We studied the utility of a trial multivitamin preparation 'NK-041' for total parenteral nutrition. This preparation was used in 260 surgical patients during total parenteral nutrition, and changes in their blood vitamin levels were analyzed. Blood levels of all vitamins administered varied within the respective normal ranges, with few exceptional patients. During the administration of NK-041, avitaminosis, hypervitaminosis and side-effects were not observed. Laboratory examinations demonstrated that no abnormalities attributable to this preparation had arisen in these patients. Thus, these results seem to warrant the conclusion that NK-041 is useful as a multivitamin preparation for total parenteral nutrition, with no adverse effects.
Most Japanese pregnant women do not take the estimated average requirement (EAR) of folate for pregnant women, which is 400 μg/d. Nevertheless, folate deficiencies have not been reported. We examined biomarkers for evaluating the status of folate in pregnant Japanese women. Apparently healthy pregnant Japanese women were cross-sectionally recruited from a private obstetric hospital. We measured nutritional biomarkers of folate in these women, as well as their folate intake. The numbers of subjects were 230 (49, 62, and 81, and 38 in the first (up to 15 wk), second (16-30 wk), and third (over 31 wk) trimesters of pregnancy, and 1 mo after delivery, respectively). Folate intakes (medians) in the first, second, and third trimesters, and 1 mo after delivery were 235±147 (194), 226±83 (218), and 256±85 (254), and 300±105 (305) μg/d, respectively. Folate concentrations in plasma and erythrocytes appeared to be valid indicators for assessing folate status, with cut-off values of 7 nmol/L and 300 nmol/L, respectively. Plasma folate concentrations (medians) in the first, second, and third trimesters, and 1 mo after delivery were 17.6±9.6 (16.7), 12.4±8.3 (9.4), and 12.1±8.4 (9.4), and 10.7±8.9 (7.9) nmol/L, respectively. Each of the folate values was over the cut-off value. Erythrocyte folate concentrations (medians) in the first, second, and third trimesters, and 1 mo after delivery were 358±108 (365), 389±154 (365), and 325±150 (315), and 308±158 (276) nmol/L, respectively. Each of the folate values was near or over the cut-off value. In conclusion, the data obtained demonstrated that the estimated average requirement of folate for pregnant Japanese women was ≍250 μg/d.
Residual amounts of 2, 2-bis-(p-chlorophyenyl), 1, 1-dicholroethylene (p,p' DDE) and the concentrations of total lipids and triglyceride were studied with respect to the internal organs of rats which had been dosed with p,p' DDE and fed on the diet free from vitamin A or containing excess of vitamin A. Either the absence or excess of vitamin A was found to increase triglyceride content and p,p' DDE storage level in the liver. However, the ratio of p,p' DDE stroage level in the liver to content of liver triglyceride was almost constant independently of the vitamin A treatment. It is suggested that the dose of vitamin A has some influences upon the metabolic processes of liver triglyceride in rats and changes the storage level of P,P'DDE in the organ.
In order to examine the effect of dietary protein and fat on DDT metabolism and liver lipid concentration, rats were supplied with calory adjusted diets consisting of various amounts of protein and fat. The results suggested that dietary protein and fat changed the liver lipid concentration. They also showed that dietary protein and fat affected the residual concentration of DDT and its metabolites in the liver and adipose tissue. The change of the concentration of lipids in liver accompanied a change of the residual concentration of DDT in liver. This fact indicates that one effect of dietary protein and fat on the metabolism of DDT is attributable to the metabolic change of lipids in liver. Dietary protein accelerated the metabolism of DDT and reduced its residual concentration in liver. The results suggest that regression equations exist between the residual concentration of DDT in liver and (1) dietary factors and (2) lipid concentration in liver; In (DDT) = a x 1n (x1) + b x 1n(x2) + c x 1n(TL) + d (1) In(DDD or DDE) = a' x 1n(TL) + b' x 1n(PL) + c' (2) where x1, x2, TL, and PL are the dietary protein, dietary fat content, total lipid, and phospholipid concentration in liver, respectively. a, a', b, b', c, c', and d are constants. The concentrations of DDT and its metabolites estimated from equations (1) and (2) agrees well with the measured concentrations in liver.
In order to understand the function of carrageenan, an indigestible polysaccharide, as a promoter of colonic tumors induced by 1,2-dimethylhydrazine (DMH), molecular weight distribution of fecal carrageenan and amounts of fecal bile acids in rats given carrageenan and DMH treatment were examined. Gel filtration pattern on Sephacryl S-300 of fecal carrageenan was very similar to that of feeding carrageenan, and carrageenan ingested was quantitatively excreted in feces. Hexafluoroisopropyl ester-trifluoroacetyl derivatives of fecal bile acids were analyzed by gas chromatography on QF-1. Although there was a decreased concentration of deoxycholic acid and total bile acids in carrageenan-fed rats compared to control rats, no difference in the daily output was found because carrageenan ingestion increases fecal output. Significant increased concentration and daily output of lithocholic acid, a tumor-promoter, by feeding carrageenan were found. Thus, it was suggested that the promoting effect of carrageenan on colon tumorigenesis by DMH may be mediated by increased excretion of lithocholic acid and may not participate in degradation of carrageenan ingested.
We investigated whether dietary wheat bran (Wb) influences the proliferating cell nuclear antigen (PCNA)-labeling index (LI) of the colorectum of rats after the injection of 1,2-dimethylhydrazine (DMH). Male Wistar/ST rats were divided into four groups and given either a fiber-free diet or diets supplemented with Wb at the expense of the whole diet (5, 10 or 20 g/100 g diet). Then, they were subcutaneously injected with a single dose of DMH (20 mg/kg body weight) or the vehicle. At four weeks after the treatment, frozen sections of the colorectum were immunostained with anti-PCNA antibody (19F4). In the fiber-free group, DMH treatment significantly increased the PCNA-LI of the distal colon and rectum. Among the DMH treatment groups, the PCNA-LI of the distal colon in the Wb 5 g/100 g diet group was significantly lower than that in the fiber-free group. The PCNA-LI of the distal colon tended to increase as the amount of Wb supplemented was increased in the DMH-treated groups except for the fiber-free group. A similar trend was observed for the rectum. In conclusion, the ingestion of Wb diminished the increase in PCNA-LI in rat colorectum induced by DMH injection.
