A method for simultaneously detecting the contents of three protein-binding toxins in a blood sample

Abstract

The present invention belongs to the technical field of protein-bound toxin detection. The present invention provides a method for simultaneously detecting the contents of three protein-bound toxins in a blood sample. The method of the present invention includes the steps of preparing a test sample solution, preparing a series of mixed reference substance solutions, performing high performance liquid chromatography determination on the series of mixed reference substance solutions, plotting the content standard curves of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid, performing high performance liquid chromatography determination on the test sample solution, and respectively calculating the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test sample solution according to the content standard curves. The method of the present invention can simultaneously detect three protein-bound toxins in a short time, with low cost, simple method, low requirements for the conditions of the detection instrument, simple sample treatment method, and can detect a large number of samples.

Description

Technical Field

[0001] The present invention relates to the technical field of protein-bound uremic toxin detection, and particularly to a method for simultaneously detecting the contents of three protein-bound uremic toxins in a blood sample. Background Art

[0002] Uremic toxins refer to substances that accumulate in the body during renal failure and have specific biochemical or biological activities, which can be excreted through a healthy kidney under normal circumstances. Currently, uremic toxins are usually classified into three categories according to molecular weight and protein-binding status: (1) small-molecule water-soluble toxins with a molecular weight less than 0.3 kD, such as urea; (2) medium- and large-molecule toxins with a molecular weight mostly in the range of 0.3 - 12 kD, mainly peptides and cytokines; (3) protein-bound uremic toxins (PBUTs), such as p-cresol sulfate (PCS), indoxyl sulfate (IS), 3-indoleacetic acid (3-IAA), etc.

[0003] PBUTs are usually low-molecular-weight compounds. Since they can bind tightly to plasma proteins (mainly albumin) and their molecular weight can reach 66 kD, they are classified as high-molecular-weight compounds among uremic toxins, namely protein-bound uremic toxins (PBUTs). Therefore, this group of toxins is difficult to be removed by conventional hemodialysis methods. PCS is a prototype protein-bound uremic toxin, originating from the intestine. Intestinal bacteria metabolize aromatic amino acids (such as tyrosine and phenylalanine) into phenolic metabolites, which are then sulfated by the liver to form PCS. PCS levels have been shown to predict the clinical outcomes of patients with chronic kidney disease and are associated with their cardiovascular and all-cause mortality. Indole and indole derivatives are produced by tryptophan metabolism. Intestinal microbial tryptophanase first converts tryptophan into indole, which is then transported to the liver, and indole is sulfated by sulfotransferase in the liver to form IS. 3-IAA is directly produced by metabolism in the intestine or in tissues through tryptamine. These tryptophan metabolites are endogenous ligands of the transcription factor aryl hydrocarbon receptor and interact with various regulatory and signaling proteins. Activation of the aryl hydrocarbon receptor mediates cardiotoxicity, vascular inflammation, and procoagulant and pro-oxidant phenotypes of vascular cells. Increasing evidence shows that PBUTs play an important role in various pathological features of uremia.

[0004] At present, p-cresol sulfate and indoxyl sulfate are the most deeply and widely studied PBUTs. The detection methods for both include ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and high performance liquid chromatography (HPLC). Among them, HPLC includes fluorescence detection method and ultraviolet detection method. 3-Indoleacetic acid is usually determined by ultraviolet detection method. The detection method of UHPLC-MS is limited by cost and detection conditions and is not widely used; existing literature usually focuses on single-component detection. For example, in the literature "Determination of indoxyl sulfate concentration in human serum by HPLC-FLU method and its application in hemodialysis patients", a fluorescence detector was used to determine the concentration of indoxyl sulfate, with a single component; in the literature "Determination of uremic solutes in biological fluids of chronic kidney disease patients by HPLC assay", although a fluorescence detector was used to determine the concentrations of phenol, p-cresol, 3-indoleacetic acid, and indoxyl sulfate, different detection wavelengths were set for the method and multiple injections were required; in the literature "Protein-bound uremic toxins in hemodialysis patients measured by liquid chromatography / tandem mass spectrometry and their effects on endothelial ROS production", a liquid chromatography-mass spectrometry method was used to detect the contents of indoxyl sulfate, hydroxyglucuronide, indoleacetic acid, p-cresol sulfate, p-methylglucuronide, phenyl sulfate, phenol glucuronide, phenylacetic acid, phenylacetylglutamine, hippuric acid, 4-ethylphenyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid in blood samples, and the chromatogram showed that the components could not be effectively separated. Summary of the Invention

