Abnormal serum lysophospholipids in multiple myeloma patients

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) mediate various kinds of biological activities and play an important role in cellular signal transduction. We analyzed serum phospholipids obtained from 16 multiple myeloma (MM) patients and observed that serum LPA level was significantly higher in MM patients (5.3±0.5 nmol/mL) than in normal controls (1.7±0.3 nmol/mL). LPC level was also higher than that in normal controls, and it correlated significantly with the concentration of LPA (r=0.678, P<0.01). In MM patients, palmitic acid/linoleic acid ratios in phosphatidylcholine and LPC were higher than those in normal controls. In the 12-mon follow-up study of two patients with the immune globulin G type, we recognized that the increase of LPC, LPA, and arachidonic acid/linoleic acid ratio in phosphatidylinositol corresponded with a decline in the serum albumin level and choline esterase activity.     

Lipase-catalyzed synthesis of lysophospholipids in a continuous bioreactor

A novel enzymatic method of lysolecithin synthesis was developed with immobilized lipase as a catalyst. The enzymatic transesterification was carried out in a number of alcohols, and the reaction was optimized with regard to the water content and temperature of the medium. Similar kinetics of transesterification were observed with several individual phospholipids. The reaction was also performed continuously in a packed-column bioreactor, which was operated for 1180 h. The lipase displayed strict regio-selectivity towardsn-1 fatty acid in the phospholipid molecule, thus yielding exclusivelysn-1 lysolecithins as the final product.sn-2 Lysophospholipids were subsequently obtained by acyl migration catalyzed by ammonia vapor. Advantages associated with the use of lipases as opposed to conventional, phospholipase-A₂ catalyzed hydrolysis are briefly discussed.     

Comparison of selective cytotoxicity of alkyl lysophospholipids

Alkyl lysophospholipids have been shown to be cytooxic to a number of neoplastic tissues. One, ET-18-OCH₃, has been used to selectively purge leukemic cells from mixtures with normal marrow progenitor cells,in vitro andin vivo. We have measured the 50% inhibitory (IC₅₀) effect of a series of ether, lipids (EL) on leukemic cells (HL60, K562, Daudi, KG-1, KG-1a) and normal marrow progenitor cells. Cells were incubated with varying concentrations of EL for 4 hr and assayed for viability, [³H]thymidine incorporation and clonogenicity in semi-solid media. The effect on protein kinase C (PKC) activity was assayed for each compound. Compounds tested included three glycerophosphocholine analogs-ET-18-OCH₃, ET-16-NHCOCH₃, and BM 41.440. In addition, a lipoidal amine, CP 46665, an ethyleneglycolphospholipid, AEPL, and four single chain alkylphosphocholine analogs, HePC₂, HePC₃, HePC₄ and HePC₆ were also tested. During the period of incubation, the cells remained viable (>70%) as judged by trypan blue dye exclusion. The glycerophosphocholines were the most active and showed the highest therapeutic index. The lipoidal amine was active, but toxic to normal marrow progenitor cells. The ethyleneglycolphospholipid was active against HL60, but not against the other cell lines. The single chain alkylphosphocholine analogs were less active. All of the compounds inhibited PKC activity; however, the glycerophosphocholines were the most inhibitory. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Modulation of chemical carcinogenesis in rats by alkyl lysophospholipids

The influence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and 1-hexadecylmercapto-2-methoxymethyl-rac-propyl-3-phosphocholine (TLP, BM41.440) on methylnitrosourea (MNU)-induced rat mammary carcinomas and of ET-18-OCH₃ on 7,12-dimethylbenzanthracene (DMBA)-induced leukemias was investigated. Both agents effectively delayed MNU-induced mammary tumor formation at high, cytotoxic dosages but TLP had no influence at low “immunomodulatory” doses. ET-18-OCH₃ also significantly protected against leukemia development in DMBA-treated Long-Evans rats.     

