Thiamine Phosphate Esters Research Articles

Thiamine status and genes encoding intestinal thiamine transporters and transcription factors in obese subjects

Background and aimsThe inconsistent data on thiamine status in obese subjects necessitates an examination of genes associated with intestinal absorption of thiamine. We aimed to reveal thiamine status in obese subjects and examine the expression of SLC19A2/3 genes encoding thiamine transporters and Sp1 transcription factor. Methods and resultsThirty-five adult obese subjects and 11 healthy controls were included in this cross-sectional study. Small intestine epithelial cells were used for quantitative RT-PCR analysis of the gene expression. The daily thiamine and energy intake were assessed with a food frequency questionnaire. Thiamine phosphate esters were hydrolyzed to free thiamine, and liquid chromatography with a tandem mass spectrometry-based method was used to measure total thiamine in whole blood. Daily energy intake according to body weight and daily carbohydrate intake were not significantly different between groups after adjustment for sex. Although daily thiamine intake was significantly lower in the obesity group (p = 0.015), obese subjects had significantly higher whole blood thiamine levels than controls (44.96 ± 14.6 ng/mL and 33.05 ± 8.6 ng/mL, p = 0.002). There was a significant positive correlation between whole blood thiamine and BMI (r = 0.342, p = 0.020). SLC19A2 gene expression was lower in those with BMI ≥35 kg/m2 (p = 0.036). A significant positive correlation was found between SLC19A2 expression and whole blood thiamine level (r = 0.310, p = 0.038). ConclusionA possible association between intestinal thiamine intake and total thiamine in whole blood was determined. The transcriptional changes of genes encoding the high-affinity membrane thiamine transporters, especially SLC19A2, probably play a role in this relationship.

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass

Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.

Thiamine tetrahydrofurfuryl disulfide promotes voluntary activity through dopaminergic activation in the medial prefrontal cortex

A physically active lifestyle is associated with better health in body and mind, and it is urgent that supporting agents for such lifestyles be developed. In rodents, voluntary locomotor activity as an active physical behavior may be mediated by dopaminergic neurons (DNs). Thiamine phosphate esters can stimulate DNs, and we thus hypothesized that thiamine tetrahydrofurfuryl disulfide (TTFD), a thiamine derivative, promotes locomotor activity via DNs in rats. Acute i.p. administration of TTFD enhanced rat locomotor activity in a normal cage. In vivo microdialysis revealed that TTFD-enhanced locomotor activity was synchronized with dopamine release in the medial prefrontal cortex (mPFC). Antagonism of the dopamine D1 receptor, but not D2 receptor, in the mPFC fully suppressed TTFD-enhanced locomotor activity. Finally, we found a TTFD dose-dependent increase in voluntary wheel running. Our findings demonstrate that DNs in the mPFC mediates TTFD-enhanced locomotor activity, suggesting the potential of TTFD to induce active physical behavior.

THIAMINASE ACTIVITY OF MYOGLOBIN OXOFERRYL FORMS

Thiamine oxidation chemistry in presence of metmyoglobin and hydrogen peroxide is quite complex and different products can be formed. Incubation of thiamine with metmyoglobin and hydrogen peroxide can result in splitting of thiamine molecule at carbon atom of the methylene bridge and production of aminopyrimidine and thiazole components as separate molecules or in formation of thiochrome, thiamine disulfide, oxodihydrothiochrome, and thiaminethiazolone. Oxidative transformation of thiamine phosphate esters in presence of metmyoglobin and hydrogen peroxide gives similar products however thiaminase activity, i.e. splitting of the molecules into aminopyrimidine and thiazole phosphate parts, is much higher in this case. Addition of tyrosine or paracetamol to incubation mixture inhibits thiaminase activity and formation of disulfides, but yield of thiochrome or thiochrome phosphates increases. Identification of products of thiamine (or its phosphate esters) oxidation in the presence of metmyoglobin and hydrogen peroxide was performed using HPLC, mass-spectrometry and spectral-fluorescent methods. Role of oxoferryl forms of myoglobin in degradation of thiaminediphosphate, cofactor of the important enzymes of carbohydrate metabolism, by thiaminase mechanism is discussed.

