Application of sphingomyelin metabolite in preparation of kit for predicting preeclampsia

Abstract

The invention relates to the technical field of biology, in particular to application of sphingomyelin metabolites as markers in preparation of a kit for predicting preeclampsia. By detecting sphingomyelin metabolites in 180 serum samples of preeclampsia patients, it is further verified that the sphingomyelin metabolites have good correlation with blood samples of the preeclampsia patients. Based on good correlation between sphingomyelin metabolites and blood samples of preeclampsia patients, it is considered that the sphingomyelin metabolites can serve as markers for predicting preeclampsia, and the sphingomyelin metabolites are used for predicting preeclampsia through quantitative determination of the content of the sphingomyelin metabolites. Therefore, the invention provides a brand new way for predicting and / or treating preeclampsia.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the application of sphingomyelin metabolites in preparing a kit for predicting the occurrence of preeclampsia. Background Art

[0002] Preeclampsia (PE) is a multi-organ damage syndrome unique to human pregnancy that seriously endangers the health of mothers and fetuses. It is characterized by new or worsening hypertension and proteinuria after 20 weeks of pregnancy, which seriously affects the morbidity and mortality of pregnant women and perinatal infants worldwide (Steegers E AP, Dadelszen PV, Duvekot JJ, et al. Pre-eclampsia [J]. Lancet, 2010, 376 (9741): 631-644.). The pathogenesis of preeclampsia is complex and involves multiple pathophysiological factors. There is still a lack of ideal predictive and early diagnostic markers (Rana S, Lemoine E, Granger JP, et al. Preeclampsia: Pathophysiology, Challenges, and Perspectives [J]. Circulation research: a journal of the American Heart Association, 2019, 124 (7): 1094-1112.). Therefore, it is necessary to seek biomarkers that can effectively predict preeclampsia early, assess the risk of pregnant women before clinical symptoms appear, intervene and prevent as early as possible, and thus reduce the mortality rate associated with preeclampsia. The discovery of the role of sphingomyelin metabolites in angiogenesis and vascular function regulation may provide new clues to the pathogenesis and prediction of preeclampsia.

[0003] Sphingomyelin is the main structural component of cell membranes. Its metabolites, sphingosine (Sph), dihydrosphinganine (dhSph) and their 1-phosphate (sphingosine-1-phosphate, S1P) and dhS1P (sphinganine-1-phosphate), are considered to be key regulatory molecules for the balance of vascular function in the body and play an important role in the pathogenesis of vascular function (Cantalupo A, Zhang Y, Kothiya M, et al. Nogo-B regulatesendothelial sphingolipid homeostasis to control vascular function and blood pressure [J]. Nature medicine, 2015, 21 (9): 1028.). S1P is produced by intracellular sphingosine kinase (SPHK) catalyzing sphingosine (SPH), and is rapidly exported to the extracellular space through the transporter SPNS2. It binds to specific G protein-coupled receptors (S1PR1-5) on the cell membranes of different tissues, activating multiple downstream signaling pathways, thereby exerting a series of biological effects, such as cell proliferation, angiogenesis, inflammation, cell migration, etc. (Maceyka M, Spiegel S. Sphingolipid metabolites in inflammatory disease [J]. Nature, 2014, 510(7503): 58-67.).

[0004] In recent years, some scholars have found that there is an imbalance in sphingolipid metabolism in the peripheral blood and placenta of patients with preeclampsia. Studies have found that the serum S1P level in patients with early-onset preeclampsia is significantly lower than that in normal pregnant women (Melland-Smith M, Ermini L, Chauvin S, et al. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia[J]. Autophagy, 2015, 11(4): 653-669.). Other studies have found that the levels of S1P and dhS1P in the placental tissue of patients with severe preeclampsia are significantly higher than those in normal pregnant women (Walsh SW, Reep DT, Alam SMK, et al. Placental Production of Eicosanoids and Sphingolipids in Women Who Developed Preeclampsia on Low-Dose Aspirin[J]. Reprod Sci, 2020, 27(12): 2158-2169.). The study also found that the dhSph level in the placental chorionic artery of patients with preeclampsia was significantly higher than that of normal pregnant women (Del Gaudio I, Sasset L, Lorenzo AD, et al. Sphingolipid Signature of Human Feto-Placental Vasculature in Preeclampsia[J]. International Journal of Molecular Sciences, 2020, 21(3): 1019.). Therefore, the inventors believe that sphingomyelin metabolites may be involved in the occurrence and development of preeclampsia.

[0005] However, due to the mutual conversion between Sph and S1P, dhSph and dhS1P, similar chemical structures, low peripheral blood concentrations, and certain technical difficulties in simultaneously measuring these markers, there is controversy about the relationship between the concentration level of sphingomyelin metabolites in peripheral blood and preeclampsia. In recent years, some scholars have used LC-MS / MS technology to detect sphingomyelin substances, but the sample pretreatment process is complicated and time-consuming, and is not suitable for rapid determination of large quantities of samples. Therefore, this field needs a method for simply, quickly, and accurately detecting multiple sphingomyelin metabolites, so as to facilitate further confirmation of specific sphingomyelin metabolites that have a good correlation with blood samples of patients with preeclampsia. Summary of the invention

[0006] The object of the present invention is to provide a new use of sphingomyelin metabolites in predicting preeclampsia.

