Obese individuals as thiamin storers.

OBJECTIVE: To investigate thiamin and its phosphoester content in plasma and erythrocytes for a complete picture of thiamin status in obese individuals. DESIGN: Comparative study of the thiamin status of obese vs normal individuals. SUBJECTS: In all, 10 healthy, overweight, fertile age women (age: 33.1+/-5.1 y; BMI: 47.0+/-0.2 kg/m(2)) and 10 normal women (age: 30.1+/-3.5 y; BMI: 22.8+/-0.2 kg/m(2)). METHODS: a high-pressure liquid chromatography (HPLC) method for the determination of thiamin and its phosphoesters in the plasma and erythrocytes of the subjects. RESULTS: The major findings were: (1) significant decrease of plasma thiamin, its monophosphate and total thiamin contents in obese vs normal women; (2) significant decrease of thiamin pyrophosphate ester and total thiamin content in obese vs normal women; (3) significant increase in plasma thiamin/thiamin monophosphate ratio (in practice, it was inverted) and corresponding decrease of the plasma thiamin monophosphate/erythrocytes thiamin pyrophosphate ratio in obese vs normal women, where plasma thiamin monophosphate and erythrocytes thiamin pyrophosphate contents are an index of thiamin status. CONCLUSIONS: This study advances the hypothesis that obese women maintain higher levels of thiamin compared to normal weight subjects by storing greater amounts of thiamin in cells through preferential intracellular thiamin recycling to compensate for relatively lower levels of thiamin.

Three thiamine analogues differently alter thiamine transport and metabolism in nervous tissue: an in vivo kinetic study using rats.

Thiamine (T) analogues pyrithiamine, oxythiamine or amprolium in amounts 10-1000 times higher than labelled T, were i.p. injected into rats together with 14C-T (30 microg; 46.25 KBq). The radioactivity associated with T and its phosphoesters in the plasma and cerebral cortex, brainstem, cerebellum, and sciatic nerve were determined at time intervals from 0.25 to 240 h from injection. The experimental data obtained were processed with a mathematical compartmental model that calculated the fractional rate constants. These are the amount of content in a given compartment that is replaced in 1 h and expressed in per hour. The results showed that all three analogues inhibited thiamine entry from plasma. Instead, oxythiamine enhanced T phosphorylation to T pyrophosphate (TPP); amprolium and oxythiamine enhanced TPP dephosphorylation to monophosphate (TMP); pyrithiamine reduced TPP dephosphorylation and TMP formation, while none of the analogues modified TMP dephosphorylation to T. In conclusion, in living rats, the action of T analogues was much more complex than could be expected from their structure and action in vitro.

A hybrid input-output approach to model metabolic systems: an application to intracellular thiamine kinetics.

Models of the dynamics of complex metabolic systems offer potential benefits to the deep comprehension of the system under study as well as for the performance of certain tasks. Unfortunately, dynamic modeling of a great deal of metabolic systems may be problematic due to the incompleteness of the available knowledge about the underlying mechanisms and to the lack of an adequate observational data set. In theory, a valid alternative to classical structural modeling through ordinary differential equations could be represented by input-output approaches. But, in practice, such methods, which learn the nonlinear dynamics of the system from input-output data, fail when the experimental data set is poor either in size or in quality. Such a situation is not rare in the case of metabolic systems. This paper deals with a hybrid approach which aims at overcoming the problems addressed above. More specifically, it allows us to solve the identification problems of the intracellular thiamine kinetics in the intestine tissue. The method, which is half way between the structural and input-output approach, uses the outcomes of the simulation of a qualitative structural model to build a good initialization of a fuzzy system identifier. Such an initialization allows us to efficiently cope with both the incompleteness of knowledge and the inadequacy of the available data set, and to derive an input-output model of the intracellular thiamine kinetics in the intestine tissue. The comparison of the predictions of the intracellular thiamine kinetics obtained by the application of such a model with those obtained by traditional approaches, namely compartmental models, neural networks, and fuzzy systems, highlighted a better performance of our model. As the structural assumptions are relaxed, we obtained a model slightly less informative than a purely structural one but robust enough to be used as a simulator. The paper also discusses the interpretative potential offered by such a model, as tested on diabetic subjects.