Sulbutiamine shows promising results in reducing fatigue in patients with multiple sclerosis

BackgroundFatigue is the most frequent and often debilitating symptom for patients with multiple sclerosis (MS). There are no available effective therapies for fatigue associated with MS, and it is unclear whether a successful therapy of MS leads to clinical improvement. Sulbutiamine is a lipophilic compound that crosses the blood–brain barrier more readily than thiamine and increases the levels of thiamine and thiamine phosphate esters in the brain. Whereas several clinical trials have demonstrated the beneficial effects of sulbutiamine in patients with asthenia, there have been no reports on the effects of sulbutiamine on fatigue in patients with MS. ObjectivesOur study was designed to evaluate the short-term effects of sulbutiamine on fatigue in patients with MS. MethodsPatients were included if fatigue was one of their three predominant symptoms. They were required to have a total score on the Fatigue Impact Scale (FIS) of >20, and on the Beck Depression Inventory of <17, and no relapse in the last 3 months prior to onset of the study. Patients were advised to receive 400mg orally of sulbutiamine once daily for two months. The outcome of the study was in the changes of FIS. ResultsTwenty-six patients with MS (18 females and 8 males) were selected. The patients were 18–57 years of age (mean:37,2). The average score of Expanded Disability Status Scale (EDSS) of the patients was 2,71. A significant number of the subjects who were on some kind of disease modifying treatment (DMT) demonstrated obvious improvement in their total FIS scores, whereas none of the subjects who were not on any DMT improved (13/23 vs. 0/5). The average fatigue score was 77 (SD:30,5) at the baseline and 60,5 (SD:29,7) on Day 60, respectively. Sulbutiamine intake resulted in a significant reduction on the total score of FIS and on all three subscales assessing physical, cognitive, and psychosocial functioning (all p-values < 0,01). There were no serious adverse events. ConclusionsSulbutiamin appears to be effective in treating fatigue in MS; particularly in patients who were on some DMT, but not on those who were not. It is well-tolerated by all. This observation may encourage further evaluations of the efficacy of sulbutiamine on fatigue in MS.

Thiamin (vitamin B1) and thiamin phosphate esters in five cereal grains during maturation

Non-phosphorylated thiamin (T), thiamin monophosphate (TMP), and thiamin diphosphate (TDP) were extracted from whole grains of wheat (Triticum aestivum, cv. Dekan), triticale (Triticosecale, cv. SW Talentro), rye (Secale cereale, cv. Agronom), oats (Avena sativa, cv. Dominik), and barley (Hordeum vulgare, cv. Merlot) at different growth stages (from inflorescence emergence to full ripeness of the grains) and determined with high-performance liquid chromatography and fluorescence detection. No thiamin triphosphate or adenosine thiamin triphosphate was found in these cereals. Total thiamin levels (the sum of T and its phosphate esters) determined on a dry matter (DM) basis were found to be quite similar in all grain samples throughout the analysis period, ranging from 5.59 (wheat) to 13.00nmol/g DM (oats). In spikelets 3–5 days after flowering, the total thiamin content in all cereals was 40–75% T, 5–13% TMP, and 18–46% TDP. Generally, T increased and TDP decreased continuously with grain development, with maximum T (88–99%), and minimum TDP (2–10%) at ripeness. No TMP was detected in mature grains. The results indicate that variation in T, TMP, and TDP levels is probably due above all to the conversion of phosphorylated thiamin phosphate esters into less highly or non-phosphorylated forms.

Degradation and conversion of thiamin and thiamin phosphate esters in fresh stored pork and in raw sausages

Pork samples were extracted with trichloroacetic acid (4%) and analyzed for thiamin (T) and thiamin phosphate esters with high performance liquid chromatography (HPLC). The following concentrations (on protein basis) were found in samples analyzed 0.5 h after slaughter: non-phosphorylated T, 1.0 nmol/g; thiamin monophosphate (TP), 1.3 nmol/g; thiamin diphosphate (TP 2), 9.1 nmol/g; and thiamin triphosphate (TP 3), 65.7 nmol/g. A fifth substance was also detected, which has not yet been identified in pork and may be related to thiamin tetraphosphate (TP 4) or adenosine thiamin triphosphate (ATP 3); when calculated as thiamin tetraphosphate the content (on protein basis) of the fifth substance was 15.0 nmol/g. After 216 h storage, the contents (on protein basis) of T and TP had changed as follows: 46.3 nmol/g T; 3.2 nmol/g TP, 5.0 nmol/g TP 2, and 12.5 nmol/g TP 3, The fifth substance completely degraded within 6 h. These changes are probably due to the instability of highly phosphorylated thiamin phosphate esters, which undergo dephosphorylation during storage. In addition, there was a decrease in total thiamin concentrations (on protein basis) during the first 24 h of storage from 92.1 nmol/g to 24.0 nmol/g, followed by an increase to 67.0 nmol/g over a 216 h storage period. One possible explanation for the decrease observed during the first 24 h might be that thiamin is converted into a reversible, binding form that cannot be extracted by trichloroacetic acid.

Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

Thiamine (vitamin B(1)) deficiency is associated with severe diseases such as beriberi and Wernicke encephalopathy. Although most Americans have sufficient dietary intake, thiamine deficiency is observed in the alcohol-dependent and elderly populations. Measurement of thiamine concentration in whole blood provides an assessment of vitamin B(1) status in at-risk individuals. We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase. The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP). The means (ranges) for an apparently healthy population were 114 (70-179) nmol/L for TDP and 125 (75-194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min. We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.

Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used

We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood.

Thiamine Status in Liver and Brain of Rats Genetically Selected for Different Sensitivity to Hypnotic Effect of Alcohol

The mechanisms of the different sensitivity or resistance of animals and humans to alcohol are still not completely understood. For further biochemical characterization of animals genetically selected for high-alcohol sensitivity (HAS) and low-alcohol sensitivity (LAS) with the hypnotic effect of alcohol, the thiamine status and thiamine metabolizing enzymes in these animals have been studied. We investigated thiamine diphosphate and thiamine triphosphate levels as well as the activity of thiamine-dependent enzyme, transketolase, and thiamine-metabolizing enzymes, thiamine kinase, and thiamine triphosphatase in the liver and brain of HAS, LAS, and CAS (control) rats by standard biochemical techniques. It was found that the activity of transketolase, and the level of the coenzyme form of thiamine, thiamine diphosphate (TDP), were significantly lower in HAS versus LAS rats. The activation of transketolase by the exogenous TDP (TDP-effect) was significantly higher in the liver and brain regions of HAS rats compared with LAS rats. The level of TDP in the liver and cerebellum of HAS rats was significantly lower compared with LAS rats. These results indicate a severe deficiency of TDP in HAS rats. HAS rats have a significantly lower activity of thiamine triphosphatase, the additional source of TDP. Accordingly, HAS rats have much higher thiamine triphosphate levels in the liver and brain, compared with LAS rats. There were no significant differences between groups with respect to the thiamine diphosphatase and thiamine kinase activity. Most of the above parameters had the intermediate values in CAS rats, compared with LAS and HAS rats. These data indicate the possible role of the thiamine phosphate esters and related enzymes in the mechanisms that bring about the differential sensitivity to the hypnotic effect of alcohol. HAS rats have the genetically mediated thiamine diphosphate deficiency and increased thiamine triphosphate levels, probably due to reduced activity of thiamine triphosphatase in the liver and brain, compared with LAS rats. It can be related with the higher initial sensitivity of HAS rats to hypnotic effect of ethanol.

Thiamin Is Decomposed Due to Anaphe spp. Entomophagy in Seasonal Ataxia Patients in Nigeria

A fairly high activity of a relatively heat-resistant thiaminase was detected and characterized from the pupae of an African silkworm Anaphe spp. which had been the putative cause of a seasonal ataxia and impaired consciousness in Nigerians. The thiaminase in the buffer extract of Anaphe pupae was type I (thiamin: base 2-methyl-4-aminopyrimidine methyl transferase EC 2.5.1.2), and the optimal temperature and pH were 70 degrees C and 8.0-8.5, respectively. Based on gel filtration chromatography, the molecules were estimated to be 200 kDa. Second substrates which could be utilized by the thiaminase were pyridoxine, amino acids, glutathione, taurine and 4-aminopyridine. Thiamin phosphate esters were inactive as substrates. This is the first report describing an insect thiaminase. Our results indicate the necessity of thorough heat treatment for the detoxification of the African silkworm, making the worm a safe source of high-quality protein.

Thiamine deficiency results in metabolic acidosis and energy failure in cerebellar granule cells: an in vitro model for the study of cell death mechanisms in Wernicke's encephalopathy.

