Longitudinal chemokine profile expression in a blood-brain barrier model from Alzheimer transgenic versus wild-type mice.

BACKGROUND: Alzheimer's disease is widely described since the discovery of histopathological lesions in Mrs. Auguste Deter in 1906. However to date, there is no effective treatment to deal with the many cellular and molecular alterations. The complexity is even higher with the growing evidence of involvement of the peripheral blood mononuclear cells (PBMCs). Indeed, monocytes and T cells are shown in the cerebral parenchyma of AD patients, and these cells grafted to the periphery are able to go through the blood-brain barrier (BBB) in transgenic mouse models. It is known that BBB is disrupted at a late stage of AD. Chemokines represent major regulators of the transmigration of PBMCs, but many data were obtained on AD animal models. No data are available on the role of AD BBB in a healthy brain parenchyma. Therefore, the purpose of this study was to analyze the longitudinal chemokine profile expression in a BBB model from AD transgenic mice versus wild-type (WT) mice. METHODS: A primary mouse BBB model was used with a luminal compartment either AD or WT and an abluminal compartment WT consisting of astrocytes and microglia. PBMCs were extracted by a ficoll gradient and incubated in the transwell with a direct contact with the luminal side, including the endothelial cells and pericytes. Then, the complete BBB model was incubated during 48 h, before supernatants and cell lysates were collected. Chemokines were quantified by X-MAP® luminex technology. RESULTS: Abluminal CX3CL1 production increased in 12-month-old AD BBB while CX3CL1 levels decreased in luminal lysates. CCL3 in luminal compartment increased with aging and was significantly different compared to AD BBB at 12 months. In addition, abluminal CCL2 in 12-month-old AD BBB greatly decreased compared to levels in WT BBB. On the contrary, no modification was observed for CCL4, CCL5, and CXCL10. CONCLUSION: These first findings highlighted the impact of AD luminal compartment on chemokine signature in a healthy brain parenchyma, suggesting new therapeutic or diagnostic approaches.

Prevention of the ß-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures.

BACKGROUND: Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD). There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by ß-amyloid (Aß) peptides, the nuclear factor-kappa B (NF-κB) pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR) controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aß42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. METHODS: Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aß neurotoxicity (72 h), co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα), interleukin (IL)-1ß, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test RESULTS: In these co-cultures, PKR inhibition prevented Aß42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα) and interleukin (IL)-1ß, and limited apoptosis. CONCLUSION: In spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.

Imipramine, in part through tumor necrosis factor alpha inhibition, prevents cognitive decline and beta-amyloid accumulation in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in the older people, is a multifactoral pathology, characterized by cognitive deficits, increase in cerebral deposition of the beta-amyloid (Abeta) peptide, neurofibrillary tangles, and neurodegeneration. Studies currently support a central role of neuroinflammation, through production of proinflammatory cytokines including excess tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of AD, especially in Abeta-induced cognitive deficits. Imipramine, a tricyclic antidepressant, has potent anti-inflammatory and neuroprotective effects. This study investigates the effect of imipramine on alterations of long-term and short-term memories, TNF-alpha expression, and amyloid precursor protein (APP) processing induced by intracerebroventricular injection of Abeta25-35 in mice. Mice were treated with imipramine (10 mg/kg i.p. once a day for 13 days) from the day after the Abeta25-35 injection. Memory function was evaluated in the water-maze (days 10-14) and Y-maze (day 9) tests. TNF-alpha levels and APP processing were examined in the frontal cortex and the hippocampus (day 14). Imipramine significantly prevented memory deficits caused by Abeta25-35 in the water-maze and Y-maze tests, and inhibited the TNF-alpha increase in the frontal cortex. Moreover, imipramine decreased the elevated levels of Abeta both in frontal cortex and hippocampus with different modulations of APP and C-terminal fragments of APP. So, imipramine prevents memory impairment through its intrinsic property to inhibit TNF-alpha and Abeta accumulation and may represent a potential candidate for AD treatment.

Identification of a muscle factor related to MyoD in a fish species.

We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes.

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.

A new in vitro approach for investigating the MPTP effect on DA uptake.

