Cholinesterase Activity In Human Serum Research Articles

A 96-well wax printed Prussian Blue paper for the visual determination of cholinesterase activity in human serum

In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2–15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method.

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.

Effect of sorbic acid and some other food preservatives on human serum cholinesterase activity

The effect of some selected food preservatives on serum cholinesterase was determined. It was found that potassium metabisulphite, methyl parabene and propyl parabene caused a significant increase while sacharine, benzoic acid, salicylic acid, copper carbonate and sorbic acid caused a significant decrease in cholinesterase activity. The behavior of serum cholinesterase activity in response to a gradual change in the incubation time as well as the sorbic acid concentration was also studied. The graphical and statistical analysis of the data showed an exponential decrease in cholinesterase activity with an increase in the concentration of sorbic acid. The cholinesterase activity was also found to be decreased exponentially with an increase in the incubation time. The regression analysis showed a good agreement between the experimental and calculated values. Keywords: Sorbic acid, enzyme activity, cholinesterase, inhibition sensitivity

Electrochemical Determination of Cholinesterase Activity and Indication of Its Inhibitors Using A Thick-Film Metallized Platinum Electrode

Abstract A thick-film metallized platinum electrode operated in the voltammetric mode is used to determine the cholinesterase activity in human serum and erythocytes. Inhibitors of the enzyme can also be quanitified by the electrode with cholinesterase either immobilized directly on the electrode surface or dissolved in the test medium.

Cholinesterase phenotyping: clinical aspects and laboratory applications.

The measurement of cholinesterase activity in human serum is an important investigation, especially in patients suspected of poisoning with organophosphate insecticides, or those experiencing prolonged paralysis following treatment with the short acting muscle relaxant succinylcholine. Appropriate clinical management of affected individuals can only be instituted if enzyme activity is measured using a method capable of clear interpretation and phenotypic ascription of cholinesterase, ascertained by use of selected enzyme inhibitors, is reliable. This review considers factors which lead to significant cholinesterase abnormalities and advises on the techniques most appropriate to their investigation. It is concluded that no one method is suitable both for measurement of activity and determination of genotype. The use of propionyl thiocholine is recommended for the first procedure and benzoyl choline for the latter. Those laboratories in which a request for the assessment of cholinesterase status is an unusual event should make greater use of assistance from colleagues whose experience with the problem is greater.

The effect of storage at different temperatures on cholinesterase activity in human serum.

The effect of storage on the catalytic concentration of cholinesterase and on the reference values of cholinesterase in human serum were studied. When serum was stored at room temperature (20 degrees C), at 4 degrees C and at - 20 degrees C (one year) there was no change in catalytic activity. Even after nine times freezing and thawing (over nine weeks) the catalytic activity was unaffected. The average reference value was significantly higher for males than for females (3.11 +/- 0.57 vs 2.50 +/- 0.43 kU/l).

A procedure for the kinetic colorimetric determination of serum cholinesterase activity.

A choline oxidase-peroxidase coupled enzyme procedures is proposed for the determination of cholinesterase activity in human serum. This system is not only kinetic and colorimetric but is also relatively quick and simple to perform. The initial comparisons suggest that this method correlates well with a commonly used propionylthiocholine-dinitrobis-(nitro-benzoic acid) technique. Large amounts of bilirubin in the sample appear to have only minor deleterious effects on the assay. Since there are only two reagents that may be premixed, the procedure appears to be amenable to automation. The use of a mixture of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate and 4-aminoantipyrene in the peroxidase catalyzed indicator reaction provides for a marked increase in sensitivity over previously reported 4-aminoantipyrene-phenol systems. This augmented sensitivity provides for a relatively large reagent to sample ratio. In addition, the reagents lend themselves toward lyophilization or "dry-fill".

Serum cholinesterase in the mothers of neural tube defect progeny

Human Serum Cholinesterase activity and polymorphism at its two gene loci, CHE1 and CHE2, were compared in maternal serum from neural tube defect pregnancies, normal pregnancies and a non-pregnant control group. Variants at the CHE1 locus were identified by dibucaine, fluoride and R02 0683 inhibition. The CHE2 phenotype was demonstrated by DISC polyacrylamide gel electrophoresis. Total HSChE activity in the pregnant groups was slightly less than in the control group but there was no difference in activity between the affected and the normal pregnancies. Three variants were identified. All were found in the non-affected pregnant group. One variant at CHE1 was identified, an I phenotype, and two C5+ phenotypes, the CHE2 variant. No obvious relationships were found between HSChE activity or a particular genetic variant and NTD progeny.

Human serum pseudocholinesterases: molecular weight estimation of a subunit structure.

Molecular weight determinations by gel filtration on Sephadex G-200 were performed on fractions of human serum cholinesterases as obtained after ultrafiltration through a semipermeable membrane. The molecular weight of the fraction filtering through a membrane with molecular weight exclusion limits of 100 000 was estimated to be 86 000, whereas that of the fraction retained by the filter was higher than 300 000, approximating the value of 348 000 previously reported in the literature. Both fractions were shown to be interconvertible under the experimental conditions used. These results provide further evidence in favor of the existence of an enzymatically active subunit structure of cholinesterases and suggest that subunits combine into tetramers to form the major component of the cholinesterase activity in human serum.

Studies on Inhibition of Human Serum Cholinesterase by Lysergic Acid and Clavine Alkaloids

The inhibition of human serum cholinesterase by lysergic acid diethylamide (LSD), lysergic acid amide (LAA), isolysergic acid amide (iso-LAA), lysergol and elymoclavine was investigated, acetylcholine was used as a substrate. The enzyme activity was assayed by colorimetric determination of residual acetylcholine in the incubation mixture. The 50 percent inhibition of the cholinesterase activity in human serum was observed with 6×10-4μM LSD, 3×10-2μM elymoclavine and 1×10-1μM lysergol. Only 20 per cent inhibition occurred with concentration of LAA or iso-LAA up to 2×10-1μM. These alkaloids were the competitive inhibitors of the enzyme to acetylcholine.