Table 1.
UV Measurement of PhIP-N -Oxidized Metabolites Bound to Commercial Human Serum Albumin and LC-ESI/MS/MS Estimates of Adducts Formed at Cys34
| pmol PhIP adduct/nmol HSA1 | ||||
|---|---|---|---|---|
| Assay method | NO2-PhIP-modified HSA | HNOH-PhIP-modified HSA | NO-PhIP-modified HSA | NO-PhIP-modified HSA pretreated with 4-CMBA |
| UV Estimate | 293 ± 23 | 82 ± 11 | 131 ± 19 | 71 ± 7 |
| pmol PhIP-Cys 34 adduct/nmol HSA1 | ||||
|---|---|---|---|---|
| LC-ESI/MS/MS | NO2-PhIP modified SA | HNOH-PhIP-modified HSA | NO-PhIP-modified HSA | NO-PhIP-modified HSA pretreated with 4-CMBA5 |
| Acid hydrolysis2 | - | 72.5 ± 8.0 | 69.5 ± 11.1 | 13.4± 1.6 |
| Trypsin/chymotrypsin3 | 21.5 ± 1.0 | 2.2 ± 0.1 | 3.5 ± 0.5 | 0.1 ± 0.0 |
| 3-enzyme digestion4 | 121 ± 31.6 | 11.8 ± 1.7 | 59.0 ± 6.1 | 0.1 ± 0.0 |
| 3-enzyme digestion4 | 85.9 ± 3.5 | 69.4 ± 8.1 | 49.1 ± 6.3 | Not assayed |
1
Each value represents the average and standard deviation of 3 experiments.
2
Measured as PhIP
3
Measured as LQQC*PF adduct following digestion with trypsin/chymotrypsin
4
Measured as C*PF following digestion with pronase E, leucine aminopeptidase, and prolidase; two digests done on different days
5
The amount of titratable Cys in β-mercaptoethanol reduced HSA was 0.95 nmol Cys/nmol SA and <0.02 nmol Cys/nmol SA in 4-CMBA-treated HSA