Previous studies have shown that dopamine (DA) uptake was decreased after preincubation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)) in in vitro slice and synaptosome models. The present study, conducted with and without preincubation, attempted to determine whether inhibition results from a direct effect of neurotoxins on neuronal DA transporter or from an alteration of the transporter secondary to other toxic events. DA uptake was inhibited about 50% in the presence of MPTP+O(2) or MPP(+) (0.1, 1 and 5 mM) in rat striatal slices and synaptosomes. Such inhibition was obtained in synaptosomes preincubated for 150 min with MPP(+) and then washed. Inhibition of DA uptake was lower in slices preincubated with MPTP (5 mM)+O(2) and then washed (30%). Experiments in synaptosomes prepared from slices preincubated with MPTP or MPP(+) showed greater inhibition of DA uptake with MPTP. The results suggest that the inhibition of DA uptake in vitro by MPTP or MPP(+) results initially from a direct effect on the transporter during its penetration in nerve endings and subsequently from a transporter alteration related to toxic events. Thus, the preincubation of striatal slices followed by DA uptake measurement in synaptosomes would appear to be a good in vitro model for studying the dopaminergic toxicity of MPTP.

Inhibitory effects of ascorbic acid on dopamine uptake by rat striatal synaptosomes: relationship to lipid peroxidation and oxidation of protein sulfhydryl groups.

Ascorbic acid is frequently added in the incubation medium to prevent oxidation of dopamine (DA) during uptake assays. However, a preliminary study showed that the presence of ascorbic acid induced a decrease of DA uptake after prolonged incubation. The purpose of this study was to determine the mechanism underlying ascorbic acid-induced alterations of DA uptake in rat striatal synaptosomes. In this context, the effects of physiological concentrations of ascorbic acid (100-500 microM) on DA uptake and Na+/K+ ATPase activity (which is essential for DA transporter function) were assessed in synaptosomes before and after incubation at 37 degrees C. The capacity of synaptosomes to take up DA was significantly decreased after incubation owing to a reduction in DA transporters (but with no modification of their affinity for DA). This partial inhibition was associated with a decrease of Na+/K+ ATPase activity, a production of thiobarbituric acid reactive substances (TBARS) and malonaldehyde (MDA), and a loss of sulfhydryl group content. Addition of Trolox C to the medium prevented the reduction of DA uptake, the inhibition of Na+/K+ ATPase activity, the decrease in sulfhydryl group content and the production of TBARS and MDA. These results suggest that ascorbic acid in the presence of contaminant ferrous ions induced a decrease in functional DA transporters, probably through a lipid peroxidation process involving oxidation of sulfhydryl groups and at least in part through a decrease of Na+/K+ ATPase activity.

Impairment of the neuronal dopamine transporter activity in MPP(+)-treated rat was not prevented by treatments with nitric oxide synthase or poly(ADP-ribose) polymerase inhibitors.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite MPP(+), damages of the nigrostriatal dopaminergic pathway, similar to those observed in Parkinson's disease. An intranigral injection of 10 microg MPP(+) in rat induced a decrease of about 30% of the neuronal dopamine transporter (DAT) activity 21 days after lesion. Based on the hypothesis that MPTP/MPP(+) neurotoxicity involves the nitric oxide (NO) production and/or an activation of poly(ADP-ribose) polymerase (PARP), we investigated the preventive effects of a treatment either with L-Name, a NO synthase (NOS) inhibitor or 3-aminobenzamide, a PARP inhibitor on the reduction of dopamine uptake induced by MPP(+). Rats received a daily injection i.p. of 50 mg/kg L-Name or 10 mg/kg 3-aminobenzamide 3 days before and during 21 days after the MPP(+) lesion. The results showed that inhibitors of NOS and PARP did not prevent the alteration of DAT activity induced by 10 microg MPP(+), indicating that NO and PARP were not involved in the biochemical cascade leading to the inhibition of rat DAT activity by MPP(+) in our experimental conditions.

Origin of 4-hydroxynonenal incubation-induced inhibition of dopamine transporter and Na+/K+ adenosine triphosphate in rat striatal synaptosomes.

