Virtual 4DCT generated from 4DMRI in gated particle therapy: phantom validation and application to lung cancer patients.

Objective. Respiration negatively affects the outcome of a radiation therapy treatment, with potentially severe effects especially in particle therapy (PT). If compensation strategies are not applied, accuracy cannot be achieved. To support the clinical practice based on 4D computed tomography (CT), 4D magnetic resonance imaging (MRI) acquisitions can be exploited. The purpose of this study was to validate a method for virtual 4DCT generation from 4DMRI data for lung cancers on a porcine lung phantom, and to apply it to lung cancer patients in PT.Approach. Deformable image registration was used to register each respiratory phase of the 4DMRI to a reference phase. Then, a static 3DCT was registered to this reference MR image set, and the virtual 4DCT was generated by warping the registered CT according to previously obtained deformation fields. The method was validated on a physical phantom for which a ground truth 4DCT was available and tested on lung tumor patients, treated with gated PT at end-exhale, by comparing the virtual 4DCT with a re-evaluation 4DCT. The geometric and dosimetric evaluation was performed for both proton and carbon ion treatment plans.Main results. The phantom validation exhibited a geometrical accuracy within the maximum resolution of the MRI and mean dose deviations, with respect to the prescription dose, up to 3.2% for targetD95%, with a mean gamma pass rate of 98%. For patients, the virtual and re-evaluation 4DCTs showed good correspondence, with errors on targetD95%up to 2% within the gating window. For one patient, dose variations up to 10% at end-exhale were observed due to relevant inter-fraction anatomo-pathological changes that occurred between the planning and re-evaluation CTs.Significance. Results obtained on phantom data showed that the virtual 4DCT method was accurate, allowing its application on patient data for testing within a clinical scenario.

Fluence-modulated proton CT optimized with patient-specific dose and variance objectives for proton dose calculation.

Particle therapy treatment planning requires accurate volumetric maps of the relative stopping power, which can directly be acquired using proton computed tomography (pCT). With fluence-modulated pCT (FMpCT) imaging fluence is concentrated in a region-of-interest (ROI), which can be the vicinity of the treatment beam path, and imaging dose is reduced elsewhere. In this work we present a novel optimization algorithm for FMpCT which, for the first time, calculates modulated imaging fluences for joint imaging dose and image variance objectives. Thereby, image quality is maintained in the ROI to ensure accurate calculations of the treatment dose, and imaging dose is minimized outside the ROI with stronger minimization penalties given to imaging organs-at-risk. The optimization requires an initial scan at uniform fluence or a previous x-ray CT scan. We simulated and optimized FMpCT images for three pediatric patients with tumors in the head region. We verified that the target image variance inside the ROI was achieved and demonstrated imaging dose reductions outside of the ROI of 74% on average, reducing the imaging dose from 1.2 to 0.3 mGy. Such dose savings are expected to be relevant compared to the therapeutic dose outside of the treatment field. Treatment doses were re-calculated on the FMpCT images and compared to treatment doses re-recalculated on uniform fluence pCT scans using a 1% criterion. Passing rates were above 98.3% for all patients. Passing rates comparing FMpCT treatment doses to the ground truth treatment dose were above 88.5% for all patients. Evaluation of the proton range with a 1 mm criterion resulted in passing rates above 97.5% (FMpCT/pCT) and 95.3% (FMpCT/ground truth). Jointly optimized fluence-modulated pCT images can be used for proton dose calculation maintaining the full dosimetric accuracy of pCT but reducing the required imaging dose considerably by three quarters. This may allow for daily imaging during particle therapy ensuring a safe and accurate delivery of the therapeutic dose and avoiding excess dose from imaging.

Integration of spatial distortion effects in a 4D computational phantom for simulation studies in extra-cranial MRI-guided radiation therapy: Initial results.