Relevance of DHA‐containing lysophospholipids

In this issue of the European Journal of Lipid Science and Technology, Jin et al. [114–122] report on the anti‐inflammatory effect of docosahexaenoyl‐lysophosphatidylcholine with even a stronger effect when the docosahexaenoic acyl is oxygenated on its carbon 17. See accompanying article http://dx.doi.org/10.1002/ejlt.201100169     

Immunomodulatory and therapeutic properties of alkyl lysophospholipids in mice

This paper describes the immunomodulatory and therapeutic properties of the alkyl lysophospholipids [ALP; 1-O-octadecyl-2-O-rac-glycero-3-phosphocholine (ET-18-OCH₃)]. ALP was able to activate macrophages both in vitro and in vivo as well as to act as an immunoadjuvant for syngeneic tumor vaccines. However, ALP appeared to be transferred, at least in part, to the macrophage membrane, and some of the tumoricidal macrophage-activating properties seem to be associated with the direct cytotoxic effect of membrane-released ALP. ALP also had some therapeutic activity for experimental and spontaneous metastases, requiring administration three but not two times weekly at near-toxic doses; this suggests that at least some of its therapeutic activity is due to direct cytotoxicity.     

Determinants of DHA Incorporation into Tumor Tissue During Dietary DHA Supplementation

Docosahexaenoic acid (DHA), upon incorporation into tumor tissue, has the potential to sensitize tumors to the effects of chemotherapy or radiation therapy. Although DHA has usually been supplied to tumor tissue in the diet, appropriate dietary conditions required to obtain optimal tumor levels have not been established. Hence, we studied mammary tumor tissue responses in rats fed various durations and doses of DHA. Rats fed a palm oil enriched diet (diet 0) were switched to diets providing either 0.8 g DHA/day (diet 1) or 1.5 g DHA/day (diet 2). Tumor tissue fatty acid composition was analysed at baseline (diet 0), at weeks 1, 4 and 9 during diet 1 and at week 4 during diet 2. Dietary DHA supplementation differentially increased DHA within phospholipids (PL) and triacylglycerol (TAG) fractions in tumors. DHA level equilibrated between 2 and 4 weeks in PL while DHA increase was more progressive in TAG and did not reach a steady state. A higher dose of DHA further increased DHA content in tumor PL and TAG (P = 0.018 and P < 0.001, respectively). DHA concentration in plasma PL was positively correlated with DHA in tumor PL (r = 0.72; P = 0.0003) and TAG (r = 0.64; P = 0.003). We conclude that dietary DHA supplementation enhances tumor content of DHA in a time- and dose-dependent manner, and that the DHA level in plasma PL could be used as a proxy for tumor DHA. These findings have implications for dietary DHA supplementations in cancer patients.     

Effect of DHA supplementation on DHA status and sperm motility in asthenozoospermic males

The effects of supplementation with docosahexaenoic acid (DHA) on DHA levels in serum, seminal plasma, and sperm of asthenozoospermic men as well as on sperm motility were examined in a randomized, double-blind, placebo-controlled manner. Asthenozoospermic men (n=28; ≤50% motility) were supplemented with 0, 400, or 800 mg DHA/d for 3 mon. Sperm motility and the fatty acid composition of serum, seminal plasma, and sperm phospholipid were determined before and after supplementation. In serum, DHA supplementation resulted in decreases in 22∶4n−6 (−30% in the 800-mg DHA group only) and total n−6 (−6 and −12% in the 400- and 800-mg DHA groups, respectively) fatty acids. Increases were noted in DHA (71 and 131% in the 400- and 800-mg DHA groups, respectively), total n−3 fatty acids (42 and 67% in the 400- and 800-mg DHA groups, respectively), and the n−3/n−6 ratio (50 and 93% in the 400- and 800-mg DHA groups, respectively). In seminal plasma, DHA supplementation resulted in a decrease in 22∶4n−6 (−31% in the 800-mg DHA group only) and an increase in the ratio of n−3 to n−6 (35 and 33% in the 400- and 800-mg DHA groups, respectively). There were insignificant increases in DHA and total n−3 fatty acids. In sperm, decreases were noted in 22∶4n−6 (−37 and −31% in the 400-and 800-mg DHA groups, respectively). There were no other changes. There was no effect of DHA supplementation on sperm motility. The results show that dietary DHA supplementation results in increased serum- and possibly seminal plasma—phospholipid DHA levels, without affecting the incorporation of DHA into the spermatozoa phospholipid in asthenozoospermic men. This inability of DHA to be incorporated into sperm phospholipid is most likely responsible for the observed lack of effect of DHA supplementation on sperm motility.     

