Reproducibility of nifedipine absorption from GITS tablets: comparison of single-dose pharmacokinetics using 10, 20, 40 and 60 mg nifedipine.

OBJECTIVE: To examine the reproducibility of nifedipine absorption from gastrointestinal therapeutic system (GITS) tablets by comparing the single-dose pharmacokinetic profiles of 4 different dosages administered orally. METHODS: Twelve healthy male volunteers, aged between 22 and 29 years were enrolled in the open, 4-way, dose escalation study with single oral doses of 10, 20, 40 and 60 mg (two 30 mg) nifedipine GITS tablets. Each administration was separated by a 1-week washout period. Coefficients of variation (CV) of dose-corrected area under the concentration-time curve (AUC) and peak plasma drug concentrations (Cmax) were calculated from the pharmacokinetic profiles. RESULTS: Mean AUC and mean Cmax were dose-proportional from 10 to 60 mg. Although the CV of 4 mean dose-corrected AUC and Cmax were 5.5% and 17.5%, respectively, CV of dose-corrected AUC and Cmax in each subject varied from 5.1 to 37.4% (mean 11.0%) and from 14.1% to 46.4% (mean 25.8%), respectively. CONCLUSIONS: Whereas mean plasma nifedipine concentration remained markedly stable over a 16- to 24-hour interval and mean dose-corrected AUC showed good reproducibility with nifedipine GITS, the CV of dose-corrected AUC of the nifedipine GITS tablets in each subject showed large variability.

Relationship between time after intake of grapefruit juice and the effect on pharmacokinetics and pharmacodynamics of nisoldipine in healthy subjects.

A clinical study was performed in eight healthy volunteers to investigate the effect of various timing of grapefruit juice intake on nisoldipine pharmacokinetics and pharmacodynamics, and to validate our pharmacokinetic model. The subjects were given 10 mg oral nisoldipine with water (control), or 5 mg oral nisoldipine with 200 mL grapefruit juice (G0) or with water at 14 (G14), 38 (G38), 72 (G72) or 96 hours (G96) after a 7-day period of thrice-daily intake of grapefruit juice. Grapefruit juice ingestion did not affect heart rate or the effect area during the first 8 hours of heart rate after nisoldipine administration, although significant decreases of systolic and diastolic blood pressure were caused in G0 by coadministration of grapefruit juice with nisoldipine. Headaches were reported by 3, 2, and 1 persons in G0, G14, and G38, respectively, but no subjects in G72 and G96 reported headaches. Compared with the control group, the maximum plasma concentration of nisoldipine was significantly increased after grapefruit juice intake in G0 and G14, and the plasma concentration was significantly increased at each time in G0 to G72. Therefore the effect of grapefruit juice decreased time dependently and lasted for at least 3 days after intake. Furthermore, our model gave predicted values in good agreement with the observed values. It is therefore necessary to withhold grapefruit juice for at least 3 days before administration of the drug to prevent grapefruit juice-nisoldipine interaction.

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen.

To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen. Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (delta UbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase. Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b to heme o electron transfer occurs with a rate constant of approximately 10(4) s-1 in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen.

To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen. Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (DeltaUbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase. Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b-to-heme o electron transfer occurs with a rate constant of approximately 1x10(4) s(-1) in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.

Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide.

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.

Case report: mucinous cholangiocarcinoma featuring a multicystic appearance and periportal collar in imaging.

A case of mucinous cholangiocarcinoma (CC), a rare histological type of CC, featuring unusual images is reported. The patient was hospitalized because of acute development of jaundice and fever. Computed tomography demonstrated multiple cystic lesions in the liver and a band-like low density area parallel to the intrahepatic portal vein, a so-called 'periportal collar'. Endoscopic cholangiography revealed a stricture of the hepatic duct with slight upstream dilatation. Cytology of the bile juice and fine-needle aspiration of the cystic lesion in the liver disclosed mucinous carcinoma. The patient died of multiorgan failure 3 weeks after admission. The autopsied liver showed that multiple mucus lakes were lined with adenocarcinoma cells and signet ring cells were floating in the mucus lakes. The cancer cells had spread along the portal tract and invaded into the hepatic parenchyma.

Invasion depth diagnosis of depressed type early colorectal cancers by combined use of videoendoscopy and chromoendoscopy.