Previous experiments reported that an incubation of striatal synaptosomes with 4-hydroxynonenal (4-HNE) resulted in an inhibition of dopamine (DA) uptake and Na+/K+ adenosine triphosphate (ATPase) activity. The present work investigated whether theses inhibitions are related to a 4-HNE binding to the DA transporter (DAT) and the Na+/K+ ATPase. The number of specific [125I]-PE21 binding sites on the DAT was significantly reduced after incubation with 4-HNE. The Na+/K+ ATPase activity decrease induced by 4-HNE was partially reversed, in a dose-dependent manner, by veratridine, a pump stimulator agent. Our previous data (Morel, P., Tallineau, C., Pontcharraud, R., Piriou, A. and Huguet, F., Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes. Neurochem. Int., 33 (1999) 531-540) combining with the data observed in this study suggest that changes in DA uptake in striatal synaptosomes are directly related to 4-HNE binding to the DAT, whereas the decrease in Na+/K+ ATPase activity resulted only partially from 4-HNE binding to the pump and is mainly secondary to membrane lipid disruption.

Membrane carbohydrate conjugates desialylation does not alter [3H]-dopamine uptake in rat striatal slices.

Incubation of rat striatal slices induced a large decrease (about 50%) of DA uptake and a slight desialylation of polysialogangliosides (GT1b, GD1b, GD1a) with an increase of monosialogangliosides (GM1). Moreover, a pretreatment of slices by exogenous added neuraminidase of Vibrio cholerae did not modify DA uptake, although the pattern of gangliosides was modified and there was considerable loss (about 45%) of sialic acid in gangliosides and glycoproteins. It was verified that neuraminidase activity occured in synaptic membrane. Thus, DA uptake was apparently not altered by desialylation of plasma membrane carbohydrate conjugates.

Autoxidation of rat brain homogenate: evidence for spontaneous lipid peroxidation. Comparison with the characteristics of Fe2+- and ascorbic acid-stimulated lipid peroxidation.

Aerobically-incubated brain homogenates are known to undergo autoxidation characterized by spontaneous TBARS production, presumably as a result of lipid peroxidation. However, TBARS measurement alone, because of its lack of specificity, is not sufficient to demonstrate the occurrence of lipid peroxidation in complex biological systems. This study, undertaken to determine whether or not spontaneous oxidation of rat brain homogenate is due to lipid peroxidation, measured different specific markers of this process (fatty acids, lipid aldehydes and the formation of fluorescence products) and studied changes in alpha-tocopherol. Incubation of rat brain homogenates at 37 degrees C under air led to spontaneous TBARS formation, which was accompanied by lipid aldehydes and lipid fluorescence products as well as polyunsaturated fatty acid (PUFA) degradation. Alpha-tocopherol was also consumed. On the whole, these results demonstrate that autoxidation of brain homogenate is a spontaneous lipid peroxidation process. When homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation was enhanced. However, spontaneous and stimulated peroxidation showed similar patterns not characteristic of classical lipid peroxidation, i.e. without the lag and accelerating phases typical of a propagating chain reaction. PUFA degradation was limited despite stimulation of peroxidation.

Evidence of lipoperoxidation induced by lactic acid on kidney homogenates.

Sawas and Gilbert (Arch. Int. Pharmacodyn. Ther., 276 (1985) 301-312) reported that the commercial solution of haloperidol induces lipoperoxidation of kidney homogenates from Sprague-Dawley rats. However, it would appear that this effect is attributable to the excipient, lactic acid, rather than to haloperidol itself. Lactic acid enhances susceptibility to lipoperoxidation of kidney homogenates in a dose- and time-dependent manner by increasing production of thiobarbituric acid-reactive substances and slightly decreasing polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid. This stimulation of lipoperoxidation may be attributed to a mechanism less dependent on enzymatic action than on Fe2+ and Fe3+. Lactic acid may facilitate iron release and formation of iron complexes, factors which increase susceptibility to oxidative stress.

Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests.

Vitis vinifera cell suspensions were used to isolate and characterize the flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes) found in red wine. Furthermore, we showed that astringin is produced although this stilbene has not previously been reported to be a constituent of V. vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical. Antioxidant activities were assessed by their capacity to prevent Fe2+-induced lipid peroxidation in microsomes and their action on Cu2+-induced lipid peroxidation in low-density lipoproteins. The results showed that astringin has an important antioxidant effect similar to that of trans-resveratrol, and a higher radical scavenger activity than the latter. Astringinin appeared to be more active. These data indicate that phenolic compounds (stilbenes, catechins, anthocyanins) exhibit interesting properties which may account in part for the so-called "French paradox," i.e. that moderate drinking of red wine over a long period of time can protect against coronary heart disease.

