to be tested:

[0040] d. Preparation of the sample solution to be tested

[0041] Take a serum sample and prepare it using the same method as in step b to obtain the sample solution to be tested;

[0042] e. Determination of muscle inositol content in the sample to be tested

[0043] Take the sample solution to be tested and measure the response current value using the same method as in step c. Calculate the muscle inositol content in the sample to be tested based on the standard curve in step (1).

[0044] The following experimental examples illustrate the beneficial effects of the present invention.

[0045] Experimental Example 1: Detection of spiked muscle inositol in serum samples

[0046] Take 200 mmol / L of undiluted muscle inositol and dilute it to prepare a series of muscle inositol standard solutions (50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, 4000.0, 5000.0) μmmol / L. Take 100 μL of each concentration of muscle inositol standard solution and mix it with 900 μL of blank serum. Vortex to prepare spiked mixed serum samples with muscle inositol concentrations of (5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 400.0, 500.0) μmol / L. Add 120 μL of the spiked mixed serum sample to a centrifuge tube, then slowly add 480 μL of acetonitrile and vortex for 30 s. Centrifuge the mixture at 13300 rpm / min for 12 min at 4 °C. The supernatant was then dried under nitrogen at 80°C; the residue was dissolved in 40 μL of glycine-NaOH buffer (550 mmol / L, pH = 10.3). 40 μL of the reconstituted solution and 5 μL of NAD were then added. + (160 mmol / L) 5 μL of IDH solution (4500 U / L) was vortexed and the enzyme reaction was carried out at room temperature for 20 min. 40 μL of the reaction mixture was added to the SPCE working electrode and connected to an electrochemical workstation (the reference electrode of this workstation was AgCl ink, and the working and counter electrodes were carbon ink). The electrochemical workstation parameters were: voltage (0.1 V to 0.73 V), increment 0.005 V, amplitude 0.10 V, pulse width 0.05 s, and pulse period 0.2 s. DPV scanning was performed, and each experiment was repeated 5 times, recording the response current. The response current in blank serum with a muscle inositol concentration of 0 was I0, and the response current in the standard sample containing the target muscle inositol was I... XThe ΔIC working curve was plotted using the increase in response current ΔI and the concentration of muscle inositol in the spiked mixed serum sample. A linear regression equation for ΔIC was obtained. Within the range of 5.0–500.0 μmol / L, the DPV peak current showed a good linear relationship with the serum muscle inositol concentration, yielding the regression equation: Y = 0.007225*X + 0.1893 (where Y is the NADH oxidation peak current in μA, and X is the muscle inositol concentration in μmol / L), with a linear correlation coefficient of 0.9981. Using a current signal greater than 3 times the noise signal as the limit of detection, and repeating the experiment more than 5 times, the limit of detection for this method was found to be 1.0 μM (S / N = 3). Using a current signal greater than 10 times the noise signal as the limit of detection, and repeating the experiment more than 5 times, the limit of quantitation was found to be 2.5 μM (S / N = 10). See details... Figure 1 .

[0047] Example 2: Precision and recovery of serum muscle inositol determined by electrochemical method

[0048] Different concentrations of muscle inositol standard solution were added to blank serum to prepare three working solutions: low (10.0 μmol / L), medium (100.0 μmol / L), and high (200.0 μmol / L). 120 μL of the mixed serum sample was added to a centrifuge tube, followed by the slow addition of 480 μL of acetonitrile and vortexing for 30 s. The mixture was centrifuged at 13300 rpm / min for 12 min at 4 °C. The supernatant was then dried under nitrogen at 80 °C; the residue was dissolved in 40 μL of glycine-NaOH buffer (550 mmol / L, pH = 10.3). 40 μL of the reconstituted solution and 5 μL of NAD+ were then added to each solution. +The NADH (160 mmol / L) solution and 5 μL of IDH (4500 U / L) solution were vortexed and the enzyme reaction was carried out at room temperature for 20 min. 40 μL of the reaction mixture was added to the SPCE working electrode and connected to the electrochemical workstation. The electrochemical workstation parameters were: voltage (0.1 V to 0.73 V), increment 0.005 V, amplitude 0.10 V, pulse width 0.05 s, and pulse period 0.2 s. DPV scanning was performed, and each experiment was repeated 5 times, recording the response current. The serum muscle inositol concentration was calculated using the regression equation obtained in Experiment 1: Y = 0.007225 * X + 0.1893 (Y is the NADH oxidation peak current in μA, X is the muscle inositol concentration in μmol / L). For the same serum sample, myoinositol was measured five times consecutively on the same day for five consecutive days. The relative recovery rate was calculated as (measured concentration of myoinositol after spiking - initial concentration of myoinositol before spiking) ÷ added concentration of myoinositol × 100%. The results showed that the intra-day precision was 3.2%–6.2%, and the inter-day precision was 7.1%–9.0%; the intra-day recovery rate was 96.6%–106%, and the inter-day recovery rate was 90.3%–103% (see Table 1).

