Choline Esters Research Articles

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

The Acetylcholine Therapy in the Treatment of Schizophrenia - The Experience of Mario Fiamberti in the Hospital of Varese (1937)

In the first half of the 20th century, in most European countries, it was thought that cholinesterase and other drugs that counteract acetylcholine should reduce the manifestations of schizophrenia. In 1937, Fiamberti (1894-1970) introduced the transorbital method of lobotomy which established the use of acetylcholine shock treatment for curing the disturbances of schizophrenia. Accepting the idea that the psychic alterations of schizophrenia were caused by a pathological interruption of nerve conduction at a presumably cortical level, Fiamberti thought he could apply this to the clinical field using the properties of acetylcholine, an acetic ester of choline. Here, we examined, in detail, the contribution of Mario Fiamberti to acetylcholine therapy.

Identification of Sinapine-Derived Choline from a Rapeseed Diet as a Source of Serum Trimethylamine N-Oxide in Pigs

Choline and its metabolites have diverse and important functions in many physiological processes, especially for anabolic metabolism in growth and reproduction. Besides endogenous biosynthesis and direct choline supplement, choline esters in the diet are another source of choline in the body. Phenolic choline esters are a group of unique dietary choline esters rich in the seeds of Brassicaceae plants, among which sinapine is a choline ester of sinapic acid abundant in rapeseed. In this study, 40 nursery pigs were fed with rapeseed-derived feed ingredients (RSF) or soybean meal for 3 weeks (20 pigs/diet). The metabolic fate of sinapine-derived choline in RSF was examined by comparing the distribution of choline and its metabolites in digesta, liver, and serum samples by liquid chromatography-mass spectrometry analysis. The results showed that choline was released from extensive hydrolysis of sinapine in the small intestine. However, sinapine-derived choline did not increase the levels of choline and its major metabolites, including betaine, phosphocholine, and glycerophosphocholine, in the liver and serum. Instead, RSF feeding increased trimethylamine (TMA), the microbial metabolite of choline, in the large intestine and further increased trimethylamine N-oxide (TMAO), the oxidation metabolite of TMA, in the liver and serum. Overall, these results suggested that sinapine-derived choline from rapeseed feeding had limited influences on the post-absorption choline pool as a result of its low bioavailability but may serve as a major source of TMAO through microbial metabolism in nursery pigs. Improving the bioavailability of sinapine-derived choline might have the potential to modify the nutritional values and functionalities of rapeseed meal in swine feeding.

In situ oxidation of canola meal sinapic acid by horseradish peroxidase (type II) and tyrosinase.

The enzymatic oxidation of sinapic acid catalyzed by horseradish peroxidase (HRP) or tyrosinase was investigated using model systems, which contained the pure compound or canola meal. Spectrophotometric scanning of pure sinapic acid solution in the presence of HRP (0.2 U) or tyrosinase (40.3 U) showed continuous decreases in absorbance at 304nm over a period of 90 and 60min, respectively. HPLC analyses of enzymatic end products, obtained by the catalysis with HRP or tyrosinase, indicated the presence of two main compounds (1 and 2). After alkaline hydrolysis of canola meal, sinapic acid that was released from sinapine was also converted to compounds 1 and 2 by HRP or tyrosinase. Enzyme reaction kinetics results indicate that the catalytic efficiency (CE=0.538), reaction velocity (Vmax =5.67 ∆A/h), and Michaelis-Menten constant (Km =926.64µM) of HRP are significantly higher than those of tyrosinase (CE=0.041, Vmax =0.41 ∆A/h, Km =173.03µM) at 50-250μM pure sinapic acid concentrations. PRACTICAL APPLICATIONS: Canola meal contains a large amount of sinapine, which is the choline ester of sinapic acid, a strong antioxidant compound. However, the oxidation or decarboxylation products of sinapic acid could add value by increasing the level of electron-dense carboxylic and carbonyl compounds. In this study, enzymatic treatment of alkaline-hydrolyzed canola meal with horseradish peroxidase (HRP) and tyrosinase was investigated and shown to be suitable for converting sinapic acid into oxidized compounds. Therefore, the enzymatic treatment is a potential application for value-added processing of canola meal.

First evidence of cholinesterase-like activity in Basidiomycota.

