Desaturation Of Essential Fatty Acids Using Stable Isotope GC/MS

在最近应用基于稳定同位素的GC/MS方法之前，对动物或人类体内必需脂肪酸代谢知之甚少。 必需脂肪酸代谢是在成年人（男性和女性）以及吸烟者和非吸烟者中进行的研究。 这是在单次口服剂量的这些前体后，氘化-LA和氘化-LNA转化的体内代谢的稳定同位素研究。 我们的研究结果表明，与非吸烟者相比，女性吸烟者的血浆剂量百分比增加了两倍，22：5 n-3转化为22：6 n-3的转化率也更高。 男性吸烟者血浆总n-3脂肪酸升高，D5-18：3 n-3转换更快，D5-20：5 n-3消失率延迟且更慢，D5 -20：5 n-3进入22：5 n-3的百分比更大。 一般来说，吸烟增加了血浆中n-3脂肪酸的生物利用度，加速了部分转化率，并增加了一些长链n-3脂肪酸的形成百分比。 在大鼠中，观察到向饮食中添加预先形成的DHA导致在几个器官中从18-C前体到DHA和DPAn 6的标记物的积累减少，即使组织DHA显著增加。 雌性大鼠积累更多的DHA和DPAn 6，但较少的AA比男性喂养时，控制饮食含有3重量%的α-亚麻酸。 一个n-3脂肪酸缺乏的饮食导致标记的肝脏22：4 n6和22：5 n6从18：2n 6前体显着下降。 一个密切相关的研究项目涉及神经系统和其他器官DHA的起源。可能的来源是从饮食中预先形成的DHA，从代谢的前体，LNA，或从身体储存的DHA。已经开发了一种新的技术，允许定量评估在各种饮食条件下LNA代谢产生的DHA的量。在这项研究中，有必要控制从出生到大脑发育显著的时期的饮食。这是通过使用新开发的人工饲养技术，使用几乎不含n-3脂肪酸的人工大鼠奶来实现的。然后将n-3脂肪酸作为氘代LNA加入，并含有不同水平的DHA。在一个主要的实验中，在出生后的第8-29天给幼鼠喂食含0或2% DHA的食物。在此期间，可以计算出，在喂食D5-LNA作为其n-3脂肪酸的唯一来源的动物中，40%的新形成的脑DHA来源于预先形成的DHA，而不是LNA代谢。这是令人惊讶的，因为饮食中没有DHA;因此，所有沉积在大脑中的DHA都必须通过血液来源于其他器官。当将DHA添加到饮食中时，LNA代谢为DHA的速率显著降低，这可能是由于终产物抑制的形式，并且88%的脑DHA来自预先形成的饮食DHA。膳食DHA的这些代谢作用的生化机制正在研究中。 在肝脏、心脏、肌肉、肾脏和睾丸中也观察到标记DHA的下降，但在脂肪组织中没有观察到这种变化。 在给予预先形成的DHA的大鼠中，脑DHA水平也较高，表明代谢不能提供足够的脑DHA来源。 另一个对婴儿喂养的配方奶粉不含DHA的结果是，当饮食中不存在预先形成的DHA时，在身体剧烈生长期间，包括心脏、肺、肾和脾在内的几个器官的DHA含量净损失。 试图确定DHA转运到大脑和其他器官的潜在机制。纯化脂蛋白并用放射性示踪剂标记，并用DHA、AA或油酸（OA）酰化的磷脂的示踪水平进行修饰。将修饰的脂蛋白静脉注射到小鼠体内。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进行了研究。我们发现，LDL中较高比例的DHA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，我们比较了静脉注射到小鼠体内的14 C-DHA和3 H-（OA）在血浆池中的分布和组织摄取。我们发现DHA被肝脏迅速吸收，选择性地酰化成甘油三酯，并以VLDL的形式释放回循环中。大部分来自VLDL和LDL的DHA似乎被肝外器官迅速吸收。这种模式似乎是DHA独有的，因为在TG和VLDL组分中没有发现大量的非必需油酸，以类似的方式追踪。 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掺入脑中的新应用已经开始。 现在已经获得了19名健康志愿者和17名酗酒者的大脑和心脏图像。 已在真实的时间内对11-C-DHA的血浆中脂肪酸输入函数进行了广泛表征。结果表明，除丘脑K* 值和灰质/白色物质比值不同外，男性和女性健康志愿者的J（in）和K* 值相似。最初的研究表明，酗酒者大脑皮层的许多区域的DHA含量可能低于对照组，但需要更多的研究对象。

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. Essential fatty acid metabolism was studies in human adults, both male and female, and those who smoked as well as non-smokers. This was a stable isotope study of in vivo metabolism of deuterated-LA and deuterated-LNA conversion after a single oral dose of these precursors. Our results indicated that female smokers had a two-fold increase in the percent of plasma dose and a higher fractional conversion rate for 22:5n-3 conversion to 22:6n-3 compared with non-smokers. Male smokers had elevated total plasma n-3 fatty acids, a more rapid turn over of D5-18:3n-3, a disappaerance rate of D5-20:5n-3 that was both delayed and slower, and a greater percentage of D5-20:5n-3 was directed into 22:5n-3 relative to non-smokers. Generally, smoking increased the bioavailablity of n-3 fatty acids from plasma, accelerated fractional conversion rates, and increased the percent formation for some long chain n-3 fatty acids. 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. Another finding of consequence for infants fed formulas without DHA was that several organs including the heart, lungs, kidney and spleen had a net loss of DHA content during a period of intense body growth when no preformed DHA was present in the diet. 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 19 healthy volunteers and 17 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.