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.
Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were treated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separated and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reverse phase technique, and then as porphyrin esters after a more laborious extraction 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 fluorescing 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-, hexa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased with increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than by reverse phase HPLC.
Gederaas
O A
OA
Department of Clinical Chemistry, University Hospital, N-7006, Trondheim, Norway.
Berg
K
K
Romslo
I
I
D000230:Adenocarcinoma / Q000737:chemistry* / Q000188:drug therapy; D000622:Aminolevulinic Acid / Q000627:therapeutic use*; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000737:chemistry* / Q000188:drug therapy; D006801:Humans; D011166:Porphyrins / Q000032:analysis*; D014407:Tumor Cells, Cultured
D011166:Porphyrins; D000622:Aminolevulinic Acid