The unusual bile acids hydroxylated at 1β-, 2β-, 4β-, 6α- and 19-positions of cholic and chenodeoxycholic acids have been found from the meconium, neonatal bile, blood and urine, and amniotic fluid and pregnant urine by GC-MS analysis. These hydroxylated bile acids and their conjugates were synthesized as their references from the corresponding usual bile acids as starting materials, and the simultaneous and high performance analytical methods were developed by GC-MS, HPLC and enzyme immunoassay. The above mentioned unusual bile acids were identified and determined in significant amounts of the total bile acids in the biological fluids from neonates and pregnant women, but not from normal adults. We, therefore, proposed that they should be called as "fetal bile acids". Application of the developed methods was performed for the studies on the dynamic profile of fetal bile acids in developing fetus and neonates, and the clinical diagnosis of the hepatobiliary diseases of infants and congenital bile acid biosynthetic disorders, Zellweger syndrome, celebrotendinus xanthomatosis, 3-oxo-Δ4-steroid 5β-reductase deficiency and congenital biliary atresia. Analyses of steroidal hormones, equine estrogens and 18-hydroxycortisol were also described.
A simple and precise method was established for the simultaneous determination of five selected marker components in oriental pharmaceutical decoction "Heii-San"( ?? ?? ?? ) by means of high-performance liquid chromatography using tetra-n-amylammonium bromide (TAA) as an ion-pair reagent. For the separation of these five components, such as hesperidin (1), 6-gingerol (2), honokiol (3), glycyrrhizin (4) and magnolol (5), an ODS column was used with multi-step gradient elution with 10 mM TAA (H3PO4, pH 4.0)-acetonitrile. The acetonitrile content linearly increased from 25 to 90%. This method was compared with other three methods, i.e. a water-acetonitrile method, a phosphate buffer method and an ion-suppression method. Five main components were eluted within 50 min without interference from co-existing components.
The antagonism of histamine H2-receptor by SWR-104SA (1'-bromo-N-[3-[3-(1-piperidinylmethyl) phenoxy] propyl]-spiro [1, 3-dioxolane-2, 9'-pentacyclo-[4. 3. 0. 0.2, 50.3, 80.4, 7] nonane]-4'-carboxamide monooxalate) was estimated using the isolated guinea-pig atrium and gastric acid secretion in rats. The concentration-response curves for the positive chronotropic effect of histamine on the atrium were displaced to the right in parallel without change in the maximum response by SWR-104SA and roxatidine acetate hydrochloride (roxatidine). The pA2 values of SWA-104SA and roxatidine acetate hydrochloride were 7.27 and 7.38, respectively. The slopes of the regression line of log (DR-1) against log SWR-104SA and roxatidine concentration were 1.00 and 0.92, respectively. There was no significant difference between the two compounds with respect to the histamine H2-receptor antagonism and/or binding manner in vitro. In the rat gastric fistula model stimulated by histamine, however, antisecretory potency of SWR-104SA was 3 times less than that of roxatidine. SWR-104SA given p.o. prevented the formation of gastric lesion induced by HCl-ethanol and indomethacin dose-dependently, roxatidine also prevented its formation by HCl-ethanol, but failed to prevent that by indomethacine. These antiulcer activities of SWR-104SA were shown at the lesser doses of antisecretory activity. On the other hand, roxatidine did not prevent the ulcer formation at the same dose level of antisecretory activity. These results indicate that the antiulcer effect of SWR-104SA is not caused by the antisecretory action alone. In addition, the mucosal protective activity of SWR-104SA for HCl-ethanol induced gastric lesion was independent of endogenous prostaglandins. Moreover SWR-104SA had inhibitory effects on indomethacin-induced gastric hypermotility in rats. These actions may partly explain the gastric protection of this compound and additional mechanisms such as mucosal blood flow could be involved in the antiulcer efficacy. Consequently, it appears that SWR-104SA is a new antiulcer drug that exerts a potent cytoprotective effect in addition to its gastric antisecretory activity.