[0005] To overcome the above-mentioned defects existing in the prior art, the present invention provides a method for simultaneously detecting the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in a blood sample more quickly, simply, and accurately, so as to realize the clinical monitoring and management of 3 protein-bound toxins, and effectively improve the efficiency of clinical research and large-scale sample investigation and comparison.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for simultaneously detecting the contents of three protein-bound toxins in a blood sample, including the following steps:

[0008] (1) Mix the blood sample with methanol, centrifuge, and take the supernatant as the test solution;

[0009] (2) Mix p-cresol sulfate, indoxyl sulfate, 3-indoleacetic acid with methanol to obtain a mixed reference solution, and then successively dilute the mixed reference solution with methanol to obtain a series of mixed reference solutions;

[0010] (3) Perform high performance liquid chromatography determination on the series of mixed reference solutions, record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the series of mixed reference solutions, use the concentration of the series of mixed reference solutions as the abscissa, and use the chromatographic peak areas measured corresponding to the concentrations as the ordinate to respectively plot the content standard curves of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid;

[0011] (4) Perform high performance liquid chromatography determination on the test solution, record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test solution, and then calculate the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test solution respectively through the content standard curves plotted in step (3).

[0012] Preferably, in step (1), the mixing volume ratio of the blood sample to methanol is 1:1 to 5.

[0013] Preferably, in step (1), the temperature of the centrifugation is 4 to 8 °C, the rotation speed of the centrifugation is 3000 to 6000 rpm, and the time of the centrifugation is 30 to 50 min.

[0014] Preferably, in the mixed reference solution in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently 350 to 450 μg / mL.

[0015] Preferably, in the series of mixed reference solutions in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently 4 to 6 μg / mL, 8 to 12 μg / mL, 24 to 26 μg / mL, 48 to 52 μg / mL, and 98 to 102 μg / mL.

[0016] Preferably, the conditions for the high performance liquid chromatography determination in steps (3) and (4) are independently:

[0017] In the mobile phase, the 0.1 vt% trifluoroacetic acid aqueous solution is component A, and acetonitrile is component B;

[0018] The gradient elution conditions are: 0 to 8 min, 85% A; 8 to 18 min, 85% A → 65% A; 18 to 23 min, 65% A → 85% A; 23 to 28 min, 85% A;

[0019] The detection wavelength is 200 to 220 nm;

[0020] The flow rate is 0.8 - 1.2 mL / min;

[0021] The column temperature is 25 - 35 °C.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. By optimizing the high-performance liquid chromatography method, the present invention has established a high-performance liquid chromatography-ultraviolet detection method capable of simultaneously detecting p-cresol sulfate (PCS), indoxyl sulfate (S), and 3-indoleacetic acid (3-IAA), and conducts qualitative and quantitative analysis. This method can simultaneously detect three compounds in a short time, with low cost, simple method, low requirements for detection instrument conditions, simple sample treatment method, and can detect a large number of samples.

[0024] 2. Usually, when using a fluorescence detector to detect three toxins, p-cresol sulfate (PCS), indoxyl sulfate (S), and 3-indoleacetic acid (3-IAA), the excitation and emission wavelengths of the three toxins are different. The emission wavelengths for detecting p-cresol sulfate and indoxyl sulfate are often around 310 or 390 nm, and the emission wavelength of 3-IAA is often around 350 nm. Therefore, only one or two toxins can be detected under the same chromatographic conditions. However, this method uses an ultraviolet detector, and three toxins can be simultaneously detected by injecting the sample once under the same wavelength condition, which is simpler and faster.

[0025] 3. The precipitant for sample pretreatment usually selects one or a combination of methanol, acetonitrile, ethanol, acetone, etc., which can adapt to different mobile phases used for detecting different protein-bound toxins. It is found in the experimental process of the present invention that when using acetonitrile as the precipitant and simultaneously using acetonitrile as the mobile phase, split peaks appear in the chromatographic peaks of the compounds. However, using methanol can make the protein precipitation more complete, effectively remove impurities, and effectively improve the detection quality, greatly improving the detection efficiency.

