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Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells

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Lipids

Abstract

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12–15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (<12 min) were assigned to the following species by virtue of comigration with authentic standards: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH), and 3β-hydroxycholest-5-ene-7α/7β-hydroperoxide (7α/7β-OOH). Formation of 5α-OOH and 6β-OOH (singlet oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18–22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18–22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts ofPCOOH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

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Abbreviations

AlPcS4 :

chloroaluminum phthalocyanine tetrasulfonate

ChOOH:

cholesterol hydroperoxide(s)

DCP:

dicetylphosphate

DFO:

desferrioxamine

EC:

electrochemical

FAOOH:

fatty acid hydroperoxide(s)

FCS:

fetal calf serum

GC/MS:

gas chromatography/mass spectrometry

GPX:

glutathione peroxidase

GSH:

reduced glutathione

HPLC-EC:

high-performance liquid chromatography with mercury drop electrochemical detection

HPLC-RC:

high-performance liquid chromatography with radiochemical detection

LOOH:

lipid hydroperoxide(s)

MC540:

merocyanine 540

MTT:

3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide

5α-OOH:

3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide

6β-OOH:

3β-hydroxycholest-4-ene-6β-hydroperoxide

7α-OOH:

3β-hydroxycholest-5-ene-7α-hydroperoxide

7β-OOH:

3β-hydroxycholest-5-ene-7β-hydroperoxide

PBS:

phosphate buffered saline (25 mM sodium phosphate, 125 mM NaCl, pH 7.4)

PC:

phosphatidylcholine

PCOOH:

phosphatidylcholine hydroperoxide(s)

PEOOH:

phosphatidylethanolamine hydroperoxide(s)

PHGPX:

phospholipid hydroperoxide glutathione peroxidase

PLOOH:

phospholipid hydroperoxide(s); PLA2, phospholipase A2

POPC-OOH:

hydroperoxide of 1-palmitoyl-2-oleoyl phosphatidylcholine

PLPC-OOH:

hydroperoxide of 1-palmitoyl-2-linoleoyl phosphatidylcholine

TGOOH:

triacylglycerol hydroperoxide(s)

TLC:

thin-layer chromatography

TMPD:

N,N,N′,N′tetramethyl-p-phenylenediamine

TPP:

triphenylphosphine

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Author notes

  1. Witold Korytowski

    Present address: Institute of Molecular Biology, Jagiellonian University, Krakow, Poland

Authors and Affiliations

  1. The Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, 53226, Milwaukee, WI

    Gary J. Bachowski, Witold Korytowski & Albert W. Girotti

Authors
  1. Gary J. Bachowski
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  2. Witold Korytowski
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  3. Albert W. Girotti
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Additional information

This paper is based on a dissertation submitted by G.J. Bachowski in partial fulfillment of the requirements for a Ph.D. degree in Biochemistry at the Medical College of Wisconsin (Milwaukee, WI).

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Bachowski, G.J., Korytowski, W. & Girotti, A.W. Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells. Lipids 29, 449–459 (1994). https://doi.org/10.1007/BF02578241

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  • DOI: https://doi.org/10.1007/BF02578241

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