Comments on the estimation of cell membrane alteration after drug treatment by LDH release

We are glad that our paper1 attracted significant attention from the journal’s readership. While we are thankful for the wise comments of Dr Jurisic about technical issues, we cannot agree with all the theses included. It is true that for our in vitro experiments the lactate dehydrogenase (LDH) microassay would have been the better choice; however, the method was not popular and we were not familiar with it at the time the experiments were performed. We also disagree with some of Dr Jurisic’s points, particularly regarding 2 issues. 1. Dr Jurisic states that the immunoprecipitation-based cytochrome c assay is more sensitive than the LDH enzymatic assay. The detection of cytochrome c by immunoprecipitation is an undeniably sensitive method, but the LDH assay as an enzymatic method is also sensitive per se. Based on unit definition, we calculated that each single molecule of the released LDH (skeletal muscle–derived isozyme) performs about 42 000 enzymatic reactions within 1 second at 25°C. The reaction velocity is determined by the decrease in absorbance at 340 nm, resulting from the oxidation of nicotinamide adenine dinucleotide, so the signal is strongly amplified. In comparison, a single molecule of cytochrome c can be detected in our immunoprecipitation assay by only a single antibody (no significant amplification of the signal). 2. One has to underline some important differences between cytochrome c and LDH. Both molecules are localized in different cellular compartments: cytochrome c in the mitochondrial intermembrane compartment and LDH in the cytoplasm. The translocation of cytochrome c to the cytoplasm is a prerequisite for the initiation of the apoptotic process. LDH is already available there, and it is separated from the extracellular space by a single lipid (cellular) membrane. Also, the mechanisms of release of both molecules may differ significantly. With a molecular weight of approximately 140 kDa, LDH is about 10 times larger than cytochrome c (molecular weight approximately 14 kDa, inclusive of the coenzyme). Even if it is considered that single subunits of LDH are released separately and reaggregate extracellularly, still, based on the significant size difference, both molecules are likely released by different, yet-to-be-elucidated mechanisms. Given the distinctions highlighted above, as well as the differences in the release kinetics (Renz et al,1 Figure 2C, in vitro data; Table 1 and Figure 4, in vivo data), extracellularly detected cytochrome c and LDH likely indicate different ongoing cellular processes. Nevertheless, both methods are valuable indicators of cell damage in the clinic and under experimental conditions.