Initial characterization of the complement activating compounds in extracts of smokeless tobacco

Aqueous extracts of smokeless tobacco (ST) have been shown to be potent act ivators of complement. However, the mechanisms by which smokeless tobacco a ctivates complement are not well understood. This study was undertaken to i dentify the complement activating compounds in ST extracts. The approximate molecular size of the activating agent(s) in smokeless tobacco was determi ned by dialyzing aqueous extracts of loose leaf chewing tobacco (1S1), dry snuff (1S2), and moist snuff (1S3). Following dialysis (total dilution effe ct of 1:10(9)), using a membrane with a molecular weight retention limit of 12-14 kDa, all extracts retained full capacity to activate serum complemen t as determined by a hemolytic assay. Fractionation of the extracts by gel filtration chromatography revealed that the complement activating agents in ST were high molecular weight compounds that eluted between 400 kDa and th e void volume (1500 kDa) of a Sephacryl S300 column. The high molecular wei ght complement-activating peak was isolated and found to be a more potent c omplement activator than the unfractionated extract. The chemical nature of the complement activating compounds was determined by subjecting the extra cts to boiling for 30 min, an organic extraction with chloroform/methanol 2 :1, or treatment with a DNAse/RNAse enzyme cocktail. None of these treatmen ts destroyed the capacity of ST extracts to activate complement, suggesting that the activating agents may be carbohydrate-like. Finally, an extractio n protocol designed to remove polyphenols significantly diminished the comp lement activating capacity of the ST extracts. These results clearly demons trate that the complement activating substances in smokeless tobacco extrac ts may be large (>400 kDa) polyphenol-containing compounds (i.e. tannins). Identification of this agent(s) will be important for distinguishing the me chanism of smokeless tobacco-induced complement activation.