Sn-2 Fatty Acid Research Articles

Rapid identification of plasmalogen molecular species using targeted multiplexed selected reaction monitoring mass spectrometry

Plasmalogens (Pls) levels are reported to be altered in several neurological and metabolic diseases. Identification of sn-1 fatty alcohols and sn-2 fatty acids of different Pls species is necessary to determine the roles and mechanisms of action of Pls in different diseases. Previously, full-scan tandem mass spectrometry (MS/MS) was used for this purpose but is not effective for low-abundance Pls species. Recently, multiplexed selected reaction monitoring MS (SRM/MS) was found to be more selective and sensitive than conventional full-scan MS/MS for the identification of low-abundance compounds. In the present study, we developed a liquid chromatography (LC)-targeted multiplexed SRM/MS system for the identification and quantification of different Pls choline (Pls-PC) and Pls ethanolamine (Pls-PE) species. We determined five precursor-product ion transitions to identify sn-1 and sn-2 fragments of each Pls species. Consequently, sn-1 and sn-2 fatty acyl chains of 22 Pls-PC and 55 Pls-PE species were identified in mouse brain samples. Among them, some species had C20:0 and C20:1 fatty alcohols at the sn-1 position. For quantification of Pls species in mouse brain samples, a single SRM transition was employed. Thus, our results suggest that the LC-targeted multiplexed SRM/MS system is very sensitive for the identification and quantification of low-abundance lipids such as Pls, and is thus expected to make a significant contribution to basic and clinical research in this field in the future.

Synthesis of symmetrical medium- and long-chain triacylglycerols rich in arachidonic acid at sn-2 position for infant formula

Medium- and long-chain triacylglycerols (MLCT) rich in arachidonic acid (ARA) at sn-2 position were synthesized by a two-step enzymatic method. Firstly, sn-2 monoacylglycerols (MAG) were synthesized at a temperature of 25 °C by enzymatic alcoholysis. The MAG with 69.42% ARA at sn-2 position were obtained by solvent extraction and low temperature solvent crystallization. Secondly, the MLCT rich in ARA at sn-2 position and capric acid (CA) at sn-1,3 positions were produced by enzymatic esterification. Under the optimal conditions (MAG:CA = 1:3 (mol/mol), 0.05 MPa vacuum, 8% Lipozyme RM IM, 5 h, 25 °C), the content of triacylglycerol was up to 93.60%. The triacylglycerol in the form of C10:0-C20:4-C10:0 (including isomers) was about 40.43%. The ARA contents in the total and sn-2 fatty acid composition of the final product were 32.35% and 51.12%, respectively. MLCT rich in ARA at sn-2 position were successfully produced and the product has the potential application for functional food and infant formula.

Continuous gradient temperature Raman spectroscopy of 1-stearoyl- 2-docosahexaenoyl, 1-stearoyl- 2-arachidonoyl, and 1,2-stearoyl phosphocholines

Mixed chain phospholipids containing a saturated fatty acid at sn1 and a polyunsaturated fatty acid in sn2 are common in the specialized biological membranes prevalent in neural, retinal and organ tissues. Particularly important are mixed lipids containing palmitic or stearic acid and arachidonic or docosahexaenoic acid. Gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements and phase transitions. Herein we utilize GTRS for 1-18:0, 2-20:4n-6 PC; 1-18:0 2-22:6n-3 PC; and 1-18:0, 2-18:0 PC from −80 to 50 °C temperatures. 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allowed detailed vibrational mode assignment and analysis. Samples were analyzed neat and with molecular hydration. Previously reported phase transitions for hydrated 18:0−20:4PC and 18:0−22:6PC and numerous spectral differences resulting from hydration and the double bond structure were clearly observed. Molecular models showed that the addition of minimal water molecules results in significant structural differences compared to the neat molecules; 18:0−22:6PC is strikingly compact with water when viewed from the hydrophilic end. This precise Raman data cannot be observed in typically utilized fully hydrated vesicle samples, however the improved GTRS will allow for more precise analysis in fully hydrated vesicles because the underlying modes in the unavoidably broadened spectra can be identified.