The frequency of apoptosis after treatment with 1,2-dimethylhydrazine (DMH) was counted in the descending colonic and rectal crypts of food-deprived and fed rats. Food-deprived or fed rats were subcutaneously injected with DMH (100 mg/kg body weight). Six hours after the injection, apoptotic cells were observed in crypt regions by light microscopy. The incidence of DMH-induced apoptosis in food-deprived rats was significantly higher than in fed rats. The incidence appeared to be higher in descending colon than in rectum. PAS staining revealed that DMH treatment lowered mucin secretion in crypts, which was substantially lowered by food deprivation. The effect of food deprivation on apoptosis induced by DMH may be due to the decrease in mucus barrier against DMH.
The effects of supplementing Bifidobacterium longum SBT 2928 and Lactobacillus acidophilus SBT 2062 to a high-fat, low-calcium diet on bile acid concentration, fatty acid concentration, cytolytic activity and intestinal alkaline phosphatase (ALP) activity of fecal water in rats injected with and without 1,2-dimethylhydrazine dihydrochloride (DMH) were examined. Male Wistar rats at 8 weeks of age were fed a diet containing 18% coconut oil, 2% corn oil and 0.1% calcium for 15 d. Lyophilized cultures were supplemented to test diets at a concentration of 1%. The feeding of a high-fat, low-calcium diet elevated the bile acid concentration, cytolytic activity and ALP activity of fecal water as compared to the AIN-76A diet, whereas the fatty acid concentration was not changed. None of the cultures had any effect on these parameters. Furthermore, 8 week-old rats were given a single subcutaneous injection of DMH at 40 mg/kg body weight, and fed the same diets for 15 d. The DMH injection had no effect on the bile acid concentration but increased the fatty acid concentration and cytolytic activity of fecal water. In contrast, ALP activity was lower in the DMH-treated rats than in the non-treated rats. The ingestion of B. longum lowered cytolytic activity but had no effect on the bile acids, fatty acids and ALP activity of fecal water. L. acidophilus had no effect on these parameters.
We investigated the influence of the administration of anti-asialo GM1 antibody on aberrant crypt foci (ACF) formation induced by a single injection of 1,2-dimethylhydrazine (DMH). At four weeks after the injection of DMH (20 mg/kg body weight), the number of ACF and aberrant crypts were counted. Most ACF appeared in the distal large bowel, accounting for approximately 60% of the total ACF in both groups. Rats administered anti-asialo GM1 had significantly more ACF in the distal colon, the rectum and the total large bowel as compared to control rats. A similar tendency was observed for the number of aberrant crypts. The increased number of ACF resulting from the administration of anti-asialo GM1 was not accompanied by the enlargement of ACF size in every part of the colon. This study demonstrated that the administration of anti-asialo GM1 at the initiation stage leads to an increase in ACF as well as aberrant crypts in the distal colon, rectum and total large bowel probably via the suppression of natural killer cells.
This study was conducted to examine the mechanisms of the anti-colon tumor effect of dietary sericin. Dietary supplementation of 3% sericin reduced colon mucosal lipid peroxide and aberrant crypt foci in 1,2-dimethylhydrazine-treated rats. The colon content from sericin-fed rats had much stronger antioxidant activity compared to that from control rats not receiving sericin. The amino acid composition of undigested proteins in the colon contents from sericin-fed rats was similar to that of sericin ingested. The results suggest that the strong antioxidant activity of undigested sericin in the colon content causes lower oxidative stress and tumorigenesis in the colon.
The effects of the dietary addition of orotic acid were studied on lipid levels in the rat liver and serum, 1,2-diacylglycerol levels in some organs, activities of antioxidant liver enzymes (superoxide dismutase, glutathione peroxidase, and catalase), and serum enzyme activities (ornithine carbamoyltransferase and alanine aminotransferase), after feeding for 0, 7, 14, and 21 d, respectively. Rats on the orotic acid diet accumulated more liver total lipids, triacylglycerol, and phospholipids than those on the basal diet. However, the levels of serum triacylglycerol and phospholipids of those rats were markedly decreased after 7, 14, and 21 d on the diet. Dietary orotic acid increased the 1,2-diacylglycerol levels in the liver of rats fed for 14 or 21 d, but not in the ileum of small intestine, vastus lateralis muscle, and heart. The addition of orotic acid lowered the activities of liver total and Cu,Zn-superoxide dismutase after feeding for 7, 14, and 21 d. The serum ornithine carbamoyltransferase activity after 14, and 21 d and that of serum alanine aminotransferase after 7, 14, and 21 d were increased. These data suggested that the increase in the activities of serum enzymes tested may result from liver damage induced by the marked accumulation of liver lipids and possibly from the increased superoxide anion because of the decreased activities of hepatic superoxide dismutase by orotic acid feeding.
Since it has been demonstrated that a high level of fat is a dietary factor in the etiology of colon cancer, the effect of carrageenan, a polysaccharide extracted from the red seaweeds, on 1,2-dimethylhydrazine-induced colonic tumors in rats fed a semipurified control diet containing an ordinary level of fat was studied. Nevertheless, the enhancing effect of carrageenan on colonic tumors was observed. The rats fed a carrageenan diet had approximately twice the fecal weight compared to the rats fed a control diet. While no significant differences were found in beta-glucuronidase activities in colonic mucosa, liver or plasma in the carrageenan-fed rats and controls, the activity in feces was significantly lower in the carrageenan-fed rats. At least, no beta-glucuronidase activity seemed to be related to the tumor-enhancing effect of carrageenan.
The combined effects of dimethylsulfoniopropionate (DMSP) (10(-3), 10(-4) and 10(-5) M) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (5 ng/mL) and the nerve growth factor (NGF) (5 ng/mL) on the outgrowth and elongation of neurites from pheochromocytoma (PC12) cells were examined on RPMI medium containing fetal bovine serum and horse serum with penicillin and streptomycin in collagen-coated dishes for 5 d. The growth was higher in increasing order of the DMSP (10(-3) M), MPTP and NGF, the DMSP (10(-5) M), MPTP and NGF, the MPTP and NGF group and the control group up to 3 d, but not in the NGF and the DMSP (10(-4) M), MPTP and NGF groups. The growth in all the experimental groups showed plateaus from days 4 to 5. The appearance of neurites from the cells in all the groups showed maxima on the 3rd day. The administration of NGF significantly stimulated the outgrowth of neurites from the cells, while the supplementation of MPTP noticeably inhibited the appearance of neurites even in the presence of NGF up to 5 d. However, the addition of DMSP (10(-3 )and 10(-4) M) to the latter group completely prevented the inhibition of the MPTP. These facts were significantly supported by the photographs of neurite-bearing cells on the 3rd day and also by the photometric analyses examining the reaction of MPTP to DMSP, NGF or Collagen IV.
