Thiamine Monophosphate Research Articles

Oral treatment of polioencephalomalacia and subclinical thiamine deficiency with thiamine propyl disulphide and thiamine hydrochloride.

Thiaminase type I production by Bacillus thiaminolyticus and activity in vitro were repressed by the primary substate thiamine and by thiamine monophosphate and thiamine propyl disulphide. At thiamine concentrations of 300-3000 mumol/l production of active enzyme by B. thiaminolyticus, and activity of purified enzyme, were totally repressed. Growth of B. thiaminolyticus was inhibited by thiamine propyl disulphide at 3000 mumol/l. Activity of purified thiaminase was lost when incubated with ruminal fluid from healthy thiaminase free sheep. Enzyme activity was also lost when exposed to mildly alkaline conditions but was stimulated and stabilized against heat denaturation if treated with dithiothreitol. Thiaminase activities in the ruminal fluids of cases of ovine polioencephalomalacia were repressed within 2 h of oral administration of thiamine propyl disulphide. Blood pyruvate levels and transketolase activities were restored to normal following treatment. Treated animals recovered clinically and were returned to pasture.

3] Determination of thiamin and its phosphate esters by high-performance liquid chromatography

This chapter describes high-performance liquid chromatography (HPLC) of thiamin and its phosphate esters, which is considered the most sensitive method to determine these compounds at the subpicomole level within lo-20 min. This method is applicable to the analysis of thiamin and its phosphates not only in animal tissues but also in body fluids, foods, and pharmaceutical preparations. This method is also useful in the study of intermediary metabolism of thiamin. In the chromatographic mode, two systems, straight-phase HPLC and reversed-phase HPLC, are used. In the former system, thiamin, thiamin monophosphate (ThMP), thiamin diphosphate (ThDP), and thiamin triphosphate (ThTP) are eluted from a column in this order. In the latter system, the elution order of thiamin phosphates is the reverse. When detection of thiamin phosphates is carried out fluorometrically, two different procedures are used—namely, pre- and postcolumn derivatization methods. The mobile phase used in the precolumn derivatization method of HPLC for thiamin and its phosphates should contain a buffer solution of pH above 8, as thiochrome fluoresces at pH values above 8. All phosphate bonds remain intact when thiamin phosphates are converted to thiochrome phosphates by alkaline oxidation.

Subcellular localization and some properties of intermediate-molecular-weight acid phosphatase from rat liver.

The distribution of acid phosphatases of intermediate molecular weight was determined in various rat tissues. The distribution study of intermediate-molecular-weight acid phosphatase (disignated P-II) in the subcellular fractions of rat liver and kidney indicated that P-II was localized in the mitochondrial fractions. The P-II partially purified from liver showed a pI value of 7.0 on isoelectric focusing, and the apparent molecular weight was estimated to be 40000 by Sephadex G-100 gel filtration or 44000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for phosphoproteins, phosphoserine, o-phosphocholine and thiamine monophosphate. The enzyme showed a high activity on pyridoxal phosphate, β-glycerophosphate, flavin mononucleotide and adenosine 2'-monophosphate. It was markedly inhibited by Hg2+ and Ag+, but not significantly by sulfhydryl blocking agents or by L-(+)-tartrate.

Cleavage of thiamine pyrophosphate by sulfite in saccharomyces cerevisiae

Sulfite at low concentrations (≧ 1 mM caused a cleavage of thiamine in vitro depending upon time, pH value, and temperature. Thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) were also cleaved, but the reaction rates were slower. For the separation and determination of thiamine, TMP and TPP in yeast high-performance liquid chromatography (HPLC) was used. Thiamine and its phosphate esters were converted into the corresponding fluorescent thiochrome derivatives by alkaline oxidation, adsorbed on an amino-phase HPLC column and eluted with acetonitrile/100 mM-potassium phosphate buffer, pH 7.5 (40+60, v/v) as solvent system. In yeast TPP represented about 73% of the sum of all thiamine compounds, thiamine and TMP 26% and 1%, respectively. During long-term incubations of yeast (up to 24 hours) 2 mM-sulfite decreased the intracellular content of TPP and also of ATP depending upon the pH value of the medium. The decreasing effect of sulfite on the ATP content was faster and stronger than on the TPP content. Two TPP-dependent enzymes, pyruvate decarboxylase [EC.4.1.1.1] and transketolase [EC.2.2.1.1] were inactivated by sulfite within 1 hour to 58% and 13% of the initial values, respectively.

