Choline Esters Research Articles

Localization and Properties of Cholinesterases in the Common Prawn (Palaemon serratus): a Kinetic-Histochemical Study

Due to the diversity in biochemical properties and tissue distribution of cholinesterase (ChE) enzymes, a characterization should be performed before they are used as biomarkers for monitoring pesticide contamination. In this study we investigate the distribution of ChEs and their kinetic properties in diverse tissues of the common prawn Palaemon serratus. The results concerning the histochemical localization of ChEs suggest that the highest amount of ChE activity occurs in prawn eyes, followed by the brain, gills, and digestive tract. Negligible staining was observed in the muscle. We describe the kinetic properties of ChEs in eyes, gills, and hepatopancreas, investigating their substrate preferences with different thiocholine esters and their sensitivity to inhibition with selective inhibitors. The results suggest that the studied enzymes are ChEs and not nonspecific esterases, due to their apparent affinity for choline esters, with a distinct preference for the substrate acetylthiocholine, and their high sensitivity to inhibition by eserine sulfate. P. serratus eyes can be considered the best tissue for recovery of ChE enzyme: this tissue is easy to isolate, it expresses high levels of ChE, and the enzyme shows properties of a vertebrate AChE.

Comparison of Two Storage Methods for the Analysis of Cholinesterase Activities in Food Animals

Cholinesterases (ChE) are specialized carboxylic ester hydrolases that catalyse the hydrolysis of choline esters. They are classified into either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Determination of ChE in the tissues is the appropriate tool for the diagnosis of organophosphorus and carbamate exposures. In general, a significant inhibition was seen in both AChE and BChE activities after 6 months of freezing at −80°C and after 3 months of freezing at −20°C. Linear regression of mean AChE and BChE was observed in all individual samples during the months of the two freezing methods. Bland and Altman plot of the ratios of the two freezing methods have showen the mean difference between the two freezing methods to be 8.8, and SD was 144.7 and −127.6 for upper and lower limits, respectively, for liver, while in muscle the mean difference was 1.5 and SD was 32.5 and −28.9 for upper and lower limits, respectively.

Baculo-expression and enzymatic characterization of CES7 esterase

The male reproductive tracts in different species are characterized by similar patterns of male-dependent overexpression of carboxylesterases. This phenomenon indicates male sex-associated functions of these enzymes for spermatogenesis, sperm maturation, and sperm use. Recently, a novel epididymis-specific gene named Ces7 was cloned and characterized, which belongs to the carboxylesterase family. To study the functions of CES7 in sperm maturation and storage, CES7 recombinant protein was expressed in baculovirus system. The recombinant protein had carboxylesterase activity hydrolyzing cholesterol ester and choline ester. CES7 as carboxylesterase might be involved in ester hydrolysis, sperm maturation, and storage in male reproductive tract.

De-oiled rapeseed and a protein isolate: characterization of sinapic acid derivatives by HPLC–DAD and LC–MS

De-oiled rapeseed is a rich source of proteins and phenolic compounds. The phenolic compounds, namely sinapic acid derivatives (SAD), could occur as free sinapic acid, esterified (as sinapine, the choline ester of sinapic acid) and decarboxylated (as canolol) forms. Rapeseed protein preparations containing very low phenolic compounds have been the focus of our ongoing research. A precipitated rapeseed protein isolate is investigated for SAD such as sinapine, sinapoyl glucose, canolol using HPLC–DAD and LC–MS. Profile of the phenolic compounds of de-oiled rapeseed, press cakes and the precipitated protein isolate are compared. HPLC–DAD analysis indicated SAD; particularly sinapine is the main phenolic compound of all the substrates. The protein derivation process did not remarkably alter the profile of the investigated protein isolate.

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐ tandem mass spectrometry

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI-QTOF-MS and direct infusion ESI-FTICR-MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision-induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl- and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed-phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI-MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class.

Serum butyrylcholinesterase activity and phenotype associations with lipid profile in stroke patients

Background Butyrylcholinesterase (BuChE) catalyzes the hydrolysis of acetylcholine and other choline esters and is also involved in lipid metabolism. The purpose of this study was to investigate any association between BuChE serum phenotype and activity and lipid profile of ischemic stroke patients. Methods We determined serum BuChE activities and phenotypes, and levels of total cholesterol (TC), LDL-C, HDL-C and triacylglyerol (TG) in 33 patients with acute ischemic stroke within 12 h of the onset of the attack and 29 controls. Results The mean (± SD) serum BuChE activity and the BuChE of U/A phenotype in the stroke individuals were significantly lower and higher than that of the control (315 (± 124) IU/L. vs. 384 (± 99) IU/L, p = 0.02, t = − 2.4 and 21.2% vs.3.4%, p = 0.026 respectively). Conclusions Our results showed that a negative correlation between BuChE activity with TC level, in addition the frequency of BuChE phenotypes with low activity is high in stroke patients, who have high levels of cholesterol, may have increased susceptibility to stroke.

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine ( n = 4) and sebacyldicholine ( n = 8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in Arabidopsis thaliana

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-beta-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations.

Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.

