Protective Action of Phospholipid Hydroperoxide Glutathione Peroxidase against Membrane-damaging Lipid Peroxidation

The general reactivity of membrane lipid hydroperoxides (LOOHs) with the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) has been investigated. When human erythrocyte ghosts (lipid content: 60 wt % phospholipid; 25 wt % cholesterol) were treated with GSH/PHGPX subsequent to rose bengal-sensitized photoperoxidation, iodometritally measured LOOHs were totally reduced to alcohols. Similar treatment with the classic glutathione peroxidase (GPX) produced no effect unless the peroxidized membranes were preincubated with phospholipase AZ (PLA*). However, under these conditions, no more than -60% of the LOOH was reduced; introduction of PHGPX brought the reaction to completion. Thin layer chromatographic analyses revealed that the GPX-resistant (but PHGPX-reactive) LOOH was cholesterol hydroperoxide (ChOOH) consisting mainly of the 5a (singlet oxygen-derived) product. Membrane ChOOHs were reduced by GSH/PHGPX to species that comigrated with borohydride reduction products (diols). Sensitive quantitation of PHGPX-catalyzed ChOOH reduction was accomplished by using [ “C]cholesterol-labeled ghosts. Kinetic analyses indicated that the rate of ChOOH decay was -‘/e that of phospholipid hydroperoxide decay. Photooxidized ghosts underwent a large burst of free radical-mediated lipid peroxidation when incubated with ascorbate/iron or xanthinejxanthine oxidase/iron. These reactions were only partially inhibited by PLA2/GSH/GPX treatment, but totally inhibited by GSH/PHGPX treatment, consistent with complete elimination of LOOHs in the latter case. These findings provide important clues as to how ChOOHs are detoxified in cells and add new insights into PHGPX’s protective role.

The general reactivity of membrane lipid hydroperoxides (LOOHs) with the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) has been investigated.
When human erythrocyte ghosts (lipid content: 60 wt % phospholipid; 25 wt % cholesterol) were treated with GSH/PHGPX subsequent to rose bengal-sensitized photoperoxidation, iodometritally measured LOOHs were totally reduced to alcohols. Similar treatment with the classic glutathione peroxidase (GPX) produced no effect unless the peroxidized membranes were preincubated with phospholipase AZ (PLA*).
However, under these conditions, no more than -60% of the LOOH was reduced; introduction of PHGPX brought the reaction to completion. Thin layer chromatographic analyses revealed that the GPX-resistant (but PHGPX-reactive) LOOH was cholesterol hydroperoxide (ChOOH) consisting mainly of the 5a (singlet oxygen-derived) product. Membrane ChOOHs were reduced by GSH/PHGPX to species that comigrated with borohydride reduction products (diols).
Sensitive quantitation of PHGPX-catalyzed ChOOH reduction was accomplished by using [ "C]cholesterol-labeled ghosts. Kinetic analyses indicated that the rate of ChOOH decay was -'/e that of phospholipid hydroperoxide decay. Photooxidized ghosts underwent a large burst of free radical-mediated lipid peroxidation when incubated with ascorbate/iron or xanthinejxanthine oxidase/iron. These reactions were only partially inhibited by PLA2/GSH/GPX treatment, but totally inhibited by GSH/PHGPX treatment, consistent with complete elimination of LOOHs in the latter case. These findings provide important clues as to how ChOOHs are detoxified in cells and add new insights into PHGPX's protective role.