[0007] The present invention utilizes high performance liquid chromatography tandem triple quadrupole mass spectrometry to establish a simple, rapid, and sensitive LC-MS / MS method that can simultaneously and quantitatively detect four sphingomyelin metabolites in the blood, and finds specific sphingomyelin metabolites that have a good correlation with blood samples of patients with preeclampsia.

[0008] The present invention further verifies the good correlation with the blood samples of preeclampsia patients by detecting sphingomyelin metabolites in 180 serum samples of preeclampsia patients.

[0009] Based on the good correlation between sphingomyelin metabolites and blood samples of patients with preeclampsia, the present invention believes that sphingomyelin metabolites can be used as a marker for predicting preeclampsia, and can be used to predict preeclampsia by quantitatively measuring its content.

[0010] The first aspect of the present invention provides the use of sphingomyelin metabolites as markers in the preparation of a kit for predicting the occurrence of preeclampsia, wherein the sphingomyelin metabolites refer to one or two of sphingosine (Sph), sphingosine-1-phosphate (S1P), dihydrosphinganine (dhSph) or dihydrosphinganine-1-phosphate (dhS1P).

[0011] The second aspect of the present invention provides the use of a reagent for detecting the expression level of sphingomyelin metabolites in preparing a kit for predicting the occurrence of preeclampsia, wherein the sphingomyelin metabolites are one or two of Sph, S1P, dhSph or dhS1P.

[0012] Furthermore, the sphingomyelin metabolite is a combination of Sph and S1P, or a combination of dhSph and dhS1P.

[0013] Furthermore, the S1P / Sph ratio is used in the preparation of a kit for predicting the occurrence of preeclampsia. When the S1P / Sph ratio is lower than the cut-off value at the same stage of pregnancy, it is predicted that preeclampsia may occur. Among them, the cut-off value of the S1P / Sph ratio before 20 weeks of pregnancy is 42.30 (AUC=0.684, 95% CI: 0.594-0.766); the cut-off value of the S1P / Sph ratio at 20-28 weeks of pregnancy is 20.60 (AUC=0.692, 95% CI: 0.599-0.775); the cut-off value of the S1P / Sph ratio after 28 weeks of pregnancy is 11.29 (AUC=0.762, 95% CI: 0.675-0.835).

[0014] Furthermore, the application is to detect sphingomyelin metabolites in serum samples by conventional methods, and to establish a qualitative or quantitative method for detecting sphingomyelin metabolites and a matching reagent or kit.

[0015] Furthermore, the reagent for detecting the expression level of sphingomyelin metabolites is a reagent for detecting the expression level of sphingomyelin metabolites in serum samples using high performance liquid chromatography tandem mass spectrometry or ultra high performance liquid chromatography tandem mass spectrometry.

[0016] In a specific embodiment of the present invention, the method for detecting sphingomyelin metabolites is high performance liquid chromatography tandem mass spectrometry, and sphingomyelin metabolites are all detected in ESI+ mode. TM HPLC chromatographic conditions are as follows: the mobile phase is 0.5% formic acid / 10mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10mmol / L ammonium formate / 90% acetonitrile solution (phase B), the flow rate is 0.2mL / min, the gradient elution program is 0-1min B phase maintained at 50%, 1-4.5min B phase from 50%→100%, 4.5-7min B phase maintained at 100%, 7-7.1min B phase from 100%→50%, 7.1-8min B phase maintained at 50%; the chromatographic analysis column is Kinetex EVO C18 (1.7μm, 2.1*100mm), the column temperature is 40℃, the injection volume is 20μL, and the detection of 4 metabolites can be completed within 8 minutes. AB SCIEX Quad TM The operating conditions of 4500MD mass spectrometer are as follows: electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, ion source temperature TEM is 600°C, ionization voltage IS is 4500V, collision gas CAD is 7psi, curtain gas CUR is 30psi, spray gas GS1 is 60psi, and auxiliary heating gas GS2 is 55psi.

[0017] Furthermore, the pretreatment method of serum samples selected methanol containing 0.1% formic acid as a precipitant to extract four sphingomyelin metabolites from the blood.

[0018] Furthermore, the detailed steps of the pretreatment method are as follows: take 100 μL of the human serum sample to be tested, mix it with 10 μL of isotope internal standard and 300 μL of precipitant (methanol containing 0.1% formic acid), vortex mix it, sonicate it for 15 minutes, centrifuge it for 10 minutes, draw the supernatant into an Agilent micro glass insert tube, and inject it on the machine.

[0019] The third aspect of the present invention provides the use of a reagent for detecting the expression level of sphingomyelin metabolites in the preparation of a kit for predicting the occurrence of severe preeclampsia, wherein the reagent for detecting the level of sphingomyelin metabolites is a reagent for detecting the expression level of dhSph and the ratio of dhS1P / dhSph in serum.

[0020] Preeclampsia is defined as severe if any of the following symptoms occur in pregnant women: (1) persistent uncontrollable blood pressure: systolic blood pressure ≥160 mmHg and / or diastolic blood pressure ≥110 mmHg; (2) persistent headache, visual disturbances, or other central nervous system abnormalities; (3) persistent upper abdominal pain and subcapsular hematoma or liver rupture; (4) elevated blood alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels; (5) urine protein >2.0 g / 24 h or oliguria (24 h urine volume <400 ml, or hourly urine volume <17 ml); (6) hypoproteinemia with ascites, pleural effusion, or pericardial effusion; (7) persistent decrease in platelet count and less than 100 × 10 9 / L, intravascular hemolysis; (8) heart failure; (9) pulmonary edema; (10) fetal growth restriction or oligohydramnios, intrauterine fetal death, placental abruption, etc.

