Gradual Change or Phase Transition: Characterizing Fluid Lipid-Cholesterol Membranes on the Basis of Thermal Volume Changes

AbstractCholesterol has been reported to govern biomembrane permeability, elasticity, and the formation of lipid rafts. There has been a controversy whether binary lipid-cholesterol membranes should better be described in terms of a phase separation (liquid-ordered and liquid-disordered phases) or of gradual changes in largely homogeneous membranes. We present a new approach for detecting and characterizing phase equilibria in colloidal dispersions using pressure perturbation calorimetry (PPC). We apply this to the study of the thermal expansivity of mixtures of 1-palmitoyl-2-oleoyl sn-glycero-3-phosphatidylcholine (POPC) and cholesterol as a function of composition and temperature. We show that cholesterol can condense lipids not only laterally (with respect to interfacial area) but also in volume. A quantitative comparison with expansivity curves simulated assuming either phase separation or random mixing within one phase reveals that the real system shows an intermediate behavior due to submicroscopic demixing effects. However, both models yield consistent system parameters and are thus found to be useful for describing the systems to a similar approximation. Accordingly, one cholesterol may condense 3±1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine molecules by ∼−(1.4±0.5) vol % at 2°C; both absolute values decrease with increasing temperature

Quantifying intermolecular interactions as a basis of domain formation in membranes

Within the last two decades the field of membrane biology has witnessed an increased interest in the function and organization of membrane lipids with a particular focus on the possibility of these to demix into separate domains. The present thesis aimed at providing quantitative information about intermolecular interactions that may be responsible for the formation of such lipid domains in membranes. Vesicular lipid model systems mimicking the composition of the plasma membrane were biophysically characterized by means of modern microcalorimetric techniques as a function of temperature and in the presence (or absence) of detergents. For the formation and/or existence of one specific type of lipid domain, so called lipid rafts, that are under intense scrutiny at present, cholesterol is reasoned to be of paramount importance. To study differential interactions of cholesterol with different lipids, three independent experimental assays for isothermal titration calorimetry (ITC) in conjunction with a novel mathematical formalism to model these were introduced. By means of reversible complexation with methylated–�–cyclodextrin (cyd), sufficient amounts of the hydrophobic cholesterol molecule can be solubilized in the aqueous phase. Thereby it became possible to study the thermodynamics of either uptake of or release of cholesterol from lipid vesicles of various compositions. As one important result a comprehensive set of quantitative data on cholesterol/lipid interactions was obtained including for the first time also information on enthalpic contributions to the differential interactions of cholesterol with different lipids. Additionally, in these studies lipid/cyd interactions could be investigated and suggestions on how to optimize cholesterol extraction from biological membranes were made that could be derived from the different stoichiometries of the complexes formed, i.e., lipid or cholesterol complexed to cyd, respectively. The possibility to isolate detergent resistant patches is commonly used to argue for the existence of (functional) domains in the original, detergent–free membrane. This kind of reasoning does, however, neglect the possibility of detergent–induced alteration or (in the worst case) induction of domains. In this context, a theoretical model suitable to describe the selective solubilization of a membrane containing two lipid domains (liquid ordered and liquid disordered) was developed. Based on equilibrium thermodynamical relations it was shown that detergent–induced formation of ordered membrane domains can occur if the detergent mixes nonideally with an order preferring lipid and/or cholesterol. Furthermore, both the composition as well as the mere existence of the liquid ordered domain was shown to be highly variable upon addition of detergent to the membrane. A experimental study was carried out in parallel to these theoretical simulations with the goal to better understand the mixing of a commonly used nonionic detergent with different lipid/cholesterol systems. In order to allow for a quantitative discussion of the experimental results obtained, a theory for nonideal mixing in multicomponent lipid/detergent system was developed that accounts for nonideality in terms of simple pair interaction statistics. The parameters collected imply that a separation of ordered from disordered membrane domains can under certain circumstances occur. A crucial parameter governing the abundance and composition of detergent–resistant membrane patches appeared to be the unfavourable interaction of cholesterol with detergent. Taken together, these two studies provided additional evidence against the simple identification of lipid rafts with detergent resistant membrane patches. The third part of this thesis was devoted to a characterization of different phase equilibria employing a rather new experimental technique, pressure perturbation calorimetry (PPC). A micellar sphere–to–rod transition was characterized in terms of a large set of structural, volumetric, and thermodynamic parameters including the first published data on the change in partial molar volume of a detergent occurring upon the transition. Subsequent to this study, the question whether binary mixtures of an unsaturated lipid and cholesterol should be better described in terms of a phase separation (liquid ordered and liquid disordered phases) or of gradual changes in largely homogenous membranes was addressed with the help of PPC experiments. The possibility of cholesterol to condense lipids not only laterally but also with respect to volume was measured in this study for the first time. Information on the number of condensed lipids per cholesterol were obtained by comparing the results of simulations of expansivity curves according to three theoretical models appropriate to be applied in this context. It was found that the behaviour of the binary mixtures investigated is best described in terms of submicroscopic demixing rather than true phase separation or random mixing

Myelodysplastic syndromes: Aspects of current medical care and economic considerations in Germany

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases mainly affecting older people. The use of an increasing number of therapeutic options depends on a systematic risk stratification of the patients. A high percentage of MDS patients need blood transfusions as supportive care, which influence quality of life and cause a great part of the costs generated by MDS therapy. In this article which is based on a workshop about the burden of MDS held in October 2006 in Munich, MDS is discussed with regard to different aspects: current therapies, transfusion medicine, geriatrics, quality of life, and health economic aspects

Titration Calorimetry Standards and the Precision of Isothermal Titration Calorimetry Data

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested as possible enthalpy standards. The performances of several commercial calorimeters, including the VP-ITC, ITC200, and Nano ITC-III, were validated using these suggested standard reactions