Fluorometric quantification of protoporphyrin IX in biological skin samples from in vitro penetration/permeation studies

A fluorometric analytical method was developed for quantification of protoporphyrin IX (PpIX) in skin samples and receptor phase solution after in vitro cutaneous penetration/permeation studies. Analytical conditions used were: excitation and emission wavelengths: 400 nm and 632 nm; bandwidth: 0.5 nm; excitation and emission slits: 10/10. PpIX was recovered from two different layers of skin, the stratum corneum (SC) and the epidermis plus dermis ([E+D]), by vortex homogenization, probe and bath sonication, using DMSO as an extraction solvent. The detection and quantification limits were 0.002 and 0.005 μg/mL, respectively. The assay was linear from 0.005 - 0.5 μg/mL. The within-day and between-day assay precision and accuracy in DMSO and receptor phase solution were each studied at the two concentration levels 0.04 and 0.2 μg/mL, and 0.01 and 0.08 μg/mL, respectively. The coefficients of variation and deviation from the theoretical values were lower than 5%. The skin recovery of PpIX from SC and [E+D] layers using two different concentrations (0.5 and 1.0 μg/mL) were all above 90.0%. The method described has potential application to in vitro penetration/permeation studies of PpIX using porcine skin as a biological membrane model.

Um método analítico por espectrofluorimetria foi desenvolvido para quantificar a protoporfirina IX (Pp IX) em amostras de pele e fase receptora após a realização de testes in vitro de penetração/permeação cutâneas. As condições analíticas utilizadas foram: comprimentos de onda de excitação e emissão: 400 nm e 632 nm; largura de banda: 0,5 nm; fendas de excitação e emissão: 10/10. A PpIX foi extraída de amostras de estrato córneo (EC) e da epiderme sem estrato córneo + derme ([E+D]) através da agitação em vórtex e sonicação por haste e banho, utilizando-se o DMSO como solvente extrator. O limite de detecção e quantificação foram, respectivamente, de 0,002 e 0,005 μg/mL. O método mostrou-se linear da faixa de 0,005 - 0,5 μg/mL. A precisão e exatidão intra e inter-ensaio em DMSO e na fase receptora foram validadas utilizando-se duas concentrações distintas, respectivamente, de 0,004 e 0,2 μg/mL, e 0,01 e 0,08 μg/mL. Os valores de coeficiente de variação e o desvio do valor teórico foram inferiores a 5%. A recuperação da PpIX das camadas da pele (EC e [E+D]) utilizando-se duas concentrações distintas (0,5 e 1,0 μg/mL) foram todas acima de 90,0%. O método descrito pode ser utilizado para determinação da PpIX após estudos de penetração/permeação cutânea in vitro utilizando pele de porco como modelo de membrana.

In vitro study of biosynthesis of protoporphyrin IX induced by Ù-Aminolevulinic acid in normal and cancerous cells of the human cervix

Bxkground. Ù-Aminolevulinic acid (ALA) is recognized as the starter in the biosynthesis of the heme group, the structural basis of cytochromes, chlorophylls, biliary pigments, and other porphyrins. It is the first intermediary in the biosynthesis of protoporphyrin IX (PpIX), and of ther heme group. PpIX is present in low concentration in normal cells, and in high concentration in tumor cells. Methods. The accumulation of protoporhyrin IX (PpIX) induced by Ù-aminolevulinic acid (ALA) was tested in two cervico-uterine cancer cell lines (HeLa and CaLo), and in normal human cervical epithelial (NHCE) cells. Results. The optimal concentration of ALA that induced maximum levels of intra- and extracellular accumulation of PpIX in both HeLa and NHCE cells was 300 µg of ALA/mL, and for CaLo cells, 150 µg/mL. The viability of HeLa, CaLo, and NHCE cells exposed to ALA measured 81, 98 and 84 percent, respectively. The optimale time for accumulation of PpIX, both intra- and extracellular, was 4 h for HeLa and NHCE cells and 5 h for CaLo cells per 24 h of expusure to optimal concentrations of ALA. After the maximum level of PpIX accumulation was reached, there was a gradual decrease until there was only a small quantity. A statistically significant difference (p <0.0001) was foundf in the accumulation of PpIX, depending on the concentrations of ALA used as well as between cervical cancer cell lines and 1:7, and for NHCE and CaLo cells. 1:5. Conclusions. These results are important for determining the usefulness of the sensitizer (PpIX)