Choline Esters Research Articles

Natural Product Research in the Australian Marine Invertebrate Dicathais orbita

The predatory marine gastropod Dicathais orbita has been the subject of a significant amount of biological and chemical research over the past five decades. Natural products research on D. orbita includes the isolation and identification of brominated indoles and choline esters as precursors of Tyrian purple, as well as the synthesis of structural analogues, bioactivity testing, biodistributional and biosynthetic studies. Here I also report on how well these compounds conform to Lipinski’s rule of five for druglikeness and their predicted receptor binding and enzyme inhibitor activity. The composition of mycosporine-like amino acids, fatty acids and sterols has also been described in the egg masses of D. orbita. The combination of bioactive compounds produced by D. orbita is of interest for further studies in chemical ecology, as well as for future nutraceutical development. Biological insights into the life history of this species, as well as ongoing research on the gene expression, microbial symbionts and biosynthetic capabilities, should facilitate sustainable production of the bioactive compounds. Knowledge of the phylogeny of D. orbita provides an excellent platform for novel research into the evolution of brominated secondary metabolites in marine molluscs. The range of polarities in the brominated indoles produced by D. orbita has also provided an effective model system used to develop a new method for biodistributional studies. The well characterized suite of chemical reactions that generate Tyrian purple, coupled with an in depth knowledge of the ecology, anatomy and genetics of D. orbita provide a good foundation for ongoing natural products research.

The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase

Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family.

Suppressive subtractive hybridisation transcriptomics provides a novel insight into the functional role of the hypobranchial gland in a marine mollusc

The hypobranchial gland present in gastropods is an organ whose function is not clearly understood. Involved in mucus production, within members of the family Muricidae it is also the source of the ancient dye Tyrian purple and its bioactive precursors. To gain further insights into hypobranchial gland biology, suppressive subtractive hybridisation was performed on hypobranchial gland and mantle tissue from the marine snail Dicathais orbita creating a differentially expressed cDNA library. 437 clones were randomly sequenced, analysed and annotated and 110 sequences had their functions putatively identified. Importantly this approach identified a putative gene involved in Tyrian purple biosynthesis, an arylsulphatase gene. Confirmation of the upregulation of arylsulphatase in the hypobranchial gland compared to the mantle was demonstrated using quantitative real-time PCR. Other genes identified as playing an important role in the hypobranchial gland were those involved in mucus protein synthesis, choline ester regulation, protein and energy production. This study confirms that the hypobranchial gland is involved in the production of mucus secretion and also identifies it as a site of chemical interaction and biosynthesis. This study lays the foundation for a better understanding of the enzymatic production of Tyrian purple precursors within the gland.

Correlation between butyrylcholinesterase variants and sensitivity to soman toxicity.

Butyrylcholinesterase (BChE) is synthesized in the liver and found in high concentrations in blood plasma, liver, heart, pancreas, vascular endothelium, skin, brain white matter, smooth muscle cells and adipocytes. BChE is a non specific enzyme that hydrolyzes different choline esters (succinylcholine, mivacurium) and many other drugs such as aspirin, cocaine and procaine. The enzyme is also considered as a bioscavenger due to its ability to neutralize the toxic effects of organophosphorus compounds (nervous system fs agents) such as soman. BChE displays several polymorphisms that influence its serum activity; therefore they could determine the individual sensitivity to chemical nerve agents. In this study, we investigated the correlation between BChE variants and the degree of enzyme inhibition and reactivation after soman application on blood samples of 726 individuals. The blood samples of individuals expressing abnormal variants, were more sensitive to soman compared to variants of homozygotes and heterozygotes for U-allele. We found significant differences in the degree of enzyme reactivation between different variants (with and without U-presence).

A Patient with Prolonged Paralysis

A 19-year-old Asian male with no notable medical history presented to the emergency department with a 12-h history of acute abdominal pain. The patient's condition was diagnosed as acute appendicitis, and he underwent an emergent laparoscopic appendectomy. A 1-mg dose of vecuronium followed by 120 mg of succinylcholine was administered to induce paralysis and facilitate endotracheal intubation. The progression of the patient's muscle relaxation was monitored intraoperatively with a train-of-four twitch monitor and was marked by fewer stimuli making it across the neuromuscular junction. In general surgeries, a neuromuscular block down to 2 twitches is adequate for rapid sequence induction. Normally, a dose of 0.5–2 mg succinylcholine per kilogram body weight completely abolishes the muscle response to nerve stimulation. Within 2 to 2.5 min, the neuromuscular junction starts to show signs of recovery, or twitches. In this case, the patient was administered 1.7 mg/kg succinylcholine. After the appendectomy was completed, however, the patient uncharacteristically remained paralyzed for 1.75 h. He showed no muscle twitches, no spontaneous inspiratory efforts, and no protective airway reflexes. He subsequently required sedation and assisted ventilatory support. ### QUESTIONS TO CONSIDER 1. What are the pharmacodynamic properties of succinylcholine? 2. What is the role of butyrylcholinesterase in the pharmacokinetics of succinylcholine? 3. What conditions can cause delayed recovery from succinylcholine administration? 4. What additional testing should be used to further evaluate this patient? Cholinesterases are enzymes that catalyze the hydrolysis of choline esters. Acetylcholinesterase is distributed in the gray matter of the central nervous system, where it terminates synaptic transmission by specifically hydrolyzing the neurotransmitter acetylcholine (1, 2). Butyrylcholinesterase (BChE),4 also known as pseudocholinesterase, is distributed in the white matter of the central nervous system and in the blood. Although it has no known physiological function, BChE is of pharmacologic and toxicologic importance (1). Unlike acetylcholinesterase, BChE is capable of hydrolyzing exogenous …