Thiamine deficiency (TD) in both humans and experimental animals results in severe compromise of mitochondrial function and leads to selective neuronal cell death in diencephalic and cerebellar structures. To examine further the influence of TD on neuronal survival in relation to metabolic changes, primary cultures of rat cerebellar granule cells were exposed to thiamine-deficient medium for up to 7 days in the absence or presence of the central thiamine antagonist pyrithiamine (Py). Exposure of cells for 7 days to thiamine-deficient medium alone resulted in no detectable cell death. On the other hand, 50 microM Py treatment led to reductions of thiamine phosphate esters, decreased activities of the thiamine-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase, a twofold increase in lactate release (P < 0.001), a lowering of pH, and significant (58%, P < 0.001) cell death. DNA fragmentation studies did not reveal evidence of apoptotic cell death. Addition of 50 microM alpha-tocopherol (vitamin E) or 100 microM of butylated hydroxyanisole (BHA) to Py-treated cells resulted in significant neuroprotection. On the other hand, addition of 10 microM MK-801, an NMDA receptor antagonist, was not neuroprotective. These results suggest that reactive oxygen species (ROS) play a major role in thiamine deficiency-induced neuronal cell death. Insofar as this experimental model recapitulates the metabolic and mitochondrial changes characteristic of thiamine deficiency in the intact animal, it might be useful in the elucidation of mechanisms involved in the neuronal cell death cascade resulting from thiamine deficiency.

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme alpha-ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.

コイのビタミンＢ１の代謝

Radioactive thiamin, C-2 of thiazole labeled with 14C, was parenterally administered to carp. After 24h, thiamin, its phosphate esters and thiazole in the eleven kinds of tissues and in the water of the aquarium, were fractionated by SP-Sephadex C25, and their radioactivities were measured. The radioactive thiamin was used to synthesize thiamin phosphate esters, mostly thiamin diphosphate (TDP), retained as a free form of thiamin, or decomposed to thiazole. The ratios of 14C-TDP to the total incorporated radioactivity in the ordinary muscle, kidney, and red muscle were 47, 42, and 34%, respectively. The eye tissue contained 14C-thiamin corresponding to over 50% of total incorporated radioactivity, which was very different from the other tissues. The ratios of 14C-thiazole, a decomposed product of 14C-thiamin, was highest in the kidney, followed by the gill and the heart. Furthermore, the amount of 14C-thiazole excreted into the water of the aquarium was ten times more than that in these tissues. To ascertain whether thiazole was truly produced by tissue such as liver, 14C-thiamin was added to cultured carp hepatocytes, and the radioactivities were analyzed both in the cell and in the medium by the same method. After 24h incubation, 14C-thiazole was mainly found in the medium, while no production of 14C-thiazole was observed in control experiments, in which 14C-thiamin was added to the cells treated with trichloroacetic acid or to the medium without hepatocytes. Furthermore, cultured hepatocytes produced 14C-TDP corresponding to 71% of total radioactivity incorporated in the cells. These results indicate that thiazole is produced by carp liver in vivo, and thiamin phosphate esters, especially TDP, are also synthesized from thiamin.

Kinetics of thiamin and thiamin phosphate esters in human blood, plasma and urine after 50 mg intravenously or orally.

The concentrations of thiamin and thiamin monophosphate and diphosphate in plasma and whole blood samples were assessed in six healthy subjects for 12 h and in urine for 24 h following an IV and PO bolus dose of 50 mg thiamin HCl. Unphosphorylated thiamin increased rapidly in plasma after IV administration and then decreased to its initial value within 12 h in all but one subject; the half-life was 96 min. Thiamin mono and -diphosphate increased moderately (56%), and decreased slowly; the half-life of diphosphate was 664 min. Within 24 h, 53% of the administered dose was recovered in the urine, indicating a restricted distribution. After oral administration, the peak thiamin concentration in plasma was reached after 53 min and the concentration then had increased to 179% of its initial value. The elimination half-life was 154 min, and only 2.5% of the given dose was recovered in the urine. The relative bioavailability of thiamin was 5.3%. A moderate amount of the administered thiamin was stored in blood. Other body tissues must play an important part, therefore, in the distribution of thiamin.

Purification and characterization of thiamine triphosphatase from bovine brain

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine bran has been purified 68 000-fold to an electropheretically homogenous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650 M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33 900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg 2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revelaed a biphasic saturation curve being described by higher-degree rational polynomials.