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

Unexpected irregular structures of poly(itaconic acid) prepared in Deep Eutectic Solvents

Poly(itaconic acid) (PIA) was synthesized by persulfate initiated free-radical polymerization of ternary Deep Eutectic Solvents (DES) containing equimolar amounts of itaconic acid (IA) as hydrogen-bond donor, quaternary ammonium salt, namely choline chloride (CC) or tetraethylammonium chloride (TEAC) as hydrogen-bonds acceptors, and water as the initiator solvent. The syntheses were carried out at 65 °C for 2 h, and polymers produced were purified by dialysis and by treatment on strong cation exchange resin. The microstructures of the polymers were analysed using 1H, 13C and 1H–13C HSQC NMR spectroscopy. The unexpected methylene and methine groups resulting from decarboxylation and/or branching as well as choline ester moieties were detected as irregular structures in PIA synthesised in IA-TEAC and IA-CC DES, respectively.

Ionic liquids with methotrexate moieties as a potential anticancer prodrug: Synthesis, characterization and solubility evaluation

The technological utility of active pharmaceutical ingredients (APIs) is enormously improved when they are converted into ionic liquids (ILs). API-ILs possess better aqueous solubility and thermal stability than that of solid-state salt or crystalline drugs. However, many such API-ILs are not biocompatible or biodegradable. In the current study, we synthesized a series of IL-APIs using methotrexate (MTX), a potential anticancer prodrug, and biocompatible IL-forming cations (choline and amino acid esters). The MTX-IL moieties were characterized through 1H NMR, FTIR, p-XRD, DSC and thermogravimetric analysis. The solubility of the MTX-ILs was evaluated in simulated body fluids (phosphate-buffered saline, simulated gastric, and simulated intestinal fluids). An assessment of the in vitro antitumor activity of the MTX-ILs in a mammalian cell line (HeLa cells) was used to evaluate their cytotoxicity. The MTX-ILs showed aqueous solubility at least 5000 times higher than that of free MTX and two orders of magnitude higher compared with that of a sodium salt of MTX in both water and simulated body fluids. Importantly, a proline ethyl ester MTX prodrug showed similar solubility as the MTX sodium salt but it provided improved in vitro antitumor activity. These results clearly suggest that the newly synthesized API-ILs represent promising potential drug formulations.

Role of serum cholinesterase in patients treated with salvage radical prostatectomy

BackgroundSerum cholinesterase (ChE) a serine hydrolase that catalyses the hydrolysis of esters of choline, is involved in cellular proliferation and differentiation, therefore affecting carcinogenesis. The aim of this study was to understand the prognostic role of preoperative serum ChE in patients with radiation-recurrent prostate cancer (CaP) treated with salvage radical prostatectomy (SRP). Material and methodsThis retrospective study included 214 patients with radiation-recurrent CaP treated with SRP from January 2007 to December 2015 at 5 academic centers. Patients were considered with abnormal/decreased ChE levels if <5 kU/l. Biochemical recurrence-free and metastases-free (MFS) survival analyses were performed. ResultsMedian serum ChE level was 6.9 (interquartile range) 6–7.7) kU/l. Serum ChE level (<5 kU/l) was decreased in 25 (11.7%) patients. Decreased serum ChE level was associated with lower body mass index (P = 0.006) and metastasis to lymph nodes (P = 0.004). In multivariable analysis, continuous ChE was an independent predictor of MFS (hazard ratio [HR] 0.48, confidence interval [CI] 0.33–0.71, P < 0.001), overall survival (HR 0.68, CI 0.48–0.96, P = 0.03) and cancer-specific survival (HR 0.41, CI 0.2–0.84, P = 0.01). Serum ChE improved the C-index (by 2.54%) to 87.8% for prediction of overall survival and (by 3%) to 92% for prediction of MFS. ConclusionPreoperative serum ChE is associated with the development of metastasis in patients with radiation-recurrent CaP who underwent SRP. The biological underpinning of this association with the biological and clinical aggressiveness of CaP needs to be further elucidated.

Robustness of the non-neuronal cholinergic system in rat large intestine against luminal challenges.

