Lipidomic analysis of human placental syncytiotrophoblast microvesicles in adverse pregnancy outcomes.

PROBLEM: Syncytiotrophoblast microvesicles (STBM) are shed from placenta into the maternal circulation. STBM circulate in increased amounts in adverse pregnancies, e.g., preeclampsia and recurrent miscarriages (RM). Recently dysregulation of lipid metabolites has been proposed to be associated with their pathogenesis. Lipid composition of STBM in healthy and adverse pregnancies remains unknown. OBJECTIVE: To determine lipid composition of STBM and whether STBM lipid composition differs in pathologic and normal pregnancies. STUDY DESIGN: Patients with Preeclampsia (n = 6) or history of RM (n = 9) (>2 consecutive losses <20 weeks) and gestational age-matched normal pregnant controls (same number as cases) were recruited. STBM were prepared from placental explant culture supernatant. Lipid profiling of STBM was performed by mass spectrometry in combination with liquid chromatography. We quantified ∼200 lipids in STBM including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucosylceramide, GluCer; ganglioside mannoside 3, GM3); (iii) free cholesterol and cholesteryl esters, CE. RESULTS: The major lipid classes in STBM were SM, Chol, PS, PC and PI, along with PA and GM3 enrichments. SM/PC ratio showed a unique reversal (3:1) compared to that normally found in human cells or plasma. Level of total PS was significantly upregulated (p < 0.005) in preeclampsia patients, while PI (p < 0.0005), PA (p < 0.005), and GM3 (p < 0.05) were significantly downregulated. Similar trends were obtained in RM. CONCLUSIONS: Differential lipid expression of STBM in preeclampsia or RM includes those that are implicated in immune response, coagulation, oxidative stress, and apoptosis.

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain - evidence of increased phospholipase A(2) activity.

Recent studies suggest that CNS phospholipase A(2) (PLA(2) ) isoforms play a role in nociception, but until now, direct evidence of increased brain PLA(2) activity during allodynia or hyperalgesia is lacking. The present study was carried out, using lipidomics or systems wide analyses of lipids using tandem mass spectrometry, to elucidate possible changes in rat brain lipids after inflammatory pain induced by facial carrageenan injection. The caudal medulla oblongata showed decreases in phospholipids including phosphatidylethanolamine and phosphatidylinositol species, but increases in lysophospholipids, including lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine, indicating increased PLA(2) activity and release of arachidonic acid after facial carrageenan injection. These changes likely occur in the spinal trigeminal nucleus which relays nociceptive input from the orofacial region. High levels of sPLA(2) -III, sPLA(2) -XIIA, cPLA(2) and iPLA(2) mRNA expression were detected in the medulla oblongata. Increase in sPLA(2) -III mRNA expression was found in the caudal medulla of carrageenan-injected rats, although no difference in sPLA(2) -III protein expression was detected. The changes in lipids as determined by lipidomics were therefore consistent with an increase in PLA(2) enzyme activity, but no change in enzyme protein expression. Together, these findings indicate enhanced PLA(2) activity in the caudal medulla oblongata after inflammatory orofacial pain.

Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (∼50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.

The effect of APOE genotype on brain levels of oxysterols in young and old human APOE epsilon2, epsilon3 and epsilon4 knock-in mice.

Despite apolipoprotein E's important role in cholesterol transport and metabolism in the brain as well as its influence on Alzheimer's disease, the impact of the human APOE genotype on cholesterol metabolism in brain has not been fully examined. This study was carried out to investigate APOE genotype effects on oxysterols measured. In this study the measurement of cholesterol and several oxysterols in the brains of human APOE epsilon2, epsilon3 and epsilon4 knock-in mice at 8 weeks and 1 year of age using gas chromatography mass spectrometry (GC-MS) demonstrated no APOE genotype or age effect on total brain cholesterol and the oxysterol 24-hydroxycholesterol. The level of 27-hydroxycholesterol was elevated in 1 year old animals for all APOE genotypes. Interestingly, lathosterol an indicator of cholesterol synthesis was significantly reduced in the 1 year old animals for all APOE genotypes. APOE epsilon4 expressing mice exhibited statistically lower levels of lathosterol compared to APOE epsilon2 in both the young and old mice. Oxidized cholesterol metabolites were significantly lower in APOE epsilon2 mice compared to other genotypes at 8 weeks old. Although minimal differences were observed between APOE E3 and E4 knock-in (KI) mice, these findings indicate that there are some clear APOE genotype specific effects on brain cholesterol synthesis and associated metabolic pathways, particularly in APOE epsilon2 KI mice.

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals.

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.

Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6-pentakisphosphate 3-phosphatase activity.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375-13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P(5) (K(m)=7.1 microM) and PtdIns(3,4,5)P(3) (K(m)=26 microM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P(5) levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P(5) to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation.

The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation.

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.

Functional characterization of a mammalian Sac1 and mutants exhibiting substrate-specific defects in phosphoinositide phosphatase activity.

The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

Essential role of phosphoinositide metabolism in synaptic vesicle recycling.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Proton induced vesicle fusion and the isothermal lalpha-->HII phase transition of lipid bilayers: a 31P-NMR and titration calorimetry study.

