First clinical evaluation of a new percutaneous optical fiber glucose sensor for continuous glucose monitoring in diabetes.

BACKGROUND: This article describes a new fiber-coupled, percutaneous fluorescent continuous glucose monitoring (CGM) system that has shown 14 days of functionality in a human clinical trial. METHOD: The new optical CGM system (FiberSense) consists of a transdermal polymer optical fiber containing a biochemical glucose sensor and a small fluorescence photometer optically coupled to the fiber. The glucose-sensitive optical fiber was implanted in abdominal and upper-arm subcutaneous tissue of six diabetes patients and remained there for up to 14 days. The performance of the system was monitored during six visits to the study center during the trial. Blood glucose changes were induced by oral carbohydrate intake and insulin injections, and capillary blood glucose samples were obtained from the finger tip. The data were analyzed using linear regression and the consensus error grid analysis. RESULTS: The FiberSense worn at the upper arm exhibited excellent results during 14 wearing days, with an overall mean absolute relative difference (MARD) of 8.3% and 94.6% of the data in zone A of the consensus error grid. At the abdominal application site, FiberSense resulted in a MARD of 11.4 %, with 93.8% of the data in zone A. CONCLUSIONS: The FiberSense CGM system provided consistent, reliable measurements of subcutaneous glucose levels in human clinical trial patients with diabetes for up to 14 days.

Blood glucose self-monitoring with a long-term subconjunctival glucose sensor.

BACKGROUND: To evaluate the feasibility of an implantable subconjunctival glucose monitoring system (SGMS) for long-term glucose monitoring, we investigated the in vivo performance of the system. METHOD: The SGMS consists of an implantable ocular mini implant (OMI) and a handheld fluorescence photometer. A clinical study was performed on 47 diabetes patients split into two cohorts. Two different types of OMI were used, with and without a biocompatible surface coating. Duration of the study was 1 year. Correlation between capillary blood glucose and SGMS-derived interstitial fluid glucose was investigated during the first 6 months of the study. RESULTS: Both OMI types were tolerated well in the eyes of the patients. At the beginning of the study, the SGMS of both cohorts revealed a high accuracy with mean absolute relative difference (MARD) values of 7-12%. The performance of the uncoated OMIs deteriorated within 3 months of wearing time, exhibiting a MARD value of 20%. The performance of the surface-coated OMIs was preserved longer. Glucose correlation measurement with reasonable results (MARD of 14%) could be performed for up to 6 months of wear. CONCLUSIONS: The biocompatible surface coating on the OMIs enabled a longer duration of action of up to 6 months compared with 3 months for uncoated implants in a clinical trial.

Specific volume and compressibility of bilayer lipid membranes with incorporated Na,K-ATPase.

Ultrasound velocimetry and densitometry methods were used to study the interactions of the Na,K-ATPase with the lipid bilayer in large unilamellar liposomes composed of dioleoyl phosphatidylcholine (DOPC). The ultrasound velocity increased and the specific volume of the phospholipids decreased with increasing concentrations of protein. These experiments allowed us to determine the reduced specific apparent compressibility of the lipid bilayer, which decreased by approx. 11% with increasing concentrations of the Na,K-ATPase up to an ATPase/DOPC molar ratio = 2 × 10⁻4. Assuming that ATPase induces rigidization of the surrounding lipid molecules one can obtain from the compressibility data that 3.7 to 100 times more lipid molecules are affected by the protein in comparison with annular lipids. However, this is in contradiction with the current theories of the phase transitions in lipid bilayers. It is suggested that another physical mechanisms should be involved for explanation of observed effect.

Energetics and kinetics of photosynthetic water oxidation studied by photothermal beam deflection (PBD) experiments.

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890­1903). Recent PBD results on formation of the Q⁻(A)/Y(•+)(Z) radical pair suggest a value of ΔH similar to the free energy change, ΔG, of -540±40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S3−−>S0, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q⁻(A)/Y(•+)(Z) formation (<1 µs) and the S3−−>S0 transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 ų per PSII unit) in the S3−−>S0 transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O2 and proton release. The observed volume changes show that the S3−−>S0 transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.

Effect of cholesterol and ergosterol on the compressibility and volume fluctuations of phospholipid-sterol bilayers in the critical point region: a molecular acoustic and calorimetric study.

