Thiamine Diphosphate in Whole Blood, Thiamine and Thiamine Monophosphate in Breast-Milk in a Refugee Population

BACKGROUND: The provision of high doses of thiamine may prevent thiamine deficiency in the post-partum period of displaced persons. METHODOLOGY/PRINCIPAL FINDINGS: The study aimed to evaluate a supplementation regimen of thiamine mononitrate (100 mg daily) at the antenatal clinics in Maela refugee camp. Women were enrolled during antenatal care and followed after delivery. Samples were collected at 12 weeks post partum. Thiamine diphosphate (TDP) in whole blood and thiamine in breast-milk of 636 lactating women were measured. Thiamine in breast-milk consisted of thiamine monophosphate (TMP) in addition to thiamine, with a mean TMP to total thiamine ratio of 63%. Mean whole blood TDP (130 nmol/L) and total thiamine in breast-milk (755 nmol/L) were within the upper range reported for well-nourished women. The prevalence of women with low whole blood TDP (<65 nmol/L) was 5% and with deficient breast-milk total thiamine (<300 nmol/L) was 4%. Whole blood TDP predicted both breast-milk thiamine and TMP (R(2) = 0.36 and 0.10, p<0.001). A ratio of TMP to total thiamine ≥63% was associated with a 7.5 and 4-fold higher risk of low whole blood TDP and deficient total breast-milk thiamine, respectively. Routine provision of daily 100 mg of thiamine mononitrate post-partum compared to the previous weekly 10 mg of thiamine hydrochloride resulted in significantly higher total thiamine in breast-milk. CONCLUSIONS/SIGNIFICANCE: Thiamine supplementation for lactating women in Maela refugee camp is effective and should be continued. TMP and its ratio to total thiamine in breast-milk, reported for the first time in this study, provided useful information on thiamine status and should be included in future studies of breast-milk thiamine

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups

Identification of Inhibitors against Mycobacterium tuberculosis Thiamin Phosphate Synthase, an Important Target for the Development of Anti-TB Drugs

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6–9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M.tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M.tuberculosis. In this study, a comparative homology model of M.tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC50 values ranging from 20 – 100 µg/ml and two of these exhibited weak inhibition of M.tuberculosis growth with MIC99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M.tuberculosis growth with an MIC99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M.tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed

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

The effect of thiamin tetrahydrofurfuryl disulfide on behavior of juvenile DBA/2J mice

Due to genetic defects or illness some individuals require higher amounts of thiamin than are typically provided by the diet. Lipid-soluble thiamin precursors can achieve high blood levels of thiamin and result in increased concentrations in the central nervous system. High intakes of thiamin have been reported as beneficial in children with autism and attention deficit/hyperactivity disorder. The current study examined the effect of thiamin tetrahydrofurfuryl disulfide (TTFD), a lipophilic precursor, on behavior in the juvenile male DBA/2J mouse. Mice given by oral gavage deionized water or deionized water providing 100mg or 340mg TTFD/kg body weight daily for 17days, starting at postnatal day 18, were tested for effects on operant learning, social interaction, general activity level, and prepulse inhibition of acoustic startle, as well as effects on growth and select organ weights. Results indicate lower activity and altered social interaction at both treatment levels and decreased acoustic startle at the 100mg/kg level. Compared to controls, percent weight gain was lower in the TTFD-treatment groups, but percent body length increase was not affected by TTFD treatment. TTFD treatment did not influence percent organ weights as percentage of body weights. TTFD treatment resulted in increased whole brain thiamin concentrations. These results support the concept that lipophilic thiamin precursors provided during early development can affect a number of behavioral parameters. In clinical trials with children with behavior disorders, attention should be given to preventing possible adverse gastrointestinal irritant effects associated with TTFD therapy

Structural Basis for the Catalytic Mechanism of Mammalian 25-kDa Thiamine Triphosphatase*S⃞

Mammalian soluble thiamine triphosphatase (ThTPase) is a 25-kDa cytosolic enzyme that specifically catalyzes the conversion of thiamine triphosphate (ThTP) to thiamine diphosphate and has an absolute requirement for divalent cations. We have investigated the kinetic properties of recombinant mouse thiamine triphosphatase (mThTPase) and determined its solution structure by NMR spectroscopy. Residues responsible for binding Mg2+ and ThTP were determined from NMR titration experiments. The binding of Mg2+ induced only a minor local conformational change, whereas ThTP binding was found to cause a more global conformational change. We derived a structural model for the mThTPase·ThTP·Mg2+ ternary complex and concluded from this that whereas free mThTPase has an open cleft fold, the enzyme in the ternary complex adopts a tunnel fold. Our results provide a functional rationale for a number of conserved residues and suggest an essential role for Mg2+ in catalysis. We propose a mechanism underlying the high substrate specificity of mThTPase and discuss the possible role of water molecules in enzymatic catalysis

At the crossroad of thiamine degradation and biosynthesis

The physiological significance of thiaminase II has escaped our understanding for many years. The recent discovery of a new thiamine salvage pathway shows that this enzyme is involved in the regeneration of precursors for thiamine biosynthesis