Feeding behaviour and food utilisation in tilapia, Oreochromis niloticus: effect of sex ratio and relationship with the endocrine status.

The feeding behaviour of male monosex, female monosex, and mixed groups of Oreochromis niloticus was studied under conditions of self-feeding. Feeding activity was observed almost exclusively during the light period. The food intake pattern was similar whatever the sex ratio, and voluntary food intake (VFI) appeared lower in the male monosex groups than in the others. Male monosex groups displayed higher specific growth rates (SGR) and a lower food conversion ratio than female monosex and mixed groups. The SGR of males was higher in the monosex than in the mixed groups, whereas females of mixed and monosex groups displayed no significant difference in SGR. The efficiency of food utilisation was also analysed: nutrient retention ratios were higher in male monosex than in female monosex and mixed groups. Males displayed a distinctly higher metabolic capacity. Differences in sex-related hormones (11 ketotestosterone = 11-KT, 17beta-Oestradiol = 17beta-E2) and a metabolic hormone (triiodothyronine = T3) were observed between males and females. The hypothesis of an involvement of these hormones in the higher metabolic capacity of males is discussed. The observed differences in feeding behaviour between the different groups also suggest an effect of social interactions on the efficiency of food conversion and thus on the differential growth of males and females.

Gentamicin-induced kidney damage and lipid peroxidation in rats.

Although it has been reported that injections of gentamicin induces lipid peroxidation in rat renal cortex (Ramsammy et al. (1985) Biochem. Pharmacol. 34, 3895-3900), our results showed no modification of thiobarbituric-reagent substances (TBARS) or in analysis of the polyunsaturated fatty acid profile. Moreover, endogenous vitamin E and glutathione were not consumed. In in vitro systems, gentamicin incubated with microsomes, homogenates and kidney slices from the normal rat failed to induce lipid peroxidation. We show that the increase in TBARS in vivo detected by Ramsammy et al. was wrongly attributed to the oxidant power of gentamicin. As this antibiotic does react positively to thiobarbituric acid in the presence of a system generating free radicals, it is possible that these authors accidentally introduced such a system into their experiments.

Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from vitis vinifera cell cultures

Suspension cultures of Vitis vinifera L. (Vitaceae) produce many hydroxylated stilbene glucosides found in red wine. From these cells, we isolated and characterized glycosylated stilbenes, (Z)-piceatannol (3,5,3',4'-tetrahydroxystilbene) -3-O-beta-d-glucopyranoside (6) and (E)- and (Z)-resveratrol (3,5, 4'-trihydroxystilbene)-4'-O-beta-d-glucopyranoside (2 and 7, respectively), which have not previously reported to be constituents of Vitis vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1 diphenyl-2-picryl-hydrazyl, a stable free radical. Antioxidant activities were assessed by their capacity to prevent Cu2+-induced lipid peroxidation in human low-density lipoprotein.

Cloning of a trout fast skeletal myosin heavy chain expressed both in embryo and adult muscles and in myotubes neoformed in vitro.

In fish, little is known about the isoforms of myosin heavy chain in developing muscle. Two cDNA libraries from whole skeletal muscle of embryo (eyed stage) (A) and from white muscle of 300 g body weight immature trout (B) were constructed. Three cDNA clones were isolated and characterised as encoding for a fast skeletal myosin heavy chain. Two cDNA clones A1 (1534 bp) and B6 (2203 bp) which were extracted from the two different libraries had the same nucleotide sequence including the 3' untranslated region. The third cDNA B8 (1606 bp) shared 98% identity with the others. The latter could possibly be an allelic isoform of the B6. Northern blot analysis revealed that the fast skeletal MyoHC transcripts were expressed throughout development from myotube appearance to the white muscle present at older stages (adult). These results suggest that this myosin heavy chain is present throughout muscle development in fish and are consistent with the hyperplastic growth of fish muscle. The amino acid sequence of the trout myosin heavy chain diverged from its mammalian and avain counterpart with respect to a higher level of glycine which could be related to an environmental adaptation by increasing thermal instability of the molecule.

Sensitivity of muscle satellite cells to pollutants: an in vitro and in vivo comparative approach.

