Models of CNS functioning: alcohol consumption, impaire

中枢神经系统功能模型：饮酒、受损

• 批准号: 6983117
• 负责人: James Dee Higley
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6983117
• 关键词: Macaca mulatta; age difference; alcoholic beverage consumption; alcoholism /alcohol abuse; alcoholism /alcohol abuse chemotherapy; animal developmental psychology; anxiety; behavior prediction; behavioral /social science research tag; behavioral genetics; buspirone; child rearing; early experience; fluoxetine; genetic mapping; genetic polymorphism; genetic susceptibility; impulsive behavior; ketamine; mental disorder chemotherapy; neurotransmitter metabolism; nutrition related tag; parent deprivation; personality; positron emission tomography; self destructive behavior; serotonin; serotonin inhibitor; serotonin transporter

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 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 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, 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. 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. 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. Physiologic compartmental models were constructed to compare the biosynthesis of 22:6n3 from 18:3n3 and 20:5n3 in plasma. Term neonates were administrated an oral dose of 20 mg of 2H5-18:3n3 and 2 mg of 13C-20:5n3 per kg of BW. Blood was then sampled at 0, 4, 8, 24, 48, 96, 168 hr after administration. 2H5-18:3n3 and 13C-U-20:5n3 as well as their metabolites were simultaneously detected in plasma. A greater rate constant coefficient for the conversion of 2H5-22:5n3 to 2H5-22:6n3 (0.05 hr-1) than for 13C20-22:5n3 to 13C20-22:6n3 (0.014 hr-1) was determined from the model calculations on seven infants. This resulted in 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)). Compartmental modeling is a useful tool for calculating biosynthetic rate parameters that are needed for determining n-3 fatty acid substrate utilization for 22:6n3 supply. 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 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, 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. 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 eight individuals have now been obtained and data anlysis has begun. Extensive characterization of the fatty acid input function in plasma has been made in real time for the 11-C-DHA.

