Influence of food on the gastric motor effect of the Kampo medicine rikkunshito in rat.

BACKGROUND: Rikkunshito, one of the Kampo medicines, is widely prescribed as a remedy for various upper gastrointestinal syndromes. The effect of rikkunshito is related to endogenous ghrelin and its active ingredient atractylodin enhances ghrelin receptor signaling. Kampo medicines are traditionally administered before or between meals; however, no definitive benefit of the timing of administration has been proven yet. To clarify the influence of food on the pharmacological action of rikkunshito, we investigated the gastric motor activity and pharmacokinetic profiles of atractylodin after the administration of rikkunshito in fasted and fed rats. METHODS: Phase III-like contractions in the gastric antrum after an injection of ghrelin were measured using a strain gauge force transducer. Rikkunshito was administered to rats during fasting or after a nutrient test meal. Ghrelin was injected 30 minutes later and gastric motility was evaluated. Furthermore, after rikkunshito administration, the pharmacokinetic profiles of atractylodin in the plasma and brain of fasted and free-fed rats were assessed. KEY RESULTS: Rikkunshito administration potentiated ghrelin-induced phase III-like contractions under fasting conditions. This effect was attenuated in animals fed a test meal. Atractylodin was detected pharmacokinetically in the plasma and brain after rikkunshito administration in rats, and free-fed rats exhibited a decreased maximum concentration of plasma atractylodin and a delayed time to reach the maximum concentration. CONCLUSIONS & INFERENCES: We show that the pharmacological action of rikkunshito is influenced by food in rats. The efficacy of rikkunshito may be associated with decreased absorption of its active ingredient atractylodin when food is in the stomach.

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection.

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20x3.9 mm I.D.) and analytical column (Symmetry C18, 75x4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile-water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40 degrees C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.

Baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form.

When baicalin was orally administered to conventional rats, it was detected in their plasma for 24 h after administration, but baicalein, the aglycone of baicalin, was not detected. However, when baicalin was given to germ-free rats, only a small amount of baicalin was detected in their plasma within 2 h after the administration, its AUC0-lim (the area under the concentration-time curve from 0 to last determination time) being 12.0% of that in conventional rats. Subsequently, a considerable amount (55.1 +/- 6.2%) of baicalin was recovered from the gastrointestinal tract even 4 h after administration. When baicalein was orally administered to conventional rats, however, baicalin appeared rapidly in their plasma at an AUC0-lim value similar to that obtained after oral administration of baicalin, despite the absence of baicalein in plasma. When intestinal absorption was evaluated by the rat jejunal loop method, baicalein was absorbed readily, but only traces of baicalin were absorbed. Moreover, in conventional rats a small amount (13.4 +/- 3.1%) of baicalin and an appreciable amount (21.9 +/- 3.4%) of baicalein were recovered from the gastrointestinal tract even 4 h after oral administration of baicalin, but only a small amount (3.93 +/- 1.43%) of baicalein was detected in the intestinal tract 1 h after administration of baicalein. Baicalin was transformed to baicalein readily by the rat gastric and caecal contents. When baicalin was administered orally to conventional rats, an appreciable amount of baicalein was recovered in their gastrointestinal tracts. Moreover, baicalein was efficiently conjugated to baicalin in rat intestinal and hepatic microsomes. These results indicate that baicalin itself is poorly absorbed from the rat gut, but is hydrolysed to baicalein by intestinal bacteria and then restored to its original form from the absorbed baicalein in the body.

In-vivo assessment of extrahepatic metabolism of paeoniflorin in rats: relevance to intestinal floral metabolism.