Acetylcholine and atypical esters of choline such as propionyl- and butyrylcholine are produced by the colonic epithelium and are released when epithelial receptors for short-chain fatty acids (SCFA) are stimulated by propionate. It is assumed that the SCFA used by the choline acetyltransferase (ChAT), the central enzyme for the production of these choline esters, originate from the colonic lumen, where they are synthesized during the bacterial fermentation of carbohydrates. Therefore, it seemed to be of interest to study whether the non-neuronal cholinergic system in the colonic epithelium is affected by maneuvers intended to stimulate or to inhibit colonic fermentation by changing the intestinal microbiota. In two series of experiments, rats were either fed with a high fiber diet (15.5% (w/v) crude fibers in comparison to 4.6% (w/w) in the control diet) or treated orally with the antibiotic vancomycin. High fiber diet induced an unexpected decrease in the luminal concentration of SCFA in the colon, but an increase in the caecum, suggesting an upregulation of colonic SCFA absorption, whereas vancomycin treatment resulted in the expected strong reduction of SCFA concentration in colon and caecum. MALDI MS analysis revealed a decrease in the colonic content of propionylcholine by high fiber diet and by vancomycin. High fiber diet caused a significant downregulation of ChAT expression on protein and mRNA level. Despite a modest increase in tissue conductance during the high fiber diet, main barrier and transport properties of the epithelium such as basal short-circuit current (Isc), the flux of the paracellularly transported marker, fluorescein, or the Isc induced by epithelial acetylcholine release evoked by propionate remained unaltered. These results suggest a remarkable stability of the non-neuronal cholinergic system in colonic epithelium against changes in the luminal environment underlying its biological importance for intestinal homeostasis.

An evaluation of neonicotinoids' potential to inhibit human cholinesterases: Protein–ligand docking and interaction profiling studies

Many so-called neuroactive insecticides target invertebrate neurotransmitter systems, including the cholinergic system. With their relatively low toxicity to vertebrates, neonicotinoids represent a new class of neuroactive insecticides that bind to nicotinic receptors for acetylcholine in the insect central nervous system and result in paralysis and eventual death due to receptor overstimulation. On the understanding that, today, cholinesterase inhibitors are used to obtain the symptomatic relief of Alzheimer disease (AD), the aforementioned direct cholinomimetic action of neonicotinoids could, perhaps, confer anti-AD drug-like attributes to these compounds. It is shown here, using protein–ligand docking and interaction profiling, that neonicotinoids penetrate deep into the active-site gorge of both acetylcholinesterase and butyrylcholinesterase and that they form relatively strong noncovalent bonds with multiple critical residues that normally bind/hydrolyze choline esters. With their gorge-spanning shape and dual-binding specificity, neonicotinoids (first-generation compounds in particular) represent promising leads for the development of reversible, mixed-type cholinesterase inhibitors in the fight against AD.

Evaluation of the inhibitory effect of abamectin on mammalian butyrylcholinesterase: Enzyme kinetic and molecular docking studies

ABSTRACTAbamectin, a blend of the natural avermectins B1a and B1b, is a widely-used insecticide/miticide with relatively low toxicity to mammals. Exposure to high doses of it, however, leads to cholinergic-like neurotoxic effects. Butyrylcholinesterase, which is best known for its abundant presence in plasma, is a serine hydrolase loosely coupled with the cholinergic system. It protects and supports the neurotransmitter function of its sister enzyme acetylcholinesterase. Here, using experimental and computational studies, we provide evidence demonstrating that abamectin is a potent (IC50 = 10.6 μM; Ki = 2.26 ± 0.35 μM) inhibitor of horse serum butyrylcholinesterase and that it interacts with the enzyme in a reversible, competitive manner predictively to block the mouth of the active-site gorge of the enzyme and to bind to several critical residues that normally bind/hydrolyze choline esters.

Assay of serum cholinesterase activity by an amperometric biosensor based on a co-crosslinked choline oxidase/overoxidized polypyrrole bilayer.

Based on choline oxidase immobilized by co-crosslinking on an overoxidised polypyrrole modified platinum electrode, a novel electrochemical assay for cholinesterase activity in human serum was developed. The assay was performed by adding an aliquot of cholinesterase standard solution or serum sample to phosphate buffer containing choline or thiocholine ester and measuring the oxidation current of hydrogen peroxide at the rotating modified electrode polarized at +0.7 V vs. SCE. The influence of some experimental parameters such as pH of the assay, mass transport at the electrode, type and concentration of the cholinesterase substrate was studied and optimised. Reversible inhibition of choline oxidase from cholinesterase substrates was evidenced for the first time, which increases in the order of acetylcholine, butyrylcholine and s-butyrylthiocholine. Wide linear range, fast response time and appreciable long-term stability were assured for both acethyl- and butyrylcholinesterase assays. On allowing the polypyrrole layer to efficiently remove interferences from the electroactive compounds in the sample, the present method revealed to be suitable for the detection of butyrylcholinesterase in human serum at activities as low as 0.5 U L-1. The validation with a reference spectrophotometric method showed no significant differences when human serum samples were analysed.

