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 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. 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. Further analyses during this reporting period have demonstrated that this effect was entirely due to females as males exhibited no such effect. The kinetic constants for this process have been obtained after extensive modeling work was performed using the SAM program. These finding are 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. In this reporting period, a novel multiple-isotope technique that we have termed MultiplE Simultaneous Stable Isotopes, or MESSI, has undergone further development and application. 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 has now been directly applied to the study of the essential fatty acid metabolism of 18- vs. 20- carbon fatty acids in human infants. Both the NIAAA IRB and the FDA have now approved the use of these multiple stable isotopes in human infants and an initial study of a group of 12 infants has been successfully completed. 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 up to a period where significant brain development has occurred. 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 levels DHA. In one major experiment, rat pups were fed diets with 0 or 2% DHA between days 8-29 of life. During this period, it could be calculated that 40% of the newly formed brain DHA in the animals fed D5-LNA as their only source of n-3 fatty acids were derived from preformed DHA and not from LNA metabolism. This was surprising as there was no DHA in the diet; thus, all preformed DHA deposited in the brain must have been derived from other organs via the blood stream. When DHA was added to the diet, there was a pronounced decrease in the rate of LNA metabolism to DHA, a type of end-product inhibition. There was also a higher level of brain DHA in the rats given preformed DHA indicating that metabolism could not provide an adequate source of brain DHA. A novel application of PET imaging for the study of C11-DHA incorporation into brain has been initiated. Five rhesus monkeys have been imaged and dosimetry data for evrry organ system calculated. Extensive ethical reviews have been completed and accumulation of the first images of DHA in the human brain is expected within the coming year.