The extraction ratios of paeoniflorin in gut wall (EG), liver (EH) and lung (EL) were assessed by comparing AUCs after various routes of its administration to estimate the first-pass effects and the metabolism by intestinal flora. Pulmonary extraction ratio of paeniflorin was assessed by comparing AUCs calculated from venous and arterial plasma concentrations after its intravenous administration (0.5 mg kg-1). The mean pulmonary extraction ratio was estimated to be 0.06. The hepatic extraction ratio (EH was assessed by comparing AUCs after intraportal and intravenous administrations (0.5 and 5 mg kg-1). The plasma concentration profiles of paeoniflorin after intraportal administration were very close to those after intravenous administration, suggesting a negligible hepatic extraction ratio of paeoniflorin. The AUC value after intraperitoneal administration (0.5 mg kg-1) was greater than that after intraportal or intravenous administration. This finding suggests that paeoniflorin is not metabolized in the gut wall. The transference of paeoniflorin from the serosal side to the mucosal side was evaluated by the in-vitro everted sac method. The low intestinal permeability (19.4% at 60 min) was demonstrated by the comparison with phenobarbital (63.1% at 60 min). We conclude that paeoniflorin is not metabolize by gut wall, liver and lung, its poor absorption from the intestine results in extremely low bioavailability and the unabsorbed fraction of paeoniflorin is degraded by the intestinal flora.

Association of restriction fragment-length polymorphisms in the alcohol dehydrogenase 2 gene with alcoholic brain atrophy.

Alcohol abuse can induce brain atrophy, but it only occurs in some alcoholics. To investigate whether genetic polymorphism of alcohol-metabolizing enzymes [including alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] was related to alcoholic brain atrophy, we determined restriction fragment-length polymorphisms of the ADH2 and ALDH2 genes in 77 male alcoholics. Computed tomography was used to determine the severity of brain atrophy. Digestion with MaeIII and MboII after polymerase chain reaction amplification showed that the ADH2(1) gene frequency was significantly higher in patients with brain atrophy than in those without brain atrophy (chi 2 = 9.274, p < 0.01), whereas no significant association was observed between brain atrophy and the ALDH2 gene Multivariate analysis (including age, total alcohol intake, liver cirrhosis, and ADH2 genotype) showed that the ADH2(1)/ADH2(1) genotype was associated with alcoholic brain atrophy. These findings suggest that the ADH2(1) allele may be associated with alcoholic brain atrophy.

[Implicit sequential learning using Stroop task].

The research investigated the implicit sequential learning under the condition in which Stroop task was used. A stimulus of color name printed in incongruent color was presented on CRT monitor on each trial. The task was naming the color in which the word was printed. The color names were presented randomly and the printed colors were presented according to either a high structured or a low structured sequence. The learning of both sequences was observed when assessed through performance measure (decrease or increase in reaction times). The learning may be said to have occurred implicitly since the subjects trained under the low structured sequence proved to be almost unaware of the nature of the sequence and they performed no better than control subjects in a cued-recall task (generate the sequence). These results are considered important in examining the conditions in which sequential events can be learned implicitly.

Polymorphisms in alcohol metabolizing enzyme genes and alcoholic cirrhosis in Japanese patients: a multivariate analysis.

Alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and P450IIE1 are the primary enzymes that catalyze the conversion of ethanol to acetaldehyde and then to acetate. Genetic polymorphisms have been reported in ADH2, ADH3, ALDH2, and the 5'-flanking region of P450IIEI. In this study, we used multivariate analysis to determine which genetic polymorphisms in alcohol metabolizing enzymes were independently associated with the development of alcoholic cirrhosis. Thirty-four noncirrhotic alcoholic patients, including 27 with fatty liver and 7 with nonspecific changes, and 46 patients with alcoholic liver cirrhosis were studied. Restriction fragment length polymorphisms (RFLPs) in the ADH2 and P450IIE1 genes were detected by digestion of polymerase chain reaction (PCR)-amplified DNA with MaeIII and RsaI, respectively. In the ALDH2 gene, RFLPs were detected by differences in the MboII site after PCR amplification. By multivariate analysis of four significant factors including total alcohol intake, ADH, ALDH, and P450IIE1 using the multiple logistic regression model, genotype ADH2(2)/ADH2(2) (P = .029) and genotype c1/c1 of P450IIE1 (P = .013) were found to be independently associated with alcoholic cirrhosis. The odds ratios for ADH2(2)/ADH2(2) genotype and the type A genotype of P450IIE1 (c1/c1) were 4.600 and 4.006, respectively. These results suggest that ADH2 and P450IIE1 gene polymorphisms may be independently associated with the development of alcoholic liver cirrhosis in Japan.