Extraction of Seabuckthorn Seed Oil and Analysis of Its Fatty Acid Composition

Seabuckthorn seed oil was extracted by fat extraction method. The effects of quality of seabuckthorn seed, breaking time, soaking time and extraction solvent on the extraction rate were investigated by single factor experiment and response surface methodology. The fatty acids in seabuckthorn seed oil were determined. The research results show that the most suitable extraction conditions for using a fat extraction instrument are: seabuckthorn seed mass is 1.40 g, crushing time is 11 s, soaking time is 26 min, extraction time is 4 h, temperature is 70?, and the extraction solvent is petroleum ether. The extraction rate can reach 6.56%; the extracted seabuckthorn seed oil mainly contains three main fatty acids: linoleic acid, linolenic acid and oleic acid, these three fatty acids are also the main sn-2 fatty acids.

A conjunction of sn-2 fatty acids and overall fatty acid composition combined with chemometric techniques increase the effectiveness of lard detection in fish feed

Fish oil is a common source of fat in fish feed production. However, there is a tendency to substitute fish oil with other fats such as lard to reduce production costs. Thus, an efficient method for lard detection is highly needed for fish feed’s authenticity. In this study, sn-2 fatty acids (sn-2 FAs) and fatty acid (FA) compositions were incorporated with chemometric techniques namely Principal Component Analysis (PCA), Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA), and Orthogonal Partial Least Square-Regression (OPLS-R) to identify lard adulteration in the fish feeds. The inclusion of sn-2 FAs into PCA model 2 exhibited a preferable variation pattern relative to PCA model 1. The PCA identified C14:0, C18:0, C18:2, C18:3, C20:0 sn-2 C16:0, sn-2 C18:0, sn-2 C18:1, and sn-2 C18:2 were the most significant FAs to discriminate the fish feeds. The inclusion of sn-2 FA composition improved the OPLS-DA model 2 performance by providing more significant class discrimination between lard-adulterated, and non-adulterated fish feeds as compared to OPLS-DA model 1. The OPLS-DA model 2 identified C18:0, C18:2, C18:3, and sn-2 C16:0 FAs as markers of lard adulteration with an increment in the value of the coefficient of determination (R2) and decrement in the Root Mean Square Error of Estimation (RMSEE) and Root Mean Standard of Cross-Validation (RMSECV) values. The Support Vector Machine (SVM), Random Forest (RF), Multilayer Perceptron-Artificial Neural Network (MLP-ANN), and internal and external validations corroborated the OPLS-DA model 2 and OPLS-R model 2 performances. Therefore, the incorporation of sn-2 FA and FA compositions coupled with the chemometric techniques had improved the detection and quantification of lard adulteration in fish feeds.

Total and Sn-2 Fatty Acid Profile in Human Colostrum and Mature Breast Milk of Women Living in Inland and Coastal Areas of China

Introduction: Although lipid is the major energy source and exerts beneficial effects on infant growth, research on the composition of fatty acid (FA) at the sn-2 position of human milk (HM) in China and abroad is limited. Objectives: This study aimed to investigate the FA positional distribution in colostrum and mature HM of women living in the inland and coastal areas of China and explore the potential influences of geographical region and lactation stage on the FA profile of Chinese women. Methods: Colostrum milk (n = 61) and mature milk (n = 56) samples were obtained longitudinally from healthy lactating women in Guangzhou and Chengdu, China. Gas chromatography was used to determine the total and sn-2 FA composition. Results: Significant differences were observed in the FA profile of HM between different regions and lactation stages, with differences in polyunsaturated FA levels being the most pronounced. Nearly 70% of sn-2 FAs were saturated FAs, of which C16:0 accounted for approximately 75%. C8:0, C10:0, C18:0, C20:0, C22:0, and all of the unsaturated FAs were mainly located at the sn-1 and sn-3 positions, while C14:0, C15:0, and C16:0 were mainly at the sn-2 position. The proportion of C12:0 and C17:0 at sn-2 was approximately equivalent to that at the sn-1, 3 positions. Conclusions: The results indicate the variability in the FA profile of HM between regions and lactation stages. The contents of polyunsaturated FAs and sn-2 FAs, especially palmitic acid, should be paid more attention when optimizing infant formula.

