Diphenylhexatriene (DPH)-labeled lipids as a potential tool for studies on lipid peroxidation in monolayer films.

Using the fluorescence of diphenylhexatriene (DPH), lipid peroxidation in monomolecular films of phospholipids has been monitored dynamically to elucidate the efficacy of such a probe and to elucidate the effects of molecular organization on such peroxidation processes. Behavior in well-controlled model systems may be used to obtain insight into oxidative processes in complex biological systems. Mixed monolayers of hexadecanoyl-DPH-phosphatidylcholine (HDPH-PC) and diarachidonoyl-PC (DAA-PC) 1/10 (mol/mol) were prepared on a Langmuir trough. With pure water as subphase, and under an atmosphere of N2/O2 (3/1, v/v), DPH fluorescence remained stable over several hours, even under continuous illumination. However, over copper sulfate solution, rapid decay in the fluorescence intensity was observed and correlated with both the copper ion concentration and film pressure. No change in fluorescence was observed in absence of subphase copper ions or under an N2 atmosphere. Substitution of dioleoyl phosphatidylcholine for DAA-PC in systems above gave no decrease in fluorescence intensity. This lipid alone is not susceptible to free radical chain reaction. Absorption spectra from HDPH-PC/DAA-PC monolayers confirm that DPH is actually destroyed during film oxidation. We thus propose this system as a new experimental model for studies on lipid peroxidation in organized systems.

Diphenylhexatriene as a fluorescent probe for monitoring low density lipoprotein peroxidation.

The use of the fluorescent probe diphenylhexatriene (DPH) for monitoring low density lipoprotein (LDL) peroxidation has been investigated. The DPH incorporation into LDL results in a high fluorescence signal which decreases with time after addition of cupric ions. A strong correlation was found between the decay of the DPH fluorescence signal and the appearance of the thiobarbituric reactive substances (TBARS). HPLC and spectrofluorometric analyses demonstrated that DPH is destroyed during the time course of the copper-induced LDL peroxidation. The decrease in DPH fluorescent signal is prevented by addition of EDTA, vitamin E and drugs which protect LDL against peroxidation such as probucol or calcium antagonists. The high fluorescence of DPH allows the use of very small quantities of LDL (less than 5 micrograms/ml LDL protein). We thus suggest that DPH could be of use for continuous monitoring of LDL autooxidation, especially for the in vitro testing of the protective effect of antioxidant compounds.

Effects of phenothiazines on low density lipoprotein metabolism in cultured human fibroblasts.

Treatment of cultured human fibroblasts with trifluoperazine or chlorpromazine resulted in a biphasic effect on low density lipoprotein (LDL) catabolism, depending upon the dose. At up to 10(-5) M, a marked increase in LDL binding, internalization and degradation was observed. This phenomenon took place within the first hours of incubation with the drugs, suggesting a direct effect on cell membrane physical characteristics, probably related to the lipophilic properties of phenothiazines. Concentrations above 2 X 10(-5) M resulted in a relative decrease in LDL binding and internalization, and in a dramatic decrease in LDL degradation, which may be related to an inhibition of calmodulin-dependent processes.