Thiamine Mono Research Articles

VITAMIN B1 BIOLOGICAL FUNCTION IMPLEMENTATION IN THE BLOOD OF PATIENTS WITH STOMACH CANCER UNDER SURGICAL INTERVENTION

Background. The probability of undergoing surgery always predetermines the state of stress in a person; therefore, it is advisable to search for ways to optimize and (or) reduce this unfavourable effect. Objective. To find out the mechanism of vitamin B1 antistress activity during surgery. Material and methods. Metabolism intensity was investigated on donors’ blood lysates (n = 19) and those of patients with stage III stomach cancer (n = 64), referred to an elective surgery, aged 51-70. The blood was taken from the cubital vein three days before the operation, after premedication, during the most traumatic moment of the operation, after extubation, as well as on the first and third days of the postoperative period. The surgery was performed under combined multicomponent anesthesia using nitric oxide, sodium hydroxybutyrate, and epidural block. Thiamine and thiamine diphosphate kinase activities were assessed by the concentration of the formed thiamine di- and triphosphates of the vitamin. The activities of thiamine mono-, di- and triphosphatases were determined by the release of inorganic phosphate. The concentration of inorganic phosphate was recorded colorimetrically. The content of B1 and its derivatives in the blood was determined by the method of ion-pair reversed-phase HPLC. Results. There has been observed an increased content of thiamine monophosphate and that of free thiamine in the blood of donors and patients with stomach cancer. The registered rate of the hydrolytic thiamine monophosphatase reaction is not high. At the stages of premedication and maximum trauma of surgical exposure, the concentration of monophosphoric ester rapidly decreases alongside with monophosphatase activation. Therefore, the thiamine monophosphate hydrolysis is the rate-limiting link of vitamin B1 metabolism. The level of free thiamine remains persistently increased at all stages of surgical treatment. Thiamine monophosphatase activity is manifested at two pH optima – of 6.0 and 9.0. Thiamine monophosphate hydrolysis at pH of 9.0 is catalyzed by alkaline phosphatase. At pH of 6.0, in addition to thiamine monophosphoric ester, the enzyme hydrolyzes only p-nitrophenyl phosphate, flavin mononucleotide and phosphotyrosine, that allows it to be classified as hepatic acid phosphatase. The noted changes in B1 metabolism under stress concern mainly non-coenzyme forms - thiamine mono-, triphosphate, and free thiamine, which are used at the stages of thiol reduction as important components of insulin synthesis. Conclusions. The use of vitamin B1 allows to optimize the development of the stress response at all stages of surgical treatment. Its protective effect is achieved through the activation of the insulin-synthetic function of the pancreas, which increases the level of immunoreactive insulin in the blood. The formation of the most favorable physiological conditions for insulin synthesis provides an increased background of free thiamine, which is created due to the hydrolysis of noncoenzyme forms of the vitamin. The relationship between thiamine metabolism and B2 exchange and regulation of intracellular signaling pathways has been traced.

Thiamine Mono- and Diphosphate Phosphatases in Bovine Brain Synaptosomes

Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.

Thiamine status and lactate concentration in sepsis: A prospective observational study.

Thiamine is an essential co-factor for aerobic metabolism. Both thiamine deficiency and sepsis may be associated with hyperlactatemia and hypotension. We assessed the relationship between thiamine compounds, lactate concentrations and clinical outcomes in septic patients.We undertook a prospective observational single-center study. Erythrocyte levels of total thiamine, free thiamine, thiamine mono, di and triphosphate (TMP, TDP, and TTP respectively), the erythrocyte transketolase activity (ETKA) and the effect of thiamine diphosphate on ETKA were measured in septic patients by high performance liquid chromatography and correlated with arterial lactate. Vital status at the end of intensive care unit stay was recorded.Overall, 28 patients suffering from sepsis were included. Median (interquartile range [IQR]) age was 60 [44-77.3] years, 15 (53.6%) patients were male, median [IQR] simplified acute physiology score II was 40 [27-50]. There was no correlation between total thiamine and lactate levels (P = .33). There was no correlation between free thiamine (P = .81), TMP (P = .71), TDP (P = .31), TTP (P = .86), and lactate levels in our population. There was no correlation between ETKA (P = .58) or the effect of TDP on ETKA (P = .40) and lactate concentration. Total thiamine and TDP concentration were significantly higher in intensive care unit (ICU) survivors than in nonsurvivors (P = .03 and P = .03). The effect of TDP on ETKA was significantly higher in nonsurvivors compared to survivors (P = .04).We found no correlation between thiamine compounds and lactate concentration in sepsis. Thiamine deficiency in sepsis may be associated with ICU-mortality.

