Blood, 1 April 2003, Vol. 101, No. 7, pp. 2894-2894
CORRESPONDENCE
To the editor:
Estimation of cell membrane alteration after drug treatment by
LDH release
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 cytochrome
c release and apoptosis by flow cytometry were detected.
The method for measuring the quantity of cytochrome
c 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.8
Safety 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-
(TNF-) 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--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
Vladimir Jurisic
Correspondence: University of Kragujevac, School
of Medicine, Pathophysiology, Serbia, Yugoslavia; e-mail:
vdvd{at}mailcity.com
References
1.
Renz A, Berdel WE, Kreuter M, Belka C, Shulze-Osthoff K, Los M.
Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo.
Blood.
2001;98:1542-1548[Abstract/Free Full Text].
2.
Jurisic V, Konjevic G, Banicevic B, Djuricic B, Spuzic I.
Different alteration in lactate dehydrogenase activity and profile of peripheral blood mononuclear cells in Hodgkin's and non-Hodgkin's lymphomas.
Eur J Haematology.
2000;64:259-266[CrossRef].
3.
Korzenievski C, Callewaert DM.
An enzyme-release assay for natural cytotoxicity.
J Immunol Methods.
1983;64:313-320[CrossRef][Medline]
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4.
Decker T, Lohmann-Matther ML.
A quick and simple method for quantitation of lactate dehydrogenase release in measurement of cellular cytotoxicity and tumor necrosis factor (TNF) activity.
J Immunol Methods.
1988;15:61-67.
5.
Konjevic G, Jurisic V, Spuzic I.
Correction of the original lactate dehydrogenase (LDH) release assay for the evaluation of NK cell cytotoxicity.
J Immunol Methods.
1997;200:199-201[Medline]
[Order article via Infotrieve].
6.
Konjevic G, Jurisic V, Spuzic I.
Association of NK cell dysfunction with changes in LDH characteristics of peripheral blood lymphocytes (PBL) in breast cancer patients.
Breast Cancer Res Treat.
2001;66:255-263[Medline]
[Order article via Infotrieve].
7.
Pfluger E, Mueller EA, Anderer FA.
Preservation of cytotoxic function during multi-cycle immunomagnetic cell separation of human NK cells using a new type of magnetic beads.
J Immunol Methods.
1990;129:165-169[CrossRef][Medline]
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8.
Konjevic G, Jurisic V, Banicevic B, Spuzic I.
The difference in NK cell activity between patients with non-Hodgkin's lymphomas and Hodgkin's disease.
Br J Haematol.
1999;104:144-151[CrossRef][Medline]
[Order article via Infotrieve].
9.
Goto N, Maeyama J, Yasuda Y.
Safety evaluation of recombinant cholera toxin B subunit producer by Bacillus brevis as a mucosal adjuvant.
Vaccine.
2000;18:2164-2171[CrossRef][Medline]
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10.
Jurisic V, Konjevic G, Spuzic I.
A comparison of NK cell cytotoxicity with effects of TNF- against K-562 cells, determined by LDH release assay.
Cancer Lett.
1999;138:67-72[CrossRef][Medline]
[Order article via Infotrieve].
11.
Jurisic V, Bogdanovic G, Kojic V, Jakimov D, Baltic V.
Kinetic study of the TNF-alpha effects on the Raji cells.
Tumor Biology.
2000;21:105.
12.
Jurisic V, Bogdanovic G, Srdic T, Kraguljac N, Baltic M, Baltic V.
Modulation of TNF-alpha effects in presence of anti-CD45 and anti-CD95 monoclonal antibody in hematological cell line.
Eur J Cancer.
2001;37:229[CrossRef].
13.
Fujishiro Y, Kishi H, Matsuda T, Fuse H, Murguci A.
Lactate dehydrogenase A-dependent surface expression of immature thymocyte antigen-1: an implication for novel trafficking function of lactate dehydrogenase-A during T cell development.
Eur J Immunol.
2000;30:516-524[Medline]
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Response:
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.
Andrea Renz and Marek Los
Correspondence: Marek Los, Institute of
Experimental Dermatology, University of Münster,
Röntgenstrasse 21, D-48149 Münster, Germany;
e-mail: los{at} uni-muenster.de.
Reference
1.
Renz A, Berdel WE, Kreuter M, et al.
Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo.
Blood.
2001;98:1542-1548[Abstract/Free Full Text].
