26, 27-Hexafluoro-1α, 25-dihydroxyvitamin D3 [F6-1, 25-(OH)2D3] is more potent than 1α, 25-dihydroxyvitamin D3 [l, 25(OH)2D3] in stimulating bone resorption in vitro and in vivo. The reason why F6-1, 25(OH)2D3 is more active remains unclear. To clarify the relationship between the bone-resorbing activity of each vitamin D3 analogue and the metabolism of each analogue, in the present study, we used an ex vivo method that was established by Reynolds et al (Calcif Tissue Res, 1974, 15, 333-339). The effect of F6-1, 25(OH)2D3 or 1, 25(OH)2D3 on 45Ca release from parietal bones, prepared at 3, 14 and 24 h after injection of 1.9, 3.8, 7.6 or 15.2 pmol vitamin D analog /g body weight, was examined. F6-1, 25(OH)2D3 was more potent than 1, 25(OH)2D3 during each in vivo time period. 1, 25(OH)2D3 at 3h after the injection was more active compared to the control (no injection of 1, 25(OH)2D3), but not at 14 and 24h. The radioactivity of the bones after the injection of [3H]-F6-, 25(OH)2D3 was retained even at 24h. In the case of [3H]-1, 25(OH)2D3, the radioactivity of bones decreased with an increase in the in vivo period. In a HPLC analysis of the lipid extract of bone homogenate, [3H]-F6-1, 25(OH)2D3 alone was detected at 3 h after the injection and both [3H]-F6-1, 25(OH)2D3 and [3H]-26, 27-hexafluoro-1α, 23S, 25-trihydroxy-vitamin D3 [F6-1, 23, 25(OH)3D3] were detected at 14 and 24h after the injection. [3H]-1, 25(OH)2D3 was highly detected at 3 h after the injection, but it decreased with an increase in the in vivo period. In the ex vivo test, the activity of F6-1, 23, 25(OH)3D3 was less than that of F6-1, 25(OH)2D3 but similar to that of 1, 25(OH)2D3. The present study indicates that F6-1, 25(OH)2D3 is more active and more long-lasting than 1, 25(OH)2D3 in the ex vivo method. A higher potency of F6-1, 25(OH)2D3 is explained, at least partly, by the results that the amounts of both F6-1, 25(OH)2D3 and its active metabolite, F6-1, 23, 25(OH)3D3, in the bones are higher than that of 1, 25(OH)2D3, and that F6-1, 25(OH)2D3 and its metabolite are retained in bones longer than 1, 25(OH)2D3.
The effects of curdlan (CD) and gellan gum (GG), bacteria-producing polysaccharides, on lipid concentrations of serum and liver, fecal bile acid composition and intestinal fermentation products were studied in rats fed diets containing cellulose powder (CP), CD or GG at 5% for 4 wk. The cecal weight of the CD group increased significantly as compared to that of the other two groups and the pH of its contents was significantly low. The gastrointestinal transit time in the GG group was significantly shorter than that in the CP and CD groups. No significant inter-group differences were observed in the serum concentrations of total cholesterol and HDL-cholesterol, but a significant decrease was observed in the hepatic total cholesterol concentration of the CD group as compared to that of the CP and GG groups. No significant difference in the total bile acid excretion in feces was observed among the groups, but signifi-cantly low values were observed in the proportion of secondary bile acids in the CD group as compared to those of the CP and GG groups. Amounts of short-chain fatty acids (acetic, propionic and butyric acid) and lactic acid in the cecal contents were significantly higher in the CD group than in the other two groups. These results reveal that dietary CD is easily degraded and fermented by intestinal bacteria in the cecum and lowers cholesterol concentration in the liver, while dietary GG shortens the gastrointestinal transit time, suggesting the promotion of evacuation.
