Femtomole isotopic-enzymatic microassay of histamine in microgram amounts of brain tissue.
We present a modification of a previously described radioenzymatic technique (Snyder et al., Journal of Pharmacology and Experimental Therapeutics, 153: 544-549, 1966) for the determination of histamine. This assay is based on the incubation of tissue histamine with a partially purified preparation of histamine-N-methyltransferase (S-adenosyl-L-methionine: histamine N-methyltransferase, E.C. 2.1.1.8; HMT) in the presence of the natural donor of methyl groups, [3H]-methyl-S-adenosyl-L-methionine ([3H]-SAMe). We have found that HMT retains its histamine N-methylating activity at 4 degrees C. Incubation at low temperature results in a considerable increase in the sample to blank ratio. The reduction of the total amount of [3H]-SAMe used and addition of anhydrous sodium sulfate before the last organic extraction step further reduced the blank levels. With these modifications, the sensitivity of the method was increased to the lower femtomole range. The present assay can be used for the determination of as little as 1 to 2 pg of histamine in samples from individual rat brain nuclei containing 2 to 10 micrograms of protein.
D000818:Animals; D001923:Brain Chemistry; D006632:Histamine / Q000032:analysis* / Q000096:biosynthesis; D006637:Histamine N-Methyltransferase / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism; D008297:Male; D008832:Microchemistry; D051381:Rats; D011919:Rats, Inbred Strains; D012436:S-Adenosylmethionine / Q000378:metabolism; D014020:Tissue Extracts / Q000032:analysis*
D014020:Tissue Extracts; D012436:S-Adenosylmethionine; D006632:Histamine; D006637:Histamine N-Methyltransferase