Oxovanadium (IV) complexes of phosphates of biological relevance: NAD +, NADP + and thiamine mono- and diphosphate

Complex formation between VO(IV) and organic phosphates, NAD + (β-nicotinamide adenine dinucleotide), NADP + (β-nicotinamide adenine dinucleotide phosphate), TDP (thiamine diphosphate) and TMP (thiamine monophosphate), has been studied by combined application of pH-potentiometric and spectroscopic (EPR and electronic absorption) techniques. The species formed in aqueous solution have been examined as a function of pH and ligand-to-metal molar ratio. Different behaviour was found for the ligands. NAD + acts as a diphosphate chelator in the acidic pH range, whereas in basic media the binding of the metal ion at the deprotonated hydroxyls of the ribose moieties is strongly favoured. The presence of a basic monophosphate group on the ribose residue of NADP + affects the speciation processes and the ligand behaves as a simple monophosphate in the acidic pH range. Also different is the behaviour of TDP and TMP. While the former behaves as a simple diphosphate ligand, the latter gives rise to monophosphate complexes. In particular, TMP yields stable dinuclear species in which the ligand acts as a bridge between two VO (IV) ions by coordinating through both phosphate and N(1′) donor groups.

Thiamine pyrophosphate-dependent and thiamine metabolizing enzymes in the deafferented cerebellum and in the intact cerebral cortex of rat.

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 compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells

Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier ( K M = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [ 14C]thiamine or [ 14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1–2 h. When preloading the cells with [ 14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.

High-performance liquid chromatographic determination of total thiamine in human plasma for oral bioavailability studies

AbstractA high-performance liquid chromatographic method for the determination of total thiamine in plasma was developed for a relative bioavailability comparison of two oral thiamine preparations. After separation of thiamine mono- and diphosphates with the phosphatase enzyme, the total thiamine was subjected to reversed-phase high-performance liquid chromatography, postcolumn oxidized to thiochrome with K3[Fe(CN)6], and detected by fluorescence. In the bioavailability study, 16 human subjects were put on a low-thiamine diet for 3 days. On the 2nd and 3rd days, 14 blood samples per subject and day were taken at the same times each day. Drug administration did not take place until the 3rd day. After deduction of the native concentrations of total thiamine detected on the 2nd day (mean value of ˜ 7 ng/mL), the post-treatment pharmacokinetic parameters were determined (two different preparations, each with 200mg of thiamine - HCI).

EFFECT OF ETHANOL ADMINISTRATION ON THE IN VIVO KINETICS OF THIAMINE PHOSPHORYLATION AND DEPHOSPHORYLATION IN DIFFERENT ORGANS. I. CHRONIC EFFECTS

The effects of chronic ethanol administration on different steps in the metabolism of thiamine (T), thiamine mono- (TMP) and pyrophosphate (TPP) were determined in vivo in the liver, kidney, heart, skeletal muscle and small intestinal mucosa. The radioactivity of T and its phosphoesters was measured in plasma and in the selected organs under steady-state conditions and at fixed time intervals (0.25-240 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in rats chronically (35 days) ethanol-treated (daily dose of 4.7 g kg-1 body wt by gastric gavage). Two types of controls were used: pair-fed rats treated with a sucrose solution isoenergetic with ethanol, and water-treated rats. A nutritionally adequate diet, which supplied an excess of thiamine, was given to the rats, producing a virtually steady content of thiamine compounds in the tissues. The analytical data obtained were elaborated using appropriate compartmental mathematical models, which allowed the fractional rate constants, turnover rates and turnover times to be calculated. Alterations in thiamine metabolism were modest and differed according to the organs. The most widespread modification was to facilitate the entry of T (small intestine, kidney and heart) or TMP (small intestine and kidney), while no significant change of T and TMP release was seen. Sucrose had minimal effect in both steps. Enzymatic transformations of thiamine were likewise marginally affected. A general trend toward a slower T pyrophosphorylation and a faster T phosphate dephosphorylation was observed in the small intestine, kidney, heart and liver. Skeletal muscle was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

High-voltage electron microscopic observation of golgi complex in the rat small ganglion cell. Thiamine metabolism.

Thiamine mono- and pyrophosphatase (TMPase, TPPase) activities were demonstrated in the rat small ganglion cell under high-voltage electron microscopy. From a series of micrographs of the same field tilted in increments, a trans tubular network (TTN) consisting of looping and anastomosing tubules was found; this network showed relatively higher intense TMPase activity, and exhibited connections with TMPase-positive vesicle-like bodies. TPPase activity was localized in the trans Golgi saccules, and the TPPase-positive transmost saccule had a structural resemblance to the TTN. These results indicate that the TTN plays an important role in thiamine metabolism.

Distribution of thiamine, thiamine phosphates, and thiamine metabolizing enzymes in neuronal and glial cell enriched fractions of rat brain.

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.