Compartmental model identification based on an empirical Bayesian approach: the case of thiamine kinetics in rats.

Compartmental models are a very popular tool for the analysis of experiments in living systems. There are three main aspects that have to be taken into account: the degree of detail of the model, its a priori identifiability and the a posteriori (numerical) identifiability. In some cases, where standard approaches are adopted, the models can be either a priori or a posteriori unidentifiable. The paper proposes model identification within a Bayesian framework, to solve a posteriori unidentifiability problems. In particular, a stochastic simulation algorithm is proposed to perform a Bayesian identification of compartmental models, and an empirical Bayesian technique is proposed to propagate information among multiple experiments. The power of this methodology was demonstrated by evaluating the kinetics of thiamine under several experimental conditions. The complexity of the existing model (nine parameters) and limited experimental data (8/12 for each model) caused a posteriori identifiability problems when standard approaches were adopted. The application of the methodology identifies all 28 models (four tissues under seven different conditions).

Lipophilic thiamine treatment in long-standing insulin-dependent diabetes mellitus.

Thiamine plays an important role in the regulation of glucose metabolism and pancreatic beta-cell functioning. A role for this vitamin in cellular glucose transport has been indicated in the literature. The aim of this study was to determine whether a lipophilic form of thiamine (benzoyloxymethyl-thiamine, BOM) was able to improve metabolic control in patients with long-standing insulin-dependent diabetes mellitus (type 1). A total of 10 children with type 1 diabetes of long duration (age 11.4 +/- 1.2 years, duration of the disease 4.5 +/- 0.7 years, means +/- SEM) were studied before and after treatment with BOM in a randomized double-blind and placebo-controlled study. Five patients were assigned to the BOM-treated group and five to the placebo-group. In all patients basal and glucagon-stimulated C-peptide secretion was undetectable. Thiamine status was assayed by measuring the plasma content of thiamine and its monophosphate form at entry and after 3 months of treatment. The blood HbA(1C) levels and the daily dose of insulin per kg body weight were assessed in both groups before treatment, after 1 month and 3 months of treatment, then 3 months following its suspension. The plasma content of thiamine + thiamine monophosphate in type 1 diabetic patients (35.3 +/- 3.6 pmol/mL) was significantly lower when compared with that measured in six age-matched normal subjects (53.2 +/- 2.3 pmol/mL, P < 0.05).

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.

Thiamine uptake in human intestinal biopsy specimens, including observations from a patient with acute thiamine deficiency.

Mucosal biopsy specimens obtained by routine endoscopy from 108 human subjects, including one patient with thiamine deficiency, were incubated at 37 degrees C in oxygenated calcium-free Krebs-Ringer solution (pH 7.5) containing tritiated thiamine and [14C]dextran as a marker of adherent mucosal water. The amount of labeled thiamine taken up was measured radiometrically. In subjects with no clinical evidence of thiamine deficiency, 1) thiamine uptake by duodenal mucosa had a hyperbolic time course, reaching equilibrium at 10 min; 2) thiamine concentrations < 2.5 mumol/L were taken up predominantly by a saturable mechanism displaying Michaelis-Menten kinetics (K(m) 4.4 mumol/L and Jmax 2.3 pmol.mg wet tissue-1.6 min-1), whereas higher concentrations were taken up by passive diffusion; 3) thiamine transport had different capacities along the gastrointestinal tract (duodenum >> colon > stomach); and 4) thiamine uptake was competitively inhibited in the duodenum by thiamine analogs, albeit with a different order of potency compared with rats, and was blocked by 2,4-dinitrophenol. In the thiamine-deficient patient, the duodenal saturable uptake was increased, with higher K(m) and Jmax values. In conclusion, physiologic concentrations of thiamine were transported in human small intestine by a specific mechanism dependent on cellular metabolism, whose transporters appear to be down-regulated.