Prolonged neuromuscular blockade in a middle-eastern female patient homozygous for atypical plasma cholinesterase.

Sir, Pseudocholinesterase (PchE, also known as “butyrylcholinesterase”) is a serine enzyme capable to hydrolyze choline esters found in succinylcholine, mivacurium, and ester local anesthetics.[1] There are several genetic variants of PchE deficiency. The most important variants are the atypical or dibucaine-resistant, the fluoride-resistant, the silent variant, and the K-variant.[2] The neuromuscular prolongation after succinylcholine in patients with heterozygous or homozygous atypical PchE is variable. Several confounding factors may play a significant role in this prolongation of effect, such as male gender, concomitant use of metoclopramide or etomidate, liver disease, and pregnancy – this latter has been found to decrease PchE activity as early as the 10th week of gestation.[3] The authors present the case of a middle-eastern patient undergoing dilation and curettage who developed prolonged muscle paralysis after single administration of succinylcholine and required postoperative mechanical ventilation. She had no documented allergies and denied prior surgeries. Given that the patient was not appropriately fasted, and due to the emergent nature of the procedure, the decision was to proceed with rapid sequence induction and intubation with propofol, fentanyl, and succinylcholine (1 mg/kg) to facilitate endotracheal intubation, which was uneventful. No further neuromuscular blocker was administered. Ten minutes after the procedure finalized (approximately 45 min after induction), the patient had a persistent neuromuscular blockade, evidenced by train-of-four of 0 and the absence of spontaneous respirations. The decision was made to deepen the anesthesia with intravenous propofol and midazolam and to take the patient sedated and intubated to the postanesthesia care unit. The patient was extubated approximately 4 h after induction in the postanesthesia care unit, after the patient showed adequate muscle strength (head lift >10 s), following verbal commands and tidal volume >5 cc/kg (375 cc/breath) with minimal pressure support from the ventilator. The patient was discharged home on the postoperative day one; on further questioning, she had no recall about the episode. Dibucaine inhibition test was performed using preoperative and postoperative blood samples with results of 20% and 19%, respectively (reference range >80%). This case highlights the importance that besides genetic factors, ethnicity also plays a significant role in the development of this clinical condition, especially in the patient described in our case. Hosseini et al. studied the differences in PchE activity between the Irish population and the Iranian population which corroborated the findings from Szeinberg et al. The differences were based on the dibucaine inhibition test and fluoride test. They concluded that the Iranian population has intrinsically lower levels of PchE activity compared with the Irish population (this latter has higher dibucaine and fluoride numbers). In addition, no difference was observed regarding PchE activity between Iranian Jewish and Muslims.[45] We emphasize the significance of providing the most appropriate management to prevent detrimental outcomes for these patients (e.g., the use of neuromuscular monitoring, mechanical ventilation to avoid respiratory distress, and sedation to avoid awareness) as well as provide adequate education and communication of the diagnosis of PchE deficiency to the patient, family and the medical team in order to prevent exposure to succinylcholine in the future surgical procedures. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Volatile and bioactive compounds in opercula from Muricidae molluscs supports their use in ceremonial incense and traditional medicines

Muricidae molluscs are the source of a valuable purple dye that was traded as a luxury item in the Mediterranean region and by the late Byzantine was reserved for royalty and priests. Less well known is the use of muricid opercula in sacred incense and traditional medicines, although they are still used as rare ingredients today. This study provides the first chemical assessment of opercula from Muricidae, based on several traditional preparation procedures. Chemical analysis of opercula smoke revealed aromatic phenols, which act as fragrance stabilisers and produce a “medicinal” odour. Analysis of lipid extracts revealed pharmaceutically active compounds, including brominated indoles, choline esters and adenosine, consistent with their traditional medical applications. Depending on the preparation procedures, toxic pyridine was also detected. ICP-MS analysis of muricid opercula shows the presence of essential macro and microelements, as well as metals, some of which exceed the recommended safe levels for human use. Nevertheless, these findings support the Muricidae as an historically important marine resource, providing Biblical dyes, medicines and perfume. The opercula contains biologically active compounds and produces smoke containing volatile scent compounds, consistent with their identification as the most likely source of onycha, a controversial ingredient in sacred incense.

