Essential Fatty Acid Desaturation w/Stable Isotope GC/MS

在最近基于稳定同位素的GC/MS方法应用之前，人们对人体必需脂肪酸的体内代谢知之甚少。我们的研究集中在婴儿在生命的第一周的代谢能力，也对人类成人的代谢能力。本研究的第一阶段确定了不同胎龄婴儿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。有些令人惊讶的结果是，几乎每个婴儿都能在体内同时进行n-3和n-6脂肪酸的相互转化。此外，胎龄与血浆DHA氘富集呈负相关，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。这与人类胎儿在最后三个月的大脑发育突增是一致的。小于胎龄的婴儿对脂肪酸的代谢能力有所降低，但大多数差异只能用胎龄来解释。目前的研究涉及将花生四烯酸（AA）和DHA添加到婴儿配方奶粉中可能抑制18碳EFA代谢的最终产物。在我们的成人工作中，我们研究了正常志愿者、吸烟者和酗酒者体内必需脂肪酸的相互转化。对照饮食研究表明，增加长链n-3脂肪酸在饮食中导致血浆中氘化脂肪酸终产物的体内增加减少。这与众所周知的终产物抑制现象相一致。与正常的非吸烟者相比，吸烟者体内氘化AA和DHA的含量更高，含量也更高。酗酒者血浆中长链多不饱和物的氘富集明显增加，特别是DHA。在伴有肝纤维化的酗酒者中，肝活检样本中DHA的氘富集也比没有肝组织病理学发现的酗酒者增加。这些结果很重要，因为它们不支持在该领域普遍持有的观点，即酒精抑制必需脂肪酸的延伸/去饱和。事实上，酒精刺激这一途径的假设更符合我们的结果。我们的假设是酒精导致长链多不饱和脂肪酸如DHA的分解代谢。当酒精刺激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。代谢过程包括伸长/去饱和和转运/酰化可能在酒精中增加，以部分补偿这些重要膜成分的损失。我们最近的研究检测了色素性视网膜炎（RP）患者体内必需脂肪酸的代谢。特别地，将患有usher II型疾病或非usher型疾病的患者与正常志愿者进行比较。我们观察到，厄舍斯患者的氘化n-3脂肪酸代谢物（如EPA或DHA）的数量或富集程度显著增加，而与非厄舍斯RP组的正常志愿者相比，这些代谢物的数量或富集程度有所下降。usher患者体内DHA代谢的增加可能令人惊讶，因为人们假设色素性视网膜炎患者的视网膜DHA浓度降低，这可能部分解释了与这种神经系统疾病相关的视觉功能丧失。然而，如上所述，对于酗酒患者，代谢增加可能是由与疾病状态相关的分解代谢增加引起的。这些研究表明，有必要分析这些患者体内脂肪酸分解代谢增加或脂质过氧化指数。非usher患者的反应方向相反，这表明这种疾病的病因非常不同。在本报告所述期间，在开发一种新的多同位素技术方面取得了进展，我们将其称为多重同步稳定同位素，简称梅西。该技术的发明是为了解决确定各种底物代谢的相对功效沿着脂肪酸代谢的途径涉及多个步骤的难题。一个古老而棘手的问题是直接比较亚油酸与γ -亚麻酸与二同型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和NCI GC/MS，可以同时对多种前体的花生四烯酸的各种同位素进行反卷积，只要选择合适的同位素来提供显着的质量差，例如5道尔顿或更多。在本实验中，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20:4 - n6，并可同时测量。在同一动物中，也可以评估n-3途径，包括18碳和20碳前体转化为22:5n3和22:6n3。因此，通过这种方法可以更好地控制，避免需要四个或更多单独的动物组，因为单独动物的条件永远不可能像同一时间同一动物内的两个比较一样相似。此外，由于使用了安全稳定的同位素，这种方法可以直接应用于人体实验。特别是，该方法将促进，实际上使研究人类婴儿中18-碳和20-碳脂肪酸的必需脂肪酸代谢成为可能。NIAAA IRB已经批准在人类婴儿中使用这些多种稳定同位素，并等待FDA的批准。已经向FDA食品安全和应用营养中心提交了申请，要求批准。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部位。然而，除了大脑之外，对脂肪酸延伸/去饱和在其他器官中的能力知之甚少。研究了n-6前体亚油酸（LA）和n-3前体α -亚麻酸（LNA）在大鼠体内各器官中的转化情况。大鼠被细分为25种器官系统/组织类型。在累积的氘标记LA和LNA中，约75%存在于白色脂肪中，25%存在于皮肤、肌肉和胴体中。肝脏似乎是脂肪酸合成代谢的主要部位，而大脑具有标记AA和DHA的高特异性积累。肾、心、肺、脾和睾丸的各种n-3和n-6代谢物的出现也表现出与局部代谢一致的时间过程。因此，这是第一次对这些器官系统参与EFA代谢的体内测量，并首次提出它们是长链代谢物产生和增加的贡献者。第二个密切相关的研究项目涉及神经系统DHA的起源。可能的来源是膳食预成型DHA、前体代谢、LNA或DHA体内储存。已经开发出一种新的技术，可以定量评估在各种饮食条件下从LNA代谢中增加的DHA的量。在本研究中，有必要从接近出生到大约6周龄时控制饮食，此时大鼠的大脑已经完成了质量增加。这是通过使用可以与我们新开发的人工喂养方法相结合的手动喂养技术来实现的。研制出一种几乎不含n-3脂肪酸的人造鼠乳。然后将n-3脂肪酸作为氘化lna加入，并含有不同的水平

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 but most of the variance could be explained by gestational age only. Present studies involve the potential end-product inhibition of 18-carbon EFA metabolism when arachidonic acid (AA) and DHA when added to infant formulas. 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. This is 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. Our recent studies have examined the in vivo metabolism of essential fatty acids in patients with Retinitis Pigmentosa (RP). In particular, patients with Ushers II disease or non-Ushers disease were compared to normal volunteers. We observed that the amount or enrichment of deuterated n-3 fatty acid metabolites such as EPA or DHA were significantly increased in Ushers patients whereas there was a decrease relative to normal volunteers in the non-Ushers RP group. The increased metabolism in the Ushers patients with respect to DHA may be surprising as it has been hypothesized that the retinal concentration of DHA is reduced in Retinitis Pigmentosa and that this may, in part, explain some of the loss in visual function associated with this neurological disease. However, as noted above for alcoholic patients, increased metabolism may be induced by increased catabolism that is associated with the disease state. These studies point to the need for analysis of increased fatty acid catabolism or indices of lipid peroxidation in vivo in these patients. The opposite direction of response in the non-Ushers patients points to a quite distinct etiology of this disease. Progress has been made during this reporting period in developing a novel multiple-isotope technique that we have termed MultiplE Simultaneous Stable Isotopes, or MESSI, for short. 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 can be directly applied to human experimentation due to the use of safe stable isotopes. In particular, the approach will facilitate, indeed make possible, the study of the essential fatty acid metabolism of 18- vs. 20- carbon fatty acids in human infants. The NIAAA IRB has approved the use of these multiple stable isotopes in human infants pending approval also by the FDA. An application has been submitted to the FDA Center for Food Safety and Applied Nutrition requesting such approval. 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 to about 6 weeks of age when the rat brain has completed its increase in mass. 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 level