ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 338, No. 1, February 1, pp. 121–127, 1997 Article No. BB969810 Effect of Trivalent Metal Ions on Phase Separation and Membrane Lipid Packing: Role in Lipid Peroxidation Sandra V. Verstraeten,* Luciana V. Nogueira,† Shirley Schreier,† and Patricia I. Oteiza* ,1 *Departamento de Quı´mica Biolo ´gica, IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113-Buenos Aires, Argentina; and †Departamento de Bioquı´mica, Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo, C.P. 26077, 05599-970, Sa ˜ o Paulo, Brazil Received August 21, 1996, and in revised form November 12, 1996 Key Words: cation – membrane interaction; phase separation; lipid peroxidation; membrane rigidifica- The capacity of Al 3/ -related cations (Sc 3/ , Ga 3/ , In 3/ , tion; fluorescent probe; spin label. Be 2/ ,Y 3/ , and La 3/ ) to promote membrane rigidifica- tion and lateral phase separation was evaluated in li- posomes containing zwitterionic (phosphatidylcho- line, PC) and negatively charged (phosphatidylserine, Sc 3/ , Ga 3/ , In 3/ , Be 2/ , and Y 3/ are closely related to PS) phospholipids. These effects were correlated with Al 3/ in their chemical and physical properties (1). All the capacity of the ions to stimulate Fe 2/ -supported are nonredox cations under physiological conditions lipid peroxidation. Al 3/ , Sc 3/ , Ga 3/ , In 3/ , Be 2/ ,Y 3/ , and and have similar ionic radii and/or charge density and La 3/ (50–200 mM) increased the order parameter of the coordination number. Although not so closely related to fluorescent probe 1,3-diphenylhexatriene incorpo- Al 3/ , La 3/ shares chemical properties with the former. rated in PC:PS membranes. In addition, the electron paramagnetic resonance spectra of spin-labeled fatty Sc 3/ , Ga 3/ , In 3/ , and Y 3/ are used in nuclear medicine acids indicated a reduction in lipid motion induced by and antitumor treatment, while Be 2/ ,Y 3/ , and La 3/ Sc 3/ ,Y 3/ , and La 3/ . The effect was found to extend are applied in the electronic industry. Intoxication with down to carbon 16 on the acyl chain. The ions (10–200 these metals has been associated with affections of mM) were also able to induce lateral phase separation, lungs, liver, kidney, bones, and the central nervous sys- as evaluated from the increase in fluorescence quench- tem, both in animal models and in exposed workers. ing of the probe 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4- Sc 3/ , Ga 3/ , In 3/ , Be 2/ , and Y 3/ cause in vitro inhibition yl)amino)dodecanoyl-1-hexadecanoyl- sn-glycero-3- of several enzymes and displacement of cations from phosphocholine. The ability of the ions to alter their binding sites in macromolecules (1). However, the membrane lipid packing and induce lateral phase sep- mechanism of their toxicity is not clearly established. aration correlated in a positive manner (r 2 Å 0.91 and All the above cations promote aggregation, fusion, 0.90, respectively) with their capacity to stimulate the and membrane permeabilization in liposomes (2 – 5). production of Fe 2/ -initiated 2-thiobarbituric-reactive Al 3/ also showed the capacity to stimulate Fe 2/ -initi- species, a measure of lipid peroxidation. These results ated lipid peroxidation in membrane models and in show that Al 3/ -related metal ions cause membrane ri- chronically intoxicated animals (5 – 7). This effect may gidification and phase separation, which could affect be caused by Al 3/ -mediated alterations of lipid packing, membrane-related processes. The results support the producing an increase in the rate of propagation of lipid hypothesis that ions without redox capacity can stimu- peroxidation (4). late Fe 2/ -initiated lipid peroxidation by increasing In the present study we have used fluorescence polar- lipid packing and by promoting the formation of rigid ization and electron paramagnetic resonance (EPR) 2 clusters. Both processes will bring phospholipid acyl chains closer together, thus favoring the propagation step of lipid peroxidation.  1997 Academic Press 2 Abbreviations used: PS, phosphatidylserine; PC, phosphatidyl- choline; TBARS, 2-thiobarbituric acid-reactive substances; C 6 -NBD- 1 To whom correspondence should be addressed at Departamento PC, 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexa- decanoyl-sn-glycero-3-phosphocholine; DPH, 1,3-diphenylhexatriene; de Quı ´mica Biolo ´gica, IQUIFIB (UBA-CONICET), Facultad de Far- macia y Bioquı ´mica, Junı ´n 956, 1113-Buenos Aires, Argentina. Fax: EPR, electron paramagnetic resonance; 5-, 12-, or 16-SASL, 5-, 12-, or 16-doxyl derivative of stearic acid. 54-1-962-5457. E-mail: oteiza@criba.edu.ar. 121 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.