Our past studies as well as those of others have indicated that alcohol abuse leads to a loss of docosahexaenoate (DHA), the major polyunsaturate in the nervous system. Nutritional inadequacies, particularly during early development, may also lead to such losses in this essential fatty acid. In following up this work, it is important to establish what losses in physiological functions are caused by the loss of DHA in various organ systems. In a collaboration with several investigators at Wayne State University, the relationships of alcohol intake during pregnancy is related to the mother's and newborn infant's essential fatty acid and vitamin status. Dietary information is collected from the mothers in order to ascertain whether alcohol affects food selection or has a more direct metabolic effect in mediating potential losses in blood stream essential fats. Data from over 400 women at their first prenatal care visit indicates that alcohol intake inversely correlates with plasma vitamin E content. Complex interactions appear to be taking place between essential fatty acid levels in the mothers plasma and her history of alcohol consumption during pregnancy. Samples obtained at the time of birth including mothers blood components and cord blood and artery/vein samples have been analyzed for essential fatty acids and an increase related to alcohol intake has been found in arachidonic acid (AA) and DHA in the vessel wall. In addition, plasma levels of folate metabolites have been measured in the pregnant women at the 24 week stage of gestation. Results indicated that dietary folate and alcohol comsumption were positively associated while exposure to smoking was inversely related to a plasma folate metabolite, 5-MTHFA. This folate metabolite was also positively associted with highly unsaturated fatty acid status. In another major line of research, a model of DHA deficiency was created in order to characterize the loss in nervous system function as well as to investigate the reversibility of function when DHA concentration is restored. A novel application to the field of essential fatty acid biology was made with the introduction of olfactory-based learning and memory-related tasks for brain function assessment and these findings were recently published. This modality was used since Slotnick has reported that rats are capable of high level learning of olfactory based tasks of a nature usually only ascribed to non-human primates or higher mammals. Our principal findings are that there is a poorer performance in the acquisition of olfactory set learning in rats where brain and olfactory bulb DHA was lowered thru dietary insufficiency. That is, after the rats had acquired the task, they were over-trained in order to determine whether they could achieve the learning set, i.e., make zero or only one mistake in the first twenty trials after an information trial in a two-odor discrimination task. Rats given a safflower oil based diet for two generations were significantly poorer in this regard than rats to which oils containing alpha-linolenate and DHA were added. Animals with lower levels of brain DHA performed more poorly on spatial maze tasks using the Morris Water Maze. The n-3 deficient rats swam longer and at a higher rate, but found the platform with a longer latency. In a memory retention trial, n-3 deficient rats performed significantly worse than the n-3 adequate group, especially when deprived for three generations. Although n-3 deficient rats perform more poorly, it cannot be ascribed to lower activity or motivation as general motor activity was not different between groups and there was no difference in a progressive-ratio licking task in which animals worked for a water reward. Also, the n-3 deficient rats sampled the odors longer than the DHA-adequate animals but still made more subsequent total errors. The n-3 deficient rats were examined for changes in brain morphology using quantitative stereological techniques. Initially, studies focused on hippocampal morphology. Previous observations included a decreased cell size in the hippocampus, hypothalamus, subfornical organ, and in the parietal and piriform cortex of animals with low brain DHA. Golgi staining revealed a loss of dendritic branching in the dentate gyrus and pyramids from layer V of the fronto-pariental cortex in rats with low brain DHA. Several collaborations were established that allowed further insight into losses in neural functions associated with low brain or retinal DHA. Retinal sensitivity was lost in third generation rats on a low n-3 fatty acid diet. Similarly, rhesus monkeys given a formula with low levels of n-3 fatty acids after birth exhibited an increase in b-wave implicit times at low light intensity levels. These infant monkeys exhibited poorer orienting and motor skills than those fed AA and DHA-supplemented formulas. Once these deficiencies in brain function were established relating to DHA status, it was of interest to determine whether they were reversible. For this purpose, n-3 deficiency was induced with a safflower oil-based diet over three generations. In this study, the degree of DHA repletion was well correlated with performance in the Morris water maze. A further study in mouse brain has shown the time course curves for recovery of brain DHA when repletion is begun either at 3 or 7 weeks of age; the half time for recovery was 1.6 and 3.6 weeks, respectively. A study was carried out to determine whether lead was more neurotoxic when combined with low brain DHA status. The poorer acquisition of an olfactory discrimination in lead exposed rats appeared to be better when brain DHA levels were supported. Lead exposure during lactation also increased liver but not brain levels of AA and DHA. However, no DHA protection could be observed in lead exposed rats on spatial tasks or in olfactory-cued reversal learning. A developmental study of rat brain indicated that there was not a reciprocal replacement of DHA with the n-6 polyunsaturate, DPAn-6 during the brain growth period, as has been the dogma for adults. At 10 or 20 postnatal days there was a significant loss of 22-C HUFAs when safflower oil was fed with respect to safflower oil plus DHA. This was most pronounced in the cerebellum of the brain areas examined likely due to the fact that this area undergoes more rapid growth in the postnatal period. In the last reporting period, great progress was made in the successful rearing of pups from the second day of life using feeding bottles developed by Hoshiba. Animals were raised on n-3 supplemented or deficient rat milks to adulthood and tested for spatial task performance. There was no difference in motor activity or in the plus maze but escape latency and memory retention were poorer in the n-3 deficient rats. This technique now makes possible many experiments where control of individual fatty acids or other nutrients in the diet and thus in tissue composition is required. In the first such application, a major experiment was performed where DPAn-6 was compared to DHA feeding for the first 10 weeks of life. The DPAn-6 feeding led to no difference in brain fatty acyl composition relative to a LA reference. In both of these cases though, the level of DHA had fallen appreciably in this first generation model. Associated with this loss in brain DHA was a loss in spatial task performance but no changes in electroretinographic parameters were observed. This experiment conclusively demonstrates that the nervous sytem has a requirement for the 22-carbon n-3 highly unsaturated fatty acids for optimal function.
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