Analytical approaches to determination of total choline in foods and dietary supplements

Choline is a quaternary amine that is synthesized in the body or consumed through the diet. Choline is critical for cell membrane structure and function and in synthesis of the neurotransmitter acetylcholine. Although the human body produces this micronutrient, dietary supplementation of choline is necessary for good health. The major challenge in the analysis of choline in foods and dietary supplements is in the extraction and/or hydrolysis approach. In many products, choline is present as choline esters, which can be quantitated individually or treated with acid, base, or enzymes in order to release choline ions for analysis. A critical review of approaches based on extraction and quantitation of each choline ester as well as hydrolysis-based methods for determination of total choline in foods and dietary supplements is presented.

Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable surface groups

Stable nano-sized vesicles with a monolayer encapsulating membrane were prepared from novel bolaamphiphiles with choline ester head groups. The head groups were covalently bound to the alkyl chain of the bolaamphiphiles either via the nitrogen atom of the choline moiety, or via the choline ester's methyl group. Both types of bolaamphiphiles competed with acetylthiocholine for binding to acetylcholine esterase (AChE), yet, only the choline ester head groups bound to the alkyl chain via the nitrogen atom of the choline moiety were hydrolyzed by the enzyme. Likewise, only vesicles composed of bolaamphiphiles with head groups that were hydrolyzed by AChE released their encapsulated material upon exposure to the enzyme. Injection of carboxyfluorescein (CF)-loaded vesicles with cleavable choline ester head groups into mice resulted in the accumulation of CF in tissues that express high AChE activity, including the brain. By comparison, when vesicles with choline ester head groups that are not hydrolyzed by AChE were injected into mice, there was no accumulation of CF in tissues that highly express the enzyme. These results imply that bolaamphiphilic vesicles with surface groups that are substrates to enzymes which are highly expressed in target organs may potentially be used as a drug delivery system with controlled site-directed drug release.

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.

A hypothesis to explain the high prevalence of pseudo-cholinesterase deficiency in specific population groups

A hypothesis to explain the high prevalence of pseudo-cholinesterase deficiency in specific population groups

Biochemical and genetic analysis of butyrylcholinesterase (BChE) in a family, due to prolonged neuromuscular blockade after the use of succinylcholine

Butyrylcholinesterase (BChE) is a plasma enzyme that catalyzes the hydrolysis of choline esters, including the muscle-relaxant succinylcholine and mivacurium. Patients who present sustained neuromuscular blockade after using succinylcholine usually carry BChE variants with reduced enzyme activity or an acquired BChE deficiency. We report here the molecular basis of the BCHE gene underlying the slow catabolism of succinylcholine in a patient who underwent endoscopic nasal surgery. We measured the enzyme activity of BChE and extracted genomic DNA in order to study the promoter region and all exons of the BCHE gene of the patient, her parents and siblings. PCR products were sequenced and compared with reference sequences from GenBank. We detected that the patient and one of her brothers have two homozygous mutations: nt1615 GCA > ACA (Ala539Thr), responsible for the K variant, and nt209 GAT > GGT (Asp70Gly), which produces the atypical variant A. Her parents and two of her brothers were found to be heterozygous for the AK allele, and another brother is homozygous for the normal allele. Sequence analysis of exon 1 including 5′UTR showed that the proband and her brother are homozygous for –116GG. The AK/AK genotype is considered the most frequent in hereditary hypocholinesterasemia (44%). This work demonstrates the importance of defining the phenotype and genotype of the BCHE gene in patients who are subjected to neuromuscular block by succinylcholine, because of the risk of prolonged neuromuscular paralysis.

Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Acetylcholine

A catalytic mechanism for the butyrylcholinesterase (BChE)-catalyzed hydrolysis of acetylcholine (ACh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy calculations on both acylation and deacylation of BChE. It has been shown that the acylation with ACh includes two reaction steps, including nucleophilic attack on the carbonyl carbon of ACh and dissociation of choline ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of the substrate and dissociation between the carboxyl carbon of the substrate and the hydroxyl oxygen of the Ser198 side chain. Notably, despite the fact that acetylcholinesterase (AChE) and BChE are very similar enzymes, the acylation of BChE with ACh is rate-determining, which is remarkably different from the AChE-catalyzed hydrolysis of ACh, in which the deacylation is rate-determining. The computational prediction is consistent with available experimental kinetic data. The overall free energy barrier calculated for BChE-catalyzed hydrolysis of ACh is 13.8 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 13.3 kcal/mol.

