Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA-depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15-min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Acinar inflammatory response to lipid derivatives generated in necrotic fat during acute pancreatitis

Lipids play a role in acute pancreatitis (AP) progression. We investigate the ability of pancreatic acinar cells to trigger inflammatory response in the presence of lipid compounds generated in necrotic areas of peripancreatic adipose tissue (AT) during AP induced in rats by 5% sodium taurocholate. Lipid composition of AT was analyzed by HPLC–mass spectrometry. Acinar inflammatory response to total lipids as well as to either the free fatty acid (FFA) fraction or their chlorinated products (Cl-FFAs) was evaluated. For this, mRNA expression of chemokine (C-C motif) ligand 2 (CCL2) and P-selectin as well as the activation of MAPKs, NF-κB and STAT-3 were analyzed in pancreatic acini. Myeloperoxidase (MPO) activity, as an inducer of Cl-FFA generation, was also analyzed in AT. MPO activity significantly increased in necrotic (AT-N) induced changes in lipid composition of necrotic fat, such as increase in FFA and phospholipid (PL) content, generation of Cl-FFAs and increases in saturated FFAs and in the poly-:mono-unsaturated FFA ratio. Total lipids from AT-N induced overexpression of CCL2 and P-selectin in pancreatic acini as well as MAPKs phosphorylation and activation of NF-κB and STAT3. FFAs, but not Cl-FFAs, up-regulated CCL2 and P-selectin in acinar cells. We conclude that FFAs are capable of up-regulating inflammatory mediators in pancreatic acini and given that they are highly produced during AP, mainly may contribute to the inflammatory response triggered in acinar cells by fat necrosis. No role is played by Cl-FFAs generated as a result of neutrophil infiltration.     

Free fatty acid effects on mitochondrial permeability: an overview

A variety of experimental conditions elicit increases in mitochondrial permeability that can be differentiated from the classic cyclosporin A (CsA)-sensitive mitochondrial permeability transition (MPT). For example, butylated hydroxytoluene, signal peptides, and the hormone thyroxine have been shown to promote increases in mitochondrial permeability that are CsA-insensitive. Our laboratory has recently demonstrated that palmitic acid, a saturated 16-carbon free fatty acid (FFA), can also open a CsA-insensitive pore. This nonclassic permeability transition (NCPT) is further distinguished by a nonselective dependence on divalent cations and by spontaneous closure. To determine if induction of the NCPT is specific to palmitic acid and to resolve conflicting reports as to the mechanisms by which FFAs alter mitochondrial permeability, we examined in detail mitochondrial swelling induced by FFAs that differ in chain length and degree of saturation. The following results were obtained: (1) In the presence of modest Ca2+ concentrations (75 nmol/mg protein), medium-chain FFAs (C12-C18) were more effective in eliciting mitochondrial swelling than were shorter or longer FFAs; medium-chain alkanols and amines had no effect. (2) Under these conditions, saturated FFAs induced CsA-insensitive swelling with all the characteristics of the NCPT, while unsaturated FFAs triggered the MPT. (3) When matrix Ca2+ concentration was further elevated, unsaturated FFAs triggered the NCPT. (4) Mitochondrial swelling induced by saturated FFAs was inhibited by unsaturated FFAs but not by other saturated FFAs or medium-chain alkanols. These results suggest that ambient conditions can greatly influence the nature of the increase in mitochondrial permeability induced by FFAs. They are also consistent with our earlier proposal that Ca2+ (or Sr2+) binding to FFAs in the inner leaflet of the inner mitochondrial membrane underlies the NCPT.     

Sarcolemmal phospholipid fatty acid composition and permeability

In this study, the mechanism of ischaemia-induced increased sarcolemmal permeability, as manifested by release of intracellular enzymes, was investigated. The role of changes in the sarcolemmal phospholipid bilayer in this process was evaluated by experimental modulation of the phospholipid fatty acid composition. The isolated perfused rat heart subjected to low-flow hypoxia, was used as a model of global ischaemia. Glucose as well as saturated (palmitate) and unsaturated (linoleate) long-chain fatty acids were used as substrates. Hearts perfused with palmitate or linoleate (1.5 mM, fatty acid/albumin ratio, 3.4) showed a significantly higher rate of lactate dehydrogenase release in both control and ischaemic conditions than hearts perfused with glucose (10 mM). Lactate dehydrogenase release in the fatty acid-perfused hearts was associated with a significant increase in the percentage unsaturation of the sarcolemmal phospholipid fatty acids. Glucose-perfused hearts, on the other hand, showed only minor changes in the sarcolemmal phospholipid fatty acid composition. Attempts to correlate enzyme release directly with an increase in the percentage unsaturation of phospholipid fatty acids failed, since enzyme release was also stimulated in control fatty-acid-perfused hearts which (when compared with glucose) contained a higher percentage saturated phospholipid fatty acids. The results suggest that myocardial ischaemia, apart from changes in the sarcolemmal phospholipid fatty acid composition, also induces several other changes in sarcolemmal composition (e.g., cholesterol loss) which may affect is permeability for macromolecules.     

Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. A role for cardiolipin

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.     

Brain free fatty acids, edema, and mortality in gerbils subjected to transient, bilateral ischemia, and effect of barbiturate anesthesia

Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono- and polyunsaturated FFAs increased approximately 12-fold in total at the end of a 30-min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic acid.     

