Kenji Kumaki

Measuring Methods for Ultra-Low Light Intensity and Their Application to Extra-Weak Spontaneous Bioluminescence from Living Tissues

This paper reports experimental and theoretical studies and their comparisons on the sensitivity of photoelectric methods for detecting very weak optical signals based on the analog and digital techniques. The single photoelectron counting and the enhanced single photoelectron counting methods were found to be advantageous over the other methods with regard to both signal-to-noise ratio and effective range for optical input powers. Furthermore, the measurements of extra-weak spontaneous bioluminescence from living tissues such as tumors and livers of mice and rats were performed as a new application of the detection methods discussed and evaluated.

DIFFERENTIAL TIME COURSE OF INDUCTION OF 1α,25-DIHYDROXYVITAMIN D3-24-HYDROXYLASE mRNA EXPRESSION IN RATS BY 1α,25-DIHYDROXYVITAMIN D3 AND ITS ANALOGS

In order to investigate the in vivo mechanisms of target gene activation by vitamin D3 analogs, we compared the effects of two vitamin D3 analogs, 22-oxa-1alpha,25-(OH)2D3 (OCT) and 2beta (3-hydroxypropoxy) -1alpha,25-(OH)2D3 (ED-71) with that of 1alpha,25-(OH)2D3 on 1alpha,25-(OH)2D3 -24-hydroxylase[24(OH)ase] mRNA expression in the kidney and intestine of normal rats. In these experiments, all three compounds induced 24(OH)ase mRNA, but the time course of induction for each respective treatment was clearly different. OCT caused the most rapid onset of increased 24(OH)ase mRNA expression and its subsequent return to pre-injection levels. In marked contrast, ED-71 was the slowest to increase expression which was prolonged over that observed with the other compounds tested. These differences probably relate to the pharmacokinetic properties of these analogs, which are mainly generated by the affinity of analogs for the vitamin D-binding protein(DBP).

Determination of 22-oxacalcitriol, a new analog of 1α,25-dihydroxyvitamin D3, in human serum by liquid chromatography–mass spectrometry

A sensitive and specific liquid chromatographic-mass spectrometric assay has been developed for the determination of 22-oxacalcitriol (OCT), which is a new analog of 1alpha,25-dihydroxyvitamin D3. The analyte was isolated from serum by two solid-phase extraction steps on a C18 cartridge and NH2 cartridge. The recovery of OCT through two extraction steps was more than 90%. A related substance (ED-94), i.e. OCT with the side-chain shortened by one carbon, was used as an internal standard. Extracts were chromatographed on a C18 reversed-phase column interfaced to the electrospray ionization source. The mass spectrometer was operated in the positive-ion mode of selected reaction monitoring. The chromatographic run-time for one injection was less than 6 min. The intra- and inter-assay coefficients of variation for the lowest concentration examined (30 pg ml[-1]) were 9.83 and 10.67, respectively. And the analytical recovery of OCT added to serum was quantitative. Assay linearity was obtained in the range of 20-640 pg ml(-1).

Characteristics of mass spectrometric analyses coupled to gas chromatography and liquid chromatography for 22-oxacalcitriol, a vitamin D3 analog, and related compounds

The characteristics of the mass spectra of vitamin D3 related compounds were investigated by GC-MS and LC-MS using 22-oxacalcitriol (OCT), an analog of 1,25-dihydroxyvitamin D3, and related compounds. Fragmentation during GC-MS (electron impact ionization) of TMS-derivatives of OCT and the postulated metabolites gave useful structural information concerning the vitamin D3-skeleton and its side-chain, especially with respect to the oxidation positions of metabolites. In contrast, few fragment ions were observed in LC-MS (atmospheric pressure chemical ionization), showing that LC-MS gave poor structural information, except for molecular mass. However, when comparing the signal-to-noise ratio (S/N) observed during GC-MS and LC-MS analysis for OCT in plasma extracts, the S/N in LC-MS was over ten-times greater than in GC-MS, possibly due to the low recovery on derivatization and thermal-isomerization in GC-MS. Furthermore, both the GC-MS and the LC-MS allowed the analysis of many postulated metabolites in a single injection without any prior isolation of target metabolites from biological fluids by LC. These results suggest that GC-MS and LC-MS analysis for vitamin D3 related compounds such as OCT each have unique and distinct advantages. Therefore, the complementary use of both techniques enables the rapid and detailed characterization of vitamin D3 related compounds.