Effect of Operating Parameters on Oil and Phenolic Extraction Using Supercritical CO 2

Supercritical fluid extraction (SFE) with carbon dioxide was used to extract oil from canola press cake. Different operating conditions, e.g. pressure, temperature, and co-solvent % were investigated to optimize extraction parameters to yield canola meal with <4% oil. The residual oil content in the extracted canola meal reduced to 2.1–2.9% in our experimental trials. Residues of the optimum conditions based on oil yield were compared for the total phenolic content and the main phenolic compounds. Sinapine (the choline ester of sinapic acid) was the major phenolic constituent in both the SFE and n-hexane extracted canola meals and press cakes. n-Hexane extracted residues showed the retention of the highest sinapic acid, sinapine, sinapoyl glucose and total phenolic contents (mg/g) while the SF-extracted residues showed the lowest values for these compounds.

Gastrin response to candidate messengers in intact conscious rats monitored by antrum microdialysis

We monitored gastrin release in response to locally applied candidate messengers in intact conscious rats. Earlier studies have been performed on anaesthetized animals, isolated pieces of antrum, or purified preparations of gastrin cells. In this study we created an experimental situation to resemble physiological conditions, using reverse microdialysis to administer regulatory peptides and amines that might affect gastrin secretion. Microdialysis probes were implanted in the submucosa of the antrum of the rat stomach. Three days later, putative messenger compounds were administered via the probe. Their effects on basal (24 h fast) and omeprazole-stimulated (400 μmol/kg/day, 4 days peroral administration) gastrin release were monitored by continuous measurement (3 h) of gastrin in the perfusate (radioimmunoassay). Fasted rats (low microdialysate gastrin, 2.1 ± 0.1 pmol l − 1 ) were used to study stimulation of gastrin release. Omeprazole-treated rats (high microdialysate gastrin, 95.8 ± 6.7 pmol l − 1 ) were used to study suppression of gastrin release. The following agents raised the concentration of microdialysate gastrin (peak response): gastrin-releasing peptide (GRP) (11-fold increase at a near-maximal dose), carbachol (5-fold increase), serotonin (2-fold increase) and isoprenaline (20-fold increase). Adrenaline and noradrenaline induced transient but powerful elevation (40- and 20-fold increase). Somatostatin, galanin and bradykinin (at near-maximal doses) suppressed omeprazole-stimulated gastrin release (50% decrease). Calcitonin gene-related peptide, ghrelin, gastric inhibitory peptide, motilin, neurotensin, neuromedin U-25, peptide YY and vasoactive intestinal peptide were without effect on gastrin release, as were aspartate, γ-aminobutyric acid, glutamate, glycine, dopamine and histamine. The results support the view that G cells operate under neurocrine/paracrine control. They were stimulated by agents present in enteric neurons (GRP, galanin, choline ester and catechol amines) and in gastric endocrine cells (serotonin). They were inhibited by somatostatin (D cell peptide), galanin (neuropeptide) and by the inflammatory agent bradykinin.

How the cholinesterases got their modern names

The classification of the cholinesterases into ‘true’ and ‘pseudo’ became obsolete when, some 60 years ago, the author and his co-workers showed that both enzymes had a broad specificity and differed mainly in their acyl group specificity. The importance of complementarity between enzyme and substrate was shown by the high rate of hydrolysis of carbon analogues of choline esters and this enabled pioneer studies of the intermolecular forces between the enzymes’ active centres and their substrates to be carried out.

Simple Choline Esters as Potential Anti-Alzheimer Agents

In this manuscript we report an integrated study to develop simple choline esters as cholinergic agents potentially useful against the Alzheimer disease. In previously reported experiments we demonstrated the capability of the pivaloylcholine to exert cholinergic effects into the Central Nervous System, so we decided to explore small variants of choline esters. The knowledge of crystallographic models of the enzymes involved in the hydrolysis of the acetylcholine allowed to consider the steric compatibility of some choline derivatives within their catalytic sites. The purpose of the work was to find out analogues with increased selectivity toward the acetylcholinesterase versus the butyrrylcholinesterase. Theoretical models were compared to enzymatic tests carried out with both enzymes and two different methods. In this screening we have selected two candidates for the in vivo experiments with pre-treated rats. Their electroencephalographic profiles were recorded and averaged before and after the intraperitoneal treatment with two compounds in comparison to the pivaloyl lead ester. The results demonstrated that one of the esters can exert biological effects similar to the parent compound.

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.