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 方法之前，人们对人体必需脂肪酸体内代谢知之甚少。我们的研究重点是婴儿出生第一周的代谢能力以及成年人的代谢能力。这项工作的第一阶段定义了不同胎龄婴儿中亚油酸向花生四烯酸的转化以及α-亚麻酸向二十二碳六烯酸的转化。令人有些惊讶的结果是，几乎每个婴儿都能够在体内进行 n-3 和 n-6 脂肪酸相互转化。此外，孕龄与血浆 DHA 氘富集度呈反比关系，特别是；也就是说，最不发达的婴儿在这方面具有最大的代谢能力。这与人类胎儿在妊娠最后三个月发生的大脑突增是一致的。小于胎龄的婴儿脂肪酸代谢能力有所下降。 在本报告期内，一种新型多同位素技术，我们称之为多重同步稳定同位素（MESSI），得到了进一步的开发和应用。发明该技术是为了解决确定涉及多个步骤的脂肪酸代谢途径中各种底物代谢的相对功效的难题。一个古老而棘手的问题是直接比较代谢，例如亚油酸与γ-亚麻酸与二高-γ-亚麻酸形成花生四烯酸的代谢。使用体内稳定同位素方法并采用NCI GC/MS，可以同时对来自多种前体的花生四烯酸的各种同位素异构体进行分析，只要选择合适的同位素以产生显着的质量差异，例如5道尔顿或更多。在本实验中，给大鼠口服含有以下同位素的油：13-C-U-18:2n6、D5-20:3n6、D5-18:3n3、13-C-U-20:5n3。结果表明，两种 n-6 脂肪酸同位素均转化为 20:4n6，并且可以同时测量。在同一动物中，还可以评估 n-3 途径，包括 18-碳和 20-碳前体转化为 22:5n3 和 22:6n3。因此，通过这种具有更好控制的方法，不需要四组或更多组不同的动物，因为不同动物的条件永远不会与同一动物同时进行的两次比较相似。与这些研究相关，重要的是确定任一稳定同位素是否会导致相对于内源化合物的代谢率降低。氘代或 13-C 标记的亚油酸或 α-亚麻酸未检测到同位素效应。这是第一个此类体内研究。 此外，这种方法现已直接应用于人类婴儿中18碳脂肪酸与20碳脂肪酸的必需脂肪酸代谢的研究。 NIAAA IRB 和 FDA 现已批准在人类婴儿中使用这些多种稳定同位素，并且对 12 名婴儿进行的初步研究已成功完成。构建生理室模型以比较血浆中 18:3n3 和 20:5n3 的 22:6n3 生物合成。足月新生儿口服剂量为每公斤体重 20 毫克 2H5-18:3n3 和 2 毫克 13C-20:5n3。然后在给药后0、4、8、24、48、96、168小时采集血液样本。血浆中同时检测到2H5-18:3n3和13C-U-20:5n3及其代谢物。从模型中确定，2H5-22:5n3 转化为 2H5-22:6n3 (0.05 hr-1) 的速率常数系数大于 13C20-22:5n3 转化为 13C20-22:6n3 (0.014 hr-1) 的速率常数系数七个婴儿的计算。这导致 18:3n3 衍生的 22:6n3 的每小时合成率为 47 nmol，而 20:5n3 衍生的 22:6n3 的每小时合成率为 17 nmol (P=0.04))。区室建模是计算生物合成速率参数的有用工具，这些参数是确定 22:6n3 供应的 n-3 脂肪酸底物利用率所需的。 第二个密切相关的研究项目涉及神经系统 DHA 的起源。可能的来源包括饮食中预先形成的 DHA、前体 LNA 的代谢或体内储存的 DHA。一种新技术已被开发出来，可以定量评估各种饮食条件下 LNA 代谢产生的 DHA 量。对于这项研究，有必要从出生前一直到大脑发生显着发育的时期控制饮食。这是通过使用手工喂养技术来实现的，该技术可以与我们新开发的人工喂养方法相结合。开发出一种几乎不含 n-3 脂肪酸的人造大鼠奶。然后将 n-3 脂肪酸作为氘化 LNA 添加，并含有不同水平的 DHA。在一项主要实验中，幼鼠在出生后第 8-29 天期间被喂食含有 0 % 或 2% DHA 的饮食。在此期间，可以计算出，在以 D5-LNA 作为 n-3 脂肪酸唯一来源的动物中，新形成的脑 DHA 的 40% 来自预先形成的 DHA，而不是来自 LNA 代谢。这是令人惊讶的，因为饮食中不含 DHA；因此，所有沉积在大脑中的预先形成的 DHA 必定是通过血流从其他器官中获得的。当 DHA 添加到饮食中时，LNA 代谢为 DHA 的速率显着降低，这是一种最终产物抑制。给予预先形成的 DHA 的大鼠脑部 DHA 水平也较高，这表明新陈代谢无法提供足够的脑部 DHA 来源。 我们试图确定 DHA 转运到大脑和其他器官的潜在机制。脂蛋白经过纯化并用放射性示踪剂标记，并用示踪剂水平的 DHA、AA 或油酸 (OA) 酰化磷脂进行修饰。将修饰的脂蛋白静脉注射到小鼠体内。研究了放射性示踪剂的血浆和组织分布作为时间和脂蛋白组成的函数。我们发现 LDL 中 DHA 的比例越高，大脑和心脏对这些脂蛋白的吸收就会增加。与对照未改变的 LDL 相比，AA 或 OA 中 LDL 的类似富集并未导致任何变化。 HDL 的组织摄取不依赖于其脂肪酸组成。接下来，我们比较了小鼠静脉注射的 14C-DHA 和 3H-(OA) 的血浆池分布和组织摄取。我们发现 DHA 迅速被肝脏吸收，选择性酰化为甘油三酯，并以 VLDL 形式释放回循环中。来自 VLDL 和 LDL 的大部分 DHA 似乎被肝外器官迅速吸收。这种模式对于 DHA 来说似乎是独特的，因为以类似的方式追踪，在 TG 和 VLDL 组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 转运至肝外组织中的重要作用，以及肝脏参与 DHA 转运的初始选择性。 PET 成像用于研究 C11-DHA 掺入大脑的新应用已经启动。现已获得八个人的大脑和心脏图像，数据分析也已开始。已对 11-C-DHA 的血浆脂肪酸输入功能进行了实时广泛表征。