Prior to the recent application of stable isotope based GC/MS methodology, little was known about in vivo essential fatty acid metabolism in animals or humans. ? ? 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 detection, one can simultaneously perform the analysis 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, human infants or 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. Our results indicated that, on a per dosage basis, the 20-C PUFAs were more efficiently converted to their end-products than were the 18-C precursors. This work also carefully established proper quantification procedures for comparing the deuterium and C-13 isotopic peaks. In connection with these studies, it was important to determine whether either of the stable isotopes led to an decreased rate of metabolism relative to the endogenous compounds. No isotope effect could be detected with deuterated or 13-C labeled linoleic or alpha-linolenic acids. This was the first such in vivo study. In separate experiments, it was shown that most of the alpha-linolenic acid label is degraded by catabolism and recyled into cholesterol and non-essential fatty acids in liver and brain.? ? 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. Physiologic compartmental models were constructed to compare the net accretion of 22:6n3 from 18:3n3 and 20:5n3 in plasma and also to compare 20:4n6 and 22:5n6 from either 13-C-18:2n6 or 2H5-20:3n6. Although a greater percentage of the labeled 18:2n6 relative to 20:3n6 was converted to 20:4n6, nevertheless, on a per dosage basis, there was greater conversion of the 20-C intermediate than the 18-C precursor. Analysis of n-3 fatty acid metabolism indicated an hourly synthetic rate of 47 nmol for the 18:3n3-derived 22:6n3 compared to 17 nmol for the 20:5n3-derived 22:6n3 (P=0.04). Hereagain, on a dosage basis, there was greater conversion of the 20-C precursor than the 18-C precursor to 22:6n3. In 11 IUGR infants, the absolute concentration of labeled-22:6n3 was lower than gestational age or birth weight matched controls and a less active 22:5n3 to 22:6n3 conversion observed was attributed to differences in peroxisomal metabolism.? ? In rats, it was observed that addition of preformed DHA to the diet leads to a decreased accumulation of label from 18-C precursors into DHA and DPAn6 in several organs even though there was a significant increase in tissue DHA. Female rats accumulated more DHA and DPAn6 but less AA than males when fed a controlled diet containing 3 wt% alpha-linolenic acid. An n-3 fatty acid deficient diet led to a marked decline in labeling of liver 22:4n6 and 22:5n6 from the 18:2n6 precursor. ? ? A closely related research project concerns the origins of nervous system and other organ 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 newly developed artifiicial rearing techniques using an artificial rat milk that was nearly devoid of n-3 fatty acids. The n-3 fatty acids are then added as deuterated-LNA and containing varying levels of 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, possibly due to a form of end-product inhibition, and 88% of brain DHA was derived from the preformed dietary DHA. The biochemical mechanisms underlying these metabolic effects of dietary DHA are being investigated. A decline in labeled DHA was also observed in liver, heart, muscle, kidney and testes but no such changes were observed in adipose tissues. 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.? ? An attempt was made to determine what the underlying mechanisms for DHA transport into brain and other organs. Lipoproteins were purified and labeled with radiotracers and modified with a tracer levels of phospholipids acylated with DHA, AA or oleic acid (OA). The modified lipoproteins were intravenously injected in mice. The plasma and tissue distribution of the radiotracers were investigated as a function of time and the lipoproteins composition. We found that higher proportion of DHA in LDL results in an enhanced uptake of these lipoproteins by brain and heart. A similar enrichment of LDL in AA or OA did not result in any changes compared to control unaltered LDL. Tissue uptake of HDL did not depend on its fatty acid composition. We next compared the distribution in plasma pools and tissue uptake of 14C-DHA and 3H-(OA) intravenously injected in mice. We found that DHA is rapidly taken up by liver, selectively acylated into triglycerides and released back into the circulation in VLDL. Most of the DHA from VLDL and LDL appeared to be rapidly taken up by extrahepatic organs. This pattern seems to be unique for DHA, because no significant amount of non-essential oleic acid, traced in a similar way, was found in TG and VLDL fractions. In summary, these results point to the important role of VLDL and LDL in transport of DHA to extrahepatic tissues, and to the involvement of liver in the initial selectivity for DHA transport.? ? A novel application of PET imaging for the study of C11-DHA incorporation into brain has been initiated. Brain and heart images from 17 healthy volunteers and 11 alcoholics have now been obtained. Extensive characterization of the fatty acid input function in plasma has been made in real time for the 11-C-DHA. Our findings thusfar are that the J(in) and K* values for male and female healthy volunteers are similar except for the K* values in the thalamus and the gray matter/white matter ratio. There is a suggestion from initial studies that alcoholics may have a lower incorporation of DHA in many areas of cortex than control subjects, but more subjects will be needed.
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