The methanolic extract from broad beans (MEBB), which is comprised of phenolic compounds, has free-radical scavenging activity. The effects of MEBB on cytosolic antioxidant enzymes and cell proliferation were examined in cultures of old (78-84% life-span completed) WI-38 human diploid fibroblasts. Because catechin is poly-phenol and has radical scavenging activity, it was used as the control in experiments. We observed that MEBB increased cellular growth when added to the cell culture. In MEBB at 40 and 120μg/mL, the cell proliferation increased by 14 and 27%, respectively, as compared to the control. In catechin, cell proliferation increased as well. Regarding cytosolic glutathione peroxidase (GSH-Px) activity, treatment of old cells with MEBB at 40 and 120μg/mL resulted in decreases as compared to the control. In contrast, catechin showed no similarities to the modification of GSH-Px activity. Cytosolic SOD activity was increased by treatment with 40μg/mL MEBB, and the activity showed a gradual decrease with increased MEBB concentrations. A similar trend occurred in the cells treated with catechin (4-20μM). These results suggest that cytosolic antioxidant enzyme activities in old cells may be modulated by MEBB treatment. We conclude that there may be a relation between the optimum MEBB concentration for the increase of cellular growth and the MEBB concentration required to exhibit a decrease in GSH-Px activity.
The combined effects of ethanol and components in fresh garlic on ethanol metabolism were investigated in the livers of mice. Male, I 1-wk-old C3H/HeNCrj mice were intragastrically administered 2g ethanol/kg body weight after being administered fresh garlic juice for 8 d (garlic group), and changes in the concentrations of ethanol, acetaldehyde and acetate in the serum, and changes in the activity of hepatic enzymes related to ethanol metabolism in mice were examined. The increases in the concentrations of acetaldehyde and acetate in the serum after ethanol administration tended to be diminished following garlic administration. The microsomel ethanol-oxidizing system (MEOS) in the livers of the garlic groups was significantly lower than that of the control microsomes at 2h after ethanol administration. It therefore seems that the decrease of MEOS in hepatic microsomes caused a smaller increase in the acet-aldehyde concentration in the serum of the garlic groups because cyto-solic alcohol dehydrogenase showed no significant difference between the control and garlic groups. After ethanol administration, the content of cytochrome P-450 in the hepatic microsomes of the control groups increased, while that of the garlic groups did not change although cytochrome P-450 (CYP) 2E1 and 1A2 in the hepatic microsomes of the garlic groups increased. These results indicate that the induction of isozymes of cytochrome P-450 other than CYP 2E1 and 1A2 was inhibited following garlic administration. Cytosolic high Km and total aldehyde dehydrogenase (AJDH) in the liver of the garlic groups tended to be lower than those activities of the control groups at 1 and 2h after ethanol administration. It therefore seems that the decreases of AIDH in the hepatic cytosols diminished the increase of acetate in the serum of the garlic groups after ethanol administration. These results suggest that the ethanol metabolism in the mouse liver is controlled by components in fresh garlic juice.
The effects of dietary soybean phospholipid, its hydrogena-tion product and safflower phospholipid on gene expression and the activity of hepatic enzymes in fatty acid biosynthesis were examined in fasted-refed rats. Phospholipid composition of soybean phospholipid and its hydrogenation product were the same, but the hydrogenation product contained negligible amounts of unsaturated fatty acids. Among phos-pholipid classes, lysophosphatidylcholine and phosphatidylinositol pro-portions were slightly higher in safflower phospholipid than in soybean phospholipid or its hydrogenation product. Rats were fasted for 2 d and refed a fat-free diet or a diet containing 4% fatty acids either as soybean oil or various phospholipid preparations for 3 d. Compared to the fat-free diet, the soybean oil diet only slightly decreased specific, but not total hepatic fatty acid synthetase and malic enzyme activity, and it was totally ineffective in modulating glucose 6-phosphate dehydrogenase and pyruvate kinase activity under our experimental conditions. The diets containing phospholipids, however, markedly decreased the activity of these enzymes. The extent of reduction was somewhat attenuated with hydrogenated soybean phospholipid as compared with soybean and safflower phos-pholipids. Dot and Northern blot hybridization using specific cDNA probes showed that, compared to a fat-free diet, diets containing phospholipids profoundly decreased the hepatic mRNA levels of enzymes in fatty acid synthesis. Soybean oil, however, only marginally affected these parameters. Hepatic mRNA levels for enzymes correlated well with enzyme activity. Dietary phospholipids therefore appear to have decreased enzyme activity in fatty acid synthesis primarily by suppressing the mRNA levels of these enzymes. Compared to soybean oil, hydrogenated soybean phospholipid is still effective in decreasing the activity and mRNA level of enzymes in fatty acid synthesis. Therefore, it is difficult to ascribe the potent physiological activity of phospholipid in reducing fatty acid synthesis entirely to polyunsaturated fatty acid moiety.