In vivo study of the kinetics of thiamine and its phosphoesters in the deafferented rat cerebellum.

The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on different steps of the metabolism of thiamine (Th), thiamine monophosphate (ThMP) and thiamine pyrophosphate (ThPP) were evaluated in vivo in rats. 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. Under steady state condition the radioactivity of Th and its phosphoesters was determined in plasma and whole cerebellum after an i.p. injection of thiazole-[2(14)C]-thiamine (30 micrograms:1.25 micro Ci). Analytical data were processed by using an improved mathematical compartmental model, which allowed the calculation of fractional rate constants (FRC), turnover rates (TR) and turnover times (TT). Both CD and SD caused a significant reduction of TR values for Th phosphorylation to ThPP, dephosphorylation of ThPP to ThMP and Th, and ThMP, but not Th, release. TT for all Th compounds were increased compared to controls, indicating a general slowing of thiamine metabolism in the deafferented cerebellum. These results indicate an imbalance in the thiamine metabolism resulting from the impaired activity of cerebellar neurons. The possible implications of the changes in rate of Th compound turnover with respect to biochemical changes in cerebellar ataxia are discussed.

Intestinal alkaline phosphatase can transphosphorylate thiamin to thiamin monophosphate during intestinal transport in the rat.

Intestinal alkaline phosphatase (IAP) purified from calf intestine and IAP present in the brush border membrane of rat small intestine effectively transphosphorylated thiamin (T) to thiamin monophosphate (TMP) using Na2-beta-glycerophosphate or Na2-creatine phosphate as phosphate donors at pH 8.5. TMP production in the brush border membrane was very small and corresponded to 0.001-0.01 percent of the total inorganic phosphate simultaneously released by the enzyme activity. This reaction, however, could account for TMP formation independently from that much more important due to the hydrolysis of thiamin pyrophosphate during T intestinal absorption.

Further studies on erythrocyte thiamin transport and phosphorylation in seven patients with thiamin-responsive megaloblastic anaemia.

Erythrocyte thiamin metabolism and transport were investigated in 7 patients from Brazil, Israel and Italy suffering from thiamin-responsive megaloblastic anaemia (TRMA) associated with diabetes mellitus and sensorineural deafness. All patients discontinued thiamin therapy for 4-7 days before the investigation. TRMA patients showed invariably reduced total thiamin levels in erythrocytes (percentage reduction compared with healthy controls, -46.8 +/- 3%; mean +/- SEM). The proportions of individual thiamin compounds, expressed as a percentage of total thiamin content, were within the normal range, whereas their absolute amounts were significantly decreased in the following order: thiamin monophosphate > thiamin pyrophosphate > thiamin. Thiamin pyrophosphokinase activity was also reduced as compared with controls (mean reduction +/- SEM, -25.9 +/- 1%). The saturable, specific component of thiamin uptake, which normally prevails at physiological concentrations of thiamin (< 2 mumol/L), was absent in erythrocytes obtained from TRMA patients, while the non-saturable (diffusive) component of uptake was normally present. These results confirm observations made previously in two patients and demonstrate that TRMA is consistently associated with a state of thiamin deficiency, which is presumably secondary to reduced thiamin cellular transport and absorption (caused by lack of a membrane-specific carrier), and to impaired intracellular pyrophosphorylation.

Effects of phenytoin on the in vivo kinetics of thiamine and its phosphoesters in rat nervous tissues.

