Improved Ellman procedure for erythrocyte cholinesterase

Area of Science:

• Biochemistry
• Clinical Chemistry
• Enzymology

Background:

• The standard method for erythrocyte cholinesterase measurement faces interference from hemoglobin.
• High background absorbance at 410 nm due to the hemoglobin Soret band limits assay precision.
• This limitation restricts the assay to specialized laboratories with high-end spectrophotometers.

Purpose of the Study:

• To develop a more precise and accessible assay for erythrocyte cholinesterase.
• To overcome the limitations of the existing method caused by hemoglobin interference.
• To enable routine laboratory measurement of erythrocyte cholinesterase for organophosphate exposure assessment.

Main Methods:

• Modified the Dietz et al. procedure by replacing quinidine sulfate with benzethonium chloride (Hyamine 1622).
• Utilized spectrophotometry to measure absorbance at 440 nm, shifting the hemoglobin interference.
• Investigated the effect of detergent micelle interactions on 5-thionitrobenzoate absorbance.

Main Results:

• Benzethonium chloride shifted the hemoglobin band to 405 nm and reduced its intensity.
• Detergent micelle interactions shifted 5-thionitrobenzoate absorbance peak to 435 nm.
• Reduced background absorbance to approximately 0.4 A, doubling assay precision.

Conclusions:

• The modified assay provides a twice-improved precision compared to the original method.
• The assay is suitable for routine clinical laboratories with moderate spectrophotometers.
• Facilitates erythrocyte cholinesterase measurement to complement plasma cholinesterase in organophosphate exposure studies.