Figure 1. Chromatograms of samples prepared by HClO4 extraction from neutrophil granulocytes.
(A) One extract (black) was spiked with ADPR (13 pmol). ADPR in extracts displayed the same retention time as ADPR in standards (shown in the Supplementary Figure S1 at http://www.BiochemJ.org/bj/398/bj3980225add.htm, displaying an HPLC analysis of a mixture of several standard nucleotides). The ADPR content in the non-spiked extract (grey chromatogram) was calculated as 7.6 pmol. The inset shows an enlarged section of the chromatogram that contained the ADPR peak. (B) Effects of pyrophosphatase incubation on the ADPR peak in extracts (and in standards, see inset). Note that the AMP peak is considerably enlarged by pyrophosphatase treatment, indicating that ADPR is not an exclusive substrate of the enzyme. The minor differences in the retention time in both panels are due to different columns (although of the same type) and different equilibration times with eluent (described in the Experimental section) (1 versus 2 days) in the chromatographies.