Analytical Detection of Sulfonated Metabolites

There are typically three methods to detect sulfonated metabolites (1) radiometric assays employing S -labeled PAPS (2) HPLC-UV detection (3) LC/MS detection. The protein source can be tissue cytosol or purified recombinant SULT protein. 

1 Radiometric Assays Traditionally, sulfonated metabolites for many small chemicals have been detected with a radiometric assay that utilizes S -labeled PAPS (Anderson and Weinshilboum, 1980 Foldes and Meek, 1973). The reaction involves incubation of the substrate, cosubstrate, and enzyme in an appropriate buffer. The incubation is terminated by the addition of barium hydroxide, barium acetate, and zinc sulfate, which cause the unreacted PAPS to precipitate out. Thus, the unprecipitated radioactivity is associated with the sulfonated product and can be quantitated with liquid scintillation counting. A variation of this assay has been developed for larger molecules such as flavonoids, where the incubation is terminated by the addition of ethyl acetate under acidic pH conditions and in the presence of an ion-pairing agent, whereby the sulfonated product can then be detected in the organic phase upon liquid-liquid phase separation (Varin et al., 1987). 

One of the first SULTlAl inhibitors identified in the rat liver was 2, 6-dichloro-4-nitrophenol (DCNP) (Mulder and Scholtens, 1977). DCNP is a dead-end inhibitor, and exhibits low IC50 values toward SULTlAl and SULT 1 A3 (Seah and Wong, 1994). Hydroxylated polychlorinated biphenyls (HPCBs) are potent inhibitors of recombinant human SULTIEI. HPCBs exhibit low micromolar IC50 values toward thyroid hormones (Schuur et al., 1998). Several dietary chemicals such as quercetin, curcumin, and flavones are known to inhibit SULTs. Some commonly used drugs that inhibit SULTlAl and SULT1A3 activity include NSAIDs such as mefenamic acid, naproxen, and salicylic acid. 

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