RESUMO
In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.
Assuntos
Apoptose , Células Hep G2/citologia , Fosfatidilcolinas/metabolismo , Ciclo Celular , Células Hep G2/metabolismo , Células Hep G2/patologia , Humanos , Potencial da Membrana Mitocondrial , Fosfatidilcolinas/toxicidadeRESUMO
Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na](+), m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC-MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida/métodos , Fosfatidilcolinas/sangue , Fosfatidilcolinas/química , Sódio/química , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Estudos de Casos e Controles , Constrição Patológica/sangue , Feminino , Humanos , Isomerismo , Masculino , Fosfatidilcolinas/isolamento & purificaçãoRESUMO
Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.
Assuntos
Ferroptose , Humanos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Fosfatidilcolinas , Células Hep G2 , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismoRESUMO
Wastewater treatment processes are believed to be anthropogenic sources of nitrous oxide (N(2)O) and methane (CH(4)). However, few studies have examined the mechanisms and controlling factors in production of these greenhouse gases in complex bacterial systems. To elucidate production and consumption mechanisms of N(2)O and CH(4) in microbial consortia during wastewater treatment and to characterize human waste sources, we measured their concentrations and isotopomer ratios (elemental isotope ratios and site-specific N isotope ratios in asymmetric molecules of NNO) in water and gas samples collected by an advanced treatment system in Tokyo. Although the estimated emissions of N(2)O and CH(4) from the system were found to be lower than those from the typical treatment systems reported before, water in biological reaction tanks was supersaturated with both gases. The concentration of N(2)O, produced mainly by nitrifier-denitrification as indicated by isotopomer ratios, was highest in the oxic tank (ca. 4000% saturation). The dissolved CH(4) concentration was highest in in-flow water (ca. 3000% saturation). It decreased gradually during treatment. Its carbon isotope ratio indicated that the decrease resulted from bacterial CH(4) oxidation and that microbial CH(4) production can occur in anaerobic and settling tanks.
Assuntos
Poluentes Atmosféricos/análise , Monitoramento Ambiental/métodos , Metano/análise , Dióxido de Nitrogênio/análise , Eliminação de Resíduos Líquidos , Poluentes Atmosféricos/química , Poluentes Atmosféricos/metabolismo , Isótopos/análise , Metano/química , Metano/metabolismo , Dióxido de Nitrogênio/química , Dióxido de Nitrogênio/metabolismo , Esgotos/química , Esgotos/microbiologia , Microbiologia da Água , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Poluentes Químicos da Água/metabolismoRESUMO
Increasing evidence of phospholipid peroxidation in the pathophysiology of various diseases demonstrates the need for pure phosphatidylcholine hydroperoxide (PCOOH) reference as a standard for quantification, and for biological studies in this field. Our previous study showed the usability of reaction between hydroperoxide and vinyl ether for preparation of PCOOH. However, the reaction has sometimes provided brown byproducts. Here, we report a method to improve reaction conditions, and demonstrate the production of 30 mg of PCOOH (a pure mixture of 13- and 9-hydroperoxyoctadecadienoyl PCOOH isomers) from 100 mg of phosphatidylcholine without byproduct formation. The resultant PCOOH will be useful as a standard for quantification studies, as well as for the evaluation of PCOOH pathogenicity.