Validation of the use of 6,6'-dithiodinicotinic acid as a chromogen in the Ellman method for cholinesterase determinations.
The Ellman method for cholinesterase determination is a spectrophotometric method which entails the use of 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as a chromogen and records the level of cholinesterase activity as the change in absorbance at 412 nm. Although this procedure commonly poses no problem, an exception arises when analyzing tissues rich in hemoglobin, because hemoglobin also optimally absorbs light at 400-430 nm. Use of 6,6'-dithiodinicotinic acid (DTNA) might be a solution because, like DTNB, it also is a chromogen for sulfhydryl groups, but with an optimal absorption wavelength of 340 nm (ie removed from the hemoglobin absorbance maximum). Our validation studies indicate that although DTNA is a slightly less efficient indicator of sulfhydryl group concentration, DTNA yields similar activity and degree of enzyme inhibition in tissues from control and treated animals. Moreover, because the assay is read at 340 nm instead of 412 nm, the DTNA assay is markedly more sensitive for determining cholinesterase activity in hemoglobin-rich tissues. Since the advantages of the DTNA method far outweigh the disadvantages, it should be regarded as a sensitive and convenient procedure for determining cholinesterase activity, especially in hemoglobin-rich tissues.
Willig
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Department of Biological Sciences, Clemson University, SC 29634, USA.
Hunter
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D000042:Absorption; D000284:Administration, Oral; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D004390:Chlorpyrifos / Q000008:administration & dosage / Q000633:toxicity*; D002800:Cholinesterase Inhibitors / Q000633:toxicity*; D002802:Cholinesterases / Q000097:blood* / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008297:Male; D009539:Nicotinic Acids / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism*
D002800:Cholinesterase Inhibitors; D006454:Hemoglobins; D009539:Nicotinic Acids; D013439:Sulfhydryl Reagents; C001102:6,6'-dithiodinicotinic acid; D004228:Dithionitrobenzoic Acid; D002802:Cholinesterases; D004390:Chlorpyrifos