[0021] Furthermore, the detection method of the sphingomyelin metabolites dhSph and dhS1P is a high performance liquid chromatography tandem mass spectrometry method, and the sample pretreatment method is the same as above.

[0022] Furthermore, when the serum dhSph level was higher than the cut-off value of 2.42 ng / mL (AUC=0.769, 95% CI: 0.669-0.852) and the dhS1P / dhSph ratio was lower than the cut-off value of 61.74 (AUC=0.757, 95% CI: 0.655-0.841) before 20 weeks of gestation, severe preeclampsia was predicted.

[0023] The fourth aspect of the present invention also provides the use of sphingomyelin metabolites in screening drugs for treating preeclampsia.

[0024] Furthermore, the application refers to using the candidate drug for patients with preeclampsia to detect whether the phosphorylation ratio of sphingomyelin metabolites increases.

[0025] A fifth aspect of the present invention provides a kit for predicting the occurrence of preeclampsia, wherein the kit comprises a reagent for detecting the expression level of one or both of Sph, S1P, dhSph or dhS1P in serum.

[0026] Furthermore, the method for using the kit is: first, in vitro detecting the content of sphingomyelin metabolites in the serum sample to be tested, and secondly, comparing the content of sphingomyelin metabolites with that in the serum of the normal group to determine whether the content of sphingomyelin metabolites in the serum sample to be tested is different from that in the normal sample.

[0027] In a sixth aspect of the present invention, a method for detecting the expression level of sphingomyelin metabolites in serum is provided, wherein the sphingomyelin metabolites are one or two of Sph, S1P, dhSph or dhS1P, and the pretreatment method of the serum sample selects methanol containing 0.1% formic acid as a precipitant to extract four sphingomyelin metabolites from the blood, and the method comprises the following steps: using high performance liquid chromatography tandem mass spectrometry, the sphingomyelin metabolites are all detected in ESI+ mode; Shimadzu Jasper TM HPLC chromatographic conditions are as follows: the mobile phase is 0.5% formic acid / 10mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10mmol / L ammonium formate / 90% acetonitrile solution (phase B), the flow rate is 0.2mL / min, the gradient elution program is 0-1min B phase maintained at 50%, 1-4.5min B phase from 50%→100%, 4.5-7min B phase maintained at 100%, 7-7.1min B phase from 100%→50%, 7.1-8min B phase maintained at 50%; the chromatographic analysis column is Kinetex EVO C18 (1.7μm, 2.1*100mm), the column temperature is 40℃, the injection volume is 20μL, and the detection of 4 metabolites can be completed within 8 minutes. AB SCIEX Quad TM The operating conditions of 4500MD mass spectrometer are as follows: electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, ion source temperature TEM is 600°C, ionization voltage IS is 4500V, collision gas CAD is 7psi, curtain gas CUR is 30psi, spray gas GS1 is 60psi, and auxiliary heating gas GS2 is 55psi.

[0028] Furthermore, the detailed steps of the pretreatment method are as follows: take 100 μL of the human serum sample to be tested, mix it with 10 μL of isotope internal standard and 300 μL of precipitant (methanol containing 0.1% formic acid), vortex mix it, sonicate it for 15 minutes, centrifuge it for 10 minutes, draw the supernatant into an Agilent micro glass insert tube, and inject it on the machine.

[0029] The advantages of the present invention are:

[0030] The present invention further verifies the good correlation between sphingomyelin metabolites and blood samples of patients with preeclampsia by detecting sphingomyelin metabolites in 180 serum samples of patients with preeclampsia. Based on the good correlation between sphingomyelin metabolites and blood samples of patients with preeclampsia, the present invention believes that sphingomyelin metabolites can be used as a marker for predicting preeclampsia, and through quantitative determination of its content, it is used to predict the occurrence of preeclampsia. Therefore, the present invention provides a new approach for the prediction and / or treatment of preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 .Chromatograms of four sphingomyelin metabolites.

[0032] Figure 2 .Chromatograms of 3 internal standards.

[0033] Figure 3 .The relationship between serum sphingomyelin metabolites and their ratios and preeclampsia.

[0034] Figure 4 .The relationship between serum sphingomyelin metabolites and their ratios and the severity of preeclampsia. Among them, CTL: pregnant women with normal pregnancy, MPE: pregnant women with mild preeclampsia, SPE: pregnant women with severe preeclampsia. DETAILED DESCRIPTION

[0035] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0036] Example 1: Establishment of a method for detecting sphingomyelin metabolites

[0037] 1. Reagents and Materials

[0038] LCMS grade ammonium formate (Anaqua Chemicals Supply, USA), HPLC grade acetonitrile (Merck, Germany), HPLC grade methanol (Merck, Germany), LCMS grade formic acid (Anaqua Chemicals Supply, USA), Watsons distilled water, Agilent micro glass insert (250 μL, 5.6*30 mm), chromatographic analysis column Kinetex EVOC18 (1.7 μm, 2.1*100 mm) (Tianjin, Aeger-Finomei Co., Ltd.).

