Author: Wang, Jianying
Title: Rapid differentiation of the plasma samples from ischemic stroke patients and health control group and investigation of the effects of hypoxia on vesicular lipid trafficking
Advisors: Yao, Zhongping (ABCT)
Degree: Ph.D.
Year: 2022
Subject: Cerebrovascular disease -- Diagnosis
Cerebral anoxia
Lipids -- Analysis
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: xxix, 177 pages : color illustrations
Language: English
Abstract: Stroke is one of the leading causes of death and disability in the world, and ischemic stroke (IS) accounts for around 85% of the total stroke cases. IS should be treated with tissue plasminogen activator within 3-4.5 hours of the stroke onset, and this time window is called the "golden window" of stroke treatment. However, conventional methods for diagnosis of IS, such as computed tomography and magnetic resonance image, are time-consuming and sometimes they cannot provide reliable results, hereby the discovery of biomarkers for rapid diagnosis of IS is a strategy to solve the problem. However, most of the biomarkers reported so far are proteins, which cannot readily cross the blood-brain barrier to cause significant changes in blood within 3-4.5-hour "golden window". Lipids account for about 10% wet weight of the brain and they alter immediately once ischemic injury happens, thus they are potential biomarkers of IS. Therefore, we aimed to discover lipid biomarkers for rapid and reliable diagnosis of IS utilizing lipidomics approach. In this project, plasma samples from IS patient group and health control (HC) group were collected and analyzed after optimization of the sample preparation procedures and liquid chromatography mass spectrometry (LC-MS) protocols. Principal component analysis (PCA) based on the obtained LC-MS data showed good distinguishment of the IS and HC groups in the discovery dataset, indicating that IS patients could be differentiated from HC based on their lipid profiles in the discovery phase. Subsequently, based on the marker discovery in discovery dataset and the evaluation of validation dataset, a total of 20 lipids were selected as the potential biomarkers. Among these 20 potential markers, the levels of FA(20:0), LPC(O-20:0), LPS(O- 15:0), nordeoxycholic acid, 23:0 sterol, and 14:0 cholesterol ester were significantly increased in the plasma of IS patients compared with their levels in the health controls. In contrast, the remaining 14 lipids showed reductions in IS patients, including n-butyl arachidonoyl amine, FA(20:3), TG(14:0/18:2/18:3), TG(15:1/19:1/20:5), PS(P-20:0/20:5), PI(17:0/22:1), PC(O-38:9), PI(O-20:0/15:1), PE(P-16:0/20:3). PC(18:2/22:6), PS(O-18:0/19:1), PS(20:0/22:4), glycinoprenol-9, and Cer(m18:1/24:1). Furthermore, after binary logistic regression analysis, a combination of three lipids, i.e., FA(20:3), PS(O-18:0/19:1) and LPS(O-15:0), showed distinctive differentiation of IS and HC with area under the curve (AUC) value, sensitivity, and the specificity of 1.000, 100% and 100%, and 0.963, 100% and 87.5% for the discovery dataset and the validation dataset, respectively. Therefore, the combination of FA(20:3), PS(O-18:0/19:1) and LPS(O-15:0) was defined as a "biomarker panel" and could be applied in the diagnosis of IS. The development of LC-MS-based methods allowed rapid analysis of plasma samples and diagnosis of IS with high accuracy, thereby shortening the diagnosis time before tpA treatment and reducing the mortality and morbidity of IS patients. In addition, we could find that the correlation network among lipid biomarkers and phospholipid and sphingolipid pathways were altered upon IS onset, indicating that these potential IS-related lipid markers may be the targets of IS prevention and treatment. Furthermore, the levels of six lipid biomarkers including GlcCer(t18:1/18:0), GlcCer(d16:2/22:0), LPE(O-14:0), PG(O-20:0/22:0), PI(O-18:0/19:1) and PC(20:5/20:4) were significantly changed in IS patients when compared to HC, which suggested that these six lipids might help with the assessment of the severity of IS patients and evaluate the prognosis of IS treatment.
To further shorten the analysis time required for IS diagnosis, direct analysis in real time-mass spectrometry (DART-MS), a rapid and simple technique that allows direct analysis of samples with minimal sample pretreatment, was attempted for direct analysis of the plasma samples from the IS patients and HC group. The spectral data from 36 IS samples and 19 health controls were used as the discovery dataset to build an orthogonal projection to latent structures discriminant analysis (OPLS-DA) model, and the result of response permutation testing indicated that the established OPLS-DA model was valid without overfitting. The score plots of OPLS-DA model showed good differentiation of the IS patient group from the HC group with prediction power for both the HC group and IS group of 100%. Subsequently, an additional spectral data from 17 IS samples and 9 HC samples were employed to further validate the established OPLS-DA model with the prediction abilities of 100% and 94.1% for the healthy group and the disease group, respectively. Therefore, the established OPLS-DA could be employed for the rapid differentiation between IS and HC groups using DART-MS.
Vesicle trafficking is closely related to the progress of diseases. However, the studies of vesicle trafficking have been mainly focused on the transport of proteins, with very few on vesicular trafficking of small molecules such as lipids. In this project, the lipidomics studies of semi-intact (SI) cells and vesicle samples were carried out to respectively reflect the alterations of lipid compositions of intracellular membrane system and vesicle after hypoxia, an important characteristic for development of tumor cell. Interestingly, the level of glycerophosphocholine (PC) was increased significantly in SI cell treated with insufficient oxygen. However, the levels of glycerophosphoglycerol (PG), glycerophosphoserine (PS) and lysophosphoscholine (LPC) decreased markedly after hypoxia, and the levels of diacylglycerol (DG) and glyceroethanolamine (PE) were not changed in SI after hypoxic cultivation, indicating that the hypoxia inhibited the catabolism pathway of PC, instead of PC anabolism pathway. Since sphingolipid and lysophospholipid, bioactive lipids that were related to the vesicular transportation, were significantly altered in the SI cell after oxygen deprivation, we further investigated the relationship between hypoxia and vesicular lipid composition. Interestingly, different from other glycerophospholipid (GP), the level of PE was decreased in vesicles from the hypoxic cells, so we presumed that the rigidity of vesicle membrane might be increased by hypoxia. Strikingly, our data showed that significantly more long fatty acyl chains (C > 40) were observed in vesicles under hypoxia compared to normal condition, which further confirmed that stimulation of hypoxia increased the stiffness of vesicle membrane. Moreover, the transmission electron microscope (TEM) result showed that membrane of hypoxic vesicle became thicker than that of normal vesicle, it might be due to the longer fatty acyl chain accumulated in hypoxic vesicle. In addition, the Nanosight and TEM results showed that vesicle size was significantly increased under hypoxia. This study revealed that lipid composition of intracellular membrane system and vesicle were significantly influenced by oxygen deprivation, and hypoxia increased the rigidity of vesicle membrane and vesicle size, which might shed a new light on the study on mechanism and treatment of cancer.
Rights: All rights reserved
Access: open access

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