Alterations in lipid and calcium metabolism associated with seizure activity in the postischemic brain

Transient ischemia is known to lead to a long-lasting depression of cerebral metabolic rate and blood flow and to an attenuated metabolic and circulatory response to physiological stimuli. However, the corresponding responses to induced seizures are retained, demonstrating preserved metabolic and circulatory capacity. The objective of the present study was to explore how a preceding period of ischemia (15 min) alters the release of free fatty acids (FFAs) and diacylglycerides (DAGs), the formation of cyclic nucleotides, and the influx/efflux of Ca(2+), following intense neuronal stimulation. For that purpose, seizure activity was induced with bicuculline for 30 s or 5 min at 6 h after the ischemia. Extracellular Ca(2+) concentration (Ca(2+)(e)) was recorded, and the tissue was frozen in situ for measurements of levels of FFAs, DAGs, and cyclic nucleotides. Six hours after ischemia, the FFA concentrations were normalized, but there was a lowering of the content of 20:4 in the DAG fraction. Cyclic AMP levels returned to normal values, but cyclic GMP content was reduced. Seizures induced in postischemic animals showed similar changes in Ca(2+)(e), as well as in levels of FFAs, DAGs, and cyclic nucleotides, as did seizures induced in nonischemic control animals, with the exception of an attenuated rise in 20:4 content in the DAG fraction. We conclude that, at least in the neocortex, seizure-induced phospholipid hydrolysis and cyclic cAMP/cyclic GMP formation are not altered by a preceding period of ischemia, nor is there a change in the influx/efflux of Ca(2+) during seizure discharge or in associated spreading depression.     

Effect of the pattern of elevated free fatty acids on insulin sensitivity and insulin secretion in healthy humans.

In order to investigate whether the pattern of elevated free fatty acids (FFAs) has any effect on insulin sensitivity and insulin secretion in humans, we produced 2 distinct serum FFA patterns (PT 1 and 2) by infusing 6 healthy volunteers with 2 different lipid emulsions plus heparin for 24 hours. A hyperglycemic clamp (approx. 8 mM, 140 min) was performed before and 5 and 24 hours after both lipid infusions to determine insulin sensitivity and insulin secretion simultaneously. Total FFAs had increased comparably by 24 hours (2020+/-268 microM in PT 1) and (1812+/-154 microM in PT 2, p =0.24). Serum PT 1 contained 66% saturated FFAs plus monoenes and 34% polyenes, while PT 2 contained 80% saturated FFAs plus monoenes and 20% polyenes. Thus, the ratio of polyunsaturated to saturated plus monoenes was about 0.5 in PT 1 vs. 0.25 in PT 2. In PT 1, the insulin sensitivity index (ISI) decreased by 20 +/- 7% and 27 +/- 10% from basal after 5 and 24 hours, respectively. In PT 2, the ISI decreased significantly more after 5 (41+/-7%, p = 0.008) and 24 hours (52+/-6%, p = 0.005). In contrast, different phases of insulin secretion did not change during the lipid infusion and did not vary between the two FFA profiles. In conclusion, these findings provide preliminary evidence that the composition of elevated serum FFAs influenced insulin sensitivity in humans. The FFA pattern low in polyunsaturated FFAs reduced insulin sensitivity more than the pattern high in polyunsaturated FFAs. In contrast, no effect on insulin secretion was observed.     

Changes of free fatty acids and acyl-CoAs in rat brain hippocampal slice with tetraethylammonium-induced long-term potentiation

We investigated the role of acyl-CoAs during induction and maintenance of long-term potentiation in rat brain hippocampus. Changes of acyl-CoA and free fatty acids (FFA) in hippocampus were measured during tetraethylammonium (TEA)-induced LTP. Results indicated that concentrations of acyl-CoAs and FFAs in slices were changed during TEA-induced LTP and 16:0-CoA and 18:0-CoA were increased in the early phase of stimulation, whereas free fatty acids in this phase were rather decreased. The increase of 20:4-CoA was delayed more than saturated acyl-CoAs. To examine the role of acyl-CoA in LTP of evoked transmitter release, we measured the glutamate release from hippocampal slice with the addition of acyl-CoA using glutamate electrode. Acyl-CoA (16:0-, 18:1-, and 20:4-CoA) could enhance glutamate release in hippocampal slice. It is suggested that saturated acyl-CoAs may play a functional role in the early phase of LTP.     

Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-βH1 beta-cells

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-βH1 beta-cells.Exposure of human EndoC-βH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs.The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

No in vitro effects of fatty acids on glucose uptake, lipolysis or insulin signaling in rat adipocytes.

Elevated plasma levels of free fatty acids (FFA) can produce insulin resistance in skeletal muscle tissue and liver and, together with alterations in beta-cell function, this has been referred to as lipotoxicity. This study explores the effects of FFAs on insulin action in rat adipocytes. Cells were incubated 4 or 24 h with or without an unsaturated FFA, oleate or a saturated FFA, palmitate (0.6 and 1.5 mM, respectively). After the culture period, cells were washed and insulin effects on glucose uptake and lipolysis as well as cellular content of insulin signaling proteins (IRS-1, PI3-kinase, PKB and phosphorylated PKB) and the insulin regulated glucose transporter GLUT4 were measured. No significant differences were found in basal or insulin-stimulated glucose uptake in FFA-treated cells compared to control cells, regardless of fatty acid concentration or incubation period. Moreover, there were no significant alterations in the expression of IRS-1, PI3-kinase, PKB and GLUT4 following FFA exposure. Insulin's ability to stimulate PKB phosphorylation was also left intact. Nor did we find any alterations following FFA exposure in basal or cAMP-stimulated lipolysis or in the ability of insulin to inhibit lipolysis. The results indicate that oleate or palmitate does not directly influence insulin action to stimulate glucose uptake and inhibit lipolysis in rat fat cells. Thus, lipotoxicity does not seem to occur in the fat tissue itself.     

Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

Background

In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.

Methods

Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.

Results

The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.

Conclusions

Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.     

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.