[0039] Standard products of four sphingomyelin metabolites: Sph (U.S., Cayman Company, purity ≥ 98%), S1P (U.S., Cayman Company, purity ≥ 98%), dhSph (U.S., Cayman Company, purity ≥ 98%), and dhS1P (U.S., AvantiPolar Lipids Company, purity ≥ 99%).

[0040] Three isotope internal standards: Sph-d7 (U.S., Cayman Company, purity ≥ 99%), S1P-d7 (U.S., Cayman Company, purity ≥ 99%), dhSph-d7 (U.S., Cayman Company, purity ≥ 99%).

[0041] Sph-d7 was used as the internal standard for Sph, S1P-d7 was used as the internal standard for S1P and dhS1P, and dhSph-d7 was used as the internal standard for dhSph.

[0042] 2. Mass spectrometry conditions

[0043] The mass spectrometer model is AB SCIEX QuadTM 4500MD triple quadrupole mass spectrometer, electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, the source parameters and compound parameters of the mass spectrometer are obtained by optimization, the ion source temperature TEM is 600 ° C, the ionization voltage IS is 4500V, the collision gas CAD is 7psi, the curtain gas CUR is 30psi, the spray gas GS1 is 60psi, and the auxiliary heating gas GS2 is 55psi. The optimized compound parameters are shown in Table 1.

[0044] Table 1 Compound parameters of four sphingomyelin metabolites and three isotopic internal standards

[0045]

[0046] In Table 1, DP is the declustering potential, EP is the entrance voltage, CE is the collision energy, CXP is the collision cell exit voltage, Q1 is the parent ion, and Q3 is the product ion.

[0047] 3. Liquid chromatography conditions

[0048] The Shimadzu JasperTM HPLC chromatographic conditions are as follows: the mobile phases are 0.5% formic acid / 10 mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10 mmol / L ammonium formate / 90% acetonitrile solution (phase B), the flow rate is 0.2 mL / min, and the gradient elution program is 0-1 min, phase B is maintained at 50%, 1-4.5 min, phase B is from 50%→100%, 4.5-7 min, phase B is maintained at 100%, 7-7.1 min, phase B is from 100%→50%, and 7.1-8 min, phase B is maintained at 50%; the chromatographic analysis column is Kinetex EVO C18 (1.7 μm, 2.1*100 mm), the column temperature is 40°C, the injection volume is 20 μL, and the detection of 4 metabolites can be completed within 8 minutes.

[0049] 4. Configuration of standard curve, quality control products and internal standard solution

[0050] Preparation of standard stock solutions: Accurately weigh appropriate amounts of Sph and dhSph standards, dissolve them with chloroform-methanol (2 / 1, V / V), make up to volume, and store them in brown glass bottles. The concentrations of Sph and dhSph stock solutions are 1.244 mg / mL and 830 μg / mL, respectively, and store them in a -80°C refrigerator. Accurately weigh appropriate amounts of S1P and dhS1P standards, ultrasonically dissolve them with chloroform-methanol-acetic acid (80 / 20 / 12, V / V / V), make up to volume, and store them in brown glass bottles. The concentrations of S1P and dhS1P stock solutions are 104 μg / mL and 100 μg / mL, respectively, and store them in a -80°C refrigerator.

[0051] Prepare the standard curve working solution: accurately pipette an appropriate amount of the above stock solution, dilute it with methanol and mix it to prepare a 20× standard curve mixed working solution. Accurately pipette 5μL of the 20× standard curve mixed working solution, add 95μL of 25% methanol, mix well, and obtain the standard curve working solution. The final standard curve concentration points of this experiment are 0.5, 1, 2.5, 5, 15, 30, 50ng / mL for Sph and dhSph; S1P and dhS1P are expanded 10 times to 5, 10, 25, 50, 150, 300, 500ng / mL.

[0052] Preparation of quality control samples: Accurately pipette an appropriate amount of the above stock solution, dilute with methanol and mix to prepare 20× quality control working solution. Accurately pipette 5μL of 20× quality control working solution and add it to 95μL of mixed serum, mix evenly, and prepare quality control samples of different concentrations. The above mixed serum comes from the mixed serum of healthy people. The low concentration (QC-L) of the quality control samples in this experiment is 1ng / mL Sph, 1ng / mL dhSph, 10ng / mL S1P and 10ng / mL dhS1P; the medium concentration (QC-M) is 5ng / mL Sph, 5ng / mL dhSph, 50ng / mL S1P and 50ng / mL dhS1P; the high concentration (QC-H) is 30ng / mLSph, 30ng / mL dhSph, 300ng / mL S1P and 300ng / mL dhS1P.

[0053] Prepare internal standard working solution:

[0054] Weigh an appropriate amount of Sph-d7 and dhSph-d7 internal standards, dissolve them with chloroform-methanol (2 / 1, V / V), make up to volume, and store them in a brown glass bottle to obtain 500 μg / mL Sph-d7 and dhSph-d7 stock solutions, which are stored in a -80°C refrigerator. Weigh an appropriate amount of S1P-d7 internal standard, ultrasonically dissolve them with chloroform-methanol-acetic acid (80 / 20 / 12, V / V / V), make up to volume, and store them in a brown glass bottle to obtain 400 μg / mL S1P-d7 stock solution, which is stored in a -80°C refrigerator. Accurately pipette an appropriate amount of the above internal standard stock solutions, dilute them with methanol, and mix them to prepare a mixed internal standard working solution with a concentration of 500 ng / mL for Sph-d7, dhSph-d7, and S1P-d7.