Aerobic cells are constantly exposed to the possibility of oxidative damage mediated by activated oxygen species such as superoxide (OF), hydrogen peroxide (HzOz), hydroxyl radical (OH'), or singlet oxygen ('02 Natural regeneration of GSH from GSSG is catalyzed by GSSG reductase (Equation 2). Previous studies clearly established that phospholipid hydroperoxides (whether membranebound or detergent-dispersed) are not susceptible to direct reduction by GPX (4)(5)(6). Instead, the oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase AP (PLAJ; GPX then acts on the liberated fatty acid hydroperoxides. On the basis of these findings, a mechanism for detoxification and repair of phospholipid hydroperoxide lesions has been proposed which involves consecutive action of PLAz and GPX on membrane phospholipid hydroperoxides, followed by reinsertion of new fatty acyl groups. Most of the early experiments leading to these conclusions were carried out with phospholipid vesicles (liposomes) that lacked cholesterol (4)(5)(6). Recent studies by Thomas and Girotti (7,8)  cholesterol hydroperoxides. We have begun to examine this question by using a recently discovered second selenoenzyme, provisionally termed "phospholipid hydroperoxide glutathione peroxidase" (PHGPX) (9,10). Unlike GPX, PHGPX can reduce membrane phospholipid hydroperoxides in situ without the necessity of prior hydrolysis by PLAz (9). Thus, a more direct protective role of PHGPX is apparent. Previous studies (10) indicated that PHGPX is relatively nonselective in its action on phospholipid hydroperoxides. However, whether it would also act on cholesterol hydroperoxides was not directly assessed. We now provide the first evidence for PHGPX-catalyzed reduction and detoxification of cholesterol hydroperoxides in a natural cell membrane.  (21).

Enzymatic Reduction of Lipid-derived
Hydroperoxides-In initial experiments, the relative abilities of GPX and PHGPX to catalyze the GSH-dependent reduction of membrane LOOHs in situ was examined by coupled enzymatic assay with GSSG-reductase. In these determinations, the rate and also extent of NADPH oxidation during the reductase-catalyzed regeneration of GSH (Equation 2) was used as a measure of peroxidatic action on LOOHs (Equation 1). The test system consisted of isolated membranes that were photoperoxidized in the presence of rose bengal, a lop-generating dye (22). As expected from previous studies (7), GPX alone (0.4 unit/ml) caused little (if any) LOOH loss when added to the peroxidized ghosts in an enzymatic assay mixture (Fig. 1, truce A). (In this experiment, the small Asdo decrement observed after the introduction of GPX is attributed primarily to dilution of the reaction mixture, with trace amounts of H202 or fatty acid hydroperoxide making a possible contribution.) When added FIG subsequently to GPX, PHGPX (0.1 unit/ml) caused an immediate and rapid increase in the rate of AsbO decay, which slowed to approximately the background rate after -1 min. Introduction of more PHGPX (0.1 unit/ml) at this point caused no further change; alternatively, introduction of a known amount of cumene hydroperoxide produced the expected decrement in As.,o, indicating that the PHGPX was still active (data not shown). A second portion of the peroxidized membranes was treated with CaC12/PLAz before being analyzed. With this preparation, GPX produced a sizeable Asa decrement (Fig. 1, truce B). However, the reaction was clearly not complete, since subsequent addition of PHGPX resulted in another decrement, the magnitude of which was approximately 2/3 of that produced by GPX. This was seen consistently in all replicate determinations. It is important to note that the Asdo value generated by PHGPX (Fig. L4) is nearly the same (<lo% difference) as that generated by successive additions of GPX and PHGPX (Fig. 1B). Moreover, there was good agreement between LOOH values calculated from these measurements (116 and 105 nmol/mg of protein, respectively) and the absolute value of total LOOH determined independently by iodometric assay (108 nmol/mg of protein). These results suggested that PHGPX could react quantitatively with all LOOHs in the membrane. By contrast, and in agreement with earlier findings (7, 8)  30 min of incubation, the LOOH had decayed to 6% of its starting value.' By contrast, incubation with GSH plus GPX produced no net effect on LOOH over that observed with GSH alone (-15% loss after 30 min). Lipid extracts from this experiment were also analyzed by TLC (Fig. 2B), which allowed hydroperoxides of cholesterol and different phospholipid classes to be scrutinized.
Whereas no TMPD-reactive hydroperoxides were detected in a non-irradiated control (lane b), these products were clearly evident in the photooxidized sample (lane c). Based on the chromatographic migration of photooxidized standards, hydroperoxides of two major membrane phospholipids (PC and PE) could be identified, along with unresolved phosphatidylserine and sphingomyelin products. In agreement with the iodometric measurements ( Fig.  2A), 30 min incubation with GSH alone or GSH plus GPX caused little perceptible change in the spot intensity of each photoproduct.
