T L Leraas | PubMed Health

PubMed1998

A fluorescence plate reader assay for monitoring the susceptibility of biological samples to lipid peroxidation.

The susceptibility of biological samples to lipid peroxidation can be determined by exposing samples to a lipid peroxidation initiator and measuring the length of time prior to the onset of lipid peroxidation. Previous studies have shown that aldehydes generated by lipid peroxidation can react with amines to produce fluorescent products. We have utilized this principle to develop a fluorescence plate reader assay for measuring susceptibility to lipid peroxidation. In this assay, samples are placed in glycine/phosphate buffer and loaded into a 96-well plate. Lipid peroxidation initiators are added, and fluorescence is monitored over time. Samples were assayed for susceptibility to lipid peroxidation by both the thiobarbituric acid reactive substances assay and the fluorescence plate reader assay. We found good agreement between these two methods in assessing relative susceptibility to lipid peroxidation in liver microsomes and mitochondria. The fluorescence assay was also used to monitor lipid peroxidation in liposomes and rat liver homogenates. Fluorescence was stable over an extended time period and could be induced by a variety of lipid peroxidation initiators. The fluorescence plate reader assay offers a rapid method for monitoring lipid peroxidation in a large number of samples.

Animals↗

PubMed1997

alpha-Tocopheryl hemisuccinate administration increases rat liver subcellular alpha-tocopherol levels and protects against carbon tetrachloride-induced hepatotoxicity.

Rats were administered a series of tocopherol analogs 18 h prior to a hepatotoxic dose of carbon tetrachloride (CCl4). Of the compounds tested, only d-alpha-tocopheryl hemisuccinate (TS) provided significant protection against CCl4-induced hepatotoxicity. No protection was observed with either d-alpha-tocopherol (alpha-T) or a tocopherol succinate ether derivative, d-alpha-tocopheryloxybutyric acid (TSE). None of the tocopherol analogs significantly inhibited CYP2E1 activity as measured by oxidation of p-nitrophenol. Liver homogenates and subcellular fractions (cytosol, nuclei, plasma membranes, mitochondria and microsomes) were collected 18 h after tocopherol analog administration in the absence of CCl4. Homogenate and subcellular alpha-T levels were not significantly increased following TSE administration but were increased 2-3 fold following TS and alpha-T administration. Total tocopherol levels (alpha-T+ TS + TSE) in liver homogenates and subcellular fractions were highest in rats supplemented with TS. In these animals, TS was detected in all subcellular fractions and total tocopherol levels were increased from 6-23 fold over those seen in controls and 2-9 fold over alpha-T treated rats. In vitro studies in which liver homogenates and subcellular fractions were peroxidized with ascorbate and ADP/Fe suggest that increasing levels of alpha-T but not TS correlates with increased protection against lipid peroxidation. These results suggest that the ability of TS to protect against CCl4-induced hepatotoxicity relates to its enhanced hepatic accumulation and subsequent hydrolysis to alpha-T.

Alanine Transaminase↗