Enzymatic Interesterification of Palm Stearin and Palm Olein Blend Catalyzed by sn-1,3-Specific Lipase: Interesterification Degree, Acyl Migration, and Physical Properties

Acyl migration of fatty acid at sn-2 is often observed alongside enzymatic interesterification (EIE), causing the loss of lipase selectivity toward the acyl group at sn-1,3. In this study, an oil blend consisting of palm stearin (PST) and palm olein (POL) was interesterified via a chemical interesterification (CIE) and enzymatic method using a packed bed reactor. Characterization in terms of the triacylglycerol (TAG) compositions, sn-2 fatty acid distributions, and solid fat content profiles was performed. In comparison to that of CIE fats, EIE fats showed different modification effects on the solid fat content. Under similar reaction conditions, different interesterification degrees (IDs) were obtained according to the various blend ratios. Using the same mass ratio of substrates (POL/PST of 9:1), the EIE reaction time and temperature affected the ID and the change in the fatty acyl group at the sn-2 position. Under the reaction time of 46 min, an ID of 94.41% was acquired, while at 80 °C, the degree of acyl migration at sn-2 was 92.87%. EIE with high acyl migration exhibited a lower crystallization rate than that of EIE with low acyl migration. However, the effect of acyl migration on crystal polymorphism and oxidative stability was insignificant. Outcomes from this study are meaningful for the establishment of a theoretical basis for a controlled positional-specific EIE that is catalyzed by sn-1,3-specific lipase.

<p>Human Secretary Phospholipase A2 Mutations and Their Clinical Implications</p>

Phospholipases A2 (PLA2s) belong to a superfamily of enzymes responsible for hydrolysis of the sn-2 fatty acids of membrane phospholipids to release arachidonic acid. PLA2s are the rate limiting enzyme for the downstream synthesis of prostaglandins and leukotrienes that are the main mediators of inflammation. The extracellular forms of this enzyme are also called the secretary phospholipase A2 (sPLA2) and are distributed extensively in most of the tissues in the human body. Their integral role in inflammatory pathways has been the primary reason for the extensive research on this molecule. The catalytic mechanism of sPLA2 is initiated by a histidine/aspartic acid/calcium complex within the active site. Though they are known to have certain housekeeping functions, certain mutations of sPLA2 are known to be implicated in causation of certain pathologies leading to diseases such as atherosclerosis, cardiovascular diseases, benign fleck retina, neurodegeneration, and asthma. We present an overview of human sPLA2 and a comprehensive compilation of the mutations that result in various disease phenotypes. The study not only helps to have a holistic understanding of human sPLA2 mutations and their clinical implications, but is also a useful platform to initiate research pertaining to structure–function relationship of the mutations to develop effective therapies for management of these diseases.

Effects of enzymatic free fatty acid reduction process on the composition and phytochemicals of rice bran oil

The effects of enzymatic free fatty acid reduction process (EFFARP) on the composition and phytochemicals of dewaxed and degummed rice bran oil (DDRBO) were investigated and compared with the effects observed using internal acyl acceptors. The acid value of DDRBO was effectively decreased from 16.99 mg KOH/g to approximately 0.36 mg KOH/g by EFFARP. EFFARP significantly decreased the moisture content and peroxide value of DDRBO and increased the induction period. The Sn-2 fatty acid comoposition of DDRBO after EFFARP was very reaching the total fatty acid composition. EFFARP significantly increased the triacylglycerol content compared to the control, while the oryzanol content was not obviously affected. The contents of free sterol, and total tocopherol and tocotrienol were increased slightly by EFFARP compared to the control. When conducted under vacuum with added nitrogen, EFFARP shows great application potential in the edible oil industry.