在最近应用基于稳定同位素的GC/MS方法论之前，对体内的人类必需脂肪酸代谢知之甚少。我们的研究集中在生命的第一周以及人类成年人的代谢能力上。这项工作的第一阶段定义了亚油酸向蛛网膜酸的转化，也定义了在不同妊娠年龄的婴儿中α-内酚酸酯转化为二十二烷酸酯的转化。令人惊讶的结果是，几乎每个婴儿都能在体内进行N-3和N-6脂肪酸互转换。此外，妊娠年龄与血浆氘富集的DHA存在反比关系。也就是说，在这方面，发育最少的婴儿具有最大的代谢能力。这与上三个月在人类胎儿中发生的大脑生长突变一致。胎龄小的婴儿的脂肪酸代谢能力有所降低。 在此报告期间，我们称之为多个同时稳定同位素或梅西的新型多异位技术已经经历了进一步的开发和应用。该技术的发明是为了解决确定各种底物的代谢相对效果的困难问题，沿脂肪酸代谢的途径涉及多个步骤。一个旧的且棘手的问题是代谢的直接比较，例如LinoLeate与γ-细烯酸盐与Dihommo-gamma-linolenate形成蛛网膜酸盐的代谢。使用体内稳定的同位素方法并采用NCI GC/MS，可以同时从多个前体中对各种蛛网酸的同位素进行分析，只要选择合适的同位素以给出明显的质量差异，例如，例如，5 daltons或更多。在目前的实验中，给大鼠口服含有以下同位素的油剂量：13-C-U-18：2N6，D5-20：3N6，D5-18：3N3：3N3，13-C-U-20：5N3。证明两个N-6脂肪酸同位素均转化为20：4N6，并且可以同时测量它们。在同一动物中，还可以评估N-3途径，包括18碳和20碳前体的转化到22：5n3和22：6n3。因此，这种方法以更好的控制来消除对四个或多个单独的动物组的需求，因为单独动物中的条件永远不会像同一动物中的​​两个比较一样相似。与这些研究有关，重要的是确定任何一个稳定的同位素是否导致相对于内源性化合物的代谢率降低。无法通过氘代或13-C标记的亚油酸或α-内酚酸检测到同位素效应。这是第一次这样的体内研究。 此外，这种方法现已直接应用于人类婴儿中18-碳脂肪酸的必要脂肪酸代谢的研究。 NIAAA IRB和FDA现在都批准在人类婴儿中使用这些多个稳定同位素，并且对一组12名婴儿的初步研究已成功完成。构建了生理隔室模型，以比较血浆中18：3n3和20：5n3的22：6n3的生物合成。新生儿任期的口服剂量为20毫克2H5-18：3N3和2毫克的13c-20：5n3：每公斤BW。然后在给药后在0、4、8、24、48、96、168小时以0、4、8、24、48、96、168小时采样。 2H5-18：3N3和13C-U-20：5N3以及它们的代谢产物在血浆中同时检测到。比13c20-22：5n3至13c20-22：6n3（0.014 hr-1）的2H5-22：5N3至2H5-22：6N3（0.05 hr-1）的转化率恒定系数更高。这导致18：3n3衍生的22：6n3的每小时合成速率为47 nmol，而20：5n3衍生的22：6n3（p = 0.04）为17 nmol。隔室建模是计算生物合成速率参数的有用工具，用于确定N-3脂肪酸底物利用率22：6N3供应。 第二个密切相关的研究项目涉及神经系统DHA的起源。可能的来源来自饮食中预先形成的DHA，来自前体，LNA的代谢或DHA的体内储存。已经开发了一种新的技术，可以在各种饮食条件下对从LNA代谢中积累的DHA量进行定量评估。对于这项研究，有必要控制饮食从近出生到发生重大大脑发育的时期。通过使用手动喂养技术可以将其与我们新开发的人工喂养方法相结合的使用实现。开发了一种几乎没有N-3脂肪酸的人造大鼠牛奶。然后添加N-3脂肪酸作为氘代LNA并含有不同水平的DHA。在一个主要实验中，在生命的第8-29天之间，大鼠幼崽的饮食为0或2％DHA。在此期间，可以计算出，在饲喂D5-LNA的动物中，有40％的新形成的脑DHA是其唯一的N-3脂肪酸来源，它来自预先形成的DHA，而不是LNA代谢。这是令人惊讶的，因为饮食中没有DHA。因此，沉积在大脑中的所有预先形成的DHA必须通过血流源自其他器官。当将DHA添加到饮食中时，LNA代谢对DHA的速率明显降低，DHA是一种末期抑制。鉴于预先形成的DHA，大鼠的大脑DHA水平也更高，表明新陈代谢无法提供足够的脑DHA来源。 试图确定DHA运输到大脑和其他器官的基本机制。将脂蛋白纯化并用放射性示例标记，并用示踪剂水平的磷脂，用DHA，AA或油酸（OA）酰化。将改性的脂蛋白静脉注射在小鼠中。研究了放射性示例的血浆和组织分布，这是时间和脂蛋白组成的函数。我们发现，LDL中DHA的比例较高会导致大脑和心脏对这些脂蛋白的摄取增强。与对照未改变的LDL相比，AA或OA中LDL的类似富集不会导致任何变化。 HDL的组织摄取不取决于其脂肪酸组成。接下来，我们比较了在小鼠中注射14C-DHA和3H-（OA）的血浆池和组织摄取的分布。我们发现DHA被肝脏迅速吸收，有选择地酰化为甘油三酸酯，并释放回VLDL的循环。来自VLDL和LDL的大多数DHA似乎被肝外器官迅速占据。对于DHA，这种模式似乎是独一无二的，因为在TG和VLDL级分中没有发现大量的非必需油酸。总之，这些结果表明，VLDL和LDL在DHA向肝外组织的运输中的重要作用，以及肝脏参与DHA转运的初始选择性。 启动了PET成像在研究C11-DHA掺入大脑中的新型应用。现在已经获得了来自八个人的大脑和心脏图像，并且已经开始了数据厌食。对于11-C-DHA，已经实时对等离子体中的脂肪酸输入功能进行了广泛的表征。