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

基本信息

批准号：
9354063
负责人：
Joseph Hibbeln
金额：
$ 15.16万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
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/9354063
关键词：
Adipose tissue Adult Affinity Agreement Alcohol consumption Alcohol withdrawal syndrome American Animal Feed Animals Arachidonic Acids Automatic Data Processing Automation Biochemical Birth Blood Brain Brain imaging Cerebrovascular Circulation Chemicals Chronic Data Deposition Development Diet Dietary Factors Dietary intake Docosahexaenoic Acids Dose Epidemiologic Studies Essential Fatty Acids Fatty Acids Female Goals Growth Heart Human Image 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 Positron-Emission Tomography Procedures Protocols documentation Rattus Recovery Reporting Research Research Project Grants Robotics Rodent Running Sampling Saturated Fatty Acids Serum Smoke Smoker Smoking Source Spleen Stream Techniques Testis Time Tissues Umbilical cord structure alpha-Linolenic Acid base cerebral atrophy cost effective data acquisition fatty acid metabolism feeding healthy volunteer heart imaging human subject in vivo male non-smoker non-smoking novel nutrition population based problem drinker pup research study stable isotope

项目摘要

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.

在最近应用基于稳定同位素的 GC/MS 方法之前，人们对动物或人类体内必需脂肪酸代谢知之甚少。对成年人（男性和女性、吸烟者和不吸烟者）进行了必需脂肪酸代谢的研究。这是一项对单次口服剂量这些前体后氘化 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，并且 22:5n-3 转化为 22:6n-3 的分数转化率更高。男性吸烟者的血浆 n-3 脂肪酸总量升高，D5-18:3n-3 的周转更快，D5-20:5n-3 的消失率延迟且更慢，D5 的百分比更高相对于非吸烟者，-20:5n-3 被定向为 22:5n-3。一般来说，吸烟会增加血浆中 n-3 脂肪酸的生物利用度，加速部分转化率，并增加某些长链 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 会导致多个器官中从 18-C 前体到 DHA 和 DPAn6 的标记积累减少，尽管组织 DHA 显着增加。当喂食含有 3 wt% α-亚麻酸的受控饮食时，雌性大鼠比雄性大鼠积累更多的 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 的标记较 18:2n6 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。可能的来源包括饮食中预先形成的 DHA、前体 LNA 的代谢或体内储存的 DHA。一种新技术已被开发出来，可以定量评估各种饮食条件下 LNA 代谢产生的 DHA 量。对于这项研究，有必要从出生前一直到大脑发生显着发育的时期控制饮食。这是通过使用新开发的人工饲养技术实现的，该技术使用几乎不含 n-3 脂肪酸的人造大鼠奶。然后将 n-3 脂肪酸作为氘化 LNA 添加，并含有不同水平的 DHA。在一项主要实验中，幼鼠在出生后第 8-29 天期间被喂食含有 0 % 或 2% DHA 的饮食。在此期间，可以计算出，在以 D5-LNA 作为 n-3 脂肪酸唯一来源的动物中，新形成的脑 DHA 的 40% 来自预先形成的 DHA，而不是来自 LNA 代谢。这是令人惊讶的，因为饮食中不含 DHA；因此，所有沉积在大脑中的预先形成的 DHA 必定是通过血流从其他器官中获得的。当 DHA 添加到饮食中时，LNA 代谢为 DHA 的速率显着降低，这可能是由于某种形式的终产物抑制，并且 88% 的大脑 DHA 来自预先形成的饮食 DHA。膳食 DHA 的这些代谢作用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。给予预先形成的 DHA 的大鼠脑部 DHA 水平也较高，这表明新陈代谢无法提供足够的脑部 DHA 来源。喂养不含 DHA 配方奶粉的婴儿的另一个后果是，在身体快速生长期间，当饮食中不存在预先形成的 DHA 时，包括心脏、肺、肾和脾在内的多个器官的 DHA 含量出现净损失。 PET 成像用于研究 C11-DHA 掺入大脑的新应用已经启动。现已获得 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实时广泛表征。我们分别使用 1-11CDHA 和 15OH2O 的 PET 测量了区域掺入系数 (K*) 和未酯化血浆 DHA 进入脑脂质的速率，以及区域脑血流量 (rCBF)。成像数据针对脑萎缩进行了校正。我们将 22 名不吸烟的健康对照受试者与 15 名不吸烟的慢性酗酒者进行了比较，并在他们最后一次饮酒后 7 天内进行了研究。与对照组相比，酗酒者大脑中 DHA 和 rCBF 的 K* 值均显着且广泛升高。两组中未酯化的血浆 DHA 相似（对照组和酗酒者分别为 2.1 和 1.8 nmol/ml），DHA 进入整个大脑的速率也相似（分别为 2.4 ± 1.6 mg/d 和 2.1 ± 0.9 mg/d）。酗酒者较高的 rCBF 表明早期戒酒期间大脑功能活动发生了变化。酗酒者中 DHA 的 K* 较高，表明大脑对 DHA 的亲和力较高，因此与血浆可用性相比，大脑可能存在 DHA 缺乏。一项人类方案正在积极评估将亚油酸膳食摄入量从 8 en% 降低至 1 en% 对 ALA 向 EPA 和 DHA 的伸长和去饱和的影响。另一项研究是开发高通量方法来量化大量人类受试者的必需脂肪酸状态。自动化高通量脂肪酸分析是从先前基于直接酯交换的程序开发出来的，包括化学程序的自动化、数据采集和自动数据处理。该方法经过验证并应用于流行病学研究中的脐带血清样本。该方法在1-600g/mL血清范围内呈线性，r20.99。 23 种脂肪酸的运行内 CV <5.4%，在低脂基质中，三个浓度的回收率范围为 76%119%（14:0 除外）。通过机器人方法测量的人血浆脂肪酸浓度与 Lepage&Roy 方法非常一致。美国受试者 (n=287) 脐带血清中的脂肪酸谱显示，饱和脂肪酸、单不饱和脂肪酸、n-6 和 n-3 多不饱和脂肪酸平均分别占总脂肪酸的 38.0%、24.9%、32.0% 和 4.6% 。这是第一份关于完整、经过验证、具有成本效益、自动化、高通量脂肪酸测量方法及其在基于人群的研究中的应用的报告。