However, similar incubation with GSH plus PHGPX resulted in an almost total disappearance of the phospholipid-derived hydroperoxides and a sizeable, albeit incomplete, loss of cholesterol hydroperoxides. Although membrane hydroperoxides were not susceptible to direct reduction by the GSH/GPX system, they did react after PLA, treatment ( Fig. 1; Ref. 7). Under these conditions, the peroxide content decreased rapidly to 35-40% of its starting value, but remained at this level after prolonged incubation with GSH/GPX (7). Trivial explanations for this incomplete reaction were ruled out, e.g. progressive inactivation of PLA, or GPX, or permeability barriers against the enzymes. Examination of TLC chromatograms clearly indicated that PLAs action had released fatty acid hydroperoxides, which were then susceptible to GPX attack. Significantly, cholesterol hydroperoxides were shown to be the only major LOOHs to resist enzymatic reduction subsequent to PLA, treatment. The poor reactivity of these peroxides with GPX could not be attributed to hindered accessibility, since solubilization of the membranes with Triton X-100 or extraction from the mem-' Unlike the experiments of Fig. 1, those of Fig. 2 were carried out in the absence of Triton X-100. Therefore, near quantitative reaction of' PHGPX with LOOHs did not require dispersal of membrane lipids. branes had no significant effect (7). It is apparent from these earlier results that the discrepancy between the iodometrically determined and the GPX-determined LOOH values for the experiment shown in Fig. 1B was due to cholesterol hydroperoxides. In contrast, to GPX, PHGPX appeared to react directly with these species in situ (within the membrane) just as it reacted with phospholipid hydroperoxides.
In subsequent experiments, we studied PHGPX-catalyzed reduction of cholesterol hydroperoxides in greater detail, focusing on (a) substrate-product relationships and (b) kinetics of substrate loss. Enzymatic Reduction of Cholesterol Hydroperoxides-Because of (a) inadequate sterol resolution and (b) possible interference with phospholipid-derived products, the solvent system used for the TLC shown in Fig. 2B (chloroform/ methanol/water, 75:25:4 (v/v)) was not suitable for examining reduction products of cholesterol hydroperoxides. For this reason, we selected a less polar system (heptane/ethyl acetate, 1:l (v/v)) which affords good resolution of cholesterol products from one another and from starting material, while leaving phospholipid species at the origin (15). As shown in Fig. 3A, photooxidized ghosts contained a prominent TMPDreactive product(s) (lane c) of the same mobility (RF -0.37) as peroxidized cholesterol (ChOOH) in a liposome standard (lane a). A minor spot closer to the solvent front (seen only in lane c) was not identified.
As in the Fig. 2 experiment, no peroxides could be detected in membranes that were dyesensitized, but not irradiated (lane b). Borohydride treatment (lane d) resulted in total disappearance of all TMPD reactivity, including that observed at the origin (phospholipid-derived), which is consistent with reduction of hydroperoxides to alcohol derivatives (20). A 30-min incubation with GSH plus PHGPX had the same effect (lane g), in general agreement with the results shown in Fig. 2B and their diol reduction products (Fig. 3B). Borohydride treatment of photooxidized Enzymatic Detoxification of Membrane Lipid Hydroperorides ghost lipids converted cholesterol hydroperoxide to 5a-cholest-6-ene-3&5-diol (5~OH), which appeared as a blue spot (RF -0.26) on the HzSO1-treated plate (Fig. 3B, cf lanes c and d). As can be seen, 5~OH migrated more slowly than parent cholesterol (RF -0.62) or its hydroperoxide (RF -0.37), but more rapidly than the reduction products of 7ketocholesterol (lane h), cholest-5-en-3/3,7cu-diol (7o(-OH; RF -0.15) and cholest-5-en-3&7&diol (7&OH; RF -0.20). The immediate precursor of 5~0H, 3B-hydroxy-5a-cholest-6-ene-5-hydroperoxide (5~OOH), is a characteristic product of singlet oxygen ('OZ) attack on cholesterol that can be used as an unequivocal indicator of IO2 intermediacy (24,25). In addition to rose bengal, several other photosensitizing agents have been shown to be good IO2 generators in ghost membranes by virtue of 5~OOH formation (15,26,27). It should be pointed out that the photoperoxidation reaction described in Fig. 3 was carried out in the presence of an antioxidant (butylated hydroxytoluene) to prevent (or at least minimize) any formation of radical-derived products, e.g. 7ol-/7P-OOH generated photochemically or by allylic rearrangement of 5a-OOH (15,24). This assured that the cholesterol hydroperoxide population would be as homogeneous as possible (consisting predominantly of 5~00H) for subsequent reactions with GPX or PHGPX.
