Anti-bis(monoacylglycero)phosphate antibody accumulates acetylated LDL-derived cholesterol in cultured macrophages

Bis(monoacylglycero)phosphate (BMP), also called lysobisphosphatidic acid, is a phospholipid highly enriched in the internal membranes of multivesicular late endosomes, in which it forms specialized lipid domains. It has been suggested that BMP-rich membranes regulate cholesterol transport. Here, we examine the effects of an anti-BMP antibody on cholesterol metabolism and transport in two macrophage cell lines, RAW 264.7 and THP-1, during loading with acetylated low density lipoprotein (AcLDL). Anti-BMP antibody was internalized and accumulated in both macrophage cell types. Cholesterol staining with filipin and mass measurements indicate that AcLDL-stimulated accumulation of free cholesterol (FC) was enhanced in macrophages that had accumulated the antibody. Unlike the hydrophobic amine U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), esterification of AcLDL-derived cholesterol by ACAT was not modified after anti-BMP treatment. AcLDL loading led to an increase of FC in the plasma membrane. This increase was further enhanced in anti-BMP-treated macrophages. However, cholesterol efflux to HDL was reduced in antibody-treated cells. These results suggest that the accumulation of anti-BMP antibody alters cholesterol homeostasis in AcLDL-loaded macrophages.     

Metabolism of bis(monoacylglycero)phosphate in macrophages

To further elucidate the role of bis(monoacylglycero)phosphate in lysosomes, its metabolism was assessed by incubation of intact and disrupted macrophages in the presence of labeled lipid precursors. In rabbit pulmonary macrophages bis(monoacylglycero)P accounted for 17.9% and acylphosphatidylglycerol for 2.6% of phospholipid phosphorus. Major fatty acids in bis(monoacylglycero)P were oleic (47%), linoleic (29%), and arachidonic (6.4%); those in acylphosphatidylglycerol were of similar distribution except for a high content of palmitic acid (20%). When homogenates of rabbit pulmonary and peritoneal macrophages, rat pulmonary macrophages, and human blood leukocytes were incubated with sn[(14)C]glycerol-3-phosphate and CDP-diacylglycerol at pH 7.4, there was labeling of bis(monoacylglycero)P and acylphosphatidylglycerol that correlated with content of bis(monoacylglycero)P. When intact rabbit pulmonary macrophages were incubated for 60 min with [(3)H]glucose and [(32)P]orthophosphate, small amounts of label appeared in bis(monoacylglycero)P and only traces in acylphosphatidylglycerol. In contrast, incubation of intact cells with the (14)C-labeled fatty acid precursors palmitic, oleic, and arachidonic acids resulted in much greater labeling of the two lipids. Labeling of phospholipids was greatest with arachidonate as precursor and least with palmitate; after 60 min, labeling of bis(monoacylglycero)P with arachidonate was 10- and 50-fold greater than with oleate and palmitate, respectively, and was exceeded only by that of phosphatidylcholine. Calculated ratios of labeling of fatty acid to P, particularly those for arachidonate, were much greater for bis(monoacylglycero)P and for acylphosphatidylglycerol than for other phospholipids. This suggests a uniquely high turnover of fatty acids in bis(monoacylglycero)P and acylphosphatidylglycerol and thus a more specific role for these compounds in metabolism of complex lipids in the lysosome.-Huterer, S., and J. Wherrett. Metabolism of bis(monoacylglycero)phosphate in macrophages.     

