Search

The membrane and intracellular steps of thiamin (T) uptake were studied in rat isolated enterocytes. The membrane step was investigated by using enterocytes deenergized with 25 μM rotenone and a short (30 s) incubation time, while the intracellular metabolic step was assessed by using nor-moenergized (normal) cells incubated for a longer (30 min) time. Evaluation of Km, Jmax and KD (apparent passive permeability coefficient) values, at 30 s incubation, indicated that deenergization slightly reduced both the affinity and the capacity of the saturable component of T uptake, as well as the capacity of the non-saturable component. At 30 min incubation, deenergization suppressed completely the saturable component, without affecting the non-saturable component. Comparison of the Km and Jmax values of the saturable components in nor-moenergized enterocytes at 30 s and 30 min indicated that intracellular metabolic phosphorylation is probably the event responsible for the active process involved in T uptake.

The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on thiamine pyrophosphate (ThPP)- dependent enzymes (transketolase, TK; pyruvate-, PDH, and alpha-ketoglutarate, alphaKGDH, dehydrogenases) and thiamine (Th) metabolizing enzymes (thiamine pyrophosphokinase, ThPK; thiamine mono-, ThMPase, and pyrophosphatases, ThPPase) were evaluated in vitro in rats in steady state conditions. The enzymes were also determined in the intact cerebral cortex of the same animals. CD was carried out by i.p. injection of 3-acetylpyridine, followed by harmaline and niacinamide. SD was carried out by complete dissection of the peduncles of the left cerebellar hemisphere. Chemical and surgical cerebellar deafferentations significantly lowered the contents of both Th-metabolizing and ThPP-dependent enzymes in the cerebellum without modifying those of the cerebral cortex. ThPK and TK, which are particularly concentrated in neurons, were the most affected enzymes. The decrease in ThPP-dependent enzymes shown here is associated with the slowing down of the Th phosphorylation-dephosphorylation cycle and with no modification in the ThPP content in the cerebellum, both of which were found in a previous study. Cerebellar deafferentation seems to only affect the apoenzyme moiety of the enzymes.

The effects of acute ethanol administration on different steps of thiamine (T), thiamine monophosphate (TMP) and pyrosphosphate (TPP) metabolism were determined in vivo in nervous tissues (cerebral cortex, cerebellum, brain stem and sciatic nerve) and in other tissues (small intestinal mucosa, kidney, heart, skeletal muscle and liver) of rats. The radioactivity of T and its phosphoesters in plasma and tissues was determined under steady-state conditions and at fixed time intervals (0.25-24 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in the presence of a constant plasma ethanol concentration (37 mM; 1.75 g.l-1) produced by repeated intragastric administration of ethanol. Control animals received water intragastrically. Ethanol-treated rats and controls were starved, with water ad libitum during the 24 hr study period. Data were evaluated by using appropriate compartmental models, which allowed calculation of fractional rate constants, turnover rates and turnover times. In nervous tissues ethanol enhanced TMP entry (without affecting T entry or T and TMP release), reduced turnover time of total T and TPP, caused an almost general enhancement of TPP dephosphorylation without affecting T pyrophosphorylation, and increased markedly T content in the mixture released by tissues. Overall, ethanol appeared to enhance exchanges of T compounds in nervous tissues. In other tissues, the effects of ethanol were less consistent. Ethanol increased T uptake in kidney and liver and T release in liver and heart, but had no effect on T exchanges in the small intestinal mucosa and in skeletal muscle. In the presence of ethanol, TMP uptake increased in heart and skeletal muscle and decreased in the small intestinal mucosa, while TMP release decreased in heart and remained unchanged in all other organs. Turnover times tended to increase for total T and to decrease for TPP. T pyrophosphorylation was generally reduced, and T phosphates dephosphorylation generally enhanced. T became the most abundant component in the mixture released from all tissues.

Ventricular myocardial cell action potentials were measuredin situ by means of intracellular microelectrodes in the heart of pyridoxine-deficient rats both at spontaneous frequency and at frequencies increased to higher levels. Pyridoxine deficiency lowered the rates of depolarization and repolarization phases of ventricular action potentials thus lengthening their duration. A noticeable bradycardia seemed to be induced by pyridoxine deficiency.

Ventricular cell action potentials were measured in situ using intracellular microelectrodes in the hearts of vitamin A-deficient rats, both at the spontaneous heart rate and at rates increased to various higher levels. The changes found indicate that a lack of vitamin A in the tissue may modify the ion permeability underlying cardiac electrogenesis, without affecting cardiac energetic metabolism. Results are discussed in the light of recent theories that vitamin A may stabilize the structure of cell membranes and control their permeability.

The myocardial fibre cell action potentials of rats, made thiamine deficient by a synthetic diet or by antivitamin treatment (pyrithiamine or oxythiamine), have been measured in Situ by means of fluctuating microelectrodes. Although the amplitude of the action potential was unchanged, its duration was distinctly increased, owing to a lessened repolarization. Bradycardia, which was also found in thiamine deficiency, was viewed essentially as a consequence of the slowed cellular membrane recovery processes.