[A sensitivity of CRP determination of cord blood and neonatal serum for neonatal infection].

CRP was determined for 110 cord bloods and peripheral blood of 36 newborns collected within 72 hours after delivery for the early diagnosis of newborn infection. The determination of CRP was done by a counting immunoassay method using PAMIA-30(Sysmex, Kobe, Japan). Sample volume needed was small and the time for determination was short. Within-run and between-run precisions were satisfactory, with CV values being approximately 6%. The CRP of healthy newborns was lower than that of cord blood, and the mean value was 33.4 +/- 4.2 ng/ml and the value was not significantly different from that obtained from the newborn babies with turbid amnionic fluid or early rupture of a sac. The CRP gradually increased after delivery had a peak at 24 to 48 hours after delivery. This tendency was observed both in healthy and infected newborns. The data were divided into 6 groups depending on the time collected after delivery (6, 12, 24, 48, and 72 hours). The CRP of blood from infected newborns tended to have higher CRP than that of healthy newborns in each group. Increased amount of CRP (ng/ml/hrs) was calculated as ((CRP of peripheral blood at time x)--(CRP of cord blood))/x, and this value was significantly higher (p < 0.05) in infected newborns than in healthy newborns 12hrs and more after delivery. Thus, CRP might be useful for monitoring the newborn infection.

Serum levels of integrins in chronic liver diseases.

The serum levels of beta 1 integrin (microgram/ml) were significantly higher in the patients with chronic persistent hepatitis (2.59 +/- 0.04), chronic active hepatitis (3.45 +/- 0.13), cirrhosis (4.77 +/- 0.30) and hepatocellular carcinoma (4.71 +/- 0.49) than in normal subjects (2.11 +/- 0.08). Serum levels of beta 3 integrin (microgram/ml) were significantly higher in the patients with chronic active hepatitis (10.48 +/- 1.22), liver cirrhosis (13.55 +/- 1.54) and hepatocellular carcinoma (14.1 +/- 1.77) when compared with normal subjects (5.51 +/- 0.52). A positive correlation was found between serum levels of beta 1 and beta 3 integrins (p < 0.001). A strong positive correlation was observed between serum levels of beta 1 integrin and histologic features, particularly in the degree of hepatic fibrosis, while no correlation was found between serum levels of beta 3 integrin and hepatic fibrosis. Immunohistochemical studies revealed that the beta 1 integrin was present on the plasma membranes of hepatocytes and sinusoidal lining cells in the normal liver, and was increased in fibrotic areas, and on the plasma membranes of hepatocytes and sinusoidal lining cells of the chronic liver disease. However, no positive staining for beta 3 integrin was observed in fibrotic area. The serum level of beta 1 integrin in patients with chronic liver diseases may therefore be a useful marker of hepatic fibrosis.

Serum tenascin levels in chronic liver disease.

To evaluate the diagnostic significance of tenascin, the extracellular matrix glycoprotein in chronic liver disease, serum tenascin levels were measured by a newly developed ELISA in 21 patients with chronic persistent hepatitis, in 55 with chronic active hepatitis, in 59 with liver cirrhosis, in 31 with hepatocellular carcinoma, in 26 with acute hepatitis and in 66 healthy subjects. The serum tenascin level was significantly elevated in the patients with chronic active hepatitis, liver cirrhosis, hepatocellular carcinoma, and acute hepatitis when compared with the healthy subjects (p < 0.001). The serum tenascin level also increased with increasing severity of chronic liver diseases. A significant correlation was observed between the serum tenascin levels and serum levels of various extracellular matrix proteins such as type III procollagen N-aminoterminal peptide (PIIIP), laminin and the 7S domain of type IV collagen (p < 0.001). A strong positive correlation was observed between the serum tenascin levels and histologic findings, particularly in the degree of hepatic fibrosis. This is the first report documenting serum tenascin level increases in patients with various chronic liver diseases. The measurement of the serum tenascin levels may provide additional information relevant to the study of connective tissue.