Composition analysis of fatty acids and stereo-distribution of triglycerides in human milk from three regions of China

Understanding the components composition characteristics of human milk is conductive for the development of infant formulas. The total fatty acids composition and the stereo-distribution of fatty acids in human milk triglycerides (TGs) from three regions (Zhengzhou, Wuhan and Harbin) of China were analyzed. The total and intrapositional fatty acids composition of human milk TGs showed significant differences among three regions. Zhengzhou, Harbin and Wuhan human milk possessed highest levels of medium-chain fatty acids (MCFA, 6.36 ± 1.46%), short-chain fatty acids (SCFA, 0.10 ± 0.03%) and monounsaturated fatty acids (MUFA, 34.61 ± 0.45%), and polyunsaturated fatty acids (PUFA, 30.34 ± 0.33%), respectively. The preference distribution of fatty acids in human milk TGs was hardly affected by regions, for the relative conservative interpositional levels of each fatty acids in three regions. Not only the specific distribution of sn-2 position, but the esterification of fatty acids in sn-1 and sn-3 positions also had preference. SCFA and MCFA were mainly esterified at sn-3 position, but LCSFA most unlikely distributed at the sn-3 position. The preference esterification of MUFA were sn-1, sn-3 > sn-2, and PUFA were sn-3 > sn-1 > sn-2. Therefore, the optimization of infant formulas not only needs considering total fatty acid composition of TGs but requires thinking over the stereo-distribution of fatty acids.

Regiospecific Analysis of Triacylglycerols by Ultrahigh-Performance-Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

An ultrahigh-performance-liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was developed for the analysis of AAB and ABC type triacylglycerol (TG) regioisomers. Excellent linear regressions were established between the ratio of [RR]+ product ions and the proportion of regioisomers of TGs. The method was further optimized by analysis of 18 regiospecific pairs of AAB type TGs and five triplets of regioisomers of ABC type TGs with acyl carbon number (ACN) ranging from 36 to 54 and the number of double bonds (DB) from 0 to 7. Reverse linear relationships were recognized between the slope of the calibration curve and the number of double bonds of the sn-2 fatty acids. Negative linear regressions were found between the intercepts of the calibration curves and the sum of ACN + DB of sn-2 fatty acids. The method was highly repeatable as shown by the low deviation and high stability of the calibration curves at different concentrations and between different periods of analysis. This is the first time that calibration curves for the ABC type TGs are reported. The results provide crucial and novel information for reliable and quantitative determination of regioisomeric TGs in natural fats and oils.

Enzymatic labelling of snake venom phospholipase A2 toxins

Almost all animal venoms contain secretory phospholipases A2 (PLA2s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA2s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2s from Bothrops asper - one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono- or bi-derivatives of these three PLA2s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.

Abstract 991: Secretory phospholipase A2 as a trigger for drug release in the treatment of triple-negative breast cancer

Abstract Triple negative breast cancer describes a subgroup of breast cancers which are negative for the estrogen and progesterone receptors as well as the HER2 protein. As such, triple negative breast cancers have seen little benefit from the targeted therapies developed for the treatment of breast cancer. To address this challenge, we examined an alternative target within the tumor as well as the tumor environment. Secretory phospholipase A2 (sPLA2) cleave phospholipids at sn-2 ester bonds, releasing lysophospholipids and fatty acids, and are over expressed in several pathologies including breast cancer. Herein, we evaluated the therapeutic activity of secretory phospholipase A2 responsive liposomes (SPRL) compared to the clinically used, sterically-stabilized, pegylated liposomes (SSL) for in vitro response in a triple-negative breast cancer (TNBC) model. In these studies, SSL and SPRL formulations were made according to previous studies and resulted in three formulations SSL, SPRL- E and SPRL-G. SPRL were made by the addition of either DSPE (SPRL-E) or DSPG (SPRL-G). Doxorubicin was used as the drug of choice and Dox-loaded liposomes were prepared by remote-loading using an ammonium sulfate gradient. Toxicity studies were performed by the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or resazurin while uptake studies were performed by fluorescence microscopy and flow cytometry. Briefly, cells were seeded at a density of 10,000 cells per well and subjected to free doxorubicin or liposomal doxorubicin in concentrations from 0 - 100 µM. At the appropriate time points, 0-72 hours, cells were harvested for studies. Treatment of breast cancer cells with doxorubicin encapsulated in SSL and SPRL resulted in cytotoxicity in the MDA-MB-231 cells line comparable to free drug with an IC50 of approximately 3 µM at the 72 hour time point. In tracking of drug and liposome delivery, we demonstrated that drug uptake was liposome-dependent, as encapsulation of doxorubicin in SPRL resulted in greater intracellular drug levels compared to SSL. These data show the therapeutic activity of SPRL compared to SSL, and suggest that SPRL may be useful for the treatment of TNBC. Two-dimensional models do not fully recapitulate the complexity of barriers to drug delivery, nor the effect of multiple cell types. Therefore, ongoing studies are examining the utility of these liposome formulations in three-dimensional co-cultures that include macrophages and fibroblasts. In this model, we hope to assess the ability of the tumor microenvironment to alter drug release. Citation Format: Shanese L. Jasper, Elena B. Skarupa, Brian S. Cummings, Robert D. Arnold. Secretory phospholipase A2 as a trigger for drug release in the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 991.