PURPOSE: Spatial distortions in magnetic resonance imaging (MRI) are mainly caused by inhomogeneities of the static magnetic field, nonlinearities in the applied gradients, and tissue-specific magnetic susceptibility variations. These factors may significantly alter the geometrical accuracy of the reconstructed MR image, thus questioning the reliability of MRI for guidance in image-guided radiation therapy. In this work, we quantified MRI spatial distortions and created a quantitative model where different sources of distortions can be separated. The generated model was then integrated into a four-dimensional (4D) computational phantom for simulation studies in MRI-guided radiation therapy at extra-cranial sites. METHODS: A geometrical spatial distortion phantom was designed in four modules embedding laser-cut PMMA grids, providing 3520 landmarks in a field of view of (345 × 260 × 480) mm3 . The construction accuracy of the phantom was verified experimentally. Two fast MRI sequences for extra-cranial imaging at 1.5 T were investigated, considering axial slices acquired with online distortion correction, in order to mimic practical use in MRI-guided radiotherapy. Distortions were separated into their sources by acquisition of images with gradient polarity reversal and dedicated susceptibility calculations. Such a separation yielded a quantitative spatial distortion model to be used for MR imaging simulations. Finally, the obtained spatial distortion model was embedded into an anthropomorphic 4D computational phantom, providing registered virtual CT/MR images where spatial distortions in MRI acquisition can be simulated. RESULTS: The manufacturing accuracy of the geometrical distortion phantom was quantified to be within 0.2 mm in the grid planes and 0.5 mm in depth, including thickness variations and bending effects of individual grids. Residual spatial distortions after MRI distortion correction were strongly influenced by the applied correction mode, with larger effects in the trans-axial direction. In the axial plane, gradient nonlinearities caused the main distortions, with values up to 3 mm in a 1.5 T magnet, whereas static field and susceptibility effects were below 1 mm. The integration in the 4D anthropomorphic computational phantom highlighted that deformations can be severe in the region of the thoracic diaphragm, especially when using axial imaging with 2D distortion correction. Adaptation of the phantom based on patient-specific measurements was also verified, aiming at increased realism in the simulation. CONCLUSIONS: The implemented framework provides an integrated approach for MRI spatial distortion modeling, where different sources of distortion can be quantified in time-dependent geometries. The computational phantom represents a valuable platform to study motion management strategies in extra-cranial MRI-guided radiotherapy, where the effects of spatial distortions can be modeled on synthetic images in a virtual environment.

12- and 18-month follow-up after residential treatment of methamphetamine dependence: Influence of treatment drop-out and different treatment concepts.

This study investigates the effects of two different residential treatments and of treatment drop-out in a German methamphetamine (MA) dependent sample. 108 subjects from two addiction treatment concepts were recruited at treatment begin and followed-up at 12 (T2) and 18 (T3) months after treatment. Based on follow-up samples (n = 38 at T2, n = 25 at T3), 77.1% at T2 and 68.0% at T3 were MA abstinent. Classifying everyone, who did not participate at follow-ups as having had a relapse, showed MA-abstinence rates of 25.0% (at T2) and 15.7% (at T3). There was no difference in MA-use between treatment conditions nor between treatment completers and drop-outs. Having injected any substance predicted MA-use at T2 (p = .03). The median time of relapse was 1.5 days after hospital release. Depression scores at T2 predicted MA-use at T3 (p = .02). T2 participants that dropped out of treatment had higher craving scores at T2, than T2 subjects who completed treatment (p = .03). The results show positive effects of current inpatient treatment programs without differences between different concepts. More research is needed to clarify the impact of treatment drop-out. Attention should be paid to a successful transition from residential to outpatient services and to a reduction of study attrition.

Improving the modelling of susceptibility-induced spatial distortions in MRI-guided extra-cranial radiotherapy.

Magnetic-resonance linear-accelerator (MR-LINAC) systems integrating in-room magnetic-resonance-imaging (MRI) guidance are a currently emerging technology. Such systems address the need to provide frequent imaging at optimal soft-tissue contrast for treatment guidance. However, the use of MRI-guidance in radiotherapy should address imaging-related spatial distortions, which may hinder accurate geometrical characterization of the treatment site. Since spatial encoding relies on well-defined magnetic fields, accurate modeling of the magnetic field alterations due to [Formula: see text]-inhomogeneities, gradient nonlinearities, and susceptibilities is needed. In this work, the modeling of susceptibility induced distortions is considered. Dedicated susceptibility measurements are reported, aiming at extending the characterization of different tissues for MRI-guided extra-cranial radiotherapy applications. A digital 4D anthropomorphic phantom, providing time-resolved anatomical changes due to breathing, is exploited as reference anatomy to quantify spatial distortions due to variations in tissue susceptibility. Sub-millimeter values can be attributed to susceptibility-induced distortions, with maximum values up to 2.3 mm at a gradient strength of 5 mT m-1. Improvements in susceptibility simulation for extra-cranial sites are shown when including specifically the contributions from lung, liver and muscular tissues.

