The authors investigated the influence of vitamin E on leucocyte counts in preserved blood. Blood were obtained from 5 normal persons, with citrate・phosphate・dextrose system. To each blood, α-tocopherylacetate was added with the concentration of 10mg%, and its solvent was added to the control blood. Leucocyte were measured once or twice aweek for one month, using micro cell counter. As a result, leucocyte decrease was less marked in tocopherol group (E) than in the control (C), that is, the reductions in E and C were 16,41% at 7 day, 29,59% at 24th day, 33,69% at 31th day, respectively. From the results it was shown that α-tocopherylacetate had a preventive action for the spontaneous destruction of leucocytes in preserved blood.
S-benzoylthiamine O-monophosphate (BTMP) is better absorbed from the intestine than thiamine and causes a higher and longer-lasting rise of cocarboxylase level in the liver and blood. In the present studies, the metabolism of BTMP by rat liver and intestine homogenates was investigated. The phosphate group of BTMP was rapidly hydrolyzed by alkaline phosphatase of the intestinal mucosa. The S-benzoyl group of BTMP was found to be hardly decomposed enzymically prior to the hydrolysis of the phosphate group. Available evidence indicated that the phosphate of BTMP inhibited the hydrolysis of S-benzoyl linkage, probably due to steric factors in enzyme-substrate association. As the principal metabolic pathway, BTMP first underwent the dephosphorylation and then S-benzoylthiamine (S-BT) thus produced was converted to thiamine by thioesterase. Due to lower activity of the thioesterase as compared with that of the phosphatase, S-BT was accumlated in the reaction mixture with intestine homogenate. As a possible mechanism of the absorption of orally administered BTMP, it was suggested that BTMP might be transported across the intestinal mucosa after the conversion to S-BT.
S-Benzoylthiamine O-monophosphate (BTMP) as well as thiamine penetrated human red cell membrane very slowly, while S-benzoylthiamine (SBT) was found to penetrate very rapidly. Thus, SBT was suspected to be an actual form in the absorption of BTMP from intestine, after dephosphorylation of BTMP by alkaline phosphatase at the intestinal mucosa. As a possible model, it was demonstrated in the red cell suspension that BTMP showed a high permeability to the cell membrane under the presence of alkaline phosphatase in the medium. In the red cell suspension, it was found that SBT was accumulated in the cells and the cell to medium concentration ratio of the total thiamine reached over 2.8. From studies on i) transport of SBT into red cell ghosts as well as the intact cells, ii) decomposition of SBT in the red cell homogenate and in an aqueous medium, iii) effect of the concentration gradient on the uptake, and iv) effect of the extracellular pH on the uptake, it was concluded that the penetration of SBT into red cells proceeds through two steps ; the first, a passive diffusion of SBT through the cell membrane, and the second, a rapid decomposition of SBT to thiamine and the resultant accumulation in the cell. The decomposition of SBT to thiamine in the cell was shown to be mainly through a non-enzymatic reaction, mostly through exchange with SH group in glutathione. Possible mechanisms for the appearence of thiamine in the incubation medium accompaning the transport of SBT were also discussed.
Mechanism of the intestinal absorption of S-benzoylthiamine O-monophosphate (BTMP) was investigated in dog. The small intestine was ligated to separate from the other part of intestine and the mesenteric vein was cannulated to collect the blood sample. After infusion of BTMP, the venous blood was analysed for total thiamine and the thiamine derivatives by means of chemical analysis and paper partitition chromatography. It was found that, 1) the absorption was detected in mesenteric venous blood immediately after the infusion and the blood thiamine concentration reached a maximum about 15 minutes after the infusion, 2) S-benzoylthiamine (SBT) was a main component of the total thiamine absorbed (ca. 60% at 15 minutes after the infusion) and the amount of free thiamine increased gradually with time, but no BTMP was detected, 3) a considerable part (30 to 40%) of the total thiamine absorbed was found in red cells already in mesenteric venous blood, 4) the total amount absorbed from the lumen in 1 hour was about 77% to the dose, 5) after 1 hour, BTMP was still detected in the lumen, while not in the intestinal wall, and 6) the absorption was significantly depressed in the presence of phosphatase inhibitors such as arsenate. From these results, it was concluded that BTMP is dephosphorylated at the intestinal mucosa and SBT is transported into the epithelial cells and transferred into mesenteric venous blood without significant change to thiamine. After infusion of BTMP to dog intestine, SBT was also detected (ca. 14% at 10 minutes after the infusion) in carotid blood, with thiamine as a main component, indicating that SBT is circulated in the body in peripheral blood.
Pyrimidines related to thiamine were detected and analyzed by gas chromatography with hydrogen flame detector. A stainless column, 0.3×150 cm, packed with Neosorb NC (Nishio Kogyo Co.) coated with 15% diethyleneglycol succinate was maintained at 190℃ with a nitorogen flow of 70 ml/min. The retention times of 2-methyl-4-amino-5-aminomethylpyrimidine (I), 2-methyl-4-amino-5-formylpyrimidine (II), 2-methyl-4-amino-5-pyrimidine carboxylic acid (III), and 2-methyl-4-amino-5-hydroxylpyrimidine (IV), trifluoroacetylated in tetrahydrofuran or pyridine were within 10 minutes and the ratio was 1.0,1.0,1.3 and 1.5 respectively. Samples for analysis were prepared about 1 mg/0.1 ml on (I), (II) and (III), and about 2 mg/0.1 ml on (IV).
