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. Essential fatty acid metabolism was studies in human adults, both male and female, and those who smoked as well as non-smokers. This was a stable isotope study of in vivo metabolism of deuterated-LA and deuterated-LNA conversion after a single oral dose of these precursors. Our results indicated that female smokers had a two-fold increase in the percent of plasma dose and a higher fractional conversion rate for 22:5n-3 conversion to 22:6n-3 compared with non-smokers. Male smokers had elevated total plasma n-3 fatty acids, a more rapid turn over of D5-18:3n-3, a disappaerance rate of D5-20:5n-3 that was both delayed and slower, and a greater percentage of D5-20:5n-3 was directed into 22:5n-3 relative to non-smokers. Generally, smoking increased the bioavailablity of n-3 fatty acids from plasma, accelerated fractional conversion rates, and increased the percent formation for some long chain n-3 fatty acids. 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. Another finding of consequence for infants fed formulas without DHA was that several organs including the heart, lungs, kidney and spleen had a net loss of DHA content during a period of intense body growth when no preformed DHA was present in the diet. A novel application of PET imaging for the study of C11-DHA incorporation into brain has been initiated. Brain and heart images from 19 healthy volunteers and 17 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. We measured regional incorporation coefficients (K*) and rates of unesterified plasma DHA entry into brain lipids, and regional cerebral blood flow (rCBF), using PET with 1-11CDHA and 15OH2O, respectively. Imaging data were corrected for brain atrophy. We compared 22 non-smoking healthy control subjects to 15 non-smoking chronic alcoholics studied within 7 days of their last drink of alcohol. Both K* for DHA and rCBF were significantly and widely elevated throughout the brain in alcoholics compared with controls. Unesterified plasma DHA was similar in both groups (2.1 and 1.8 nmol/ml in controls and alcoholics, respectively) as was the rate of DHA incorporation into the brain as a whole (2.4 1.6 mg/d and 2.1 0.9 mg/d, respectively). Higher rCBF in alcoholics suggests altered brain functional activity during early withdrawal from alcohol. Higher K* for DHA in alcoholics indicates higher brain affinity for DHA and thus a potential brain DHA deficit vis--vis plasma availability. A new human protocol has been initiated this year to asssess the effects of lowering dietary intake of linoleic acid from 8 en% to 1 en% on the elongation and desaturation of ALA to EPA and DHA. A separate line of investigation has been to develop high throughput methods of quantifying essential fatty acid status among large numbers of human subjects. An automated high throughput fatty acid analysis was developed from a previous procedure based on direct transesterification including the automation of chemical procedures, data acquisition and automatic data processing. The method was validated and applied to umbilical cord serum samples in an epidemiological study. The method was linear in the range of 1-600g/mL serum with r2≥0.99. The within-run CV was <5.4% for 23 fatty acids and a range of recoveries over three concentrations were 76%119% in a low-lipid matrix with the exception of 14:0. The fatty acid concentration as measured by the robotic method for human plasma was in good agreement with the Lepage&Roy method. The fatty acid profile in umbilical cord serum from American subjects(n=287) showed an average of 38.0%, 24.9%, 32.0% and 4.6% of total fatty acids for saturates, monounsaturates, n-6 and n-3 polyunsaturates, respectively. This is the first report of a complete, validated, cost-effective, automated, high throughput fatty acid measurement method along with application to a population-based study.

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Project End
Budget Start
Budget End
Support Year
31
Fiscal Year
2013
Total Cost
$230,617
Indirect Cost
Name
National Institute on Alcohol Abuse and Alcoholism
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