[0055] Example 2: Establishment of a pre-treatment method for serum sphingomyelin metabolites

[0056] 1. Pretreatment and extraction of four metabolites of serum sphingomyelin

[0057] Accurately pipette 100 μL of standard curve working solution or quality control sample or human serum sample to be tested, add 10 μL of internal standard working solution and 300 μL of precipitant (methanol containing 0.1% formic acid), vortex mix, ultrasonicate for 15 min, centrifuge for 10 min (15,000 rpm, 4°C), pipette the supernatant into Agilent micro-glass insert, and inject on the machine.

[0058] 2. Linear range, correlation coefficient and minimum quantification limit of four metabolites of serum sphingomyelin

[0059] The chromatograms of sphingosine Sph, dihydrosphingosine dhSph and their 1-phosphate metabolites S1P and dhS1P are shown in the figure. The lower limit of quantification is between 0.5-5ng / mL, the linear range is 100 times, the weight is 1 / x, and the correlation coefficient r is greater than 0.99. Both clinical patient and healthy human samples can be quantitatively detected within the linear range (see Table 2). The chromatogram of the actual serum sample shows that the four sphingomyelin metabolites can be well separated from the interference peaks on this chromatographic analysis column (see Figure 1 ), the chromatograms of the three internal standards are shown in Figure 2 .

[0060] Table 2 Linear range, correlation coefficient and minimum quantification limit for the detection of four metabolites of serum sphingomyelin

[0061]

[0062] 3. Precision and spike recovery of four metabolites of serum sphingomyelin

[0063] Six parallel quality control samples of low (QC-L), medium (QC-M), and high spike concentrations (QC-H) were prepared. After pretreatment to extract the four metabolites of sphingomyelin, each metabolite was determined under the optimal conditions of high performance liquid chromatography tandem mass spectrometry, and the precision and spike recovery were calculated. As shown in Table 3, the average spike recovery of the four metabolites of sphingomyelin detected by this experimental method was 86.5%-107.3%, and the relative standard deviation was 1.90%-7.00%, which were all within the acceptable range, indicating that the results of the detection of the four metabolites of sphingomyelin by this experimental method are stable and reliable.

[0064] Table 3 Spiked recovery and precision of four metabolites of serum sphingomyelin

[0065]

[0066] Example 3: Study on the relationship between serum sphingomyelin metabolites and preeclampsia

[0067] 1. Preeclampsia group: 180 serum samples from patients with preeclampsia were collected, of which 61 serum samples were collected at a gestational age of less than 20 weeks (including 31 serum samples from patients with mild preeclampsia and 30 serum samples from patients with severe preeclampsia), 59 serum samples were collected at a gestational age between 20 and 28 weeks (including 31 serum samples from patients with mild preeclampsia and 28 serum samples from patients with severe preeclampsia), and 60 serum samples were collected at a gestational age greater than 28 weeks (including 30 serum samples from patients with mild preeclampsia and 30 serum samples from patients with severe preeclampsia).

[0068] 2. Control group: 176 serum samples from normal pregnant women were collected, of which 60 serum samples were collected at a gestational age of less than 20 weeks, 56 serum samples were collected at a gestational age between 20 and 28 weeks, and 60 serum samples were collected at a gestational age greater than 28 weeks. The serum samples were all fasting blood collected from pregnant women during regular prenatal examinations. The serum was separated after centrifugation and stored at -80°C until testing. There were no significant differences in age, gestational age, and preeclampsia diagnosis week between the control group and the preeclampsia group. The systolic blood pressure, diastolic blood pressure, pre-pregnancy body mass index (BMI), and first delivery rate of the preeclampsia group were significantly higher than those of the control group, as shown in Table 4.

[0069] 3. The diagnostic criteria for preeclampsia in the above experimental groups are: refer to the version of "Guidelines for the Diagnosis and Treatment of Hypertensive Disorders Complicating Pregnancy (2020)" issued by the Hypertensive Disorders Complicating Pregnancy Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association. Preeclampsia: After 20 weeks of pregnancy, pregnant women have systolic blood pressure ≥140mmHg and (or) diastolic blood pressure ≥90mmHg, accompanied by any one of the following: urine protein quantification ≥0.3g / 24h, or urine protein / creatinine ratio ≥0.3, or random urine protein ≥+; no proteinuria but accompanied by any one of the following organs or systems involved: important organs such as the heart, lungs, liver, and kidneys, or abnormal changes in the blood system, digestive system, and nervous system, and the placenta and fetus are involved. Based on the severity of symptoms, the preeclampsia group was further divided into two experimental subgroups: mild preeclampsia group and severe preeclampsia group. Severe preeclampsia is defined as: (1) persistent uncontrolled blood pressure: systolic blood pressure ≥160 mmHg and / or diastolic blood pressure ≥110 mmHg; (2) persistent headache, visual disturbances, or other central nervous system abnormalities; (3) persistent upper abdominal pain and subcapsular hematoma or liver rupture; (4) elevated blood alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels; (5) urine protein >2.0 g / 24 h or oliguria (24-h urine volume <400 ml, or hourly urine volume <17 ml); (6) hypoproteinemia with ascites, pleural effusion, or pericardial effusion; and (7) a persistent decrease in platelet count below 100 × 10 9 / L, intravascular hemolysis; (8) heart failure; (9) pulmonary edema; (10) fetal growth restriction or oligohydramnios, intrauterine fetal death, placental abruption, etc.

