Thiaminylated adenine nucleotides. Chemical synthesis, structural characterization and natural occurrence.

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

Thiamin phosphates in egg yolk granules and plasma of regular and embryonated eggs of hens and in five- and seven-day-old embryos.

The thiamin (TH), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) content was analyzed in yolk plasma and yolk granules of unincubated and incubated chicken eggs and in 5- and 7-day-old chick embryos. Analytes were extracted from samples weighing approximately 1 g, with trichloroacetic acid (0.4 g/L), isolated with solid-phase extraction, and determined with HPLC. Before solid-phase extraction with a C-18 cartridge and a strong anion exchange cartridge, analytes were converted to thiochromes with cyanogen bromide. For HPLC, a C-8 pre-column and a C-8 column connected to a NH(2) column were used with a fluorescence detector. No thiamin phosphates were found in egg yolk plasma or granules in native and embryonated eggs. The TH content in DM was 3,400 +/- 700 ng/g for yolk plasma, 1,500 +/- 140 ng/g for yolk granules of unincubated eggs, and 4,400 +/- 1,100 ng/g for yolk plasma and 2,100 +/- 450 ng/g for yolk granules of 5-d embryonated eggs. In chick embryos, TH as well as thiamin phosphates were found. The mean content in 5-d-old embryos was 1,200 +/- 400 ng of TH and 8,000 +/- 1,000 ng of TDP/g of DM. In 7-d-old embryos, the content of TH was 900 +/- 200 ng, TMP was 400 +/- 200 ng, and TDP was 5,500 +/- 1,300 ng/g of DM. Thiamin triphosphate was not detected, and the minimum detectable limit for T was 0.3 ng/mL, for TMP 0.5 ng/mL, and for TDP 0.9 ng/mL, respectively. Recovery was 71% for TMP and 64% TDP in plasma and 58% TMP and 47% TDP in granules. It was demonstrated that only TH is present in egg yolk fractions in native and embryonated eggs of hens and that thiamin phosphates can be found in 5- and 7-d-old embryos, which indicates that they are synthesized by the chick embryo. According to its assumed unique role in highly specialized tissues, TTP was not found in early embryos.

Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

Simultaneous detection of thiamine and its phosphate esters from microalgae by HPLC.

We present an easy and sensitive method for measuring thiamine and its phosphate esters in small biological samples of microalgae (Amphidinium carterae Hulburt and Nitzschia microcephala Grun). The method consists of extraction of thiamine and its derivatives in acid solution, followed by liquid chromatography with fluorescence detection. The detection limit is as low as 15 fmol of thiamine. For comparison to microalgae, the method has been applied to evaluate thiamine levels in the crustacean Artemia salina Leach and is suitable for nutritional studies of the food web of the Baltic salmon, which suffers from thiamine deficiency. This method of HPLC analysis can be readily utilized to follow uptake and interconversion of thiamine and its phosphate esters in many micro- and macroalgae.

Thiamine, thiamine phosphates, and their metabolizing enzymes in human brain.

Total thiamine (the sum of thiamine and its phosphate esters) concentrations are two- to fourfold lower in human brain than in the brain of other mammals. There were no differences in the total thiamine content between biopsied and autopsied human brain, except that in the latter, thiamine triphosphate was undetectable. The main thiamine phosphate-metabolizing enzymes could be detected in autopsied brain, and the kinetic parameters were comparable to those reported in other species. Thiamine diphosphate levels were lowest in hippocampus (15 +/- 4 pmol/mg of protein) and highest in mammillary bodies (24 +/- 4 pmol/mg of protein). Maximal levels of thiamine and its phosphate ester were found to be present at birth. In parietal cortex and globus pallidus, mean levels of total thiamine in the oldest age group (77-103 years) were, respectively, 21 and 26% lower than those in the middle age group (40-55 years). Unlike cerebral cortex, the globus pallidus showed a sharp drop in thiamine diphosphate levels during infancy, with concentrations in the oldest group being only approximately 50% of the levels present during the first 4 months of life. These data, consistent with previous observations conducted in blood, suggest a tendency toward decreased thiamine status in older people.

