Fatty Acid-free Albumin Research Articles

Advances in the use of the fluorescent probe fura-2 for the estimation of intrasynaptosomal calcium.

Fura-2 has been used to measure intracellular Ca2+ with great success in a variety of cell and subcellular preparations, including synaptosomes. There is, however, a great deal of variability in the reported estimates of resting intrasynaptosomal Ca2+ ([Ca2+]i). Fura-2 AM is highly lipophilic and passes readily across the plasma membrane into the cytoplasm, where it is de-esterified and trapped. The lipophilicity of fura-2, however, promotes the formation of micelles in aqueous media, which may impede the passage of the probe across cell membranes. Our results suggest that some of the variability in the reported [Ca2+]i estimates may be related to fura-2 de-esterification and loading efficiencies. The use of the nonionic detergent pluronic F-127 is recommended to prevent the formation of fura-2 micelles. The use of a detergent is not always an acceptable practice, however, especially in studies in which detergent-lipid interactions may influence membrane parameters. We found that fatty acid free bovine serum albumin (BSA) (0.25%) greatly increases the intrasynaptosomal concentration of the probe, resulting in a significant increase in the signal-to-noise (S/N) ratio. The mechanism appears to be independent of effects of BSA on synaptosomal integrity and directly related to the prevention of fura-2 micelle formation, as evidenced by light spectroscopic scattering measurements. Thus, BSA appears to keep the probe in a form that crosses the synaptic plasma membrane more readily. The effectiveness of BSA in improving the loading of fura-2 into synaptosomes was comparable to the detergent pluronic F-127, making it possible to measure [Ca2+]i without compromising membrane integrity.

Nonesterified fatty acids and lipid peroxidation.

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidation of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.

Death of serum-free mouse embryo cells caused by transforming growth factor beta 1 and effects of nutritional factors.

Transforming growth factor beta 1 (1 ng/ml) caused death of serum-free mouse embryo cells cultured in a medium consisting of a 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's F12 medium supplemented with fibronectin, insulin, transferrin, epidermal growth factor, and high density lipoprotein. Cell death occurred in the presence of polyunsaturated fatty acids including linoleic acid in the absence of selenium. The death could be reversed by adding alpha-tocopherol to the culture indicating a mechanism involving fatty acid peroxidation. Butylated hydroxytoluene was a poor suppressor of cell death in contrast to alpha-tocopherol. High density lipoprotein and fatty acid-free albumin also suppressed cell death at the level of 20 micrograms/ml and 1 mg/ml, respectively. Transforming growth factor beta 1 also caused a low rate of cell growth after heat treatment of the cells at 45 degrees C.

Arachidonate Transfer Between Platelets and Lipoproteins

Although platelets have specific bindingsites for LDL and HDL, it is doubtful whether lipoproteins modulate platelet functions via receptor-mediated processes. We investigated platelet-lipoprotein interaction during prolonged incubation with concentrations of LDL and HDL that saturate the bindingsites within a few minutes. When [3H]arachidonate-labeled human platelets were incubated for 4 h with lipoproteins, part of the 3H-radioactivity transferred to LDL and to a lesser extent to HDL. The transfer was temperature-sensitive, unaffected by modification of lysine in LDL or indomethacin treatment of the platelets, and almost irreversible. [3H]arachidonate transfer to lipoproteins could be mimicked by incubating platelets with a high concentration of fatty acid free albumin. This showed, that the loss of 3H-radioactivity reflected a decrease in endogenous arachidonate, leading to impaired aggregation, secretion and thromboxane B2 formation in platelets after stimulation with thrombin but not with arachidonate. Thus, the decrease in platelet functions seen after long incubation with HDL is caused by depletion of platelet arachidonate. Despite an even stronger arachidonate depletion by LDL, this lipoprotein initiated arachidonate metabolism and secretion independent of specific binding sites for LDL on the platelet. Surprisingly, the major part of the secretion was preserved when the formation of prostaglandin endoperoxides/thromboxane A2 was inhibited with indomethacin. These findings argue against a role for LDL and HDL receptors in the modulation of platelet functions and are more in favor of lipid exchange processes between platelets and lipoproteins.

Role of phospholipids in the lipophorin particles of Rhodnius prolixus.

The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.