BACKGROUND: Depressed type early colorectal cancers are found less frequently than other polypoid cancers although they have a higher submucosal invasion rate. Recently videocolonoscopy and chromoendoscopy have become available and precise descriptions of these lesions are now routine. Because endoscopic mucosal resection is designated for intramucosal and focally extended submucosal (m-sm1) cancers, an evaluation of the characteristic findings indicating invasion depth with these modalities is important. METHODS: Between January 1991 and March 1996, 64 depressed type early colorectal cancers were detected and treated. When a faint abnormality of the mucosa was suspected by routine videocolonoscopy, 0.1% of indigo carmine solution was sprayed on the mucosal surface (chromoendoscopy). Colonoscopic findings of m-sm1 cancers and moderately and massively extended submucosal (sm2-3) cancers were retrospectively reviewed and compared with confirmed histologic findings. RESULTS: Characteristic colonoscopic findings needed for surgical operation were as follows: (1) expansion appearance, (2) deep depression surface, (3) irregular bottom of depression surface, and (4) folds converging toward the tumor. By using these findings, the invasion depth of depressed type early colorectal cancers could be correctly determined in 58 of 64 lesions (91%). CONCLUSIONS: Characteristic colonoscopic findings obtained by a combination of videocolonoscopy and chromoendoscopy are useful for determination of the invasion depth of depressed type colorectal cancers, an essential factor in choosing a treatment modality.

Determination and novel features of the absolute absorption spectra of the heme a moieties in cytochrome c oxidase.

The absolute absorption spectra of the two heme a moieties in cytochrome c oxidase were determined in the Soret region where spectral contributions from copper ions are negligible. This determination employs a set of absorption spectra of the enzyme recorded during anaerobic reduction with sodium dithionite, and does not require any other spectral data. The unique feature of the component spectra revealed in the present study suggests the existence of a specific interaction of heme a with the immediate environment as its origin. The usefulness of the absolute spectra in quantitative analysis of the spectral data is presented.

Site-directed mutagenesis of bacterial hemoglobin: the role of glutamine (E7) in oxygen-binding in the distal heme pocket.

The bacterial and yeast hemoglobins have a glutamine instead of histidine in the E7 position of the distal heme pocket. The recently determined crystal structure of Vitreoscilla hemoglobin (VHb) indicates that this residue is oriented out of the heme pocket and may not ligand the bound oxygen. This is in contrast to elephant myoglobin which also has a Gln(E7) but which does ligand the bound oxygen. This residue was changed in VHb using site-directed mutagenesis to leucine (VHbL) or to histidine (VHbH). Spectral and kinetic studies of the binding of oxygen and CO to VHbL showed that this substitution had little effect on the ligand-binding properties of this protein, evidence that Gln(E7) does not H-bond the bound ligand, in agreement with the findings of the crystallographic study of VHb. In contrast, the functional properties of VHbH were drastically altered in a way suggesting that the E7His may itself be liganded to the heme iron. These studies are further evidence that the distal heme pocket in VHb and related microbial hemoglobins differs from that in mammalian hemoglobins and may resemble in some ways the heme pocket in cytochrome b5.

Early nonpolypoid colorectal cancer: radiographic diagnosis of depth of invasion.

PURPOSE: To diagnose depth of invasion of early flat and depressed nonpolypoid colorectal cancers with double-contrast barium enema examination. MATERIALS AND METHODS: Ninety-seven early nonpolypoid colorectal cancers, initially identified with colonoscopy, were investigated with double-contrast barium enema examination. Depth of invasion in resected specimens obtained with endoscopic mucosal or surgical resection was determined. Cancer confined to the mucosal layer or with focal extension to the submucosal layer was defined as Ca-m; moderate to massive extension of cancer into the submucosal layer was classified Ca-sm. RESULTS: In flat elevated and depressed cancers, converging folds and semilunar deformity were observed significantly more frequently in Ca-sm than in Ca-m tumors (P < .05 and P < .01, respectively). Sensitivities and specificities of these findings for Ca-sm tumor were 42%, 93% and 86%, 85%, respectively. Deep depression, irregular surface of the depression, and tumor size larger than 20 mm were also predictive findings for depressed Ca-sm tumors (P < .05); the specificity of each was 100%. With use of these radiographic predictors, overall accuracy for diagnosing depth of invasion was 85%. CONCLUSION: Findings on double-contrast barium enema study are highly predictive of depth of invasion of early nonpolypoid colorectal cancer. Radiographic findings of converging folds, semilunar deformity, deep depression, irregular surface of the depression, and tumor size are predictors of Ca-sm tumor.

