Gold nanoparticle–antibody conjugates for specific extraction and subsequent analysis by liquid chromatography–tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk

Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24 ± 0.04 μg mL⁻¹ GNP suspension for MDA-LDL with an affinity K_d of 5.25 ± 0.11 × 10⁻¹⁰ M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC–MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.     

从微藻中提取多不饱和脂肪酸

初步探测了用尿素包合法从一种海洋微藻中提取出的脂肪中富集16∶2,16∶3,18∶2,20∶5 (分别指碳和双键数,16∶2指十六碳两双键)四种多不饱和脂肪酸(PUFAs)。海洋微藻富含PUFAs,实验研究得出用无水乙醚和石油醚混合溶剂在体积比1∶2、温度20℃、提纯时间5h的实验条件下萃取海藻中的脂肪质量最高。皂化过程选取甲醇作为溶剂要明显优于以乙醇作为溶剂的结果,实验确定采用KOH 甲醇作为皂化试剂;研究了作为关键因素的尿素在尿素包合过程中的影响,得出质量分数为20%的尿素试验提纯效果较好 ,富集所得的多不饱和脂肪酸总量占脂肪酸的89%.     

Analysis of the Dynamic Decomposition of Unsaturated Fatty Acids and Tocopherols in Commercial Oils During Deep Frying

The decomposition of unsaturated fatty acids and tocopherols in 10 commercial edible oils during deep frying was investigated. The dominant tocopherol in oils rich in polyunsaturated fatty acids (PUFAs) was γ-tocopherol, except for natural perilla oil (δ-tocopherol dominant), and the main tocopherol in oleic acid-rich oils was α-tocopherol. The PUFA-rich oils had higher tocopherol contents than the oleic acid-rich oils. Both the reduction rate of total unsaturated fatty acid (TUFA) and total tocopherol (TToc) were linear with frying time (t). The decomposition rate of TToc is faster than that of TUFA since the slope values obtained from fitting equations (Y?=?k t) k_TToc (1.520–14.483) were obviously larger than for k_TUFA (0.155–0.270). By establishing a dynamic decomposition index, unsaturated fatty acids and tocopherol in oils showed dynamic decomposition over multiple frying cycles. The obtained results showed that decomposition characteristics of oils are related to their fatty acid compositions.     

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs’ production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs’ accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs’ profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs’ extraction and purification from microalgal biomass.     

高产PUFAs多形单毛孢菌株的获取及碳源选择

从土壤中筛选出一株产多不饱和脂肪酸的真菌,经鉴定为多形单毛孢,该菌株能产多种不饱和脂肪酸.碳源优化选择表明葡萄糖和蔗糖复合碳源为最佳碳源,当葡萄糖和蔗糖以质量比9∶1复合,添加总糖量为100 g/L时,培养10 d后,生物量达到15.45 g/L,产油率达到77.1%,总多不饱和脂肪酸产量达到8450.1mg/L.其中,十八碳三烯酸、二十碳四烯酸、二十碳五烯酸和二十二碳六烯酸产量分别能达到640.9,1947.3,333.4和292.3 mg/L.     

High Endogenously Synthesized N-3 Polyunsaturated Fatty Acids in Fat-1 Mice Attenuate High-Fat Diet-Induced Insulin Resistance by Inhibiting NLRP3 Inflammasome Activation via Akt/GSK-3β/TXNIP Pathway

