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Reduction of ferricyanide by thiamine or thiamine pyrophosphate.

The colorimetric assay for the activity of pyruvate dehydrogenase (EC 1.2.4.1) developed by Itokawa is based on a coupled reduction of ferricyanide and the formation of Prussian blue (Brian Research 94, 475-484). In this assay system, we found that the coenzyme, thiamine pyrophosphate, itself reduced ferricyanide independent of both the enzyme and the substrate. Similar effect was also observed with thiamine, but not with thiochrome. The reduction of ferricyanide by thiamine or thiamine pyrophosphate was blocked by trichloroacetic acid. Measurement of the activity of purified pyruvate dehydrogenase based on monitoring the reduction of ferricyanide as described by Schwartz et al (Biochem. Biophys. Res. Commun. 31, 495-500) has been widely used. Our findings clearly disprove the assumption that reduction of ferricyanide is dependent on pyruvate dehydrogenase and invalidate assays for thiamine-dependent dehydrogenases based on the reduction of ferricyanide.

Animals↗

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals.

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.

Amino Acid Sequence↗

thiBPQ encodes an ABC transporter required for transport of thiamine and thiamine pyrophosphate in Salmonella typhimurium.

In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli.

ATP-Binding Cassette Transporters↗

[The content of thiamine, its coenzyme form and activity of thiamine diphosphate-dependent enzymes in ontogenesis].

In experiments on rats of different age it is shown that the lowest content of thiamine and its phosphoric ethers in the liver is typical of the early periods of embryonal development and the highest one of the first days after birth. A direct dependence is established between the content of thiamine, its coenzyme form, enzyme of thiamine diphosphate synthesis (thiamine pyrophosphokinase) and thiamine diphosphate-containing enzyme (transketolase) in the rat liver in different periods of ontogenesis.

Animals↗

Comparison of three methods of thiamine supplementation by measurement of urinary thiamine excretion in sheep.

The relative efficacy of thiamine supplementation to sheep by injection, subcutaneous implant and orally administered protected thiamine bolus was compared in two experiments using a grass and hay ration and a ration containing bracken rhizomes to induce thiamine deficiency. In both experiments, urinary excretion of thiamine was significantly higher in supplemented sheep than in the controls, and in sheep supplemented by injection than in sheep supplemented by implant or protected boluses. Thiamine excretion was lower in sheep fed the ration containing bracken rhizomes than in sheep fed the grass ration.

Administration, Oral↗

Thiamine deficiency in black male hostel-dwellers. The need for thiamine supplementation of sorghum beer.

Some indices of nutrition have been examined in hostel- and non-hostel-dwelling groups of industrially employed black males. Hostel-dwellers in the large metropolitan areas have to prepare their own food and many are accustomed to excessive alcohol intake, especially of sorghum beer. In the two groups studied, blood levels of vitamin B12, folate, pyridoxal and albumin were similar, but erythrocyte thiamine levels were significantly lower in the hostel-dwellers. Although the proportion of subjects with elevated levels of gamma-glutamyltransferase, an index of alcoholic liver disease, was similar in the two groups, thiamine-deficient hostel-dwellers had a greater proportion of elevated values suggesting that thiamine deficiency was related to both inadequate diet and excessive alcohol consumption. Fortification of sorghum beer with thiamine might prevent or reduce thiamine deficiency in this group. The cost would not materially affect the price of the beer.

Adult↗

Metabolism and biological activity of a thiamine new salt. I. Urinary excretion and tissue content of vitamin B1 in rats treated with thiamine chloride ester monophosphate of Bis-glucosamine.

Research has been carried out on the metabolism of thiamine chloride ester monophosphate of bis-D-glucosamine (TCMPG) evaluating the urinary excretion, the blood and the hepatic content of thiamine in the rat after intraperitoneal injection of this compound. The results obtained have shown a greater retention of vitamin B1 when it is administered as glucosamine salt. In fact, the amount of thiamine excreted by the rats which have received the TCMPG is lower while the blood and liver levels of vitamin are higher, compared with the control rats treated with thiamine monophosphate only, or with thiamine and glucosamine. These results permit the conclusion that glucosamine increases the utilization of the vitamin and its penetration into the cells.

