As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4′-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4′-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.
Fast 8 MHz polarization modulation
coupled with analytical modeling,
fast beam-scanning, and synchronous digitization (SD) have enabled
simultaneous nonlinear optical Stokes ellipsometry (NOSE) and polarized
laser transmittance imaging with image acquisition rates up to video
rate. In contrast to polarimetry, in which the polarization state
of the exiting beam is recorded, NOSE enables recovery of the complex-valued
Jones tensor of the sample that describes all polarization-dependent
observables of the measurement. Every video-rate scan produces a set
of 30 images (10 for each detector with three detectors operating
in parallel), each of which corresponds to a different polarization-dependent
result. Linear fitting of this image set contracts it down to a set
of five parameters for each detector in second harmonic generation
(SHG) and three parameters for the transmittance of the incident beam.
These parameters can in turn be used to recover the Jones tensor elements
of the sample. Following validation of the approach using z-cut quartz,
NOSE microscopy was performed for microcrystals of both naproxen and
glucose isomerase. When weighted by the measurement time, NOSE microscopy
was found to provide a substantial (>7 decades) improvement in
the
signal-to-noise ratio relative to our previous measurements based
on the rotation of optical elements and a 3-fold improvement relative
to previous single-point NOSE approaches.
Synthesis of various cyclic carbonates with yield up to 100% and turn over frequency (TOF) of 351 h -1 using CO 2 and epoxides and a cobalt (III) complex of tetraamidomacrocyclic ligand is described. The catalyst was characterized by single crystal X-ray crystallography. A study of reaction conditions indicates that 2 MPa pressure of CO 2 without any co-solvent is sufficient to achieve the desired product.
Second
harmonic generation (SHG) microscopy measurements indicate
that inkjet-printed racemic solutions of amino acids can produce nanocrystals
trapped in metastable polymorph forms upon rapid solvent evaporation.
Polymorphism impacts the composition, distribution, and physico-kinetic
properties of organic solids, with energetic arguments favoring the
most stable polymorph. In this study, unfavored noncentrosymmetric
crystal forms were observed by SHG microscopy. Polarization-dependent
SHG measurement and synchrotron X-ray microdiffraction analysis of
individual printed drops are consistent with formation of homochiral
crystal production. Fundamentally, these results provide evidence
supporting the ubiquity of Ostwald’s Rule of Stages, describing
the hypothesized transitioning of crystals between metastable polymorphic
forms in the early stages of crystal formation. Practically, the presence
of homochiral metastable forms has implications on chiral resolution
and on solid form preparations relying on rapid solvent evaporation.
Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.
Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen-rich biological tissues. NOSE is based on polarization-dependent second harmonic generation imaging. NOSE was used to access the molecular-level distribution of collagen fibril orientation relative to the local fiber axis at every position within the field of view. Fibril tilt-angle distribution was investigated by combining the NOSE measurements with ab initio calculations of the predicted molecular nonlinear optical response of a single collagen triple helix. The results were compared with results obtained previously by scanning electron microscopy, nuclear magnetic resonance imaging, and electron tomography. These results were enabled by first measuring the laboratory-frame Jones nonlinear susceptibility tensor, then extending to the local-frame tensor through pixel-by-pixel corrections based on local orientation.
Digital lock-in amplification (LIA) with synchronous digitization (SD) is shown to provide significant signal to noise (S/N) and linear dynamic range advantages in beam-scanning microscopy measurements using pulsed laser sources. Direct comparisons between SD-LIA and conventional LIA in homodyne second harmonic generation measurements resulted in S/N enhancements consistent with theoretical models. SD-LIA provided notably larger S/N enhancements in the limit of low light intensities, through the smooth transition between photon counting and signal averaging developed in previous work. Rapid beam scanning instrumentation with up to video rate acquisition speeds minimized photo-induced sample damage. The corresponding increased allowance for higher laser power without sample damage is advantageous for increasing the observed signal content.
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