Although sterol-phospholipid interactions have been of interest for many years now, a complete thermodynamic profile of these systems is still missing. To contribute to a better understanding of the thermodynamic functions of these systems, we determined isothermal compressibility coefficient data for dipalmitoylphosphocholine (DPPC) and DPPC-containing cholesterol and ergosterol vesicles by means of molecular acoustics (ultrasound velocimetry and densimetry) and differential scanning and pressure perturbation calorimetric techniques. A particular focus was on the influence of the differential structural properties of the two sterols on the thermodynamic properties of lipid bilayers, and on the nature of the critical point region of phospholipid-sterol systems by determining thermodynamic fluctuation parameters. Contrary to significant changes in conformational and dynamical properties of the DPPC-sterol membranes, no marked differences were found in the various thermodynamic properties studied, including the adiabatic (beta(S)(lipid)) and isothermal (beta(T)(lipid)) compressibility, as well as the volume fluctuations. Differences in beta(T)(lipid) and beta(S)(lipid) become dramatic in the gel-fluid transition region only, due to a significant degree of slow relaxational processes in the microsecond time range in the transition region. Our data show no evidence for the existence of a typical critical point phenomenon in the concentration and temperature range where a critical point in the DPPC-sterol phase diagram is expected to appear. Hence, on a macroscopic level, it seems more appropriate to describe the sterol-phospholipid binary mixtures in the liquid-ordered/liquid-disordered coexistence region as a phase region consisting essentially of small nanodomains only. Such small-domain dimensions, with a series of particular properties such as increased line energy, spontaneous curvature, and limited lifetime, seem also to be typical of raftlike domains in cell membranes.

Enthalpy changes during photosynthetic water oxidation tracked by time-resolved calorimetry using a photothermal beam deflection technique.

The energetics of the individual reaction steps in the catalytic cycle of photosynthetic water oxidation at the Mn(4)Ca complex of photosystem II (PSII) are of prime interest. We studied the electron transfer reactions in oxygen-evolving PSII membrane particles from spinach by a photothermal beam deflection technique, allowing for time-resolved calorimetry in the micro- to millisecond domain. For an ideal quantum yield of 100%, the enthalpy change, DeltaH, coupled to the formation of the radical pair Y(Z)(.+)Q(A)(-) (where Y(Z) is Tyr-161 of the D1 subunit of PSII) is estimated as -820 +/- 250 meV. For a lower quantum yield of 70%, the enthalpy change is estimated to be -400 +/- 250 meV. The observed nonthermal signal possibly is due to a contraction of the PSII protein volume (apparent DeltaV of about -13 A(3)). For the first time, the enthalpy change of the O(2)-evolving transition of the S-state cycle was monitored directly. Surprisingly, the reaction is only slightly exergonic. A value of DeltaH(S(3)-->S(0)) of -210 meV is estimated, but also an enthalpy change of zero is within the error range. A prominent nonthermal photothermal beam deflection signal (apparent DeltaV of about +42 A(3)) may reflect O(2) and proton release from the manganese complex, but also reorganization of the protein matrix.

Study of the interaction of an alpha-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry.

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic alpha-helical transmembrane peptide, acetyl-K(2)-L(24)-K(2)-amide (L(24)), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L(24) decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L(24) in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L(24):PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L(24). As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and (2)H NMR spectroscopy study of the interaction of the L(24) and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between (2)H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L(24) suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus.

The recent crystallographic structure at 3.0 A resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q(B). The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O(2) evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q(A) and Q(B) were found in the crystallized PSII. We propose that the extra quinones are located in the Q(B) cavity and serve as a PSII intrinsic pool of electron acceptors.

Intermediates in assembly by photoactivation after thermally accelerated disassembly of the manganese complex of photosynthetic water oxidation.

The Mn4Ca complex bound to photosystem II (PSII) is the active site of photosynthetic water oxidation. Its assembly involves binding and light-driven oxidation of manganese, a process denoted as photoactivation. The disassembly of the Mn complex is a thermally activated process involving distinct intermediates. Starting from intermediate states of the disassembly, which was initiated by a temperature jump to 47 degrees C, we photoactivated PSII membrane particles and monitored the activity recovery by O2 polarography and delayed chlorophyll fluorescence measurements. Oxidation state and structural features of the formed intermediates of the Mn complex were assayed by X-ray absorption spectroscopy at the Mn K-edge. The photoactivation time courses, which exhibit a lag phase characteristic of intermediate formation only when starting with the apo-PSII, suggest that within approximately 5 min of photoactivation of apo-PSII, a binuclear Mn complex is formed. It is proposed that a MnIII2(di-mu-oxo) complex is a key intermediate both in the disassembly and in the assembly reaction paths.

Bromide does not bind to the Mn4Ca complex in its S1 state in Cl(-)-depleted and Br(-)-reconstituted oxygen-evolving photosystem II: evidence from X-ray absorption spectroscopy at the Br K-edge.

Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn(4)Ca complex of PSII in its dark-stable S(1) state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl(-)-depleted and Br(-)-substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br(-). EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of approximately 5 A to Br(-). The native Cl(-) ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S(1) state.