As shown in lane g (Fig. 3B), incubation with GSH/PHGPX resulted in a clean reduction of 5~00H to 5~OH, with no evidence of other diols (e.g. 7a-OH or 7/3-OH) under the conditions used. As expected from peroxide visualization (Fig. 3A), neither GSH alone nor GSH/GPX caused any significant conversion of 5~00H to 501-OH (lanes e and f).
To enhance the sensitivity of cholesterol product detection and to allow quantitation as well, we used membranes that were radiolabeled with ['4C]cholesterol. Prior to dye-sensitized photooxidation, cholesterol itself was the only detectable radioactive species (Fig. 4, lane A). Immediately after irradiation, a peak corresponding to [Wlcholesterol hydroperoxide was observed (10% of the total radioactivity), with lesser amounts of 7~0H and 5a-OH (-5% collectively). As in the experiment shown in Fig. 3 (cf lanes c, e, and f) Fig. 3 with those of Fig. 4, one should note that butylated hydroxytoluene was added before irradiation in the former case, but not in the latter. Previous studies (27) have shown that butylated hydroxytoluene inhibits free radicalmediated formation of 7a-/7/3-OOH from cholesterol and also the slow allylic rearrangement of 5a-OOH to 7~OOH that may accompany '02-mediated photooxidation.
The inferred presence of significant 7a-OOH in the product profile shown in Fig. 4 (CL lanes B and C) is attributed primarily to allylic rearrangement of 5~OOH. It is apparent from Fig. 4 that PHGPX can reduce not only membrane-bound 5a-OOH but also 7a-OOH. Any kinetic differences in these reactions remain to be determined.
Additional insights into LOOH removal by PHGPX were gained by comparing the time course of cholesterol hydroperoxide decay with that of the overall lipid population. (similarly to the one described in Fig. 4), peroxide disappearance was accompanied by the formation of increasing amounts of 7a-OH as well as 5~OH.
As indicated above, partial rearrangement of 5~OOH to 7~00H (during irradiation as well as enzyme treatment) probably accounts for the appearance of 7a-OH. As shown in Fig. 5B Ghost membranes (1 mg of protein/ml) were photooxidized as described in Fig. 2  drop over the first 5 min (estimated k -19 h-l), followed by a terminal slow reaction (k -3.6 h-l). In the absence of PHGPX (but presence of GSH and desferrioxamine), peroxide loss was relatively slow (Iz < 0.2 h-r), as seen in other experiments (cfi Fig. 2). On the basis of the TLC observations in Fig. 2 (lane f) and similar chromatograms showing time courses for the decay of different LOOH classes (data not shown), the rapid loss of total LOOH depicted in Fig. 5B is ascribed primarily to phospholipid hydroperoxides and the slow reaction to cholesterol hydroperoxides. Consistent with this assignment, the apparent rate constant for the slow reaction approximated that of ['4C]cholesterol hydroperoxide disappearance (see above).

