1999; Nagar et al., 2006). Genetic polymorphisms have been reported in
SULT1A1, SULT1A2, SULT2A1, SULT1C2, andSULT1E1genes (Table 3.5). The
functional significance of this genetic variation has been studied, and there are
several reports of association, for example, betweenSULT1A1genotype and
breast or colorectal cancer (Bamber et al., 2001; Zheng et al., 2001).
3.2.5 Analytical Detection of Sulfonated Metabolites
There are typically three methods to detect sulfonated metabolites: (1)
radiometric assays employing S^35 -labeled PAPS; (2) HPLC-UV detection; (3)
LC/MS detection. The protein source can be tissue cytosol or purified
recombinant SULT protein.
3.2.5.1 Radiometric Assays Traditionally, sulfonated metabolites for many
small chemicals have been detected with a radiometric assay that utilizes
S^35 -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).
3.2.5.2 High Pressure Liquid Chromatography (HPLC) Sulfonated drug
conjugates have also been detected with HPLC and LC/MS techniques. Since
authentic standards are not available for most sulfonated products, reversed-
phase HPLC assays have typically relied on deconjugation of the product with
the enzyme sulfatase, detection of the parent compound, and subsequent
calculation of the product formed. An example of sulfonate detection with
HPLC is an assay developed for curcuminoid sulfate quantitation (Asai and
Miyazawa, 2000). Here, the parent compounds (curcumin, demethoxycurcu-
min, and bidemethoxycurcumin) were separated on a TSKgel-ODS 80Ts
250 mm 4.6 mm column at 40C. The mobile phase consisted of acetonitrile/
water (48 : 52 v/v), containing 50 mM phosphoric acid at 1 mL/min. The eluent
was monitored with a UV–VIS detector at 425 nm. For detection of sulfated
curcuminoids, the sample was incubated with 100 U sulfatase type VIII at 37C
for 4 h. Quercetin sulfates have similarly been quantitated with HPLC with
detection at 370 nm (van der Woude, et al., 2004). Deconjugation of sulfates
was achieved enzymatically by the use of sulfatase. Separation of chemicals was
CYTOSOLIC SULFOTRANSFERASES 67