Improved high performance liquid chromatographic determination of thiamin diphosphate in erythrocytes

Thiamine and its phosphate esters play vital physiological roles and thiamine deficiency causes deleterious effects on human body. It is important to quantify accurately the thiamine metabolites in body fluids. However, due to the lack of appropriate internal standards, poor inter-laboratory standardization and time-consuming pretreatment procedure, the existing methods are limited in clinical applications. Hence, we developed a single-step HPLC-MS/MS method for accurate and precise measurement of thiamine and its phosphate esters in human whole blood. Whole blood samples were deproteinized and the supernatants were collected. The levels of thiamine diphosphate (TDP), thiamine monophosphate (TMP), and thiamine were determined by HPLC-MS/MS method after adding isotopic internal standards. The method was linear from 15.625–3.125-1.563 nmol/L to 1000–200-100 nmol/L for TDP-TMP-thiamine. The lower limit of quantification was 15.625–3.125–1.563 nmol/L. The intra-day and inter-day precisions and accuracy for all QCs samples were ≤15.9% and ≤11.1%, respectively. The matrix effect was not significant. Recoveries were 103.7% for TDP, 102.7% for TMP, and 105.3% for thiamine. All QCs were stable for three freeze-thaw cycles, or at room temperature for 3 h, or at −80 °C for 15 days. We compared this new method with an established HPLC method based on derivatization of thiamine metabolites. It is found that this method correlated well with HPLC method for TDP determination (R2 = 0.93). However, the correlation was not ideal for TMP (R2 = 0.40) or thiamine (R2 = 0.72) determination. Subject's diet was shown to have no significant effect on the concentrations of thiamine metabolites in their blood samples. To conclude, we developed a single-step, non-derivatization HPLC-MS/MS method that can detect thiamine and its phosphate esters in human whole blood accurately and quickly.

X-ray studies on metal ion interactions with vitamins. 1. Crystal and molecular structure of the tetrakis(.mu.-acetato)bis(thiamin monophosphate)dirhodium(II) sesquihydrate complex: thiamin base-metal bonding

X-ray studies on metal ion interactions with vitamins. 1. Crystal and molecular structure of the tetrakis(.mu.-acetato)bis(thiamin monophosphate)dirhodium(II) sesquihydrate complex: thiamin base-metal bonding

Determination of thiamine and its phosphate esters in human and rat blood by high-performance liquid chromatography with post-column derivatization

A method for the determination of thiamine and thiamine phosphate esters in human and rat blood by liquid chromatography with post-column derivatization, and distribution of thiamine and its phosphate esters in human and various animals is demonstrated. Blood is deproteinized and centrifuged. Aliquots of the samples are applied to μBondapak C 18 column attached to a high-performance liquid chromatograph. Addition of potassium ferricyanide sodium hydroxyide solution to the column effluent with a proportioning pump converts thiamine phosphate into fluorophores, the intensities of which are measured with a spectrofluorophotometer. In human blood, thiamine pyrophosphate was present in the greatest concentration, followed by thiamine triphosphate; thiamine monophosphate and thiamine were present in small amounts. In rat blood, thiamine pyrophosphate was found in the largest amount, followed by thiamine monophosphate. Thiamine triphosphate and thiamine were present in small amounts.

Synthesis of thiamine triphosphate in rat brain in vivo.

Thiamine metabolism in vivo was studied by intracerebroventricular injection of labeled thiamine in rat brain. Labeled thiamine was found to be rapidly converted to the phosphorylated thiamine esters. The distribution of the radioactive thiamine compounds was reached to steady state at 3 hr after injection: thiamine, thiamine monophosphate, thiamine pyrophosphate, and thiamine triphosphate were 8-12%, 12-14%, 72-74%, and 2-3%, respectively, in cerebral cortex. The presence of labeled thiamine triphosphate in the brain was further confirmed by the treatment with thiamine triphosphatase which had an absolute substrate specificity for thiamine triphosphate. These results suggest that thiamine triphosphate is synthesized in vivo in rat brain.

