In this paper, we presented a new procedure to determine daily minimum energy expenditure (MEE) in free-moving rats and mice kept in a chamber. Energy expenditure was measured for 23h period and averaged every 10min. Data were sorted in ascending order. MEE was estimated from the regression line with the smallest slope and the biggest intercept among the regression lines calculated between the sorted energy expenditure and the data ranking. Among three duplicate measurements in individual animals, MEE gave the smallest coefficient of variation (2.2%) as compared with actual measured-values: either the single lowest value (4.0%) or the average of the 6 lowest values (2.5%). Judging from diurnal patterns of energy expenditure and locomotor activity and from video tape observation of the rat's performance, it was confirmed that MEE represented an energy expenditure at rest. MEE decreased with fasting from days 1 to 5. MEE per body weight also declined with age, but stayed around 71-72kcal/day/kg3/4 at 18 and 34 weeks of age in male Wistar rats. MEE in mice increased at lower ambient temperatures between 16 and 32°C, but stayed fairly constant at the same temperature in repeated experiments. Thus, MEE estimated by the present regression procedure was highly reproducible and valid to determine the fundamen-tal value of daily energy expenditure under free-moving conditions.
The content of nucleotides and nucleosides of human milk was analyzed using a newly developed method for high performance liquid chromatography. By this method it is possible to analyze nucleo-tides and nucleosides simultaneously. Human milk was pooled according to season, lactation period, and geographical area. Three kinds of nucleosides-cytidine, uridine, and adenosine-and 6 kinds of nucleotides -5'-CMP, 5'-UMP, 5'-AMP, 5'-GMP, 5'-IMP, and 5'-CDP-were detected. Cytidine, 5'-CMP, and 5'-CDP predominated throughout lactation. Also there seemed to be geographical differences in nucleoside composition. The overall amounts of nucleotides and nucleosides were higher in winter than in summer. No nucleosides were detected in bovine milk, nor in bovine milk-based infant formula, and bovine milk contained much less nucleotides than human milk. These results suggest that nucleosides and nucleotides found in human milk may play some important roles in the development of infants.
Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine were studied. Specific Fe(II) and Fe(III) binding sites were detected by iron binding assay using immobilized BBM from the upper intestine on a nitrocellulose sheet and the binding to Fe(II) was considerably higher than that to Fe(III) . The dissociation constants for Fe(II) were 0.26±0.02 nM (M±SE) for high-affinity binding sites and 2.70±0.26 nM for low-affinity binding sites. For Fe(II), the number of high-affinity binding sites was 0.35±0.04×1016/mg of protein (M±SE) and that of low-affinity binding sites was 1.07±0.11×1016/mg of protein. Bound Fe(II) could not be replaced with Fe (II), Fe(III) or transferrin-bound iron and approximately 50% of bound Fe(II) was removed by chelators. The apparent molecular weights of Fe (II) binding peaks were 250 and 120 kDa by gel filtration. SDS-polyacryl-amide gel electrophoresis (SDS-PAGE) detected three bands with iron-binding activity. The distribution of the Fe(II) binding sites showed that the number of low-affinity sites was significantly higher in the proximal third of the intestine compared with the rest of the intestine. Iron deficiency increased the number of Fe(II) binding sites. These findings suggest that the Fe(II) binding sites might play a physiological role in iron absorption in the rat.
The metabolic fates of the carbon skeletons of essential amino acids were investigated in growing rats fed on diets containing graded percentages of protein calories (5, 10, 15, 20, 30, and 40 PC%) using soybean protein isolate at 4, 100 kcal of metabolizable energy per kg of diet. The incorporation of 14C into the body protein 12h after the intraperitoneal injection of labeled essential amino acids was more than 60% of the dose in the 5 to 10 PC% groups, but it began to decline gradually in the higher PC% groups. The expired 14CO2 production from labeled threonine, tryptophan, leucine, isoleucine, valine, phenylalanine, and lysine increased linearly with increasing levels of dietary soybean protein, but the rate of increase in the 14CO2 production was lower in the higher PC% groups. In comparison, the 14CO2 production from methio-nine, histidine, and arginine decreased in the 5 to 15 PC% groups, and it increased linearly in the higher PC% groups. The extent of 14CO2 production varied among the essential amino acids in each dietary group. These results indicate that the carbon skeletons of essential amino acids are mainly utilized for body protein synthesis, but significant amounts of their carbons are oxidized to expired carbon dioxide for energy produc-tion, and that the metabolic responses of these amino acids to dietary protein level change at around 20 PC%, where the growth rate reached its approximate maximum. The utilization of individual essential amino acids in rats fed on the soybean protein diets changed markedly as compared to those on purified whole egg protein diets, the results of which have been reported elsewhere.
