Search

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development., (Copyright © 2017. Published by Elsevier Inc.)

The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the development of novel strategies to modulate oocyte membrane dynamics and structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Beef cows were treated to ovulate a larger (LF-LCL group, associated with greater receptivity) or smaller (SF-SCL group) follicle and, consequently, to present greater or smaller plasma concentrations of estradiol during proestrus-estrus, as well as progesterone during early diestrus. Oviduct and uterus (4 days after gonadotropin-releasing hormone-induced ovulation; D4) as well as the uterus (D7) were collected, and lipid profiles were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This technique allowed the identification and tissue localization of sphingomyelins (SM), phosphatidylcholines (PC), ceramides (Cer), and phosphatidylethanolamines (PE). Multivariate statistics were used to separate samples into groups with distinctly different phospholipid profiles in the uterus at D4 and D7. Different abundance of ions corresponding to specific lipids were detected on D4 (Cer [42:1], PC [31:0], PC [32:1], PC [34:4], and PC [36:4] greater for LF-LCL group; and PC [38:7], PC [38:5], PC [38:4], PC [40:7], and PC [40:6] greater for SF-SCL group) and D7 (SM [34:2], SM [34:1], PC [32:1], and PC [35:2] greater for LF-LCL group). The MALDI-MS imaging showed the spatial distributions of major phospholipids. In conclusion, distinct phospholipid profiles were associated with animals treated to show contrasting receptivity to the embryo. Functional roles of the identified phospholipids on uterine function and preimplantation embryo development deserve further studies., (© 2016 by the Society for the Study of Reproduction, Inc.)

Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This work presents an in vitro investigation of the effect of protein restriction on the metabolism of albendazole (ABZ). This study was conducted using liver microsomal fractions obtained from Wistar rats. For the quantitative analysis, a multidimensional High Performance Liquid Chromatography (2D HPLC) method was fully validated for the determination of the ABZ metabolites: albendazole sulfoxide, albendazole sulfone and albendazole 2-aminesulfone. The target compounds were directly extracted using a C8-RAM-BSA column (5.0x0.46 cm i.d.) and analyzed on a chromatographic chiral column containing amylose tris(3,5-dimethylphenylcarbamate) (150x4.6 mm i.d.). The in vitro biotransformation results showed that the protein restriction influenced the oxidative metabolism of ABZ. The production of R-(+)-ABZ-SO (1309 nmol/L) and S-(-)-ABZ-SO (1456 nmol/L) was higher in the control animals than in the animals fed with a diet containing 6% protein, which produced 778.7 nmol/L and 709.5 nmol/L for R-(+) and S-(-)-ABZ-SO enantiomers, respectively. These results were statistically inspected by Student´s t test and the results showed a significant difference between the two means (p<0.05). Moreover, the production of ABZ-SO enantiomers was enantioselective where the S-(-)-ABZ-SO was formed in greater amounts than the R-(+)-ABZ-SO in control animals (p=0.0231). However, the enantioselectivity was not observed when the in vitro biotransformation of ABZ was conducted using the microsomal fractions obtained from protein restriction animals (p>0.05). Furthermore, animal nutritional condition could affect the pattern of ABZ sulphoxidation indicating that the protein nutrition affect primarily the formation of R-(+)-ABZSO and S-(-)-ABZ-SO enantiomers.

