A membrane-associated iron-binding complex was isolated from rat intestinal mucosa during iron absorption. Two 59Fe peaks (peaks 1 and 2) were separated on Sepharose 6B gel filtration from detergent solubilized 20, 000 × g precipitate of upper intestinal mucosal homogenate after administration of 59Fe-labelled ferrous materials. Peak 1 was a membrane iron-binding complex whose apparent molecular weight was over 106 Da estimated by gel filtration, while peak 2 was identified as ferritin. Three major bands were detected on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of peak 1. The treatment of mucosal homogenate with 5mM ethylenediaminetetraacetic acid released 59Fe binding from peak 1. Incorporation of 59Fe to peak 1 showed the maximum at 1 h and then reduced, while [59Fe] ferritin showed reciprocal behavior, which suggested that peak 1 may be a rapid turnover iron pool and transfer 59Fe to ferritin. Peak 1 was also isolated from the brush border membrane and showed similar SDS-PAGE pattern to that from the 20, 000 ×g precipitate of mucosal homogenate. Western blot analysis did not reveal immunoreactive transferrin in peak 1. Those findings suggest that peak 1 may be a non-ferritin, non-transferrin iron-binding complex located on the brush border membrane and accept iron from the intestinal canal during iron absorption.
To investigate the roles of the autonomic nervous system in the thermic effects of protein and carbohydrates in rats, we determined the urinary excretion of catecholamines and the resting oxygen consump-tion by means of HPLC-fluorometry and open-circuit respirometry, re-spectively. Protein administration significantly increased the urinary excretion of norepinephrine and epinephrine over those on water admin-istration. The thermic effect of protein was 16.6% of the basal metabolic rate and was inhibited by phentolamine, prazosin, or atropine, but not by propranolol. These results suggest that the sympathetic nervous system via α1-adrenoceptors and the parasympathetic nervous system are in-volved in the thermic effect of protein. The administration of carbo-hydrates such as glucose, sucrose, and fructose significantly enhanced the urinary excretion of norepinephrine, but only glucose administration increased the urinary excretion of epinephrine. The thermic effects of carbohydrates were 8-9% of the basal metabolic rate and were inhibited by propranolol, but not by phentolamine or atropine. These findings suggest that the sympathetic nervous system via β-adrenoceptors, but not the parasympathetic nervous system, contributes to the thermic effect of carbohydrates. Thus, we conclude that the autonomic nervous system is involved in the thermic effects of protein and carbohydrates by different mechanisms.
The effects of dietary soybean protein isolate intakes on the metabolic fates of L-[U-14C]methionine, L-[U-14C]serine, and L-[U-14C]-alanine were investigated in growing rats. In the growth experiment for 21 days, body weight gain reached a plateau at more than 20 protein calorie percent (PC%) in the diet, and the protein efficiency ratio attained maximum in the 10 PC% group. Carcass and liver lipid contents increased greatly in the lower levels of dietary soybean protein, which was associated with their decrease in moisture contents. In the isotope experiments, the incorporation of 14C into the body protein 12h after the injection of [14C]methionine was extremely high (more than 80% of the dose) in the 5 to 15 PC% groups, but decreased thereafter in the higher PC% groups. The expired 14CO2 production from [14C]methionine was depressed in the lower PC% groups, and thereafter it increased with increasing levels of dietary protein, showing a break point at around 20 PC%. The carbon skeleton of [14C]alanine was extensively oxidized to 14CO2, even in protein-depleted rats, while serine carbon was utilized for body protein synthesis rather than for energy production. These results indicate that methionine, which is a limiting amino acid of soybean protein for rats, is preferentially utilized for body protein synthesis especially in protein-depleted rats, and the metabolic responses of methi-onine, serine, and alanine are quite different from each other.
