Messenger ribonucleic acid (mRNA) levels and activities of renal 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) were determined in 3 groups of rats: vitamin D-deficient, normal, and 1α-hydroxyvitamin D3 (1αOHD3)-administered rats. To measure renal 24-OHase mRNA, a DNA probe complementary to the reported sequence of the recently cloned P450 component was employed. The DNA probe hybridized with 24-OHase mRNA. Northern blot analysis indicated that the size of the transcript was approximately 3.4kb, which was similar in size to that of the previous report. Oral administration of 1αOHD3 (2μg/kg bw/day×7 days) markedly increased the plasma concentration of 1, 25-dihydroxyvitamin D (1, 25 (OH)2D), renal 24-OHase activity and the renal 24-OHase mRNA level. However, the levels of 24-OHase mRNA were undetectable in vitamin D-deficient and normal rat kidneys, while a significant amount of 24-OHase activity was observed in the normal rat kidney. In vitamin D-deficient rats, renal 25-hydroxyvitamin D3-1α-hydroxylase activity was markedly elevated and 24-OHase activity was completely abolished. When an oligonucleotide probe complementary to coding sequence of heme binding region was used for dot blot analysis, the mRNA was clearly detected in normal rat kidney. Furthermore, activity of the enzyme was attenuated by human parathyroid hormone (1-34) but the mRNA abundance did not change with 2 nmol (every 4 h, 5 times) of hormone treatment in 1αOHD3-dosed thyroparathyroidectomized rats. The present study demonstrates that induction of 24-OHase activity by 1, 25(OH)2D3 occurs at least in large part through increase of the gene expression in the kidney. Furthermore, these findings suggest that 2 forms (inducible and constitutive) of 24-OHase exist in rat kidney, and that the previously cloned P450 component is the inducible form of 24-OHase.
Determination of 1α, 25-dihydroxyvitamin D [1, 25(OH)2D, gross amounts of 1, 25(OH)2D2 and 1, 25(OH)2D3] and separative deter-mination of 1, 25(OH)2D2 and 1, 25(OH)2D3 in plasma using calf thymus receptor have been investigated. A lipid extract from 1 ml of plasma is applied to a Bond Elut C18OH column and an eluate corresponding to 1, 25-(OH)2D including both 1, 25(OH)2D2 and 1, 25(OH)2D3 is applied to calf thymus receptor to assay a gross amount of the two compounds. On the other hand, when separative assay of the two compounds is performed, the 1, 25 (OH)2D eluate obtained from the Bond Elut C18OH column is further applied to HPLC using a Zorbax SIL column with 5% isopro-panol in methylene chloride as a developing solvent to separate the two compounds from one another. The separated eluates are independently applied to the receptor to assay the two compounds. Since less amounts of unknown components non-specifically bound to interfering concomi-tants besides 1, 25(OH)2D exist in the calf thymus receptor, complicated purification steps to eliminate the concomitants are unnecessary. The detection limit by this method is 1.25 pg/tube which is sensitive enough for a routine method to assay 1, 25(OH)2D in plasma.
Three analogs of adenosylcobalamin were synthesized and their coenzymic properties in the diol dehydrase reaction were studied. Neither adenosylcobinamide nor adenosylcobinamide phosphate was active as coenzyme and showed very low affinity for apoenzyme, irrespec-tive of the presence of nucleotide loop fragments, such as 5, 6-dimethyl-benzimidazole, α-D-ribazole, or α-D-ribazole-3'-phosphate. The coordina-tion of pyridine to the cobalt atom neither confers the coenzymic function upon adenosylcobinamide nor strengthens the inhibitory effect of cyano-aquacobinamide and methylcobinamide significantly. The analog of adenosylcobalamin in which the N-3 position of 5, 6-dimethylbenz-imidazole is methylated was also not active as coenzyme and showed very low affinity for apoenzyme. Since 3, 5, 6-trimethylbenzimidazole in this analog is no longer coordinated to the cobalt atom, these results show that at least a part of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin is essential for tight binding to the apoenzyme and therefore for manifestation of coenzymic function.
To investigate the nutritional condition in a hilly village (Kotyang) and a suburban village (Bhadrakali) in Nepal and to clarify the possible cause of the difference in total serum cholesterol level between the two groups of villagers habitually eating low fat diets, we carried out a nutrition survey using the 24-h recall method and blood sampling in 403 subjects (204 men and 199 women) in the hilly village and 466 (244 men and 222 women) in the suburban village. Total serum cholesterol was statistically significantly lower in the hilly villagers than in the suburban villagers for both sexes, but HDL-cholesterol was not. In both villages, 82% of the total energy was taken from carbohydrate, 7-8% from fat and 10% from protein. Energy, protein, fiber, potassium, magnesium, mono-unsaturated fatty acid, polyunsaturated fatty acid, and vitamin A in the hilly villagers were significantly higher than those in the suburban villag-ers. Total serum cholesterol was significantly associated with age and body fat percentage, suggesting that total serum cholesterol level was not directly associated with total fat intake in these Nepalese people.
The effects of dietary protein restriction on protein synthe-sis were investigated in perfused rat hindlimb. The fractional rate of protein synthesis was measured with [ 3H ] phenylalanine in young adult (7-week-old) rats fed a low protein (5% casein) diet and a protein-free diet for 3 weeks. The low protein diet (LPD) allowed a moderate gain in body weight. The fractional rate of protein synthesis fell to 70% of the control value in LPD group and further fell to less than a half in the protein-free diet (PFD) group. Thus, the protein synthesis rate decreased as the dietary protein content was reduced. The fall of protein synthesis was mainly accompanied by the reduction of RNA activity (mg pro-tein/mg RNA/day) rather than RNA concentration (RNA/protein). The rate of protein breakdown was calculated by subtracting growth rate from protein synthesis rate. The breakdown rate was decreased in LPD group and increased slightly in PFD group. From the low rates of protein synthesis and breakdown, it appears that dietary protein restriction, at least allowing a gain in body weight, makes the turnover rate slow down. The overall changes in protein synthesis obtained in the perfused hindlimb are consistent with the reported results in vivo.