Separation of isomeric lysophospholipids by reverse phase HPLC

A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 orsn-2 position) and the position (Δ⁶ or Δ⁹) or geometric configuration (cis ortrans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at thesn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both thesn-1 andsn-2 isomers of monounsaturated species increased in the order Δ⁹-cis < Δ⁹-trans < Δ⁶-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ⁹-cis and Δ⁶-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.     

Production and Characterization of DHA and GLA-Enriched Structured Lipid from Palm Olein for Infant Formula Use

Palm olein was modified via lipase-catalyzed acidolysis reaction to obtain fatty acid composition and positional distribution similar to human milk fat. In the reaction, a free fatty acid mix containing 23.23 % docosahexaenoic (DHA), 31.42 % gamma-linolenic (GLA), and 15.12 % palmitic acid was employed. The DHA and GLA were incorporated into the structured lipid (SL) product to improve its nutritional value. Response surface methodology (RSM) was used to investigate the effects of reaction time and substrate mole ratio (palm olein to a free fatty acid mix) on the amount of palmitic acid at the sn-2 position of SL triacyglycerols (TAG), and on the total DHA and GLA incorporation. Gram-scale production of SL was performed using the conditions predicted by RSM to maximize the content of palmitic acid at the sn-2 position. Verification of the predictions from RSM confirmed its practical utility. The resulting SL had 35.11 % palmitic acid at the sn-2 position, with 3.75 % DHA and 5.03 % GLA. Differential scanning calorimetry and HPLC analyses of the TAG revealed changes in their polymorphic profiles and TAG molecular species of SL compared to palm olein. The SL from this study can potentially be used in infant formula formulations.     

The structure of DHA in phospholipid membranes

Early experiments and molecular simulations of PUFA favored a rigid arrangement of double bonds in U-shaped or extended conformations such as angle-iron or helical. Although results of recent solid-state NMR measurements and molecular simulations have confirmed the existence of these structural motifs, they portray an image of DHA (22∶6n−3) as a highly flexible molecule with rapid transitions between large numbers of conformers on the time scale from picoseconds to hundreds of nanoseconds. The low barriers to torsional rotation about C-C bonds that link the cis-locked double bonds with the methylene carbons between them are responsible for this unusual flexibility. Both the amplitude and frequency of motion increase toward the terminal methyl group of DHA.     

Enzymatic acylation of ether and ester lysophospholipids in rat liver microsomes

The acylation of lysophospholipids by rat liver acyltransferases was studied. A comparison between ester and ether lysophospholipids as substrates revealed large differences in substrate properties. For instance, oleic acid from oleoyl-CoA and arachidonic acid from arachidonoyl-CoA were not incorporated into 1-O-octadecyl-sn-glycero-3-phosphocholine under experimental conditions that allowed an optimal transfer of oleic acid and arachidonic acid to 1-O-palmitoyl-sn-glycero-3-phosphocholine. However, we observed an acyl-CoA-independent transfer of arachidonic acid from 1-O-stearoyl-2-O-arachidonoyl-sn-glycero-3-phosphoinositol to 1-O-octadecyl-sn-glycero-3-phosphocholine.     

Production of structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol from borage oil

γ-Linolenic acid (GLA) has the physiological functions of modulating immune and inflammatory responses. We produced structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol (CGC) from GLA-rich oil (GLA45 oil; GLA content, 45.4 wt%), which was prepared by hydrolysis of borage oil with Candida rugosa lipase having weak activity on GLA. A mixture of GLA45 oil/caprylic acid (CA) (1∶2, w/w) was continuously fed into a fixed-bed bioreactor (18×180 mm) packed with 15 g immobilized Rhizopus oryzae lipase at 30°C, and a flow rate of 4 g/h. The acidolysis proceeded efficiently, and a significant decrease of lipase activity was not observed in full-time operation for 1 mon. GLA45 oil contained 10.2 mol% MAG and 27.2 mol% DAG. However, the reaction converted the partial acylglycerols to structured TAG and tricaprylin and produced 44.5 mol% CGC based on the content of total acylglycerols. Not only FFA in the reaction mixture but also part of the tricaprylin and partial acylglycerols were removed by molecular distillation. The distillation resulted in an increase of the CGC content in the purified product to 52.6 mol%. The results showed that CGC-rich structured TAG can efficiently be produced by a two-step process comprising selective hydrolysis of borage oil using C. rugosa lipase (first step) and acidolysis of the resulting GLA-rich oil with CA using immobilized R. oryzae lipase (second step).     