Effectiveness of methamphetamine abuse treatment: Predictors of treatment completion and comparison of two residential treatment programs.

BACKGROUND: There is an increasing demand of evidence-based treatment options for methamphetamine users, but research in this field is limited. This study therefore evaluates the efficacy of two residential treatment programs for methamphetamine users. METHOD: A total of 108 patients with a history of methamphetamine abuse from two inpatient rehabilitation centers were studied for psychiatric symptoms, craving, psychosocial resources, and cognitive functioning at the start and end of therapy. Patients from one center ("amphetamine type stimulant group") received conventional group therapy plus an additional 10 h of group therapy focusing on stimulant use. Patients from the other center ("treatment as usual") received conventional group therapy only. Predictors of drop-out were estimated. RESULTS: A drop-out rate of 40.7% was observed without a significant difference between both centers. Patients remained significantly longer in treatment as usual compared to amphetamine type stimulant treatment. Irrespective of treatment program, craving and psychiatric symptoms significantly decreased while psychosocial resources, processing speed, and cognitive flexibility improved over time. Other cognitive measures yielded mixed results. History of injection drug use was a significant predictor for treatment drop-out. CONCLUSIONS: Existing treatments are effective in reducing craving and psychiatric symptoms. Additional stimulant specific groups do not appear to influence treatment completion and secondary outcome measures. Institutions should therefore offer treatment for methamphetamine users, even if they do not provide a therapy content focusing on methamphetamine. History of injection drug use should receive attention in treatment to prevent drop-out. Changes in cognitive functioning need to be further explored.

Rapid implementation of the repair-misrepair-fixation (RMF) model facilitating online adaption of radiosensitivity parameters in ion therapy.

INTRODUCTION: Treatment planning for ion therapy must account for physical properties of the beam as well as differences in the relative biological effectiveness (RBE) of ions compared to photons. In this work, we present a fast RBE calculation approach, based on the decoupling of physical properties and the [Formula: see text] ratio commonly used to describe the radiosensitivity of irradiated cells or organs. MATERIAL AND METHODS: In the framework of the mechanistic repair-misrepair-fixation (RMF) model, the biological modeling can be decoupled from the physical dose. This was implemented into a research treatment planning system for carbon ion therapy. RESULTS: The presented implementation of the RMF model is very fast, allowing online changes of [Formula: see text]. For example, a change of [Formula: see text] including a complete biological modeling and a recalculation of RBE for [Formula: see text] voxel takes 4 ms on a 4 CPU, 3.2 GHz workstation. DISCUSSION AND CONCLUSION: The derived decoupling within the RMF model allows fast changes in [Formula: see text], facilitating online adaption by the user. This provides new options for radiation oncologists, facilitating online variations of the radiobiological input parameters during the treatment plan evaluation process as well as uncertainty and sensitivity analyses.

Quantification of the uncertainties of a biological model and their impact on variable RBE proton treatment plan optimization.

PURPOSE: In proton radiation therapy, a relative biological effectiveness (RBE) equal to 1.1 is currently assumed, although biological experiments show that it is not constant. The purpose of this study was to quantify the uncertainties of a published biological model and explore their impact on variable RBE treatment plan (TP) optimization. METHODS: Two patient cases with a high and a low (α/ß)x tumor were investigated. Firstly, intensity modulated proton therapy TPs assuming constant RBE were derived, and subsequently the variable RBE weighted dose (RWD), including the uncertainty originating in the fit to the experimental data and the uncertainty of the (α/ß)x, were calculated. Secondly, TPs optimized for uniform biological effect assuming a variable RBE were created using the worst case tissue specific (α/ß)x. RESULTS: For the nasopharyngeal cancer patient, the uncertainty of (α/ß)x corresponded to a CTV D98 confidence interval (CI) of (-2, +4)% while for the fit parameter CI was (-2,+1)%. For the standard fractionation prostate case the (α/ß)x CI was (-7,+5)% and the fit parameter CI was (-3,+3)%. For the hypofractionated case both CIs were (-1,+1)%. In both patient cases, the RBE in most organs at risk (OARs) was significantly underestimated by the constant RBE approximation, whereas the situation was not as definite in the target volumes. Overdosage of OARs was reduced by using the biological effect optimization. CONCLUSION: For the two patient cases, the RWD uncertainty from the fit parameter in the biological model contributed non-negligibly to the total uncertainty, depending on the patient case and the organ. The presented optimization strategy is a basic method for robust biological effect optimization to reduce potential consequences caused by the (α/ß)x uncertainty.