[0026] 4. After the sample is precipitated and separated, the present invention takes the supernatant as the sample to be detected, reducing excessive dilution processes, ensuring the concentration of toxins in the sample, and avoiding the situation where the concentration is lower than the quantification limit due to too many dilution steps, resulting in undetectable results, effectively reducing the detection error. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative efforts.

[0028] Figure 1Chromatogram peak shape comparison between acetonitrile precipitant (left figure) and methanol precipitant (right figure);

[0029] Figure 2 High performance liquid chromatogram of the blank serum (Figure A) and the mixed standard solution (Figure B) of the present invention;

[0030] Figure 3 High performance liquid chromatogram of the test sample of the present invention. Detailed implementation mode

[0031] The present invention provides a method for simultaneously detecting the contents of three protein-bound toxins in a blood sample, comprising the following steps:

[0032] (1) After mixing the blood sample with methanol, centrifuge and take the supernatant as the test sample solution;

[0033] (2) Mix p-cresol sulfate, indoxyl sulfate, 3-indoleacetic acid with methanol to obtain a mixed reference solution, and then serially dilute the mixed reference solution with methanol to obtain a series of mixed reference solutions;

[0034] (3) Perform high performance liquid chromatography determination on the series of mixed reference solutions, record the chromatogram peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the series of mixed reference solutions, use the concentration of the series of mixed reference solutions as the abscissa, and the chromatogram peak areas measured at the corresponding concentrations as the ordinate to respectively plot the content standard curves of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid;

[0035] (4) Perform high performance liquid chromatography determination on the test sample solution, record the chromatogram peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test sample solution, and then calculate the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test sample solution respectively through the content standard curves plotted in step (3).

[0036] In the present invention, the preferred mixing volume ratio of the blood sample to methanol in step (1) is 1:1 to 5, more preferably 1:2 to 4, and even more preferably 1:3.

[0037] In the present invention, the preferred centrifugation temperature in step (1) is 4 to 8 °C, more preferably 5 to 7 °C, and even more preferably 6 °C; the preferred centrifugation speed is 3000 to 6000 rpm, more preferably 3500 to 4500 rpm, and even more preferably 4000 rpm; the preferred centrifugation time is 30 to 50 min, more preferably 35 to 45 min, and even more preferably 40 min.

[0038] In the present invention, in the mixed reference substance solution in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently preferably 350-450 μg / mL, and more preferably 400 μg / mL.

[0039] In the present invention, in the series of mixed reference substance solutions in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently preferably 4-6 μg / mL, 8-12 μg / mL, 24-26 μg / mL, 48-52 μg / mL, 98-102 μg / mL, and more preferably 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL.

[0040] In the present invention, the conditions for high performance liquid chromatography determination in steps (3) and (4) are independently preferably:

[0041] In the mobile phase, an aqueous solution of 0.1 vt% trifluoroacetic acid is component A, and acetonitrile is component B;

[0042] The gradient elution conditions are: 0-8 min, 85% A; 8-18 min, 85% A → 65% A; 18-23 min, 65% A → 85% A; 23-28 min, 85% A;

[0043] The detection wavelength is 200-220 nm, and more preferably 210 nm;

[0044] The flow rate is 0.8-1.2 mL / min, and more preferably 1 mL / min;

[0045] The column temperature is 25-35 °C, and more preferably 30 °C.

[0046] The technical solutions provided by the present invention will be described in detail below in conjunction with experimental examples, but they should not be construed as limiting the protection scope of the present invention.

[0047] The instruments and sources used in the following experimental examples: Agilent 1260 type high performance liquid chromatograph-ultraviolet detector (Agilent Technologies, USA); SQP type electronic balance (Sartorius Scientific Instruments Co., Ltd.); SN-LSC-40S type centrifuge (Shanghai Shangpu Instrument Equipment Co., Ltd.); WZ-100SP type water bath (Shanghai Shensheng Technology Co., Ltd.).

[0048] Experimental Example 1

[0049] A method for simultaneously detecting the contents of three protein-bound toxins in a blood sample, the steps are as follows:

[0050] (1) Selection of precipitant: In this invention, the effects of acetonitrile and methanol as precipitants on sample detection were investigated, and the results are as Figure 1 shown. According to the experimental results, it is known that when acetonitrile is used as the precipitant, it has a great influence on the peak shape of the target compound, showing phenomena such as split peaks and tailing, while when methanol is used as the precipitant, the peak shape of the target compound is smooth and the resolution is good.