Production of Structured Phosphatidylcholine with High Content of DHA/EPA by Immobilized Phospholipase A1-Catalyzed Transesterification

This paper presents the synthesis of structured phosphatidylcholine (PC) enriched with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) by transesterification of DHA/EPA-rich ethyl esters with PC using immobilized phospholipsase A₁ (PLA₁) in solvent-free medium. Firstly, liquid PLA₁ was immobilized on resin D380, and it was found that a pH of 5 and a support/PLA₁ ratio (w/v) of 1:3 were the best conditions for the adsorption. Secondly, the immobilized PLA₁ was used to catalyze transesterification of PC and DHA/EPA-rich ethyl esters. The maximal incorporation of DHA and EPA achieved was 30.7% for 24 h of reaction at 55 °C using a substrate mass ratio (PC/ethyl esters) of 1:6, an immobilized PLA₁ loading of 15% and water dosage of 1.25%. Then the reaction mixture was analyzed by ³¹P nuclear magnetic resonance (NMR). The composition of reaction product included 16.5% PC, 26.3% 2-diacyl-sn-glycero-3-lysophosphatidylcholine (1-LPC), 31.4% 1-diacyl-sn-glycero-3-lysophosphatidylcholine (2-LPC), and 25.8% sn-glycerol-3-phosphatidylcholine (GPC).     

Protectin DX, a Double Lipoxygenase Product of DHA, Inhibits Both ROS Production in Human Neutrophils and Cyclooxygenase Activities

Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits in an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX)—a docosahexaenoic acid di-hydroxylated product which inhibits blood platelet aggregation—on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B₄ (LTB₄). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases.     

DHA与EPA的研究进展

二十二碳六烯酸(Docosahexaenoic acid,22∶6n3,DHA)和二十碳五烯酸(Eicosapentaenoic acid,20∶5n3,EPA)均属于n3类多不饱和脂肪酸(Polyunsatumted fatty acids,PUFAs),能够在一定程度上预防和治疗糖尿病、类风湿性关节炎、自身免疫紊乱等疾病,对人体健康非常重要,因而受到越来越多的关注。本文对DHA与EPA的生理功能、合成机制及其在微藻和真菌中合成的研究现状进行了综述,并对利用转基因技术在哺乳动物体内生产EPA和DHA的前景进行了展望。     

破囊壶菌发酵生产DHA的研究进展

二十二碳六烯酸（DHA）是一种非常重要的多不饱和脂肪酸,能参与人及动物体内多个生理过程。破囊壶菌具有生长迅速、细胞内DHA含量高的特点,是工业化生产DHA的潜力菌。本文主要介绍破囊壶菌DHA代谢通路、影响破囊壶菌生产DHA的因素以及破囊壶菌中试发酵的研究现状。首先,对破囊壶菌合成DHA的两个代谢途径,即脂肪酸合成酶（fatty acid synthesis pathway,FAS）途径和聚酮合酶（polyketide synthesis pathway,PKS）途径进行总结和描述;其次,对影响破囊壶菌发酵生产DHA的三个主要因素（碳氮源、溶氧和温度）进行综述;随后,阐述了破囊壶菌发酵生产DHA的中试放大工艺的研究现状;最后,提出破囊壶菌发酵生产DHA过程中存在的问题,并指出进一步分离获得优质的破囊壶菌菌株、对其代谢途径和关键酶的研究以及中试放大工艺的研究是下一步研究的重点。通过对上述一系列问题进行综述,旨在为利用破囊壶菌工业化生产DHA提供一定的参考。     

DHA合成条件探究

以乙酰乙酸乙酯为原料合成脱氢醋酸,并通过熔点测定、IR、MS、1HNMR对产物进行简单的表征。探讨了催化剂的种类和用量、反应时间以及反应温度等对产物产率的影响。结果表明：当选用碳酸氢钠做催化剂合成DHA时,催化剂用量为0.11%,反应釜温度控制在180℃～190℃,时间为120 min,产物产率最高为51.3%,所得产物为浅黄色晶体;通过薄层色谱分析,所含杂质少。