Urinary level of L-fucose as a marker of alcoholic liver disease.

The urinary levels of L-fucose were measured in 93 alcoholics; 20 of these were without liver disease, 57 with noncirrhotic alcoholic liver disease, and 16 with alcoholic liver cirrhosis. In addition, patients with cirrhosis due to viral infection, and healthy subjects were evaluated. The mean urinary L-fucose concentration showed significantly higher values in patients with alcoholic liver disease and alcoholic liver cirrhosis when compared with the healthy subjects or the chronic alcoholics without liver disease (p < 0.001). The urinary L-fucose level was also significantly higher (p < 0.001) in cases of alcoholic liver cirrhosis than in noncirrhotic alcoholic liver disease (384 +/- 97 vs. 240 +/- 95 mumol/g of creatinine). No difference was observed between the healthy subjects and chronic alcoholics without liver disease (143 +/- 29 vs. 155 +/- 60 mumol/g of creatinine). The urinary level of L-fucose was significantly higher with alcoholic cirrhosis (384 +/- 97 mumol/g of creatinine) than with viral cirrhosis (265 +/- 42 mumol/g of creatinine) (p < 0.001). The measurement of urinary L-fucose may be a useful marker of alcoholic liver disease.

Prevalence of hepatocellular carcinoma in patients with alcoholic cirrhosis and prior exposure to hepatitis C.

Sixty-three patients with alcoholic cirrhosis were retrospectively studied for the prevalence of antibodies to core (P22) and nonstructural (C100) region of hepatitis C virus (HCV). The prevalence rate of anti-P22 antibodies in patients with alcoholic cirrhosis was higher than that of anti-C100 antibodies (63.5% vs. 54.9%). The positivity rate of anti-C100 and/or anti-P22 antibodies was 73.0% (46/63) in alcoholic cirrhosis. We performed a multivariate analysis on the effects of age, sex, cumulative alcohol intake, anti-HCV antibodies, indocyanine green excretion test, and serum albumin on the development of hepatocellular carcinoma HCC in patients with cirrhosis, using Cox's proportional-hazards model, which revealed that anti-HCV positivity was the only independent prognostic variable for HCC in patients with alcoholic cirrhosis. The probability of HCC was significantly higher in the anti-HCV-positive patients than in the negative patients with alcoholic cirrhosis (p < 0.05). The 3-, 5- and 10-yr cumulative occurrence rate of HCC was, respectively, 13.3%, 41.3%, and 80.7% for anti-HCV-positive patients with alcoholic cirrhosis, compared with 0%, 8.3%, and 18.5% for anti-HCV-negative patients. In nonalcoholic patients with type C cirrhosis, the 3-, 5-, and 10-yr cumulative occurrence rate of HCC was 7.3%, 23.1%, and 56.5%, respectively. The follow-up studies indicate that hepatocarcinogenesis is hastened significantly in patients with alcoholic cirrhosis if they are positive for anti-HCV antibody, and that heavy alcohol consumption also is a risk factor for the development of HCC in patients with type C cirrhosis.

Serum vitronectin receptor in alcoholic liver disease: correlation with fibronectin receptor and morphological features.

