A General Method for Quantification and Discovery of Acyl Groups Attached to Acyl Carrier Proteins in Fatty Acid Metabolism using LC-MS/MS.
利用 LC-MS/MS 定量和发现脂肪酸代谢中酰基载体蛋白附着的酰基的一般方法。
- 作者列表："Nam JW","Jenkins LM","Li J","Evans B","Jaworski JG","Allen DK
:Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2, 3-trans-enoyl-ACPs), and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain acyl-ACPs (C16:3), indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to Type II fatty acid synthase systems found in plants, bacteria, as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using LC-MS/MS with de novo generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feed stocks, and medical studies in health and disease.
: 酰基载体蛋白 (ACPs) 是生物系统中脂肪酸生物合成的支架，使其对全面了解脂质代谢至关重要。然而，评估个体酰基-ACPs 的准确定量方法并不存在。我们开发了一种稳健的方法来定量酰基-ACPs 到皮克水平。我们成功鉴定了酰基-ACP 延伸中间体 (3-羟基酰基-acps 和 2,3-反式-烯基-acps) 和意外的中链 (C10: 1，C14: 1) 和多不饱和长链酰基-ACPs (C16: 3)，表明该方法的敏感性以及目前对脂质代谢和 ACP 功能的描述是不完整的。这样的 ACPs 可能对燃料的中链脂质生产很重要，并且突出了叶绿体中知之甚少的脂质重塑事件。该方法广泛适用于在植物、细菌、以及来自哺乳动物和真菌的线粒体，因为它利用了 ACPs 中高度保守的 Asp-Ser-Leu-Asp (DSLD) 氨基酸序列，酰基附着在该序列上。我们的方法允许使用 LC-MS/MS 与从头生成的标准和同位素稀释策略进行敏感定量，并将填补我们理解的空白, 通过对最佳生物燃料、可再生饲料库存以及健康和疾病医学研究的脂肪酸生物合成过程的定量探索提供见解。
METHODS:BACKGROUND:Given the importance of habitual dietary protein intake, distribution patterns and dietary sources in the aetiology of age-related declines of muscle mass and function, the present study examined these factors as a function of sex and age in Irish adults aged 18-90 years comprising The National Adult Nutrition Survey (NANS). METHODS:In total, 1051 (males, n = 523; females, n = 528) undertook a 4-day semi-weighed food diary. Total, body mass relative intake and percentage contribution to total energy intake of dietary protein were determined in addition to protein distribution scores (PDS), as well as the contribution of food groups, animal- and plant-based foods to total protein intake. RESULTS:Total and relative protein intake [mean (SD)] were highest in those aged 18-35 years [96 (3) g day , 1.32 (0.40) g kg day ], with lower protein intakes with increasing age (i.e. in adults aged ≥65 years [82 (22) g, 1.15 (0.34) g kg day , P < 0.001 for both]. Differences in protein intake between age groups were more pronounced in males compared to females. Protein distribution followed a skewed pattern for all age groups [breakfast, 15 (10) g; lunch, 30 (15) g; dinner, 44 (17) g]. Animal-based foods were the dominant protein source within the diet [63% (11%) versus 37% (11%) plant protein, P < 0.001]. CONCLUSIONS:Protein intake and the number of meals reaching the purported threshold for maximising post-prandial anabolism were highest in young adults, and lower with increasing age. For main meals, breakfast provided the lowest quantity of protein across all age categories and may represent an opportunity for improving protein distribution, whereas, in older adults, increasing the number of meals reaching the anabolic threshold regardless of distribution pattern may be more appropriate.
METHODS:BACKGROUND:Low cardiorespiratory fitness (CRF) increases risk of all-cause mortality and cardiovascular events. Periodic CRF assessment can have an important preventive function. OBJECTIVE:To develop a protocol-free method to estimate CRF in daily life based on heart rate (HR) and body acceleration measurements. METHODS:Acceleration and HR data were collected from 37 subjects (M=49%) while performing a standardized laboratory activity protocol (sitting, walking, running, cycling) and during a 5-days free-living monitoring period. CRF was determined by oxygen uptake (VO2max) during maximal exercise testing. A doubly-labeled water validated equation was used to predict total energy expenditure (TEE) from acceleration data. A fitness index was defined as the ratio between TEE and HR (TEE-pulse). Activity recognition techniques were used to process acceleration features and classify sedentary, ambulatory and other activity types. Regression equations based on TEE-pulse data from each activity type were developed to predict VO2max. RESULTS:TEE-pulse measured within each activity type of the laboratory protocol was highly correlated to VO2max (r from 0.74 to 0.91). Averaging the outcome of each activity-type specific equation based on TEE-pulse from the laboratory data led to accurate estimates of VO2max (RMSE: 300.0 mlO2/min or 10%). The difference between laboratory and free-living determined TEE-pulse was 3.7 ± 11% (r =0.85). The prediction method preserved the prediction accuracy when applied to free-living data (RMSE: 367 mlO2/min or 12%). CONCLUSIONS:Measurements of body acceleration and HR can be used to predict VO2max in daily life. Activity-specific prediction equations are needed to achieve highly accurate estimates of CRF.
METHODS:OBJECTIVE:Postprandial dyslipidemia is a common feature of insulin resistant states and contributes to increased cardiovascular disease risk. Recently, bile acids have been recognized beyond their emulsification properties as important signaling molecules that promote energy expenditure, improve insulin sensitivity, and lower fasting lipemia. While bile acid receptors have become novel pharmaceutical targets, their effects on postprandial lipid metabolism remain unclear. Here we investigated the potential role of bile acids in regulation of postprandial chylomicron production and triglyceride excursion. Approach and Results: Healthy C57BL/6 mice were given an intraduodenal infusion of taurocholic acid (TA) under fat-loaded conditions and circulating lipids were measured. Targeting of bile acid receptors was achieved with GW4064, a synthetic agonist to the farnesoid X receptor (FXR), and with deoxycholic acid (DCA), an activator of the Takeda G-protein-coupled receptor 5. TA, GW4064, and DCA treatments all lowered postprandial lipemia. FXR agonism also reduced intestinal triglyceride content and activity of microsomal triglyceride transfer protein, involved in chylomicron assembly. Importantly, TA effects (but not DCA) were largely lost in FXR knockout mice. These bile acid effects are reminiscent of the anti-diabetic hormone glucagon-like peptide-1 (GLP-1). While the GLP-1 receptor agonist exendin-4 retained its ability to acutely lower postprandial lipemia during bile acid sequestration and FXR deficiency, it did raise hepatic expression of the rate limiting enzyme for bile acid synthesis. CONCLUSIONS:Bile acid signaling may be an important mechanism of controlling dietary lipid absorption and bile acid receptors may constitute novel targets for the treatment of postprandial dyslipidemia.