Bis(monoacylglycero)phosphate inhibits TLR4-dependent RANTES production in macrophages

Toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharide (LPS) triggering production of pro-inflammatory cytokines which help eradicate the bacteria but could also be harmful when overproduced. The signaling activity of TLR4 is modulated by cholesterol level in cellular membranes, which in turn is affected by bis(monoacylglycero)phosphate (BMP), a phospholipid enriched in late endosomes. We found that exogenously added BMP isomers become incorporated into the plasma membrane and intracellular vesicles of macrophages and strongly reduced LPS-stimulated production of a chemokine RANTES, which was correlated with inhibition of interferon regulatory factor 3 (IRF3) controlling Rantes expression. To investigate the mechanism underlying the influence of BMP on TLR4 signaling we applied Laurdan and studied the impact of BMP incorporation on lipid packing, a measure for membrane order. Enrichment of model and cellular membranes with BMP significantly reduced their order and the reduction was maintained during stimulation of cells with LPS. This effect of BMP was abolished by enrichment of macrophages with cholesterol. In parallel, the inhibitory effect of BMP exerted on the TLR4-dependent phosphorylation of IRF3 was also reversed. Taken together our results indicate that BMP reduces the order of macrophage membranes which contributes to the inhibition of TLR4-dependent RANTES production.     

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.     

Formation of bis(monoacylglycero)phosphate by a macrophage transacylase

Formation of bis(monoacylglycero)phosphate (BMP) from lysophosphatidyl[U-14C]glycerol was studied in rabbit pulmonary alveolar macrophages. The majority of the activity was found in the particulate fraction (lysosome-enriched) sedimenting between 2000 and 12,000 rpm and it was maximal at pH 4.5. The activity in this fraction was stimulated by 2-mercaptoethanol and additional lipids from the fraction and inhibited by 5 mM CaCl2, 0.5 mM acyl-CoA, 1.0 mM chlorpromazine and by detergents, whereas chloroquine, cholesterol and butanol had no effect. The activity was retained by the particles after repeated freezing and thawing. After treatment with n-butanol, most of the activity was lost, but 84% could be recovered in the aqueous phase if the butanol-extracted lipids were added back giving an activity of 266 nmol/h per mg of protein. Lipids most effective in restoring activity were the total lipids extracted by butanol from the particulate fraction, fractions of the total lipids containing phospholipids and phosphatidylcholine from both native and commercial sources, with native BMP and commercial phosphatidylglycerol and sphingomyelin having a much smaller effect. The complexity of the lipid requirements was further indicated by the finding that addition of pure lipids to the total lipid extract reduced the efficacy of the latter. A direct transfer of [14C]oleic acid to BMP from labelled macrophage microsomal lipids was catalyzed by the soluble enzymes as was transfer from dioleoylphosphatidylcholine in the presence of lysophosphatidylglycerol. The particulate enzyme also catalyzed the transfer of [14C]oleic acid from 2-oleoylphosphatidylcholine to BMP in the presence of lysophosphatidylglycerol. These findings indicate that the transacylase involved in conversion of lysophosphatidylglycerol to BMP utilizes complex lipids other than phosphatidylinositol as acyl donors and has complex requirements for lipids as physicochemical activators. They further suggest that the transacylation might be catalyzed by lysosomal phospholipase A2.     

Degradation of bis(monoacylglycero)phosphate by an acid phosphodiesterase in rat liver lysosomes.

Bis(monoacylglycero)phosphate was purified from the livers of chloroquine-treated rats and labeled with tritium by a nonreductive catalytic exchange procedure. The mechanism of its degradation by rat liver lysosomes has been examined. A substantial amount of bis(monoacylglycero)P is degraded to monoglyceride and lysophosphatidic acid by a lysosomal phosphodiesterase having an acid pH optimum. Some bis(monoacylglycero)P is degraded to lysophosphatidylglycerol by lysosomal phospholipase A. In contrast, other phosphoglycerides have been reported to be degraded by sequential deacylation in lysosomes. The initial rate of breakdown of bis(monoacylglycero)P is only 10% of the rate observed for dioleoylphosphatidylcholine. [3H]Lysophosphatidylglycerol conversion to [3H]bis(monoacylglycero)P is stimulated by unlabeled bis(monoacylglycero)P, resulting in a futile cycle which allows the resynthesis of bis(monoacylglycero)P from its breakdown product, lysophosphatidylglycerol. This futile cycle and the unusual sn-1-glycerophospho-sn-1'-glycerol stereoconfiguration of the water-soluble backbone (Joutti, A., Brotherus, J., Renkonen, O., Laine, R., and Fischer, W. (1976) Biochim. Biophys. Acta 450, 206-209) may be important factors in the marked resistance of bis(monoacylglycero)P to degradation by lysosomal acid hydrolases.     