Enzymatic Protection against Free Radical-mediated Lipid
Peroxidation-Earlier studies have shown that photoperoxidized ghosts undergo a large burst of free radical-mediated lipid peroxidation (as detected by TBARS formation) when exposed to ascorbate and ferric iron (15,16,18,27). A similar effect was observed when the membranes were treated with xanthine and xanthine oxidase in the presence of iron. Nonphotooxidized membranes showed essentially no reaction with ascorbate/iron and relatively little reaction with xanthine/ xanthine oxidase/iron. In either system, stimulation of lipid peroxidation was attributed to LOOH-dependent initiation, i.e. ferrous iron-induced reduction of LOOHs to lipid oxyl radicals (LO'), which trigger chain reactions via H abstraction (1). Classical GPX had no effect on these reactions unless the membranes were first treated with Ca2+/PLA2. While this treatment alone had a sizeable inhibitory effect, the combination of Ca2+/PLA2 and GSH/GPX was even more (but not completely) inhibitory. TLC analyses indicated that liberated fatty acid hydroperoxides were totally reduced by GPX, leaving cholesterol hydroperoxides as the only probable initiating species. With this information at hand, it was of obvious  Fig. 2), 1 mg of protein/ml, were incubated in the dark at 37 "C with 0. interest to determine how PHGPX would affect these reactions. As shown in Fig. 6A, incubation of photoperoxidized ghosts with 0.5 mM ascorbate and 0.05 mM FeCls resulted in a strong surge of lipid peroxidation (TBARS formation) which leveled off at -11 nmol/mg protein after 15 min. Little peroxidation (if any) was seen with non-photooxidized ghosts. Low concentrations of butylated hydroxytoluene (e.g. 25 pM) inhibited the ascorbate/iron-stimulated reaction (data not shown), indicating that it was free radical-mediated. The small zerotime level of TBARS (-1 nmol/mg protein) represents partial degradation of starting photoperoxides that occurred during the TBA assay (18). Significantly, preincubation of the photooxidized membranes with GSH/PHGPX resulted in essentially total inhibition of the ascorbate/iron-stimulated lipid peroxidation, i.e. TBARS formation was little different from that seen in the dark control. GSH alone had relatively little effect, if any. Prevention of peroxidation by GSH/PHGPX is consistent with the fact that all measurable initiating LOOHs, including cholesterol hydroperoxides, were removed by GSH/ PHGPX treatment (Fig. 2). Similar results were obtained in the case of xanthinelxanthine oxidase-driven lipid peroxidation (Fig. 6B). Thus, when photoperoxidized ghosts were incubated with xanthine, xanthine oxidase, and FeCl,, they underwent a burst of O;-dependent (superoxide dismutaseinhibitable) lipid peroxidation, which was almost totally nullified by prior treatment with GSH/PHGPX (Fig. 6B). The small residual reaction after such treatment was identical in magnitude to the reaction of the non-irradiated control.

DISCUSSION
Membrane lipid peroxidation is one of the most prominent forms of cellular damage induced by conditions of oxidative stress. Aerobic cells are equipped with a battery of defenses against the deleterious effects of lipid peroxidation. Primary defense is based on prevention of initiating reactions. This can be achieved by agents such as (a) enzyme scavengers of reactive oxygen species, e.g. superoxide dismutase, catalase, peroxidases, (b) chemical antioxidants, e.g., a-tocopherol, @carotene, ascorbate, and (c) iron-sequestering proteins, e.g., apoferritin, apolactoferrin. A second mode of protection involves enzymatic removal of lipid-derived hydroperoxide intermediates. These reactions are typically catalyzed by GSHrequiring enzymes which fall into two classes: Se-dependent GSH-peroxidases and certain Se-independent enzymes, e.g. GSH-S-transferase B (28). This latter pathway can be considered as a back-up to the various primary lines of defense that involve iron inactivation or oxyradical/H202 scavenging.
Early studies on a thermolabile cytosolic factor capable of inhibiting microsomal lipid peroxidation in the presence of GSH suggested that this factor might be GPX (29). Inasmuch as GPX was known to catalyze the reduction of a wide range of hydraperoxides, including fatty acid hydroperoxides, it was inferred that direct reduction of membrane lipid hydroperoxides in situ might also take place. However, subsequent work clearly indicated that this could not be the case. For example, Grossman and Wendel (4) and Sevanian et al. (5) reported that phospholipid hydroperoxides in micelles or unilamellar liposomes are poor substrates for GPX unless first acted upon by PLAP. Similar observations were made in connection with GSH-S-transferase action on phospholipid hydroperoxides (30). The proposed mechanism in each case involved (i) PLAncatalyzed hydrolysis of m-2 fatty acyl hydroperoxide groups, (ii) "release" of the fatty acid hydroperoxide, with subsequent reinsertion of a new unsaturated fatty acyl group, and (iii) GPX-or transferase-catalyzed reduction of the hydroperoxide.