Biologically optimized helium ion plans: calculation approach and its in vitro validation.

Treatment planning studies on the biological effect of raster-scanned helium ion beams should be performed, together with their experimental verification, before their clinical application at the Heidelberg Ion Beam Therapy Center (HIT). For this purpose, we introduce a novel calculation approach based on integrating data-driven biological models in our Monte Carlo treatment planning (MCTP) tool. Dealing with a mixed radiation field, the biological effect of the primary (4)He ion beams, of the secondary (3)He and (4)He (Z = 2) fragments and of the produced protons, deuterons and tritons (Z = 1) has to be taken into account. A spread-out Bragg peak (SOBP) in water, representative of a clinically-relevant scenario, has been biologically optimized with the MCTP and then delivered at HIT. Predictions of cell survival and RBE for a tumor cell line, characterized by [Formula: see text] Gy, have been successfully compared against measured clonogenic survival data. The mean absolute survival variation ([Formula: see text]) between model predictions and experimental data was 5.3% ± 0.9%. A sensitivity study, i.e. quantifying the variation of the estimations for the studied plan as a function of the applied phenomenological modelling approach, has been performed. The feasibility of a simpler biological modelling based on dose-averaged LET (linear energy transfer) has been tested. Moreover, comparisons with biophysical models such as the local effect model (LEM) and the repair-misrepair-fixation (RMF) model were performed. [Formula: see text] values for the LEM and the RMF model were, respectively, 4.5% ± 0.8% and 5.8% ± 1.1%. The satisfactorily agreement found in this work for the studied SOBP, representative of clinically-relevant scenario, suggests that the introduced approach could be applied for an accurate estimation of the biological effect for helium ion radiotherapy.

Variance-based sensitivity analysis of biological uncertainties in carbon ion therapy.

PURPOSE: Biological models to estimate the relative biological effectiveness (RBE) or the equivalent dose in 2 Gy fractions (EQD2) are needed for treatment planning and plan evaluation in carbon ion therapy. We present a model-independent, Monte Carlo based sensitivity analysis (SA) approach to quantify the impact of different uncertainties on the biological models. METHODS AND MATERIALS: The Monte Carlo based SA is used for the evaluation of variations in biological parameters. The key property of this SA is the high number of simulation runs, each with randomized input parameters, allowing for a statistical variance-based ranking of the input variations. The potential of this SA is shown in a simplified one-dimensional treatment plan optimization. Physical properties of carbon ion beams (e.g. fragmentation) are simulated using the Monte Carlo code FLUKA. To estimate biological effects of ion beams compared to X-rays, we use the Local Effect Model (LEM) in the framework of the linear-quadratic (LQ) model. Currently, only uncertainties in the output of the biological models are taken into account. RESULTS/CONCLUSIONS: The presented SA is suitable for evaluation of the impact of variations in biological parameters. Major advantages are the possibility to access and display the sensitivity of the evaluated quantity on several parameter variations at the same time. Main challenges for later use in three-dimensional treatment plan evaluation are computational time and memory usage. The presented SA can be performed with any analytical or numerical function and hence be applied to any biological model used in carbon ion therapy.

Energy cost of walking in children with spastic cerebral palsy: relationship with age, body composition and mobility capacity.

The energy cost (EC) of walking is different for typically developing (TD) and children with cerebral palsy (CP). The associated factors of EC are not fully understood in children with CP. We assessed the relationship between EC and age, body surface area (BSA), and gross motor function measure (GMFM). We retrospectively examined data collected between 2003 and 2011 on 276 children aged 4-18 years who were classified as Gross Motor Function Classification System level I, n=79; II, n=123; and III, n=74. Energy cost was assessed while children walked 6-8 min at a comfortable, self-selected speed using their typical walking aids and/or orthoses as part of a clinical gait analysis. During the test, participants wore a breath-by-breath portable gas analysis system, measuring oxygen consumption. To calculate EC (J/kg/m), oxygen consumption was converted to J/kg/min and divided by walking speed. Data were analyzed using linear regression model. Energy cost correlated inversely with age (ß=-0.16, R2=0.02, P=0.01), BSA (ß=-3.35, R2=0.11, P<0.0001), and GMFM (ß=-0.12, R2=0.42, P<0.0001). In the multiple linear regression model, GMFM was the most potent correlate of EC, BSA explained another 10% of the variance (R2=0.53), and age was a marginally significant correlate of EC (P=0.08). In summary, in children with CP in our study, EC decreased as GMFM and BSA increased, and GMFM was the most potent correlate of EC.