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%–90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed.     

Metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin

Polyglycerophospholipids (PGPLs) such as bismonoacylglycerophosphate (BMP) and cardiolipin are important membrane phospholipid species for the maintenance of membrane integrity. While BMP serves as membrane curvature regulator in multivesicular bodies for efficient lysosomal enzyme function, cardiolipin stabilizes the electron transfer complex in the inner mitochondrial membrane, which is crucial for physiological ATP production. Beside their membrane modulatory functions PGPLs play an important role in various signaling events. Although a number of disease associations were found for PGPL species, detailed information about their molecular role still remains unknown. This article reviews the known biological functions of PGPLs and the existing mass spectrometric methods. We discuss the different analytical strategies and how ESI–MS/MS can expand our understanding of PGPL homeostasis.     

Synthesis of the unusual lipid bis(monoacylglycero)phosphate in environmental bacteria

The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole-time-of-flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes.     

Formation of acylphosphatidylglycerol by a lysosomal phosphatidylcholine:bis(monoacylglycero)phosphate acyl transferase

A delipidated soluble fraction prepared from a mitochondrial-lysosomal fraction of rabbit alveolar macrophages that catalyzes transacylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate was also found to transfer oleic acid from [14C]dioleoyl phosphatidylcholine to form acylphosphatidylglycerol. The reaction was dependent on the presence of bis(monoacylglycero)phosphate and was maximal at a concentration of 44 microM when the ratio of fatty acid transferred to fatty acid released was 0.28. Addition of phosphatidylglycerol had only a small effect. Homogenates of rat liver also catalyzed the reaction and after subcellular fractionation the activity was localized to lysosomes. The lysosomal activity was solubilized by delipidation with butanol to give a preparation with a specific activity 2462 times that of the homogenate. Optimal activity of soluble preparations from both macrophages and liver was at pH 4.5, with little activity above 6.0. Release of free fatty acid was also stimulated under conditions of optimal acyl transfer. Both acyl transfer and release of fatty acid were inhibited by Ca2+, detergents, chlorpromazine, lysophosphatidylcholine, and oleic acid. When there was disproportional inhibition, acyl transfer was always more affected. These results suggest that sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate and then acylphosphatidylglycerol constitute a mechanism in the lysosome for the transport and partition of fatty acids released by the lysosomal phospholipases.     

Promotion of vesicular stomatitis virus fusion by the endosome-specific phospholipid bis(monoacylglycero)phosphate (BMP)

Vesicular stomatitis virus (VSV) is a prototypic virus commonly used in studies of endocytosis and membrane trafficking. One proposed mechanism for VSV entry involves initial fusion with internal vesicles of multivesicular endosomes followed by back-fusion of these vesicles into the cytoplasm. One feature of endosomal internal vesicles is that they contain the lipid bis(monoacylglycero)phosphate (BMP). Here, we show that the presence of BMP significantly increases the rate of VSV G-mediated membrane fusion. The increased fusion was selective for VSV and was not evident for another enveloped virus, influenza virus. Our data provide a biological rationale for a two-step infection reaction during VSV entry, and suggest that BMP preferentially affects the ability of VSV G to mediate lipid mixing during membrane fusion.     

