Desaturation Of Essential Fatty Acids Using Stable Isoto

使用稳定 Isoto 使必需脂肪酸去饱和

• 批准号: 6818611
• 负责人: Norman Salem
• 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/6818611
• 关键词: Macaca mulatta; age difference; alcoholism /alcohol abuse; arachidonate; brain metabolism; clinical research; deuterium; developmental neurobiology; developmental nutrition; dietary lipid; dietary supplements; eicosanoid metabolism; essential fatty acids; ethanol; fatty acid metabolism; gas chromatography mass spectrometry; human subject; laboratory rat; linoleate; nutrient drug interaction; nutrition related tag; omega 3 fatty acid; positron emission tomography; radiation dosage; saturated fatty acids; smoking; stable isotope

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 our adult work, normal volunteers, smokers and alcoholic smokers were studied for essential fatty acid interconversions in vivo. Controlled diet studies indicated that increasing the long chain n-3 fatty acids in the diet led to a decrease in the in vivo accretion of the deuterated fatty acid end products in plasma. Further analyses during this reporting period have demonstrated that this effect was entirely due to females as males exhibited no such effect. The kinetic constants for this process have been obtained after extensive modeling work was performed using the SAM program. These finding are consistent with the well known phenomenon of end product inhibition. Smokers produced increased amounts and had greater enrichments of deuterated AA and DHA relative to normal non-smokers. Alcoholic-smokers had a marked increase in deuterium enrichments of long chain polyunsaturates in plasma, particularly DHA. In alcoholics with liver fibrosis, deuterium enrichment of DHA in liver biopsy samples was also increased relative to alcoholics without liver histopathological findings. These results are significant as they do not support the commonly held notion in the field that alcohol inhibits elongation/ desaturation of essential fatty acids. In fact, a hypothesis where alcohol stimulates this pathway would be more consistent with our results. Our hypothesis is that alcohol leads to catabolism of long chain polyunsaturates like DHA. When the alcohol challenge is of sufficient intensity and duration, this will lead to a decrease in the tissue concentration of DHA. Metabolic processes including elongation/desaturation and transport/acylation may be increased in the alcoholic in partial compensation for the loss of these important membrane constituents. 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 deconvolution 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. 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. It has long been assumed that the liver is the principal site of essential fatty acid anabolism. However, there is little knowledge of the capacities for fatty acid elongation/ desaturation in various other organs except for the brain. The conversion of the both the n-6 precursor, linoleic acid (LA) and the n-3 precursor, alpha-linolenic acid (LNA) has been assessed in various rat organs in vivo. The rat has been subdivided into 25 organ systems/tissue types. Of the accumulated deuterium labeled LA and LNA, about 75% was found in the white adipose while 25% was in the skin, muscle or carcass. Liver appeared to be the primary site for fatty acid anabolism and the brain had a high specific accumulation of labeled AA and DHA. The kidney, heart, lung, spleen and testis also exhibited time courses for the appearance of various n-3 and n-6 metabolites that were consistent with local metabolism. Thus, these were the first measurements on the in vivo participation of these organ systems for EFA metabolism and the first suggestion that they are contributors to long chain metabolite production and accretion. 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 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. A novel application of PET imaging for the study of C11-DHA incorporation into brain has been initiated. Five rhesus monkeys have been imaged and dosimetry data for evrry organ system calculated. Extensive ethical reviews have been completed and accumulation of the first images of DHA in the human brain is expected within the coming year.

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。 在我们的成人工作中，研究了正常志愿者、吸烟者和酗酒者体内必需脂肪酸的相互转化。控制饮食研究表明，增加饮食中的长链 n-3 脂肪酸会导致血浆中氘化脂肪酸终产物的体内积聚减少。本报告期间的进一步分析表明，这种影响完全是女性造成的，而男性则没有表现出这种影响。使用 SAM 程序进行大量建模工作后，获得了该过程的动力学常数。这些发现与众所周知的终产物抑制现象一致。与正常不吸烟者相比，吸烟者产生的氘化 AA 和 DHA 的量更高，并且含量更高。吸烟者血浆中长链多不饱和化合物（尤其是 DHA）的氘含量显着增加。在患有肝纤维化的酗酒者中，相对于没有肝脏组织病理学发现的酗酒者，肝活检样本中 DHA 的氘富集也有所增加。 这些结果很重要，因为它们不支持该领域普遍持有的观点，即酒精抑制必需脂肪酸的伸长/去饱和。事实上，酒精刺激这一途径的假设与我们的结果更加一致。我们的假设是，酒精会导致 DHA 等长链多不饱和脂肪酸的分解代谢。当酒精挑战足够强度和持续时间时，这将导致组织中 DHA 浓度下降。酒精中的代谢过程（包括延伸/去饱和和运输/酰化）可能会增加，以部分补偿这些重要膜成分的损失。 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解卷积，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。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。 长期以来人们一直认为肝脏是必需脂肪酸合成代谢的主要场所。然而，除了大脑之外，人们对其他各种器官的脂肪酸延伸/去饱和能力知之甚少。已在体内各种大鼠器官中评估了 n-6 前体亚油酸 (LA) 和 n-3 前体 α-亚麻酸 (LNA) 的转化。大鼠被细分为 25 个器官系统/组织类型。在标记为 LA 和 LNA 的累积氘中，约 75% 存在于白色脂肪中，而 25% 存在于皮肤、肌肉或屠体中。肝脏似乎是脂肪酸合成代谢的主要部位，而大脑中标记的 AA 和 DHA 具有高特异性积累。肾脏、心脏、肺、脾和睾丸也表现出各种 n-3 和 n-6 代谢物出现的时间进程，与局部代谢一致。因此，这是对这些器官系统参与 EFA 代谢的体内参与的首次测量，并且首次表明它们是长链代谢物产生和积累的贡献者。 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 脂肪酸以氘化 LNA 的形式添加，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。 PET 成像用于研究 C11-DHA 掺入大脑的新应用已经启动。对五只恒河猴进行了成像，并计算了每个器官系统的剂量测定数据。广泛的伦理审查已经完成，预计在来年积累第一批 DHA 在人脑中的图像。