Fatty acid transport: the diffusion mechanism in model and biological membranes.

The transport of fatty acids (FA) across membranes can be described by three fundamental steps: adsorption, transmembrane movement, and desorption. In model membranes, these steps are all rapid and spontaneous for most fatty acids, suggesting that FA can enter cells by free diffusion rather than by protein-mediated mechanisms. Here we present new fluorescence approaches that measure adsorption and transmembrane movement of FA independently. We show that FA adsorb to the plasma membrane of adipocytes and diffuse through the membrane by the flip-flop mechanism within the time resolution of our measurements (approximately 5 s). Thus we show that passive diffusion is a viable mechanism, although we did not evaluate its exclusivity. Important implications of the diffusion mechanism for neural cells are that all types of FA could be available and that selectivity is controlled by metabolism. Studies of FA uptake into brain endothelial cells and other brain cell types need to be performed to determine mechanisms of uptake, and metabolism of FA must be separated in order to understand the role of membrane transport in the overall uptake process.

How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?

Although transport of long-chain free fatty acids (FFAs) into cells is often analyzed in the same way as glucose transport, we argue that the transport of the lipid-soluble amphipathic FFA molecule must be viewed differently. The partitioning of FFAs into phospholipid bilayers and their interfacial ionization are particularly relevant to transport. We summarize new data supporting the diffusion hypothesis in simple lipid bilayers and in plasma membranes of cells. Along with previous supporting data, the new data indicate that transport of FFAs through membranes could occur rapidly by flip-flop of the un-ionized form of the FFA. It appears that, at least for the adipocyte, passive diffusion guarantees fast entry and exit of FFAs at both low and high concentrations. Although there are several candidate proteins for the membrane transport of FFAs, most of these proteins have other established functions. Thus, unlike the glucose transporters, these proteins would not be single-function proteins. Definitive proof of their function as FFA transporters awaits their reconstitution into simple model systems.

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.

Dissociation of long and very long chain fatty acids from phospholipid bilayers.

Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar phosphatidylcholine vesicles (SUV) were monitored by measuring the pH inside the vesicle with an entrapped water-soluble fluorophore, pyranin. With a pH gradient imposed upon SUV preloaded with FA, the rate of flip-flop of saturated very long chain FA (C20:0, C:22:0, and C24:0) was shown to be fast (t1/2 < 1 s); previously, we showed by stopped flow measurements that flip-flop of long chain (14-18 carbons) FA is very fast [t1/2 < 10 ms; Kamp, F., et al. (1995) Biochemistry 34, 11928-11937]. The rates of dissociation of FA from SUV were evaluated by incorporating FA into donor vesicles and measuring transfer to acceptor vesicles. The transfer was followed by changes in internal pH of either donor or acceptor vesicles with stopped flow (C14:0, C16:0, C17:0, C18:0, C18:1, and C18:2) or on-line (C20:0, C22:0, and C24:0) fluorescence. All FA showed a single-exponential transfer process that was slower than the lower limits established for the rate of flip-flop, with t1/2 of dissociation ranging from 20 ms for C14:0 to 1900 s for C24:0. The pseudo-unimolecular rate constant (koff) for dissociation of C14:0 to C26:0 showed a 10-fold decrease for each addition of two CH2 groups to the acyl chain and a delta (delta G) of -740 cal/CH2. The dissociation rate constants for oleic acid (18:1) and linoleic acid (18:2) were 5 and 10 times faster, respectively, than that of C18:0. The rates of dissociation for typical dietary FA are sufficiently rapid that complex mechanisms (e.g. protein-mediated) may not be required for their desorption from biological membranes. The very slow dissociation rates for C24:0 and C26:0 may accentuate their pathological effects in diseases in which they accumulate in tissues.

Fatty acid flip-flop in phospholipid bilayers is extremely fast.

