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Distribution of thiamine, thiamine phosphates, and thiamine metabolizing enzymes in neuronal and glial cell enriched fractions of rat brain.
Full text linkThe distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions
A thiamine/H+ antiport mechanism for thiamine entry into brush border membrane vesicles from rat small intestine.
Full text linkOutwardly oriented H+ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pHin5:pHout7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10-13% of intravesicular transport. At the same gradient, the Km and Jmax values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 +/- 1.4 microM and 14.9 +/- 3 pmol.mg-1 protein.6 sec-1 respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H+ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H+ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H+ antiport, and imipramine. Conversely, the guanidine/H+ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H+ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9
Effects of acute and chronic ethanol administration on thiamine metabolizing enzymes in some brain areas and in other organs of the rat.
No full textThe effect of ethanol (4.7 g/kg body wt intragastrically as a single dose or once daily for 35 days) on the levels of the thiamine metabolizing enzymes (thiamine pyrophosphokinase, TPKase; thiamine pyrophosphatase, TPPase; and monophosphatase, TMPase) was studied in different organs (liver, kidney, small intestine, heart and skeletal muscle) and nervous regions (cerebral cortex, cerebellum, medulla oblongata, pons, corpus callosum, hypothalamus and sciatic nerve) of the rat. In order to evaluate the non-specific effects of the stress of gastric gavage and of the additional caloric intake, appropriate control groups of animals were treated intragastrically with water or with a saccharose solution isoenergetic with ethanol respectively. All animals were reared on a nutritionally adequate diet supplying amounts of thiamine higher than the recommended daily requirement. Enzymatic activities were determined quantitatively by biochemical methods. Tissue TPKase levels were generally reduced by both acute and chronic ethanol administration. TPPase levels were generally reduced after acute and increased after chronic ethanol treatment. Changes in brain TMPase levels were similar to those observed for TPPase. In visceral organs and skeletal muscle TMPase activity was increased by chronic ethanol treatment as compared to acute ethanol administration. In conclusion, ethanol exerts a marked influence on the tissue levels of the thiamine metabolizing enzymes: the activity of the enzymes dephosphorylating thiamine phosphates is increased whereas the activity of the thiamine pyrophosphate synthesizing enzyme is reduced. These changes may contribute to an important extent to the disturbances in thiamine cellular uptake and metabolism observed in alcoholism
Three thiamine analogues differently alter thiamine transport and metabolism in nervous tissues: an in vivo kinetic study using rats
No full textEffect of acute and chronic ethanol administration on the transport of thiamine from plasma to different brain regions of the rat.
No full textThe effect of ethanol (4.7 g/kg body wt intragastrically as a single dose or once daily for 35 days) on the transport of thiamine from plasma to four brain regions (cerebellum, cerebral cortex, pons and medulla) was studied in albino rats. Animals were given an intravenous injection of labelled thiamine with a sampling procedure which allowed the determination of regional blood flow and tissue thiamine uptake. Regional blood flow was found to be enhanced after acute, but non chronic, ethanol administration. The magnitude of increase ranged from 13 to 35% depending on the brain region being considered. Thiamine was transferred from plasma to cerebral tissue by a saturable process with a non-saturable component prevailing at thiamine concentrations above 10-15 microM. Three main modifications in the thiamine transport were found as a result of ethanol treatment: a reduction in affinity for the carrier (Km increased), an increase in maximal transport rate (Jmax) and an increase in non-saturable diffusion (KD constant increased). The effects were more pronounced after acute ethanol administration. As a consequence of these modifications both acute and chronic ethanol treatment caused an increase in thiamine transport rate at high plasma concentrations. On the contrary, at low (physiological) plasma concentrations, thiamine transport was little increased by acute ethanol administration and virtually unaffected by chronic ethanol intoxication
Effect of ethanol administration on the in vivo kinetics of thiamine phosphorylation and dephosphorylation in different organs. I. Chronic effects.