[0070] Table 4 Clinical data of the experimental group

[0071]

[0072]

[0073] Note: All data are expressed as mean ± SD, *P < 0.05 indicates statistical significance.

[0074] 4. Detection method of sphingomyelin metabolites: refer to Example 1.

[0075] 5. Extraction of serum samples: refer to Example 2.

[0076] 6. Statistical methods: SPSS 20.0 was used as the statistical software, and Graphpad prism 5.0 was used as the drawing software. Data were expressed as median (interquartile range, IQR 25-75%) or mean ± standard deviation (mean ± SD). One-way analysis of variance was used for data with normal distribution and homogeneity of variance; Kruskal-Wallis H test was used for multiple independent sample data with unequal variance, and Mann-Whitney U test was used for two independent samples. P < 0.05 indicated statistically significant differences.

[0077] 7. Relationship between serum sphingomyelin metabolites and preeclampsia: The levels of sphingomyelin metabolites in the peripheral blood serum of normal pregnant women and preeclampsia patients at different gestational stages were compared. The specific results are shown in Tables 5 and Figure 3 . It was found that before 20 weeks of pregnancy and after 28 weeks of pregnancy, the serum Sph and dhSph levels of patients with preeclampsia were significantly higher than those of normal pregnancy at the same stage, and the dhS1P / dhSph ratio was significantly lower than that of normal pregnant women at the same stage. However, there were no significant differences in the serum Sph level, dhSph level and dhS1P / dhSph ratio between patients with preeclampsia and normal pregnant women at 20-28 weeks of pregnancy. There was no significant difference in the serum S1P level between patients with preeclampsia and normal pregnant women at the same stage before 20 weeks of pregnancy, while the serum S1P level between patients with preeclampsia and normal pregnant women at the same stage after 20 weeks of pregnancy was significantly lower than that of normal pregnant women at the same stage. At any stage of pregnancy, the S1P / Sph ratio of patients with preeclampsia was significantly lower than that of normal pregnant women at the same stage. At any stage of pregnancy, there was no significant difference in the serum dhS1P level between patients with preeclampsia and normal pregnant women at the same stage. ROC curve analysis found that the AUC of the S1P / Sph ratio was significantly higher than that of other metabolites at any stage of pregnancy. The AUC of the S1P / Sph ratio before 20 weeks of pregnancy was 0.684 (95% CI: 0.594-0.766), and the cut-off value was 42.30; the AUC of the S1P / Sph ratio during 20-28 weeks of pregnancy was 0.692 (95% CI: 0.599-0.775), and the cut-off value was 20.60; after 28 weeks of pregnancy, the AUC of the S1P / Sph ratio was 0.762 (95% CI: 0.675-0.835), and the cut-off value was 11.29.

[0078] Table 5 Relationship between serum sphingomyelin metabolites and preeclampsia during different gestational periods

[0079]

[0080]

[0081] Note: Data are expressed as median (interquartile range, IQR 25-75%), and P < 0.05 indicates statistical significance.

[0082] 8. Relationship between serum sphingomyelin metabolites and the severity of preeclampsia: The levels of sphingomyelin metabolites in the peripheral blood serum of normal pregnant women at different stages of pregnancy and patients with preeclampsia of different severity were compared. The specific results are shown in Tables 6 and Figure 4 . Results: Compared with normal pregnant women at the same stage, there were no significant differences in serum Sph and dhSph levels before 28 weeks of pregnancy in patients with mild preeclampsia, and serum Sph and dhSph levels after 28 weeks of pregnancy were significantly higher than those in normal pregnancy at the same stage. There were no significant differences in serum S1P levels, dhS1P levels, and dhS1P / dhSph ratios at any stage of pregnancy, and the S1P / Sph ratio at any stage of pregnancy was significantly lower than that in normal pregnancy at the same stage. Compared with normal pregnant women at the same stage of pregnancy, serum dhSph levels were significantly increased in patients with severe preeclampsia before 20 weeks of pregnancy and after 28 weeks of pregnancy, while S1P / Sph ratio and dhS1P / dhSph ratio were significantly decreased. The serum S1P levels of patients with severe preeclampsia were lower than those of normal pregnant women after 20 weeks of pregnancy, and there were no significant differences in serum Sph levels and dhS1P levels at any stage of pregnancy compared with those of normal pregnant women. Compared with patients with mild preeclampsia at the same stage, the serum dhSph level of patients with severe preeclampsia before 20 weeks of gestation was significantly increased, and the dhS1P / dhSph ratio was significantly decreased. ROC curve analysis before 20 weeks of gestation found that the AUC of serum dhSph and dhS1P / dhSph ratio were significantly higher than those of other metabolites, with the AUC of serum dhSph being 0.769 (95% CI: 0.669-0.852) and the cut-off value being 2.42 ng / mL; the AUC of dhS1P / dhSph ratio was 0.757 (95% CI: 0.655-0.841) and the cut-off value being 61.74.

[0083] Table 6 Relationship between serum sphingomyelin metabolites and severity of preeclampsia during different gestational periods

[0084]

[0085]

[0086] Note: Data are expressed as median (interquartile range, IQR 25-75%), P < 0.05 indicates statistical significance. Comparison between the experimental group and the normal pregnancy group at the same gestational stage: *P < 0.05, **P < 0.01, ***P < 0.001. Comparison between the mild eclampsia group and the severe eclampsia group at the same gestational stage: #P < 0.05, ##P < 0.01.

