Changes In Membrane Composition Research Articles

Omega-3 Fatty Acid Treatment and T2 Whole Brain Relaxation Times in Bipolar Disorder

OBJECTIVE: The authors hypothesized that changes in brain membrane composition resulting from omega-3 fatty acid administration in patients with bipolar disorder would result in greater membrane fluidity, as detected by reductions in T2 values. METHOD: Women with bipolar disorder (N=12) received omega-3 fatty acids for 4 weeks. A cohort of bipolar subjects (N=9) and a group without bipolar disorder (N=12) did not receive omega-3 fatty acids. T2 values were acquired at baseline and after 4 weeks. RESULTS: Bipolar subjects who received omega-3 fatty acids had significant decreases in T2. There was a dose-dependent effect when the bipolar omega-3 fatty acid group was subdivided into high- and low-dose cohorts. CONCLUSIONS: Omega-3 fatty acids lowered T2 values, consistent with the hypothesis that the fluidity of cell membranes was altered. Further studies are needed to clarify the significance of alterations in brain physiology induced by omega-3 fatty acids, as reflected in T2 values.

Mycobacterium tuberculosis and Mycobacterium avium modify the composition of the phagosomal membrane in infected macrophages by selective depletion of cell surface-derived glycoconjugates

The growth of pathogenic mycobacteria in phagosomes of the host cell correlates with their ability to prevent phagosome maturation. The underlying molecular mechanism remains elusive. In a previous study, we have shown that Mycobacterium avium depletes the phagosome membrane of cell surface-derived glycoconjugates (de Chastellier and Thilo, Eur. J. Cell Biol. 81, 17-25, 2002). We now extended these quantitative observations to the major human pathogen, Mycobacterium tuberculosis (H37Rv). At increasing times after infection of mouse bone marrow-derived macrophages, cell-surface glycoconjugates were labelled enzymatically with [3H]galactose. Subsequent endocytic membrane traffic resulted in a redistribution of this label from the cell surface to endocytic membranes, including phagosomes. The steady-state distribution was measured by quantitative autoradiography at the electron microscope level. Relative to early endosomes, with which phagosomes continued to fuse and rapidly exchange membrane constituents, the phagosome membrane was depleted about 3-fold, starting during infection and in the course of 9 days thereafter. These results were in quantitative agreement with our previous observations for Mycobacterium avium. For the latter case, we now showed by cell fractionation that the depletion was selective, mainly involving glycoproteins in the 110-210 kDa range. Together, these results indicated that pathogenic mycobacteria induced and maintained a bulk change in phagosome membrane composition that could be of special relevance for survival of pathogenic mycobacteria within phagosomes.