The in vivo effects of chronic (30 days) and subchronic (10 days) intragastric treatment with phenytoin (PHT) (500 mg/kg) b.wt., suspended in 10% arabic gum water solution) on the uptake and metabolism of thiamine (T), T monophosphate (TMP) and T pyrophosphate (TPP) were evaluated in rat nervous regions (cerebral cortex, brainstem, cerebellum and sciatic nerve) by determining the radioactivity of T and its phosphoesters in plasma and tissues at fixed time intervals (0.25-240 h) after an i.p. injection of thiazole-[2-14C]thiamine (30 micrograms: 1.25 microCi). A nutritionally adequate diet containing T in excess was given to the animals in order to produce a virtually stable content of T compounds in the tissues. Analytical data were processed by using a compartmental model which allowed the calculation of fractional rate constants (FRC), turnover rates (TR) and turnover times. Compared with vehicle-treated controls, animals treated chronically with PHT exhibited lower levels of radiolabelled T compounds in all nervous regions except for the cerebral cortex. These alterations were not found in animals receiving subchronic treatment. Evaluation of FRC values indicated that PHT-induced effects on T metabolism differed depending on the length of PHT treatment and the nervous region considered. Overall, PHT appeared to interfere mainly with T and TMP uptake, TPP dephosphorylation to TMP and TPP turnover times, these effects being particularly prominent in the cerebellum and in the brainstem of chronically treated animals. Since all changes in T uptake and metabolism were observed in the absence of overt behavioural toxicity, these findings may have potential clinical relevance in highlighting possible mechanisms by which PHT therapy can alter brain metabolism.

Effect of ethanol on the in vivo kinetics of thiamine phosphorylation and dephosphorylation in different organs of rat--II. Acute effects.

The effects of acute ethanol administration on different steps of thiamine (T), thiamine monophosphate (TMP) and pyrosphosphate (TPP) metabolism were determined in vivo in nervous tissues (cerebral cortex, cerebellum, brain stem and sciatic nerve) and in other tissues (small intestinal mucosa, kidney, heart, skeletal muscle and liver) of rats. The radioactivity of T and its phosphoesters in plasma and tissues was determined under steady-state conditions and at fixed time intervals (0.25-24 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in the presence of a constant plasma ethanol concentration (37 mM; 1.75 g.l-1) produced by repeated intragastric administration of ethanol. Control animals received water intragastrically. Ethanol-treated rats and controls were starved, with water ad libitum during the 24 hr study period. Data were evaluated by using appropriate compartmental models, which allowed calculation of fractional rate constants, turnover rates and turnover times. In nervous tissues ethanol enhanced TMP entry (without affecting T entry or T and TMP release), reduced turnover time of total T and TPP, caused an almost general enhancement of TPP dephosphorylation without affecting T pyrophosphorylation, and increased markedly T content in the mixture released by tissues. Overall, ethanol appeared to enhance exchanges of T compounds in nervous tissues. In other tissues, the effects of ethanol were less consistent. Ethanol increased T uptake in kidney and liver and T release in liver and heart, but had no effect on T exchanges in the small intestinal mucosa and in skeletal muscle. In the presence of ethanol, TMP uptake increased in heart and skeletal muscle and decreased in the small intestinal mucosa, while TMP release decreased in heart and remained unchanged in all other organs. Turnover times tended to increase for total T and to decrease for TPP. T pyrophosphorylation was generally reduced, and T phosphates dephosphorylation generally enhanced. T became the most abundant component in the mixture released from all tissues.

Thiamin mono- and pyrophosphatase activities from brain homogenate of Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia patients.

Thiamin-pyrophosphatase (TPPase) and thiamin-monophosphatase (TMPase) were determined using a spectrophotometric method at various pH values (5.5, 7.5, and 9.0) in brain tissue obtained at autopsy from amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) patients from Guam and from Guamanian patients who died from other diseases (controls). TPPase separation by thin-layer polyacrylamide gel isoelectric focusing (IEF) was also performed using both gray and white matter. TPPase content, chemically determined at pH 9.0, was found to be significantly reduced in the frontal cortex of ALS and PD patients compared to controls. TMPase content, on the contrary, was unchanged. IEF analysis showed 9 clear-cut bands with TPPase activity in the pH range 5.4-7.2 and a broad band at pH 4.7-5.2. The enzymatic activity was higher in gray than in white matter. In one patient the pattern was clearly different, with two additional bands observed at pH 7.1 and 6.7, and thought to be due to genetic microheterogeneity.