In vivo metabolism off the vitamin D analog, 22-oxacalcitriol: Evidence for side-chain truncation and 17-hydroxylation

After intravenous administration of the vitamin D3 analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24R(OH)OCT, 24S(OH)OCT, (25R)-26(OH)OCT, (25S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1alpha,25(OH)2 vitamin D3] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20S(OH)-hexanor-OCT and 17,20S(OH)2-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C23-O22 cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1alpha,25-(OH)2D3.

Binding of the carboxyl-terminal fragments of human parathyroid hormone to rat osteoblastic cells ROS 17/2.8

We have recently demonstrated that the carboxyl-terminal (C-terminal) PTH fragments increase or decrease alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells, depending on the length of deletion of amino-terminal amino acids of the PTH molecule, and interact with amino-terminal (N-terminal) PTH fragment [Acta Endocrinol 128:367]. In the present study, we examined individual and combined inhibitory effects of N-terminal and a series of truncated C-terminal PTH fragments [PTH (1-34), (35-84), (53-84) and (71-84)] on the binding of intact PTH molecule [PTH (1-84)] to ROS 17/2.8 cells.The C-terminal PTH fragments, as well as the N-terminal PTH fragment, partially inhibited the binding of [35S]-labeled PTH (1-84) to the cells. The inhibitory effect of C-terminal PTH fragments was reduced along with the deletion of aminoterminal amino acids of the PTH molecule, but still retained in the shortest C-terminal PTH fragment, PTH (71-84). When added together, PTH (1-34) reinforced the inhibitory effect of each C-terminal PTH fragment. The combination of PTH (1-34) and the complementary C-terminal PTH fragment, PTH (35-84), resulted in inhibition of [35S] PTH (1-84) binding to the level obtained by addition of the same concentration of unlabeled PTH (1-84).These findings suggested that the region relatively close to the C-terminal end of the PTH molecule might be essential for the binding of C-terminal PTH fragment could be responsible for the modification of the binding affinity of the peptide to the receptor and the action of the peptide.

The [53–63] amino acid portion of the parathyroid hormone molecule is essential for the stimulatory effect of carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8

The effect of a series of truncated carboxyl-terminal parathyroid hormone (PTH) fragments on alkaline phosphatase (ALP) activity was further examined in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8. As we previously reported, dexamethasone-induced ALP activity was inhibited not only by hPTH(1-84) and aminoterminal PTH fragment hPTH(1-34), but also by carboxyl-terminal PTH fragment hPTH(69-84). The longer carboxyl-terminal PTH fragment hPTH(53-84) stimulated ALP activity, and the shorter carboxyl-terminal PTH fragment hPTH(71-84) did not affect ALP activity.The longest newly synthesized carboxyl-terminal PTH fragment hPTH(35-84), which is complementary to amino-terminal PTH fragment hPTH(1-34), stimulated ALP activity as potently as hPTH(53-84), but not more potently than hPTH(53-84). Another newly synthesized carboxyl-terminal PTH fragment hPTH(64-84), which has an intermediate peptide length between hPTH (53-84) and hPTH(69-84), inhibited ALP activity as potently as hPTH(69-84).These results suggest that the 35-52 amino acid portion of the PTH molecule might not be crucial for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity, and that the 53–63 portion, but not the 64–68 portion, of the PTH molecule might be essential for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity. Furthermore, the importance of the 69th and 70th amino acid of the PTH molecule for the inhibitory effect of carboxyl-terminal PTH fragments on ALP activity was confirmed.