To investigate the effects of different routes of alimentation on lipid metabolism, lipid-free nutrients with the same amount of energy and composition were continuously administered via the oral cavity (oral group), directly to the stomach (enteral group), or into the superior caval vein (parenteral group) of unrestrained rats. The body weight gain 1 week after continuous nutrition was greater in enteral and parenteral groups than in the orally-fed group. In comparison with the orally-fed group, the enterally-fed group had significantly greater liver and retro-peritoneal adipose tissue weights and hepatic lipid content, whereas the parenterally-fed group produced similar changes to those in the enteral group without significant accumulation of hepatic lipid. The rate of fatty acid synthesis after 1 week of alimentation was 3-fold higher in the liver in enterally-fed group, and approximately 10-fold higher in white adipose tissue in both enterally- and parenterally-fed groups than in orally-fed group. Plasma concentrations of catecholamines after 6 h were signifi-cantly higher in the orally-fed group than in either the enteral or paren-teral group. However, plasma insulin concentrations were not signifi-cantly different among the three groups. The results indicate that lipid synthesis and its deposition in the liver and adipose tissue are greatly influenced by the route of alimentation, possibly owing to difference in the early neuro-hormonal responses to different routes of alimentation.
The effects of vegetable oils (soybean oil and coconut oil) and C18-unsaturated fatty acids (oleic acid, C18:1; linoleic acid, C18:2; linolenic acid, C18:3) on plasma ethanol levels in male rats (6 weeks old) were investigated. Vegetable oils decreased and delayed the peak of plasma ethanol concentration: a dose-response to vegetable oils was observed in the concentration and time to maximum concentration of plasma ethanol but no change in disappearance time. These phenomena were observed in two conditions: 1) oral administration of vegetable oils before oral intubation of ethanol and 2) simultaneous oral administration of vegetable oils and ethanol. Similar responses were obtained in three C18-unsaturated fatty acids. No changes in hepatic alcohol and aldehyde dehydrogenase isozyme (high Km and low Km) activities were observed. The remaining rate of ethanol in stomach was significantly higher with administration of vegetable oils or linoleic acid. A high negative correla-tion between the maximum plasma ethanol concentration and the remain-ing rate of ethanol in stomach was found. These results suggest that the slowing of the gastric emptying is a major mechanism for the decreasing and delaying effects on plasma ethanol levels by vegetable oils. The present paper also suggests that fatty acids may participate in the decreas-ing and delaying actions on the peak of plasma ethanol concentration by vegetable oils.
The teratogenic effects of triethylene tetramine dihydro-chloride (Trien-2HC1) on fetal mouse brain were studied on gestational day 19. Trien-2HC1 was given throughout pregnancy at levels of 0 (control), 3, 000, 6, 000, or 12, 000 mg/liter as drinking water, ad libitum. Mean litter size and live fetus per dam at birth were not significantly different among the four groups. The frequency of gross brain ab-normalities in live fetus at birth such as hemorrhages, delayed ossification in cranium, hydrocephaly, exencephaly, and microcephaly increased with increasing levels of the drug. Microscopically, dysorganization of neuro-nal cell layers, spongiform changes in white matter, and reduced myelin development were noted in the coronally sectioned cerebrum from Trien-2HC1-treated fetus. These abnormal findings increased dose-dependently in regard to the extent and severity at the levels of 6, 000 and 12, 000 mg/ liter. No such changes were observed in the cerebrum of controls. These results suggest that microscopic changes in fetal brain caused by Trien-2HC1 may be in part similar to those in brindled mutant mouse. Special attention should be paid to the developing fetal brain when Trien-2HC1 is used during pregnancy.
To evaluate the changes in zinc and copper concentrations in breast milk and maternal blood during lactation, milk and blood samples were obtained from 80 lactating women during the period be-tween 2 and 201 days of lactation. Zinc and copper concentrations were measured by atomic absorption spectrophotometry. Breast milk zinc and copper concentrations markedly decreased during the first few weeks of lactation and gradually declined for the remaining period. Mean values of milk zinc and copper levels were 1.76 and 0.29g/ml, respectively, be-tween 15 and 84 days after parturition and were 0.76 and 0.19μg/ml between 85 and 201 days of lactation. Calculated daily intakes of these minerals for infants from breast milk were markedly lower than those of U5 Recommended Dietary Allowances. Plasma zinc levels of lactating mothers increased as lactation progressed, whereas erythrocyte zinc and plasma copper concentrations decreased. Plasma zinc and copper and erythrocyte zinc values returned to normal approximately three months after parturition.
The localization of rat small intestinal microvillous sucrase-isomaltase was studied by immunoelectron microscopy to investigate its intracellular transport to the microvillous membrane. At the same time, the usefulness of a rapid embedding method of tissues in Lowicryl K4M for immunocytochemistry of sucrase-isomaltase was examined. Sucrase-isomaltase was present not only in the microvillous membrane, but also in the apical vesicles and the apical plasma membrane invaginations. Negli-gible labeling was observed in the other portions of the absorptive cells. These findings suggest that the final step of intracellular transport of sucrase-isomaltase to the microvillous membrane is via smooth apical vesicles. The rapid immunoelectron microscopic method adopted in this study seemed to be a useful technique for the study of the intracellular localization of sucrase-isomaltase.