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。 在我们的成人工作中，研究了正常的志愿者，吸烟者和酗酒者的体内脂肪酸互续。受控饮食研究表明，饮食中长链N-3脂肪酸增加导致血浆中氘化脂肪酸最终产物的体内积聚减少。在此报告期间的进一步分析表明，这种影响完全归因于女性，因为男性没有这种影响。在使用SAM程序进行大量建模工作后，已经获得了此过程的动力学常数。这些发现与最终产品抑制的现象一致。吸烟者产生的量增加，并且相对于普通非吸烟者而言，氘化的AA和DHA的富集更大。酒精 - 吸烟者的长链多不饱和度的氘富集显着增加，尤其是DHA。在肝纤维化的酒精中毒中，相对于没有肝组织病理学发现的酒精中毒，肝活检样品中DHA的氘富集也增加了。 这些结果很明显，因为它们不支持酒精抑制必需脂肪酸的伸长/去饱和的田间普遍持有的概念。实际上，酒精刺激该途径的假设将与我们的结果更加一致。我们的假设是，酒精会导致长链多不饱和度等多不饱和的分解代谢。当酒精挑战具有足够的强度和持续时间时，这将导致DHA组织浓度的降低。在酒精中毒中，包括伸长/去饱和和转运/酰基在内的代谢过程可能会增加这些重要膜成分的部分补偿。 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的多个前体进行各种同位素的反卷积，但只要选择合适的同位素以给出显着的质量差异，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。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。 长期以来，人们一直认为肝脏是必需脂肪酸合物的主要部位。但是，除了大脑以外，其他各种器官中脂肪酸伸长/去饱和的能力几乎没有知识。 N-6前体，亚油酸（LA）和N-3前体的转化已在体内的各种大鼠器官中进行了评估。大鼠已细分为25种器官系统/组织类型。在标有LA和LNA的累积氘中，在白脂肪中发现了约75％，而皮肤，肌肉或car体中有25％。肝脏似乎是脂肪酸合成代谢的主要部位，大脑具有高特异性的标记为AA和DHA。肾脏，心脏，肺，脾脏和睾丸还展示了与局部代谢一致的各种N-3和N-6代谢产物出现的时间课程。因此，这些是这些器官系统用于EFA代谢的体内参与的第一个测量，也是第一个建议，即它们是长链代谢物生产和积聚的贡献者。 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。 启动了PET成像在研究C11-DHA掺入大脑中的新型应用。已经成像了五只恒河猴，并计算出EVRRY器官系统的剂量测定数据。已经完成了广泛的道德综述，并且预计DHA在来年内的第一批DHA图像的积累。