Specific detection of lipid hydroperoxides using HPLC-EC method.
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Animals↗
Biomedical subjects
S S Shibata
Publications and source records attributed to S S Shibata.
Selective quantification of arachidonic acid hydroperoxides and their hydroxy derivatives in reverse-phase high performance liquid chromatography.
For the quantification of lipid hydroperoxides by high performance liquid chromatography (HPLC), it has been necessary to improve the detection system specific to the hydroperoxy group. We first developed a technique which combined detection by uv absorption due to conjugated diene and detection based on electrochemical (EC) reduction in reverse-phase HPLC for the selective determination of arachidonic acid hydroperoxides (hydroperoxyeicosatetraenoic acid, HPETE) and its reduced derivative, hydroxyeicosatetraenoic acid (HETE). 15-HPETE was quantified selectively by EC detection, although both 15-HPETE and 15-HETE were detected by uv absorption and were hardly resolved in the chromatogram. Isomers in HPETE obtained from autoxidized arachidonic acid were partially separated in the chromatogram and seem to have been quantified similarly to 15-HPETE. The application of this analytical system to the analysis of 15-HPETE added in human plasma has demonstrated that the recovery of HPETE extracted from human plasma is much lower than that from normal saline and that HPETE is reduced to HETE by incubation at 37 degrees C. The fact that a high concentration of glutathione accelerated this reduction may indicate that human plasma possesses a glutathione-dependent HPETE-reducing ability as a defense system against excess accumulation of lipid hydroperoxides. Blood plasma effectively suppressed the decomposition of HPETE induced by ferrous ion indicating the presence of factors which prevent the action of ferrous ion on HPETE.
Arachidonic Acids↗
Limitations of the hemoglobin method for detecting lipid hydroperoxides.
The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides, trilinoleoylglycerol hydroperoxides and phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were different. Some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.
Animals↗
Limitations of the method using peroxidase activity of hemoglobin for detecting lipid hydroperoxides.
The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides was examined by using pure methyl linoleate hydroperoxides, trilinoleoylglycerol hydroperoxides and egg yolk phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were quite varied. Furthermore, some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, it seems the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.
Animals↗