The effect of dimethylsulfoniopropionate (DMSP) on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease (PD) of mice was examined for 5 d. The distilled water (the control group) and the DMSP solution at 5 x 10(-4) M (the DMSP group) were supplemented ad libitum to six mice each in two groups for 2 wk. An appropriate amount of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) solution (20 mg/kg body wt) was then intraperitoneally injected into all the test mice once a day initially for 3 d, which definitely made the control mice similar to the PD-model mice. The moving ability (running power) of the mice in both groups was measured using an automatic Wheel Running Instrument. The immobility duration of the upside-down mice in both groups was estimated by a newly developed polygraph (RMP-6008M, Nihon Koden Co., Ltd., Japan). The results indicated that the mice in the DMSP group showed a stronger moving ability and a shorter immobility duration compared to the mice in the control group during the experimental period. Furthermore, the amounts of catecholamines (dopamine and norepinephrine) in the brains except for the cerebellums of all the test mice were estimated 2 d after the last MPTP injection, which demonstrated that the brains of the mice in the DMSP group accumulate larger amounts of catecholamines, especially dopamine, than them in the control group. Accordingly, the administration of low concentrations of DMSP proved to prevent and/or ameliorate the decreased mobility and the typical immobility (Akinesia) of the MPTP-induced PD-model mice probably due to increased amounts of dopamine in the brains of the DMSP group mice.
The induction of Parkinson's disease (PD) in senescence-accelerated mice (SAMP8) by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the effects of dimethylsulfoniopropionate (DMSP) on induced PD model mice of SAMP8 were investigated for 5 wk. After many trials, the tail suspension test determining the PD symptoms indicated that an appropriate amount of MPTP clearly raises the SAMP8 mice to the PD-model mice. Moreover, DMSP administration to the PD-SAM model mice proved to completely reduce the PD symptoms in the mice and to accumulate large amounts of norepinephrine, dopamine and dioxyphenylacetate in the mouse brains without cerebellums. These results suggest that catecholamines accumulated in large quantities by the supplementation of DMSP to the double-diseased mice, PD-SAMP8 model mice, completely ameliorated the PD symptoms in these model mice.
Polyacrylamide disc gel electrophoresis of n-butanol solubilized alkaline phosphatase from chick duodenum revealed that the change of alkaline phosphatase induced by 1, 25-(OH)2D3 involved the transformation of desialoenzyme to sialoenzyme. The initial stimula-tion by 1, 25-(OH)2D3 of the incorporation of sialic acid into duodenal microsomes corresponded with the initial increase in calcium absorption. After this initial stimulation, there was a rapid decline in sialic acid incorporation into microsomes decreasing below control levels at 24hr. Calcium concentration in the microsomes followed a pattern similar to the incorporation of sialic acid into microsomes. The depressed sialic acid incorporation was reversed by the addition of calcium in vitro.
These results suggest that the initial action of 1, 25-(OH)2D3 is to change the membrane permeability to calcium and to change the subcellular distribution of calcium in the small intestine. The accumulated calcium in the microsomes then stimulates the sialic acid incorporation into desialoenzyme. This results in the changes of isozyme pattern of alkaline phosphatase, viz, the transformation of desialoenzyme to sialoenzyme. The transformed alkaline phosphatase might be one of the factors in-volved more directly in the regulation of calcium transport in intestine.
The mechanism by which resistance to 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) occurs in patients with chronic renal failure was studied. This agent induces differentiation and 1,25-(OH)2D3-24-hydroxylase activity in the mitochondria of the human promyelocytic leukemia cell line, HL-60, via a steroid-hormone receptor mechanism. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% normal or uremic serum. Treatment of these cells with 10(-8)M 1,25-(OH)2D3 for 5 days in a medium containing 10% uremic serum from 4 patients with chronic renal failure resulted in a maturation of the cells amounting to 30.3 +/- 18.7% (mean +/- SD) and 32.5 +/- 11.2%, as obtained by NBT reduction assay and NSE assay, respectively. These values were significantly lower than those obtained with 10% serum from 3 normal controls (66.6 +/- 12.8%, 58.3 +/- 10.9%, p less than 0.02). The treatment of HL-60 cells with 1,25-(OH)2D3 in a mixture of 5% normal plus 5% uremic serum caused cell differentiation to an extent similar to that in 10% uremic serum, which suggests the presence of a substance(s) having 1,25-(OH)2D3-inhibitory activity in the uremic serum. Exposure of HL-60 cells to uremic serum significantly impaired their responsiveness to 1,25-(OH)2D3 as assessed by the induction of the cell's ability to hydroxylate the C-24 position of 1,25-(OH)2[3H]D3. The mechanism by which uremic serum confers an impaired cellular response to 1,25-(OH)2D3 seemed to be due, in part, to a decrease in 1,25-(OH)2D3 receptor levels. A significant positive correlation was observed between intracellular cAMP levels and 1,25-(OH)2D3-induced HL-60 cell maturation. In summary, the mechanism by which uremic serum confers 1,25-(OH)2D3 resistance upon HL-60 cells seemed to be due to the presence of 1,25-(OH)2D3-inhibitory activity in uremic serum, which may modulate cellular responsiveness to 1,25-(OH)2D3 by such mechanisms as reducing 1,25-(OH)2D3 receptor levels in the cells, in part through alteration in cAMP metabolism.
The effect of pyridoxal 5'-phosphate on the 1,25-dihydroxyvitamin D3 receptor system has been studied by using pig intestinal chromatin. Pyridoxal 5'-phosphate did not affect the binding of 1,25-dihydroxyvitamin D3 to its receptor extracted from chromatin with hypertonic KCl, although in the presence of pyridoxal 5'-phosphate 1,25-dihydroxyvitamin D3-receptor complexes were not readily precipitated with polyethylene glycol. In contrast, pyridoxal 5'-phosphate showed a potency to dissociate the 1,25-dihydroxyvitamin D3 receptor from chromatin in a dose-dependent manner. A low concentration of pyridoxal 5'-phosphate was as effective as hypertonic KCl in dissociating the receptor from chromatin, while pyridoxine, p-nitrophenyl phosphate, or inorganic phosphate was much less effective. These observations suggest the inhibitory effect of pyridoxal 5'-phosphate on the recognition of 1,25-dihydroxyvitamin D3 by its receptor system.
