DESATURATION OF ESSENTIAL FATTY ACIDS USING STABLE ISOTO

Alcohol Abuse And Alcoholism

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Abstract

Prior to the recent application of stable isotope based GC/MS methodology, little was known about human essential fatty acid metabolism in vivo. Our studies have focused on the metabolic capacities of infants in the first week of life and also on that of human adults. The first phase of this work defined the conversion of linoleic acid to arachidonate and also the conversion of alpha-linolenate to docosahexaenoate in infants of varying gestational ages. The somewhat surprising results were that essentially every infant was capable of both n-3 and n-6 fatty acid interconversions in vivo. Moreover, there was an inverse relationship of gestational age with plasma deuterium enrichment of DHA, in particular; i.e., the least developed infants had the greatest metabolic capability in this respect. This is consistent with the brain growth spurt that occurs in human fetuses during the last trimester. Infants who were small for gestational age had a somewhat diminished metabolic capacity for fatty acids but most of the variance could be explained bygestational age only.In our adult work, normal volunteers, smokers and alcoholic smokers were studied for essential fatty acid interconversions in vivo. Controlled diet studies indicated that increasing the long chain n-3 fatty acids in the diet led to a decrease in the in vivo accretion of the deuterated fatty acid end products in plasma. This is consistent with the well known phenomenon of end product inhibition. Smokers produced increased amounts and had greater enrichments of deuterated AA and DHA relative to normal non-smokers. Alcoholic-smokers had a marked increase in deuterium enrichments of long chain polyunsaturates in plasma, particularly DHA. In alcoholics with liver fibrosis, deuterium enrichment of DHA in liver biopsy samples was also increased relative to alcoholics without liver histopathological findings. These results are significant as they do not support the commonly held notion in the field that alcohol inhibits elongation/desaturation of essential fatty acids. In fact, a hypothesis where alcohol stimulates this pathway would be more consistent with our results. Our hypothesis is that alcohol leads to catabolism of long chain polyunsaturates like DHA. When the alcohol challenge is of sufficient intensity and duration, this will lead to a decrease in the tissue concentration of DHA. Metabolic processes including elongation/desaturation and transport/acylation may be increased in the alcoholic in partial compensation for the loss of these important membrane constituents. Our recent studies have examined the in vivo metabolism of essential fatty acids in patients with Retinitis Pigmentosa (RP). In particular, patients with Ushers II disease or non-Ushers disease were compared to normal volunteers. We observed that the amount or enrichment of deuterated n-3 fatty acid metabolites such as EPA or DHA were significantly increased in Ushers patients whereas there was a decrease relative to normal volunteers in the non-Ushers RP group. The increased metabolism in the Ushers patients with respect to DHA may be surprising as it has been hypothesized that the retinal concentration of DHA is reduced in Retinitis Pigmentosa and that this may, in part, explain some of the loss in visual function associated with this neurological disease. However, as noted above for alcoholic patients, increased metabolism may be induced by increased catabolism that is associated with the disease state. These studies point to the need for analysis of increased fatty acid catabolism or indices of lipid peroxidation in vivo in these patients. The opposite direction of response in the non-Ushers patients points to a quite distinct etiology of this disease. Progress has been made during this reporting period in developing a novel multiple-isotope technique that we have termed the MultiplE Simultaneous Stable Isotopes, or MESSI, for short. This technique was invented to address the difficult problem of determining the relative efficacy of metabolism of various substrates along a pathway of fatty acid metabolism involving multiple steps. An old and intractable problem has been the direct comparison of metabolism, for example, of linoleate vs. that of gamma-linolenate vs dihommo-gamma-linolenate to form arachidonate. Using the in vivo stable isotope approach and employing NCI GC/MS, one can simultaneously perform the deconvolution of various isotopomers of arachidonate from multiple precursors providing that suitable isotopes are selected to give a significant mass difference, eg, 5 daltons or more. In the present experiments, rats were given an oral dose of oil containing the following isotopes: 13-C-U-18:2n6, D5-20:3n6, D5-18:3n3, 13-C-U-20:5n3. It was demonstrated that both n-6 fatty acid isotopes were converted to 20:4n6 and that they could be simultaneously measured. In the same animal, the n-3 pathway could also be assessed, both with respect to the 18-carbon and 20-carbon precursor conversions to 22:5n3 and 22:6n3. Thus, the need for four or more separate groups of animals are obviated by this approach with better control since the conditions in separate animals can never be as similar as two comparisons within the same animal at the same time. Moreover, this approach can be directly applied to human experimentation due to the use of safe stable isotopes. In particular, the approach will facilitate, indeed make possible, the study of the essential fatty acid metabolism of 18- vs. 20- carbon fatty acids in human infants.