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Biomedical subjects

T Okano

Publications and source records attributed to T Okano.

At least 19 recordsLinked to original sources
PubMed1979

A rapid and precise method for the determination of vitamin D3 in rat skin by high-performance liquid chromatography.

In order to develop the investigations into photobiogenesis of vitamin D3, a rapid and precise method for the determination of the vitamin in rat skin was established by using high-performance liquid chromatography (HPLC). The proposed method included saponification of small pieces of rat skin, extraction of the unsaponifiable matter and application to HPLC using "Zorbax SIL" (straight-phase) as an adsorbent and 0.5% isopropanol in n-hexane as a mobile phase. The applicable lower limit of the method was 2ng of vitamin D3/cm2 of subcutaneous tissue-removed skin and it was possible to assay a concentration higher than 2 ng/cm2. The proposed method was applied to determine the content of vitamin D3 in rat skin obtained from in vivo and in vitro irradiation experiments. In the in vitro experiment, the yield of vitamin D3 increased in proportion to the irradiation time. On the other hand, the yield in the in vivo experiment showed a proportional increase similar to the in vitro experiment until 60 min irradiation, while a nearly constant value was obtained by irradiation for longer than 60 min. When the rat skin obtained from the in vitro experiment was irradiated with monochromatic UV rays in the range s60-350 nm, the most effective wavelength for the formation of vitamin D3 was confirmed to be 303 nm, which differs from the result obtained from the experiment in a test tube (295 nm). Moreover, the yield of vitamin D3 by irradiation with UV rays below 288 nm was extremely low, which again differed from the results of a test tube experiment. These differences were thought to be due to the filter effect of the malpighian layer in the epidermis of rat skin.

Animals↗
PubMed1979

Assay of 25-hydroxyvitamin D3 in human plasma by high-performance liquid chromatography.

A method using preparative and analytical high-performance liquid chromatography (HPLC) is proposed for the assay of 25-hydroxyvitamin D3 (25-OH-D3) in human plasma. A constant volume (0.2--2.0 ml) of a plasma sample was saponified. The unsaponifiable matter was first applied to preparative HPLC using a column of the straight-phase type (Zorbax SIL) in order to separate a 25-OH-D3 fraction from lipophilic concomitants giving ultraviolet-absorbing noise. Then, the separated 25-OH-D3 fraction was applied to analytical HPLC using a column of the reversed-phase type (Zorbax ODS) in order to measure the content of 25-OH-D3 from the peak height. This is a revised method from Jones (1978): Clin. Chem., 24, 287--298). The results showed that the clean-up procedure by the first preparative HPLC was successfully performed because the peak corresponding to 25-OH-D3 on the chromatogram of the second analytical HPLC was not disturbed by any other interfering peaks. Moreover, recovery through the whole procedure was satisfacotry (about 100%) and the procedures of saponification and isolation of the unsaponifiable matter diminished the overload to the columns. These are the revised points of Jones' method. When two determinations were performed on 12 samples of plasma taken from normal adults in October, the values were 22.6 +/- 4.8 and 21.0 +/- 3.6 (mean +/- SD) ng/ml, respectively.

Adult↗
PubMed1979

Isolation and purification of in vivo-generated 25-hydroxyvitamin D2 by preparative high-performance liquid chromatography.

In order to obtain a standard compound of 25-hydroxyvitamin D2 (25-OH-D2), a method for isolating in vivo-generated 25-OH-D2 from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D2 was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D2 from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D2. The proposed method for isolating in vivo-generated 25-OH-D2 is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.

Animals↗
PubMed1979

Relationship of carcinogenicity, mutagenicity, and K-region reactivity in benz[c]acridines.

Benz[c]acridine and its 10 methyl-substituted derivatives were examined for chemical reactivity with osmium tetroxide and mutagenic activity on Salmonella typhimurium, and the results were contrasted with the electronic charge in the K region and the carcinogenic activity of benz[c]acridines. The addition of osmium tetroxide took place at the K region of benz[c]acridines. A linear relationship was established between the charge in the K region and the rate constant of the second-order reaction between osmium tetroxide and benz[c]acridines except the 5,7-dimethyl derivative whose substituent in the 5-position sterically hindered the reaction. Benz[c]acridines showed mutagenic activity in the presence of S-9 Mix, but not in the absence of S-9 Mix. There was a corresponding relationship among the K-region reactivity, mutagenic activity, and carcinogenic activity in benz[c]acridines. The only exception for this was the 7,11-dimethyl derivative in which the 11-methyl group had a steric effect on the ring-nitrogen atom. It was suggested that a common mechanism with regard to the reactivity of the K region is working in both carcinogenesis and mutagenesis. It was concluded that benz[c]acridines are activated, before they display a carcinogenic or mutagenic activity, to a proximate form such as 5,6-epoxides, through a metabolic process in which the nucleophilic property of the K region to react with electrophilic reagents plays an important role.