292CHANGES IN MEMBRANE SULFHYDRYL STATUS OF BOAR SPERMATOZOA BY FREEZING

The processes of cooling and freezing/thawing produce physical and chemical stresses on the sperm membrane that reduce the viability and fertilizing capacities. The cold shock and freezing of spermatozoa are associated with an oxidative stress, with reactive oxygen species (ROS) generation (Chatterjee et al., 2001, Mol. Reprod. Dev. 60, 498–506) and with a significant reduction of the GSH content (Gadea et al., in press). In the processes of capacitation, fertilization and freezing, qualitative and quantitative changes in protein membrane composition occurs, including changes in distribution of sulphydryl groups on the sperm membrane. The aim of this work was to evaluate the changes in the sulfhydryl groups of proteins from the sperm surface after cooling and freezing procedures as a marker of membrane changes. Ejaculate-rich fractions from three mature Pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15°C over 2h (control). Thereafter sperm were centrifuged and diluted in lactose/egg-yolk extender cooled to 5°C over 2h and later frozen with glycerol and equex by classic methodology (Westendorf et al., 1975, Dtsch. Tierärztl. Wschr. 82, 261–267). Sperm parameters were measured in extended semen (control) at 0, 1 and 2h after cold shock at 5°C and after freezing-thawing. The structure of the sperm membrane was evaluated with carboxyfluorescein diacetate/propidium iodide (DCF) (Harrison and Vickers, 1990, J. Reprod. Fert. 88, 343–352), and the sulfhydryl status of proteins from spermatozoa surface are evaluated with fluorescent-staining 5-iodoacetamidofluoresceine (5-IAF) and by acrosome integrity (normal apical ridge, NAR). Some seminal parameters to evaluate functionality such as motility (MOT), forward progressive motility (FPM, 0–5), and mitochondria activity with Rhodamine 123(MIT) were also evaluated. Data from 11 freezing batches were analyzed by one-way ANOVA. When ANOVA revealed a significant effect, values were compared by the Tukey test. The freezing process significantly affected all the sperm parameters studied. Motility was negatively affected from the onset of cooling to 5°C. However, DCF, NAR and 5-IAF were only affected after freezing process. Mitochondria activity decreased in the last period of the cooling procedure (2h) and it was lower after freezing. An inverse significant relation was found between 5-IAF and motility, viability, NAR and mitochondria functionality (P<0.01). These results show that freezing damage produces an alteration in the structure of the sperm membranes (DCF, NAR, 5-IAF) and sperm functionality (motility and mitochondrial). However, only motility (MOT and FPM) was affected by cold shock when lactose/egg-yolk extender was used. Previous studies of cold shock with no cryo-protective medium (BTS) showed a marked effect on sulphydryl membrane characteristics (Marco and Gadea, 2003). These preliminary results in the use of 5-IAF in boar semen showed that freezing produces an alteration in the structure of the sperm membranes, which could be detected by simple fluorescent staining. This research was supported by grant AGL 2000-0485-C02-01.

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

In adult systems, high homocysteine (HoCys) levels inhibit methylation reactions and can induce apoptosis in the central nervous system. In embryos, exogenous HoCys is teratogenic and is associated with neural tube defects. Because, methylation inhibitors and inducers of apoptosis can influence membrane composition, we have studied whether or not embryonic exposure to HoCys influenced membrane phospholipid levels, membrane fatty acid composition, and Caspase-3 activities in embryonic chick brains. Embryonic exposure to HoCys caused reduced brain phosphatidylcholine levels and increased levels of brain phosphatidylethanolamine. Exogenous HoCys also promoted decreased levels of long-chain, unsaturated membrane fatty acids and increased levels of saturated short-chain membrane fatty acids. These HoCys-induced brain membrane changes correlated with HoCys-induced increases in brain Caspase-3 activities, HoCys-induced reductions in brain mass, HoCys-induced reductions in embryo mass, and HoCys-induced reductions in the percentage of embryos that survived to 11 days of development (theoretical stage 37). Thus, HoCys-induced changes in brain membrane composition correlated with HoCys-induced apoptosis and reduced embryo viability.

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

Dietary fatty acids alter blood pressure, behavior and brain membrane composition of hypertensive rats

The beneficial effect of dietary n-3 polyunsaturated fatty acids (PUFAs) on developing hypertension has been repeatedly demonstrated. However, related changes in brain membrane composition and its cognitive correlates have remained unclear. Our study aimed at a comprehensive analysis of behavior and cerebral fatty acid concentration in hypertension after long-term PUFA-rich dietary treatment. Hypertensive and normotensive rats were provided a placebo, or one of two PUFA-enriched diets with a reduced (n-6)/(n-3) ratio for 75 weeks. Exploratory behavior and spatial learning capacity were tested. Systolic blood pressure (BP) was repeatedly measured. Finally, brain fatty acid composition was analyzed by gas chromatography. Hypertensive rats exhibited more active exploration but impaired spatial learning compared to normotensives. Both diets reduced BP, increased PUFA and monounsaturated fatty acid (MUFA) concentration, and reduced saturated fatty acid content in brain. The level of cerebral PUFAs and MUFAs was lower in hypertensive than in normotensive rats. Furthermore, BP positively, while spatial learning negatively correlated with cerebral (n-6)/(n-3) PUFA ratio. We concluded that regular n-3 PUFA consumption could prevent the development of hypertension, but reached only a very delicate improvement in spatial learning. Furthermore, we consider a potential role of metabolically generated MUFAs in the beneficial effects of PUFA supplementation.