We studied age-related alteration of inhibitory effects of a Ca2+ channel antagonist, isradipine, on α1-adrenoceptor mediated responses in 6-, 10-, and 40- week-old rats. Age-related changes were observed in the inhibitory effects of isradipine on the norepinephrine-induced maximum contractions in the isolated thoracic aorta, the amplitude of the Ca2+-evoked increase of intracellular Ca2+ concentration and sustained contraction in fura-2-loaded preparations and the maximum number of binding sites (Bmax) of [3H] (+)-isradipine to aortic membranes. The dissociation constant (KD) of [3H] (+)-isradipine did not alter with age. In anesthetized rats, isradipine inhibited the dose-response curves of norepinephrine on the blood pressure in 6-and 40-week-old animals more effectively than those in 10-week-old animals. An inverse relationship between the potency of norepinephrine in the isolated thoracic aorta, the inhibitory effects of isradipine on the norepinephrine-induced maximum contractions and the logarithm of Bmax obtained in the [3H] (+)-isradipine binding experiment were found. These results suggest that the age-related alteration of inhibitory effects of isradipine on α1-adrenoceptor mediated contractile responses and the increase of blood pressure are due to changes in the α1-adrenoceptor density and the population of voltage-dependent L-type Ca2+ channels, rather than changes in the affinity of drug to the α1-adrenoceptor or Ca2+-sensitivity of contractile elements of aortic smooth muscle.
Two inclusion modes were realized in the complexation between phenobarbital (PHB) and 2-hydroxypropyl-β-cyclodextrin (HPCyD) in aqueous solution using 13C-NMR spectroscopic and isothermal titration microcalorimetric studies. The geometries of the complexes were estimated by molecular dynamics calculations. Results of 13C-NMR spectrometry showed that the chemical shifts of barbituric acid and phenyl rings were significantly shifted upfield due to penetrating both rings into HPCyD cavity. And the affinity constants of the carbons of the phenyl ring were higher than those of the barbituric acid ring. The signals of ethyl side-chain contrarily moved downfield by the repulsions with the carbons situated around the rim of HPCyD. The calorimetric data indicates that two different types of PHB-HPCyD complexes at 1 : 1 stoichiometry are formed in unionized PHB, whereas, single type of inclusion at ionized PHB. The first type of inclusion complexes was formed with high affinity by an entropy-driven reaction associated with nearly constant values of -ΔG1 (26.9±1.0kJ/mol), -ΔH1 (3.73±0.86kJ/mol), and large positive ΔS1 (77.5±1.5J/mol/K) in all pH. It seems that the phenyl ring gets included within the HPCyD cavity and that the hydrophobic interaction dominates the stabilization of the complex. However, the second type of inclusion complexes with lower affinity was characterized by large values of -ΔH2 and small values of -ΔS2 at pH lower than 7.0, reflecting the van der Waals'or electrostatic interactions. The large inflections around pKa 7.4 were observed in all thermodynamic parameters. Especially, the values of -ΔG2 were lessened at pH > 8.0 due to decreases in the stability constants to the order of 102M-1. The barbituric acid ring seems to penetrate the cavity at lower pH than pKa. In both cases, the ethyl side-chain rests outside of the cavity.
A new solid-phase extraction procedure for urinary 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) was established. Sep-Pak Diol cartridge was used, because MHPG is a neutral and alcoholic compound. Aqueous samples were adsorbed to the cartridge, then MHPG was eluted by the addition of ethyl acetate. After the eluate was evaporated, the residuum was dissolved with HPLC mobile phase and injected into HPLC. The extraction procedure was highly specific to MHPG, and none of other acidic catecholamine metabolites, such as vanillylmandelic acid (VMA), homovanillic acid (HVA) and 3, 4-dihydroxy-phenylacetic acid (DOPAC), was extracted. The recovery of MHPG using this method was over 90% and higher than those using previously described methods such as liquid-liquid extraction with ethyl acetate. Of the three vanillyl alcohol isomers, isovanillyl alcohol was the most suitable as an internal standard for the correction of column-to-column variation of the recovery. Human urinary unconjugated MHPG extracted by the new procedure could be measured by HPLC with a fluorescence detection. Further complicated derivatization and more sensitive detection systems, such as GC-MS and HPLC-electrochemical detector (ECD), were not needed due to high selectivity and high recovery of the extraction procedure. In addition, the urinary total (conjugated plus unconjugated) MHPG content could also be determined by the same procedure after an enzymatic hydrolysis of conjugated MHPG. The newly developed extraction procedure was simple, rapid and highly specific, and might be applicable to the analysis of MHPG in various body fluids.
Polarizing photomicroscopy using the immersion method were bound to be useful to clarify the relationship between crystal habits and characteristics such as key refractive indices or a birefringence. It was found that there was similarity in the crystal habits among crystals in the same lot which have the same key refractive indices especially when they have similar habit coefficients each other. Using the average habit coefficients as well as the obtained parameter b-values from traces of a proper interference photo under crossed polars, the distributions of particle sizes and specific surface areas are easily calculated using a personal computer. The results of the several products of phenytoin, dygitoxin and lanatoside C crystals were graphically shown and discussed, compared with the results obtained by the sieving method.