Changes In The Metabolic Footprint Of Placental Explant-Conditioned Culture Medium Related To Hypoxia And Severe Preeclampsia

Preeclampsia (PE) is a common pregnancy-associated disorder that complicates 8% of pregnancies. The disease is responsible for 60-70,000 maternal deaths worldwide per annum and 16% of intra-uterine fetal deaths in the world. Early onset severe preeclampsia (ES-PE) happened before 34 weeks, which is associated with increased fetal and maternal morbidity and mortality. Late-onset severe preeclampsia(LS-PE) happened after 34 weeks. Most of fetuses were in good condition. The pathogenesis of preeclampsia is not clear. The clinical doctors paid much attention on how to explore the incidence of severe preeclampsia and how to prevent and monitor preeclampsia to minimize its risks.The pathogenesis of severe preeclampsia is thought to involve gene, immunology, oxidative response and other factors. The placenta is proposed to be the source of these causative factors. Placental changes which are evidence in the 1st trimester happened before the maternal clinical symptoms. Placental changes include inadequate trophoblast remodeling of maternal uterine spiral arteries which limits placenta blood flow. The clinical syndrome of preeclampsia is temporally characterized by inadequate placental-maternal adaptation followed by the release of endothelial activating factors and subsequent systemic maternal endothelial dysfunction. The villous trophoblast, which has an extensive interface with the maternal circulation, exhibits several changes in PE including:increased apoptosis and degeneration of the syncytiotrophoblast with increased shedding of trophoblast-derived material into the maternal circulation. It is hypothesized that this placental phenotype results from reduced utero-placental blood flow causing hypoxic stress within the placenta, which in turn leads to the release of pathological factors into the maternal circulation. Current research on early-onset and late-onset severe preeclampsia pathogenesis is controversial. Compared with late-onset severe preeclampsia, the apoptosis and degeneration of the syncytiotrophoblast of early-onset severe preeclampsia are much more serious. The etiology and pathogenesis may be different between early-onset and late-onset severe preeclampsia. However, the mechanism of placenta pathophysiological changes is not clear.Metabolomics is a newly developed approach, as an integral part of the systems biology encompassing a number of omics sciences such as genomics, transcriptomics and proteomics, and has become one of the hottest subjects worldwide. Metabonomics uses multivariate statistical technique to analyze highly complex data sets generated by high-throughput spectroscopy such as mass spectrometry (MS) of biological samples to capture metabolic variations in response to genetic modifications and environmental stimuli and track the metabolic pathway. Metabolic products are the end products of gene expression. The products level is determined by the metabolic pathways and the role of the enzyme activity of enzymes. They are associated with the body's physiology, pathology, and development states. Therefore, metabolomics provides a way to study diseases from other side.Metabolic footprint is a technology to analyze the metabolic products secreted by cells and tissues. The cells and tissues were cultured in the medium. We control and monitor the whole process of the consumption of nutrients and secretion of metabolites. We can know the structure of metabolic products through metabolic footprint technology. Inductive metabolomic strategies have been successfully employed to indentify metabolic differences in maternal plasma obtained from PE pregnancies and conditioned culture medium from villous trophoblast. Metabolic footprint technology provides a new means of research to predict the incidence of disease and understanding disease mechanisms.The technology of metabnomics is developing rapidly. Liquid chromatography-mass spectrometry is efficient, sensitive and accurate. It is widely used in the analysis of hard, polar compounds, thermally unstable compounds and macromolecular compounds (including proteins, peptides, polysaccharides, polymers, etc). It is one of the most promising metabolomics technologies.Liquid chromatography can reduce ion suppression effects. The products can be well separated by liquid chromatography through selecting appropriate chromatographic conditions according to the same physical and chemical properties of isomers. The appropriate method of liquid chromatography can get lower detection limits and reduce background noise. We could get higher quality mass spectrometry data by using appropriate method. There are few chromatography libraries which can be used currently. However, we can build our own database and analytical spectrum by using standard materials. The unknown metabolites can be identified relying on comparision with the standard materials.The human pregnancy needs sufficient amino acids to support the fetal growth and development. The provision of amino acids to the fetus in support of growth and development