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The leucine content in rat liver and blood serum under acute oxythiamine avitaminosis is decreased. Simultaneously oxythiamine inhibits the activity of alpha-ketoisocaproate dehydrogenase resulting in a decrease of leucine catabolism in the liver. The level of total tRNA in hepatocyte cytosol is lowered, the radioactivity of leucine in aminoacyl-tRNA on the 6th min after injection of [1-14C]leucine is increased approximately 1.5-fold as compared to normal. The radioactivity of leucine in blood serum and aminoacyl-tRNA is directly significantly correlated with the content of leucine incorporated into the proteins, while the level of intracellular [14C]leucine is not correlated with the protein radioactivity (r=-0.154,p greater than 0.5) or with blood serum amino acid radioactivity (r=-0.184, p greater than 0.5). The results obtained thus indicate that 1) in acute oxythiamine avitaminosis when the labelled amino acid is incorporated within 3-6 min the label is "concentrated" in blood serum resulting in increased specific radioactivity of the protein in rat liver; 2) in blood serum leucine is predominantly utilized during protein biosynthesis; 3) acute oxythiamine avitaminosis has no significant effect on protein biosynthesis in rat liver; 4) specific radioactivity of the protein cannot serve as an index of protein synthesis rate.

The purpose of this study was to evaluate the possibility of using leukocyte transketolase activity (TKA) as an index of thiamin status. It was shown previously that hepatic TKA in rats is altered by changes in the type and amounts of carbohydrate, protein, and fat in the diet. It was, therefore, essential to determine whether leukocyte TKA in rats is affected by dietary factors other than thiamin intake. Hepatic TKA and leukocyte TKA both varied with dietary thiamin intake; maximum activity was attained when the diet contained 3 to 4 mg thiamin/kg diet. Administration of oxythiamin resulted in decreases in TKA in both liver and leukocytes. The changes in leukocyte and hepatic TKA induced by varying degrees of thiamin deficiency were closely correlated. The responses of thiamin content were also closely correlated in liver and leukocytes. However, only hepatic TKA was altered by increasing the fat or protein content of the diet or by substituting fructose for glucose. None of the dietary manipulations studied except the production of thiamin deficiency had any effect on leukocyte TKA. We conclude from our results that leukocyte TKA is a sensitive and specific indicator of thiamin nutriture in rats.

The effect of the thiamine deficiency on the H+ secretion in an isolated gastric mucosa and also on the functional properties of the mitochondria was investigated. The thiamine deficiency was called forth through introduction of oxythiamine or by exclusion of thiamine from the ration. It is shown that the thiamine deficiency in an isolated gastric mucosa of the frog produced inhibition of the H+ secretion and in the gastric mitochondria of rats--a higher activity of succinate-dehydrogenase and a diminution of the exogenous cytochrome oxidation rate.

Open-field testing has proven useful for evaluation of the effects of drugs on behavior. We now present detailed methods for subdividing open-field behaviors into four categories: sniffing, grooming, resting, and staring. Upon initial exposure to the open field, sniffing is the predominant behavior. With habituation, sniffing and grooming decrease, and resting and staring increase. Treatment with a thiamin-deficient diet and pyrithiamin, a centrally acting thiamin antagonist, markedly increases staring behavior by day 3 of treatment. Animals that are treated with a thiamin-deficient diet and oxythiamin, a peripherally acting thiamine antagonist, do not have increased staring behavior. Therefore, increased staring behavior is an early behavioral change in central nervous system thiamin deficiency. Staring and other spontaneous open-field behaviors may be useful variables to monitor in thiamin deficiency and in other metabolic encephalopathies.

14C-thiamine transport across the membranes of erythrocytes obtained from blood of normal rats was studied. The active transport was observed at less than 0.5 microM thiamine concentration. An increase in the thiamine concentration resulted in a decrease in the active transport which was accompanied by increased diffusion. Thiamine analogs inhibited its transport into erythrocytes with different efficiency. The rate of thiamine phosphate esters formation in the course of incubation characterized the state of functional activity of the kinase and phosphatase systems. The data obtained suggest that transport and phosphorylation are independent processes in erythrocytes.