In different animal species, including man, thiamin is absorbed in vivo from the small intestine by means of two mechanism: at low intraluminal concentrations (less than 1.5 microM), absorption shows a saturation kinetics; at higher concentrations, passive transport (diffusion) takes place. The absorption is maximal in the duodenum, its rate decreasing caudally along the small intestine. In vitro at low concentrations, the transcellular transport system of thiamin shows the following general features: it takes place against a chemical concentration gradient, is easily saturable (Km = 0.16-0.63 microM; Vmax = 5.2 mumol X g-1 X h-1), is blocked by cellular metabolic inhibitors, anoxia, low temperature and thiamin structural analogues. It is Na+-dependent and operates mainly from mucosa to serosa. In contrast, distinctive features of the thiamin transfer are the following: entry in the enterocyte in the free form (sodium-independent); pyrophosphorylation by means of a cytoplasm thiamin-pyrophosphokinase; the dephosphorylation of thiamin-pyrophosphate by means of particulate intracellular phosphatases; the cellular exit mainly in the free form, through the action of the controluminal Na-K activated ATPase. A working scheme for thiamin intestinal transport is proposed and discussed.

Starting from mucosal cells, isolated from rat small intestine, subcellular fractionation was carried out. Four fractions were prepared and characterized by marker enzymes and electron microscopic examination: nuclei plus microvilli, mitochondria, microsomes and supernatant. Thiamine-pyrophosphokinase activity was localized mainly in the supernatant fraction, with minor amount in mitchondria and microsomes. On the contrary thiamine-pyrophosphatase activity was present only in the particulate fractions (especially in microsomes), being completely absent from the supernatant.

Rat everted jejunal sacs were incubated for 15 and 30 min at 37 degrees C in oxygenated Krebs-Henseleit buffer, pH 7.4, containing 0.2 microM [3H]-thiamin (3H-T) or [3H]-thiamin monophosphate (3H-TMP) with and without 10 mM 1-phenylalanine (PAL) or 2.5 mM levamisole (LEV). The concentrations of 3H-T and its phosphoesters in sac wall and serosal fluid were determined by a radiometric method after electrophoretic separation. In separate experiments, thiamin pyrophosphokinase (TPKase) and thiamin pyrophosphatase (TPPase) activities were determined in mucosal scrapings, with and without PAL or LEV, by using a radiometric and a colorimetric method, respectively. 3H-TMP was transported partly unchanged by an active mechanism similarly to 3H-T, but less efficiently. During transport, 3H-TMP was also enzymatically transformed to thiamin (T) and thiamin pyrophosphate, which accumulated in the tissue. In the serosal fluid, the concentration of 3H-TMP exceeded that of 3H-T. Presence of PAL or LEV with 3H-T or 3H-TMP in the incubation medium reduced the serosal transport and the tissue content of T compounds. LEV caused a dose-dependent inhibition of TPKase without affecting TPPase, whereas PAL inhibited both activities to about the same extent. These results indicate that the transport of TMP involves a number of different processes similar to those responsible for T transport. The effects of PAL and LEV underline the importance of phosphorylation-dephosphorylation coupling.

The hypothesis that the corticosterone-induced enhancement of D-glucose uptake by brush-border membrane vesicles from the small intestine of adult rats is caused by an alteration in vesicle volume was tested by means of structural morphometric computerized analysis. By using a Ca2+ precipitation method, microvillous membrane vesicles were prepared from different groups of adult rats: normal controls; animals sacrificed 8 days after adrenalectomy; sham-operated pair-fed controls; animals treated with corticosterone (1 or 10 mg x 100 g b.wt.-1 daily i.p. for 8 days); corticosterone vehicle-treated controls. The vesicles were processed by conventional electron microscopy techniques. Morphometric computerized analysis was carried out on electron micrographs at a 20,000 x magnification, after selecting by an interactive method the vesicles showing certain morphologic features. The morphometric parameters determined for the vesicles from each experimental group were diameter, shape factor (minimal to maximal diameter ratio), surface area and volume. The following main findings were obtained: the vesicles were consistently spherical, with the glycocalyx on the outer side; sham-operation and pair-feeding with adrenalectomized rats, as well as corticosterone-vehicle injection, significantly reduced vesicular volume as compared to normal controls; adrenalectomy significantly decreased vesicular volume as compared to pair-feeding; excess of corticosteroids caused opposite changes (i.e. a significant increase of vesicle volumes as compared to vehicle injection), which were apparently dose-dependent. A comparison with D-glucose vesicular transport data previously obtained under the same experimental conditions revealed the existence of a positive relationship between vesicular volume and D-glucose half-filling time (t 1/2). A positive relationship between vesicular volume and D-glucose vesicular space was found for all experimental conditions except vehicle injection. As a whole, the morphometric results were consistent with functional findings, supporting our previous hypothesis that the corticosterone-induced increase in D-glucose transport by small intestinal microvillous vesicles is due, at least in part, to enlargement of the vesicle.