The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes

Quaternary ammonium compounds, such as choline and acetylcholine significantly inhibited thiamine uptake in isolated rat hepatocytes. Kinetic analysis using Lineweaver-Burk and Dixon plots of inhibition experiments revealed that choline and acetylcholine were purely competitive inhibitors for thiamine uptake with K i values of 0.61 mM and 0.31 mM, respectively. Among quaternary ammonium compounds, hemicholinium-3 and curare were the strongest inhibitors, and kinetic studies showed that these compounds were also purely competitive inhibitors with K i values of 12.5 μM and 4.3 μM, respectively. These results indicate that choline, acetylcholine and their structural analogs share a common binding site with thiamine in isolated rat hepatocytes. On the other hand, choline uptake by isolated rat hepatocytes occurred by a saturable mechanism with a K t of 162 ± 3.85 μM and V max of 80.1 ± 1.30 pmol/10 5 cells per min as well as by a nonsaturable mechanism. Thiamine, pyrithiamine, oxythiamine, chloroethylthiamine and dimethialium inhibited choline uptake, while thiamine phosphates such as thiamine monophosphate and thiamine pyrophosphate insignificantly inhibited uptake. Although a Lineweaver-Burk plot of choline uptake in the presence of thiamine showed that thiamine also competitively inhibited choline uptake, a Dixon plot of the inhibition experiment was hyperbolic and indicated that the inhibition of choline uptake by thiamine was ‘pseudo-competitive’. On the basis of these results, it is suggested that in isolated rat hepatocytes thiamine and choline do not share common transport sites.

Selective medium for isolation of Haemophilus somnus from cattle and sheep.

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.

Endocytosis and the lysosomal apparatus of rat Leydig cells

The Leydig cells of the rat testis which are involved in testosterone production contain an abundance of smooth endoplasmic reticulum and mitochondria (Figs. 2,6). These cells also possess many peroxisomes, lysosomes and multivesicular bodies (MVB's). On the cell surface, the plasma membrane contains numerous short microvilli, small invaginations and large plasmalemmal folds which appear to engulf extracellular fluid. There are also many large dilated vacuoles adjacent to the cell surface. The purpose of the present study is to determine if these cells show endocytic activity and to differentiate by various cytochemical means lysosomal elements from peroxisomes.To identify lysosomes, tissue chopper sections of 2% glutaraldehyde-fixed testes (containing 2.5% dextran) were incubated in media containing thiamine monophosphate as a substrate (Lalli, 1983) to demonstrate the presence of acid phosphatase or in media containing P-nitrocatechol sulfate for the demonstration of arylsulfatase (Hopsu-Havu et al., 1967).

Nervous tissue thiamine metabolism in vivo. I. Transport of thiamine and thiamine monophosphate from plasma to different brain regions of the rat

The transport of thiamine (T) and thiamine monophosphate (TMP) across the blood-brain barrier was measured in vivo in the rat. Different doses of [ 14C]T (15–550 nmol) and [ 14]TMP (11–110 nmol) were injected into the femoral vein. The content of T and its phosphoesters in blood and brain tissue (cerebellum, pons, medulla and cerebral cortex) 20 s after the injection was determined radiometrically after electrophoretic separation. Blood flow and blood volume in the same regions of the brain was also determined. Both T and TMP entered rapidly the cerebral tissue, where they were found chemically unmodified. The cerebral tissue extracted less than 7% of plasma T. At physiological plasma T concentrations, the rate of transport ranged from 0.43 to 0.65 nmol·g −1·h −1 with only minor differences among the various regions. T was transported into the nervous tissue by two separate mechanisms: one saturable, that at physiological plasma T levels accounted for 95% (cerebellum) to 91% (cerebral cortex) of the total T taken up, and one non-saturable, that was most efficient in the cerebral cortex. The K m (half-saturation constant) of the former transport mechanism ranged from 1.95 to 2.75 nmol·ml −1 in the 4 areas investigated. V max (maximal transport rate) values ranged from 6 to 9 nmol·g −1·h −1, the highest value being found in the cerebellum. The overall transport rate of TMP was on average 5–10 times as low as that of T and also showed a saturable and a non-saturable component. Both components were slower than those observed for T.

