As a severe pregnancy complication, the pathophysiology of preeclampsia is not fully understood and the only effective treatment is delivery . In the present study, we applied high performance liquid chromatography coupled with quadrupole-time-of light mass spectrometry (HPLC-QTOF-MS) to investigate the metabolic changes in women with preeclampsia. Ninety serum samples and nine placentas tissue were used in the above metabolomic analyses. Sixteen metabolites in serum were identified as the differential metabolites and the area under ROC curves suggested that pyroglutamic acid (pGlu), methionine, glutamine and taurocholic acid were potentially valuable for PE diagnosis. Furthermore, metabolic pathways analysis was performed on web-based Metaboanalyst4.0 and it revealed that the metabolisms of linoleic acid and alpha-linolenic acid, phenylalanine, tyrosine and tryptophan biosynthesis, D-glutamine/D-glutamate, phenylalanine, glutathione, tyrosine and tryptophan were significantly altered and might be involved in the PE pathogenesis.
Pyroglutamic acid, a natural amino acid derivative, can be synthesized in living cells enzymatically and non-enzymatically. It has been reported that pGlu could efficiently inhibit the catalytic activity of human angiotensin-converting enzyme (ACE) . For instance, at the concentration of 20 µg/mL, pGlu was found to inhibit 98.2% of the activity of human ACE in vitro. ACE plays a central function of converting angiotensin Ⅰ (Ang Ⅰ) to Ang Ⅱ and it has been shown to contribute to hypertension via the renin-angiotensin system (RAS) [19–21]. However, the circulating and intrarenal RAS was supposed to be down-regulated to compensate the up-regulated local uteroplacental RAS in preeclampsia [22, 23].In our study, we found that the serum pGlu was increased in PE group with a fold-change value of 1.3, which may be associated with the downregulation of intrarenal RAS in preeclampsia.
As an essential amino acid, methionine is required for protein synthesis. In the methionine cycle, it can be regenerated from homocysteine (Hcy) and transformed into S-adenosylmethionine (SAM), which is the universal methyl donor in many cellular methylation reactions . In a case-control study, the SAM level was increased in the PE group although this difference was not statistically significant . In another study with 32 PE patients and 64 controls, maternal plasma Hcy and folate were significantly elevated in patients in the third trimester . In our analysis, the levels of methionine were much higher in PE patients than those in normal pregnancy. In addition, among those identified differential metabolites, the methionine showed the best performance for PE diagnosis with the AUC of 0.909. However, an opposite change of methionine has been reported in another metabolomics study . Therefore, further studies are warranted to better understand the role of methionine in the PE pathogenesis.
Similarly, the glutamine serum level was also increased in the PE group. In a metabolomic study with placental tissues, it was reported that the concentrations of glutamine were elevated in severe PE patients . Interestingly, there were other studies in which the glutamine in both placenta and serum were found at a lower concentration in PE women [29, 30]. What’s more, the low concentration of glutamine may increase the expression of intercellular cell adhesion molecules-1 in human umbilical vein endothelial cells, enhance migration of neutrophils across the endothelial cells, and cause tissue destruction eventually .
Emerging metabolomic studies suggested that the dysregulation of lipid metabolism played an important role in the development of preeclampsia [28, 32, 33]. The lipid metabolism changes in PE could be characterized by increased levels of serum triglyceride (TG), low-density lipoprotein (LDL), and circulating free fatty acids (FFAs), and accompanied with decreased level of high-density lipoprotein (HDL). In a study focused on the components of esterified and free fatty acids, it has been reported that the levels of palmitic, oleic and linoleic acids were significantly increased in women with PE . In the present study, the linoleate which could be consumed to derive linoleic acid was decreased and the metabolic product of alpha-linolenic acid such as (9Z,12Z,15Z)-octadecatrienoic acid was severely increased in the placenta of PE women. What was more, the levels of arachidonic acid and its derivative such as 5,6-epoxyeicosatrienoic acid (EET) in placenta was higher in the PE group compared with the normal pregnancy in our study. Linoleic acid is the precursor of endogenous arachidonic acid (AA) which could be further converted to EETs by the cytochrome P-450 (CYP) epoxygenase. Herse et al. reported that the EETs including 5,6-EET, 14,15-EET, and the dihydroxyeicosatrienoic acids, were elevated in the preeclamptic women due to the up-regulated expression of the CYP subfamily 2J polypeptide 2 (CYP2J2) . More importantly, the supplement of linoleic acid during pregnancy has been reported to be beneficial to the prevention and management of PE .
This was the first metabolomics study of human placenta which reported the levels of glutathione (GSH), its oxidized form (glutathione disulfide, GSSG) and the GSSG/GSH ratio were all increased in PE patients. The change of placental GSH was consistent with previous study conducted by Knapen et al . The decrease of GSH levels in placenta and serum was also has been reported in several studies [38–40]. As observed in our study, it has been reported that the GSSG/GSH ratio was significantly increased in the placenta of PE patients in other researches [39, 41].
In order to better understand the GSH metabolism disturbance in PE, the RT-qPCR experiments were performed to examine the gene expression level of relevant enzymes in the pathway, such as the expression of GCLC, GCLM, GSS, GPx1, GPx4 and GSR. Although not statistically significant, the increased expression of GCLM may have reflected the cellular reducing power demands. However, excessive production of reactive oxygen species (ROS) depleted the GSH pool and resulting in high-level GSSG/GSH ratio (51.8 in PE vs 17.3 in control, extracted from raw data of MetDNA analyses with placenta) and decreased overall antioxidants. Interestingly, the GPx-1 mRNA expression showed a mild reduction in PE in our study. Bilodeau et al proposed that GPx-1/3/4 deficiency might promote the synthesis of vasoconstrictive eicosanoids such as F2-isoprostanes and thromboxanes, which are known to be up-regulated in PE placentas . The mRNA expression of COX-1 and COX-2 that are directly involved in the production of thromboxanes , was significantly elevated in the PE placenta (Fig. 4). The induction of the COX enzymes has been reported closely related to excessive oxidative stress in rat cytotrophoblast, spongiotrophoblast and glycogen cells and might be regulated through activation of the p38MAPK and the NF-κB transcription factor .
As a part of tryptophan metabolic pathway, the major catabolic route is the oxidation of tryptophan to kynurenine by the hepatic enzyme tryptophan 2,3-dioxygenase or ubiquitous indoleamine 2,3- dioxygenase (IDO) . In normal pregnancy, the IDO is highly expressed within placental and contributed to an increase of plasma kynurenine/tryptophan ratio. In preeclampsia, the levels of plasma tryptophan and kynurenine/tryptophan ratio were vastly decreased compared with normal pregnancy due to reduction of placental IDO expression . Santillan et al. found that the IDO deficiency could lead to pathognomonic renal glomerular endotheliosis, proteinuria, pregnancy-specific endothelial dysfunction, intrauterine growth restriction, and mildly elevated blood pressure in IDO knockout mice models . Thus, it was clear that the downregulation of tryptophan metabolism and reduction of IDO activity were involved in the pathogenesis of PE.