The effects of acute exercise and starvation on hepatic branched-chain α-keto acid dehydrogenase (BCKDH) complex activity were examined in female rats fed high (30%)- or low (8%)-protein diets. The total activity of the complex was significantly higher in the high protein-fed rats than in the low protein-fed rats but was not affected by acute exercise and starvation in either diet group. The proportion of the active form of BCKDH complex was less than 10% in both diet groups. Acute exercise and starvation markedly increased the active form of the complex in both diet groups. The activity of BCKDH kinase, which is responsible for inactivation of the BCKDH complex by phosphorylation, tended to be decreased by acute exercise and starvation in both diet groups. These results suggest that the activity of the BCKDH kinase is an important factor determining the proportion of the active form of BCKDH complex in exercise and starvation, and that the female rat is a useful model for studying the regulation of hepatic BCKDH complex activity.
We evaluated the bioavailability of two types of calcium from milk in two experiments. One was a micellar calcium phosphate-phosphopeptide (MCP-PP) complex in which the chemical form was similar to the original form of milk, and the other was a commercial whey calcium in which the chemical form was different from that of milk. In experiment 1, the calcium absorption, bone mineral density, and bone strength were examined when growing female rats were fed either MCP-PP complex or whey calcium as the sole source of calcium for 46 d. In experiment 2, the calcium solubility in the small intestine was measured when female rats were meal-fed either MCP-PP complex or whey calcium. The apparent calcium absorption rate in both groups decreased time-dependently during the experimental period, but the time-dependent change in the apparent calcium absorption rate was statistically different. It decreased more slowly in rats fed the MCP-PP diet than in rats fed the whey calcium diet. The bone mineral density of the femur in rats fed the MCP-PP diet was significantly higher than that of the rats fed the whey calcium diet. The bone strength (breaking force and energy) of the femur in rats fed the MCP-PP diet was higher than in the rats fed the whey calcium diet. The amount of soluble calcium in the small intestinal contents in rats at 2.5 h after ingestion of the MCP-PP diet was approximately three times higher than that found in rats fed the whey calcium diet. These results indicate that the calcium bioavailability of MCP-PP complex is higher than that of whey calcium, and this difference is due in part to the solubility in the intestine.
We studied the inhibitory effect of Cladosiphon fucoidan on the attachment of Helicobacter pylori (H. pylori), a gastroduodenal pathogen, to human gastric cell lines. The bacterial binding in these cell lines was inhibited more by Cladosiphon fucoidan (IC50=16-30mg/mL), than by the fucoidan from Fucus (IC50>30mg/mL). Dextran sulfate, another sulfated polysaccharide, did not inhibit the binding at all. Pre-incubating the bacterial suspension with fucoidans reinforced the inhibitory ability of these components, and reduced the IC50 value of Cladosiphon fucoidan to approximately 1mg/mL. However, the binding was not inhibited by pre-treatment of gastric cells with these components. It was also shown that this fucoidan blocks both Leb-and sulfatide-mediated attachment of H. pylori to gastric cells. Furthermore, fucoidan-binding proteins were found on the H. pylori cell surface by Western blot analysis. Thus, the inhibitory effect exerted by Cladosiphon fucoidan on binding between H. pylori and gastric cells might result from the coating with this component of the bacterial surface.