Enterococcus faecalisPlasmid pAD1-Encoded Fst Toxin Affects Membrane Permeability and Alters Cellular Responses to Lantibiotics

Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 22

Experimental diabetic neuropathy has a number of features in common with the human disease, including a reduction of nerve conduction velocity (NCV) and Na+, K+‐ATPase activity in nerve fibers, and alteration of the essential fatty acid metabolism leading to changes in membrane composition. N‐3 polyunsaturated fatty acids (PUFA) are beneficial in different pathological conditions including diabetic complications. Since the PUFA composition of cell membranes largely depends on the diet, most studies have supplemented the diet with fish oil (FO), particularly rich in docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or DHA and EPA ethyl esters. We have prepared a new formulation of n‐3 PUFA wax esters (WE) enriched in DHA and EPA that have a bioavailability comparable to oil and esters, are easily digestible and are free from most of the previous drawbacks. The present study was designed to measure Na+, K+‐ATPase activity, NCV, and FA content in plasma phospholipids (PL) and to assess the preventive effect of newly formulated n‐3 PUFA‐WE, and individual EPA and DHA‐WE (as compared with standard and FO‐supplemented diets) on these abnormalities. Diabetes was induced by streptozotocin (STZ, 60 mg/kg/ip). After STZ injection, diabetic and non‐diabetic control rats were fed the standard diet (St) without supplementation or supplemented with either FO or n‐3 PUFA‐WE or individual EPA and DHA‐WE, to reach a daily dose of 0.15 g/kg, the experiment continued for five weeks. Analysis of the FA composition of plasma PL, which better reflects FA intake and content in biological membranes, showed that the sums of n‐3 PUFA were double the St diet after n‐3 supplement of independent of the dietary regimen, whereas those of FA of the n‐6 family were not affected by n‐3 supplementation. Thus, the n‐6/n‐3 ratios were reduced from 15:1 with the St diet to 7:1 for the FO and WE diets. Na+, K+‐ATPase activity was significantly lower in diabetic rats (−30–35%), significantly restored in diabetic rats that received FO, n‐3 PUFA and EPA‐WE supplementation, and partially restored by DHA‐WE (9% lower than with non‐diabetic controls). These effects were partially associated with a significant beneficial effect on NCV which was reduced by 20% in diabetic rats. FO protected against this decrease (3%), n‐3 PUFA‐WE were only partially protective (11%), and EPA and DHA‐WE had scant effect. This study indicates that, like FO, n‐3 PUFA‐EPA and DHA‐WE have beneficial effects on diabetic‐induced alterations in sciatic nerve ATPase+‐ATPase activity and, partially, also on NCV. The newly formulated n‐3 PUFA WE offer a potential advantage over products in current use, on account of their greater stability.

Membrane Pathology in Schizophrenia: Implication for Arachidonic Acid Signaling

Schizophrenia is a major mental disorder with no clearly identified pathophysiology. A variety of theories have been proposed to explain the pathophysiology of schizophrenia. One approach that is finding empirical support is the investigation of membrane composition and function. Evidence to date suggests that there are defects in phospholipid metabolism and cell signaling in schizophrenia. Specifically, low levels of arachidonic acid (AA)-enriched phospholipids have been observed in both central and peripheral tissues. It is well known that changes in membrane composition are associated with a variety of functional consequences. Since AA has many key roles in neural functioning, understanding its significance for the pathophysiology of schizophrenia may lead to novel approaches to improving treatment of schizophrenia. The purpose of this review is thus to explore some of the roles of AA signaling in biological, physiological, and clinical phenomena observed in schizophrenia. Keywords: Schizophrenia, arachidonic acid, phospholipid metabolism, neurotransmission, phosphoinositide signaling system, eicosanoids, endocannabinoids, ethyl eicosapentaenoate supplementation

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema.

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml. kg(-1). min(-1) saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (approximately 4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

Membrane pathology in schizophrenia: implication for arachidonic acid signaling.

Schizophrenia is a major mental disorder with no clearly identified pathophysiology. A variety of theories has been proposed to explain the pathophysiology of schizophrenia. One approach that is finding empirical support is the investigation of membrane composition and function. Evidence to date suggests that there are defects in phospholipid metabolism and cell signaling in schizophrenia. Specifically, low levels of arachidonic acid (AA)–enriched phospholipids have been observed in both central and peripheral tissues. It is well known that changes in membrane composition are associated with a variety of functional consequences. Since AA has many key roles in neural functioning, understanding its significance for the pathophysiology of schizophrenia may lead to novel approaches to improving treatment of schizophrenia. The purpose of this review is thus to explore some of the roles of AA signaling in biological, physiological, and clinical phenomena observed in schizophrenia.