Our previous experiments have shown that the appetite or preference for alcohol is affected by the rat strain and nutritional status, such as dietary protein levels. To determine the affected factors in alcohol preference, the alcohol metabolism in SHRSP (stroke-prone spontaneous-ly hypertensive rats) and WKY (Wistar-Kyoto) rats fed with the stan-dard level (15%) or low level (5%) purified egg protein diet (PEP) was investigated. The animals were kept on the experimental diets for 4 weeks. After 12 h fasting, a 15% ethanol solution was given in a dose of 100mg ethanol per 100g body weight with a gastric probe to all animals and the blood ethanol and acetaldehyde levels were determined. Compa-red with 15% PEP diet-fed SHRSP, WKY showed higher levels of blood ethanol and acetaldehyde. Furthermore, the same results were also observed in SHRSP and WKY fed with 5% PEP diet. On the other hand, regardless of the rat strain, rats fed a low level protein diet showed higher blood ethanol and acetaldehyde levels. We also found that there was no significant change in alcohol dehydrogenase (ADH) activity and acetalde-hyde dehydrogenase (ALDH) activity between SHRSP and WKY. How-ever, both SHRSP and WKY fed a 15% PEP diet showed higher ADH and ALDH activity compared with rats fed the 5 % PEP diet. These results suggested that the affected factors of preference for alcohol may be correlated with blood ethanol and acetaldehyde levels after alcohol intake.
The daily intakes of various lipids by 72 Japanese women (40-59 years of age) were measured directly from mock samples of food actually consumed (this method is similar to the duplicate portion method). One sample was collected from each of 12 subjects every 2-months for a period of 1 year. The daily intakes of total fatty acid (FA), cholesterol, plant sterol, phospholipid (PL), and phosphatidylcholine (PC) were 37.9g, 300mg, 152mg, 3.1g, and 1.7g, respectively. No effect of the sampling period was found for any lipid measured. The combined eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids showed marked individual variation, and ranged from 0 to 4.3 g, and the average was 0.8g. The n6/n3 polyunsaturated fatty acids (PUFA) ratio ranged from 0.9 to 19.1 (average, 4.2). There was a strong correlation only between cholesterol and PL intakes (γ=0.796, p<0.05). The mean serum cholesterol, which was known in 42 subjects was 190mg/dl, and showed no relation to their daily intakes of any of the measured lipids.
Effects of dietary fats consisting of different fatty acids on lipoprotein lipase activities in the interscapular brown adipose tissue, heart, and soleus muscle, and on body fat accumulation were studied in rats. Sprague-Dawley male rats were meal-fed an isoenergetic diet based on either beef tallow or safflower oil for 8 weeks. Lipoprotein lipase activities in the interscapular brown adipose tissue, heart, and soleus muscle before and after a meal were lower in the beef tallow diet group than in the safflower oil diet group. Body fat accumulation was greater in the beef tallow diet group than in the safflower oil diet group. The norepinephrine turnover rates in the interscapular brown adipose tissue, heart, and soleus muscle were lower in the beef tallow diet group. β-Adrenergic receptor bindings were determined with [125I] iodocyano-pindolol. Binding affinities of β-adrenergic receptor in the interscapular brown adipose tissue, heart, and soleus muscle were lower in the beef tallow diet group probably resulted from lower membrane fluidities. These results suggest that intake of the beef tallow diet promotes body fat accumulation by reducing the lipoprotein lipase activities resulting from lower sympathetic activities in the brown adipose tissue, heart, and skeletal muscle.
The effects of low-meat-protein diets on hypercholesterol-emia and proteinuria were studied in rats with nephrotoxic serum nephri-tis. After an injection of nephrotoxic serum, rats were given either a 20% meat-protein diet (20M), an 8.5%-meat-protein diet (8.5M), or a valine-(0.05%)-supplemented 8.5%-meat-protein diet (8.5MV) for 12 days. Urinary protein excreted from the 20M-fed, nephritic control rats in-creased rapidly and linearly during the initial 3 days, and thereafter the high excretion rate was maintained for up to 12 days. Two low-meat-protein diets (8.5M, 8.5MV) commenced to suppress proteinuria 3 days after feeding and the suppression was preserved during the rest of the experimental periods. Compared with the 20M, both low-meat-protein diets significantly improve hypercholesterolemia induced in this nephrit-ic model. These two diets significantly enhanced the fecal excretion of neutral sterols. They caused neither fatty liver nor severe growth retarda-tion. These effects of 8.5MV were identical to those of 8.5M. The results suggest that low-meat-protein feeding, without amino acid supplementa-tion, improves hypercholesterolemia and proteinuria in nephritis without severe protein malnutrition. The results also suggest that the hypochole-sterolemic effect of the low-meat-protein diets may be, at least in part, attributed to increased fecal excretion of steroids.