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Analytical and semipreparative high performance liquid chromatography methods using polysaccharide-based chiral stationary phases were developed for the enantiomeric resolution of albendazol sulfoxide. The enantioseparation of this compound was evaluated with four chiral stationary phases: cellulose and amylose tris(3,5-dimethylphenylcarbamate), amylose tris[(S)-1-phenylethylcarbamate] and amylose tris(3,5-dimethoxyphenylcarbamate), under three elution conditions: normal, reversed-phase and polar organic mode. The influences of the mobile phase and of the structure of the chiral stationary phase on the enantiomeric separation are discussed. The best chiral performances were achieved on an amylose tris(3,5-dimethylphenylcarbamate) phase under normal (R(s)=4.96) and polar organic mode (R(s)=2.60 and 3.09). A polar organic condition using methanol as mobile phase offered shorter retention factors (k(1)=0.34) and was scaled up to semipreparative HPLC to obtain milligram quantities of both albendazole sulfoxide enantiomers for further in vitro studies. Optical rotation and circular dichroism of both enantiomers of albendazole sulfoxide was determined. The compounds ABZ, ABZ-SO, (R)-(+)-ABZ-SO and (S)-(-)-ABZ-SO were all evaluated regarding their capacity to inhibit the in vitro growth of three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma). In addition, the effect of the (R)-(+)-ABZ-SO compound in the cell cycle profile and apoptosis of MCF-7 cells were also studied. Results indicated that compound ABZ was the most potent regarding cell growth inhibition and that the (+)-(R)-ABZ was a more potent inhibitor of cell growth than the (S)-(-)-ABZ-SO, particularly in the MCF-7 cell line. In addition, the (R)-(+)-ABZ-SO significantly increased the levels of apoptosis of the MCF-7 cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Many chiral pesticides are introduced into the environment as racemates, although their pesticidal activity is usually the result of preferential reactivity of only one enantiomer, while the other enantiomer may have toxic effects against nontarget organisms. Methamidophos (O,S-dimethyl phosphoramidothioate), a chiral compound, is an insecticide widely used in agriculture in both developed and developing countries. However, this pesticide has a high toxicity not only to targeted insects but also to human and animals. In the present study, the enantiomers of methamidophos were enantiomerically separated by a semipreparative chiral liquid chromatography at the multimilligram scale on a polysaccharide-based chiral stationary phase and a preliminary evaluation of their in vitro inhibition of plasma butyrylcholinesterase (BChE) of hens was performed. In the present study, our first effort was to resolve the racemic mixture of methamidophos and to that end reversed-phase, normal-phase, and polar organic elution conditions were investigated in four different polysaccharide-based chiral phases. The best performance was achieved on a cellulose tris(3,5-dimethylphenylcarbamate) phase under normal phase. This chromatographic condition allowed the separation of 225 mg of methamidophos enantiomers with a high degree of chiral purity (>98%) in a short analysis time. Significant differences were found between the concentration that causes 50% of enzyme inhibition (IC50) of the three isoforms of methamidophos. (-)-Methamidophos showed an IC50 approximately three times larger than the (+)-enantiomer for plasma BChE of hens., (Copyright © 2011 SETAC.)

The development and validation of a fully automated achiral-chiral high performance liquid chromatography (HPLC) method for the simultaneous determination of albendazole metabolites: enantiomers of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO(2)) and albendazole 2-aminosulphone (ABZ-SO(2)NH(2)) in bovine plasma are described. This method involves an octyl restricted access media bovine serum albumin column (C(8)-RAM-BSA) (50 mm x 4.6 mm I.D.) for sample clean-up, followed by enantioselective analysis on a column containing an amylose tris(3,5-dimethylphenylcarbamate) stationary phase (150 mm x 4.6 mm I.D.). The chromatographic separations of all target compounds were performed at 30 degrees C using a mobile phase composed of phosphate buffer (10 mmol L(-1); pH 7.5):acetonitrile (60:40, v/v), flow rate of 0.5 mL min(-1) and fluorescence detection at 290 nm and 320 nm, excitation and emission, respectively. The influence of different organic modifiers and chiral selector of the stationary phase on enantioseparation of ABZ-SO was investigated. The method developed was fully validated. The calibration curves were linear in the concentration range of 40.00-1280 ng mL(-1) for each albendazole sulphoxide enantiomer, 10.0-320 ng mL(-1) for albendazole sulphone and 20.0-320 ng mL(-1) for albendazole 2-aminosulphone. The inter- and intra-day precision ranged from 0.760% to 7.79% relative standard deviation (R.S.D.), and the accuracy ranged 101% from 114% of the nominal values while the transfer efficiency was in the range of 84.4-103%. The method showed good linearity, precision, accuracy, sensitivity and selectivity allowing it to be appropriate for further pharmacokinetics and metabolism studies of albendazole.

The enantiomers of sulfoxide proton pump inhibitors--omeprazole, lansoprazole, rabeprazole and Ro 18-5364--were enantiomerically separated by liquid chromatography at multimilligram scale on a polysaccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantiomeric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose.

Kielcorins are xanthonolignoids with protein kinase C inhibition and antitumor activities. Four racemates were enantioresolved at a multimilligram scale on tris-3,5-dimethylphenylcarbamate amylose phase using polar organic conditions as mobile phase. The low-solubility of these compounds conditioned the injection amount and consequently the productivity. A solid-phase injection system was developed to increase the production rate of the semipreparative process. The effects of the racemates and the related enantiomers on the in vitro growth of human breast cancer cell line MCF-7 were compared. Differences in their growth inhibitory activity were observed.