Routine clinical determination of carotene, vitamin E, vitamin A, 25-hydroxy vitamin D3 and trans-vitamin K1 in human serum by straight phase HPLC

A universal extraction procedure is described for fat-soluble vitamins in human serum. Methods are presented for routine quantitative analysis by isocratic straight phase HPLC with UV-detection of (alpha + beta)-carotene, vitamin E (alpha-tocopherol) and vitamin A (all-trans-retinol) in one single run, and of vitamin K1 (trans-phylloquinone) and 25-hydroxy vitamin D3 after sample clean-up using disposable reversed-phase cartridges. The limits of detection, precisions and selectivities of the developed assays are shown to be satisfactory after more than three years' experience. The routine clinical determination of fat-soluble vitamins can be performed in less than 5 mL of serum. Analyses of external quality control and randomly taken outpatient samples are shown to be of great value in assessing laboratory performance.     

Single-shot gradient-assisted photon echo electronic spectroscopy

Two-dimensional electronic spectroscopy (2D ES) maps the electronic structure of complex systems on a femtosecond time scale. While analogous to multidimensional NMR spectroscopy, 2D optical spectroscopy differs significantly in its implementation. Yet, 2D Fourier spectroscopies still require point-by-point sampling of the time delay between two pulses responsible for creating quantum coherence among states. Unlike NMR, achieving the requisite phase stability at optical frequencies between these pulse pairs remains experimentally challenging. Nonetheless, 2D optical spectroscopy has been successfully demonstrated by combining passive and active phase stabilization along with precise control of optical delays and long-term temperature stability, although the widespread adoption of 2D ES has been significantly hampered by these technical challenges. Here, we exploit an analogy to magnetic resonance imaging (MRI) to demonstrate a single-shot method capable of acquiring the entire 2D spectrum in a single laser shot using only conventional optics. Unlike point-by-point sampling protocols typically used to record 2D spectra, this method, which we call GRadient-Assisted Photon Echo (GRAPE) spectroscopy, largely eliminates phase errors while reducing the acquisition time by orders of magnitude. By incorporating a spatiotemporal encoding of the nonlinear polarization along the excitation frequency axis of the 2D spectrum, GRAPE spectroscopy achieves no loss in signal while simultaneously reducing overall noise. Here, we describe the principles of GRAPE spectroscopy and discuss associated experimental considerations.     

Coupled liquid chromatography-gas chromatography for the rapid analysis of gamma-oryzanol in rice lipids

An approach based on on-line coupled liquid chromatography-gas chromatography (LC-GC) was developed for the rapid analysis of gamma-oryzanol in rice. Total lipids were extracted from rice and subjected to LC-GC without any prior purification. gamma-Oryzanol was pre-separated by HPLC from rice lipids and transferred on-line to GC analysis in order to separate its major constituents. 24-methylenecycloartanyl ferulate, cycloartenyl ferulate, campesteryl ferulate, beta-sitosteryl ferulate and campestanyl ferulate. The identities of the compounds were confirmed by off-line GC-MS analysis. Total gamma-oryzanol content could be quantified by HPLC-UV detection and the distribution of gamma-oryzanol constituents could be determined by on-line coupled GC analysis. The proposed methodology paves the way for high-throughput investigations providing information on natural variations in gamma-oryzanol content and its composition in different rice varieties.     

Fractional revivals in the rovibrational motion of I2

Motivated by pump-probe experiments of I(2) in a room-temperature sample, the detection of fractional revivals is investigated using full-dimensional quantum wave packet calculations. It is shown that the structures observed in the pump-probe signal depend sensitively on the probe parameters employed and that the observed signal reflects a particular phase effect between fractional revivals.     

MECHANISM OF PHOTOOXYGENATION OF THE CD (II) COMPLEX OF PYROPHEOPHORBIDE A METHYL ESTER

Photooxygenation of (pyropheophorbidato a methyl ester)cadmium (II) was studied using ^18,18O₂ labeling of the molecular oxygen required for cleavage of the macrocycle. After reductive demetallation of the primary oxidation product (4,5-dioxo-4,5-secopyropheophorbidato a methyl ester)cadmium (II), the isotope content of formylbilinone 4a was analyzed by repeated-scan fast atom bombardment mass spectrometry. Comparison of the spectroscopic data of the labeled pigment 4a with the statistical probabilities of¹⁸ O isotope incorporation calculated for four possible reaction mechanisms clearly proves that photooxidative ring cleavage occurred by the one-molecule mechanism, i.e. the terminal oxygen atoms of 4a were derived from one oxygen molecule. Furthermore, a study of the exchange of the¹⁸ O-labeled atoms revealed that no exchange occurs within the pH 4.5–9.5 range. In stronger alkaline or acidic solutions, only the oxygen atom of the formyl group is exchanged. Hydrolysis of the methyl ester group of 4a was achieved, without loss of the¹⁸ O label on the formyl group, at pH 7.2 in the presence of pig liver esterase.     