Determination of thiamin and thiamin phosphate esters in blood by liquid chromatography with post-column derivatization.

We demonstrate a liquid-chromatographic method involving post-column derivatization for determining the concentration of thiamin and its phosphate esters in human blood. Blood, erythrocytes, or plasma is deproteinized and centrifuged. Aliquots of the samples are applied to a mu Bondapak C18 column attached to a "high-performance" liquid chromatograph. Addition of potassium ferricyanide/sodium hydroxide solution to the column effluent with a proportioning pump converts thiamin phosphates into fluorophores, the intensities of which are measured with a spectrofluorophotometer. Thiamin, thiamin monophosphate, thiamin pyrophosphate, and thiamin triphosphate eluted as single peaks; no coeluting substances were detected. Thiamin pyrophosphate was the ester present in greatest concentration, followed by thiamin triphosphate; thiamin monophosphate and thiamin were present in slight amounts. This method allows easy determination of thiamin and its phosphate esters in 0.1 mL of blood.

Effects of lithium on tryptophan transport into plasma-membrane vesicles derived from rat brain synaptosomes

during the period 5-120min after injection of thiamin. Most of the thiamin in the brain was in the phosphorylated form: free (unphosphorylated) thiamin 3%. thiamin monoand pyrophosphates 90%. and thiamin triphosphate 2%. The ratio of distribution of thiamin pyrophosphate to thiamin monophosphate was I : I . but values of 3.5 : 1 have also been obtained. The factors responsible for these variations require investigation. The radioactivity in the rain at 5min after injection of 1I4Clthiamin was found only in thiamin and thiamin pyrophosphate. giving evidence of phosphorylation of thiamin directly t o thiamin pyrophosphate. It increased linearly with time from 10 to 30min and thereafter showed a steady decline in the rate of uptake of I i4CIthiamin. Free thiamin accounted for 20% of the total radioactivity in the brain at Smin, 30% at IOmin. 24% at 20min. 20% at 30min, 1 I % at 60min and 8% at 120min. Thiamin monoand pyro-phosphates together contained 72-82% and thiamin triphosphate 4% of the radioactivity in the brain at times from 10 to 120min after injection of 1 14C Ithiamin. Thiamin showed the highest specific radioactivity (d.p.m./ nmol) at all times: its phosphate esters appeared to approach this value at 120min (Fig. I). In view of the fact that thiamin pyrophosphate is the precursor of the other two thiamin phosphates, the observed specific radioactivities were contrary to expectation: thiamin triphosphate > thiamin monophosphate > thiamin pyrophosphate. Moreover, the specific-radioactivity-time curves did not intersect during the period 5-120min after injection of 1Clthiamin. These findings are at variance with the precursor-product relationship and indicate the presence of more than one compartment of the thiamin pyrophosphate pool.

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase.

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.

Morphological, biochemical, antigenic, and cytochemical relationships among Haemophilus somnus, Haemophilus agni, Haemophilus haemoglobinophilus, Histophilus ovis, and Actinobacillus seminis.

Morphology, biochemical reactions, pigmentation, antigens, and cell envelope proteins were examined in 12 strains of Haemophilus somnus, Haemophilus agni, Histophilus ovis, and Actinobacillus seminis. All of the strains except A. seminis are related and are considered as a single Haemophilus-Histophilus (HH) group. In immunodiffusion tests, HH group bacteria had at least two antigens common to all members of the group, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that they have similar cell envelope protein profiles. A quantitatively variable yellow pigment with absorption maxima of 430 to 435 nm was present in strains of H. somnus and H. agni. The HH group did not produce catalase and grew only in air containing 10% CO2. Of 10 HH group bacteria, 9 required thiamine monophosphate for growth. A. seminis was distinguished from the HH group by its lack of yellow pigment, production of catalase, growth in air, lack of a thiamine monophosphate requirement, and different cell envelope protein profile. In gel immunodiffusion tests, A. seminis antigens produced two lines of partial identity with the HH group when antiserum against H. somnus was used. Reference strains of Haemophilus influenzae, Actinobacillus lignieresii, and Haemophilus haemoglobinophilus were compared with the test strains. In immunodiffusion tests, a single antigen was found to be common to H. haemoglobinophilus, A. seminis, and the HH group. No similarities between any of the test strains and H. influenzae or A. lignieresii were noted. The close relationship of H. somnus, H. agni, and Histophilus ovis suggests that these unofficially named bacteria may belong to a single taxon.