requires a balance between uteroplacental uptake, metabolism and transport, the degree of which varies considerably between amino acid types. Furthermore, for glutamine and glutamate, there is an interorgan exchange between the placenta and fetal liver that exists throughout most of gestation. Provision of glutamine to the fetus results from the combination of uterine uptake and transfer coupled with placental production. There is considerable fetal hepatic uptake of glutamine, with approximately 50% exiting the fetal liver after conversion to glutamate. Hepatic production is the primary source of fetal glutamate, as well as determining the supply to the placenta. Of the glutamate perfusing the placenta, approximately 90% is extracted by the placenta. Placental glutamate extraction, coupled with placental ammonia production associated with branched-chain amino acid transamination, completes the interorgan cyclicity of glutamine/glutamate during pregnancy.Tryptophan (Trp) is an essential amino acid which is important for keeping the activation and proliferation of cells. It is also an important component of proteins. Tryptophan participates in varieties of pathophysiological processes of the body. In mammals, most of the tryptophan changed into kynurenine (Kyn) with the help of the indoleamine 2,3-dioxygenase (IDO). Indoleamine 2,3-dioxygenase catalyzes extrahepatic tissues by the pathway of kynurenine. Indoleamine 2,3-dioxygenase is the first rate-limiting enzyme, which is widely expressed in the maternal-fetal interface, trophoblast and placenta. The expression of indoleamine 2,3 dioxygenase is enhanced with the development of placenta of health pregnant women. The level of tryptophan in maternal plasma is lower in normal pregnancy. The level of kynurenine in maternal plasma is higher in normal pregnancy. Indoleamine 2,3-dioxygenase plays an important role in regulating the maternal-fetal immune tolerance. These amino acids play a crucial role in the phathological changes of human placenta. The changes of these amino acids's concentration are related to the pathogenesis of preeclampsia.In the current investigation we aimed to further develop placental metabolic footprinting to investigate changes in cultured villous tissue from early-onset severe preeclampsia, late-onset severe preeclampsia and uncomplicated pregnancies cultured under normoxic and hypoxic conditions. We received the placentas within 20 min of delivery. The objective was to investigate the differences in the metabolic footprint associated with PE and to determine whether hypoxic conditions have a role to play in the metabolic processes of placental tissue in PE by using high performance liquid chromatography-mass spectrometry (HPLC-MS). The other objective is to explore the different pathogenesis of early-onset severe preeclampsia and late-onset severe preeclampsia. This study was also to identify metabolites or metabolic pathways which merit further investigation. At the same time, we use standard materials to establish the glutamate, glutamine, tryptophan and kynurenine chromatography library. By analyzing the concentration of glutamate, glutamine, tryptophan and kynurenine in the culture medium, we explore the effect of these amino acids on the pathogenesis of severe preeclampsia. This study is meaningful to provide treatment targets, reduce morbidity, reduce infant and maternal complications and reduce maternal and perinatal mortality. CHAPTERⅠCHANGES IN THE METABOLIC FOOTPRINT OF PLACENTAL EXPLANTS-CONDITONED CULTURE MEDIUM OF SEVERE PREECLAMPSIA IN HYPOXIA CONDITIONOBJECTIVEThe first objective was to investigate the differences in the metabolic footprint associated with early-onset severe preeclampsia and late-onset severe preeclampsia and to determine whether hypoxic conditions have a role to play in the metabolic processes of placental tissue in early-onset severe preeclampsia and late-onset severe preeclampsia. The second objective is to explore the different pathogenesis of early-onset severe preeclampsia and late-onset severe preeclampsia. The third objective is to study the unknown metabolites in the culture medium of placenta at hypoxia condition.METHODS1 Culture of placenta villous explants:the pregnant women were separated into three groups:uncomplicated pregnancy (n=27), early-onset severe preeclampsia (n=13) and late-onset severe preeclampsia (n=14). Placenta was received within 20 min of delivery. The explants were cultured in medium for 48 h at 37℃in an atmospheric oxygen tension of 1% or 6% O2. After 48 h, culture medium was replaced with fresh equilibrated medium, and after a further 48 h, conditioned culture medium was collected and immediately frozen at-80℃refrigerator.2 Pretreatment of medium sample:culture medium samples thawed at room temperature, drawing 100μL medium samples in the centrifuge tube, add 300μL protein precipite with methanol, vortex mixing for 1 minute, using high-speed refrigerated centrifuge, at 4℃Conditions, the 12,000 rpm centrifugation for 15 minutes, the supernatant filtered through water filter needle. After filtration, the samples were analyzed by HPLC-MS. 3 