Nervous tissue thiamine metabolism in vivo. III. Influence of ethanol intake on the dynamics of thiamine and its phosphoesters in different brain regions and sciatic nerve of the rat

The effects of chronic ethanol administration on different steps of the metabolism of thiamine (T), thiamine mono- (TMP) and thiamine pyrophosphate (TPP) in the cerebellum, brainstem, cerebral cortex and sciatic nerve were evaluated in vivo. The radioactivity of T and its phosphoesters was determined in plasma and in the selected nervous structures under steady-state conditions and at fixed time intervals (0.5–192 h) after an i.p. injection of [ 14C]T (30 μg: 1.25 μCi) to rats chronically (35 days) ethanol-treated (daily dose of 4.7 g· kg −1 b.wt. by gastric gavage) and pair-fed controls similarly treated with a sucrose solution isoenergetic with ethanol. All rats were given a nutritionally adequate diet supplying an excess of thiamine, which produced a virtually steady content of thiamine compounds in the tissues. By using a compartmental mathematical model, fractional rate constants, turnover rates and turnover times were calculated. Ethanol caused a reduction of the rate of thiamine compound enzymatic transformations (T phosphorylation to TPP, TPP dephosphorylation to TMP and TMP to T), and a facilitation of the regional uptake of T and TMP, associated to a less relevant influence on their release. Isoenergetic sucrose prevailingly caused an increased rate of thiamine metabolic steps (except phosphorylation in the brainstem and cerebral cortex), with negligible modifications of T and TMP uptake and release. Thus the changes induced by ethanol were virtually opposite to those caused by sucrose.

Nervous tissue thiamine metabolism in vivo. II. Thiamine and its phosphoesters dynamics in different brain regions and sciatic nerve of the rat

Different steps of the metabolism of thiamine (T), thiamine mono- (TMP), pyro- (TPP) and triphosphate (TTP) in the cerebellum, brainstem, cerebral cortex and the sciatic nerve were evaluated in the rat in vivo. The radioactivity of T and its phosphoesters was determined at fixed time intervals (0.5–240 h) after an intraperitoneal injection of [ 14C]T (30μg:1.25 μCi), under steady state conditions. The dynamics of thiamine compounds was evaluated using a compartmental mathematical model that allowed the fractional rate constants (FRC), turnover rates (TR) and turnover times to be calculated. The phosphorylation of T to TPP and the dephosphorylations of TPP to TMP and TMP to T could be estimated in all the structures investigated. Their turnover rates were found to be ordered in the sequence: cerebellum > brainstem > cerebral cortex > sciatic nerve. The transphosphorylation of TPP to TTP was so small that it could not be determined in a reliable way. Regional differences were found both in the rate and in the composition of T and TMP mixture released from nervous structures. The shortest turnover time of TPP was found in the cerebellum, while the sciatic nerve exhibited the fastest renewal of T and TMP. In all the structures investigated TPP had a rather short turnover time, suggesting that its function might be associated to a rapid conversion into chemically different forms. The possible relationships between the rates of turnover of T compounds are the sensitivity of the nervous structures to T deficiency are discussed.

Formation of a thiamine artifact during chromatography: A single column procedure for the separation of thiamine and the thiamine mono-, di-, and triphosphate esters

Chromatography of a preparation of [ 14C]thiamine pyrophosphate (thiamine-PP) on Dowex 1X8 in the formate form produced an unexpected peak, X-1, which was eluted just prior to the thiamine-PP peak. An ammonium formate, pH 4.5, gradient was the eluant. Rechromatography of either peak X-1 or thiamine-PP produced the same two peaks. The radioactive specific activity per micromole labile phosphate was the same for the two peaks. Peak X-1 appears to be a thiamine compound formed in the presence of formate solutions. This procedural artifact was circumvented by the substitution of acetate for formate. By varying the pH as well as the ionic strength a single column procedure has been developed that separates thiamine and the three phosphate esters quantitatively in micromolar amounts.

Thiamine mono- and diphosphate splitting enzymes in the pre- and postmetamorphic Pelobates syriacus

1. 1. The ontogenic development of enzymes participating in the dephosphorylation of thiamine mono- and diphosphates was investigated in Pelobates syriacus. The developmental stages examined were between tadpoles possessing hind limbs and animals of about 20 days after the end of metamorphosis. 2. 2. The enzymatic activity was determined by incubating thiamine monophosphate and thiamine diphosphate with liver tissue, under standard conditions and by identifying the products by paper chromatography. 3. 3. Both thiamine mono- and diphosphate splitting enzymes were present in all the pre- and postmetamorphic stages. 4. 4. The results obtained seem to indicate that the activity of the investigated enzymes at pH 9 is higher after the completion of metamorphosis than before and continues to rise during early postmetamorphic life. 5. 5. A general developing line of the activity of different phosphatases according to the metamorphic stage of the tadpoles has been suggested.

Thiamine metabolism in wild-type and mutant strains of Neurospora crassa

Bioautography has been used to identify the thiamine-active substances accumulated in Neurospora mycelia and culture media of wild type and thi-1 strains. These strains have been compared at different stages of growth to detect differences in thiamine-active compounds. The nature of the thi-1 block has been investigated. The bioautographic characteristics of thiamine-active substances from thi-1 strains were indistinguishable from those of thi-1 + strains in these experiments. Thiamine phosphates are nutritionally relatively unavailable to thi-1 strains. The presence of high levels of thiamine phosphates in extracts of thi-1 mycelia explains the poor growth of thi-1 strains on these extracts. Thiamine mono-, di-, and triphosphates have been identified by their properties in two chromatographic systems and auxanographic assays by three mutant strains. The main component in these extracts is thiamine diphosphate.