The stereoconfiguration of newly formed molecules of bis(monoacylglycero)phosphate in BHK cells

Newly formed molecules of bis(monoacylglycero)phosphate (known also as lysobisphosphatidic acid), which were labeled with 32Pi in cultured BHK cells during relatively short pulses, were subjected to stereoanalysis. In contrast to the high proportion of sn-1-glycerophosphate residues in the bulk of the bis(monoacylglycero)phosphate molecules, the newly formed molecules were rich in sn-3-glycerophosphate residues.     

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd−/− and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd−/− and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd−/− mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd−/− mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd−/− mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.     

Incorporation of polyunsaturated fatty acids into bis(monoacylglycero)phosphate and other lipids of macrophages and of fibroblasts from control and Niemann-Pick patients

On the basis of earlier studies of rabbit pulmonary alveolar macrophages, the incorporation of 14C-labelled polyunsaturated fatty acids into the lipids of human fibroblasts from patients with various phenotypes of Niemann-Pick disease was examined in order to define further the disturbance in metabolism of bis(monoacylglycero)phosphate occurring in these disorders. Docosahexaenoic acid, which had not been studied previously, was found to be incorporated by macrophages into bis(monoacylglycero)phosphate in a highly selective fashion and was therefore used along with arachidonic acid for studies of fibroblasts. Following incubation of fibroblasts in serum-free medium for 60 min, the distribution of arachidonic acid label in lipids was: phosphatidylcholine, 51%; phosphatidylethanolamine, 12%; phosphatidylinositol, 9.5%; and bis(monoacylglycero)phosphate, 2.3%; and of docosahexaenoic acid label was 36, 20, 2.6 and 10.3% respectively. Phosphatidylinositol had the highest specific activity of arachidonic acid label and bis(monoacylglycero)phosphate of docosahexaenoic acid label. Prolongation of incubation to 21 h, with or without removal of label remaining in the medium at 1 h, resulted in proportional redistributions with phosphatidylcholine decreasing and phosphatidylethanolamine increasing. In bis(monoacylglycero)phosphate and phosphatidylinositol, the proportions of arachidonic acid label decreased and increased respectively, whereas the proportions of docosahexaenoic acid label in these lipids were unchanged. As virtually all label taken up by cells was esterified, these redistributions are taken to reflect transacylations. In Niemann-Pick cells, the expected redistribution of arachidonic acid label in bis(monoacylglycero)phosphate failed to occur with cell types A and B which are deficient in sphingomyelinase-phospholipase C, and excess label accumulated after a 21-h incubation. Excess docosahexaenoic acid label also accumulated in the bis(monoacylglycero)phosphate of these cells. The highly selective incorporation of docosahexaenoic acid in two cell types suggests a special role for bis(monoacylglycero)phosphate in the metabolism of n-3 polyunsaturated fatty acids. A high specific activity found early in incubations of macrophages suggests that polyunsaturated fatty acids may be incorporated into phospholipids during de novo synthesis of phosphatidic acid.     

Trans-4-hydroxy-2-hexenal is a neurotoxic product of docosahexaenoic (22:6; n-3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA) is elevated in the CNS in patients with Alzheimer's disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans -4-hydroxy-2-hexenal (HHE), a six carbon analog of the n-6 fatty acid derived trans -4-hydroxy-2-nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD₅₀'s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration- and time-dependent manners. The time-dependent formation of HHE differed from that of F₄-neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5-fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.     

The stereoconfiguration of bis(monoacylglycero)phosphate synthesized in vitro in lysosomes of rat liver: comparison with the natural lipid.

