Studies of vitamin E metabolism, particularly in subjects undergoing exercise and patients with peroxisomal and mitochondrial disorders
Vitamin E (a-tocopherol) is the major lipid soluble secondary antioxidant in vivo and is important for maintaining the integrity of cell membranes. It may also have more specific functions in vivo including roles in cell signaling and gene expression. Relatively little is known, however, about the details and dynamics of vitamin E metabolism, which may be important in fully understanding its role(s) in vivo. Vitamin E metabolites detected in urine include a-tocopheronolactone (a- TL), with an oxidized chroman ring, and compounds with successively shortened phytyl side chains, including the carboxyethyl-hydroxychromans (CEHC) and carboxymethylbutyl-hydroxychromans (CMBHC). As a-TL can be produced from a-tocopherol by oxidation it may be a potential biomarker of in vivo oxidative stress. The CEHCs are the major urinary metabolites but the cellular localisation of the side-chain shortening process (mitochondria and/or peroxisomes) remains undefined. The aims of my studies were to determine whether a-TL could be a useful biomarker of oxidative stress and define the cellular localisation of the side-chain shortening process of the tocopherols. Initially methods were established and validated for the measurement of urinary vitamin E metabolites and the F2-isoprostane, 8isoPGF2a(a non-enzymatically derived isomers of the prostaglandins, formed in vivo by free radical mediated oxidation of arachidonic acid and an accepted biomarker of oxidative stress) by gas chromatography-mass spectrometry (GC-MS). To investigate whether a-TL was a potential biomarker of in vivo oxidative stress, urinary a-TL and 8-isoprostane were measured in healthy trained and untrained males before and after a standardised endurance exercise regimen. A significant correlation (p<0.0001) was found between the concentrations of a-TL and 8- isoPGF2a. There was, however, no evidence of an increase in oxidative stress following exercise in the subjects studied. Studies to investigate the role of peroxisomes and mitochondria in vitamin E metabolism utilized two approaches. The first involved the analysis of urinary vitamin E metabolites of patients with peroxisomal (n=5) and mitochondrial (n=3) disorders, and those suspected of peroxisomal or mitochondrial disorders (n=5) compared to age matched controls (n=19). No consistent and significant differences in metabolite profiles were found between the groups. The second approach involved tissue culture studies to investigate peroxisomal and mitochondrial function. These studies suggested that both organelles played a role in tocopherol metabolism. To conclude, firstly a-TL was not shown conclusively, by this thesis to be a biomarker of oxidative stress, but its correlation with the independent biomarker, 8-isoPGF2a, may suggests that there is still potential in it being a biomarker in other states of oxidative stress such as in sepsis or disease. Therefore the techniques developed and used here may be used in further investigations to answer the question whether a-TL is a biomarker of oxidative. Secondly, the localization of Y-tocopherol metabolism has been shown to involve the peroxisome and mitochondria, in human liver HepG2 cells, with effects at different stages of metabolism showing similarity to the branched chain fatty acid metabolism pathway.