More recent studies by Thomas and Girotti (7,8), using photoperoxidized erythrocyte membranes, indicated that phospholipid-derived hydroperoxides were completely eliminated by Ca2'/PLA2 treatment followed by GSH/GPX, whereas cholesterol hydroperoxides were unaffected. Extracted cholesterol products (mainly 5a-OOH in 50 mM Tris-HCl, pH 7.4, 20% ethanol) were resistant to GSH/ GPX, thereby ruling out physical inaccessibility in the membrane as a possible reason for nonreactivity.
The only other known investigation of GPX activity on cholesterol hydroperoxides was that of Little (31), who showed that free radicalderived products, e.g. 7/l-OOH and 25-OOH, are also poor substrates for GPX, the reaction rates being less than 5% of the rate observed with H,Oz or linoleic acid hydroperoxide.
These findings prompted us to carry out the present study with PHGPX, the second selenium-requiring peroxidase to be isolated and characterized (9,10). Although PHGPX also contains an active site selenocysteine group, it differs from classical GPX in several respects (28), e.g. (i) relatively high membrane affinity (hydrophobic character), (ii) functional molecular weight (-20 kDa for monomeric PHGPX uersus -85 kDa for tetrameric GPX), (iii) lack of absolute specificity for GSH as the reducing substrate, (iv) a broad specificity for hydroperoxides, including phospholipid hydroperoxides. PHGPX was first isolated from rat and porcine liver by Ursini et al. (9) and characterized in terms of its ability to inhibit free radical-mediated lipid peroxidation in phosphatidylcholine liposomes and microsomes.
Chromatographically distinct from any known GPX or GSH-S-transferase, the enzyme was provisionally termed a "Peroxidation Inhibiting Protein" (PIP) and later given its present designation, PHGPX.3 It is likely that the previously described cytosolic factor with peroxidation inhibiting properties (29) was, in fact, PHGPX (28). Maiorino et al. (14) reported that the GSH/PHGPX system, coupled with NADPH/glutathione reductase, could be employed for accurate determinations of membrane LOOH content. Using peroxidized microsomal membranes as a test system, they observed excellent agreement between enzymatically determined LOOH values and values obtained by iron/thiocyanate assay. It was deduced, therefore, that all classes of membrane LOOHs were accessible to and were reacting quantitatively with GSH/PHGPX.
Since microsomes contain only small amounts of cholesterol (typically <lo% of the total lipid weight), it was not clear from these and related studies (10,14) whether cholesterol hydroperoxides (in addition to phospholipid hydroperoxides) were substrates for PHGPX. Consequently, the present work has provided the first direct evidence for PHGPX-catalyzed reduction of cholesterol hydroperoxides in a biological membrane. Relatively little else of related interest has been done in this area, other than the one study already mentioned (31), an earlier report on the peroxidatic action of cytochrome P-450 on steroid hydroperoxides (32), and a more recent study dealing with the metabolism of 5a-OOH and 7cu-OOH by Staphylococcus typhimurium (33). In the latter case, evidence was presented for a slow isomerization and/or reduction of the hydroperoxides, but the putative enzymes involved were not identified.
While PHGPX was capable of reacting with both cholesterol hydroperoxides and phospholipid hydroperoxides in the membranes studied (erythrocyte ghosts), the rates of these reactions were found to be significantly different. Thus, under 3 Despite its limitations, the term phospholipid hydroperoxide glutathione peroxidase (PHGPX), has been retained in this publication. This has been done for convenience and to minimize confusion in terminology.
the conditions described (CL Fig. 5), cholesterol species were reduced at a first order rate which was only -15% of that ascribed to phospholipid species. It is not clear at this point whether this difference is an intrinsic one (i.e. based on structural properties of the hydroperoxides per se) or whether other factors (e.g. substrate arrangement in the bilayer, interaction with other membrane elements) are more important. It should be noted that both classes of hydroperoxides disappeared completely after sufficiently long periods of exposure to GSH/PHGPX (cf Figs. 2 and 4), indicating that there were no absolute permeability barriers to PHGPX, i.e. that LOOHs in both membrane leaflets were accessible to externally added enzyme. This was not unexpected, however, since the ghost membranes were known to be unsealed, i.e. leaky to macromolecules at least as large as hemoglobin. Similar results were reported earlier for GPX-catalyzed reduction of phospholipid-derived hydroperoxides in photooxidized, PLAptreated ghosts (7).