The effects of 1,25-dihydroxycholecalciferol(1,25-(OH)2-D3) injection into developing chick embryos on bone gamma-carboxyglutamic acid-containing protein (BGP) levels in bone and serum were observed in relation to those on calcium metabolism using chick embryos and chicks aged from 13 days' incubation to 7 days of age. Chick BGP was determined by radioimmunoassay using antiserum to purified chick BGP. The injection of 1,25-(OH)2-D3 into the developing chick embryos of 13 days' incubation resulted in the increases of bone BGP levels at hatching and at 2 days of age. However, the distribution of bone BGP at hatching was not influenced by 1,25-(OH)2-D3. On the other hand, serum BGP levels were significantly increased by 1,25-(OH)2-D3 at 14, 15, 18 days' incubation and at 2 days of age. The early changes of serum BGP levels after 1,25-(OH)2-D3 treatment were correlated with the increases of serum calcium concentrations and the decreases of inorganic phosphorus concentrations. These results suggest that 1,25-(OH)2-D3 may directly or indirectly mediate BGP synthesis or secretion associated with calcium metabolism.
We examined the effects of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its synthetic analogue, 22-oxa-1,25-(OH)2D3, on differentiation of U937 cells by studying the cellular growth, surface marker expression and cytosolic free Ca2+ concentration ([Ca2+]i). RA inhibited cellular growth but did not induce expression of Mo2 (CD14), a monocyte/macrophage specific surface marker. To the contrary, 1,25-(OH)2D3 did not inhibit cellular growth, but increased CD 14-positive cells. Simultaneous addition of 1,25-(OH)2D3 and RA had no additive effect on cellular growth inhibition or CD14 expression. With regard to [Ca2+]i, however, 5 days' incubation with either of them increased the basal [Ca2+]i level and induced U937 cells to respond to formyl-methionyl-leucyl-phenylalanine (FMLP). When the cells were incubated with both 10(-6) M RA and 10(-8) M 1,25-(OH)2D3, basal [Ca2+]i was higher and FMLP caused a greater increase in [Ca2+]i than when only RA or 1,25-(OH)2D3 was added. These data suggest that RA and 1,25-(OH)2D3 induce monocytoid differentiation in U937 cells through different pathways and act synergistically in the differentiation process. The 22-oxa-1,25-(OH)2D3 induced CD14 expression, basal [Ca2+]i increase and [Ca2+]i response to FMLP, but did not cause cellular growth inhibition in U937 cells, and in these points, 22-oxa-1,25-(OH)2D3 exhibited no significantly different effects from 1,25-(OH)2D3. Thus, 22-oxa-1,25-(OH)2D3 has the same potent activity as 1,25-(OH)2D3 in inducing differentiation of U937 cells.
Lymphatic recovery of 1(3)-stearoyl-2,3(1)-dilinoleoylglycerol (SLL) and 2-linoleoyl-1,3-distearoylglycerol (SLS) which had been enzymatically synthesized were compared with those of trilinoleoylglycerol (LLL) and the randomly esterified triacylglycerol which contained stearic acid and linoleic acid at 1:2. Recoveries of linoleic acid in all of the triacylglycerols were more than 94.0%. Lymphatic 24 h-recoveries of stearic acid given as SLL and SLS were significantly lower than that of stearic acid given as the randomly esterified triacylglycerol. Recoveries of stearic acid from SLL, SLS and the randomly esterified triacylglycerol were 88.49%, 68.3% and 101%. respectively.
The synergistic effect of food additives or food colors on the toxicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was investigated using primary cultured rat hepatocytes. When hepatocytes from rats fed a standard diet were treated with a mixture of four major food additives (sorbitol, sodium L(+)-glutamate, benzoic acid, and propylene glycol) or a mixture of six typical artificial food colors (erythrosine, allura red, new coccine, brilliant blue, tertrazine, and fast green), the in vitro treated food-color mixture itself showed cytotoxicity: the reduction of cell viability and decreases in the activities of gluconeogenesis and ureogenesis. The food-color mixture enhanced cytotoxicity of Trp-P-1 obviously. We then investigated the effects of in vivo-dosed food additives or food colors on Trp-P-1-caused toxicity. Hepatocytes were isolated and cultured from rats fed a diet containing a mixture of food additives or a mixture of food colors with half the amount of their respective acceptable daily intake for 4 wk. Trp-P-1 was administered to the hepatocytes at various concentrations for 12 h. Synergistic effects of in vivo-dosed food additives and food colors were not observed on Trp-P-1-caused cytotoxicity as estimated by a loss of cell viability and the reductions of DNA and protein syntheses. On the contrary, we have observed that in vivo administered food colors synergistically facilitated to reduce the activities of gluconeogenesis and ureogenesis in Trp-P-1-treated hepatocytes. These results suggest that the daily intake of artificial food colors may impair hepatic functions such as gluconeogenesis and ureogenesis, when dietary carcinogens are exposed to the liver cells.
L-Gulono-1,4-lactone oxidase activity was detected in G. frondosa: therefore its properties were studied after purification. A 766-fold purified preparation of the enzyme from fresh fruit bodies was obtained by means of a seven-step procedure, the overall yield being 14%. The purified enzyme gave a single band on polyacrylamide gel electrophoresis and its absorption spectrum exhibited the characteristic of a flavoenzyme. The enzyme produced L-ascorbic acid and H2O2, with L-gulono-1,4-lactone (GL) as the substrate and oxygen as the electron acceptor, and was optimally active at around pH 7.0 and 45 degrees C. Its molecular mass was determined to be 250 kDa on gel filtration, while the dissociated enzyme exhibited a molecular mass of 69 kDa on SDS-polyacrylamide gel electrophoresis, but the true molecular weight is unknown because of the trypsin treatment in the purification process. The apparent Km value for GL was 24+/-1 mM. Its substrate specificity was extremely high and, assuming that for GL to be 100, the following results were obtained: D-mannono-, 25: D-glucono-, 4; L-idono-, 3; L-galactono-1,4-lactone, 2; and 15 other lactones tested, 0. It is presumed that this enzyme is similar to animal GL-oxidase, ascomycetes D-arabinonolactone oxidase, etc.