Pseudocholinesterase, aspartate transaminase, alanine transaminase as markers for cervical cancer: a study in a tertiary care hospital

Background: Cervical cancer is a malignant neoplasm arising from cells originating in the cervix, almost always caused by human papillomavirus (HPV). Butyrylcholinesterase is a non-specific cholinesterase enzyme that hydrolyses different choline esters. PCHE levels were lower than the normal at all the stages of cancer cervix. The present study was attempted to find out the role of some of the biochemical markers like serum pseudocholinesterase (PCHE), serum aspartate transaminase (AST), and serum alanine transaminase (ALT), in malignancy of the uterine cervix.Methods: 60 patients aged between 30 -70 years who were having cervical cancer were included into the study and 30 healthy patients within the same age group were included as controls. 5ml of venous blood was collected from all the study participants under aseptic conditions in a plain tube. Serum Pseudocholinesterase, Serum Aspartate transaminase and Serum Alanine transaminase was estimated for all the patients in both the groups.Results: The mean value of pseudocholinesterase in cervical cancer patients was lower in the cases when compared to controls and it was statistically significant (P value 0.0005), while the mean value of serum AST and ALT were higher in cases when compared to controls and it were also statistically significant. When compared with Pearson’s coefficient, the serum PChE had a significant negative correlation with AST and ALT, while the serum AST had a significant positive correlation with ALT and a negative correlation with PChE.Conclusions: Among the women with cervical cancer, it was observed that the enzyme activity of PChE was lower than normal, while that of SGPT and SGOT were higher. PCHE, AST, and ALT can be used as tumor markers in the management of malignancy of the uterine cervix.

1H NMR-based Investigation of Metabolic Response to Electro-Acupuncture Stimulation

Acupuncture is a traditional Chinese medicine therapy that has been found useful for treating various diseases. The treatments involve the insertion of fine needles at acupoints along specific meridians (meridian specificity). This study aims to investigate the metabolic basis of meridian specificity using proton nuclear magnetic resonance (1H NMR)-based metabolomics. Electro-acupuncture (EA) stimulations were performed at acupoints of either Stomach Meridian of Foot-Yangming (SMFY) or Gallbladder Meridian of Foot-Shaoyang (GMFS) in healthy male Sprague Dawley (SD) rats. 1H-NMR spectra datasets of serum, urine, cortex, and stomach tissue extracts from the rats were analysed by multivariate statistical analysis to investigate metabolic perturbations due to EA treatments at different meridians. EA treatment on either the SMFY or GMFS acupoints induced significant variations in 31 metabolites, e.g., amino acids, organic acids, choline esters and glucose. Moreover, a few meridian-specific metabolic changes were found for EA stimulations on the SMFY or GMFS acupoints. Our study demonstrated significant metabolic differences in response to EA stimulations on acupoints of SMFY and GMFS meridians. These results validate the hypothesis that meridian specificity in acupuncture is detectable in the metabolome and demonstrate the feasibility and effectiveness of a metabolomics approach in understanding the mechanism of acupuncture.

A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.

Pseudocholinesterase Deficiency in an Indian Community

Background: India, the seventh largest country, 3rd largest standing army and one of the oldest civilizations in the world, with a literacy rate of 74.04%. Indian population was a mixture of 46,73,034 categories of different castes and sub-caste each related to a specific occupation. Arya Vysya is a Hindu Indian caste with a total population of 22,952,000. Methods: Pseudocholinesterase is a glycoprotein enzyme with complex molecular structure synthesized in the liver and specifically hydrolyzes certain exogenous cholinesters. Pseudocholinesterase deficiency is a genetic enzyme abnormality or acquired alteration in the metabolism of choline esters in this patient may experience a paralysis of the respiratory muscles, to overcome this, required more time mechanically-assisted breathing. The presence of the genetic defect is not realized until one is exposed to succinylcholine or mivacurium. Results: Arya Vysya community people of India having deficiency of pseudocholinesterase is the most affected people than any other with homozygous mutation incidence rate of 2-4%. Very recent studies in 2016 reveal that malnutrition-induced pseudocholinesterase deficiency is also a possible etiology. Conclusion: It was suggested that the Arya Vysya Community people with a history of pseudocholinesterase deficiency should tell his or her doctor if when they undergo anesthesia for surgery to avoid potentially serious unwanted adverse effects. Key words: India, Arya Vysya, Pseudocholinestase, Pseudocholinestrase deficiency, Choline esters, Acholest test paper.