A novel chloride-binding site modulates the heme-copper binuclear center of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping. Previous studies [Y. Orii, T. Mogi, M. Sato-Watanabe, T. Hirano, and Y. Anraku (1995) Biochemistry 34, 1127-1132] suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer. To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties. UQO-412, which exhibits the Soret peak at 412 nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift. UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center. Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a K(d) value of 3.5 mM. Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable. The present studies demonstrated that the E. coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center. Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion.

Oxidation process of bovine heart ubiquinol-cytochrome c reductase as studied by stopped-flow rapid-scan spectrophotometry and simulations based on the mechanistic Q cycle model.

Stopped-flow rapid-scan spectrophotometry was employed to study complicated oxidation processes of ubiquinol-cytochrome c reductase (QCR) that was purified from bovine heart mitochondria and maximally contained 0.36 mol of ubiquinone-10/mol of heme c1. When fully reduced QCR was allowed to react with dioxygen in the presence of cytochrome c plus cytochrome c oxidase, the oxidation of b-type hemes accompanied an initial lag, apparently low potential heme bL was oxidized first, followed by high potential heme bH. Antimycin A inhibited the oxidation of both b-type hemes. The oxidation of heme c1 was triphasic and became biphasic in the presence of antimycin A. On the other hand, starting from partially reduced QCR that was poised at a higher redox potential with succinate and succinate-cytochrome c reductase, the b-type hemes were oxidized immediately without a lag. When the ubiquinone content in QCR was as low as 0.1 mol/mol heme c1 the oxidation of the b-type hemes was almost suppressed. As the Q-deficient QCR was supplemented with ubiquinol-2, the rapid oxidation of b-type hemes was restored to some extent. These results indicate that a limited amount of ubiquinone-10 found in purified preparations of QCR is obligatory for electron transfer from the b-type hemes to iron-sulfur protein (ISP) and heme c1. The characteristic oxidation profiles of heme bL, heme bH, and heme c1 were simulated successfully based on a mechanistic Q cycle model. According to the simulations the two-electron oxidation of ubiquinol-10 via the ISP and heme c1 pathway, which is more favorable thermodynamically than the bifurcation of electron flow into both ISP and heme bL, does really occur as long as heme bL is in the reduced state and provides ubiquinone-10 at center i. Mechanistically this process takes time, thus explaining the initial lag in the oxidation of the b-type hemes. With the partially reduced QCR, inherent ubisemiquinone at center i immediately oxidizes reduced heme bH thus eliminating the lag. The mechanistic Q cycle model consists of 56 reaction species, which are interconnected by the reaction paths specified with microscopic rate constants. The simulations further indicate that the rate constants for electron transfer between the redox centers can be from 10(5) to 10(3) s-1 and are rarely rate-limiting. On the other hand, a shuttle of ubiquinone or ubiquinol between center o and center i and the oxidation of heme c1 can be rate-limiting. The interplay of the microscopic rate constants determines the actual reaction pathway that is shown schematically by the "reaction map." Most significantly, the simulations support the consecutive oxidation of ubiquinol in center o as long as both heme bL and heme bH are in the reduced state. Only when heme bL is oxidized and ISP is reduced can SQo donate an electron to heme bL. Thus, we propose that a kinetic control mechanism, or "a kinetic switch," is significant for the bifurcation of electron flow.

[Evaluation of early colorectal cancer with submucosal invasion based on histological architecture].

To clarify the clinicopathological features of tumors with submucosal invasion, especially superficial elevated and sessile ones, histological architectures of 32 cases of submucosal invasive cancers were analyzed. They were classified into 3 types based on cross-section view: PG and NPG of Shimoda's classification and PG'. Histological architectures were drawn in accordance with these findings. In 7 PG-Ca which consisted of PG only, no apparent correlation was found between the tumor sizes and invasion depth of submucosal layer. However, 12 NPG-Ca which consisted of NPG only and 12 Mixed-Ca which include various cross-section views both showed massive invasion into the submucosa with 1 cm or more in tumor size. Therefore these two types were similar in biological behavior in terms of invasion depth. And degree of submucosal invasion tended to increase in the order of PG-Ca, Mixed-Ca and NPG-Ca. Examinating histological architectures of the Mixed-Ca tumors in details, all of these cancers were consisted of both PG and PG'. Of the 12 Mixed-Ca, 91.7% were proved to be PG dominant type. Macroscopically, I s and II a contained 88.9% and 40.0% of Mixed-Ca, respectively. In conclusion, these results suggest that PG' is a subtype of PG, and PG-Ca have a correlation between tumor invasion and alteration from PG to PG' in histological architectures among submucosal cancers. It is important to clarify morphological features of PG' in margin of I s and II a tumors in diagnosing the depth of early colorectal cancers.

