We have not tested this assay with blood samples. In the literature search I found the following reference:
Plasma levels in young women: 456.1 +/- 53.1 (SEM) pg/mL which equals 3.1 nM. (Kawashima et al. Neuroscience Letters 80:(3), 339-342; 1987.) If this is indeed a representative study, it would mean that our assay is not sensitive enough to detect acetylcholine in plasma or serum samples. Furthermore, plasma levels of choline are much higher (~10 µM) and will thus have to be removed before assaying.
The assay is based on the enzymatic degradation of acetylcholine to choline and acetate and the subsequent detection of choline.
We have not tested acetylcholine assay specifically with brain tissue samples, but believe it should work. Typical concentrations of choline and acetylcholine in brain tissue are 10-70 nmoles/g. So if the tissue sample is suspended in ice-cold PBS at 500 mg/mL, we should expect 5-35 µM choline or acetylcholine in the supernatant.
Suggested protocol: weigh brain tissue (approx. 50 mg) and add 100 µL ice-cold PBS, lyze tissue by homogenization or sonication. Centrifuge at 14,000 rpm for 10 min at 4°C. Carefully transfer the supernatant to a clean tube and perform the assays for choline and acetylcholine.
Due to the low concentration range, the fluorescence assay procedure is recommended.
Ahmad, A., Ramasamy, K., Jaafar, S. M., Majeed, A. B. A., & Mani, V. (2014). Total isoflavones from soybean and tempeh reversed scopolamine-induced amnesia, improved cholinergic activities and reduced neuroinflammation in brain. Food and Chemical Toxicology 65: 120-128. Assay: Acetylcholine in rat brain.
Baitharu, I., Jain, V., Deep, S. N., Hota, K. B., Hota, S. K., Prasad, D., & Ilavazhagan, G. (2013). Withania somnifera root extract ameliorates hypobaric hypoxia induced memory impairment in rats. Journal of ethnopharmacology 145(2): 431-441. Assay: Acetylcholine in rat hippocampus.
Chen L et al (2012). Acute exposure to DE-71: Effects on locomotor behavior and developmental neurotoxicity in zebrafish larvae. Environ Toxicol Chem. 31(10):2338-44. Assay: Acetylcholine in fish.
He C, et al (2012). Embryonic exposure to benzo(a)pyrene influences neural development and function in rockfish (Sebastiscus marmoratus). Neurotoxicology 33(4):758-62. Assay: Acetylcholine in fish.
He C, et al (2012). Exposure of Sebastiscus marmoratus embryos to pyrene results in neurodevelopmental defects and disturbs related mechanisms. Aquat Toxicol 15(116-117):109-15. Assay: Acetylcholine in fish.
Mani V, et al (2012). Effects of the Total Alkaloidal Extract of Murraya koenigii Leaf on Oxidative Stress and Cholinergic Transmission in Aged Mice. Phytother Res. 27(1):46-53. Assay: Acetylcholine in mouse brain.
To find more recent publications, please click here.
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