In order to clarify the significance of serum level of vitronectin receptor (VNR) in alcoholic liver disease (ALD), we have investigated the relationship with fibronectin receptor (FNR) and histological liver features in 21 ALD patients. Serum level of VNR and FNR was measured by enzyme immunoassay. Liver disease activity was scored based on levels of fibrosis and focal intralobular necrosis and degeneration. The serum level of VNR (micrograms/ml) was significantly higher in the patients with hepatic fibrosis (9.87 +/- 2.51) and liver cirrhosis (10.80 +/- 1.52) than in normal subjects (5.51 +/- 0.52, P < 0.01) and fatty liver subjects (6.58 +/- 0.58, P < 0.05). A positive correlation was found between serum levels of VNR and fibronectin receptor (FNR) (P < 0.05). A positive correlation was observed between the serum level of FNR and the degree of hepatic fibrosis or focal intralobular necrosis and degeneration, while no correlation was found between the serum level of VNR and the degree of histological features. A positive correlation was also noted between the serum level of FNR and N-terminal type III procollagen aminopeptide (PIIIP) (P < 0.001), while no correlation was observed between the serum level of VNR and PIIIP. We conclude that the serum level of VNR is increased in patients with advanced alcoholic liver disease. However, the mechanism by which serum levels of beta subunit of VNR and FNR are increased may be different.

Serum level of carbohydrate-deficient transferrin as a marker of alcoholic liver disease.

Serum levels of carbohydrate-deficient transferrin (CDT) were assayed in 87 patients with alcoholic liver disease, 25 alcoholics without liver disease, 25 cases with viral liver disease and 37 healthy subjects, by two different methods (Pharmacia CDT RIA kit and Axis % CDT kit). The serum level of Pharmacia-CDT was significantly higher in the patients with alcoholic liver disease (38.9 +/- 2.8 U/l) compared to the normal subjects (18.9 +/- 0.2 U/l), alcoholics without liver disease (21.7 +/- 1.5 U/l) and non-alcoholic liver disease (viral liver disease) (23.4 +/- 1.6 U/l) (P < 0.001). The serum level of Axis-CDT was also significantly higher in the patients with alcoholic liver disease (4.22 +/- 0.48%) compared to the normal subjects (0.84 +/- 0.14%), alcoholics without liver disease (1.14 +/- 0.23%) and non-alcoholic liver disease (1.84 +/- 0.29%) (P < 0.001). A significant correlation was found between serum levels of CDT determined by the two kits (r = 0.718, P < 0.001). The serum level of Axis-CDT was significantly higher in patients with alcoholic hepatitis compared to the normal subjects (P < 0.005), while the serum level of Pharmacia-CDT was not increased in the patients with alcoholic hepatitis. These results indicate that determination of serum CDT levels is a useful marker of alcoholic liver disease, not a marker for alcohol consumption. Axis-CDT is more useful than Pharmacia-CDT for assaying the serum level of CDT in patients with alcoholic liver disease.

Detection of fibronectin receptor in sera: its clinical significance as a parameter of hepatic fibrosis.

Pooled sera collected from cirrhotic patients was fractionated by affinity chromatography with a fibronectin receptor monoclonal antibody against the beta-subunit of fibronectin receptor. Eluates were assayed using Western immunoblotting. The relative mobility of the protein reactive with fibronectin receptor antibody was nearly identical to that of the beta-subunit of fibronectin receptor, confirming that fibronectin receptor is present in human serum. Serum levels of the beta-subunit of fibronectin receptor were analyzed by sandwich enzyme-linked immunosorbent assay in patients with various liver diseases. The serum level of fibronectin receptor (micrograms/ml) was significantly higher in patients with chronic hepatitis (inactive, 2.59 +/- 0.04; active, 3.45 +/- 0.13), cirrhosis (4.77 +/- 0.30), alcoholic liver disease (2.96 +/- 0.16) and hepatocellular carcinoma (4.71 +/- 0.49) than in normal subjects (2.11 +/- 0.08). Strong positive correlation was observed between serum levels of fibronectin receptor and histological findings, particularly in the degree of hepatic fibrosis. Immunohistochemical studies with fibronectin receptor antibody revealed that the beta-subunit of fibronectin receptor was present on the plasma membrane of hepatocytes and sinusoidal lining cells in the normal liver and was increased in fibrotic areas and on the plasma membrane of hepatocytes and sinusoidal lining cells of fibrotic liver. The serum level of fibronectin receptor in patients with chronic liver diseases may therefore be a useful marker of hepatic fibrosis.