CuB promotes both binding and reduction of dioxygen at the heme-copper binuclear center in the Escherichia coli bo-type ubiquinol oxidase.

A CuB-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of CuB seems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that CuB plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.

Kinetic characterization of the redox components in solubilized membranes from porcine neutrophils: reduction with dithionite and photoexcited NAD(P)H.

Cytochrome b558 in solubilized membranes prepared from porcine neutrophils was reduced by dithionite with a second-order rate constant of 2.5 x 10(6) M-1 s-1 at pH 7.4 and 20 degrees C accompanied by spectral changes with peaks at 428 nm and 560 nm and isosbestic points at 420 and 441 nm. When an anaerobic mixture of solubilized membranes and NAD(P)H was exposed to a white light, cytochrome b558 was reduced biphasically but with almost the same spectral profiles as in the dithionite reduction. Thus, participation of redox component(s) of unknown nature in the photochemical reduction was suggested. The NAD(P). radical generated by photoexcitation of NAD(P)H with a 355 nm laser pulse under anaerobic conditions also reduced cytochrome b558 with a high rate constant of 4.3 x 10(8) M-1 s-1 at pH 7.4 and 20 degrees C. The reduction of cytochrome b558 accompanied a simultaneous reduction of a component having an absorption band around 420 nm, suggesting participation of an iron-sulfur (Fe-S) cluster. The cytochrome b558 reduction was followed by its reoxidation by another component with an apparent second-order rate constant of 6.5 x 10(5) M-1 s-1. During the reoxidation, the Fe-S-like component remained in the reduced state, and thus its role other than as electron mediator in neutrophils NADPH oxidase is suggested. Not only the rate constant but also the extent of cytochrome b558 reoxidation decreased as the same reaction mixture was exposed to the laser pulse repeatedly. This result clearly indicates that an electron accumulates in this electron-accepting component designated tentatively as the omega component.

Facilitated intramolecular electron transfer in the Escherichia coli bo-type ubiquinol oxidase requires chloride.

Previous flow-flash measurements using the bo-type ubiquinol oxidase of Escherichia coli have revealed that facilitated heme B-heme O intramolecular electron transfer initiated upon reaction of the fully-reduced enzyme with dioxygen proceeds with a rate constant higher than 5 x 10(4) s-1 at pH 7.4 and 20 degrees C. Depletion of chloride anions from the enzyme by HPLC performed in the present study considerably decreased the rate constant to approximately 700 s-1, but the reaction of either dioxygen or carbon monoxide at the binuclear center was not affected at all kinetically. These results strongly suggest that Cl- is essential in maintaining a subtle molecular structure around the heme B and heme O that enables facilitated intramolecular electron transfer. Furthermore, a series of absorption spectra of the enzyme collected on time scales from microseconds to milliseconds during its single turnover indicate that as heme-heme intramolecular electron transfer is retarded by depletion of Cl-, an alternative electron transfer pathway is invoked. We discuss a possible role of novel bound Cl- in electron transfer from bound quinol to the binuclear center to accomplish dioxygen reduction.

Facilitated intramolecular electron transfer in cytochrome bo-type ubiquinol oxidase initiated upon reaction of the fully reduced enzyme with dioxygen.

Flow-flash and double-flash studies of the reaction of fully reduced bo-type quinol oxidase with oxygen have revealed that a single turnover of the enzyme proceeds much faster than mammalian cytochrome c oxidase. Facilitated intramolecular electron transfer in the bo-type oxidase with k > 5 x 10(4) s-1 at pH 7.4 and 20 degrees C is responsible for this fast turnover. The kinetics of this reaction indicates that the oxygen reduction does not require electron exchange between quinol oxidase molecules, each having three metal centers. Thus, a bound quinol in the fully reduced enzyme is suggested to be an electron source for complete reduction of dioxygen into water supplementing electrons provided by the metal centers. A single turnover of the quinol oxidase yields a novel spectral species with a Soret maximum at 415 nm corresponding to a 'pulsed' state of mammalian cytochrome c oxidase.