Desaturation Of Essential Fatty Acids Using Stable Isotope GC/MS

使用稳定同位素 GC/MS 进行必需脂肪酸的去饱和

• 批准号: 9568234
• 负责人: Joseph Hibbeln
• 金额: $ 12.65万
• 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/9568234
• 关键词: Adipose tissue; Adult; Affinity; Agreement; Alcohol withdrawal syndrome; Alcohols; American; Animals; Arachidonic Acids; Automatic Data Processing; Automation; Bioavailable; Biochemical; Birth; Blood; Brain; Brain imaging; Cerebrovascular Circulation; Chemicals; Chronic; Data; Deposition; Development; Diet; Dietary Factors; Dietary intake; Docosahexaenoic Acids; Dose; Essential Fatty Acids; Fatty Acids; Female; Goals; Growth; Heart; Human; Image; Impairment; Infant; Investigation; Kidney; Label; Life; Linoleic Acids; Lipids; Liver; Lung; Mammals; Mass Fragmentography; Measurement; Measures; Metabolic; Metabolism; Methodology; Methods; Milk; Molecular; Myocardium; Nervous system structure; Oils; Omega-3 Fatty Acids; Oral; Organ; Plants; Plasma; Population Study; Positron-Emission Tomography; Procedures; Protocols documentation; Rattus; Recovery; Reporting; Research; Research Project Grants; Robotics; Rodent; Running; Sampling; Serum; Smoke; Smoker; Smoking; Source; Spleen; Stream; Techniques; Testis; Time; Tissues; Umbilical cord structure; alpha-Linolenic Acid; base; cerebral atrophy; cost effective; data acquisition; epidemiology study; experimental study; healthy volunteer; heart imaging; human subject; imaging study; in vivo; male; non-smoker; non-smoking; novel; nutrition; problem drinker; pup; stable isotope

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. 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 plasmahas been made in real time for the 11-C-DHA. We measured regional incorporation coefficients (K*) and rates of unesterified plasma DHA entry into brain lipids, and regional cerebral blood flow (rCBF), using PET with 1-11CDHA and 15OH2O, respectively. Imaging data were corrected for brain atrophy. We compared 22 non-smoking healthy control subjects to 15 non-smoking chronic alcoholics studied within 7 days of their last drink of alcohol. Both K* for DHA and rCBF were significantly and widely elevated throughout the brain in alcoholics compared with controls. Unesterified plasma DHA was similar in both groups (2.1 and 1.8 nmol/ml in controls and alcoholics, respectively) as was the rate of DHA incorporation into the brain as a whole (2.4 1.6 mg/d and 2.1 0.9 mg/d, respectively). Higher rCBF in alcoholics suggests altered brain functional activity during early withdrawal from alcohol. Higher K* for DHA in alcoholics indicates higher brain affinity for DHA and thus a potential brain DHA deficit vis--vis plasma availability. A human protocol is active to assess the effects of lowering dietary intake of linoleic acid from 8 en% to 1 en% on the elongation and desaturation of ALA to EPA and DHA. A separate line of investigation has been to develop high throughput methods of quantifying essential fatty acid status among large numbers of human subjects. An automated high throughput fatty acid analysis was developed from a previous procedure based on direct transesterification including the automation of chemical procedures, data acquisition and automatic data processing. The method was validated and applied to umbilical cord serum samples in an epidemiological study. The method was linear in the range of 1-600g/mL serum with r20.99. The within-run CV was <5.4% for 23 fatty acids and a range of recoveries over three concentrations were 76%119% in a low-lipid matrix with the exception of 14:0. The fatty acid concentration as measured by the robotic method for human plasma was in good agreement with the Lepage&Roy method. The fatty acid profile in umbilical cord serum from American subjects(n=287) showed an average of 38.0%, 24.9%, 32.0% and 4.6% of total fatty acids for saturates, monounsaturates, n-6 and n-3 polyunsaturates, respectively. This is the first report of a complete, validated, cost-effective, automated, high throughput fatty acid measurement method along with application to a population-based study.

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。 必需的脂肪酸代谢是对男性和女性的成年人以及吸烟和非吸烟者的研究。 这是对这些前体单一口服剂量后的氘化la和氘化LNA转化率的体内代谢的稳定同位素研究。 我们的结果表明，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，而22：5N-3转化率的分数转化率更高，为22：6n-3。 雄性吸烟者的总血浆N-3脂肪酸升高，更快地转移了D5-18：3N-3，不赞成率为D5-20：5N-3，既延迟又慢，而D5-20：5N-3的比例更高，将其定向到22：5n-3中，相对于非熟练者而言。 通常，吸烟增加了血浆中N-3脂肪酸的生物可利用性，分数转化率加速，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。 在饮食中没有预先形成的DHA时，对未经DHA的配方喂养的婴儿的后果的另一个发现是，包括心脏，肺，肾脏和脾脏在内的多个器官净损失了DHA含量。 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19位健康志愿者和17名酗酒者的大脑和心脏图像。 为11-C-DHA实时对脂肪酸输入功能进行了广泛的表征。我们分别使用具有1-11CDHA和15OH2O的PET测量了区域掺入系数（K*）和未酯化等离子体DHA进入脑脂质和区域脑血流（RCBF）的速率。校正了脑萎缩的成像数据。 我们将22名非吸烟的健康对照受试者与15名非吸烟的慢性酒精饮料进行了比较。与对照组相比，在酗酒中，DHA和RCBF的K*在整个大脑中均显着升高。 两组的未酯化等离子体DHA（分别为2.1和1.8 nmol/ml在对照和酒精中测）和整个大脑中的DHA率（分别为2.4 1.6 mg/d和2.1 0.9 mg/d）也是如此。 酗酒者中较高的RCBF表明在早期退出酒精期间，大脑功能活性改变了。 DHA在酒精中毒中的K*较高表明对DHA的大脑亲和力较高，因此潜在的脑DHA缺陷相对于VIS血浆可用性。 人类方案是有效的，可以评估将亚油酸饮食摄入从8％％降低到1 EN％对ALA对EPA和DHA的延长和去饱和的影响。 单独的研究线是开发出量化大量人类受试者必需脂肪酸状态的高通量方法。 自动高通量脂肪酸分析是根据基于直接式式式静止化的先前程序开发的，包括化学程序的自动化，数据采集和自动数据处理。 该方法在流行病学研究中验证并应用于脐带血清样品。 该方法在R20.99的1-600G/mL血清范围内线性。对于23个脂肪酸，运行的CV <5.4％，在低脂质基质中，三个浓度的一系列回收率为76％119％，但14：0除外。 通过机器人方法测量的人血浆的脂肪酸浓度与Lepage＆Roy方法非常吻合。 来自美国受试者的脐带血清中的脂肪酸谱（n = 287）的平均脂肪酸平均为38.0％，24.9％，32.0％和4.6％的总脂肪酸，分别为饱和，单不饱和，N-6和N-3多饱和度。这是一项完整，经过验证，成本效益，自动化，高吞吐量脂肪酸测量方法以及对基于人群的研究的应用。