Inhibition of CD3-induced Ca 2+ signals in Jurkat T-cells by myristic acid

Stimulation of T lymphocytes with antibodies against the T cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca 2+. Here we show that treatment with 20 μM free myristic acid completely inhibits this Ca 2+ signal and the cellular proliferation in Jurkat T cells. Also lauric acid inhibited cell growth while its blocking effect on the Ca 2+ signal was weaker than that of myristic acid. Other saturated free fatty acids were inactive. The inhibitory effect of myristic acid could be reversed by the addition of fatty acid free albumin, which will bind the fatty acid. Myristic acid, but not its methyl ester, inhibited both the anti-CD3-induced Ca 2+ influx across the cell membrane and Ca 2+ release from intracellular stores, but not the formation of inositol phosphates. In contrast, thapsigargin-induced release of Ca 2+ from the same intracellular stores was unaffected by myristic acid. Thus, myristic acid specifically blocks T cell antigen receptor-CD3 induced Ca 2+ mobilization in T cells.

Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: Comparison between gas chromatographic and radiometric assays

A simple gas chromatographic method for the assay of phospholipase A 2 (PLA 2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA 2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [ 3H]- or [ 14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA 2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA 2 activity in cell-free preparations derived from physically disrupted human neutrophils.

Ca 2+ entry in T cells is activated by emptying the inositol 1,4,5-trisphosphate sensitive Ca 2+ pool

Using α-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca 2+ from the inositol 1,4,5-trisphosphate (IP 3)-sensitive Ca 2+ pool independently of IP 3 production [10] and inhibit Ca 2+ influx [11], the relationship between Ca 2+ mobilization from intracellular stores and Ca 2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 μM ALA to deplete the IP 3-sensitive Ca 2+ pool. When the intracellular free Ca 2+ concentration ([Ca 2+] i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca 2+] i in the absence of inositol phosphates' formation. This sustained increase in [Ca 2+] i was insensitive to protein kinase C activation but was inhibited by Ni 2+ ions. The extent of Ca 2+ inflex was found to be correlated to the amount of Ca 2+ initially discharged from the IP 3-sensitive Ca 2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex or the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca 2+] i increase (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca 2+ entry in T cells is activated by emptying of the IP 3-sensitive Ca 2+ pool which can be dissociated from inositol phosphate production. The rate of Ca 2+ influx appears to be closely correlated to the initial discharge of Ca 2+ from the IP 3-sensitive Ca 2+ pool, suggesting that Ca 2+ may first enter the depleted pool and then is released into the cytosol.

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with cristalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.

Effects of phospholipases, proteases and neuraminidase on ?-hydroxybutyrate binding sites

gamma-Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A2 and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.

Electron transfer facilitated by superoxide dismutase: A model for membrane redox systems?

Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms. Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer. In the presence of albumin:fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron. To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase ( Cu Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide. Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C. By contrast, neither inactive Cu Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C. These results suggest that, in some circumstances, Cu Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu Zn SOD. If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide ‘detoxify-ing’ agent.

A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine

The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.

Metmyoglobin Promotes Arachidonic Acid Peroxidation at Acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.

Hemodialysis and red cell cation transport in uremia: Role of membrane free fatty acids

Active and facilitated cation transport in erythrocytes of uremic patients may be improved acutely by hemodialysis, although the mechanisms remain unknown. As nonesterified fatty acids (NEFA) can affect Na+ pump activity in vitro, changes in plasma and red cell membrane NEFA content following a single hemodialysis procedure were examined and compared with acute changes in erythrocyte cation flux rates in 34 hemodialysis patients. In nonsodium-loaded cells, small changes in Na+ pump flux with dialysis did correlate with changes in intracellular Na+ content (r = 0.59; N = 17; P less than 0.01). On average, neither maximal Na+ pump activity nor Na+/Li+ counter-transport flux improved with dialysis, but Na+/K+/Cl- cotransport rates rose 25% post-dialysis (P less than 0.02). Plasma NEFA levels rose 87% following hemodialysis but erythrocyte membrane NEFA content declined by 23% (P less than 0.001). Importantly, 24 of the 34 subjects studied had a decrease in erythrocyte membrane NEFA content of greater than 10%, and in these patients, the fall in membrane NEFA correlated with an increase in ouabain-sensitive Na+ efflux (r = 0.564; P less than 0.01). The effects of hemodialysis on both erythrocyte NEFA content and Na+ pump flux could be reproduced by incubating pre-dialysis cells in fatty acid-free albumin. We conclude that acute changes in membrane NEFA may modulate active cation transport in uremic erythrocytes.

Demonstration of the requirements for cholesterol or low density lipoprotein in the antibody response of murine lymphocytes in serum-free culture.