No full textThe effects of chronic ethanol administration on different steps in the metabolism of thiamine (T), thiamine mono- (TMP) and pyrophosphate (TPP) were determined in vivo in the liver, kidney, heart, skeletal muscle and small intestinal mucosa. The radioactivity of T and its phosphoesters was measured in plasma and in the selected organs under steady-state conditions and at fixed time intervals (0.25-240 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in rats chronically (35 days) ethanol-treated (daily dose of 4.7 g kg-1 body wt by gastric gavage). Two types of controls were used: pair-fed rats treated with a sucrose solution isoenergetic with ethanol, and water-treated rats. A nutritionally adequate diet, which supplied an excess of thiamine, was given to the rats, producing a virtually steady content of thiamine compounds in the tissues. The analytical data obtained were elaborated using appropriate compartmental mathematical models, which allowed the fractional rate constants, turnover rates and turnover times to be calculated. Alterations in thiamine metabolism were modest and differed according to the organs. The most widespread modification was to facilitate the entry of T (small intestine, kidney and heart) or TMP (small intestine and kidney), while no significant change of T and TMP release was seen. Sucrose had minimal effect in both steps. Enzymatic transformations of thiamine were likewise marginally affected. A general trend toward a slower T pyrophosphorylation and a faster T phosphate dephosphorylation was observed in the small intestine, kidney, heart and liver. Skeletal muscle was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS
Blood-brain transport of thiamine monophosphate in the rat: a kinetic study in vivo.
Full text linkTo calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue
Age-related thiamin transport by small intestinal microvillous vesicles of rat.
No full textThe effect of aging on the intestinal transport of thiamin was studied using small intestinal microvillous vesicles prepared from groups of rats aged 1, 2, 6, 12 and 24 months, respectively. The vesicles (enrichment 14.6-17.8-fold) were incubated with 0.125 to 12.5 microM tritiated thiamin and the radioactivity taken up was measured radiometrically after rapid filtration. The time course and cumulative uptake curves of thiamin and the inhibiting potency of the thiamin structural analogs pyrithiamin, amprolium and oxythiamin on the saturable component of thiamin transport were determined. The vesicle diameter was measured by using a computerized morphometric procedure, and found to be decreased in aged rats. The Km and Jmax values of the saturable component of transport increased with increasing age, the difference with younger groups being statistically significant at 24 and 12 months. The inhibitory potencies of pyrithiamin and amprolium gradually decreased with increasing age, while oxythiamin was devoid of significant inhibitory activity. Passive permeability coefficients decreased with increasing age, reaching their lowest value at 24 months. These results show that aging is associated with intrinsic alterations of the enterocytic plasma membrane resulting in a decrease of the affinity for thiamin, associated with a faster rate of the saturable component of thiamin transport, and with a significant depression of the non-saturable component
Trasporto della tiamina in vescicole di membrane microvillari di rene di ratto.
No full textMicrovillous membrane vesicles prepare from the cortex of rats, were suspended in different media, controlling the composition of the intravesicular fluid by prolonged equilibration at 4°C in preincubation buffers of desired composition. Until used they were held on ice. For the uptake experiments the vesicles were incubated at 25°C with tritiated thiamin for suitable times. Then, they were separated with a rapid filtration procedure for the radiometric measusement of the radioactivity taken up. Thiamin uptake was Na+-independent and 53% membrane bound. The concentration curve had a biphasic course which was hyperbolic (saturable) at physiological concentrations (<1.25 uM) and linear at higher ones. Thiamin uptake was three times increased by an outwardly directed H+ gradient (pHin 6 : pHout 7.5), which enhanced only thiamin translocation and maintained biphasic the concentration curve. However, the saturable component displayed kinetic costants 12-fold higher than those in the absence of gradient. Moreover, the saturable component remained unmodified by typical organic cations transported by renal microvillous vesicles, but inhibited by inhibitors of intestinal thiamin/H+, renal guanidine/H+ and Na+/H+ antiporters
Compartmental model identification based on an empirical Bayesian approach: the case of thiamine kinetics in rats
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