For experiments with FAAH, rat liver homogenates, mouse brain homogenates and membranes from COS7 cells transfected with the human enzyme are used. Frozen (?80°C) livers from adult C57BL/6 mice and frozen brains (minus cerebella) from adult Wistar or Sprague-Dawley rats are thawed and homogenized in 20 mM HEPES, 1 mM MgCl₂, pH 7. The homogenates are centrifuged at ~35000×g for 20 min at 4°C. After resuspension in buffer followed by recentrifugation and a second resuspension in buffer, the pellets are incubated at 37°C for 15 min. This incubation is undertaken in order to hydrolyse all endogenous FAAH substrates. The homogenates are then centrifuged as above, recentrifuged and resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 3 mM MgCl₂. The homogenates are then frozen at ?80°C in aliquots until used for assay. FAAH is assayed in the homogenates and in the COS7 cell membranes using 0.5 μM (unless otherwise stated) [³H]AEA labelled in the ethanolamine part of the molecule. Blank values are obtained by the use of buffer rather than homogenate. In the experiments comparing effects of Biochanin A upon FAAH and FAAH-2, the same assay is used but with 16 nM [³H]oleoylethanolamide ([³H]OEA) as substrate and with an incubation phase at room temperature. The choice of OEA rather than AEA for FAAH-2 is motivated by the relative rates of hydrolysis: OEA is metabolized four times faster than AEA by FAAH-2, whereas for FAAH the rate of hydrolysis of OEA is about a third of that for AEA. When 0.5 μM [³H]AEA is used as substrate, assay conditions for rat brain and mouse liver are chosen so that <10% of added substrate is metabolized. For the human FAAH samples, <5% of the [³H]AEA is metabolized in all cases. For 16 nM [³H]OEA, a limited supply of an expensive ligand meant that optimization is not possible, and the amount of substrate utilized is higher (34±1 and 0.5±0.1% for FAAH and its corresponding mock-transfected, respectively; 40±2 and 21±0.4 for FAAH-2 and its corresponding mock-transfected respectively)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
ICR mice are used for the behavioural tests measuring spontaneous activity (over a 10 min testing period), rectal temperature, ring immobility (over a 5 min testing period) and nociceptive threshold (tail flick tests). AEA and Biochanin A are dissolved in a vehicle consisting of ethanol, Emulphor-620 and physiological saline in a ratio of 1:1:18 v/v, and administered i.v. to the animals via the tail vein (injection volume 10 μL/g body weight). The degree of antinociception is expressed as percentage of maximum possible effect (%MPE), defined as [(test-control time)/(10-control time)]×100.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Thors L, et al. Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase. Br J Pharmacol. 2010 Jun;160(3):549-60.  [Content Brief]