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: 7317404
• 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/7317404
• 关键词: Desaturation; Essential; Fatty; Acids; Using

Prior to the recent application of stable isotope based GC/MS methodology, little was known about in vivo essential fatty acid metabolism in animals or humans. 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 detection, 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, human infants or 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. Our results indicated that, on a per dosage basis, the 20-C PUFAs were more efficiently converted to their end-products than were the 18-C precursors. This work also carefully established proper quantification procedures for comparing the deuterium and C-13 isotopic peaks. 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. In separate experiments, it was shown that most of the alpha-linolenic acid label is degraded by catabolism and recyled into cholesterol and non-essential fatty acids in liver and brain. 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. Physiologic compartmental models were constructed to compare the net accretion of 22:6n3 from 18:3n3 and 20:5n3 in plasma and also to compare 20:4n6 and 22:5n6 from either 13-C-18:2n6 or 2H5-20:3n6. Although a greater percentage of the labeled 18:2n6 relative to 20:3n6 was converted to 20:4n6, nevertheless, on a per dosage basis, there was greater conversion of the 20-C intermediate than the 18-C precursor. Analysis of n-3 fatty acid metabolism indicated 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). Hereagain, on a dosage basis, there was greater conversion of the 20-C precursor than the 18-C precursor to 22:6n3. In 11 IUGR infants, the absolute concentration of labeled-22:6n3 was lower than gestational age or birth weight matched controls and a less active 22:5n3 to 22:6n3 conversion observed was attributed to differences in peroxisomal metabolism. In rats, it was observed that addition of preformed DHA to the diet leads to a decreased accumulation of label from 18-C precursors into DHA and DPAn6 in several organs even though there was a significant increase in tissue DHA. Female rats accumulated more DHA and DPAn6 but less AA than males when fed a controlled diet containing 3 wt% alpha-linolenic acid. An n-3 fatty acid deficient diet led to a marked decline in labeling of liver 22:4n6 and 22:5n6 from the 18:2n6 precursor. A closely related research project concerns the origins of nervous system and other organ 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 newly developed artifiicial rearing techniques using an artificial rat milk 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, possibly due to a form of end-product inhibition, and 88% of brain DHA was derived from the preformed dietary DHA. The biochemical mechanisms underlying these metabolic effects of dietary DHA are being investigated. A decline in labeled DHA was also observed in liver, heart, muscle, kidney and testes but no such changes were observed in adipose tissues. 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 17 healthy volunteers and 11 alcoholics have now been obtained. Extensive characterization of the fatty acid input function in plasma has been made in real time for the 11-C-DHA. Our findings thusfar are that the J(in) and K* values for male and female healthy volunteers are similar except for the K* values in the thalamus and the gray matter/white matter ratio. There is a suggestion from initial studies that alcoholics may have a lower incorporation of DHA in many areas of cortex than control subjects, but more subjects will be needed.

在最近使用稳定基于同位素的GC/MS方法论之前，关于动物或人类中的体内必需脂肪酸代谢知之甚少。 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检测，可以同时从多个前体中对蛛网膜的各种同位素进行分析，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，为人类婴儿或大鼠提供了含以下同位素的口服剂量：13-C-U-18：2N6，D5-20：3N6，D5-18：3N3，13-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。我们的结果表明，与18-C前体相比，在每剂量的基础上，20-C PUFA被更有效地转化为最终产物。这项工作还精心建立了适当的定量程序，以比较氘和C-13同位素峰。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。在单独的实验中，结果表明，大多数α-烯醇酸标签被分解代谢降解，并在肝脏和脑中回收为胆固醇和非必需的脂肪酸。 此外，这种方法现已直接应用于人类婴儿中18-与20-碳脂肪酸的必需脂肪酸代谢的研究。构建了生理隔室模型，以比较血浆中18：3n3和20：5n3的22：6n3的净积聚，并比较20：4N6和22：5N6的净增值，从13-C-18：2N6或2H5-20：3N6进行比较。尽管相对于20：3n6的标记为18：2n6的百分比更大，但以每剂量为基础，相对于20：4n6，比18-C前体的转化率更高。 N-3脂肪酸代谢的分析表明，在18：3N3衍生的22：6n3中，每小时合成速率为47 nmol，而20：5n3衍生的22：6n3（p = 0.04）的小时合成速率为17 nmol。在剂量的基础上，与18-C前体相比，20-C前体的转化更大，而22：6n3的转化更高。在11个IUGR婴儿中，标记的22：6n3的绝对浓度低于胎龄或出生体重匹配的对照，而活跃的22：5n3至22：6n3转换观察到的转换归因于过氧化物异构代谢的差异。 在大鼠中，观察到，在饮食中添加预先形成的DHA会导致在几个器官中从18-C前体中累积到DHA和DPAN6的标记降低，即使组织DHA显着增加。雌性大鼠积累了更多的DHA和DPAN6，但与男性相比，喂食含有3 wt％α-亚麻酸的受控饮食时的AA。 N-3脂肪酸缺乏饮食导致肝脏的标记22：4n6和22：5n6的标记下降显着下降。 密切相关的研究项目涉及神经系统和其他器官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。通过使用新开发的人工饲养技术，使用几乎没有N-3脂肪酸的人造大鼠牛奶来实现这一目标。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，这可能是由于最终产物抑制的形式，而88％的脑DHA源自预先形成的饮食DHA。正在研究这些代谢性DHA代谢作用的生化机制。在肝，心脏，肌肉，肾脏和睾丸中也观察到标记的DHA的下降，但在脂肪组织中未观察到这种变化。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。现已获得来自17位健康志愿者和11名酗酒者的大脑和心脏图像。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。我们的发现因此，男性和女性健康志愿者的J（IN）和K*值除外，丘脑中的K*值和灰质/白质的比例相似。最初研究的建议是，酗酒者在许多皮质领域的掺入可能要比对照受试者较低，但需要更多的受试者。