The purpose of this study was to investigate the effects of dietary green tea catechin on phospholipase A2 (PLA2) activity and the antithrombotic reaction of platelets in streptozotocin (STZ)-diabetic rats. Sprague-Dawley male rats weighing 100±10g were randomly divided into one normal and three STZ-diabetic groups, which were subdivided into catechin-free group (DM-OC), 0.5% catechin group (DM-0.5C) and 1% catechin group (DM-1C). The activity level of platelet phospholipase A2 was higher in the diabetic groups than in the normal group, while it was lower in DM-0.5C and DM-1C than in DM-OC. The activity of platelet cyclooxygenase in DM-OC was 1.1-fold as high as in the normal group, but was significantly reduced by catechin supplementation. The platelet thromboxane A2 (TXA2) formation became higher in DM-OC as compared to the normal group, but not in DM-0.5C and DM-1C. The synthesis of aortic prostacyclin (PGI2) was lower in DM-OC and DM-0.5C than in the normal group. The PGI2/TXA2 ratio was decreased to 55% in DM-OC, but was restored by catechin supplementation. These results indicate that STZ-diabetic rats are sensitive to platelet aggregation and thrombosis, and that the abnormality can be improved by dietary catechin.
The study was designed to test the ability of sequential applications of biotin-containing ointment to increase serum biotin levels. Twenty atopic dermatitis patients (mean age, 20.5 yr) and 11 healthy subjects (mean age, 25.5 yr) volunteered to participate in this study. The diagnosis of atopic dermatitis was established dermatologically. Seven grams per day of ointment containing 0.3% biotin and 1-4 g per day of steroid ointment were both applied sequentially. The healthy subjects applied only biotin ointment. The biotin concentration was determined microbiologically. Before biotin treatment, the average serum biotin level was significantly lower in atopic dermatitis patients than in healthy subjects. The percutaneous application of biotin-containing ointment caused a significant increase in the serum biotin concentration in both healthy subjects (from 41.5±10.0 to 50.2±9.2 nmol/L) and in atopic dermatitis patients (from 27.9±17.4 to 50.7±21.6 nmol/L), especially in patients whose initial level was low, and also could be effective in regulating the atopic allergic response involving eosinophils. In conclu-sion, biotin appears to be readily absorbed through both normal and dermatitis-affected human skin.
Because retinyl palmitate was reported to be more stable to oxidation than retinol, we wondered if retinyl palmitate was also more resistant to photolysis as compared to free alcohol. We investigated the resistance of ethanolic solutions of retinol, retinyl palmitate, or both to air oxidation and (or) photolysis using fluorescent light. The initial concentrations were all-trans-retinol, 14μmol/L, and all-traps-retinyl palmitate, 14μmol/L. The concentrations of retinol and retinyl palmitate were determined by HPLC and are expressed as a percentage of their original concentrations. After 4 h of exposure to an 18 W fluorescent lamp at 15 cm from the solution, the means (SD) of the surviving analytes were 64% (3%) for retinol and 5% (2%) for retinyl palmitate in a solution containing both retinol and retinyl palmitate. Taking account of the cis isomer arising from retinyl palmitate, 29% (3%) of the retinyl palmitate survived after 4 h of photolysis. Degradation of retinyl palmitate might occur after the conversion of trans isomer to cis isomer during pho-tolysis, however, trans isomer could be degraded with a lesser extent of isomerization. After 4 h of bubbling air through the solution in the dark, 49% (6%) of retinol and 69% (4%) of retinyl palmitate survived. Exposing retinol or retinyl palmitate separately to air oxidation, bubbling air through the solution, or photolysis, exposing them to light, we found that retinyl palmitate could retard the air oxidation of retinol (p<0.001), but it had no effect on the light-induced degradation of retinol. We also studied the effect of the addition of 1, 560μmol/Lα-tocopherol, 190μmol/L β-carotene and 2, 000μmol/L ascorbic acid as anti-oxidants. In the presence of 156 μmol/L α-tocopherol, 87% (1%) of the retinol and 91% (4%) of the retinyl palmitate remained after air oxidation. Although the photolysis of retinol and retinyl palmitate was also inhibited by 190 μmol/L β-carotene, α-tocopherol and ascorbic acid did not exert inhibiting effects. We conclude that retinyl palmitate is physico-chemically more labile to photolysis but is more resistant to air oxidation than retinol.