Thiamine transport by erythrocytes and ghosts in thiamine-responsive megaloblastic anaemia.

A 9-year study of thiamine metabolism and cellular transport was performed in two patients with thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of thiamine and thiamine compounds were reduced by about 40% as compared to controls. Thiamine pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of thiamine and thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, less than 2 mumol/L, physiological concentrations of thiamine) and the non-saturable component of thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from thiamine-responsive megaloblastic anaemia patients contain low levels of thiamine compounds, probably due to their inability to take up and retain physiological concentrations of thiamine, as a result of the lack of the saturable, specific component of transport and reduced thiamine pyrophosphokinase.

Thiamin contents of cerebrospinal fluid, plasma and erythrocytes in cerebellar ataxias.

Free thiamin and thiamin monophosphate have been found in the cerebrospinal fluid, plasma and in erythrocytes of patients suffering from ataxia of different origins. In erythrocytes, thiamin pyrophosphate was also measured. In a limited number of cases, uptake of 14C-thiamin by erythrocytes was found as well. Controls were hospitalized patients affected by chronic neurological diseases without any clinical sign of thiamin deficiency. The results showed a significant decrease in thiamin and thiamin monophosphate in the cerebrospinal fluid and in the plasma of ataxic subjects, in comparison to controls. In erythrocytes, only thiamin pyrophosphate levels had decreased. The uptake of 14C-thiamin by erythrocytes was similar in both ataxic and control groups. These results were comparable to those observed in thiamin-deficient individuals, like alcoholic patients, and prompted further investigation into thiamin metabolism in these diseases.

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)

Thiamin transport by human erythrocytes and ghosts.

Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20 degrees C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 microM and at 37 degrees C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes, Km = 0.12, 0.11 and 0.10 microM and Jmax = 0.01, 0.02 and 0.03 pmol.microliter-1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts, Km = 0.16 and 0.51 microM and Jmax = 0.01 and 0.04 pmol.microliter-1 intracellular water after 15 and 30 min incubation times, respectively. At 20 degrees C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence of D-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein- (probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN- showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.

Different distribution of thiaminpyrophosphatase activity in neuronal and glial cell enriched fractions from human and rat brain: an isoelectric focusing investigation.

Enriched fractions of neuronal and glial cells from rat and human (autoptical specimens of frontal cortex) brains were assayed for thiaminpyrophosphatase (TPPase) activity by isoelectric focusing (IEF). Glial and neuronal fractions yielded about 0.16 and 0.03 g/g of wet tissue respectively. Purity was estimated to be 85-90%. The isolated fractions were homogenized in the presence of 1% Triton X-100, and IEF was carried out on thin layer polyacrylamide gel in a pH range of 3.5-9.5, using Ampholine PAG plates. TPPase activity of the protein bands was assayed by laser-densitometry, after incubation with thiaminpyrophosphate in an appropriate buffer and PbS staining. IEF analysis showed that TPPase activity was present almost exclusively in the neuronal fraction (at levels 16-fold higher than those found in the glial fraction). After IEF, TPPase activity was present in 10 distinct protein bands with different isoelectric points. These preliminary data suggest that TPPase may be involved in the neuronal activity of rat and human brain.

Thiamine, thiamine phosphates and thiamine metabolizing enzymes in synaptosomes of rat brain.

Thiamine and thiamine mono-, pyro- and triphosphate were found at detectable levels in synaptosomes isolated from whole rat brain. Synaptosomes prepared from whole brain, cerebellum and medulla were also found to contain uridine and inosine mono- and diphosphatases as well as the thiamine pyrophosphate synthetizing and hydrolyzing enzymes, but no thiamine monophosphatase. By isoelectric focusing on thin layer polyacrylamide gel of Triton X-100 homogenates of synaptosomes, thiamine pyrophosphatase activity could be separated into 10 bands with different isoelectric points. The contents of thiamine compounds and enzymes in synaptosomes were generally lower than those found in neuronal cell bodies.