The hydrolyzing activities in rat small intestine for newly developed sugar substitutes, alpha-D-glucopyranosyl-1,6-sorbitol (GPS) and alpha-D-glucopyranosyl-1,6-mannitol (GPM), both of which are produced by hydrogenation of palatinose, were characterized. GPS and GPM were hydrolyzed in mucosal homogenate as well as in brush border membranes of rat small intestine at a slower rate than the rate of hydrolysis of palatinose (30%) and sucrose (6-7%). Gel filtration column chromatography of disaccharidases solubilized from brush border membranes revealed that GPS and GPM were hydrolyzed mainly by sucrase-isomaltase complex and its degradation product, i.e. isomaltase monomer. The isomaltase monomer, purified from small intestine of rats, possessed similar Km values for GPS (2.47 mM) and GPM (5.38 mM) as compared to those of purified sucrase-isomaltase complex. The Vmax values of isomaltase monomer for GPS and GPM were twice as high as those of sucrase-isomaltase, suggesting that GPS and GPM are hydrolyzed by the active site of isomaltase. On the other hand, a small amount (up to 17%) of GPS- and GPM-hydrolyzing activities was ascribed to glucoamylase, which possessed relatively high Km values for GPS (18.7 mM) and GPM (32.9 mM). To examine a physiological significance of GPS- and GPM-hydrolyzing activities, the transmural potential difference (delta PD) evoked by Na+-dependent active transport of glucose, produced by the hydrolysis of these disaccharide alcohols, was measured in everted segments of rat jejunum. The relative rates of absorption of glucose produced by the hydrolysis of GPS and GPM were 36% and 27% of that of palatinose, directly reflecting the hydrolyzing activities determined in jejunal homogenate. These results suggest that the process of hydrolysis is the rate limiting step in digestion-absorption process of palatinose, GPS and GPM in small intestine.
We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] as a metabolite of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] produced in rat osteosarcoma cells (UMR 106). We now report the isolation of 24R,25-dihydroxy-3-epi-vitamin D3 [24R,25(OH)2-3-epi-D3] as a metabolite of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] by high-performance liquid chromatography (HPLC) with chiral column and its structure assignment by proton nuclear magnetic resonance (1H-NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. We also demonstrated the production of 24R,25(OH)2-3-epi-D, in two other cell lines [human colon carcinoma cells (Caco-2) and porcine kidney cells (LLC-PK1)] which were previously shown to convert 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3. It can be seen that the production of 24R,25(OH)2- 3-epi-D3 from 24R,25(OH)2D3 is lower than that of 1alpha,25(OH)2-3-epi-D3 from 1alpha,25(OH)2D3 in all the cells studied. 24R,25(OH)2-3-epi-D3 was found to be inactive in terms of its ability to bind to the vitamin D receptor (VDR), in inhibiting proliferation and in inducing differentiation of human promyelocytic leukemia cells (HL-60). Thus, our study indicates that the C-3 epimerization pathway is common to both 1alpha,25(OH)2D3 and 24R,25(OH)2D3 and may play an important role in modulating the concentration and the biological activity of these two major vitamin D3 metabolites in target tissues.
We investigated the effect of Lactobacillus crispatus KT-11 (KT-11) on intestinal immune systems in C3H/HeN mice. The level of intestinal total immunoglobulin (Ig) A was significantly higher in mice given KT-11 than in mice not given KT-11. Gene expression relating to antibody production and innate immune response increased more than 2-fold in the former compared with the later. Moreover, the number of IL-6(+)CD11b(+) cells was significantly higher in Peyer's patch cells cultured with KT-11 than in those cultured without KT-11, although the number of CD4(+) cells and the cell ratio of CD4(+)/CD8(+) were remarkably lower in the culture with KT-11. These results indicate that KT-11 enhances intestinal IgA production and innate immune response in C3H/HeN mice.
This study was undertaken to investigate the effects of isoenergetic and increased amounts of egg white protein one hour before a run on the changes in the post-exercise blood biochemistry and the rating of the perceived exertion (RPE). Twenty-four male distance runners were divided into four groups. Venous blood samples were collected at three time points: just before the experiment (Pre), just after a 12,000 m run (Post 0 h) and one hour after the run (Post 1 h). After the first blood sampling, each participant consumed one of the four isoenergetic supplements (86 kcal); 0 g, 5 g, 10 g, or 20 g of egg white protein. The blood glucose, free amino acid, and branched chain amino acid (BCAA) levels in the 0 g, 5 g, and 10 g protein groups were higher at Post 0 h than at Pre. The pre-exercise intake of the 20 g protein group showed the smallest changes in the blood biochemicals. The RPE scores were significantly higher at Post 0 h, and did not vary among the four protein groups. Accordingly, the pre-exercise carbohydrate intakes significantly altered the post-exercise blood biochemisty findings, but the pre-exercise protein intake did not. Furthermore, the changes in the RPE scores in our present study were not explained by changes in the serum free tryptophan or the BCAA levels, and an increased dietary intake of egg white protein might not prevent post-exercise increases in the RPE scores.
In this study, the differences between the pattern of stained proteins from genetically obese Zucker rats and those from lean Zucker rats were analyzed using SDS-polyacrylamide gel electrophoresis. The level of the 120 kDa protein in the liver cytosol fraction of these obese rats was several times higher than that found in the lean rats. This protein was not present in the mitochondrial fractions of either strain. The level of the 120 kDa protein was decreased drastically during a 72-h fast in the obese Zucker rats. An increase in the level of this protein was induced in the lean Zucker rats through a 48-h fast followed by refeeding with a high-carbohydrate diet. However, when the lean Zucker rats were refed with a high-fat diet following the fast, no significant change was observed in the level of the 120 kDa protein. These observations strongly suggest that the elevated levels of the 120 kDa protein seen in the liver cytosol fraction of obese Zucker rats may be responsible for the observed increases in lipogenesis and pathogenesis of obesity in these rats.
This paper describes the synthesis and structural assignment of noval 125I-1 alpha,25-dihydroxyvitamin D3 derivatives (1a) and (1b) labeled with 125I-Bolton-Hunter reagent [N-succinimidyl 3-(4-hydroxy-3-iodo[125I]phenyl)propionate] (1), which is known as a protein-labeling reagent, as tracers for radioimmunoassay (RIA). The radiospecific activities of these tracers (1a) and (1b) were calculated as 2,200 Ci/mmol (81.4 TBq/mmol).