This study was designed to examine the differences in the effect of an iron-deficient diet on iron metabolism in Fischer-344 (FC), Sprague-Dawley (SD) and Wistar (WT) rats based on hemoglobin (Hb), hematocrit (Hct), serum iron levels, growth rate and organ weight. Hb concentration was higher in FC rats (14mg/100mL) on the initial day than in SD (10) and WT (10) rats. Although the Hb level was significantly decreased in FC rats fed an iron-deficient (ID, 8mg/kg) diet for 33 d compared to the FC rats fed an iron-adequate (IA, 50mg/kg) diet, the relative concentration of Hb was high in FC rats fed the ID diet as compared to the SD and WT rats fed the same diet. A similar relationship was detected between Hct and serum iron concentrations. Although serum triglycerides (TG) were significantly increased in each rat strain fed the ID diet as compared to the IA diet, the percentage of the value for the IA diet was lowest in FC rats (119%) fed the ID diet as compared to the SD (328) and WT (394) rats fed the same diet. Retroperitoneal fat pad was decreased in FC, SD and WT rats fed the ID diet as compared to the IA diet. SD rats were particularly sensitive to the reduction of retroperitoneal fat pad. The results suggested that rat strains responded differently to dietary iron inadequacy, and that FC rats were less sensitive to an iron-deficient diet as compared to the SD and WT rats.
In order to clarify whether or not a decrease in serum albumin concentration contributes to a low level of blood L-tryptophan (Trp) in nephrosis, blood Trp concentration at 30, 60, 90, and 120 min after oral administration of Trp (100μmol/kg body weight) in the same rats injected once with puromycin aminonucleoside (PAN) (100mg/kg body weight, i.p.), an inducer of nephrosis, was examined at different stages of nephrosis. The increase and decrease in blood Trp concentration after Trp administration were similar in the PAN-treated rats without nephrosis, the PAN-treated rats recovered from nephrosis, and untreated control rats. The maximum increase in blood Trp concentration at 30 min after Trp administration was lower in nephrotic rats than in control rats. In all rats treated with and without PAN, increased blood L-tryptophan concentrations at 30 min after L-tryptophan administration were positively correlated well with serum albumin concentrations (r=0.88, p<0.001). There was no difference in the intestinal absorption of the same dose of orally administered Trp between nephrotic and control rats. These results suggest that a decrease in serum albumin concentration may contribute to a low level of blood L-tryptophan in nephrosis.
The inhibitory activity of angiotensin I-converting enzyme (ACE) was extracted with 80% ethanol from the leaves of Ashi-taba (Angelica keiskei). The present ACE inhibitor was fractionated and separated with various chromatographies. The antihypertensive effects of the sample (G fraction) from Ashitaba on spontaneously hypertensive rats (SHR) were observed by long-term administration for 10 wk. Another sample (S fraction) from Ashitaba also had antihypertensive effects after a single intravenous administration to SHR. The sample was further purified by using several chromatographies. The ACE inhibitor fraction was characterized as follows: no significant absorbance, a zwitterion, a water-soluble substance and a positive ninhydrin reaction. According to a mass spectrum analysis, the molecular weight of the ACE inhibitor was determined to be 303 and Na-salt ions of carboxyl groups were detected. The ACE inhibitor from Ashitaba contained in the anti-hypertensive fraction was speculated to be very similar to authentic nicotianamine based on a comparative study of inhibitory activity, mass spectrum analysis and thin-layer chromatographies.
We investigated the effect of maitake (Grifola frondosa) water extract on inhibiting the conversion of C3H10T1/2B2C1 cells into adipocytes. Maitake water extract was fractionated by molecular sieve. Heat-labile compounds strongly inhibiting adipocyte conversion proved to occur in fractions of molecular weight of more than 10, 000 on the basis of activity measurement of glycerol-3-phosphate dehydrogenase.