The rate of movement of fatty acids (FA) across phospholipid bilayers is an important consideration for their mechanism of transport across cell membranes but has not yet been measured. When FA move undirectionally across phospholipid bilayers, the rapid movement of un-ionized FA compared to ionized FA results in transport of protons. We have previously used this property to show that FA move spontaneously ("flip-flop") across the bilayer of small unilamellar vesicles within approximately 1 s (Kamp & Hamilton, 1992, 1993). This work extends the time resolution of this assay into the millisecond time range by use of stopped flow fluorometry. In small unilamellar vesicles (diameter, approximately 25 nm) at neutral pH, flip-flop of all fatty acids studied (lauric, myristic, palmitic, oleic, and stearic) was > or = 80% complete within 5-10 ms. In large unilamellar vesicles (diameter, approximately 100 nm), the same fatty acids exhibited fast flip-flop but with a measureable rate (t 1/2 = 23 +/- 12 ms). The calculated pseudounimolecular rate constant of the un-ionized FA (kFAH) approximately 15 s-1. There was no dependence of the flip-flop rate on the fatty acid chain length or structure. We also monitored the rate of desorption and transbilayer movement of (anthroyloxy)stearic acid in small unilamellar vesicles. Whereas previous studies suggested slow flip-flop of this FA analogue, the present studies suggest that (anthroyloxy)stearic acid flip-flops rapidly and that earlier studies did not truly measure the transbilayer movement step. These findings further support the view that proteins are not required for translocation of FA across cell membranes.

Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT).

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.

Magainin oligomers reversibly dissipate delta microH+ in cytochrome oxidase liposomes.

Magainin peptides present in the skin of Xenopus laevis and identified as antimicrobial agents are shown to decrease the membrane potential in cytochrome oxidase liposomes. They also released respiratory control with a third or higher order concentration dependence. Respiratory control was restored by proteolytic digestion of the added magainin. The amount of magainin required for half-maximal stimulation of respiration was proportional to lipid concentration. At appreciably higher concentrations magainins inhibited uncoupled respiration. The results are discussed in terms of a model in which most of the added magainin adsorbs as a monomer to the membranes but equilibrates with a multimeric pore that causes rather general permeability of membranes. The ensuing ion permeation dissipates membrane potential and stimulates respiration.

Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers.

How lipophilic acids move across membranes, either model or biological, is the subject of controversy. We describe experiments which better define the mechanism and rates in protein-free phospholipid bilayers. The transbilayer movement of lipophilic acids [fatty acids (FA), covalently-labeled FA, bile acids, and retinoic acid] was monitored by entrapping pyranin, a water-soluble, pH-sensitive fluorescent molecule to measure pH inside unilamellar vesicles [Kamp, F., & Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Equations for the pseudo-unimolecular rate constants for transbilayer movement of un-ionized (kappa FAH) and ionized (kappa FA-) acids are derived. All FA studied (octanoic, lauric, myristic, palmitic, stearic, oleic, elaidic, linoleic, linolelaidic, and arachidonic) and retinoic acid exhibited rapid transbilayer movement (t 1/2 < 1 s) via the un-ionized form across small unilamellar egg phosphatidylcholine (PC) vesicles. FA produced by phospholipase A2 in the outer leaflet of PC vesicles equilibrated rapidly to the inner leaflet. Ionized FA showed enhanced transbilayer movement (kappa FA- = 0.029 s-1) in the presence of equimolar valinomycin. The three FA analogues [12-(9-anthroyloxy)stearic acid, 5-doxylstearic acid, and 1-pyrenenonanoic acid] moved across PC bilayers via the un-ionized form; except for the anthroyloxy FA (kappa FAH = 4.8 x 10(-3) s-1), the rates were too fast to measure (t 1/2 < 1 s). The rate for cholic acid (CA) transbilayer movement was slow (kappa CAH = 0.056 s-1) compared to that of the more hydrophobic bile acids, deoxy- and chenodeoxycholic acid (t 1/2 < 1 s). The taurine conjugates of the three bile acids did not cross the bilayer (t 1/2 > 1 h). A further application of the pyranin method was to measure the partitioning of FA and bile acids among water, albumin, and PC vesicles. Our results show that the ability of lipophilic acids to permeate a PC bilayer rapidly is dependent on the presence of the un-ionized acid in the membrane interface. Considering the fast unfacilitated movement of FA across protein-free phospholipid bilayers, it is unlikely that there is a universal need for a transport protein to enhance movement of FA across membrane bilayers. Physiological implications of proton movement accompanying fast movement of un-ionized lipophilic acids (and the consequent generation of a pH gradient) are discussed.