An Improved Direct Oxidation of Alkyl Halides to Aldehydes

The oxidation of alkyl chlorides and bromides with dimethyl sulfoxide has been performed in the presence of sodium iodide. This method allows a convenient one-step procedure for the preparation of aldehydes from alkyl chlorides and bromides.     

Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels

Thin hydrogel films based on an ABA triblock copolymer gelator [where A is pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) and B is biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)] were used as a stimulus-responsive substrate that allows fine adjustment of the mechanical environment experienced by mouse myoblast cells. The hydrogel film elasticity could be reversibly modulated by a factor of 40 via careful pH adjustment without adversely affecting cell viability. Myoblast cells exhibited pronounced stress fiber formation and flattening on increasing the hydrogel elasticity. As a new tool to evaluate the strength of cell adhesion, we combined a picosecond laser with an inverted microscope and utilized the strong shock wave created by the laser pulse to determine the critical pressure required for cell detachment. Furthermore, we demonstrate that an abrupt jump in the hydrogel elasticity can be utilized to monitor how cells adapt their morphology to changes in their mechanical environment.     

Adsorption of bovine alpha-lactalbumin on suspended solid nanospheres and its subsequent displacement studied by NMR spectroscopy

Detailed knowledge of the adsorption-induced conformational changes of proteins is essential to understand the process of protein adsorption. However, not much information about these conformational changes is available. Here, the adsorption of calcium-depleted (APO)- and calcium-containing (HOLO)-bovine alpha-lactalbumin (BLA) on suspended solid polystyrene nanospheres and their subsequent displacement by a surfactant are studied by NMR spectroscopy. To our knowledge, this is the first time that adsorption of proteins on solid nanospheres, with both components present in the NMR sample, is studied by this method. High-quality one-dimensional and two-dimensional 1H NMR spectra of nonadsorbed APO- and HOLO-BLA in the presence of BLA- and/or surfactant-covered solid polystyrene nanospheres in suspension are obtained using standard NMR procedures. BLA and surfactant molecules that are adsorbed on the polystyrene nanospheres give rise to extremely broadened proton resonances. This can be exploited to determine the amount of adsorbed protein and of adsorbed surfactant in a system containing protein, nanospheres, and surfactant, without disturbing the equilibrium of the system. Two-dimensional 1H NMR spectroscopy shows that the chemical shifts of the backbone amide protons of HOLO-BLA after its adsorption and subsequent displacement from polystyrene nanospheres by the surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) are identical to those of native HOLO-BLA. The adsorption-induced unfolding of BLA to a molten globule state on polystyrene nanospheres is thus fully reversible at the residue level upon CHAPS-induced displacement of BLA. The latter is the now fulfilled essential requirement that enables the future indirect study, at the residue level, of the conformational characteristics of BLA adsorbed on polystyrene nanospheres by hydrogen/deuterium exchange and NMR spectroscopy. The results presented show that NMR spectroscopy is clearly feasible to study the adsorption of BLA on suspended polystyrene nanospheres. This technique should be applicable to the study of the adsorption of other proteins on other surfaces as well.     

Random-phase-approximation-based correlation energy functionals: benchmark results for atoms

The random phase approximation for the correlation energy functional of the density functional theory has recently attracted renewed interest. Formulated in terms of the Kohn-Sham orbitals and eigenvalues, it promises to resolve some of the fundamental limitations of the local density and generalized gradient approximations, as, for instance, their inability to account for dispersion forces. First results for atoms, however, indicate that the random phase approximation overestimates correlation effects as much as the orbital-dependent functional obtained by a second order perturbation expansion on the basis of the Kohn-Sham Hamiltonian. In this contribution, three simple extensions of the random phase approximation are examined; (a) its augmentation by a local density approximation for short-range correlation, (b) its combination with the second order exchange term, and (c) its combination with a partial resummation of the perturbation series including the second order exchange. It is found that the ground state and correlation energies as well as the ionization potentials resulting from the extensions (a) and (c) for closed subshell atoms are clearly superior to those obtained with the unmodified random phase approximation. Quite some effort is made to ensure highly converged data, so that the results may serve as benchmark data. The numerical techniques developed in this context, in particular, for the inherent frequency integration, should also be useful for applications of random phase approximation-type functionals to more complex systems.