Thiamin triphosphate (ThTP) in early stationary phase cells of Escherichia coli grown in nutrient broth with 0.1% yeast extract was found to constitute approximately 5-7% of cellular thiamin diphosphate (ThDP) or around 5 nmol/g cell. Nearly the same level of. ThTP was obtained in a Bacillus strain. When E. coli was loaded with an excess of ThTP or ThDP, cellular ThTP was found to be controlled in the course of the long term to maintain its ratio to the amount of cellular ThDP. The ThTP vs. ThDP ratio in E. coli cells after short-term ThDP uptake was found to be a function of the cellular growth phase. The ratio in early exponential phase E. coli cells was found to be approximately 4% and it became lower (less than 3%) when cell growth proceeded to the late exponential stage. Two phosphatases specific for ThTP (ThTPase) among thiamin phosphates were detected in E. coll. One required Mg2+ and was found mainly in the soluble fraction, while the other was Mgt2+independent and originated from the membrane. The two ThTPases were similar to their rat tissue counterparts.
The effect of the quality of dietary protein on the initiation of temporary anemia during vigorous physical training (sports anemia) was studied in dogs and rats. In the dog experiment, one group of dogs was fed a crude animal protein (AP) diet and the other a crude vegetable protein (VP) diet. After 6 weeks on the diets in a sedentary state (rest period), all the dogs were forced to run every day for two weeks (exercise period). The rat experiment was carried out using purified nutrient mixtures. Casein (C) was used as AP and gluten (G) as VP. Feeding was done for two weeks in two series with two diet groups of rats. One series was 15% protein feeding (15% C and 15% G) groups and the other 24 protein feeding (24% C and 24% G) groups. In each group, one group remained in a sedentary state (rest group), and the other ran vigorously on a treadmill every day for one week (exercise group). In a sedentary state, there was a slight tendency for the hemoglobin content or erythrocyte count to be reduced, even when the values remained within the normal range, in dogs and rats fed VP. On the other hand, after vigorous running, significant anemia (reduction of hemoglobin) appeared in the VP diet dogs and in all exercise rat groups except the 24% C group. It was confirmed that the anemia was caused by a reduction of erythrocyte resistance to hemolysis, which was closely related to changes in the lipid composition of blood (serum and especially erythrocytes). The change in lipid profile revealed by the experiments was a reduction of free cholesterol in blood associated with an increase of lysolecithin in dogs during the exercise period and in the rat exercise groups. It was suggested that repeated physical exercise increased the activity of LCAT (lecithin-cholesterol-acyl-transferase) in the liver, spleen, etc., resulting in the above changes in lipid patterns in the blood. In dogs of AP and rats of 24% C, however, those changes in lipid pattern caused by exercise and sports anemia did not appear significantly. The different effects of the AP diet seemed to be due to the antagonistic effects of lysine, which was present in sufficient amounts in the diet. Thus the theoretical basis for our recommendation of a high amount of AP in the diet to prevent sports anemia was clarified by the present experiments. The mechanisms of the different effects of AP and VP, especially those due to their different amino acid compositions, were discussed from recent studies on the effects of protein quality and physical training on lipid metabolism.
A reliable high-performance liquid chromatographic method that utilizes a reversed-phase separation with ion-pairing and postcolumn fluorescence derivatization for the analysis of Nτ-methylhistidine in food, chicken excreta, and rat urine is described. Nτ-Methylhistidine in the hydrolyzed sample is first roughly separated from acid and neutral amino acids by an ion-exchange column. The Nτ-methylhistidine fraction is then evaporated and the residue is dissolved in the mobile phase (15mM sodium octane sulfonate in 20mM potassium phosphate), and subjected to high-performance liquid chromatography.