Synthesis of novel cationic bolaamphiphiles from vernonia oil and their aggregated structures

The present study describes the synthesis of a novel class of vesicle-forming bolaamphiphiles with choline ester head groups. These bolaamphiphiles were derived from vernonia oil, whose main constituent is vernolic acid, a fatty acid with a unique combination of epoxy, carboxy and unsaturated double bonds. A series of bolaamphiphiles containing amido or ester groups within the hydrophobic domain were synthesized from N, N′-alkylenebis (vernolamides) and α,ω-alkylene divernolate ester in a two-stage synthesis comprising opening of the epoxy ring with chloroacetic acid, followed by quaternization with N, N-dimethylaminoethyl acetate to form choline ester head groups. The products were characterized by FT-IR, 1H and 13C NMR, and ESI-MS. Vesicles prepared from these bolaamphiphiles have the potential to serve as a targeted drug delivery systems with selective decapsulation in the presence of the enzyme acetylcholine esterase, resulting in site-specific release of the drug.

Glycoproteomic characterization of butyrylcholinesterase from human plasma

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.

Role of a GDSL lipase‐like protein as sinapine esterase in Brassicaceae

The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase-like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser-Asp-His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase-like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1-O-sinapoyl-beta-glucose, to sinapoyl-l-malate in the seedlings.

α,β-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84–93%. The potency of the amino acid ester derivatives was tested by measuring K i values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH 3O(C 6H 4)CONHC(CHC 6H 5)COOCH 2CH 2N +(CH 3) 3. This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K i values of 10 μM and 0.08 μM, respectively. These K i values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 Å from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.

Limitation of the Ellman method: Cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306 M −1 min −1) than the oximate ion of HI-6 (22 M −1 min −1). However, the rate constants obtained with the Ellman method were 84 M −1 min −1 for K027 and 22 M −1 min −1 for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.

Screening of Antioxidant Compounds During Sprouting of Brassica oleracea L. var. costata DC

The changes in antioxidant compounds of Brassica oleracea L. var. costata DC seeds were monitored during the first twelve days of seedling development. Sprouts were screened at time intervals of two days for phenolic compounds and organic acids. The identified phenolic compounds included esters of sinapic acid with glucose, gentiobiose and kaempferol, as well as sinapoylcholine. The organic acids were oxalic, aconitic, citric, pyruvic, malic, shikimic, and fumaric acids. During germination, a depletion of phenolic compounds was observed, although no qualitative changes were seen. Among individual compounds, kaempferol, choline and glucose esters of sinapic acid showed a marked decrease between days two and six, whereas the changes in gentiobiose esters of sinapic acid were smaller. The total organic acids content increased rapidly during the first four days, with less significant variations thereafter. Malic acid, the major organic acid found in sprouts, greatly contributed to this result though oxalic, pyruvic, and fumaric acids also increased in the same manner. In contrast, aconitic, citric and shikimic acids showed decreases between days two and twelve of germination.

Gene expression signature of parathion-transformed human breast epithelial cells

Environmental substances seem to be involved in the etiology of breast cancers. Many studies have found an association between human cancer and exposure to agricultural pesticides such as the organophosphorous pesticides. Parathion is a cholinesterase inhibitor that induces the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. The primary target of action in insects is the nervous system whereby pesticides inhibit the release of the enzyme acetylcholinesterase at the synaptic junction. Atropine is a parasympatholytic alkaloid used as an antidote to acetylcholinesterase inhibitors. The aim of this study was to determine the effect of parathion and atropine on cell transformation of human breast epithelial cells in vitro. These studies showed that parathion alone was able to induce malignant transformation of an immortalized human breast epithelial cell line, MCF-10F as indicated by increased cell proliferation, anchorage independency and invasive capabilities. There was also an increase in c-kit, Trio, Rho-A, Rac-3, EGFR, Notch-4, Dvl-2, Ezrin, beta catenin and mutant p53 protein expression in the parathion-treated cells. However, atropine significantly inhibited this increase. In a human cell cycle array of 96 genes, 13 of them were altered by parathion treatment. Among the genes affected were the cyclins, such as cyclin D3, the cyclin-dependent kinases (CDKs) such as CDK41 and the minichromosome maintenance deficient (MCM) MCM2 and MCM3. It is suggested that parathion influences human breast epithelial cell transformation and is an initiator factor in the transformation process in breast cancer.

Evaluation of nitrate-substituted pseudocholine esters of aspirin as potential nitro-aspirins

Herein we explore some designs for nitro-aspirins, compounds potentially capable of releasing both aspirin and nitric oxide in vivo. A series of nitrate-bearing alkyl esters of aspirin were prepared based on the choline ester template preferred by human plasma butyrylcholinesterase. The degradation kinetics of the compounds were followed in human plasma solution. All compounds underwent hydrolysis rapidly ( t 1/2 ∼ 1 min) but generating exclusively the corresponding nitro-salicylate. The one exception, an N-propyl, N-nitroxyethyl aminoethanol ester produced 9.2% aspirin in molar terms indicating that the nitro-aspirin objective is probably achievable if due cognisance can be paid to the demands of the activating enzyme. Even at this low level of aspirin release, this compound is the most successful nitro-aspirin reported to date in the key human plasma model.

The miracle at St Alfege's: seventy years on.

The miracle at St Alfege's: seventy years on.

The miracle at St Alfege's: seventy years on

The miracle at St Alfege's: seventy years on

Synthesis of new esters and amides of cephalosporin G

Synthetic methods for cephalosporin G derivatized at the carboxyl were developed. Dinitroglycerine, acetoxymethyl, and choline esters of cephalosporin G and its amide in addition to amides of cephalosporin G with glutamic acid and arginine were synthesized.