[0049] Table 1. Precision and recovery rate of electrochemical method for determining serum muscle inositol

[0050]

[0051] Experimental Example 3: Effects of Hemoglobin and Bilirubin on the Electrochemical Detection of Serum Myositol

[0052] Hemoglobin and bilirubin standards were added to serum samples of the same concentration to prepare mixed serum samples containing 0.0, 2.0, 4.0, 8.0, 12.0, and 16.0 mg / ml hemoglobin and 0.0, 40.0, 80.0, 160.0, 180.0, and 200.0 μmol / L bilirubin, respectively. 120 μL of the mixed serum sample was added to a centrifuge tube, followed by the slow addition of 480 μL of acetonitrile and vortexing for 30 s. The mixture was centrifuged at 13300 rpm / min for 12 min at 4 °C. The supernatant was then dried under nitrogen at 80 °C; the residue was dissolved in 40 μL of glycine-NaOH buffer (550 mmol / L, pH = 10.3). 40 μL of the reconstituted solution and 5 μL of NAD+ were then added to the solution. +(160 mmol / L) and 5 μL of IDH solution (4500 U / L) were vortexed and the enzyme reaction was carried out at room temperature for 20 min. 40 μL of the reaction mixture was added to the SPCE working electrode and connected to the electrochemical workstation. The electrochemical workstation parameters were: voltage (0.1 V to 0.73 V), increment 0.005 V, amplitude 0.10 V, pulse width 0.05 s, and pulse period 0.2 s. DPV scanning was performed, and each experiment was repeated 5 times, recording the response current. The regression equation obtained from Experiment 1 was: Y = 0.007225 * X + 0.1893 (Y is the NADH oxidation peak current μA, X is the muscle inositol concentration μmol / L). The serum muscle inositol concentrations before and after the addition of hemoglobin or bilirubin were defined as X. C and X T Calculate the interference value (X). T -X C A value below 1.96 s indicates no significant interference, denoted as N; a value above 1.96 s indicates significant interference, denoted as I. The results show that serum hemoglobin and bilirubin at concentrations of (0.0–4.0) mg / mL and (0.0–160.0) μmol / L, respectively, did not significantly interfere with the determination of serum muscle inositol (Table 2).

[0053] Table 2. Effects of hemoglobin and bilirubin on the electrochemical detection of serum muscle inositol

[0054]

[0055] Note: The serum muscle inositol concentrations before and after the addition of hemoglobin or bilirubin are defined as XC and XT, respectively. When the calculated interference value XT-XC is less than 1.96 s, it indicates no significant interference, denoted as N; when the calculated interference value is greater than 1.96 s, significant interference exists, denoted as I.

[0056] In summary, the method of this invention exhibits high sensitivity, fast analysis speed, wide linear range, good reproducibility, and strong anti-interference ability. The detection limit is as low as 1.0 μM (S / N = 3), the quantitation limit is 2.5 μM (S / N = 10), and the analytical detection range is 5.0–500.0 μmol / L. Three working solutions (low (10.0 μmol / L), medium (100.0 μmol / L), and high (200.0 μmol / L)) were prepared for different concentrations of muscle inositol standard solution. Five consecutive measurements were performed on the same day for five consecutive days. The intra-day precision was 3.2%–6.2%, and the inter-day precision was 7.1%–9.0%. The intra-day recovery rate was 96.6%–106%, and the inter-day recovery rate was 90.3%–103%.

