Renz et al1 analyzed rapid release of cytochrome c after treatment of the Jurkat cells with agonistic anti–CD-95 monoclonal antibody, staurosporin, etoposide, and doxorubicine. Authors estimated cell death by release of large amounts of intracellular cytochrome c simultaneously with lactate dehydrogenase (LDH) in supernates of treated cells. LDH release in supernates of treated cells was not detected, although cytochromec release and apoptosis by flow cytometry were detected.
The method for measuring the quantity of cytochromec release was based on sensitive immunoprecipitation and subsequent immunoblotting. Contrary to this, LDH release in supernates from treated tumor cells was analyzed by routine LDH method, commonly used in clinical laboratory, based on consideration that LDH is a good clinical marker for estimation of tumor burden.
Determination of LDH activity by routine method for cell culture experiments is not suitable, due to its too-low sensitivity.2 Microassay for estimation of cell death process in vitro, by LDH release, was recommended. The assay is based on substrate mixture, using the small volume of culture cells or their supernates. The results are expressed as absorbance, not in international units, for better interpretation.3-5
Determination of LDH release from cultured cells by microassay shows values that were significantly different, depending on cell type (tumor or normal),5 cell number,6 cell activation status, and separation process.7 Using corrections, LDH release assay also can be used as a sensitive indicator for natural killer cell activity estimation.8Safety of evaluation of the vaccine and virus toxicity effects were performed by LDH microassay as well.9
Contrary to data reported by Renz et al, tumor necrosis factor-α (TNF-α) induced LDH release significantly from tumor, K-562, and Raji cells in a dose- and time-dependent manner rapidly after 2 hours, measured by a microassay.10 The percentage of LDH release from TNF-α–treated tumor cells, expressed in terms of total intracellular enzyme activity, correlates with a decrease of intracellular enzyme activity, representing metabolic alteration; with a decrease of cell growth by [3H]thymidine incorporation into DNA11; and with a decrease of antigen expression, determined by flow cytometry.12
The phenomenon of the cell membrane permeability for LDH was based on the high intracellular LDH values as well as on alteration in transport channels or pore forming during cell activation or the apoptosis process. The cytochrome c request transports through 2 intracellular compartments, including mitochondria membrane to cytoplasm and from cytoplasm to extracellular space, for detection. Cytochrome c, a marker of mitochondrial alteration during apoptosis if it is released whenever the cell membrane is disintegrated, LDH as intracellular enzymes should be released as well.
The mechanisms for LDH and cytochrome c release during the apoptosis process were different and very complex. Although these 2 processes indicated diverse transports, probably including additional secretion or disintegration of the LDH molecule from cytoplasm to cell surface membrane,13 which were not definitively examined, exact measurements using sensitive and highly recommended assays are suggested.3,4-6,8,10
Comments on the estimation of cell membrane alteration after drug treatment by LDH release
We are glad that our paper1-1 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 ccan 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 cytochromec 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,1-1 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.
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