A procedure for stereoanalysis of radiochemically labeled glycerophospholipids is described. It is based on the study of the labeled alpha-glycerophosphate which retains its original configuration when liberated upon alkaline hydrolysis of the lipids. The labeled alpha-glycerophosphate is oxidized enzymatically with sn-3-glycerophosphate dehydrogenase and the product, dihydroxyacetone phosphate, is degraded with alkali to inorganic phosphate. The nonoxidizable alpha-glycerophophate (sn-1-glycerophosphate), the beta-glycerophosphate, and the inorganic phosphate derived from sn-3-glycerophosphate are quantitated after separation by thin-layer chromatography. The procedure gave the expected results when applied to [3H]glycerol-and 32P-labeled phosphatidylcholine, bis( monoacylglycero)phosphate, and phosphatidylglycerol from natural resources. Bis(monoacylglycero)phosphate, known also as lysobisphosphatidic acid, was synthesized from ]32P]diphosphatidylglycerol and from phosphatidyl[1',3'-3H]glycerol in lysosomal preparations of rat liver according to Poorthuis and Hostetler (1978. J. Lipid Res. 19: 309-315). Stereoanalysis proved that the product was in both cases a derivate of sn-1-glycerophospho-sn-1'-glycerol.     

A new synthesis of bis(diacylglycero)phosphate

The chemical synthesis of bis(diacylglycero)phosphate previously named bisphosphatidic acid, starting with a diacylglycerol and phosphatidic acid, is described. The phosphodiester bond formation is catalyzed by triisopropylbenzenesulfonylchloride. This simple approach allows the preparation of saturated as well as unsaturated bis(diacylglycero)phosphate species in one step without the use of any protecting group. The methods used until now yield only mono-acid species, or mixed-acid unsaturated species after many steps involving the introduction and the removal of protecting groups. The synthetic products have been characterized by component analysis and NMR-techniques.     

Conversion of phosphatidylglycerol to lyso(bis)phosphatidic acid by alveolar macrophages

We report here studies of the synthesis of lyso(bis)phosphatidic acid [L(b)PA] by normal and BCG-elicited rabbit alveolar macrophages. This study was prompted by our earlier observations that 1) alveolar macrophages did not synthesize L(b)PA de novo despite its abundance in these cells, 2) BCG-elicited cells contained only one-quarter the amount of L(b)PA as normal cells, and 3) the turnover of arachidonate in L(b)PA led to hydroxyeicosatetraenoic acid and leukotriene synthesis. We found that exogenous phosphatidylglycerol (PG) was specifically converted to L(b)PA by both types of cells although BCG-elicited cells had only one-quarter the synthetic capacity of normal cells. Other phospholipids were found to become cell associated but were not significantly metabolized. Both glycerol moieties and the phosphate were incorporated into the product L(b)PA. However, substitution of the ester with an alkyl linkage in position 1 blocked the conversion of PG to L(b)PA. Most of the alkylphosphatidylglycerol was converted to phosphatidylcholine and phosphatidylethanolamine. This result implied that catabolism of the acyl group in position 1 was essential for L(b)PA synthesis. Because alveolar macrophages are present in a surfactant-rich milieu, we suggest that surfactant provides a source of PG for macrophage synthesis of L(b)PA in situ. It is interesting that the surfactants from rabbits challenged with BCG have a significant decrease in PG content.     

Effect of docosahexaenoic acid intake on lipid peroxidation in diabetic rat retina under oxidative stress

Docosahexaenoic acid (DHA) plays an important role in visual function but has a highly oxidation-prone chemical structure. Therefore, we investigated how dietary DHA affects the generation of lipid peroxides in rat retina under oxidative stress in diabetes with/without vitamin E (VE) deficiency. Streptozotocin-induced (50 mg i.p./kg B.W.) diabetic Sprague-Dawley (SD) rats were assigned to four groups: (i) control/VE(+), (ii) DHA/VE(+), (iii) control/VE( - ) and (iv) DHA/VE( - ), and raised for 28 days. We then measured lipid peroxide levels in the retina, serum and liver. With a normal intake of VE, dietary DHA increased only the retinal level of thiobarbituric acid-reactive substances (TBARS) slightly. In contrast, in rats with VE deficiency, dietary DHA increased serum and liver lipid peroxide levels but not in the retina. These results suggest that dietary DHA does not necessarily promote lipid peroxidation in the retina even under high oxidative stress.