This study examined whether the Pro12Ala polymorphism of the PPARgamma2 gene is associated with obesity, hypertension and cardiovascular risk profiles in Korean adult women. We studied 129 Korean women (aged 42.71 +/- 8.56 y) who were divided into 2 groups as a Pro12Pro homozygous group and a Pro12Ala heterozygous or Ala12Ala homozygous group based upon PPARy2 genotype. Anthropometric parameters, blood pressure, abdominal fat area and blood lipid profiles were compared between the 2 groups, and the association of Ala allele frequency in PPARgamma2 gene with obesity or hypertension was evaluated. Most anthropometric parameters and blood lipid profiles did not differ significantly between the genotypes. However, all variables of skinfold thickness, body circumference and abdominal fat area of Pro12Ala heterozygous were consistently higher compared to the Pro12Pro homozygous subjects without a significance differences. The hypertensive group had significantly higher (p = 0.004) Ala12 allele frequency than the normotensive group whereas allele frequencies did not differ significantly between the obese group and non-obese group. Ala allele carriers had a significantly higher risk of hypertension than non-carriers in logistic regression analysis. There was no evidence that the Ala allele can be regarded as an independent risk factor for obesity. In conclusion, all variables related to obesity showed a consistently higher trend in Pro12Ala heterozygous subjects compared to Pro12Pro homozygous subjects. Pro12Ala heterozygous subjects showed an increasing trend of elevated blood pressure compared to Pro12Pro homozygous subjects. Ala12 variant as well as BMI and TG were regarded as independent risk factors for hypertension in our subjects.
In diabetic patients, glucolipotoxicity induces multiple defects in β-cells. Furthermore, increasing evidence also confirms the direct interaction between the adipocytes and β-cells. The beneficial efficacy of vitamin C (Vc) on β-cells is rarely investigated. In this study, INS-1 832/13 β-cells were cultured with high levels of glucose and free fatty acid (FFA) or cocultured with 3T3-L1 adipocytes in the presence or absence of Vc. Vc decreased glucolipotoxicity-induced cell mass loss by reducing apoptosis and reactive oxidative species (ROS). After treatment with elevated glucose and FFA, β-cell secretion dysfunction was evidenced and was partially improved by 1, 10 and 50 μg/mL Vc treatment. In the coculture system, impaired secretion function was also moderately normalized upon addition of 10 and 50 μg/mL Vc to the coculture medium (p<0.05). Vc at 50 μg/mL significantly (p<0.05) inhibited the fatty acid release from adipocytes to the coculture medium. Meanwhile, the elevated ROS of cocultured β-cells was decreased in the presence of Vc (1 to 50 μg/mL). In both induction methods, intracellular TGs in both β-cells and adipocytes were decreased by Vc treatment; however, Vc did not affect the intracellular insulin level. Moreover, IL-6 and adiponectin levels in the coculture medium remained under the levels of the control group. The positive effects of Vc might be due to the antioxidant capacity and TG inhibitory effect of Vc.
1. The natural abundance carbon-13 nmr of vitamins D (D2 and D3) and several isomers (5, 6-trans-vitamin D2, isotachysterol2 and isovitamin D2) have been completely assigned by employing off-resonance noise-decoupling, acetylation shifts, and lanthanide-induced shifts experiments. The last two techniques were especially useful for the present study. 2. Carbon-13 nmr spectral characteristics of the three main conjugated triene moieties (SE-Z-SZ, SE-E-SZ, or SE-E-SE), involved in the molecules of vitamin D and its isomers, were revealed. Thus, the striking dependence of the shieldings on molecular geometries and high sensitivity of the resonances to the environments of conjugated systems were surveyed. 3. Conformational preferences in solutions of the hydroxyl groups in vitamins D2 and D3 as well as 5, 6-trans-vitamin D2 were conveniently determined.
Radiolabeled tracer [3H] very low density lipoprotein (VLDL)-triglyceride (TG) and non-tracer (Triton WR 1339) methods were used to determine VLDL-TG kinetics in normal rats and in rats given either a 10% glucose or a 10% fructose drinking solution for 16 h ad libitum. The carbohydrate-fed rats were hypertriglyceridemic compared to control animals. VLDL-TG was endogenously prelabeled with [3H] glycerol in control, glucose- and fructose-fed rats, and injected into identically treated recipients. Triton WR 1339, a potent inhibitor of VLDL-TG catabolism, was injected into the same animal 30 min after the end of the tracer method to measure TG secretion rate (TGSR). The tracer and non-tracer methods showed that fructose-fed rats had a significantly lower fractional catabolic rate (FCR) than either control or glucose-fed rats. In contrast, glucose-fed rats had a TGSR greater than control without a reduction in FCR. For all treatments, TG concentration correlated with FCR for the tracer method and with TGSR for the non-tracer method. There was good correlation of TGSR determined by the tracer and non-tracer method for control rats despite the substantial difference in the absolute values. No such relationship was observed in carbohydrate-fed rats. Control rats were made hypertriglyceridemic by Intralipid infusion. A lower FCR was observed when determined by the tracer method, but this was not observed by the triton method. These results suggest that TG kinetics determined by the tracer and the triton methods cannot be readily correlated, especially in hypertriglyceridemic state. However, the two kinetic studies both suggested that overproduction of VLDL-TG in glucose-fed rats and impaired VLDL-TG catabolism in fructose-fed rats were the primary cause for their hypertriglyceridemia, respectively.
The production of vitamin B12 from carbohydrates, peptone, casamino acid, etc., by intestinal bacteria was investigated. Klebsiella pneumoniae IFO 13541 was the most efficient strain for vitamin B12 production, which depended exclusively on the concentration of yeast extract added to the medium. A concentrated solution of yeast extract (1 ml) was chromatographed on a Sephadex G-25 column (1 x 180 cm) and eluted with H2O (eighty fractions of 3 ml each were collected). It was found that fractions in which bacterial growth was most prevalent also exhibited the highest amount of vitamin production. The effectiveness of yeast extract was shown by the participation of pyrroloquinoline quinone and aspartic acid in the growth stimulation and in the vitamin B12 production in this strain.
We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP(+)-dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K.pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124 kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive.
Vitamin B12 production by Gram-negative facultative anaerobic intestinal bacteria, members of the family Enterobacteriaceae, was examined. Klebsiella pneumoniae IFO 13541 was the most effective strain with regard to such production. The growth of the strain and its production of vitamin B12 depended exclusively on the concentration of yeast extract added to the medium. The yeast extract components required for the stimulation of bacterial growth and or vitamin B12 production were identified as aspartic acid and pyrroloquinoline quinone (PQQ) and the relationship between vitamin B12 production and these two components was examined. The metabolism of aspartic acid in this process was also investigated; the major metabolites were alanine, glutaminic acid, and valine. The formation of alanine depended on dehydrogenase, the activity of which was greatly increased with increasing PQQ concentration.
