A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells
Oa. Gederaas et al., A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells, CANCER LETT, 150(2), 2000, pp. 205-213
Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were tr
eated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separate
d and quantified by high performance liquid chromatography (HPLC), both as
free porphyrin acids after an easy extraction method with a subsequent reve
rse phase technique, and then as porphyrin esters after a more laborious ex
traction method and subsequent normal phase technique. The porphyrins were
detected by means of a fluorescence detector. Analysis by normal phase HPLC
indicated that 81% (739 pmol/mg protein) of the total amounts of fluoresci
ng porphyrins accumulated was protoporphyrin IX, while similar analysis by
reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein
) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, h
exa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated
cells by both methods. The discrepancy between the two methods increased w
ith increasing hydrophilicity of the analysed porphyrins, with uroporphyrin
estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than b
y reverse phase HPLC. (C) 2000 Elsevier Science Ireland Ltd. All rights res
erved.