Quantitative analysis of total thiamine in human blood, milk and cerebrospinal fluid by reversed-phase ion-pair high-performance liquid chromatography

Whole blood hemolysed by freezing, serum, cerebrospinal fluid, and milk of human origin were deproteinized by perchloric acid. Thiamine pyrophosphate and thiamine monophosphate were hydrolysed to thiamine by acid phosphatase. Chromatography was performed on C 18-coated silica using an n-octanesulfonate containing mobile phase methanol—aqueous citrate buffer pH 4.0 (45:55, v/v). In a post-column reaction K 3Fe(CN) 6 is used to oxidise thiamine to thiochrome, which is detected by fluorometry. Two ml blood is needed. The minimum detectable amount is 60 femtomol of thiamine. The intra-assay coefficient of variation (C.V.) is 2.3% and the inter-assay C.V. is 3.9%. The recovery of added thiamine pyrophosphate to blood samples was 98.7%. The reference range was found to be 88–157 nmol/l whole blood. Examples of the analysis of cerebrospinal fluid, serum and milk are given.

Thiamin monophosphate in the CSF of patients with amyotrophic lateral sclerosis.

Free thiamin and thiamin monophosphate levels were determined by an electrophoretic fluorometric micromethod in plasma and CSF of patients with amyotrophic lateral sclerosis (ALS), alcoholics, and controls. In plasma of patients with ALS as well as in plasma and CSF of alcholics, both thiamin and thiamin monophosphate concentrations were decreased so that the thiamin-thiamin monophosphate (T/TMP) ratio remained unchanged compared with that of controls. In CSF of patients with ALS, however, thiamin monophosphate values decreased much more than thiamin levels, so that the T/TMP ratio was significantly increased. The selective impairment of thiamin monophosphate production by nerve cells is likely to result from the reduction of the activity of thiamin pyrophosphatase, an enzyme synthetized and highly concentratd in the Golgi complex. Thiamin pyrophosphatase is known to diminish in ALS as well as in experimental motor neuronal degeneration or axotomy. Thus, the T/TMP ratio could be taken as an index of the impairment of neuronal protein synthesis in ALS.

ThiK and thiL loci of Escherichia coli.

Mutants of Escherichia coli K-12 auxotrophic for thiamine phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate production of thiamine auxotrophs that had lost the enzymatic activity of either phosphomethylpyrimidine kinase or thiamine phosphate pyrophosphorylase. They include two types: one responds to thiamine monophosphate or thiamine pyrophosphate, and the other responds to thiamine pyrophosphate only; the former lacks thiamine kinase activity, and the latter lacks thiamine monophosphate kinase activity, in addition to the enzymatic defects caused by the first mutations. We found two genes, for which we propose the designations thiK and thiL, which govern the activities of thiamine kinase and thiamine monophosphate kinase, respectively. By conjugation and P1 transduction, the thiK locus was mapped at about 25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-ptsG-fabD-thiK-purB. In the case of thiL, the locus was situated at about 9 min, between tsx and acrA and probably 0.2 min clockwise from the former.

The isolation and preliminary characterization of apotransketolase from human erythrocytes

Human erythrocyte apotransketolase (EC 2.2.1.1) has been isolated with greater than 400 fold purification, and free of glyceraldehyde-3-phosphate dehydrogenase. The preparation has an absolute requirement for thiamin pyrophosphate in order to exhibit enzyme activity. Neither thiamin nor thiamin monophosphate could substitute for this requirement, nor were they inhibitory separately or together at concentrations of 1 mM. The K m for thiamin pyrophosphate was 0.4 μM. The K m for ribose-5-phosphate was 3 × 10 −4M and for xylulose-5-phosphate 1.8 × 10 −4M.