The role of macrophages (MØ) in the enhancement of lymphocyte proliferation by α-tocopherol (VE) was investigated using rat splenocytes. The proliferation of whole splenocytes was significantly higher than that of MØ-depleted splenocytes at all concentrations of concanavalin A (Con A; 0.5-10μg/ml). When whole and MØ-depleted splenocytes were preincubated with YE (2μg/ml) for 24h, the prolifera-tion of whole splenocytes was significantly enhanced compared to that of whole splenocytes preincubated with medium alone. In contrast, MØ-depleted splenocytes did not show any increase of proliferation following in vitro pretreatment with VE. When the splenic MØ pretreated with both YE (2μg/ml) and Con A (10μg/ml) for 24h were further incubated with splenic lymphocytes, their proliferation was significantly enhanced compared to that of splenic lymphocytes cultured with splenic MØ pretreated with Con A. In this experiment, the medium containing 2-mercapto-ethanol (2-ME) had the ability to enhance splenic lymphocyte prolifera-tion, which masked the enhanced effect of YE on splenic lymphocyte proliferation. Furthermore, in vitro treatment of VE could not decrease the production of prostaglandin E2, but could enhance the production of interleukin 1 from splenic MØ. These results suggest that MØ play an important role in the proliferation of splenic lymphocytes following in vitro incubation with VE, which is closely associated with the action of VE as an immunomodulator rather than antioxidant.
We investigated whether the expression of growth-related genes could be changed in primary cultured hepatocytes in response to changes in the nutritional environment. Hepatocytes were isolated from the liver of growing rats after collagenase perfusion and cultured in Williams' E medium (WE medium) containing 5% calf serum, 10-7 M insulin and 10-6 M dexamethasone for 24h. When amino acids were removed from the culture, the level of c-myc mRNA increased more than 18-fold within 2-3h, whereas replenishment of the amino acids to the medium caused rapid decrease in the mRNA level. We found that the half-life of the c-myc mRNA was prolonged more than 6-fold in the absence of amino acids. The mRNA levels of other proteins, such as ornithine decarboxylase, c-Ha-ras and actin, and their half-lives were not affected by amino acids. It is known that a short-lived protein is involved in the degradation of c-myc mRNA. In fact, the addition of cyclohexi-mide to cultured hepatocytes increased the level of c-myc mRNA either in the presence or absence of amino acids, though the levels of other mRNAs were not changed significantly. These results suggest that the synthesis of the short-lived protein is suppressed and the c-myc mRNA is thereby stabilized in the absence of amino acids.
The effect of Jew's mellow leaf powder and its water soluble viscous substance on cholesterol metabolism in rats fed a high cholesterol diet was examined. When compared to the controls, total serum and liver cholesterol concentrations were significantly decreased or tended to decrease in the groups given dry powder of fresh Jew's mellow leaves, dry powder purchased from the market or residual powder after extracting with ethanol, whereas no difference was observed in those given residual powder after extracting with water. There were significant increases or increasing tendencies in the fecal excretion of bile acids, total neutral sterols and cholesterol in those fed the experimental diets when compared to the control group. Rats fed a diet containing a water-soluble viscous substance (1.7%, about 1% as dietary fiber) obtained from the dry powder of Jew's mellow leaves showed significant decreases in serum and liver cholesterol concentrations and increases in fecal excretions of bile acids and neutral sterols. Based on the above, the component of dry powder of Jew's mellow leaves that is effective in decreasing serum and liver cholesterol concentrations was found to be a soluble dietary fiber, and the mechanism was assumed to be largely due to the increased excretion of bile acids and neutral sterols.
The influence of docosahexaenoic acid (DHA) on the ultrastructure of hepatocytes was studied. Adult male mice of Crj: CD-1 (ICR) strain were fed a fat-free purified diet supplemented with 5% (by weight) of purified DHA, refined sardine oil, and palm oil. The mice were fed the DHA diet or the palm oil diet for 7 days, and the sardine oil diet or the palm oil diet for one month. There were significant ultrastructural changes in the hepatocytes between the mice fed palm oil diet and the animals fed DHA and sardine oil diets. Many lipid droplets in the tissues of mice fed the palm oil diet were observed. Few lipid droplets were contained in the hepatocytes from the mice fed DHA and sardine oil diets, but electron-dense bodies were found in their tissues. These electron-dense bodies were mainly found near the region of the nucleus, blood sinusoids and bile canaliculi. These results suggest that the dense bodies found in the DHA and sardine oil diet groups may appear as a result of acceleration of lipid metabolism in the liver of mice.
To study the effect of Platycodon grandiflorum (P.g.) feeding on serum and liver lipid concentrations, diet-induced hyperlipidemic rats were fed diets containing 5% and 10% P.g. powder for 3 weeks. The P.g. feeding markedly decreased both serum and liver lipid concentrations in hyperlipidemic rats. Especially, 5% P.g. diet significantly decreased the concentrations of total cholesterol and triglycerides in serum and liver as compared with those of the hyperlipidemic control group. Dietary P.g. also induced a reduction in low-density lipoprotein (LDL)-cholesterol as well as an increase in the concentration of high-density lipoprotein (HDL)-cholesterol in serum. Furthermore, the atherogenic index was also low in rats fed P.g. diet. These results indicated that dietary P.g. may have a beneficial effect on preventing hypercholesterolemia and hyperlip-idemia.
Therapy with all-trans retinoic acid (ATRA) achieves complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), but the efficacy is reported to relate to plasma ATRA level after oral administration. The pharmacokinetics of ATRA and 4-oxo all-trans retinoic acid (4-oxo ATRA), a metabolite of ATRA, were studied in four children with APL at the time of initial oral administra-tion. After administration of ATRA at a dose of 30 mg/m2, the peak plasma ATRA level was 20-741ng/ml and was reached at 60-120min. The patient with the lowest peak plasma level did not achieve complete remission and had a very high 4-oxo ATRA level compared to the patients with complete remission. These findings suggest that accelerated metabo-lism of ATRA plays a role in the failure of this agent in the patients without remission.