[0087] The present invention uses high performance liquid chromatography tandem mass spectrometry to detect four sphingomyelin metabolites in the serum of patients with preeclampsia, and analyzes the relationship between serum sphingomyelin metabolites at different stages of pregnancy and the severity and progression of preeclampsia. The present invention is the first to systematically analyze the relationship between sphingomyelin metabolites and preeclampsia. The results show that:

[0088] 1) The content of serum sphingomyelin metabolites changes dynamically with the stage of pregnancy (before 20 weeks of pregnancy, 20-28 weeks of pregnancy, and after 28 weeks of pregnancy). The relationship between serum sphingomyelin metabolites and preeclampsia is different in different stages of pregnancy.

[0089] 2) After 20 weeks of pregnancy, the serum S1P levels in patients with preeclampsia (especially severe preeclampsia) were significantly lower than those in normal pregnancy at the same stage.

[0090] 3) After 28 weeks of pregnancy, the serum Sph and dhSph levels in patients with preeclampsia (especially mild preeclampsia) were significantly higher than those in normal pregnancy at the same stage.

[0091] 4) At any stage of pregnancy, the S1P / Sph ratio of patients with preeclampsia was significantly lower than that of normal pregnant women at the same stage.

[0092] 5) The serum dhSph level of severe preeclampsia patients before 20 weeks of gestation was significantly higher than that of normal pregnant women and patients with mild preeclampsia at the same stage, and the dhS1P / dhSph ratio was significantly lower than that of normal pregnant women and patients with mild preeclampsia at the same stage.

[0093] For the first time, we found that dhSph levels were significantly increased and dhS1P / dhSph ratio was significantly decreased in patients with severe preeclampsia before 20 weeks of gestation. For the first time, we found that S1P / Sph ratio was decreased in the serum of patients with preeclampsia regardless of the gestational stage.

[0094] Based on the research results of the present invention, the following conclusions are drawn:

[0095] Sph, S1P, dhSph, dhS1P and their corresponding metabolic enzymes may play an important role in the occurrence and development of preeclampsia, and are expected to provide new clues for the etiology of preeclampsia.

[0096] Embodiment 4:

[0097] The specific use process of the kit of the present invention in clinical testing:

[0098] 1) Sample pretreatment: Accurately pipette 100 μL of standard curve working solution or quality control sample or human serum sample to be tested, add 10 μL of internal standard working solution and 300 μL of precipitant (methanol containing 0.1% formic acid), vortex mix, sonicate for 15 min, centrifuge for 10 min (15,000 rpm, 4°C), pipette the supernatant into Agilent micro-glass insert, and inject on the machine.

[0099] 2) Instrumental analysis: The mass spectrometer model is AB SCIEX QuadTM 4500MD triple quadrupole mass spectrometer, electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, the source parameters and compound parameters of the mass spectrometer were obtained by optimization, the ion source temperature TEM was 600°C, the ionization voltage IS was 4500V, the collision gas CAD was 7psi, the curtain gas CUR was 30psi, the spray gas GS1 was 60psi, and the auxiliary heating gas GS2 was 55psi.

[0100] Shimadzu Jasper TM The HPLC chromatographic conditions were as follows: the mobile phases were 0.5% formic acid / 10 mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10 mmol / L ammonium formate / 90% acetonitrile solution (phase B), the flow rate was 0.2 mL / min, the gradient elution program was 0-1 min, phase B was maintained at 50%, 1-4.5 min, phase B was from 50% to 100%, 4.5-7 min, phase B was maintained at 100%, 7-7.1 min, phase B was from 100% to 50%, and 7.1-8 min, phase B was maintained at 50%; the chromatographic analysis column was Kinetex EVO C18 (1.7 μm, 2.1*100 mm), the column temperature was 40°C, the injection volume was 20 μL, and the detection of 4 metabolites could be completed within 8 minutes.

[0101] 3) Sample detection and result calculation: Take the pre-treated standard curve, quality control samples and serum samples to be tested and inject them into the high performance liquid chromatography-tandem mass spectrometer for detection, and record the chromatographic peak area of ​​sphingomyelin metabolites and the corresponding internal standard peak area. Use the labeled concentration of the standard curve as the horizontal coordinate (x), and the ratio of the actual detection peak area of ​​the standard curve to the peak area of ​​each internal standard as the vertical coordinate (y) to draw the standard curve, and you can get the linear regression equation: y = a + bx, where a is the intercept and b is the slope. Substitute the ratio of the actual peak area of ​​Sph, S1P, dhSph, and dhS1P in the serum sample to be tested to the internal standard peak area into the above standard curve equation to calculate the concentration of Sph, S1P, dhSph, and dhS1P in the serum sample.

[0102] 4) Compare the content of sphingomyelin metabolites in normal serum at the same stage of pregnancy to determine whether there is a difference in the content and ratio of sphingomyelin metabolites in the tested serum samples. When the S1P / Sph ratio is lower than the cut-off value, it is predicted that preeclampsia may occur (the cut-off value of the S1P / Sph ratio before 20 weeks of pregnancy is 42.30, the cut-off value of the S1P / Sph ratio between 20 and 28 weeks of pregnancy is 20.60, and the cut-off value of the S1P / Sph ratio after 28 weeks of pregnancy is 11.29). When the dhSph level in serum before 20 weeks of pregnancy is higher than the cut-off value of 2.42 ng / mL and the dhS1P / dhSph ratio is lower than the cut-off value of 61.74, it is predicted that severe preeclampsia may occur.