In the nervous system thiamin could have two main functions: coenzymatic, as thiamin pyrophosphate, and/or non-coenzymatic, but related to the nervous activity. In order to investigate the thiamin role we studied the turnover and metabolism of thiamin and its phosphoesters (mono-, pyro-, and tri-phosphate) in different regions (cerebellum, brain stem, cerebral cortex and sciatic nerve) of the nervous system, in a steady state condition. Practically, we determined the regional radioactivity of thiamin and its phosphoesters at different times (up to 240 hrs) from labeled thiamin administration, analyzing the obtained data by suitable compartmental models. From the results of two different types of experiment and from their mathematical elaboration, the cerebellum, with the highest thiamin compound content, appeared to be the most active region of the brain in different steps of thiamin metabolism (phosphorylation, dephosphorylation, uptake and release). The sciatic nerve, however, stood out for only thiamin and thiamin monophosphate metabolism, in spite of its very low total thiamin content.

The relationship between thiamin intestinal transport and phosphorylation in vitro was investigated using everted jejunal sacs from normal rats, rats affected with dietary thiamin deficiency at different degrees and relative pair-fed controls; recovered rats, i.e. severely thiamin-deficient animals, 6 hours after the intravenous injection of 1 mg of thiamin chloride/100 g body weight. The sacs were incubated at 37 degrees for 30 min with 0.2 microM 14C-labeled thiamin. During incubation, the endogenous phosphorylated thiamin undergoes a dephosphorylation, which is lowest in thiamin deficient rats. The jejunal sacs from all experimental groups take up and phosphorylate labeled thiamin: the rate is inversely proportional to the endogenous cellular content of phosphorylated thiamin. The net transport of labeled thiamin is enhanced in thiamin-deficient rats. The labeled thiamin net transport after incubation is linearly related to the labeled phosphorylated thiamin content of intestinal tissue. Thiamin repletion of severely thiamin-deficient rats restore the normal levels of endogenous free and phosphorylated thiamin in the intestinal within 6 hours. In addition after incubation, the labeled phosphorylated thiamin content reverts to the levels of the control, while thiamin net transport remains high.

An improved method for electrophoretic separation and fluorometric (or radiometric) determination of thiamine and thiamine mono-, di- and tri-phosphates in animal tissues (liver, small intestine, kidney, heart, brain) is described. The amount of total thiamine handled was 1.5-3.5 microgram. The procedure includes: acid excretion of the compounds from the tissue, deproteinization with TCA, purification of the extract on partially deactivated charcoal, elution with 10% n-propanol in 0.1 N formic acid, lyophilization, electrophoresis on gelatinized cellulose acetate strips, elution of the thiaminic bands with 50% ethanol and finally oxidation to thiochromes and reading of eluate fluorescence in a spectrofluorophotometer. The mean internal recovery was 83.3% and the external one 83.5%. Dephosphorylation of the T phosphates was rather scanty and each thiamine compound was comparably recovered (82.0-85.7%), apparently TTP being the worst and T the best recovered compound.

Ventricular cell action potentials were measured in situ using intracellular microelectrodes in the hearts of hypervitaminotic A rats, both at the spontaneous rate and at rates externally increased to various higher levels. The action potentials were much modified (decrease in amplitude, in depolarization rate, in fast and slow repolarization rate, and increase in partial and total duration). Excess of vitamin A also appeared to bring about a rise in spontaneous heart rate. Ion fluxes in myocardial cells at high levels of vitamin A seemed to be somewhat uncoupled from cardiac metabolism. The results obtained indicate that the changes in cardiac electrogenesis may be specifically related to the effects of an excess of vitamin A on cell membrane structure and consequently on permeability and ion fluxes through the membranes.

L'impiego di tiamina-14C, con la tecnica dei sacchetti rovesciati di intestino di ratto, ha permesso di dimostrare che, durante il trasporto contro gradiente in vitro, la tiamina viene fosforilata dal tessuto intestinale. Gli inibitori metabolici e gli analoghi strutturali della tiamina che ne inibiscono la fosforilazione del pari ne riducono il trasporto netto. Cio dimostra che il trasporto intestinale della tiamina e strettamente connesso con la sua fosforilazione.

In rats, the action potentials of myocardial fibre-cells in avitaminosis B1, induced by a deficient diet or by pyrithiamine administration, were measured by fluctuating microelectrodes on hearts in situ at spontaneous and at externally increased heart rate. The increasing of the heart rate enhanced the modifications of the action potentials of spontaneously beating thiamine-deficient hearts. This suggests that the increased rate may determine a reduction in ion gradients on both sides of the cell membrane. The slowed spontaneous heart rate of thiamine-deficient rats seems to allow full recovery of the membrane ionic distribution, even if the underlying ion processes were greatly reduced.

Countertransport experiments on rings of everted jejunum of rats suggest that thiamine is actively transported by a mobile carrier in intestinal epithelial cells and that pyrithiamine, but not oxythiamine or deshydroxythiamine, has the common carrier with thiamine.

IT is now generally accepted, especially through the work of Evans and Lepkovsky1, that a diet of carbohydrates accelerates the appearance of the symptoms of polineuritis in animals suffering from avitaminosis B1, whereas an excess of fats delays it, revealing an effect which is considered as a sparing action. This has been confirmed by many other workers.