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes.

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios > or = 1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia.

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation.

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.

Mitochondrial adaptation to in vivo polyunsaturated fatty acid deficiency: increase in phosphorylation efficiency.

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (delta psi), mitochondrial and cytosolic ATP/ADP-Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP-Pi ratio being maintained despite the lower delta psi. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

Thyroid hormones and their effects: a new perspective

ABSTRACTThe thyroid hormones are very hydrophobic and those that exhibit biological activity are 3′,5′,3,5‐Ltetraiodothyronine (T4), 3′,5,3‐L‐triiodothyronine (T3), 3′,5′,3‐L‐triiodothyronine (rT3) and 3,5,‐Ldiiodothyronine (3,5‐T2). At physiological pH, dissociation of the phenolic ‐OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non‐ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein‐bound T4 and T3 are maintained in the nanomolar range. The function of thyroid‐hormone‐binding plasma proteins is to ensure an even distribution throughout the body. Various iodothyronines are produced by three types of membrane‐bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones have many effects in vertebrates. It is proposed that there are several modes of action of these hormones.(1) The nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones.(2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes.(3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid‐hormone‐induced membrane rigidification. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellular matrix. The effects of thyroid hormones are reviewed and appear to be combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re‐examination of data from the early 1960s supports a membrane action. Findings from receptor ‘knockout’ mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as ‘vitamone’ ‐like molecules than traditional hormonal messengers.

Thyroid hormones and their effects: a new perspective.

The thyroid hormones are very hydrophobic and those that exhibit biological activity are 3',5',3,5-L-tetraiodothyronine (T4), 3',5,3-L-triiodothyronine (T3), 3',5',3-L-triiodothyronine (rT3) and 3,5',-L-diiothyronine (3,5-T2). At physiological pH, dissociation of the phenolic -OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non-ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein-bound T4 and T3 are maintained in the nanomolar range. The function of thyroid-hormone-binding plasma proteins is to ensure an even distrubtion throughout the body. Various iodothyronines are produced by three types of membrane-bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones. (1) the nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones. (2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes. (3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid-hormone-induced membrane rigidificataion. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellulara matrix. The effects of thyroid hormones are reviewed and appear to b combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re-examination of data from th early 1960s supports a membrane action. Findings from receptor 'knockout' mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as 'vitamone'-like molecules than traditional hormonal messengers.

Inhibition of nitric oxide synthesis increases erythrocyte membrane fluidity and unsaturated fatty acid content

Changes in the lipid composition of the membrane affect its fluidity and function. These variables are altered in various forms of hypertension. Our hypothesis was that the rapid increase in blood pressure (BP) caused by inhibition of nitric oxide production would lead to alterations in membrane fluidity similar to those observed in genetic hypertension. We used N ω-nitro l-arginine methyl ester (L-NAME) and vehicle-treated (3 weeks) Wistar-Kyoto rats to study the effects of nitric oxide synthase (NOS) inhibition on membrane fluidity and lipid composition. Erythrocyte membrane fluidity was measured by fluorescence anisotropy. Membrane lipids were separated using Sep-Pak and thin-layer chromatography. Fatty acid methyl esters were produced and analyzed by gas chromatography–mass spectrometry. N ω-nitro l-arginine methyl ester treatment increased BP and erythrocyte membrane fluidity. The phospholipid and unsaturated fatty acid levels in the membranes from the L-NAME–treated rats were consistent with the increase in fluidity (ie, more unsaturated fatty acid, in particular, arachidonic and docosahexaenoic acid) and a reduction in membrane sphingomyelin content. Fatty acid analysis of individual lipid groups suggested the changes in membrane fatty acid composition may be asymmetric, with the majority of the changes occurring in the outer leaflet. Inhibition of NOS results in changes in membrane composition that may explain the concurrent changes in fluidity. The increased membrane fluidity observed here contrasts with the reduced fluidity observed in genetic hypertension or unchanged fluidity in secondary hypertension. The effects could be related to NOS inhibition or may be a direct effect of L-NAME.

