Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange

Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 microM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4-20 microM cholesterol with the exception of PG at above 8 microM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO-CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 microM NAO provides an inverse linear fluorescence response at 0.2-10 microM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 microM and the limit of quantification is 0.6 microM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 microM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.