High-affinity Thiamine Transporter Research Articles

Thiamine-responsive megaloblastic anemia syndrome: A case report

Thiamine-responsive megaloblastic anemia (TRMA) syndrome is a rare autosomal recessive disorder characterized by a cardinal triad consisting of megaloblastic anemia, sensorineural deafness, and diabetes mellitus. TRMA is caused by mutations in the gene SLC19A2 encoding a high-affinity thiamine transporter, which disturbs the active thiamine uptake into cells. We report here on a 1-year and 9-month-old female baby with megaloblastic anemia, thrombocytopenia, and diabetes mellitus. Our patient had significant sensorineural hearing loss that was late to appear. Diagnosis was based on clinical features and dramatic response of anemia, thrombocytopenia, and glycemic control to thiamine therapy. In view of the clinical history of the patient, targeted gene sequencing of genes causing monogenic diabetes was performed. The genes selected comprised 40 gene loci and were sequenced by Illumina sequencing platform. We found a novel homozygous deletion mutation of complete exon 2 of the SLC19A2 gene (ENS0236137), which we believe has not been described to be associated with TRMA. Exon 2 of SLC19A2 gene includes amino acid from 69 to 269. Thiamine resulted in rapid normalization of the hemoglobin level with improvement in glycemic control. TRMA syndrome should be kept in mind in the differential diagnosis of megaloblastic anemia, deafness, and diabetes mellitus. Early introduction of high-dose thiamine can reverse anemia and allow more glycemic control for diabetes. We conclude that genetic analysis confirms the diagnosis of TRMA. As exogenous thiamine is shown to reverse some of the clinical features of the disease, a genetic diagnosis of TRMA syndrome is extremely important.

PB1977 LATE DIAGNOSIS IN SYRIAN IMMIGRANT PATIENT; THIAMINE-RESPONSIVE MEGALOBLASTIC ANEMIA SYNDROME

Background: Thiamine-responsive megaloblastic anemia (TRMA) also known as Rogers syndrome was first described by Porter et al in 1969. It is characterized by diabetes mellitus, megaloblastic anemia and progressive sensorineural hearing loss. TRMA is a rare autosomal recessive disorder. Aims: This syndrome is seen especially in consanguineous marriages and isolated communities. İt has been reported in fewer than 80 families worldwide. Diagnosis relies on uncovering genetic variations (alleles) in the SLC19A2 gene, encoding for a high affinity thiamine transporter. This transporter is essentially present in hematopoietic stem cells, pancreaticc beta cells and inner ear cells, explaining the clinical manifestations of the disease. The disease is usually diagnosed in childhood period. Methods: We presented a 21-year-old patient who was diagnosed with TRMA with the findings of pancytopenia. Results: Case Report: A 21-year-old male patient was referred to our center upon the symptoms of fatigue, hearing loss and pancytopenia. He was emigrated from Syria to Turkey for years ago because of the Syrian War. Mother and father are first-degree relatives. He has hearing loss and inarticulated since hewas 1 year old. He was diagnosed as type 1 diabetes mellitus at 10 years old and treated with insulin. Erythrocyte transfusion had been performed several times due to the anemia. In laboratory tests; Wbc:2480 10^3 /μL, Hgb:4.3 g/dL Plt: 8000 10^3 /μL, MCV:81 fL, Glukoz: 345 mg/dL, Crea1nin:0,7 mg/dL, ldh:92 U/L, Ferri1n:1140 ug/L, VitB12: 702 ng/L, Folat: 5,2 ug/L were found. There was hepatomegaly and splenomegaly in abdominal ultrasonography. Megaloblastic changes were seen in peripheral blood smear, but there was no atypical cells. Bone marrow biopsy and flow cytometric evaluation were normal. Audiologic evaluation showed bilateral severe sensorineural hearing. Considering the laboratory test and clinical findings, a diagnosis of TRMA was suspected and the patient was started on thiamine hydrochloride (100 mg orally daily). An increase in leukocyte and platelet counts was observed on the 5th day of thiamine treatment. On the 10th day of treatment, an increase in hemoglobin level was observed. His pancytopenia was completely resolved. WBC: 7000 10^3 /μL, Hgb: 15 g/dL Plt: 234000 10^3 /μL, mcv: 91 were detected afer two months of treatment. At the same time genetic evaluation was initiated. Homozygous mutation in the SLC19A2 gene (c.242_243insA (p.Y81 ∗) (p.Tyr81 ∗) was detected as a result of genetic evaluation. Summary/Conclusion: Literature showed that the cases of TRMA are mostly diagnosed during childhood period. Our case was diagnosed at the age of 21. He is different from the others with this respect. Pancytopenia has been described several times in Rogers syndrome. There were serious cytopenia in our case at the time of diagnosis. Rapid improvement in cytopenia was seen afer the treatment with thiamine. In conclusion TRMA is a rare autosomal recessive disorder which typically has a clinical triad: megaloblastic anemia, diabetes mellitus and sensorineural hearing loss. Thiamine treatment should be continued for lifelong. Family screening should be done and genetic counseling should be offered when diagnosed.