The proton-induced isothermal fusion of unilamellar lipid vesicles (Duzgunes et al., Biochemistry 24 (1985) 3091-3098) is compared with the lamellar (Lalpha)-->hexagonal (HII) phase transition of multilamellar lipid dispersions. Both lipid systems are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and oleic acid (OA) at a 7:3 molar ratio. Using solid-state phosphorus-31 nuclear magnetic resonance (31P-NMR) it is demonstrated that the multilamellar lipid dispersions are in the bilayer state at physiological pH and undergo a Lalpha-->HII phase transition between pH 6.3 and 5.7. This phase transition can also be induced at constant pH by increasing the temperature. The midpoint of the temperature-induced Lalpha-->HII transition is Th=56 degrees C (at pH 7.4) and the corresponding transition enthalpy is DeltaH=0. 7+/-0.1 kcal/mol as determined with differential scanning calorimetry. Both the proton-induced and the temperature-induced phase transition can be completely inhibited by addition of 30 mol% of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC). In a second set of experiments unilamellar vesicles are prepared either by sonication or by extrusion through polycarbonate filters at pH 7. 4 and are titrated into buffer at pH 5.7. The proton-induced fusion of the lipid vesicles is monitored with isothermal titration calorimetry, light scattering and fluorescence spectroscopy. The fusion reaction is characterized by an endothermic enthalpy of DeltaH=0.5+/-0.2 kcal/mol (at 28 degrees C). The fusion enthalpy is independent of the vesicle diameter and is only slightly reduced by an increase in temperature to 50 degrees C. Vesicle fusion is accompanied by an increase in light scattering, indicating the formation of larger lipid structures. The transition from unilamellar vesicles to fused lipid structures occurs in the same narrow pH range of 6.3-5.7 as observed for the Lalpha-->HII transition of multilamellar dispersions. Vesicle fusion can be inhibited with 30% LPC. The virtually identical set of parameters found for the Lalpha-->HII phase transition and the vesicle fusion reaction suggests that vesicle fusion also entails a Lalpha-->HII phase transition.

Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation.

The interaction of the antibiotic magainin 2 amide (M2a) with lipid bilayers was investigated with high-sensitivity titration calorimetry. The enthalpy of transfer of the cationic M2a to negatively charged small unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (75:25, mol/mol) was measured as delta H = -17.0 +/- 1 kcal/mol of peptide. The adsorption isotherm was determined by injecting lipid vesicles into peptide solutions at low peptide concentrations (cPo < 7 microM). The apparent partition coefficient was Kapp approximately 1.2 x 10(4) M-1 at a peptide equilibrium concentration of 1 microM but decreased with increasing peptide concentration. The hydrophobic partitioning of M2a into the lipid membrane is modulated by electrostatic effects that arise from the attraction of the positively charged peptide to the negatively charged membrane. Using the Gouy-Chapman theory to correct for electrostatic attraction, the experimental binding isotherms can be explained with an intrinsic (hydrophobic) partition coefficient of K = 55 +/- 5 M-1 and an effective peptide charge of z = 3.7-3.8. The free energy of binding is delta G = -4.8 kcal/mol. At peptide concentrations cPo > approximately 7 microM, a second effect comes into play, and the titration enthalpies can no longer be explained exclusively by peptide partitioning. The first few injections produce enthalpies of reaction which are distinctly smaller than expected from a pure partition equilibrium, followed by a series of injections with reaction heats larger than expected. After subtracting the enthalpic contribution due to partitioning, the residual enthalpies are endothermic for the first few injections, and exothermic for the consecutive steps. Furthermore, the endothermic excess heat is compensated exactly by the exothermic excess heat; i.e., the excess heat consumed in the first part of the titration experiment is returned during the second part. Endothermic excess enthalpies are observed for total molar peptide-to-lipid ratios of P/L > approximately 3.0%, whereas exothermic excess heats were seen for 0.7% < P/L < 3.0%. Below P/L < approximately 0.7%, the binding follows the partition equilibrium. Based on earlier spectroscopic evidence, it is suggested that magainin 2 amide binds to the lipid membrane and forms pores at high peptide-to-lipid ratio, this process being characterized by an endothermic reaction enthalpy. Pore formation is reversed with increasing lipid concentration, and the peptide pores disintegrate. The limiting peptide-to-lipid ratio deduced from titration calorimetry for M2a pore formation is in excellent agreement with spectroscopic methods. The enthalpy of pore formation amounts to delta H = +6.2 +/- 1.6 kcal/mol peptide or delta H approximately 25-45 kcal/mol pore if the pore is comprised of 4-7 peptide molecules.

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.

Paclitaxel partitioning into lipid bilayers.

Paclitaxel (taxol) is diterpenoid anticancer drug with a new mechanism of cytostatic action. It is under investigation in clinical trials for treatment of various types of human cancer. A major difficulty in developing paclitaxel as a chemotherapeutic agent in its poor water solubility. In order to improve the bioavailability of paclitaxel, novel vehicle systems such as mixed micelles or liposome-based formulations are being developed. In this study we determined the partition coefficient of paclitaxel partitioning into small unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine using two different methods, namely high-sensitivity titration calorimetry and fluorescence spectrometry. We measured a partition coefficient of Kp approximately equal to 9,500 M-1, a partition enthalpy of Delta H = -25 +/- 3 kcal mol-1 and a free energy of binding of Delta G = -7.9 kcal mol-1. The binding reaction is enthalpy-driven, which can be explained by van der Waals interactions between the hydrophobic drug and the strong temperature dependence of the partition equilibrium. A temperature increase of 10 degrees C reduces the paclitaxel solubility in the lipid phase by a factor of 4.