Desaturation Of Essential Fatty Acids Using Stable Isotope GC/MS

在基于稳定同位素的GC/MS方法学应用之前，对动物或人类体内必需脂肪酸代谢知之甚少。 人体必需脂肪酸代谢的研究对象包括成年男性和女性、吸烟者和不吸烟者。这是一项稳定的同位素研究，研究了单剂量口服这些前体后，代氢-LA和代-LNA转化的体内代谢。我们的结果表明，女性吸烟者的血浆剂量百分比是非吸烟者的两倍，22：5N-3转化为22：6N-3的分数转化率高于非吸烟者。与非吸烟者相比，男性吸烟者血浆总n-3脂肪酸水平升高，D5-18：3n-3翻转速度加快，D5-20：5n-3消失率延迟和减慢，D5-20：5n-3进入22：5n-3的比例较大。总的来说，吸烟增加了血浆中n-3脂肪酸的生物利用度，加速了分馏转化率，并增加了一些长链n-3脂肪酸的形成百分比。 在大鼠中，观察到在饮食中添加预制DHA导致从18-C前体到DHA和DPAn6中的标记在几个器官中的积累减少，尽管组织中的DHA显著增加。雌性大鼠摄入含有3wt%α-亚麻酸的对照饲料时，雌性大鼠比雄性大鼠积累了更多的DHA和DPAn6，但积累的AA更少。缺乏n-3脂肪酸的饮食导致肝脏22：4n6和22：5n6的标记明显下降，而不是18：2n6。 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。可能的来源来自饮食中预制的DHA，来自前体LNA的新陈代谢，或来自体内储存的DHA。一种新的技术已经开发出来，它允许定量评估在不同饮食条件下从LNA代谢中积累的DHA的量。对于这项研究，有必要控制饮食，从接近出生到大脑显著发育的时期。这是通过使用新开发的人工饲养技术实现的，这种人工饲养技术使用的是几乎不含n-3脂肪酸的人造老鼠奶。然后，将n-3脂肪酸作为氚-LNA加入，并含有不同水平的DHA。在一个重要的实验中，小鼠在出生8-29天期间被喂以0%或2%DHA的饲料。在此期间，可以计算出，在喂食D5-LNA作为n-3脂肪酸唯一来源的动物中，40%的新形成的脑DHA来自预先形成的DHA，而不是来自LNA的代谢。这是令人惊讶的，因为饮食中没有DHA；因此，所有储存在大脑中的预制DHA肯定都是通过血液从其他器官获得的。当在饮食中添加DHA时，LNA代谢与DHA的比率明显下降，这可能是由于一种最终产物抑制的形式，大脑中88%的DHA来自预先形成的饮食DHA。膳食DHA的这些代谢效应背后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。给予预制DHA的大鼠脑内DHA水平也较高，表明代谢不能提供足够的脑DHA来源。喂养不含DHA配方奶粉的婴儿的另一项发现是，在饮食中没有预制DHA的身体剧烈生长期间，包括心脏、肺、肾和脾在内的几个器官的DHA含量净减少。 有人试图确定DHA转运到大脑和其他器官的潜在机制。纯化脂蛋白，用放射性示踪剂标记，并用示踪剂修饰用DHA、AA或油酸(OA)酰化的磷脂。将修饰后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随时间和脂蛋白组成的变化。我们发现，低密度脂蛋白中DHA的比例越高，大脑和心脏对这些脂蛋白的摄取就越强。与对照未改变的低密度脂蛋白相比，AA或OA中类似的低密度脂蛋白的浓缩不会导致任何变化。组织对高密度脂蛋白的摄取与其脂肪酸组成无关。接下来，我们比较了小鼠静脉注射~(14)C-DHA和~3H-(OA)的血浆池分布和组织摄取。我们发现DHA迅速被肝脏摄取，选择性地酰化成甘油三酯，然后在极低密度脂蛋白中释放回循环中。极低密度脂蛋白和低密度脂蛋白中的大部分DHA似乎很快被肝外器官摄取。这种模式似乎是DHA独一无二的，因为在TG和VLDL组分中没有发现显著数量的非必需油酸，以类似的方式进行追踪。综上所述，这些结果表明极低密度脂蛋白和低密度脂蛋白在DHA向肝外组织运输中的重要作用，以及肝脏参与了DHA运输的最初选择性。 在研究C11-DHA脑内掺入的研究中，一种新的PET成像技术已经开始应用。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。已对11-C-DHA的血浆脂肪酸输入功能进行了广泛的实时表征。到目前为止，我们的发现是，男性和女性健康志愿者的J(In)和K*值相似，但丘脑的K*值和灰质/白质比除外。虽然最初的研究表明，酗酒者大脑皮质许多区域的DHA含量可能比对照组低，但由于资金限制，足够的受试者无法参加这项研究以进行这种测定。

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 thus far 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. Although there was a suggestion from initial studies that alcoholics may have a lower incorporation of DHA in many areas of cortex than control subjects, sufficent subjects could not be enrolled in the study to make this determeination due to funding constraints.