L’anémie mégaloblastique thiamine dépendante ou syndrome de Rogers : une revue de la littérature

Thiamine-responsive megaloblastic anemia (TRMA), also known as Rogers syndrome, is a rare autosomal recessive disease characterized by three main components: megaloblastic anemia, diabetes mellitus and sensorineural deafness. Those features occur in infancy but may arise during adolescence. Diagnosis relies on uncovering genetic variations (alleles) in the SLC19A2 gene, encoding for a high affinity thiamine transporter. This transporter is essentially present in hematopoietic stem cells, pancreatic beta cells and inner ear cells, explaining the clinical manifestations of the disease. Based on a multidisciplinary approach, treatment resides on lifelong thiamine oral supplementation at pharmacological doses, which reverses anemia and may delay development of diabetes. However, thiamine supplementation does not alleviate already existing hearing defects.

THIAMINE-RESPONSIVE MEGALOBLASTIC ANEMIA WITH EPILEPSY: A CASE REPORT

Abstract Background: Thiamine-responsive megaloblastic anemia (TRMA) syndrome is a rare autosomal recessive disorder characterized by cardinal triad features of megaloblastic anemia, sensorineural deafness and diabetes mellitus. TRMA manifestation is caused by mutations in the gene SLC19A2 encoding a high-affinity thiamine transporter, which disturbs the active thiamine uptake into cells. Case presentation: We herein report a 16-year-old Egyptian male with megaloblastic anemia, deafness and diabetes in addition he had epilepsy and hepatosplenomegaly. Diagnosis was based on clinical features and dramatic response of anemia to thiamine therapy. Thiamine in a dose of 250 mg/day resulted in rapid normalization of the hemoglobin level; which lasted for the whole 12-month follow up period; with partial improvement in glycemic control as reflected by decrease in glycosylated hemoglobin and regression of hepatosplenomegaly. Deafness and seizures showed no response to thiamin. Conclusions: TRMA syndrome should be ...

Novel nonsense mutation (p.Ile411Metfs*12) in the SLC19A2 gene causing Thiamine Responsive Megaloblastic Anemia in an Indian patient

Thiamine-responsive megaloblastic anemia (TRMA), an autosomal recessive disorder, is caused by mutations in SLC19A2 gene encodes a high affinity thiamine transporter (THTR-1). The occurrence of TRMA is diagnosed by megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Here, we report a female TRMA patient of Indian descent born to 4th degree consanguineous parents presented with retinitis pigmentosa and vision impairment, who had a novel homozygous mutation (c.1232delT/ter422; p.Ile411Metfs*12) in 5th exon of SLC19A2 gene that causes premature termination of hTHTR-1. PROSITE analysis predicted to abrogate GPCRs family-1 signature motif in the variant by this mutation c.1232delT/ter422, suggesting uncharacteristic rhodopsin function leading to cause RP clinically. Thiamine transport activity by the clinical variant was severely inhibited than wild-type THTR-1. Confocal imaging had shown that the variant p.I411Mfs*12 is targeted to the cell membrane and showed no discrepancy in membrane expression than wild-type. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of hTHTR-1 causing TRMA in an Indian patient through functionally impaired thiamine transporter activity.

Recurrent psychiatric manifestations in thiamine‐responsive megaloblastic anemia syndrome due to a novel mutation c.63_71 delACCGCTC in the gene SLC19A2