We report the essential role of lipid in the development of a serum-free culture medium that supports the primary antibody response by murine lymphocytes in vitro. In our preceding paper, we have reported that β-cyclodextrin (β-CD) is effective as a serum-substitute in murine lymphocyte cultures [H. Ohmori and I. Yamamoto, Eur. J. Immunol., 17, 79 (1987)]. At first, we employed RPMI-1640 that was supplemented with fatty acid-free albumin, insulin, transferrin, and β-CD as the basal serum-free medium (BSF medium). BSF medium supported the antibody response less than 30 % as efficiently as 10% fetal calf serum (FCS)-containing medium. When ether-extracts of FCS (FCS ex.) was added to BSF medium, the antibody response was enhanced by approximately 2-fold. FCS ex. was purified by silica gel thin-layer chromatography. The analyses of the purified active component by high performance liquid chromatography and mass spectrometry revealed it to be cholesterol. Authentic cholesterol or low density lipoprotein enhanced the antibody response as efficiently as FCS ex. in BSF medium. The same level of the antibody response as that in 10 % FCS-containing medium was attained when low density lipoprotein was added to BSF medium in combination with i -a la nine, putrescine, and pyruvate.

Effects of hypoxia on lipolysis in isolated rat myocardial cells

The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO4, 1 mM DL-carnitine, 1.5 mM CaCl2 and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N2-5% CO2. Control (normoxic) incubations were carried out under air-5% CO2 atmosphere. Basal glycerol release increased from 46.6 +/- 3.0 nmol/10(6) cells.30 min in normoxia to 64.5 +/- 4.3 nmol/10(6) cells.30 min in hypoxia (p less than 0.05). Addition of isoprenaline (10 microM) resulted in a significant (p less than 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 +/- 2.5 nmol/10(6) cells.30 min) than in normoxia (12.2 +/- 2.7 nmol/10(6) cells.30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 microM), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.

Influence of albumin and non-esterified fatty acids on serum prostacyclin binding in pregnancy

We investigated the etiology of the previously documented decrease in serum prostocyclin binding during pregnancy. Addition of albumin to the serum of pregnant women failed to raise binding to non-pregnant levels. Pregnancy serum bound significantly more prostacyclin following the removal of non-esterified fatty acids and the addition of fatty acid free albumin resulted in a rise in binding to non-pregnant levels. We conclude that serum protein prostacyclin binding is affected by both albumin concentration and non-esterified fatty acids.

Lack of evidence for a hepatic peroxisome proliferator receptor and an explanation for the binding of hypolipidaemic drugs to liver homogenates

The existence of a postulated hepatic receptor responsible for the peroxisomal proliferation induced in rodents by hypolipidaemic drugs has been investigated. [ 3H]-nafenopin and [ 3H]-ciprofibrate were used as labelled ligands and two competitive binding assays, using either a charcoal-dextran or a hydroxylapatite method, were developed to investigate potential binding. In both assay systems, specific displaceable binding of either nafenopin or ciprofibrate to whole homogenate, microsomal and cytosolic fractions of rat liver could not be detected in a variety of buffer systems. A positive control of ligand binding to bovine serum albumin indicated the validity of the binding assays used. In addition, both nafenopin and ciprofibrate exhibited displaceable binding to serum albumin using the hydroxylapatite binding assay and a Scatchard analysis of the binding of [ 3H]-nafenopin to fatty acid free rat serum albumin yielded a dissociation constant of 5.2 × 10 −7 M and 86 pmol of ligand bound per mg protein. Taken collectively, our data strongly argues against the existence of a specific hepatic peroxisome proliferation receptor and indicates that the peroxisome proliferating hypolipidaemic drugs bind to serum albumin and possibly to other cellular proteins not involved in the activation of genes necessary for peroxisome proliferation.

Extraction of an erythrotropin-like factor from bovine serum albumin (Cohn fraction V).

Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted in the isolation of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors.

Effects of therapeutic agents in a rabbit model of Reye syndrome

Six potential therapeutic agents were evaluated in an experimental model simulating Reye syndrome produced by infusion of the short-chain fatty acid, sodium octanoate, into rabbits. Administration of carnitine, dexamethasone, or fatty acid-free albumin resulted in prolongation of survival, less rise in intracranial pressure, and amelioration of some of the metabolic abnormalities found typically during octanoate infusion. Treatment with pentobarbital or dimethyl sulfoxide prevented intracranial pressure elevations but had no protective effect on survival. Hypertonic glucose administration produced no improvement in any of the parameters studied.