Choline is an essential nutrient, commonly grouped with the B complex vitamins, that plays key roles in epigenetic regulation. Alongside its pleiotropic role in cellular homeostasis, choline influences many cellular processes, including lipid metabolism, membrane fluidity and mitochondrial bioenergetics. Choline is a precursor for the synthesis of acetylcholine, a critical neurotransmitter in both the peripheral and central nervous systems. Dysregulation of acetylcholine homeostasis is associated with various diseases, such as multiple sclerosis, Alzheimer’s disease, and myasthenia gravis.

Choline/Acetylcholine Assay Kit provides a simple, reproducible, and sensitive tool of measuring Choline/Acetylcholine concentration in plasma, serum, cell lysates, saliva, urine, and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. Initially, the acetylcholine converter enzyme catalyzes the conversion of acetylcholine to choline. Then choline is further converted to betaine and hydrogen peroxide by the choline detection enzyme. Peroxidase then catalyzes the reaction of hydrogen peroxide with the Colorimetric Probe to form a pink colored product. The optical density measured at 570 nm is directly proportional to the concentration of Choline/Acetylcholine present in the sample.

• Microplate reader capable of measuring OD 570 nm or Ex/Em = 530/590 nm
• 96 well black plate for fluorometric assay
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C
• 5 mL tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted Choline Standards or samples to the 96-well microtiter plate.
2. For measuring Total (Choline + Acetylcholine) in the sample: initiate the reaction by adding 50 µL Acetylcholine Working Solution to acetylcholine sample wells. Carefully shake the plate for a few seconds to mix.
3. For measuring Choline in the sample: initiate the reaction by adding 50 µL Choline Working Solution to choline sample wells. Carefully shake the plate for a few seconds to mix.
4. For measuring Acetylcholine in the sample: for each sample, two pairs of wells are needed, one for Choline measurement, the other for Acetylcholine measurement. Initiate the reaction by adding 50 µL Acetylcholine Working Solution to acetylcholine sample well. Add 50 µL Choline Working Solution to Choline sample well. Carefully shake the plate for a few seconds to mix.
5. Incubate the plate for 30 minutes at 37°C with mixing in the dark.
6. Measure the absorbance at 570 nm using a plate reader.