Thiamine-responsive megaloblastic anemia syndrome (TRMA) is a recessive condition that mainly manifests as megaloblastic anemia, hearing loss, and diabetes.1, 2 It is caused by mutations in the gene SLC19A2, which codes for a high-affinity thiamine transporter.3 Our male proband had sensorineural deafness diagnosed at 4 years of age and insulin-dependent diabetes diagnosed at 5 years. At 19 he was found to have myopia, astigmatism and decreased peripheral visual fields, as well as megaloblastic anemia with low serum thiamine level. He was started on 100 mg thiamine daily, which was later decreased to 50 mg. The proband's brother had similar features. He died at 18 years, reportedly from a heart problem. The proband had a history of inappropriate sexual behavior since the age of 13 and a long history of explosive/aggressive/assaulting behavior and transient paranoid ideations. Aggressive behavior was triggered by frustration around food items and by misperceptions and beliefs that others were ‘bad-mouthing’ him. He communicated through sign language and a few spontaneous verbalizations and was oriented x3. Irritable mood, impulsivity, and paranoid ideations were noted. Insight was impaired and judgment was fair for his mental age. A gradual decline from low–normal to mild intellectual disability was noted. Thiamine treatment during 18 months substantially decreased the frequency and severity of his explosive/aggressive episodes but did not affect his transient paranoid ideations. Multiple psychotropic medications were previously ineffective at controlling these episodes. Testing for mutations in SLC19A2 revealed homozygosity for the c.63_71delACCGCTC mutation, resulting in a frameshift and truncated protein. This mutation was not previously reported.2 To our knowledge, one other patient with TRMA and a psychotic episode was previously reported.4 This female patient had only one documented psychotic episode with mood changes. Neuropsychological manifestations related to thiamine deficiency have been well established in alcoholic populations known for nutrient deficits.5 Thiamine administration controlled the progressive encephalopathy and the explosive/aggressive behavior in our patient, but his recurrent mood disorder with paranoid ideations did not resolve with thiamine supplementation. In addition to the thiamine deficiency, the novel SLC19A2 mutation reported here may have contributed to the patient's psychotic manifestations by an unknown mechanism. No conflict of interest is reported for any of the authors. This report was partially supported by The Office of People with Developmental Disabilities of New York State. The sponsor did not have any role in the conducting or the reporting of the study. Consent was obtained from the patient's legal guardian.

Inner Hair Cells Are Not Required for Survival of Spiral Ganglion Neurons in the Adult Cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.

Thiamine-Responsive Megaloblastic Anemia Syndrome With Atrial Standstill

Thiamine-responsive megaloblastic anemia (TRMA) syndrome is an uncommon autosomal recessive disorder. The disease is caused by mutations in the gene, SLC19A2, encoding a high-affinity thiamine transporter, which disturbs the active thiamine uptake into cells. Major features include megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Cardiac malformations with conduction defects and/or dysrhythmias, have also been described in some patients. To our knowledge, only 13 TRMA patients with cardiac defects have been reported. Here, we describe the first case of TRMA syndrome with atrial standstill, probably caused by a 2 base-pair deletion in exon 4 (1147delGT) of the gene SLC19A2.

Thiamine-responsive megaloblastic anaemia: a cause of syndromic diabetes in childhood

Thiamine-responsive megaloblastic anaemia (TRMA) is a rare autosomal recessive condition, characterized by megaloblastic anaemia, non-autoimmune diabetes mellitus, and sensorineural hearing loss. We describe three infants with TRMA from two consanguineous Pakistani families, who were not known to be related but originated from the same area in Pakistan. All children were homozygous, and the parents were heterozygous for a c.196G>T mutation in the SLC19A2 gene on chromosome 1q23.3, which encodes a high-affinity thiamine transporter. The result is an abnormal thiamine transportation and vitamin deficiency in the cells. Thiamine in high doses (100-200 mg/d) reversed the anaemia in all our patients. Two patients discontinued insulin treatment successfully after a short period, while the third patient had to continue with insulin. The hearing loss persisted in all three children. The diagnosis of TRMA should be suspected in patients with syndromic diabetes including hearing loss and anaemia, even if the latter is only very mild and, particularly, in the case of consanguinity.

Deletion of SLC19A2, the High Affinity Thiamine Transporter, Causes Selective Inner Hair Cell Loss and an Auditory Neuropathy Phenotype

Mutations in the gene coding for the high-affinity thiamine transporter Slc19a2 underlie the clinical syndrome known as thiamine-responsive megaloblastic anemia (TRMA) characterized by anemia, diabetes, and sensorineural hearing loss. To create a mouse model of this disease, a mutant line was created with targeted disruption of the gene. Cochlear function is normal in these mutants when maintained on a high-thiamine diet. When challenged with a low-thiamine diet, Slc19a2-null mice showed 40-60 dB threshold elevations by auditory brainstem response (ABR), but only 10-20 dB elevation by otoacoustic emission (OAE) measures. Wild-type mice retain normal hearing on either diet. Cochlear histological analysis showed a pattern uncommon for sensorineural hearing loss: selective loss of inner hair cells after 1-2 weeks on low thiamine and significantly greater inner than outer hair cell loss after longer low-thiamine challenges. Such a pattern is consistent with the observed discrepancy between ABR and OAE threshold shifts. The possible role of thiamine transport in other reported cases of selective inner hair cell loss is considered.