A method is described for isolation of orotic acid from rat liver tissue for study of the kinetics of its radioisotopic labelling. Within 24 hrs after injection of 400 mg hydroxylamine per kg of body mass, the concentration of hepatic orotate decreased, while its transport into liver tissue and the kinetics of labelling of its endogenous pool remained unaffected.

The monamine oxidase (MAO) inhibitors harmaline, tranylcypromine, deprenyl, clorgyline and iproniazid were injected intraperitoneally for five days to rabbits at doses that produced significant MAO inhibition. The first three inhibitors raised the concentrations of pyruvate and lactate in blood, decreased the activity of erythrocyte transketolase (TK) and increased the thiamin pyrophosphate (TPP) effect. The drugs also produced anorexia and loss of body weight. The changes were suggestive of an adverse effect on the thiamin status. Clorgyline and iproniazid, however, raised the blood concentrations of pyruvate and lactate but did not affect significantly erythrocyte TK activity or TPP effect. Treatment of rabbits for 14 days with the thiamin antagonists pyrithiamine (20 micrograms kg-1) or oxythiamine (0.8 mg kg-1) produced a significant drop in TK activity and increase in the lactate and pyruvate concentrations as well as an increase in TPP effect. Pyrithiamine (5, 10 or 20 micrograms kg-1, 14 days) lowered significantly the activity of MAO in the liver and brain of rabbits. Treatment with the other antagonist, oxythiamine, at doses of 0.2 or 0.4 mg kg-1 for 14 days had no significant effect on MAO activity. At a dose of 0.8 mg kg-1 a significant drop in MAO activity occurred. A pair-feeding trial indicated that the biochemical changes produced in animals treated with MAO inhibitors were attributable to the drugs per se, and not to the ensuing anorexia. Thiamin (100 micrograms kg-1, subcutaneously) when given concomitantly with pyrithiamine, oxythiamine, harmaline, deprenyl and tranylcypromine was effective in preventing the development of thiamin deficiency.

Glucose-6-phosphatase (G-6-Phase) activity in the rat liver is established to correlate directly with the content of corticosteroids in blood: it lowers with hypocorticoidism (adrenalectomy) and rises with hypercorticoidism (stress). The highest G-6-Phase activity in the liver of the intact animals is observed in autumn, the lowest one in spring, i.e. in the periods when the adrenals function has minimal and maximal values, respectively. Thiamin (0.4 g/kg) is shown to cause a decrease in the G-6-Phase activity in the liver of the intact rats and adrenalectomized or hypophysectomized animals. An assumption is advanced that the found in the experiment a pronounced insulin-like effect of vitamin B with respect to the G-6-Phase activity is connected with an intensified synthesis of the corresponding hormone in the pancreas.

Oxythiamine B1-hypovitaminosis in adrenalectomized male rats is not accompanied by acute thymus involution. This is not so much related to hypocorticoidism per se, as to a decrease in metabolic potencies of the antivitamin, particularly to a negligible depression of thymocyte tolerance to hydrocortisone. Sex differences in morphological parameters of thymus lymphopoiesis have been found after adrenalectomy.

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.

1. A study has been made of the nature of the delayed stimulation caused by external aldosterone in barnacle fibres pre-exposed to aldosterone. 2. (i) Microinjection of 0-5 M-ATPMg2- caused only a small but prompt rise in the Na efflux. (ii) Microinjection of 0-5 M-ATPMg2- followed by external application of 10(-5)M aldosterone greatly augmented the magnitude of the delayed stimulation. The response was dose-dependent, as well as dependent on the concentration of external K+ and H+, but not Na+, Ca2+ or Mg2+. (iii) External application of 10(-5) M aldosterone for 30 min followed by its withdrawal from the bathing medium failed to bring about delayed stimulation. By contrast, fibres into which ATP had been injected showed delayed stimulation under these conditions. 3. Microinjection of actinomycin-D or spironolactone SC-14266 into fibres into which ATP had been injected followed by external application of aldosterone resulted in complete abolition of the delayed stimulation. 4. Delayed stimulation was reduced whether ATP had been injected or not by prior external application of 10(-4)M ouabain or internal application of 8 x 10(-2)M ethacrynic acid. It was completely abolished by prior application of ouabain externally and ethacrynic acid internally, or only 10(-4)M diphenylhydantoin externally. 5. (i) Microinjection of atractyloside or bongkrekic acid caused a substantial fall in the resting Na efflux. Bonkrekic acid proved more powerful than atractyloside. Microinjection of 0-05 M-ATPMg2- into fibres poisoned with 2-0 x 10(-2)M bongkrekic acid completely restored the Na efflux.