[0051] (2) Sample pretreatment: An in vitro dialysis model was established. The in vitro dialysis model used 1× phosphate buffer solution (PBS, pH = 7.4) as the solvent, and a 40 g / L bovine serum albumin solution was prepared as artificial plasma. Standard p-cresol sulfate (PCS) with a concentration of 37.60 mg / L, indoxyl sulfate (IS) with a concentration of 37.65 mg / L, and 3-indoleacetic acid (3-IAA) with a concentration of 2.625 mg / L were added to the artificial plasma to simulate the plasma of uremic patients. A regenerated cellulose membrane with a molecular cut-off of 10 KD was used, filled with 20 mL of artificial plasma, immersed in 60 mL of peritoneal dialysis fluid, and dialyzed at 37 °C in a water bath for 4 h. After that, samples were taken inside the dialysis membrane. 1 mL of blood sample was taken, 3 mL of methanol was added, shaken, and centrifuged at 6 °C and 4000 rpm for 40 min. The supernatant was used as the test solution;

[0052] (3) Accurately weigh p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid respectively and place them in 10 mL volumetric flasks. Dissolve them with methanol and make up to the mark to obtain stock solutions of 2 mg / mL respectively. Then, take 2 mL each of the p-cresol sulfate stock solution, indoxyl sulfate stock solution, and 3-indoleacetic acid stock solution into the same 10 mL volumetric flask, and dilute with methanol to obtain a mixed reference solution with a final concentration of 400 μg / mL for each. Then, use methanol to serially dilute the 400 μg / mL mixed reference solution into a series of mixed reference solutions with concentrations of 5.0, 10.0, 25.0, 50.0, and 100.0 μg / mL;

[0053] (4) Perform high performance liquid chromatography determination on the series of mixed reference solutions. The conditions for the high performance liquid chromatography determination are as follows: Chromatographic column: C 18 chromatographic column; Detector: UV detector; Mobile phase A is 0.1 vt% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile; Flow rate: 1 mL / min; Column temperature: 30 °C; Detection wavelength: 210 nm, injection volume: 20 μL, and the elution program is shown in Table 1:

[0054] Table 1 High performance liquid chromatography elution program

[0055]

[0056]

[0057] Record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the series of mixed reference substance solutions. Taking the concentration of the series of mixed reference substance solutions as the abscissa and the chromatographic peak areas measured at the corresponding concentrations as the ordinate, respectively plot the content standard curves of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid, and calculate the detection limit with S / N = 3. The results are shown in Table 2 as follows:

[0058] Table 2 Standard Curves

[0059]

[0060] As can be seen from Table 2, the three compounds have good linear relationships within the concentration range of 5.0 - 100.0 μg / mL.

[0061] (5) Perform high performance liquid chromatography determination on the test sample solution under the same conditions as in step (4). Record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test sample solution, and then calculate the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test sample solution respectively through the content standard curves plotted in step (4). The final results are as follows in the test sample solution: the concentration of p-cresol sulfate is 23.53 mg / L; the concentration of indoxyl sulfate is 26.25 mg / L; the concentration of 3-indoleacetic acid is 1.50 mg / L.

[0062] Experimental Example 2

[0063] Detect the specificity of the method in Experimental Example 1:

[0064] Compare the blank serum without the addition of the three target compounds with the serum sample added with the three target compounds. The results are as Figure 2 shown. As can be Figure 2 seen, the method of the present invention has good specificity for detecting the three toxins, with good resolution and little interference.

[0065] Experimental Example 3

[0066] System suitability test:

[0067] Precisely measure 0.25 mL of the mixed reference substance solution and place it in a 10 mL volumetric flask, dissolve and make up the volume with methanol to obtain the system suitability test solution. Determine according to the chromatographic conditions in Experimental Example 1, record the chromatogram, and the elution order is as follows: indoxyl sulfate, p-cresol sulfate, 3-indoleacetic acid. Under the above chromatographic conditions, each component can be well separated, the blank solvent has no interference, and the retention times and resolutions of each component are shown in Table 3.