Analysis of triacylglycerols molecular species composition, total fatty acids, and sn-2 fatty acids positional distribution in different types of milk powders

Triacylglycerols (TAGs) are considered the main component of milk fat; possess significant functional roles from the technological, nutritional and physiological points of view. In this study, an ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was applied to identify the molecular species composition of TAGs in buffalo, camel, and ewe milk powders. The separation of TAGs was achieved by using two mobile phases; acetonitrile/isopropanol (1:9, v/v) as mobile phase A, and 40% acetonitrile as mobile phase B, the binary gradient elution allowed the separation of milk TAGs in consistent with the increase in partition number. More than 180 TAGs in buffalo milk, 90 TAGs in camel milk, and 129 TAGs in ewe milk were unambiguously identified, and the most abundant molecular species in the three types of milk powders were detected at mass to charge (m/z) values of 682.53, 822.67, and 656.53, respectively. The total acyl carbon numbers of buffalo milk TAGs ranged between 26 and 54, with double bonds from 0 to 6. While, the total acyl carbon numbers of camel milk TAGs ranged between 42 and 54, with double bonds from 0 to 3. The total acyl carbon numbers of ewe milk TAGs ranged between 26 and 52, with double bonds from 0 to 2. The major fatty acids (FAs) in the different types of milk powders were myristic, palmitic, stearic, oleic, and linoleic. The positional distribution of saturated FAs at the sn-2 position was higher in buffalo milk powder as compared to camel and ewe milk powders. This study reports a comprehensive identification of TAGs molecular species in different types of milk powders.

Evaluation model for cocoa butter equivalents based on fatty acid compositions and triacylglycerol patterns.

An effective evaluation model was established to digitize the quality of cocoa butter equivalents (CBEs) based on determinations of total and sn-2 fatty acid compositions and triacylglycerol (TAG) profiles and the "deducting score" principle. Similarity scores for selected fats and oils calculated from the model revealed differences between them and parallel cocoa butter compositions. For CBE1 and CBE2, total similarity scores were 90.6 and 90.0, whereas those of mango (76.3), dhupa (84.1), sal fat (84.7), kokum (78.3), palm mid fractions (PMF, 77.9), shea butter (64.0), illipe butter (89.7) and Pentadesma butyracea butter (67.2), respectively. Similarity scores were found to agree with physical properties, including polymorphism, crystal morphology, crystallization or melting behaviors, and solid fat content. The present study provides an accurate means of assessing CBE quality and hopefully will contribute to the development of commercial CBEs.

Enzymatic Modification of Menhaden Oil to Incorporate Caprylic and/or Stearic Acid