Inhibition of rat liver transketolase under conditions of hydroxythiamin or alimentary B1 avitaminosis was accompanied by an increase in intracellular concentration of 6-phosphogluconate and xylulose-5-phosphate. The alterations in their content was inversely correlated with the transketolase activity. The dehydrogenases involved in the pentose phosphate shunt exhibited various unspecific sensitivity to hydroxythiamin. The doses of the antimetabolites up to 0.4 mg per rat did not alter the activity of the pentose phosphate shunt dehydrogenases; an increase in the dose from 4 up to 20 mg led to an inhibition of the enzymes. Possible biochemical alteration, occurring in response to various degree of transketolase inhibitions, are discussed.

The effects of thiamine and oxythiamine on glucoso-6-phosphate, fructoso-6-phosphate, ATP, aldopentoses, glucose and glycogen levels were studied in the liver of rats with different hormonal levels. Thiamine and insulin were shown to produce a similar effect on the liver levels of hexosomonophosphates, aldopentoses and glucose. Oxythiamine effects as compared to those elicited by thiamine and insulin are always contrasting. It is suggested that the insulin-like action of thiamine is consequent on the primary effect the vitamin exerts on the functional activity of the insular apparatus of the pancreas.

Thiamine pyrophosphokinase (EC 2.7.7.2) isolated from dry brewing yeast has been purified 20-fold with a 70% yield. Certain properties of the enzyme have been determined: pH and temperature optima, donor and acceptor concentrations, and relationship between the rate of cocarboxylase biosynthesis and the incubation time and the enzyme quantity. The effects of concentrations of bivalent metal ions Co2+, Mg2+ and Mn2+ on the rate of the enzymic reaction has been studied. A change in the pH optimum as a function of the nature of the ion-activator has been investigated. It has been shown that neopyrithiamin is a competitive inhibitor and oxythiamin inhibits the enzymic reaction insignificantly. Thiamine phosphate cannot be transformed into thiamine diphosphate by the purified enzyme.

A method for determination of pyruvate dehydrogenase activity in mitochondria and tissue homogenates was developed. The method was based on a spectrophotometric monitoring of p-nitroaniline acetylation under conditions required to ensure correct stoichiometric course of the reaction. In rat tissues absolute amounts of the enzyme activity, determined by the method, were found to be several-fold lower as compared with the values determined by the conventional ferricyanide method. Within 24 hrs after a single administration of hydroxythiamin into rats (400 mg per 1 kg of body weight) the pyruvate dehydrogenase activity was decreased 1.5-fold in heart (as estimated by the reaction of p-nitroaniline acetylation) and did not alter in liver tissue. While if the determinations were carried out by the method of terricyanide reduction the enzyme activity was decreas 7- and 2-fold, respectively, in heart and liver tissue. The data obtained suggest that hydroxythiamin impaired reactions of electron transport in tissues; on the other hand, the data obtained showed that the method for determination of the pyruvate dehydrogenase activity, based on the acetylation reaction, was more specific than the conventional method which involved measuring of ferricyanide reduction.

Regional cerebral acetylcholine (ACh) levels and utilization rate were assessed in vivo in rats rendered thiamin deficient using the thiamin antagonists pyrithiamin or oxythiamin. ACh levels were significantly reduced in all brain regions of pyrithiamin treated rats and in the medulla-pons and striatum of oxythiamin treated rats compared to controls. ACh utilization was significantly reduced in the midbrain, striatum and hippocampus of pyrithiamin treated rats, but was reduced only in the striatum of oxythiamin treated rats compared to controls. Thus, there are some reductions in ACh levels and utilization that are unique to pyrithiamin induced deficiency and as such are distinct from oxythiamin/undernutrition related reductions. Since only pyrithiamin produces neurological symptoms, its unique ACh effects may be related to these symptoms.

