Aminolaevulinic acid-induced protoporphyrin IX pharmacokinetics in central and peripheral arteries of the rat[para]

Photochemistry and Photobiology, Jul 2003 by Gabeler, Edward E E, Sluiter, Wim, van Hillegersberg, Richard, Edixhoven, Annie, Et al

Laboratory assays

Chemicals. PpIX disodium salt, zinc PpIX and porphobilinogen (PBG) were obtained from Porphyrin Products (Logan, UT). Uroporphyrin (URO) and Triton X-100 were obtained from Sigma-Aldrich Chemie BV. Tris was obtained from Boehringer Mannheim (Mannheim, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany).

Sample preparation. The arterial samples were kept on ice and were homogenized in water (1:5, wt/wt) using a Potter Elvehjem homogenizer (Kontess Glass Co, Vineland, NJ). The protein content of the arterial samples was determined according the method of Lowry et al. (12).

Determination of PpIX and its precursors in plasma and arterial samples. Determination of ALA and PBG in plasma and arterial samples was performed by a fiuorimetric enzyme assay as described previously (10). This assay is based on the conversion of PBG or ALA into uroporphyrinogen (UROgen) I overnight at 37[degrees]C, respectively, by the addition to the samples of PBG deaminase (PBG-D) or ALA dehydratase (ALA-D) and PBG-D. For the determination of UROgen, no enzymes were added to the samples. Next, the samples were exposed to UV light (350 nm) for 5 min to convert porphyrinogens into porphyrins, followed by centrifugation for 30 min at 1500 g. The fluorescence of the supernatant was measured at an excitation wavelength of 410 nm and an emission wavelength of 656 nm using a LS-50B spectrofluorometer with a red-sensitive photomultiplier (Perkin Elmer, Nieuwerkerk a/d IJssel, The Netherlands). Values were calculated according to a standard line of ALA or PBG converted to UROgen or of URO. We found that if no enzymes were added to the tissue or plasma samples, no UROgen could be detected.

PpIX and zinc PpIX were extracted by adding to 25 [mu]L of plasma or tissue homogenate 50 [mu]L of Tris-HCl (50 mM, pH 8.0) and 425 [mu]L of a mixture of dimethylsulfoxide and methanol (30:70, vol/vol). The samples were mixed vigorously using a vortex and left for about 30 min at room temperature. After centrifugation for 30 min at 1500 g, 100 [mu]L of the supernatant was injected onto a 10 cm Lichrosorb RP18 column (Chrompack, Middelburg, The Netherlands) for reversed-phase high-performance liquid chromatographic analysis using acetone-methanol-water-formic acid (560:240:200:1.75, by vol/vol/vol/vol) as the mobile phase (13). Elution of the porphyrins was detected using a fluorescence detector (LS240, Perkin Elmer) with a red-sensitive photomultiplier operating at excitation and emission wavelengths of 410 and 625 nm, respectively. Sporadically, a peak preceded the zinc PpIX peak that could be identified as most likely being coproporphyrin (COPRO). Recovery of porphyrins during the extraction was determined by adding a PpIX or zinc PpIX standard to the samples and amounted to 90-100%. The concentrations of porphyrins in the samples were calculated according to standard lines of zinc PpIX and PpIX. The porphyrin levels of plasma were expressed in micromoles per liter and of arterial samples in picomoles per milligram of protein.