High-fat (HF) diets and low-grade chronic inflammation contribute to the development of insulin resistance and type 2 diabetes (T2D), whereas n-3 polyunsaturated fatty acids (PUFAs), due to their anti-inflammatory effects, protect against insulin resistance. Interleukin (IL)-1β is implicated in insulin resistance, yet how n-3 PUFAs modulate IL-1β secretion and attenuate HF diet-induced insulin resistance remains elusive. In this study, a HF diet activated NLRP3 inflammasome via inducing reactive oxygen species (ROS) generation and promoted IL-1β production primarily from adipose tissue preadipocytes, but not from adipocytes and induced insulin resistance in wild type (WT) mice. Interestingly, endogenous synthesized n-3 polyunsaturated fatty acids (PUFAs) reversed this process in HF diet-fed fat-1 transgenic mice although the HF diet induced higher weight gain in fat-1 mice, compared with the control diet. Mechanistically, palmitic acid (PA), the main saturated fatty acid in an HF diet inactivated AMPK and led to decreased GSK-3β phosphorylation, at least partially through reducing Akt activity, which ultimately blocked the Nrf2/Trx1 antioxidant pathway and induced TXNIP cytoplasm translocation and NLRP3 inflammasome activation, whereas docosahexaenoic acid (DHA), the most abundant n-3 PUFA in fat-1 adipose tissue, reversed this process via inducing Akt activation. Our GSK-3β shRNA knockdown study further revealed that GSK-3β played a pivot role between the upstream AMPK/Akt pathway and downstream Nrf2/Trx1/TXNIP pathway. Given that NLRP3 inflammasome is implicated in the development of most inflammatory diseases, our results suggest the potential of n-3 PUFAs in the prevention or adjuvant treatment of NLRP3 inflammasome-driven diseases.     

Chemical Characterization,Antiproliferative and Antioxidant Activities of Polyunsaturated Fatty Acid-Rich Extracts from Chlorella sp. S14

Microalgae is a rich source of polyunsaturated fatty acid. This study was conducted to identify and isolate microalgal strain with the potentials for producing polyunsaturated fatty acids (PUFAs) and determine its cytotoxic effect on some cancer cells. The algal strain (Chlorella sp. S14) was cultivated using modified BG-11 media, and algal biomass obtained was used for fatty acid extraction. Gas chromatographic–mass spectrometry was used to identify and quantify the levels of the fatty acid constituents. The total content of monounsaturated fatty acids (1.12%) was low compared to polyunsaturated fatty acids (PUFAs) (52.87%). Furthermore, n-3 PUFAs accounted for (12.37%) of total PUFAs with the presence of α-linolenic acid (2.16%) and cis-11,14,17-eicosatrienoic acid (2.16%). The PUFA-rich extract did not exhibit a cytotoxic effect on normal cells. Treatment with the PUFA-rich extract (150 µg/mL) significantly reduced cell viability in MCF-7 (31.58%) and A549 (62.56%) cells after the 48 h treatment. Furthermore, treatment of MCF-7 with fatty acid extracts (125 and 150 µg/mL) showed a significant reduction in MDA levels, increase in catalase activities and decrease in GSH level compared to untreated cells. However, a slight decrease in MDA level was observed in A549 cells after the 48 h treatment. There are no significant changes in catalase activities and GSH level in treated A549 cells. However, a slight reduction of NO levels was observed in treated MCF-7 and A549 cells. These results indicate the potentials of PUFA-rich extracts from Chlorella sp. S14 to reduce viability and modulate redox status in A549 and MCF-7 cells.     

两种海洋微藻脂肪酸组成研究

为了筛选多不饱和脂肪酸含量高的微藻,对两种海洋微藻,三角褐指藻（Phaeodactylum triorrutum）和等鞭金藻（Isochrysis gal-bana）进行了室内培养,并对其脂肪酸组成进行了研究。结果表明：三角褐指藻含有较高的C16:O、C16:1和C20:5（EPA）,仅含有微量的C20:4不含有C22:6（DHA）,多不饱和脂肪酸占总脂肪酸的23.8％。而等鞭金藻主要脂肪酸为C14:0、C16:0、C18:2、C18:3、C22:6（DHA）,不含有C20:4,仅有少量的EPA,多不饱和脂肪酸占总脂肪酸的比例高达49.8％。两种微藻脂肪酸组成显示出明显的种间差异。     

超临界萃取法提取海带多不饱和脂肪酸的研究

对以海带（Laminaria japonica)为原料,采用超临界CO2萃取技术提取海藻多不饱和脂肪酸的可行性进行了研究,结果表明,当CO2流量2．5－3．5L／h,萃取压力25MPa,萃取温度30℃,萃取时间3h,原料粒度40－60目,分离压力5MPa和分离温度35℃时最利于海带脂质的提取,此萃取条件可使海带总脂肪酸中多不饱和脂肪酸含量达到67．2％。