There is little evidence regarding the associations between bone growth and environmental factors among growing children, especially in Asians. The aim of this cross-sectional study was to search for the promotion factors of bone growth in Japanese children during growth. The study subjects were male (n=333) and pre/post-menarcheal female (n=179/n=68) school children aged 8-14 y. Bone status at the calcaneus was evaluated by quantitative ultrasound (Benus III), and the bone area ratio (BAR) was used as an evaluation index. Dietary intakes were assessed via brief self-administered diet history questionnaires. The participants were asked to record all of their activities for 3 d (2 weekdays and 1 holiday). They were also required to provide the most recent anthropometric measurement records at their schools and answer questions about the frequency of fractures and, for females, the length of time since menarche. Multiple regression analysis with dummy variables demonstrated that age, magnesium (more than the RDA), vitamin B(1) (more than the RDA), mean physical activity intensity per day (more than 1.7 METs), vitamin C (more than the RDA) and calcium (more than the RDA) were significantly positive influential factors of BAR for males. For premenarcheal females, age, vitamin A (more than the RDA), BMI, and mean physical activity intensity per day (more than 1.7 METs) were significantly positive influential factors of BAR, and for postmenarcheal females, only BMI and age were significantly positive influential factors of BAR. The results suggest that several manageable factors correlate with the bone mass, and the associations differ depending on gender and menarcheal status.
The radical scavenging effect and protective potential from oxidative damage by radical generator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), in renal epithelial LLC-PK1 cell of broccoli (Brassica oleracea) were investigated and identified the active components under the bioassay-linked fractionation method. The MeOH extract, and fractions of CH2Cl2, BuOH and H2O from broccoli showed the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect in a dose-dependent manner. In addition, they exerted the protective effect against LLC-PK1 cellular damage induced by AAPH dose-dependently. In particular, the BuOH fraction was evaluated as the most active fraction, indicating that the BuOH fraction contains the active components with antioxidative capacity. Employing a bioassay-linked fractionation method, the active principles were isolated and characterized as 1,2-disinapoylgentiobiose and 1-sinapoyl-2-feruloylgentiobiose from the BuOH fraction. These two compounds from broccoli displayed potent antioxidant effects against the DPPH radical, showing the IC50 values of 5.18 and 7.52 microg/mL, respectively. Moreover, the compounds significantly and dose-dependently recovered cell viability lowered by AAPH treatment, suggesting the protective roles from cellular oxidative damage. The present study suggests that broccoli has excellent antioxidative potential and the hydroxycinamic acid esters from broccoli, 1,2-disinapoylgentiobiose and 1-sinapoyl-2-feruloylgentiobiose, are considered as the active components with antioxidative effect.
The blood level of [14C]coenzyme Q10 and the redox levels of [14C]coenzyme Q10 in the liver and heart were measured after intravenous injection of [14C]coenzyme Q10 solubilized in multilamellar liposomes into guinea pigs. The blood level of radioactivity declined biexponentially with half-lives of 11.5 min and 15.6 h in the first and second phases, respectively. The levels of reduced [14C]coenzyme Q10 in the liver and heart reached 55.8 and 46.4%, respectively, of the labeled compound in the tissues at 30 min after the injection. Coenzyme Q10-reducing activity in cytosol, microsomes and mitochondria was also investigated. This activity was found in all the fractions. The total activity was the highest in the liver cytosol. Moreover, the results of experiments using a purified enzyme suggested that one of the coenzyme Q10-reducing enzymes was NAD(P)H: quinone oxidoreductase [EC 184.108.40.206, DT-diaphorase]. These results are discussed in relation to the protective effect of reduced coenzyme Q10 against lipid peroxidation in membranes.
The transfer of menaquinone-4 (vitamin K2(20] to the fetus and milk was studied in pregnant and lactating rats, respectively, after oral administration (4 mg/kg) of [3'-14C]menaquinone-4. Intestinal absorption of menaquinone-4 was rapid and the highest level of radioactivity in each tissue except guts of fetal rats was observed at 4h after dosing. The level in the fetal homogenate was low. At that time, the concentration of menaquinone-4 in the fetal liver was 84 ng/g, corresponding to 9% of the value found in the placenta. Therefore, we conclude that the transfer of menaquinone-4 to the developing rat fetus is restricted by the blood-placenta barrier, but that a sufficient amount of menaquinone-4 (more than the essential amount of vitamin K to ensure full carboxylation) can be transferred into the fetal liver. It was also observed that the radioactivity was transferred to milk after oral administration to lactating rats. Milk/blood concentration ratios at 6 and 24h after dosing were 13.8 and 65.1, respectively. The elimination half-life of radioactivity in milk was about 17h. Eighty-four percent of milk of radioactivity was due to menaquinone-4. These results suggest that the prophylactic maternal oral administration of menaquinone-4 may be efficacious for a prophylaxis of neonatal and infantile vitamin K deficiency.
The effects of protein and/or energy deficiency on 14C incorporation into body constituents and 14C output in expired air and urine were investigated in adult rats using 14C-Chlorella protein hydrolysate. Rats were given a protein-free diet (PFD) for 2 weeks and control rats were fed ad libitum or pair-fed with the PFD group on a 12% lactalbumin diet (LA and Pair-fed, respectively). On the 15th day, animals received 14C-Chlorella protein hydrolysate with 5g of their respective diet. One group of PFD animals was given tracer by stomach tube without food (PFD-fast). Normal control rats ate about twice as much diet as the PFD group. The respiratory 14C output in the PFD group was identical with those in the LA and Pair-fed groups and was less than that in the PFD-fast group, The rate of protein synthesis, provisionally expressed as relative specific radioactivity, was more in the PFD group than in the normal group in the liver and less than the latter in the muscle. The LA group retained less total radioactivity in the body than the Pair-fed or PFD group, indicating high capability to hold the body protein in protein deficiency. In addition, decreased conversion of amino acids to lipids and glycogen was observed in the PFD group. All these differences are interpreted as adaptations to protein shortage. On prolonged fasting (PFD-fast group), gluconeogenesis in the liver increased to provide energy, despite the protein deficiency. The relative importances of protein and energy for tissue protein synthesis are briefly discussed.
