Development of scattering near-field optical microspectroscopy apparatus using an infrared synchrotron radiation source

We report the status of a scattering near-field microspectroscopy apparatus developed at SPring-8 using an infrared synchrotron radiation (IR-SR) source. It consists of a scattering type scanning near-field optical microscope and a Fourier transform infrared spectrometer. The IR-SR is used as a highly brilliant and broad-band IR source. This apparatus has potential for application in near-field spectroscopy with high spatial resolution beyond the diffraction limit. In order to eliminate background scatterings from the probe shaft and/or sample surface, we used higher harmonic demodulation method. The near-field spectra were observed by 2nd harmonic components using the lock-in detection. The spatial resolution of about 300 nm was achieved at around 1000 cm^? 1 (10 μm wavelength).     

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis

Blood levels of the vitamin D₃ (D₃) metabolites 25-hydroxyvitamin D₃ (25(OH)D₃), 24R,25-dihydroxyvitamin D₃, and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are recognized indicators for the diagnosis of bone metabolism-related diseases, D₃ deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D₃ metabolites, such as 20-hydroxyvitamin D₃ and lactone D₃, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D₃ metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D₃ (2), 25(OH)D₃-23,26-lactone (6), and 1,25(OH)₂D₃-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)₂D₃-23,26-lactone (7) in human serum.     

Characterization of Hyaluronidase 4 Involved in the Catabolism of Chondroitin Sulfate

Hyaluronidases (HYALs) are endo-beta-N-acetylhexosaminidases that depolymerize not only hyaluronan but also chondroitin sulfate (CS) at the initial step of their catabolism. Although HYAL1 hydrolyzes both CS and HA, HYAL4 is a CS-specific endoglycosidase. The substrate specificity of HYAL4 and identification of amino acid residues required for its enzymatic activity have been reported. In this study, we characterized the properties of HYAL4 including the expression levels in various tissues, cellular localization, and effects of its overexpression on intracellular CS catabolism, using cultured cells as well as mouse tissues. Hyal4 mRNA and HYAL4 protein were demonstrated to be ubiquitously expressed in various organs in the mouse. HYAL4 protein was shown to be present both on cell surfaces as well as in lysosomes of rat skeletal muscle myoblasts, L6 cells. Overexpression of HYAL4 in Chinese hamster ovary cells decreased in the total amount of CS, suggesting its involvement in the cellular catabolism of CS. In conclusion, HYAL4 may be widely distributed and play various biological roles, including the intracellular depolymerization of CS.     

Determination of three C18-oxygenated steroids in adrenal lesion segments in primary aldosteronism by super-selective adrenal venous sampling and LC/ESI–MS/MS

Super-selective adrenal venous sampling (ssAVS) can collect the adrenal tributary venous blood in the aldosterone (ALD)-hypersecreting segments in primary aldosteronism. The concentrations of the C18-oxygenated steroids, especially 18-oxocortisol (18-oxoF), in the lesion segments might be more useful indices than those in the peripheral or adrenal central veins (current candidate indexes) for the differential diagnosis of unilateral ALD-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). To verify this hypothesis, we developed a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI–MS/MS) method for simultaneously quantifying ALD, 18-oxoF and 18-hydroxycortisol in the adrenal tributary venous serum sample collected by ssAVS (ssAVS serum) and compared their concentrations between APA and BAH patients. Only deproteinization was required for a 10 μl sample prior to the LC/ESI–MS/MS analysis. Endogenous corticoids did not interfere with the quantifications, and the intra-assay and interassay precisions (≤ 8.3%) and accuracies (94.2–102.7%) were acceptable. The clinical study revealed that the 18-oxoF concentration was significantly higher in the ALD-producing tumor tissues (from APA patients) than in the hyperplastic tissues (from BAH patients). However, in conclusion, the 18-oxoF concentration in the ssAVS serum sample can be a rough indication but cannot be decisive for the differential diagnosis between APA and BAH owing to the significant individual difference.