High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis:we separately set the condition of liquid chromatography, mass spectrometry conditions and operating modes. Samples were adding automatic by the machine.4 Statistical methods:LC-MS raw data files were inputed into the MarkerView TM software for data extraction and pre-treatment, using principal component analysis (PCA) method to observe the overall distribution of samples in each group. We use the principal component discriminant analysis (PCA-DA) to identify metabolic profiles contributed greatly to the metabolites between the groups. We can find the most relevant differences metabolites in severe preeclampsia by the principal component discriminant analysis. The data use T test which is in MarkerViewTM package. Some of the data were inputed into EXCEL to analyze the variance by SPSS13.0.RESULTS1 No discernable differences existed between samples taken from the edge, centre or midpoint within each placenta of uncomplicated pregnancy, early-onset severe preeclampsia and late-onset severe preeclampsia which were cultured at oxygen tension of 1% or 6%O2 (P>0.05). Therefore, data for all three sampling positions were included as replicates for further data analysis.2 There are 545 metabolite features showed a statistically significant difference between 1% and 6% O2 in uncomplicated pregnancies (P<0.05). There are 291 metabolite features showed a statistically significant difference between 6% O2 in uncomplicated pregnancies and 6% O2 early-onset severe preeclampsia (P <0.05). There are 36 metabolite features showed a statistically significant difference between 1% O2 in early-onset severe preeclampsia and 6% O2 uncomplicated pregnancies(P<0.05). There are 528 metabolite features showed a statistically significant difference between 1% O2 in late-onset severe preeclampsia and 6% O2 uncomplicated pregnancies (P<0.05). We selected a metabolite which has XIC and EMS pictures in Analyst software and EPI picture in Marker ViewTM software.3 The results of principal component analysis (PCA):There are no statistically significant difference between 1% O2 in uncomplicated pregnancies and 6% O2 late-onset preeclampsia. There are no statistically significant difference between 1% O2 in uncomplicated pregnancies and 6% O2 early-onset severe preeclampsia. There are no statistically significant difference between 1% O2 in early-onset severe preeclampsia and 6% O2 late-onset severe preeclampsia. There are no statistically significant difference between 1% O2 in uncomplicated pregnancies and 6% O2 late-onset severe preeclampsia. There are no statistically significant difference between 6% O2 in uncomplicated pregnancies and 6% O2 late-onset severe preeclampsiaCONCLUSIONWe could analyze the metabolic footprint changes in culture medium of uncomplicated and severe preeclampsia pregnancies at hypoxia and normoxia conditions by HPLC-MS technology, indicating that the method is suitable for studying the pathogenesis of preeclampsia. We found that the metabolic footprint changes were different between early-onset severe preeclampsia and late-onset severe preeclampsia, showing that the pathogenesis of early-onset severe preeclampsia and late-onset severe preeclampsia is different. We selected a metabolite which has XIC and EMS pictures in Analyst software and EPI picture in MarkerViewTM software. These pictures provide a lot of information of metabolites structure which can help us to identify the metabolites in further study. CHAPTERⅡGLUTAMATE, GLUTAMINE, TRYPTOPHAN AND KYNURENINE METABOLIC CHANGES IN THE PLACENTAL EXPLANTS-CONDITIONED CULTURE MEDIUM OF SEVERE PREECLAMPSIA IN HYPOXIA CONDITION.OBJECTIVEWe used standard materials to establish the glutamate, glutamine, tryptophan and kynurenine chromatography library by HPLC-MS technology. By analyzing the concentration of glutamate, glutamine, tryptophan and kynurenine in the culture medium of uncomplicated pregnancy, early-onset severe preeclampsia and late-onset severe preeclampsia in hypoxic and normoxia conditions, to explore the effect of these amino acids on the pathogenesis of severe preeclampsia and metablolic pathway of these amino acids.METHODS1 The groups of culture medium of placenta:uncomplicated pregnancy (n=22), early-onset severe preeclampsia (n=12) and late-onset severe preeclampsia (n=14).2 Pretreatment of medium sample:As shown in previous experiments.3 Establishment chromatograph library of standard materials:Take standard materials (glutamate, glutamine, kynurenine and tryptophan) and the standard materials were prepared by chromatography of pure methanol standard solution, respectively. They were detected by HPLC-MS under experimental conditions according to their molecular weight. And then take 100ul mixture of four standard solutions which were detected by tandem mass spectrometry to establish chromatography library of standard materials.4 High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis:we separately set the condition of liquid chromatography, mass spectrometry conditions and operating modes. Samples were adding automatic by the machine.5 Statistical methods:LC-MS raw data files were imputed