Essential Fatty Acid Desaturation w/Stable Isotope GC/MS

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 nearly 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 by gestational age only. Present studies involve the potential end-product inhibition of 18-carbon EFA metabolism when arachidonic acid (AA) and DHA when added to infant formulas. 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 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. The NIAAA IRB has approved the use of these multiple stable isotopes in human infants pending approval also by the FDA. An application has been submitted to the FDA Center for Food Safety and Applied Nutrition requesting such approval. It has long been assumed that the liver is the principal site of essential fatty acid anabolism. However, there is little knowledge of the capacities for fatty acid elongation/ desaturation in various other organs except for the brain. The conversion of the both the n-6 precursor, linoleic acid (LA) and the n-3 precursor, alpha-linolenic acid (LNA) has been assessed in various rat organs in vivo. The rat has been subdivided into 25 organ systems/tissue types. Of the accumulated deuterium labeled LA and LNA, about 75% was found in the white adipose while 25% was in the skin, muscle or carcass. Liver appeared to be the primary site for fatty acid anabolism and the brain had a high specific accumulation of labeled AA and DHA. The kidney, heart, lung, spleen and testis also exhibited time courses for the appearance of various n-3 and n-6 metabolites that were consistent with local metabolism. Thus, these were the first measurements on the in vivo participation of these organ systems for EFA metabolism and the first suggestion that they are contributors to long chain metabolite production and accretion. A second closely related research project concerns the origins of nervous system DHA. Possible sources are from dietary preformed DHA, from metabolism of the precursor, LNA, or from body stores of DHA. A novel technique has been developed that allows for the quantitative assessment of the amount of DHA accreted from LNA metabolism under various dietary conditions. For this study, it is necessary to control the diet from near birth to about 6 weeks of age when the rat brain has completed its increase in mass. This has been accomplished thru the use of hand feeding techniques that may be combined with our newly developed artificial feeding approach. An artificial rat milk was developed that was nearly devoid of n-3 fatty acids. The n-3 fatty acids are then added as deuterated-LNA and containing varying level