The extent of incorporation of exogenously administered bromodeoxyuridine (BrdU) into DNA is an indicator of the rate of DNA synthesis and cell proliferation. We developed and validated an HPLC method for quantification of BrdU in DNA, based on the separation of nucleosides and their detection by an online UV detector. The baseline separation of BrdU from both normal deoxyribonucleosides and ribonucleosides was achieved in a 60-min run on a reversed-phased C18 column, using a methanol/ammonium formate buffer, as the eluent. The limit of detection was about 0.01 nmole BrdU using a standard BrdU solution. DNA was enzymatically hydrolyzed to deoxyribonucleosides prior to HPLC. The method allowed the detection of background levels of BrdU incorporation into DNA (45 μg) of urinary bladder and duodenum of control rats after a continuous administration of a BrdU solution (50 mg/ml) at a flow rate of 10 μl/h for 4 d, using a subcutaneously implanted osmatic minipump. Compared with the levels obtained by this continuous dosing, BrdU incorporation was much lower in rats dosed intraperitoneally (IP) 120 mg/kg BrdU at 19 h and 4 h prior to sacrifice. A substantial increase (about 10-fold) in BrdU incorporation was seen in the urinary bladder DNA of rats fed a diet containing 3% uracil for 4 d, as compared with the corresponding values obtained for rats fed normal rodent diet (Purina® Chow). BrdU incorporation was much higher (about 7-fold) using the continuous dosing regimen than the two-IP injection regimen. These results were consistent with those obtained by the immunohistochemical staining of BrdU-labeled nuclei with anti-BrdU antibody. Feeding rats 3% uracil in the diet rapidly induces urinary calculi and bladder hyperplasia, which progresses with continued treatment to papillomatosis and transitional cell carcinoma. An HPLC-based 32P-postlabeling assay was also developed for the measurement of BrdU in small amounts of DNA (1 μg). 32P-labeled nucleotides were prepared as the monophosphates, resolved using a 20-min run on a reversed-phase column with a methanol/ammonium formate buffer, and detected using an online radiochemical detector. The assay had similar sensitivity to the UV/HPLC method using about 5 μg DNA, but the latter method allowed the use of more DNA, resulting in higher sensitivity. In conclusion, we developed and validated HPLC/UV and HPLC/32P-postlabeling methods for the quantification of BrdU in DNA and demonstrated their application to quantify the incorporation of BrdU into DNA of rat tissues.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]