Animals↗

Determination of thiamine and thiamine phosphates in excitable tissues as thiochrome derivatives by reversed-phase high-performance liquid chromatography on octadecyl silica.

The analysis of thiamine and thiamine phosphates by high-performance liquid chromatography owes its high sensitivity to the fluorescent derivatives or thiochromes obtained by chemical oxidation in alkaline medium. The possibility of performing precolumn oxidation with potassium ferricyanide instead of using the hazardous cyanogen bromide has been investigated. The derivatization step has been optimized with respect to the following parameters: concentration of alkali and oxidant, presence of methanol and stability of the thiochromes . A gradient separation with 25 mM phosphate buffer (pH 8.4) and methanol as mobile phase components and an octadecyl silica column as stationary phase has been set up. The analytical run takes 14 min with the following elution order: thiochrome triphosphate, thiochrome pyrophosphate, thiochrome monophosphate and thiochrome. The minimum detectable amount is 0.05 pmol. The method was found suitable for the determination of thiamine compounds in excitable tissues such as nerves and electric organs as well as in proteins extracted from membranes of these organs. It may be useful to study the role of thiamine in the electrical activity of these tissues at the molecular level.

Animals↗

[Growth kinetics of thiamine requiring Candida lipolytica during thiamine limitation: the existence of a linear growth phase].

The growth kinetics of batch cultures of the thiamine requiring Candida lipolytica 695 was investigated. It was established that a phase of logarithmic growth is followed by a linear growth phase. The time length of the linear phase and the relative increasing of the biomass during the linear phase are nearly constant and independend of the concentration of thiamine. The existence of the linear phase is discussed in connection with a constant activity of one of the two 2-ketoacid dehydrogenases as a bottle neck enzyme which needs thiamine pyrophosphate as coenzyme. Critical intracellular thiamine concentrations were calculated necessary for transition from the logarithmic to the linear phase and from the logarithmic to the stationary growth phase. Without the existence of the second critical thiamine concentration the linear growth would continue infinitely.

Candida↗

Thiamine content and turnover rates of some rat nervous regions, using labeled thiamine as a tracer.

The content of total thiamine radioactivity in some nervous structures and liver of the rat was determined in a steady state condition, using [thiazole-2-14C]thiamine as a tracer. The contents were analyzed by a mamillary type compartmental model which enabled us to calculate the influx and efflux fractional rate constants, turnover times, turnover rates and relative accuracy. Total thiamine turnover rates of the central nervous system regions were found to be ordered in the following sequence: cerebellum (0.55 microgram/g.h) greater than medullar and pons greater than spinal cord and hypothalamus greater than midbrain (plus thalamic area) and corpus striatum greater than cerebral cortex (0.16 microgram/g.h). Sciatic nerve turnover rate was 0.58 microgram/g.h. The turnover times were mainly between 5 and 10 h (range 2.4--16.4 h). The influx rate constants could be ordered as follows: cerebellum greater than hypothalamus, pons and medulla greater than corpus striatum, spinal cord, midbrain (plus thalamic area) and sciatic nerve greater than cerebral cortex. The results show in general a good agreement between turnover rate values and brain regional sensitivity to thiamine deficiency, the most vulnerable areas to thiamine depletion being those with the highest turnover rates.

Animals↗

The effect of thiamin supplementation on the thiamin status of turkey poults with ethanol-induced cardiomegaly.

1. Turkey poults were treated with graded doses (0-5% v/v) of ethanol for 6 weeks. Some birds were concomitantly injected intramuscularly with thiamin. 2. Erythrocyte transketolase (TK) activity was significantly decreased and the erythrocyte pyrophosphate (TPP) effect and blood concentrations of lactate and pyruvate were statistically greater in all ethanol-treated poults by 42 days. 3. Thiamin restored TK activity to normal levels and decreased the TPP effect and blood concentrations of lactate and pyruvate in ethanol poults. 4. Thiamin mitigated ethanol-induced cardiomyopathy in some 4-5% ethanol poults. 5. Thiamin had no significant effect on any of these parameters in control birds treated with thiamin.