AbstractMenhaden oil was enzymatically modified with caprylic (8:0) and/or stearic acid (18:0) to produce structured lipids (SL). The goal was to produce SL with high amounts of polyunsaturated fatty acids (PUFA), a low level of saturation, and a melting point of 25–35 °C. Substrate (menhaden oil to acyl donor) molar ratios were 1:1, 1:3, and 1:5 for 8:0, and 1:1, 1:2, and 1:3 for 18:0. Enzyme load was 10% of the total weight of substrates. Time course study determined optimal time for maximum acyl donor incorporation. Linear interpolation estimated molar ratios that yielded SL with 20 or 30 mol% incorporation of 8:0 or 18:0. Enzymatic reactions were also conducted with molar ratios of menhaden oil to acyl donors:8:0:18:0 (1:1:3, 1:2:2, and 1:3:1). Lipases from Candida antarctica, Lipozyme® 435, and Rhizomucor miehei, Lipozyme® RM IM (Novozymes North America, Inc., Franklinton, NC, USA), were compared for all reactions. Total and sn‐2 fatty acid compositions, triacylglycerol (TAG) molecular species, thermal behavior, volatile lipid oxidation products, solid fat contents, and oxidative stability were compared. When 8:0 was the acyl donor, the 1:3.03 and 1:4.58 ratios resulted in incorporation of 20 and 30 mol% 8:0, respectively. With 18:0 as the acyl donor, the 1:1.32 and 1:2.41 ratios led to incorporation of 20 and 30 mol% 18:0, respectively. The 1:3:1 ratio SL had a crystallization onset (C0) of 15.3 °C and a melting completion (Mc) of 33.1 °C. The physicochemical properties of these SL suggest that some may be useful in formulating food products such as margarines and spreads.

Fatty Acids, Triacylglycerol and Sn -2 Fatty Acids Distributions Variations in Seed Oil from Camellia Cultivars

Camellia seed oil is widely used in the food, health, cosmetics and medicine industries in China. The present study aimed to investigate fatty acids, triacylglycerols (TGAs) and sn-2 fatty acids distributions variations in seed oil from 46 kinds of Camellia cultivars. The predominant fatty acids was oleic acid (18:1ω9) with 71.30% (average). The sn-2 position was mainly occupied by oleic acid, linoleic acid and palmitic acid. Fifteen TAGs species were found and the main TAGs were OOO + SLO, OOP and OOL+SLL. The trisaturated TAGs species were not detected. These results indicated significant changes in the profiles of fatty acids, sn-2 position fatty acids and TGAs, and in contents of these in seed oils from different Camellia cultivars (P< 0.05). Saturated fatty acids is not positively related to its distribution in sn-2 position. The data in present paper may be important as a reference for adulteration of camellia seed oil with other oils.

Enzymatic preparation of structured triacylglycerols with arachidonic and palmitic acids at the sn-2 position for infant formula use

Structured TAGs with palmitic acid and polyunsaturated fatty acid (PUFA) at the sn-2 position have various health benefits for infants. In this study, we first compared two enzymatic routes for preparation of the structured TAGs. Results showed that the one-pot and two-step syntheses led to 37.6% and 55.4% oleic acid incorporation, respectively, after 10 h and reaction route had little effect on the sn-2 fatty acid composition. Subsequently, reaction variables of the two-step synthesis were optimized. Under the optimal conditions, 53.5% oleic acid was incorporated into the structured TAGs after 6-h acidolysis. Major fatty acids at the sn-2 position were palmitic acid (68.7%), ARA (9.8%) and oleic acid (7.9%). This is the first study reporting a two-step enzymatic method for structured TAGs preparation. Compared to the one-pot synthesis, current method significantly improves the efficiency of the acidolysis by product inhibition elimination. The synthetic TAGs have potential use in infant formulas.

Chemical composition and antioxidant capacity of extracts from the whole berry, pulp and seed of Hippophae¨ rhamnoides ssp. yunnanensis

In the present study, profiles of fatty acid, sn-2 fatty acid, triglyceride, phytochemical (including tocopherol/tocotrienol, phytosterol, flavonoid, carotenoid and polyphenol) and antioxidant capacity of extracts from the whole berry, pulp and seed of Hippophae¨ rhamnoides ssp. yunnanensis were investigated and compared. The distributions of fatty acid, sn-2 fatty acid and triglyceride that was identified using ultra-performance liquid chromatography tandem quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) showed obvious differences among the different parts of the ssp. yunnanensis. The whole berry and pulp extracts exhibited high flavonoid, carotenoid and polyphenol contents, whereas, the seed portion expressed high tocopherol/tocotrienol and phytosterol concentrations. Results deduced from this study demonstrated that ssp. yunnanensis is a rich source of unsaturated fatty acid and bioactive minor component, which should be further developed and utilized by breeders and planters.

Cellular Trafficking of Sn-2 Phosphatidylcholine Prodrugs Studied with Fluorescence Lifetime Imaging and Super-resolution Microscopy.

While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.