[0068] Table 3 Retention Times and Resolutions of Each Component

[0069] Component Name Retention Time (min) Resolution Indoxyl Sulfate 7.05 - p-Cresol Sulfate 10.61 7.91 3-Indoleacetic Acid 20.31 19.4

[0070] Experimental Example 4

[0071] Method accuracy detection:

[0072] Prepare samples with mixed reference solution concentrations of 10.0, 50.0, and 100.0 μg / mL in blank serum. After pretreatment, each concentration level was measured in parallel 6 times. Calculate the recovery rate and relative standard deviation (RSD) of the method in Experimental Example 1. The results are shown in Table 4.

[0073] Table 4 Results of method accuracy detection

[0074]

[0075] As can be seen from Table 4, the accuracy of the method of the present invention is good.

[0076] Experimental Example 5

[0077] Investigate the clearance efficiency of peritoneal dialysis fluid on three protein-bound toxins in the in vitro dialysis model of Experimental Example 1:

[0078] Clearance rate = (toxin addition concentration - post-dialysis concentration) / toxin addition concentration * 100%

[0079] According to the clearance rate formula, the clearance rates of peritoneal dialysis fluid on three toxins, p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid, were calculated to be 37.4%, 30.3%, and 42.9% respectively. According to data statistics, the concentrations of indoxyl sulfate, p-cresol sulfate, and 3-indoleacetic acid in normal human bodies are 0.53 mg / L, 1.9 mg / L, and 0.5 mg / L respectively. From the data obtained by the method of the present invention, it can be seen that the clearance effect of peritoneal dialysis fluid on protein-bound toxins fails to reach the clinically effective treatment effect, and the clearance effect of conventional peritoneal dialysis fluid on protein-bound toxins is limited.

[0080] In summary, the present invention has established a more rapid, simple, and accurate high-performance liquid chromatography method for simultaneously determining the contents of PCS, IS, and 3-IAA in plasma samples to achieve clinical monitoring and management of three protein-bound toxins, thereby providing a basis for clinical diagnosis and optimization of treatment plans, and can effectively improve the efficiency of clinical research and large-scale sample investigation and comparison.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for simultaneously detecting the contents of three protein-binding toxins in a blood sample, characterized in that, It includes the following steps: (1) Mix the blood sample with methanol, centrifuge, and take the supernatant as the test solution; (2) Mix p-cresol sulfate, indoxyl sulfate, 3-indoleacetic acid with methanol to obtain a mixed reference solution, and then serially dilute the mixed reference solution with methanol to obtain a series of mixed reference solutions; (3) Perform high performance liquid chromatography (HPLC) determination on the series of mixed reference solutions, record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the series of mixed reference solutions, use the concentration of the series of mixed reference solutions as the abscissa, and the chromatographic peak areas measured at the corresponding concentrations as the ordinate to respectively plot the content standard curves of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid; (4) Perform high performance liquid chromatography (HPLC) determination on the test solution, record the chromatographic peak areas of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test solution, and then calculate the contents of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid in the test solution respectively through the content standard curves plotted in step (3); In step (1), the mixing volume ratio of the blood sample to methanol is 1:1 - 5; The conditions for the high performance liquid chromatography determination are as follows: chromatographic column: C 18 chromatographic column; Detector: ultraviolet detector; mobile phase A is 0.1 vt% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile; flow rate: 0.8 - 1.2 mL / min; column temperature: 25 - 35 °C; detection wavelength: 200 - 220 nm; The elution program is shown in the following table: 。 2. The method for simultaneously detecting the contents of three protein-binding toxins in a blood sample according to claim 1, characterized in that, In step (1), the temperature of the centrifugation is 4 - 8 °C, the rotation speed of the centrifugation is 3000 - 6000 rpm, and the time of the centrifugation is 30 - 50 min.

3. A method for simultaneously detecting the contents of three protein-binding toxins in a blood sample according to claim 1, characterized in that, In the mixed reference solution in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently 350 - 450 μg / mL.

4. A method for simultaneously detecting the contents of three protein-binding toxins in a blood sample according to claim 1, characterized in that, In the series of mixed reference solutions in step (2), the final concentrations of p-cresol sulfate, indoxyl sulfate, and 3-indoleacetic acid are independently 4 - 6 μg / mL, 8 - 12 μg / mL, 24 - 26 μg / mL, 48 - 52 μg / mL, 98 - 102 μg / mL.

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