Oxythiamine injections to rats (400 mg/kg of body mass, subcutaneously, 2 injections with 48 hrs interval) caused 70% involution of thymus within 72 hrs after the first injection. The transketolase activity was inhibited by 70%, that of glucose-6-phosphate dehydrogenase by 15%, while the aldopentose level was decreased by 56% in the thymus. Inhibition of DNA and RNA synthesis was directly dependent on the dose and duration of the oxythiamine effect on the gland. Reduction of transketolase activity was accompanied by an adaptive; increase in glucose-6-phosphate dehydrogenase activity as well as by a decrease in levels of nicotinamide coenzymes (NAD, NADP) in spleen.

A rate of lipogenesis from 1-14C-acetate and 2-14C-pyruvate as well as total cholesterol, diene conjugates, malonic dialdehyde were increased in liver tissue of mice db/db strain with genetically determined diabetes, whereas content of free fatty acids was not distinctly altered despite of the marked elevation of corticosterone in blood plasma. Ratio of atherogenic lipoproteins VLDL and LDL was increased in blood plasma of these mice with diabetes. As oxythiamine, administered into the mice, exhibited hypolipidemic and antioxidant effects, TDP-dependent reactions appear to be among the factors responsible for deterioration of lipid metabolism in the diabetes.

The ratio of two substrates (pyruvate and lactate), activity of enzymes, responsible for their turnover in liver tissue, sceletal muscles and blood, were studied simultaneously in experiments on the effect of hydroxythiamin, which was administered at doses, causing a state of hypovitaminosis B1. Hydroxythiamin was shown to inhibit specifically the activity of thiamin-dependent enzymes (transketolase and pyruvate dehydrogenase). It affected also the metabolism of triose phosphates, pyruvate and lactate. At the same time, hydroxythiamin was responsible for changes in activities of other enzymes, participating in turnover of the substrates studied.

Kinetic analysis permitted to determine two sites of hydroxythiamine diphosphate binding in apotransketolase. The Ki values for these sites differed significantly: (7-22) X 10(-9) M and (13.0-19.7) X 10(-8) M. The rate of thiamine diphosphate turnover within holotransketolase in rat liver tissue was studied by the radioisotope method, using [14C]thiamine as a labeled precursor. The absolute values of half-substitution time and the rate constant of coenzyme degradation in the transketolase molecule are close to those for the protein moiety of the enzyme and are 153 hours and 0.108 days-1, respectively. In vivo rat liver transketolase exists in a substituted alpha-carbanion form. Within the holoenzyme molecule substitution of thiamine diphosphate for hydroxythiamine diphosphate does not influence the formation of an intermediate alpha-carbanion form of the enzyme.

The experiments on mice have shown that oxythiamine disulphide derivatives with the branched hydrocarbon chains are less toxic in the organism as compared to oxythiamine and corresponding disulphides with the unbranched hydrocarbon chains and also induce a more pronounced inhibition of transketolase in the liver and other tissues. It is found that under the effect of the above substances the recovery of enzymic activity is slower than in the case with the oxythiamine application.

Hydoxythiamine injections (400 mg/kg for 72 hours) resulted in the inhibition of transketolase activity and of 14C-orotic acid incorporation into RNA in rat liver tissue. At the same time, the decrease of RNA and phosphoribosylpyrophosphate levels has been observed, which suggests a regulation effect (via pentospohosphates) of hydroxythiamine on nucleotide and nucleic acid metabolism.

Levels of thiamin and its derivatives in brain, heart and liver were determined in control, thiamin-deficient, pyrithiamin-treated and oxythiamin-treated rats at various times after the start of treatment. With 10 micrograms of thiamin/100 g/day subcutaneously, rats rapidly lost thiamin from the tissues until growth maintenance levels were reached. Oxythiamin had a minimal further effect on tissue thiamin while pyrithiamin caused further marked reduction. With higher levels of thiamin intake, tissue thiamin levels increased with intakes of up to 150-200 micrograms/100 g/day after which they did not show any further significant increase. Levels of pyrithiamin above 50 microgram/100 g/day for 20 days had only minimal further effect upon tissue thiamin levels over that with 50 micrograms.