Acridines↗
PubMed1978

In vivo and in vitro conversion of 7-dehydrocholesterol into vitamin D3 in rat skin by ultraviolet ray's irradiation.

In order to confirm the photochemical conversion of 7-dehydrocholesterol (7-DHC) into vitamin D3 in rat skin, the following in vitro and in vivo experiments were carried out. In the first (in vitro) experiment, the skin stripped off from a sacrificed normal rat was irradiated with an ultraviolet (UV) lamp for a constant period. In the second (in vivo) experiment, the normal rat, irradiated under the same condition mentioned above, was sacrificed and then the skin was stripped off. Lipids were individually extracted with chloroform-methanol (1:1) from the skin obtained in the two experiments and the solvent was evaporated. The resulting residue was saponified and the unsaponifiable matter extracted with benzene was purified by application to hydroxyalkoxy-propyl (HAP) Sephadex column chromatography. The resulting purified vitamin D3 fraction was applied to high-performance liquid chromatography (HPLC) in order to estimate vitamin D3. No peak, aside from that of alpha-naphthol as an internal standard, was observed in the HPLC chromatogram on the skin obtained from the non-irradiated rat, whereas the peak corresponding to vitamin D3 was observed in each HPLC chromatogram on both the irradiated skin (in vitro experiment) and the skin obtained from the irradiated rat (in vivo experiment). The peaks, confirmed to be due to vitamin D3 by the results of co-chromatography, were increased according to the increase of irradiation energy and there were little differences between the corresponding estimated values of vitamin D3 in the two experiments. These results prompted the conclusion that 7-DHC in rat skin was photochemically converted into vitamin D3 by UV irradiation and that the in vivo conversion mechanism might be the same as the in vitro one.

Animals↗
PubMed1978

Identification and determination of 25-hydroxyvitamin D3 in the blood and liver of vitamin D-deficient rats irradiated with ultraviolet light.

The intestinal calcium transport activity and serum calcium and phosphorous concentrations of vitamin D-deficient rats were increased by irradiation with an ultraviolet (UV) lamp. The existence of 25-hydroxyvitamin D3(25-OH-D3) in their bloods and livers was physicochemically confirmed by high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) and mass fragmentography, whereas the compound could not be detected in the tissues of non-irradiated rats. The results strongly suggested that vitamin D3 in vivo generated in irradiated rat skin might be normally metabolized and utilized to prevent rickets. The level of 25-OH-D3 in the tissues was determined by a HPLC method.

Animals↗
PubMed1978

Formation of free radicals from carcinogenic benz[c]acridines in the presence of proteins.

Formation of a free radical from carcinogenic and noncarcinogenic benz[c]acridine derivatives in the presence of proteins was examined. When aqueous mixture of benz[c]acridine and protein was stirred for a long period, shielded from light, benz[c]acridines were converted into free radicals. Albumin had the greatest effect in accelerating the free radical formation, and the effect was smaller in globulin, histone, and deoxyribonuclease. The g-value of the free radicals thus obtained was 2.005. Intensity of the electron spin resonance (ESR) signals of the free radical from carcinogenic derivatives was higher than those of the free radical from noncarcinogenic derivatives. There was a corresponding correlation among the ESR signal intensity of the free radical formed from the mixed system of benz[c]acridine and protein, charge of the K-region or ring nitrogen of the compound, and carcinogenicity of benz[c]acridines.

Acridines↗
PubMed1977

Immunohistological study on the ACTH producing islet cell carcinoma of the pancreas.

ACTH producing cells in islet cell carcinoma associated with Cushing's syndrome were studied by an improved immunoperoxidase as well as immunofluorescence technique. The cells which contained ACTH antigen were distributed throughout the tumor tissue of both primary and metastatic lesions in rather small numbers. In the small cancer nests they were present in an irregular or mosaic pattern and, on the contrary, they tend to line along the margin in the larger nests. The intracytoplasmic localization of the ACTH antigen was characteristically demonstrated in the infranuclear area of the tumor cells, closely simulating that of the argentaffin granules of the enterochromaffin cells. The electron microscopic observation identified membrane enclosed, cored granules, 200-300 nm in diameter, present in clusters in the infranuclear area of some tumor cells, which were considered corresponding well to the ACTH positive cells in immune staining.

Adenoma, Islet Cell↗