Possible protective effect of kolaviron on CCl4-induced erythrocyte damage in rats.

The possible protective effect of kolaviron on rat erythrocytes following simultaneous administration of kolaviron (100 mg/kg of body weight/day) with carbon tetrachloride CCl4 (1.195 g/kg of body weight/day) by separate intraperitoneal injections was investigated. Kolaviron, a biflavonoid fraction of the defatted alcoholic extract of Garcinia kola seed, inhibits the accumulation of lipid peroxidation products in erythrocytes. A significant reduction (p < 0.05) by about 34%, of lipid peroxidation products was observed in erythrocytes of rats treated simultaneously with CCl4 and kolaviron when compared to CCl4-treated rats. Similarly, the significant increase (p < 0.05) in membrane cholesterol observed in CCl4-treated rats was significantly decreased (p < 0.05) in rats treated simultaneously with CCl4 and kolaviron. Therefore, there was no significant difference (p < 0.05) in cholesterol phospholipid ratio (C/P) of rats treated simultaneously with CCl4 and kolaviron, and the controls. Thus, kolaviron normalizes the CCl4-induced change in erythrocyte membrane composition. In addition, kolaviron antagonizes the effect of CCl4 on the activity of the membrane bound enzyme, Ca2+-ATPase. These results suggest that kolaviron protects erythrocyte membranes from free radical attack, on both lipids and proteins.

Essential fatty acids and the brain: possible health implications

Linoleic and α-linolenic acid are essential for normal cellular function, and act as precursors for the synthesis of longer chained polyunsaturated fatty acids (PUFAs) such as arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), which have been shown to partake in numerous cellular functions affecting membrane fluidity, membrane enzyme activities and eicosanoid synthesis. The brain is particularly rich in PUFAs such as DHA, and changes in tissue membrane composition of these PUFAs reflect that of the dietary source. The decline in structural and functional integrity of this tissue appears to correlate with loss in membrane DHA concentrations. Arachidonic acid, also predominant in this tissue, is a major precursor for the synthesis of eicosanoids, that serve as intracellular or extracellular signals. With aging comes a likely increase in reactive oxygen species and hence a concomitant decline in membrane PUFA concentrations, and with it, cognitive impairment. Neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease also appear to exhibit membrane loss of PUFAs. Thus it may be that an optimal diet with a balance of n-6 and n-3 fatty acids may help to delay their onset or reduce the insult to brain functions which these diseases elicit.

Influence of preculture on the prefreeze and postthaw characteristics of hepatocytes.

Recent studies performed in our laboratory have shown that a brief period of preculture prior to cryopreservation improves the postthaw viability of hepatocytes. The purpose of this investigation is to characterize specific metabolic and biochemical characteristics of the hepatocytes (both frozen and nonfrozen) to help elucidate the role of preculture on the postthaw viability. Fresh and thawed hepatocytes were cultured in a bioartificial liver (BAL) to determine albumin secretion as a function of time in culture. In addition, cell extracts were analyzed using nuclear magnetic resonance (NMR) spectroscopy to quantify changes in cell membrane composition and energetics as a function of time in culture prefreeze and postthaw. The results of these studies showed an increase in albumin concentration in the culture medium with time in culture for the period tested for both fresh and frozen and thawed hepatocytes. NMR spectroscopy of lipid extracts indicates that in vitro culture of hepatocytes results in an increase in cholesterol relative to membrane phospholipid. Moreover, the NMR results also indicate phospholipid interconversion, via specific lipases in cultured hepatocytes, and these changes are consistent with water permeability measurements performed previously. Significant changes in phosphoenergetics were also observed, with the net energy charge for the cells increasing significantly with time in culture. In addition, NMR spectra show increased levels of 6-phosphogluconate, another indicator of the cellular response to the stresses of isolation and ex vivo culture. These results suggest that energetic considerations may be a significant factor in the ability of hepatocytes to survive the stresses of freezing and thawing. Significant shifts in membrane phospholipids may also influence membrane permeability and postthaw survival.