Characterization of a high-resolution breath acetone meter for ketosis monitoring.

BACKGROUND: The ketone bodies beta-hydroxybutyrate (BHB) and acetone are endogenous products of fatty acid metabolism. Although ketone levels can be monitored by measuring either blood BHB or breath acetone, determining the precise correlation between these two measurement methods has been challenging. The purpose of this study is to characterize the performance of a novel portable breath acetone meter (PBAM) developed by Readout, Inc., to compare single versus multiple daily ketone measurements, and to compare breath acetone (BrAce) and blood BHB measurements. METHODS: We conducted a 14-day prospective observational cohort study of 21 subjects attempting to follow either a low-carbohydrate/ketogenic or a standard diet. Subjects were asked to concurrently measure both blood BHB and BrAce five times per day and report the results using an online data entry system. We evaluated the utility of multiple daily measurements by calculating the coefficient of variation (CV) for each daily group of measurements. We calculated the correlation between coincident BrAce and blood BHB measurements using linear ordinary least squares regression analysis. We assessed the ability of the BrAce measurement to accurately predict blood BHB states using receiver operating characteristic (ROC) analysis. Finally, we calculated a daily ketone exposure (DKE) using the area under the curve (AUC) of a ketone concentration versus time graph and compared the DKE of BrAce and blood BHB using linear ordinary least squares regression. RESULTS: BrAce and blood BHB varied throughout the day by an average of 44% and 46%, respectively. The BrAce measurement accurately predicted whether blood BHB was greater than or less than the following thresholds: 0.3 mM (AUC = 0.898), 0.5 mM (AUC = 0.854), 1.0 mM (AUC = 0.887), and 1.5 mM (AUC = 0.935). Coincident BrAce and blood BHB measurements were moderately correlated with R 2 = 0.57 (P < 0.0001), similar to literature reported values. However, daily ketone exposures, or areas under the curve, for BrAce and blood BHB were highly correlated with R 2 = 0.80 (P < 0.0001). CONCLUSIONS: The results validated the performance of the PBAM. The BrAce/BHB correlation was similar to literature values where BrAce was measured using highly accurate lab instruments. Additionally, BrAce measurements using the PBAM can be used to predict blood BHB states. The relatively high daily variability of ketone levels indicate that single blood or breath ketone measurements are often not sufficient to assess daily ketone exposure for most users. Finally, although single coincident blood and breath ketone measurements show only a moderate correlation, possibly due to the temporal lag between BrAce and blood BHB, daily ketone exposures for blood and breath are highly correlated.

Chemical vapor discrimination using a compact and low-power array of piezoresistive microcantilevers.

A compact and low-power microcantilever-based sensor array has been developed and used to detect various chemical vapor analytes. In contrast to earlier micro-electro-mechanical systems (MEMS) array sensors, this device uses the static deflection of piezoresistive cantilevers due to the swelling of glassy polyolefin coatings during sorption of chemical vapors. To maximize the sensor response to a variety of chemical analytes, the polymers are selected based on their Hildebrand solubility parameters to span a wide range of chemical properties. We utilize a novel microcontact spotting method to reproducibly coat a single side of each cantilever in the array with the polymers. To demonstrate the utility of the sensor array we have reproducibly detected 11 chemical vapors, representing a breadth of chemical properties, in real time and over a wide range of vapor concentrations. We also report the detection of the chemical warfare agents (CWAs) VX and sulfur mustard (HD), representing the first published report of CWA vapor detection by a polymer-based, cantilever sensor array. Comparisons of the theoretical polymer/vapor partition coefficient to the experimental cantilever deflection responses show that, while general trends can be reasonably predicted, a simple linear relationship does not exist.

Quantifying growth of symmetric and asymmetric lipid bilayer domains.

Here, we examine by atomic force microscopy (AFM) the kinetics and morphology of lipid domain growth during lipid phase separation by rapid thermal cooling of fully mixed two-component supported lipid bilayers. At the undercooled temperatures chosen, symmetric 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-rich domains favored slower reaction-limited growth whereas asymmetric galactosylceramide (GalCer)-rich domains favored faster diffusion-limited growth, indicated by shape factors and kinetic exponents. Because kinetically limited conditions could be accessed, we were able to estimate the activation energy barrier (approximately 16kT) and lateral diffusion coefficient (approximately 0.20 microm2/s) of lipid molecular addition to a growing domain. We discuss these results with respect to transition states, obstructed diffusion, and the necessity for coordinating growth in both leaflets in a symmetric lipid domain.

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide.

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.

Lipid domains in supported lipid bilayer for atomic force microscopy.

Phase-separated supported lipid bilayers have been widely used to study the phase behavior of multicomponent lipid mixtures. One of the primary advantages of using supported lipid bilayers is that the two-dimensional platform of this model membrane system readily allows lipid-phase separation to be characterized by high-resolution imaging techniques such as atomic force microscopy (AFM). In addition, when supported lipid bilayers have been functionalized with a specific ligand, protein-membrane interactions can also be imaged and characterized through AFM. It has been recently demonstrated that when the technique of vesicle fusion is used to prepare supported lipid bilayers, the thermal history of the vesicles before deposition and the supported lipid bilayers after formation will have significant effects on the final phase-separated domain structures. In this chapter, three methods of vesicle preparations as well as three deposition conditions will be presented. Also, the techniques and strategies of using AFM to image multicomponent phase-separated supported lipid bilayers and protein binding will be discussed.

Using nucleation rates to determine the interfacial line tension of symmetric and asymmetric lipid bilayer domains.

This work presents a novel method for experimentally quantifying interfacial line tension, which can be readily applied to study a wide variety of different lipid mixtures exhibiting phase coexistence. The method combines AFM imaging of lipid domain nucleation with classical nucleation theories. The results, using symmetric and asymmetric domains, permit the prediction of key physical parameters (critical nuclei size and nucleation rate) in multicomponent bilayer systems with implications toward understanding the dynamic nature of submicrometer domains (i.e., lipid rafts) in cell membranes.

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions.

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.

Nonlinearly additive forces in multivalent ligand binding to a single protein revealed with force spectroscopy.

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between concanavalin A (ConA) and alpha-D-mannose but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 68, and 85 pN at a loading rate of 650 +/- 25 pN/s, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and nonspecific binding. We analyze the binding configuration (i.e., serial vs parallel connections) through fitting the polymer stretching data with modified wormlike chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study.

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

The AFM measured force required to rupture the dithiolate linkage of thioctic acid to gold is less than the rupture force of a simple gold-alkyl thiolate bond.

We show with atomic force microscopy that thioctic acid, a spatially constrained system with two sulfur linkages to gold, is less stable to tensile stress than a thiolate with a single attachment to gold. The force required to remove the dithiolate-linked thioctic acid was 0.31+/-0.13 nN, whereas the force required to remove a simple thiolate from the gold substrate was 1.05+/-0.29 nN. These results suggest that SAMs of densely packed or polypodal thiols may be substantially less stable under tensile stress than previously recognized and that the additional thiolate linkages may not only fail to increase the overall strength of attachment but could actually reduce it.

Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds.

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.

Force spectroscopy of the double-tethered concanavalin-A mannose bond.

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach.

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.