THE CONCENTRATION OF THIAMIN AND THIAMIN PHOSPHATE ESTERS IN PATIENTS WITH ALCOHOLIC LIVER CIRRHOSIS

The blood and plasma concentrations of thiamin and thiamin phosphate esters were determined concomitantly by high-performance liquid chromatography (HPLC) in 22 patients with alcoholic liver cirrhosis, and also in 10 of them 24 hr after a 100 mg thiamin i.m. injection. Sixteen patients were abstaining from alcohol at the time of the study, 6 were currently misusing alcohol. The control group included 30 healthy volunteers, of whom 10 were given the same thiamin injection as the patients. Blood thiamin diphosphate was the only compound decreased in the abstaining patients compared to controls (70.9 +/- 21.9 nmol/l vs. 84.4 +/- 19.0 nmol/l), but all thiamin compounds in blood and plasma were decreased in the misusing patients. All thiamin compounds (except blood monophosphate) were also significantly lower in the misusing than in the abstaining patients (plasma thiamin: 5.3 +/- 1.3 vs. 11.7 +/- 8.3 nmol/l; plasma monophosphate: 1.0 +/- 1.1 vs. 4.1 +/- 2.9 nmol/l; blood diphosphate: 45.7 +/- 18.3 vs. 70.9 +/- 21.9 nmol/l). Thiamin phosphorylation ratio was decreased in the patients after thiamin administration compared to controls (2.83 +/- 0.74 vs. 3.68 +/- 0.58). Plasma thiamin was higher in the abstaining patients than in the controls (11.7 +/- 8.4 nmol/l vs. 7.3 +/- 2.5 nmol/l), and above the mean + 2 SD of the controls in 31% of the abstaining patients. In conclusion, current ethanol misuse is associated with low thiamin concentrations, and liver cirrhosis is associated with a decreased thiamin diphosphate concentration and thiamin phosphorylation.

Blood and serum thiamin and thiamin phosphate esters concentrations in patients with alcohol dependence syndrome before and after thiamin treatment.

The blood and serum concentrations of free thiamin and its three phosphate esters were determined concomitantly by a new high-performance liquid chromatography (HPLC) method in 30 patients with alcohol dependence syndrome on admission to hospital and 24 hr after thiamin injection. We studied 24 men and 6 women; mean age, 50 years (range 21 to 69); mean ethanol consumption during the last 30 days, 164 +/- 119 g/day. A control group included 40 healthy volunteers (25 men, 15 women), of whom 10 were given the same thiamin injection as were the patients. Thiamin monophosphate was significantly reduced in the patients compared with controls before treatment (men 2.9 +/- 2.3 and 5.9 +/- 3.1 nmol/liter) and after (8.1 +/- 5.1 and 19.5 +/- 8.1 nmol/liter). On admission, free thiamin and thiamin diphosphate were similar in controls and in patients in whole blood (B) and serum (S) and increased similarly after treatment (mean B-thiamin diphosphate in male patients: 149 +/- 64 to 238 +/- 88 nmol/liter, in controls: 179 +/- 40 to 289 +/- 18 nmol/liter). However, seven patients had extremely high free thiamin values. The phosphorylation ratio was lower in patients than in controls (p less than 0.05), before and after treatment. Finally, the mean B-diphosphate was lower in patients not taking vitamins (116 +/- 48 nmol/liter and 172 +/- 57 nmol/liter) and in patients with polyneuropathy (118 +/- 54 nmol/liter and 173 +/- 52 nmol/liter), compared with the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Concomitant determination of thiamin and its phosphate esters in human blood and serum by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of thiamin and thiamin phosphate esters in human blood or serum has been developed. The eluent consists of acetonitrile and phosphate buffer, in the ratios 90:10 (v/v) for the elution of thiamine and 60:40 (v/v) for the phosphate esters. The four compounds are eluted within 15 min. The detection limit is 13-16 fmol. Between-assay variation is 5-11%. Samples of whole blood and serum from 30 healthy adults were analysed. The following reference values were obtained for 15 females 15 males (nM, mean +/- S.D.). In serum: thiamin, 10.9 +/- 2.9/16.9 +/- 3.3; thiamin monophosphate, 8.3 +/- 1.5/3.7 +/- 1.5. In whole blood: thiamin, 29.6 +/- 10.0/33.4 +/- 10.4; thiamin monophosphate, 9.7 +/- 2.3/10.9 +/- 5.1; thiamin diphosphate, 121 +/- 29.6/165 +/- 40.4.

Effect of chronic alcohol administration on transketolase in the brain and the liver of rats

To estimate the nutritional and the pathological states in thiamin-deficiency-related diseases, especially Wernicke-Korsakoff syndrome, we studied the relationship among transketolase activity, transketolase concentration, and thiamin phosphate esters in rats chronically fed alcohol. In the brain of alcohol-fed rats, the enzyme activity and concentration decreased although there was no positive correlation between the two. On the contrary, transketolase activity in the liver correlated positively with concentration, and both transketolase activity and concentration were decreased in the thiamin-deficient groups. These findings suggest that transketolase in the brain may be different from that in the liver and that the alteration of the enzyme activity in the brain may be based on the conformational change of the protein molecule caused by chronic alcohol administration.