The inhibitory specificity and stability of ovomucoid from Japanese quail egg white (OMJPQ) were examined to understand its nutritional significance. OMJPQ showed strong inhibitory activities toward trypsins from various origins including human, and the trypsin inhibitions occurred at molar ratios of enzyme to inhibitor between 1/1 and 2/1. On the other hand, an equimolar mixture of the second and third domains of OMJPQ inhibited bovine trypsin more strongly than the corresponding native OMJPQ did. This distinction was partly explained by the presence of steric hindrance on the formation of a 2:1 trypsin-OMJPQ complex. OMJPQ retained about 100% of its original activity over a pH range from 1 to 12 after a 24-h incubation at 37°C. The inhibitor was most thermostable between pH 2 and 5, where more than 70% of its original activity was maintained after a 1-h incubation at 100°C and about 25% of the activity even after a 30-min incubation at 121°C. OMJPQ was also considerably resistant to pepsin attack. Pepsin digestion of the protein resulted in only about 40% loss of the original trypsin-inhibitory activity even after a 24-h digestion. Furthermore, the addition of bovine serum albumin to the digestion mixture brought about rapid elevation in the trypsin-inhibitory activity during an initial 30-min digestion. SDS-PAGE and immunoblot suggested that this was due to the liberation of active inhibitory domains from the native molecule by inter-domain proteolysis.
A fraction which had calcium binding activity was detected from the brush border membrane during intestinal calcium absorption in the rat. In situ duodenal loop experiment showed that 45Ca activity in mucosal homogenate, detergent-solubilized whole particulate fraction and detergent-solubilized brush border membrane was maximal at 30min after the radioactive calcium solution was injected in the loop. The calcium radioactivities did not co-precipitate with anti-IMCaI (integral membrane calcium binding protein) antibody. Gel permeation HPLC showed three calcium peaks from both solubilized whole particulate fraction and brush border membrane. In vitro calcium binding assay using the solubilized brush border membrane from the pylorus to proxi-mal ileum showed the existence of specific binding sites with a dissociation constant of 2.19±0.27mM (mean±SE) while the number of binding sites was 3.4±0.4 nmol/mg of protein. After in vitro binding, the solubilized brush border membrane was analyzed by gel filtration chromatography. Two calcium peaks, apparently 200 and 1kDa, were separated and the former peak seemed to be the specific binding fraction but did not contain IMCal. This fraction may bind luminal calcium during intestinal calcium absorption.
The effect of Lactobacillus acidophilus on iron bioavail-ability in rats was examined by the hemoglobin regeneration method. For hemoglobin depletion, female Wistar rats were fed an iron-deficient diet at 3 weeks of age for 13 days. Rats were then assigned to one of four groups, such that average blood hemoglobin value and average body weight were similar among the groups. For hemoglobin regeneration, they were fed one of two ferrous sulfate-supplemented diets that contained the following iron levels (mg/kg): 13.7 for two groups; 21.7 for the other two groups. In the two groups fed the same diet, one group additionally received oral administration of 2ml of skim milk fortified with 0.3% yeast extract once or twice a day for 7 days, and other rats were administered with skim milk fermented by L. acidophilus SBT 2062 in the same manner. Hemoglobin regeneration efficiency (HRE) was significantly higher in the fermented product-given rats than in the skim milk-supplied rats. There was no significant interaction in HRE between the dietary iron group and the oral administration group. These results indicate that L. acidophilus SBT 2062 is effective for increasing of iron bioavailability in rats.
Effects of skim milk and its fermented product by Lacto-bacillus acidophilus on plasma and liver triglyceride and cholesterol levels were examined in diet-induced hypertriglyceridemic rats. Male Sprague-Dawley rats at 4 weeks of age were fed a hypertriglyceridemic diet that contained 20% coconut oil, 17.5% fructose, and 17.5% sucrose for 14 days. The test diet was supplemented with either 20% skim milk powder or 20% powder of skim milk fermented by L. acidophilus SBT 2062. Hypertriglyceridemia was observed in the control group, but plasma cholesterol levels were not increased. Skim milk suppressed the elevation of plasma triglyceride levels, while its fermented product had no signifi-cant effect. Both dairy products prevented the elevation of liver trigly-ceride and cholesterol levels, but had no effect on plasma cholesterol levels.