Extraction and Quantification of Bioactive Tyrian Purple Precursors: A Comparative and Validation Study from the Hypobranchial Gland of a Muricid Dicathais orbita.

Muricidae are marine molluscs known for the production of Tyrian purple and bioactive precursor compounds. A validation study for the extraction and analysis of secondary metabolites found in the hypobranchial gland of the muricid Dicathais orbita is reported, using high performance liquid chromatography–mass spectrometry (HPLC-MS) with diode array detector (DAD). Quantification of the dominant secondary metabolites from D. orbita is described, followed by a comparison of solvent extraction procedures and stability studies. The intra- and inter-day relative standard deviation (RSD) for tyrindoxyl sulphate was 0.46% and 0.17%, respectively. The quantification was linear for standards murexine, 6-bromoisatin, and tyrindoxyl sulphate. The limits of detection were 0.03, 0.004, and 0.07 mg/mL, respectively, and the limits of quantification were 0.09, 0.01, and 0.22 mg/mL, respectively. The results showed that alcoholic solvents were better for extracting choline ester and indoxyl sulphate ultimate precursors, while chloroform was more suitable for the extraction of the intermediate precursors. Multivariate analysis revealed significant differences in extract composition according to the solvent used. Stability testing showed an increase of the oxidative compounds 6-bromoisatin and putative tyrindoxyl S-oxide sulphate in the ethanol extracts while more degradation products were seen in the chloroform extracts after months of cold storage. The validated method was found to be simple, reproducible, precise, and suitable for quantification of the secondary metabolites of muricid molluscs for dye precursor and nutraceutical quality control, as well as applications in marine chemical ecology.

The frequency of incidental findings in expanded carrier screening

To examine the frequency of incidental findings in expanded carrier screening - diseases where carriers may also have clinical health implications (CHI). Expanded carrier screening (ECS) identifies couples at risk for transmitting a genetic condition to their offspring. Patients may be counseled during the informed consent process that carriers of recessive conditions usually do not experience symptoms. Yet, there are several conditions on ECS panels where carriers may have CHI. We analyzed Counsyl laboratory’s experience of over 346,790 expanded carrier screens to calculate this frequency. Individuals were tested for carrier status in up to 108 genes by either targeted genotyping (TG) of up to 417 sites or next-generation sequencing (NGS) of the exons and selected introns of the chosen genes. Carrier frequencies for each condition were calculated as the higher of TG or NGS frequencies. Five autosomal recessive diseases were identified for their association with CHI: Nijmegen breakage syndrome (NBN) for cancer risk; ataxia-telangiectasia (ATM) for breast cancer risk; pseudocholinesterase deficiency (BCHE) for increased risk for prolonged period of breathing paralysis after receiving choline ester drugs; hereditary thymine-uraciluria (DPYD) for risk for toxicity following 5-fluorouracil chemotherapy treatment; and factor XI deficiency (F11) for risk for bleeding problems. Carrier frequencies for each condition were tabulated per ethnic group, weighted by US Census Data 2010, and summated to represent the ethnic distribution in USA. 346,790 patients indicating “routine carrier testing” as the reason for testing were selected from a total population of 430,584 (all indications). From this subset, 308,668 (89%) received TG and 38,122 (11%) received NGS test. The carrier frequencies for each condition were 1/280 (ATM), 1/24 (BCHE), 1/109 (DPYD), 1/150 (F11), and 1/560 (NBN). This summates to an overall frequency of 6.2% for CHI on ECS. BCHE accounted for 67% of the frequency of CHI and is more common in individuals of Ashkenazi Jewish (4.5%) and European descent (4.9%) than those of African (1.0%) or Asian descent (1.2%). 6.2% of individuals (1 in 16) screened for these diseases will have an incidental finding - where carriers identified may experience CHI. Pre-test counseling, informed consent, reporting, and post-test counseling should educate patients and providers regarding this possibility.