Muscle satellite cells from rainbow trout were exposed in vitro to increasing concentrations of different xenobiotics: copper, dichloroaniline, prochloraz, nonyl-phenol polyethexylate. Mortality and proliferation rate were measured by Hoechst binding and BrdU incorporation. Dose dependent effect of copper on survival and proliferation was observed with an EC50 at 100 microM. A dose dependent effect of nonyl-phenol diethoxylate was observed on survival with an EC50 at 100 microM. This was associated with a biphasic effect on proliferation rate observed both for nonyl-phenol di and poly-ethoxylate: a stimulation of proliferation at low concentration and an inhibition proliferation at large concentration. These effects were related to the inhibition of cells adhesion through the detergent capacity of nonyl-phenol polyethoxylate. The effects of prochloraze and dichloroaniline on cells mortality (EC 50 > 500 microM) and proliferation rate (LOEC: 100 microM) were limited. Whole fish growth, muscle fibre size distribution and satellite cells survival and proliferation were measured on rainbow trout (60-80 g BW) exposed to two concentrations of prochloraze (10 and 100 microg/ml) or nonyl phenol diethoxylate IGEPAL 210 (100 and 400 microg/ml) during 14 and 10 days exposure, respectively. Muscle fibre size distribution and satellite cells proliferation were affected by prochloraz exposure in vivo and this could be related to the alteration in fish feeding status. The exposure to IGEPAL 210 affected the number of satellite cells extracted and induce a biphasic effect on satellite cells proliferation similar to that observed in vivo. The combined exposure to IGEPAL 210 and prochloraze revealed additive effects of these two compounds. The in vivo and in vitro comparison demonstrated that in vitro satellite cells system could be used as a valuable tool to test the effects of pollutants.

Biological levels of aluminium after use of aluminium-containing bone cement in post-otoneurosurgery.

Use aluminium-containing biomaterials in otoneurosurgery for reconstitution of bone in contact with cerebrospinal fluid (CSF) also led to cases of encephalopathy and death. We report aluminium (Al) concentrations in the biological fluids of six French patients following use of Al-containing bone cement in otoneurosurgery. In five patients, the mean plasma Al levels (microgram/L) were: 1.20 +/- 0.05 (case 2), 9.20 +/- 0.10 (case 3), 1.00 +/- 0.05 (case 4), 2.80 +/- 0.05 (case 5) and 2.00 +/- 0.05 (case 6). In case 1, Al concentrations were 176 micrograms/L in the postauricular CSF accumulation, 34 micrograms/L in the pontocerebellar angle and 4 and 6 micrograms/L in the lumbar shunt. As a precautionary measure, in the first three cases the biomaterial was removed soon after the intervention, and no increase in plasma or CSF Al was observed. In the other cases, absence of neurobiological symptoms and normal concentrations of Al in plasma led neurosurgeons not to extract this biomaterial. Al assay thus may be considered to be a complementary and at times a decision-generating factor. Care is needed at all stages from sampling through analysis because Al is ubiquitous and factually high results may be clinically misleading. Herein, such considerations are discussed in conjunction with the neurotoxicity of this metal in man. In addition, the authors call for in-depth preliminary trials of these biomaterials in animals prior to introduction on the market.

Lack of correlation between TBARS production and PUFA degradation during incubation of membrane erythrocytes in an OH. (Fe2+/H2O2) generator system.

We investigated the effects of an OH. (Fe2+/H2O2) generator system on erythrocyte membrane, particularly the time-course of lipid peroxidation as estimated by measurement of conjugated dienes, thiobarbituric reactive substances (TBARS), lipofuscin-like pigments, and alpha-tocopherol. Polyunsaturated fatty acids (PUFAs), especially arachidonic acid (20:4 omega 6) and docosahexenoic acid (22:6 omega 3), were also measured. Erythrocyte membranes were suspended in phosphate buffer containing Fe2+ (200 microM) and H2O2 (1.42 mM), and incubated in a shaking water bath at 37 degrees C. Initially, there was an increase in TBARS and lipofuscin-like pigments, two well-known end products of PUFA oxidative degradation, whereas PUFAs remained unchanged (incubation time: 1 h). After two or more hours of incubation, marked lipid peroxidation was noted, with the appearance of conjugated dienes and a decrease of PUFAs, indicating that lipid peroxidation had occurred after a lag phase during which TBARS were not produced from PUFAs. This suggests that another OH. target was involved.