Subcellular localization and compartmentation of thiamine derivatives in rat brain.

The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.

Immunohistochemical demonstration of a new thiamine diphosphate-binding protein in the rat digestive tract.

We purified a new thiamine diphosphate-binding protein (ThDP-BP), produced an antiserum against it, and examined its immunohistochemical distribution in the rat gastrointestinal tract using the avidin-biotin complex method. Positive staining for ThDP-BP was found in the epithelial glands of the stomach, small intestine and large intestine, and in the nuclei of hepatocytes. Measurement of the content of thiamine and its phosphate esters in extracts from the gastric and intestinal mucosa also indicated the presence of ThDP-BP in the stomach and intestinal mucosa. ThDP-BP may be useful for investigating the absorption and metabolism of the thiamine in metabolic disorders.

Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method.

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.

Tissue difference in cellular localization of thiamine phosphate esters.

1. Thiamine phosphate levels were determined in the soluble and particulate fractions of various rat tissues. 2. There was marked tissue difference in the cellular localization of thiamine phosphates. 3. Brain thiamine triphosphate was localized only in the particulate fraction, whereas skeletal muscle thiamine triphosphate was in the soluble fraction as a protein-unbound form.

Difference in thiamine metabolism between extensor digitorum longus and soleus muscles.

1. Thiamine diphosphate level was higher in soleus muscle than in extensor digitorum longus muscle in various animals, whereas thiamine triphosphate level was less in the former muscle than in the latter except for mouse. 2. 2-Oxoglutarate dehydrogenase, transketolase and thiamine pyrophosphokinase activities were higher in soleus muscle than in extensor digitorum longus in rat and guinea pig. 3. The differences between rat two muscle phenotypes in thiamine diphosphate, but not thiamine triphosphate, level and the thiamine-related enzyme activities disappeared after denervation. 4. Tenotomy had little effect on thiamine phosphate levels and the thiamine-related enzyme activities in rat skeletal muscles.

Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues.

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.

Thiamine triphosphate and membrane-associated thiamine phosphatases in the electric organ of Electrophorus electricus.

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.

Content of thiamin phosphate esters in mammalian tissues--an extremely high concentration of thiamin triphosphate in pig skeletal muscle.

In relation to a high activity of thiamin diphosphate (TDP) kinase (Koyama, S. et al. (1985) Biochem. Int. 11, 371-380) in the skeletal muscle of pigs and guinea pigs, the content of thiamin phosphate esters in tissues of these animals has been determined by the method of high-performance liquid chromatography. An extremely high concentration of thiamin triphosphate (TTP), 69.2% of the total thiamin (26.1 nmol/g wet weight), was detected in adult pig skeletal muscles. One extreme case contained TTP as 88.7% of the total thiamin (19.6 nmol/g wet weight). TTP in pig skeletal muscle was found solely in cytosol fraction. This is the first report showing an unusually high level of TTP in mammals and may give a clue as to the physiological functions of TTP.

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.

An improved method for the electrophoretic separation and fluorometric determination of thiamine and its phosphates in animal tissues.

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.

Concentrations of thiamine and its phosphate esters in rat tissues determined by high-performance liquid chromatography.

Thiamine and its phosphate esters in rat tissues were determined by high-performances liquid chromatography after conversion to thiochrome and its phosphate esters as described previously (Ishii, K. et al. (1979) Anal. Biochem., 97, 191--195). Total thiamine concentrations determined in the brain, liver, heart and kidney were 6.39, 25.5, 15.7, and 14.9 nmol/g wet weight, respectively. In all the tissues tested, thiamine, its monophosphate, pyrophosphate, and triphosphate were found to be present at the rates of 1.2--7.7, 5.9--11.0, 85.7--90.0, and 0.7--1.6%, respectively. Although these percentages of thiamine phosphates were similar to those reported previously using different procedures, the percentage of thiamine triphosphate content in rat tissues was found to be fairly lower than that reported. The time required for the whole process of analysis averaged 90 min after isolation of the organs from the animal.