Animals↗

Effect of a high sulfur diet on rumen microbial activity and rumen thiamine status in sheep receiving a semi-synthetic, thiamine-free diet.

A semi-synthetic thiamine-free diet was used on weaned lambs to test the effect of a high sulfur level on the rumen, microbial activity and on the microbial production of thiamine. In vivo and in vitro kinetic studies, as well as the determination of the thiamine concentrations and thiaminase activity in the rumen, were performed during the 16 week experiment. A high sulfur level (0.6%) in the diet, in comparison with a normal sulfur level (0.2%), did not modify the microbial activity of the rumen with the exception of a slightly retarded decrease in the volatile fatty acid (VFA) rumen concentration. The rumen thiamine level and the thiaminase activity were not modified by the dietary sulfur level. In contrast, the rate of sulfate reduction into sulfide in the rumen increased progressively with the 0.6% sulfur diet. In conclusion, a high sulfur level (0.6%) in the diet of sheep did not modify the thiamine status of the rumen. It strongly increased the production of sulfides but an adaptation period of several weeks was required by the rumen microflora to reduce sulfate at a maximal rate.

Animals↗

The stability of thiamine and thiamine tetrahydrofurfuryl disulfide added to table wines.

Both thiamine hydrochloride and thiamine tetrahydrofurfuryl disulfide were added separately to table wines at concentrations equivalent to 0.3 and 1.5 microgram of free thiamine per kJ of caloric energy. The resultant mean increments in thiamine activity, measured by Lactobacillus fermenti microbiological assay after 21 months of storage, were in the range 55 to 103% of the added vitamin, indicative of high bioavailability of thiamine from this source.

Biological Assay↗

[Phosphorylation of some thiamine analogs by yeast thiamine pyrophosphokinase].

A rapid efficient method of separation of the thiamine pyrophosphokinase reaction products (ATP: thiamine pyrophosphotransferase) on the column packed with DEAE-Sephadex A-25 and their subsequent identification by direct spectrophotometry is suggested. Phosphorylation of some thiamine analogs substituted at the second position of the pyrimidine ring was studied. It was shown that in addition to thiamine, the enzyme transfers the pyrophosphate group to some of its derivatives. The vitamin analogs devoid of quaternary nitrogen in the thiazole cycle, do not form pyrophosphate ethers (thus being unable to act as substrates), whereas 2'-phenoxythiamine, 2'-methoxythiamine and especially 2'-phenylthiamine are phosphorylated at a greater rate than does the "true" substrate, thiamine, under similar conditions.

Phosphotransferases↗

Kinetics of thiamine-polyphenol interactions and mechanism of thiamine disulphide formation.

Interactions between thiamine and antithiamine polyphenolic compounds exhibit similar pseudo-first order rate constants and Arrhenius activation energy. Probably, hydroxide ions open the thiazole moiety of thiamine to give a sulphydryl derivative existing in equilibrium with thiamine. Ionization and air oxidation of the polyphenols yield oxidized forms which may be quinones and others. Catalytic and rapid oxidation by the quinones lead to formation of thiamine disulphide from the sulphydryl derivative shifting the equilibrium away from thiamine.

3,4-Dihydroxyphenylacetic Acid↗

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions.

Culture of neuroblastoma cells in the presence of low thiamine concentration (16 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 microM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional.

Electron Transport↗

[Cytosolic thiamine triphosphatase from bovine brain. 2. Interaction of thiamine triphosphate ester with the enzyme].

The analysis of the steady-state kinetics of the thiamine triphosphate ester hydrolysis reaction catalyzed by homogeneous thiamine triphosphatase (EC 3.6.1.28; thiamine triphosphate phosphohydrolase) from bovine brain enables us to suggest, that the ThTP binding to the catalytic site of the ThTPase active centre takes place by the phosphate radical. The correct orientation of the substrate molecule occurs by means of the contact of the thiamine component. The crucial role in this process belong to the amino group of the pyrimidine ring and hydrophobic forces. The quaternary nitrogen of thiazole is important for the hydrolytic splitting of the substrate. The hydrolysis of thiamine triphosphate ester occurs through the formation of the ternary enzyme-substrate complex, with the Mg2+ and Mg.ThTP adding being random.

Animals↗