Examinations were made on the stabilities of thiamine (B_1) and three ethoxycarbonyl-thiamines (3mM) in aqueous ethanol of different concentrations ranging from 20 to 99.5% (v/v) at 100℃ in ampules in comparison with those in aqueous solution. Each of the compounds was degraded by a pseudo first-order reaction, regardless of the presence of ethanol. A decrease in stability of B_1 was found by increasing the ethanol concentration, the rate showing a maximum in 80% ethanol. O-Ethoxycarbonylthiamine (OCET) in aqueous ethanol was decomposed showing a maximum rate in 60〜80% ethanol, where B_1 was not given even after 10 hours, while in the aqueous solution, the amount of B_1 produced from OCET was 1.7%. An increase in stability of S-ethoxycarbonylthiamine was found in proportion to the increase in ethanol concentration. The decomposition rate constant in 99.5% ethanol was less than 1/20 of the rate in aqueous solution. The degradation rate of O, S-bis (ethoxycarbonyl) thiamine in aqueous solution was little influenced by the addition of ethanol, but the percentages of the formation of thiochrome-reaction-positive compounds decreased with the increase of ethanol concentration.
Sorbyl-CoA formation method (Wakil and Hubscher, 1960) was quite specific for determination of CoA, and neither panthetheine, phosphopantetheine nor dephospho-CoA disturbed its determination. The amount of pantetheine in normal rat liver was much smaller than those of both free pantothenic acid and CoA. It was recognized that the content of dephospho-CoA plus phophopantetheine is next to that of CoA in rat liver by combined using the sorbyl-CoA method and the microbiological assay of pantetheine after treatment with intestinal phosphatase. The normal distribution pattern of the various intermediates was described.
It was reported that thiamine kinase in the membrane fraction participated in the transport of thiamine by Escherichia coli. This fact led me to study on riboflavin uptake and localizations of riboflavin kinase and FAD pyrophosphorylase in E. coli. Riboflavin uptake by growing and resting cells of E. coli K_<12> was not observed by fluorometrical measurement as already reported by Wilson and Pardee. Riboflavin kinase and FAD pyrophosphorylase were easily extracted from the sonicated cells and was not localized in the membrane fraction differing from thiamine kinase of E. coli. No changes of the both enzyme activities by riboflavin addition to the growth medium were also observed.
In the course of studies on thiamine transport by several strains of Escherichia coli, the thiamine was accumulated by the cells as thiamine diphosphate (TDP) even at the earliest time of thiamine transport. This fact led me to study on the existence and peoperties of thiamine kinase in E. coli which probably participated in the transport of thiamine by phosphorylating thiamine. The thiamine kinase activity was detected in the membrae fraction separated from the sonicated cells. Substitution of ATP by ADP in the reaction mixture of membrane fraction was observed. The enzyme activity in the presence or absence of ADP was markedly decreased by addition of 3.3 mM KCN. This result showed that the enzyme reaction required ATP which was apparently generated from endogenous or exogenous ADP through oxidative phosphorylation associated with the membrane fraction. Furthermore, it was found that the thiamine kinase activity was located in the spheroplast membrane fraction separated by treatment with lysozyme and EDTA. The reaction velocities by the membrane fraction were proportional to the amounts of enzyme proteins substrates and reaction time. The optimum pH was given 7.0 and apparent Km for thiamine was 3.0×10^<-7> M. The activities were inhibited by the addition of thiamine antagonists such as pyrithiamine, oxythiamine and dimethialium, but the degrees of inhibition were far less than that of purified yeast thiamine kinase. Evidence for the regulation of thiamine kinase by TDP in the cells was obtained by using thiazoleless strain of E. coli.
Absorption studies of α-lipoic acid-^<35>S and its derivative, thiamine-8-(methyl-6-acetyldi-hydrothioctate) disulfide (TATD), labeled in the portion of α-lipoic acid with ^<35>S were carried out using a everted sack method of isolated rat intestine. α-Lipoic acid absorption was more rapid than that of TATD at the beginning of the reaction until 45 minutes, while after that the both absorption rates were almost same. TATD which was transported from mucosa to serosa side of intestinal segments was splitted to lipoic acid and thiamine moieties of the compound and the lipoic acid was partly converted to β-lipoic acid and an unknown compound responding to Corynebacterium bovis, not to Streptococcus faecalis. α-Lipoic acid absorption was markedly high compared with that of thiamine hydrochloride, but the absorption rate of thiamine part of TATD was stimulated five times as much as that of thiamine-HCl and became a half of absorption of lipoic acid part of TATD.
Intestinal absorption of a α-lipoic acid derivative, thiamine-8-(methyl-6-acetyldihydrothioctate) disulfide (TATD) was investigated using the ligated dog intestine in order to compare with the absorption of TATD by in vitro method of isolated rat intestine, previously reported. The compound in the mesenteric venous blood was found mainly as free thiamine and lipoic acid like a result observed in the in vitro experiment. However, 1 to 30 minute bloods after infusion of TATD contained the compound of disulfide form which was detected by bioautography using a thiamine-less mutant Esherichia coli 70-23 or Streptococcus faecalis R and was determined by fluorometric method for thiamine disulfide assay, showing absorption below 10% of TATD absorbed. A small part of TATD in the blood was converted to a bound form of lipoic acid with plasma protein.