The effect of protein deficiency on the rate of loss of radioactivity from body constituents was studied in adult rats administered 14C-Chlorella protein hydrolysate or 14C-lysine. Rats were kept on a protein-free diet for 3 weeks and then injected with labelled amino acids and fed on a protein-free diet for 3 more days to allow 14C deposition in tissues. Then they were given experimental diets (protein-free diet, 1% and 10% wheat gluten diets pair-fed with the protein-free diet, and 10% wheat gluten diet ad libitum) for 7 days and sacrificed. The rates of loss of radioactivity from tissue proteins became low in general with the extent of protein deficiency. This increased capacity of tissues to retain 14C-amino acids may result from higher efficiency of protein utilization in protein deficiency. The reutilization of free amino acids and the rate of catabolism of tissue proteins are discussed on the basis of the results. The half-life of muscle protein was too long to observe the effects of experimental diets given for 7 days on the rate of loss of radioactivity.
Feeding of rats on a low casein diet supplemented with a small amount of methionine caused an enlargement of liver size and an accumulation of lipids in the liver. Experiments were conducted to examine the effects induced by the amino acid-imbalanced diet on the metabolism of dietary amino acids with special reference to protein synthesis. Protein and DNA contents of the liver per 100 g body weight with rats fed on the imbalanced diet were apparently higher than those of rats fed on the basal diet. Ingestion of the imbalanced diet clearly stimulated hepatic ribosome aggregation but not skeletal muscle ribosome aggregation. Incorporation of [14C]leucine into liver protein was markedly increased in the group consisting of rats fed on the imbalanced diet (imbalanced group) and that into skeletal muscle was similar to the result with the group receiving the basal diet (basal group). Relative content of [14C]leucine in plasma albumin was higher in rats of the imbalanced group, whereas that in plasma of very low-density lipoprotein was reduced. These results indicate that the unbalanced inter-organ or intra-organ utilizations of dietary amino acids for protein synthesis were produced by the condition of the amino acid imbalance. Such metabolic effects of the amino acid imbalance may result in the enlargement of liver size and the accumulation of lipids in the liver.
Six healthy male subjects were given a single oral dose of 0.4 g of maltitol per kg body weight containing [U-14C]-maltitol (50 microCi) and their breath, urine, feces and blood were collected at appropriate intervals for 48 h to measure the recovery of administered radioactivity. Further, to measure breath hydrogen, 15 healthy male subjects ingested 30 g of maltitol and samples of their breath were collected for a 10-h period. The expired 14CO2 showed a wide peak about 3 h after ingestion and 56% of the administered radioactivity was recovered in 48 h. An additional 0.2% of administered radioactivity was recovered as expired 14CH4, 2.6% in urine and 14.3% in feces, respectively. The obvious increase of breath hydrogen was detected at 1 h after maltitol ingestion and then a big peak at 3.5 h, whereas maltose ingestion did not increase breath hydrogen. Both excretion profiles of breath 14CO2 and hydrogen coincided well. These results demonstrate that a greater part of ingested maltitol is fermented by intestinal microbes than is hydrolyzed by digestive enzymes. Although maltitol is catabolized to carbon dioxide via intestinal microbes, the available energy is much lower than that of digestible sugars such as sucrose.
O-beta-D-Galactopyranosyl-(1----4)-O-beta-D-galactopyranosyl- (1----4)-D-glucopyranose (designated as 4'GL) are produced from lactose with Cryptococcus laurentii OKN-4. Excretion and metabolism of 4'GL in rats were examined using a radioisotope technique. [U-14c]4'GL was synthesized from [U-14C]lactose by Cryptococcus laurentii OKN-4. The 14CO2 in expired air was counted after oral administration of [U-14C]4'GL or [U-14C]lactose in conventional rats, rats treated with antibiotics and germ-free rats. The rate of 14CO2 excretion from conventional rats given [U-14C]4'GL was slower than that from those administered [U-14C]lactose. When [U-14C]4'GL was orally administered to rats given antibiotics, there was a 2-h delay in 14CO2 excretion, as compared to conventional rats. In germ-free rats, total excretion of 14CO2 from [U-14C]-4'GL decreased to about one-third of that of conventional rats during a 24-h period. Radioactivities in the serum, liver, and carcass of the [U-14C]4'GL oral administration group were lower than those of the [U-14C]lactose oral administration group. Radioactivities in the feces and urine however, were higher in [U-14C]4'GL group than in [U-14C]lactose group.
Studies were conducted to investigate the in vivo and in vitro effects of retinol and retinoic acid on the synthesis of mannolipids and mannopeptides in rat liver. The incorporation of 14C-mannose into glycolipids and glycoproteins showed a decrease in vitamin A-depleted rats as compared with vitamin A-fed rats. By means of DEAE-cellulose, silicic acid and thin-layer chromatography, the mannose-containing lipids were separated into mannosyl retinyl phosphate (MRP, Rf 0.2) and dolichyl mannosyl phosphate (DMP, Rf 0.4), respectively. A rapid increase in the synthesis of labelled MRP was observed, exhibiting a peak between 25 and 60 min after intraperitoneal administration of retinol to vitamin A-depleted rats. Similarly, administration of retinoic acid brought about elevation of 14C-mannolipid (Rf 0.2) synthesis with a peak at 60 min after injection. On the other hand, the incorporation of 14C-mannose into DMP (Rf 0.4) remained unchanged by such treatment. In vitro addition of retinyl phosphate, but not retinoyl phosphate, markedly stimulated the synthesis of 14C-mannolipid (Rf 0.2), using crude membrane of rat liver and GDP-14C-mannose as the donor. These findings strongly suggest that not only retinol but also retinoic acid plays an important biological role in mannosyl transfer reaction in rat liver. However, the molecular participation of a metabolite of retinoic acid in the formation of a mannolipid and the structure of such a metabolite remain to be established.
The metabolism of flavins in mouse was studied with [F-(2)-14C, A-(2,8)-14C]FAD and [F-(2)-14C, 32P]FMN. Ninety minutes after injection, radioactive isoalloxazine nucleus of double-labeled FAD was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas a small amount of radioactive adenine nucleus of double-labeled FAD was found in FAD in the liver. In the case of FMN, radioactive isoalloxazine nucleus of double-labeled FMN was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas only a minute amount of radioactive phosphorus was incorporated into FMN and FAD in the same organ. These results indicate that FMN and FAD injected are rapidly hydrolyzed and resynthesized in animal body.