[0057] The concentrations of muscle inositol in serum samples without bilirubin and hemoglobin, as well as in serum samples with different concentrations of bilirubin (0.0–200.0) μmol / L and hemoglobin (0.0–16.0) mg / mL, were measured. The results showed that hemoglobin and bilirubin in serum at concentrations of (0.0–4.0) mg / mL and (0.0–160.0) μmol / L, respectively, did not significantly interfere with the determination of serum muscle inositol. The results were accurate and reliable and can be used for clinical blood testing.

Claims

1. A method for quantitatively detecting muscle inositol in blood, characterized in that: It specifically includes the following steps: (1) Establishment of the muscle inositol standard curve a. Preparation of a series of standard working solutions: Take muscle inositol, dissolve it in water, and dilute it to prepare a series of standard solutions. Take each series of standard solutions, add blank serum and mix well, then add acetonitrile and mix well. Centrifuge, take the supernatant and dry it. Dissolve the residue in glycine-NaOH buffer to obtain a series of standard working solutions. b. Preparation of blank sample solution: Take blank serum, add acetonitrile, mix well, centrifuge, take the supernatant, dry it, and dissolve the residue in glycine-NaOH buffer to obtain the blank sample solution; c. Take blank sample solution and series of standard working solutions of different concentrations respectively, add oxidized nicotinamide adenine dinucleotide solution (NAD+) and muscle inositol inositol dehydrogenase solution (IDH) and mix well. React for 10-30 min. Take the reaction mixture, attach it to the screen-printed carbon electrode, and then connect it to the electrochemical workstation. Use differential pulse voltammetry (DPV method) to measure the response current value. Plot the muscle inositol standard curve with muscle inositol concentration as the X-axis and the difference between the response current values ​​of the series of standard working solutions and the blank sample solution as the Y-axis. (2) Determination of muscle inositol content in the sample to be tested: d. Preparation of the sample solution to be tested Take a serum sample and prepare it using the same method as in step b to obtain the sample solution to be tested; e. Determination of muscle inositol content in the sample to be tested Take the sample solution to be tested and measure the response current value using the same method as in step c. Calculate the muscle inositol content in the sample to be tested based on the standard curve in step (1).

2. The method according to claim 1, characterized in that: The volume ratio of the series of concentration standard solutions, blank serum and acetonitrile in step a is 1~3:8~10:30~50; the concentration of the series of concentration standard solutions is 50~5000.0 μmmol / L.

3. The method according to claim 1, characterized in that: The volume ratio of blank serum to acetonitrile in step b is 9~13:30~50.

4. The method according to claim 1, characterized in that: The mixing in steps a and b is vortex mixing; the centrifugation temperature is 2~8℃, the rotation speed is 10000~20000 rpm / min, and the time is 8~15min; the drying is nitrogen gas drying; the temperature of the nitrogen gas drying is 60~100℃.

5. The method according to claim 1, characterized in that: The amount of glycine-NaOH buffer added in steps a and b is 1 / 10 to 1 / 15 of the volume of acetonitrile; the pH value of the glycine-NaOH buffer is 9 to 11, and the glycine content is 500 to 600 mmol / L.

6. The method according to claim 1, characterized in that: The blank sample solution or series of concentration standard working solutions described in step c, with NAD + The volume ratio of the solution to the IDH solution is 25~45:3~7:3~7; the NAD... + The concentration of the solution is 100~200 mmol / L, and the concentration of the IDH solution is 4000~5000 U / L.

7. The method according to claim 1, characterized in that: The reaction temperature in step c is 20~30°C and the time is 20 min; the volume of the reaction mixture attached to the screen-printed carbon electrode is 20~60 μL.

8. The method according to claim 1, characterized in that: The parameters of the DPV method described in step c are: voltage 0.1~0.73V, increment 0.005V, amplitude 0.10V, pulse width 0.05s, and pulse period 0.2s.

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