Enzymatic preparation of food-grade l-α-glycerylphosphorylcholine from soy phosphatidylcholine or fractionated soy lecithin.

l-α-Glycerylphosphorylcholine (l-α-GPC) is a biosynthetic precursor for the neurotransmitter acetylcholine in humans, making it a useful as a cognitive enhancer for treating patients with stroke and dementia, including Alzheimer's disease. The aim of this study was to prepare l-α-GPC via Novozym 435 (an immobilized Candida antarctica lipase B)-catalyzed hydrolysis of soy phosphatidylcholine or a fractionated soy lecithin, from which triacylglycerols were completely removed, followed by food-grade solvent extraction of l-α-GPC from the reaction products. The reaction was performed in n-hexane-water biphasic media in a stirred-batch reactor. Phosphatidylcholine was completely hydrolyzed to l-α-GPC under optimal conditions: temperature, 55°C; water content, 100 wt% of the substrate weight; enzyme loading, 10 wt% of the substrate weight; and reaction time of 6 hr (for soy phosphatidylcholine) or 8 hr (for fractionated soy lecithin). Water-soluble fractions of the reaction products containing 98.6 area% l-α-GPC (from soy phosphatidylcholine) or 52.4 area% glycerophosphodiesters, including l-α-GPC (from fractionated soy lecithin), were obtained after phase separation of the media. The resulting products would be suitable for use as food-grade cognitive enhancers because of the use of enzymatic reaction and food-grade solvent extraction.

Effective Liquid Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry Separation of Isomeric Lipid Species.

Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to ∼410 ( R ∼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Purification of L-alpha glycerylphosphorylcholine by column chromatography.

Colorless L-alpha glycerylphosphorylcholine (L-α-GPC) was obtained at 99.8% purity, 69.8% recovery, and with a specific rotation of -2.5° via a five-step procedure. L-α-GPC was first produced by phospholipase A(1) hydrolysis of soy lecithin powder. Ca(2+) and Cl(-) were then effectively removed using two successive 001×7 cation and D311 anion exchange resin column chromatography procedures. Silica gel column chromatography and decoloration with active carbon were then applied to remove remaining impurities and colorant. Characterization of the L-α-GPC product was well in agreement with the standard. The resin and silica gel showed remarkable ability for L-α-GPC isolation after 10 uses. Thus, this study presents a simple and cost-effective method for preparing L-α-GPC with high yield and purity, low cost, and environmental friendliness, and encourages future investigation into its adaptation for industrial applications.

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids.

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.

[Proton magnetic resonance spectroscopy of excised pig liver].

After evaluating ex vivo pig liver by 1H magnetic resonance spectroscopy (1H-MRS) using a 1.5 Tesla super-conducting unit, the assignment of peaks was reevaluated in vitro using a 9.4 Tesla superconducting unit. The portal vein was cannulated and perfused by cooled Euro-Collins solution, and pig liver was removed and preserved in the solution. Five to 8 g of the liver was excised before and after preservation, then extracted by perchloric acid (PCA). In 1H-MRS of PCA extracted pig liver, large peaks of fatty acid disappeared, while peaks of the choline group from 3.2-3.3 ppm were clearly observed. Using high performance liquid chromatography (HPLC) and 31P (phosphorus)-MRS, the 3.23 ppm peak was determined as glycerophosphorylcholine (GPC), which diminished after preservation. The chronological change of GPC in PCA extracted pig liver was able to be observed by 1H-MRS. GPC peak may play a role as an indicator in evaluating preserved liver by 1H-MRS.

Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography. Bombesin and TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells by a common mechanism.

A method for the rapid and quantitative separation of glycerophosphocholine, choline phosphate and choline upon ion-exchange columns is described. The method has been utilized to examine the stimulation of phosphatidylcholine breakdown in quiescent Swiss 3T3 cells in response to bombesin and 12-O-tetradecanoylphorbol 13-acetate (TPA). The stimulated generation of choline is shown to precede that of choline phosphate, with no effect upon glycerophosphocholine levels; but was attenuated in cells in which protein kinase C activity was down-regulated. The results thus suggest that stimulation of the cells with either bombesin or TPA activates phospholipase D-catalysed phosphatidylcholine breakdown by a common mechanism involving the activation of protein kinase C.

Phospholipase activity in Candida albicans.

Phospholipase A and lysophospholipase have been identified as the enzymes responsible for phospholipid hydrolysis by Candida albicans. The method used to identify and measure the activity of these enzymes is described, and the probable significance of phospholipase in the invasion of the epithelium by Candida albicans discussed.

Studies on human serum high-density lipoproteins (HDL). IV. Isolation of lipoprotein families after incubation of HDL.

The high-density lipoproteins (HDL) of human serum appear to be unstable and easily exposed to chemical changes during isolation. In earlier studies we have isolated and purified HDL subfractions either in the presence of an SH-blocking agent, DTNB, or in the cold. By both procedures reproducible lipoprotein subfractions could be recovered by hydroxyl apatite column chromatography at the elution steps 0.03-0.05 mol/l (subfraction II) and 0.05-0.15 mol/l phosphate buffer (subfraction III). The protein moiety of both lipoprotein subfractions contained polypeptides A-I , A-II, thin line (TL), C-I and C-II, and the protein moiety of subfraction III contained also C-III. The incubation at 37 degrees C of these HDL subfractions gave reproducible daughter lipoprotein fractions that could be recovered by subsequent rechromatography on hydroxyl apatite. At each of the elution steps 0.05-0.075 mol/l and 0.075-0. mol/l one daughter fraction was recovered, the protein moiety of which was composed of polypeptide A-I, as judged by polyacrylamide gel electrophoresis, immunodiffusion, and amino acid analysis. The incubation of parent subfractions II and III caused also the appearance at elution step 0.001-0.01 mol/l of a daughter lipoprotein fraction - lipoprotein A (Lp-A) - that was characterized by a protein moiety with polypeptides A-I and A-II in equal amounts. The 'release' of lipoprotein A-I (Lp-A-I) and Lp-A was shown to be due rather to the incubation than to the column chromatography as such. The chemical changes occurring during the incubation of HDL suggested a degradation of phosphatidylcholine (PC) to lysophosphatidylcholine (lyso-PC) and glycerylphosphorylcholine (GPC). It is suggested that the degradation of PC might interfere with the interaction between the lipoprotein families composing HDL.