Role of Defective High-Affinity Thiamine Transporter slc19a2 in Marrow from a Mouse Model of Thiamine-Responsive Anemia Syndrome: Evidence for Defective Deoxyribose and Heme Synthesis.

The rare autosomal recessive human disorder, TRMA syndrome (thiamine-responsive anemia with diabetes and deafness, OMIM 249270 ), is caused by mutations in the high affinity vitamin B1 transporter, SLC19A2. In human TRMA, ringed sideroblasts and megaloblastic changes are found in the marrow, suggesting potential defects in heme synthesis or mitochondrial function, and DNA synthesis, respectively. The mechanism whereby defective marrow thiamine transport causes these findings in the marrow was examined in a mouse model for TRMA. We previously demonstrated in fibroblasts from humans with TRMA a defect in the rate and pathway used for de novo ribose synthesis using Stable Isotope Dynamic Metabolic Profiling (SIDMaP analysis - Boros et al, Blood 102: 3556, 2003).To examine whether this fibroblast defect accurately defined the marrow pathophysiology, we have now examined this pathway in detail in mice defective for the orthologous slc19a2 gene by targeted gene disruption (Fleming et al, Mol Genet Metab 80:234, 2003). Slc19a2 mutant (−/−) or wildtype (+/+) mice were subjected to normal mouse chow (22 mg/kg thiamine), or thiamine deficient chow diet. Diets were confirmed thiamine deficient by chemical analysis. To examine heme biosynthesis, we used 55Fe-transferrin, ex vivo, to label marrow from +/+ or −/− mice maintained for 10 days in thiamine replete or deficient chow. Mutant mice had cell surface labeling indistinguishable from +/+ at 0°C (P>0.3), reflecting available transferrin receptors. Total cellular 55Fe uptake at 37 C was equal to or slightly greater than that of +/+ marrow in thiamine depleted state. However, incorporation of 55Fe into heme lagged in −/− cells (7.3 +/− 0.4 pmol/106cells/7 min) compared to +/+ (8.6 +/− 0.5 pmol/106 cells, P =0.018 in triplicate experiments). To examine deoxyribose (dRibose) synthesis, mice were maintained on thiamine replete or depleted chow for 10 days, then [1,2 13C] glucose was administered intraperitoneally overnight and 3 hours before sacrifice and marrow harvest. DNA from whole marrow and from sorted basophilic erythroblasts (CD71+/Ter119+) was isolated, hydrolized, derivatized, and dRibose analyzed by GC-MS. The initial rate of incorporation of 13C into DNA dRibose, reflecting de novo synthetic rate, was reduced 34% in basophilic erythroblasts of −/− mice after thiamine depletion (n=3 in duplicate experiments, P<0.001). dRibose enrichment at position C1 and C2 (m2) compared to C1 onl1 (m1) reflects synthesis via transketolase (thiamine dependent, non-oxidative, m2), vs G6PD (oxidative, non-thiamine dependent, m1). The m2/m1 ratio was 0.73 in +/+ cells, but only 0.36 in −/− erythroblasts, demonstrating defective non-oxidative branch of the pentose phosphate pathway. Thus, defective slc9a2 leads to two distinct erythropoietic defects. De novo DNA dRibose synthesis via transketolase is markedly impaired. Further, while iron uptake by marrow cells is maintained at normal levels, the heme biosynthetic rate is decreased in −/− mice in thiamine-depleted status. This is the first direct demonstration of a role for thiamine in normal erythrocyte heme and DNA metabolism. Together, these defects can account directly for the meglaoblastosis and sideroblasts of human SLC19A2 defects in TRMA.

Direct genomic PCR sequencing of the high affinity thiamine transporter (SLC19A2) gene identifies three genetic variants in Wernicke Korsakoff syndrome (WKS)

Direct genomic PCR sequencing of the high affinity thiamine transporter (SLC19A2) gene identifies three genetic variants in Wernicke Korsakoff syndrome (WKS)

Male infertility and thiamine-dependent erythroid hypoplasia in mice lacking thiamine transporter Slc19a2

Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity thiamine transporter gene SLC19A2. To study the role of thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 −/− mice are viable and females are fertile. Male −/− mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 ± 0.04 knockout vs. 0.35 ± 0.05 wild type, P<0.000005). The lack of developing germ cells beyond primary spermatocytes suggests an arrest in spermatogenesis prior to meiosis II. Nuclear chromatin changes indicative of apoptosis are present. No mature sperm are found in the tubules or epididymis. This phenotype suggests a previously unknown role for thiamine transport in spermatogenesis and male fertility. Slc19a2 −/− mice on a pure 129/Sv background develop reticulocytopenia after two weeks on thiamine-depleted chow with a virtual absence of reticulocytes in the peripheral blood (0.12% knockout vs. 2.58% wild type, P=0.0079). Few erythroid precursors are found in the bone marrow. Contrary to human TRMA syndrome, we see no evidence of megaloblastosis or ringed sideroblasts in the bone marrow of Slc19a2 −/− mice in thiamine-replete or thiamine-deficient dietary states. Phenotypic differences between TRMA patients and Slc19a2 −/− mice might be explained by dissimilar tissue expression patterns of the transporter, as well as by differing metabolic needs and possible different species-specific contributions of the related thiamine transporter Slc19a3.

TRMA syndrome (thiamine-responsive megaloblastic anemia): a case report and review of the literature.

Thiamine-responsive megaloblastic anemia syndrome (TRMA) is an autosomal recessive disorder with features that include megaloblastic anemia, mild thrombocytopenia and leukopenia, sensorineural deafness and diabetes mellitus. In this disease, the active thiamine uptake into cells is disturbed. Treatment with pharmacological doses of thiamine ameliorates the megaloblastic anemia and diabetes mellitus. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We present a 5-yr-old-boy with TRMA and, because of its rarity, we review the literature.

Characterization of a Murine High-Affinity Thiamine Transporter, Slc19a2

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.

Identification and Characterization of the Human and Mouse SLC19A3 Gene: A Novel Member of the Reduced Folate Family of Micronutrient Transporter Genes

We report here the isolation, characterization, and chromosomal localization of the genes encoding the human and corresponding murine orthologue of solute carrier family 19A member 3 (SLC19A3). Human SLC19A3 encodes a 496-amino-acid residue protein with a predicted molecular weight of 56 kDa that shares sequence similarity to both SLC19A1 (reduced folate transporter (RFC-1)) and SLC19A2 (high affinity thiamine transporter (THTR-1)). Like the SLC19A1 and SLC19A2 proteins, SLC19A3 contains 12 putative transmembrane domains. The human SLC19A3 gene is widely expressed, with the most abundant expression observed in placenta, kidney, and liver, and has been mapped to chromosome 2q37. The murine SLC19A3 gene maps to central chromosome 1 in the region defined as a seizure susceptibility locus in the DBA/2J mouse strain. This article describes the identification of SLC19A3, a gene encoding a novel solute transporter, and establishes murine SLC19A3 as a candidate gene for seizures in the DBA/2J mouse.

Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts.

We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5-14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10-30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400-550 nM; Vmax 11 pmol/min/10(6) cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death.

Thiamine homeostasis in neuroblastoma cells

We recently showed that thiamine uptake by neuroblastoma cells is mediated by two saturable transport systems: the first with high affinity for thiamine ( K m = 35 nM) is blocked by veratridine; the other, with low affinity is blocked by Ca 2+. The driving force for thiamine uptake is its phosphorylation to thiamine diphosphate (TDP) by thiamine pyrophosphokinase and subsequent binding of this cofactor to apoenzymes. Our results suggest that cells of neuronal origin possess mechanisms regulating the intracellular concentration of thiamine. At low external thiamine, the vitamin is taken up by a high-affinity transporter and pyrophosphorylated in thiamine diphosphate (TDP) : this is the TDP pool of slow turnover. An intraover extracellular concentration gradient of free thiamine is observed at low external concentration of the vitamin. At higher external thiamine concentration, TDP accumulation is limited by the binding capacitiy to the apoenzymes and unbound TDP (i.e. a small pool of fast turnover) is quickly hydrolyzed. Thiamine is slowly released by the cells by at least two different mechanisms. The first, accounting for a maximum of 50% of total thiamine release, is stimulated by external thiamine and is blocked by veratridine, suggesting that it is a self-exchange mechanism catalyzed by the high affinity thiamine transporter. The remaining thiamine efflux is neither sensitive to veratridine nor to Ca 2+ and its mechanism is unknown. About 25% of intracellular thiamine is not released, even after treatment of the cells with digitonin, thus maintaining an apparent gradient. This suggests a binding or sequestration in intracellular compartments. In neuroblastoma cells, the affinity of the transporter for thiamine is the highest reported so far, but the rate of transport is 2–3 orders of magnitude lower than in hepatocytes. In vivo, liver may be a thiamine reservoir which is replenished at high plasma thiamine concentrations, while the brain would remain able to pump thiamine (albeit slowly) and maintain steep gradients even when the external thiamine concentration becomes very low.