The major cholesterol photoproduct generated by rose bengal-sensitized photooxidation of erythrocyte membranes is the IO2 adduct, 5a-OOH (26,27). When generated in the presence of a free radical trap which prevented (or at least minimized) its isomerization to 7a-OOH, 5a-OOH was the principal steroid-based substrate for PHGPX (Fig. 3). Under these conditions, a clean conversion of 5a-OOH to ~cz-OH was observed, with no evidence of other diol products. In other experiments (cfi Fig. 4), allylic rearrangement was allowed to occur during irradiation and subsequent incubation steps. In these instances, GSH/PHGPX treatment produced significant amounts of 7ar-OH (and traces of 7/3-OH epimer) in addition to 5a-OH, indicating that membrane-bound 7a-OOH and 7/3-OOH are also substrates. However, any kinetic differences in the reduction of the three different hydroperoxides remain to be elucidated.
The present findings add further support to the proposal of Ursini et al. (28) that PHGPX plays a unique role in protecting cells against the damaging effects of lipid peroxidation. We have shown that erythrocyte ghosts primed with LOOHs by dye-sensitized photooxidation produce large amounts of TBARS when exposed to ascorbate or xanthinelxanthine oxidase as a source of 0;. Earlier work with resealed ghosts (16,18) indicated that because of their propagative nature, these reactions cause far more lytic damage than photooxidation alone. One of the most significant findings of the present work is that pretreatment of LOOH-containing ghosts with GSH/PHGPX prevented ascorbate-or O;-stimulated lipid peroxidation from occurring. Under similar reaction conditions, GSH/GPX had no effect. Even after PLAZ treatment, GSH/GPX afforded only partial protection against peroxidation, the residual reaction being ascribed to reductive decomposition of cholesterol hydroperoxides (7). The relative superiority of PHGPX as a direct inhibitor of lipid peroxidation is clearly evident from the present study. These results are consistent with previous ones (9), which showed that GSH/PHGPX can totally inhibit NADPH/iron-ADP-driven lipid peroxidation in mitoplasts and microsomes. Thus, PHGPX is seen to be highly effective in protecting plasma membranes as well as subcellular membranes against the damaging effects of LOOH-mediated lipid oxidation. By comparison with GPX, PHGPX is a relatively lipophilic enzyme. In the large number of tissues from which PHGPX has been isolated (28), significant amounts of its activity have been shown to be associated with subcellular membranes. This could explain its ability to act directly on membrane LOOHs, whereas GPX, having limited ability to interact with membranes, may be more important in removing cytosolic hydroperoxides, e.g. H202 and certain fatty acid hydroperoxides. According to this idea, the functional significance of PHGPX uersus GPX in any given tissue would depend on the nature of the incident oxidative stress and the hydroperoxides arising therefrom (28). Although hydrolysis of phospholipid hydroperoxides by PLA, is not a prerequisite for PHGPX action, such hydrolysis may occur secondarily.
Thus, it is reasonable to expect that in cellular systems, fatty acyl alcohols generated by PHGPX will be cleaved by PLA2 as part of the repair process. Subsequent insertion of a new fatty acyl group into the lysolipid sn-2 position would regenerate the glycerophospholipid. This has been proposed as a more logical mechanism for damage prevention and repair than one involving consecutive action of PLA, and GPX (28). As the physiological role of PHGPX unfolds, it will be important to understand how the processes of reduction, excision, and reacylation are coordinated in tissues. It will be equally important to understand how the enzymatic reduction products of cholesterol hydroperoxides (dials) are metabolized.
Although we have been primarily concerned with cell membranes in this work, in certain tissues removal of LOOHs from other cellular structures could be equally important, e.g. internalized lipoproteins in the vascular wall (34). In this regard, we have recently shown that the GSH/PHGPX system can readily reduce hydroperoxides of cholesterol, cholesteryl esters, and phospholipids in low density lipoproteins.