[0103] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of sphingomyelin metabolites as markers in the preparation of a kit for predicting the occurrence of preeclampsia, characterized in that: The sphingomyelin metabolites are one or two of sphingosine, sphingosine-1-phosphate, dihydrosphingosine or dihydrosphingosine-1-phosphate.

2. Use of a reagent for detecting the expression level of sphingomyelin metabolites in the preparation of a kit for predicting the occurrence of preeclampsia, characterized in that: The sphingomyelin metabolites are one or two of sphingosine, sphingosine-1-phosphate, dihydrosphingosine or dihydrosphingosine-1-phosphate.

3. The use according to claim 1 or 2, characterized in that: The sphingomyelin metabolite is a combination of sphingosine and sphingosine-1-phosphate, or a combination of dihydrosphingosine and dihydrosphingosine-1-phosphate.

4. The use according to claim 1 or 2, characterized in that: The application is to detect sphingomyelin metabolites in serum samples by conventional methods, and to establish a qualitative or quantitative method for detecting sphingomyelin metabolites and a matching reagent or kit.

5. The use according to claim 4, characterized in that: The reagent for detecting the expression level of sphingomyelin metabolites is a reagent for detecting the expression level of sphingomyelin metabolites in serum samples by high performance liquid chromatography tandem mass spectrometry or ultra high performance liquid chromatography tandem mass spectrometry.

6. The use according to claim 5, characterized in that: The method for detecting sphingomyelin metabolites is high performance liquid chromatography tandem mass spectrometry, and sphingomyelin metabolites are all detected in ESI+ mode; Shimadzu Jasper TM HPLC chromatographic conditions are as follows: mobile phase is 0.5% formic acid / 10mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10mmol / L ammonium formate / 90% acetonitrile solution (phase B), flow rate is 0.2mL / min, gradient elution program is 0-1min B phase maintained at 50%, 1-4.5min B phase from 50%→100%, 4.5-7min B phase maintained at 100%, 7-7.1min B phase from 100%→50%, 7.1-8min B phase maintained at 50%; chromatographic analysis column is Kinetex EVO C18: 1.7μm, 2.1*100mm, column temperature is 40℃, injection volume is 20μL, complete the detection of sphingomyelin metabolites; AB SCIEX Quad TM The operating conditions of 4500MD mass spectrometer are as follows: electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, ion source temperature TEM is 600°C, ionization voltage IS is 4500V, collision gas CAD is 7psi, curtain gas CUR is 30psi, spray gas GS1 is 60psi, and auxiliary heating gas GS2 is 55psi.

7. Use of a reagent for detecting the expression level of sphingomyelin metabolites in the preparation of a kit for predicting the occurrence of severe preeclampsia, characterized in that: The reagent for detecting the level of sphingomyelin metabolites is a reagent for detecting the expression level of dihydrosphingosine in serum and the ratio of dihydrosphingosine-1-phosphate / dihydrosphingosine.

8. Use of sphingomyelin metabolites in screening drugs for treating preeclampsia, characterized in that: The sphingomyelin metabolites are one or two of sphingosine, sphingosine-1-phosphate, dihydrosphingosine or dihydrosphingosine-1-phosphate.

9. A kit for predicting the occurrence of preeclampsia, characterized in that: The kit comprises a reagent for detecting the expression level of one or two of sphingosine, sphingosine-1-phosphate, dihydrosphingosine or dihydrosphingosine-1-phosphate in serum by high performance liquid chromatography tandem mass spectrometry.

10. A method for detecting the expression level of sphingomyelin metabolites in serum, characterized in that: The sphingomyelin metabolites are one or two of sphingosine, sphingosine-1-phosphate, dihydrosphingosine or dihydrosphingosine-1-phosphate. The pretreatment method of the serum sample uses methanol containing 0.1% formic acid as a precipitant to extract four sphingomyelin metabolites from the blood. The method comprises the following steps: using high performance liquid chromatography tandem mass spectrometry, the sphingomyelin metabolites are all detected in ESI+ mode; Shimadzu Jasper TM HPLC chromatographic conditions are as follows: mobile phase is 0.5% formic acid / 10mmol / L ammonium formate / water solution (phase A) and 0.5% formic acid / 10mmol / L ammonium formate / 90% acetonitrile solution (phase B), flow rate is 0.2mL / min, gradient elution program is 0-1min B phase maintained at 50%, 1-4.5min B phase from 50%→100%, 4.5-7min B phase maintained at 100%, 7-7.1min B phase from 100%→50%, 7.1-8min B phase maintained at 50%; chromatographic analysis column is Kinetex EVO C18: 1.7μm, 2.1*100mm, column temperature is 40℃, injection volume is 20μL, complete the detection of sphingomyelin metabolites; AB SCIEX Quad TM The operating conditions of 4500MD mass spectrometer are as follows: electrospray ionization source ESI ionization, positive ion scanning, multiple reaction monitoring MRM mode, ion source temperature TEM is 600°C, ionization voltage IS is 4500V, collision gas CAD is 7psi, curtain gas CUR is 30psi, spray gas GS1 is 60psi, and auxiliary heating gas GS2 is 55psi.

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