The photobleaching of protoporhyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (lambda < or = 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluorescence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregated. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37 degrees C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.
We have shown that membrane sphingomyelin (SM) is an independent predictor of the variance of fasting plasma insulin (FPI) concentrations and the homeostasis model assessment (HOMA) estimate of insulin resistance in obese women. The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a key component in adipocyte differentiation that may also contribute to the sensitivity of cells to insulin. PPAR-gamma is activated by fatty acids, and the membrane composition may have an impact on the activity of PPAR-gamma and thus on the sensitivity of adipocytes to insulin. We investigated these possible links by determining the phospholipid contents of adipocyte membranes, the mRNA expression of PPAR-gamma, and the FPI and HOMA estimate of insulin resistance in obese women. The mRNA levels of tumor necrosis factor-alpha (TNF-alpha), which is suspected to play a role in insulin resistance and which downregulates PPAR-gamma expression, were also quantified. FPI and HOMA were strongly positively correlated with membrane SM (P < 0.005) and cholesterol (P < 0.005). PPAR-gamma mRNA levels were negatively correlated with FPI (P < 0.05) and HOMA (P < 0.05) and positively correlated with high-density lipoprotein (HDL) cholesterol (P < 0.05), membrane SM (P < 0.05), and cholesterol contents (P < 0.05). TNF-alpha mRNA levels were not correlated with membrane parameters. In stepwise multiple regression analysis, the variations in PPAR-gamma mRNA levels were mainly explained by HDL cholesterol (31.9%) and membrane SM (17.7%). Our study shows that the expression of PPAR-gamma, a major factor controlling adipocyte functions, the lipid composition of the membrane, and insulin sensitivity are probably closely associated in the adipose tissue of obese women.
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