into the Analyst software for data extraction and pre-treatment and then the data was exported to EXCEL. According to the formula:sample concentration (ug/ml)= sample peak area×standard concentration/standard peak area to calculate the concentration of each sample. The data was analyzed with T test and Mann-Whitney test by applying software SPSS13.0.RESULTS1 Compared with 1% O2 late-onset severe preeclampsia:the concentration of tryptophan was significantly lower and the concentration of kynurenine was significantly higher in 6% O2 late-onset severe preeclampsia; the concentration of tryptophan was significantly lower and the concentration of kynurenine was significantly higher in 6% O2 uncomplicated pregnancies.2 Compared with 6% O2 early-onset severe preeclampsia:the concentration of glutamate was lower, the concentration of tryptophan was significantly lower and the concentration of kynurenine was significantly higher in 1% O2 early-onset severe preeclampsia; the concentration of kynurenine was significantly lower in 6% O2 late-onset severe preeclampsia; the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 late-onset severe preeclampsia; the concentration of kynurenine was significantly lower in 6% O2 uncomplicated pregnancies; the concentration of glutamate was lower, the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 uncomplicated pregnancies.3 Compared with 1% O2 uncomplicated pregnancies:the concentration of tryptophan was significantly lower and the concentration of kynurenine was significantly higher in 6% O2 uncomplicated pregnancies; the concentration of tryptophan was significantly lower and the concentration of kynurenine was significantly higher in 6% O2 late-onset severe preeclampsia.4 Compared with 6% O2 uncomplicated pregnancies:the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 early-onset severe preeclampsia; the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 late-onset severe preeclampsia.5 Compared with 6% O2 late-onset severe preeclampsia:the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 early-onset severe preeclampsia; the concentration of tryptophan was significantly higher and the concentration of kynurenine was significantly lower in 1% O2 late-onset severe preeclampsia.CONCLUSIONThe method of using standard materials to establish the chromatography library of glutamate, glutamine and tryptophan can be used for studying the metabolic pathway of severe preeclampsia. Our research found the concentration of glutamate, glutamine, kynurenine and tryptophan had significantly changes in the uncomplicated pregnancies and severe preeclampsia at hypoxic condition. The study demonstrated that hypoxic plays an important role in the pathogenesis of severe preeclampsia. The changes of tryptophan and kynurenine in the culture medium of placental villous explants meet the law of metabolic pathways. They are also closely related to the pathogenesis of severe preeclampsia. The concentration of glutamate, tryptophan and kynurenine in the culture medium of placental villous explants were significantly different between early-onset severe preeclampsia and late-onset severe preeclampsia. The research suggested that the pathogenesis maybe different between early-onset severe preeclampsia and late-onset severe preeclampsia.SUMMARY1 We could analyze the metabolic footprint changes in culture medium of uncomplicated and severe preeclampsia pregnancies at hypoxia and normoxia conditions by HPLC-MS technology, indicating that the method is suitable for studying the pathogenesis of preeclampsia.2 We found that the metabolic footprint changes were different between early-onset severe preeclampsia and late-onset severe preeclampsia, showing that the pathogenesis of early-onset severe preeclampsia and late-onset severe preeclampsia is different.3 We selected a metabolite which was significant difference between different groups. The metabolite had XIC and EMS pictures in Analyst software and EPI picture in MarkerViewTM software. These pictures provided a lot of information of metabolites structure which could help us to identify the metabolites in further study.4 The method of using standard materials to establish the chromatography library of glutamate, glutamine, tryptophan and kynurenine can be used for studying the metabolic pathway of severe preeclampsia.5 Our research found the concentration of glutamate, glutamine, kynurenine and tryptophan had significantly changes in the uncomplicated pregnancies and severe preeclampsia at hypoxic condition. The study demonstrated that hypoxic plays an important role in the pathogenesis of severe preeclampsia. The changes of tryptophan and kynurenine in the culture medium of placental villous explants meet the law of metabolic pathways. They are also closely related to the pathogenesis of severe preeclampsia.6 The concentration of kynurenine in the culture medium of placental villous explants were significantly different between early-onset severe preeclampsia and late-onset severe preeclampsia. The research suggested that the pathogenesis maybe different between early-onset severe preeclampsia and late-onset severe preeclampsia.