Low expression of thiamine pyrophosphokinase-1 contributes to brain susceptibility to thiamine deficiency.

Thiamine deficiency is a well-known risk factor for the development of severe encephalopathy, such as Wernicke encephalopathy and Korsakoff syndrome, but the underlying mechanism is still mysterious. This study aims to investigate the expression levels of thiamine metabolism genes in different tissues and their impact on brain susceptibility to thiamine deficiency. The mRNA and protein levels of four genes known to be associated with thiamine metabolism: thiamine pyrophosphokinase-1 ( Tpk ), Solute carrier family 19 member 2 ( Slc19a2 ), Slc19a3 , and Slc25a19 , in the brain, kidney, and liver of mice were examined. Thiamine diphosphate (TDP) levels were measured in these tissues. Mice were subjected to dietary thiamine deprivation plus pyrithiamine (PTD), a specific TPK inhibitor, or pyrithiamine alone to observe the reduction in TDP and associated pathological changes. TPK mRNA and protein expression levels were lowest in the brain compared to the kidney and liver. Correspondingly, TDP levels were also lowest in the brain. Mice treated with PTD or pyrithiamine alone showed an initial reduction in brain TDP levels, followed by reductions in the liver and kidney. PTD treatment caused significant neuron loss, neuroinflammation, and blood-brain barrier disruption, whereas dietary thiamine deprivation alone did not. TPK expression level is the best indicator of thiamine metabolism status. Low TPK expression in the brain appears likely to contribute to brain susceptibility to thiamine deficiency, underscoring a critical role of TPK in maintaining cerebral thiamine metabolism and preventing thiamine deficiency-related brain lesions.

TPK1 as a predictive marker for the anti-tumour effects of simvastatin in gastric cancer.

The potential anti-tumour role of statins has been reported in various cancer types, including gastric cancer (GC). However, there are no biomarkers to identify patients who may benefit most from this treatment. We tested the effects of statins on 8 GC cell lines. Genes differentially expressed in simvastatin-sensitive and -resistant cell lines were used to identify potential biomarkers of simvastatin sensitivity. Patient-derived cell lines were used to mimic in vivo conditions. In simvastatin-sensitive SNU-5 cells, the levels of the PARP and cleaved caspase-3 apoptosis markers increased upon exposure to simvastatin. The levels of the PARP and cleaved caspase-3 levels were unchanged by simvastatin exposure in simvastatin-resistant SNU-668 cells. The proportion of apoptotic cells was increased in SNU-5 cells but not in SNU-668 cells under the same drug exposure conditions. Comparison of differentially expressed genes (DEGs) in sensitive and resistant cell lines identified 31 genes potentially involved in the cellular response to simvastatin. We confirmed that RNA expression of the TPK1 DEG was significantly increased in simvastatin-sensitive cell lines. TPK1 knockdown in a simvastatin-sensitive GC SNU5 cell line, decreased the anti-tumour effects of simvastatin, while TPK1 overexpression enhanced the anti-tumour effect of simvastatin. Therefore, TPK1 expression can be used as a predictive marker of the anti-tumour effects of statin treatment in patients with cancer, especially in those with GC.

Thiamine phosphokinase deficiency and mutation in TPK1 presenting as biotin responsive basal ganglia disease.

The product of thiamine phosphokinase is the cofactor for many enzymes, including the dehydrogenases of pyruvate, 2-ketoglutarate and branched chain ketoacids. Its deficiency has recently been described in a small number of patients, some of whom had a Leigh syndrome phenotype. The patient who also had a Leigh phenotype was initially found to have a low concentration of biotin in plasma and massive urinary excretion of biotin. Despite treatment with biotin and thiamine, her disease was progressive. Mutations c.311delG and c.426G > C were found in the TPK1 gene.

Thiamin transport in Helicobacter pylori lacking the de novo synthesis of thiamin.

Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [3H]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a Km value of 28 µM. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 µM, which is much lower than the Km value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20 % of that in the wild-type strain at extracellular thiamin concentration of 1 µM, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [3H]thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the high-affinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.

The uremic toxin oxythiamine causes functional thiamine deficiency in end-stage renal disease by inhibiting transketolase activity.

Decreased transketolase activity is an unexplained characteristic of patients with end-stage renal disease and is linked to impaired metabolic and immune function. Here we describe the discovery of a link to impaired functional activity of thiamine pyrophosphate cofactor through the presence, accumulation, and pyrophosphorylation of the thiamine antimetabolite oxythiamine in renal failure. Plasma oxythiamine was significantly increased by 4-fold in patients receiving continuous ambulatory peritoneal dialysis and 15-fold in patients receiving hemodialysis immediately before the dialysis session (healthy individuals, 0.18 [0.11-0.22] nM); continuous ambulatory peritoneal dialysis patients, 0.64 [0.48-0.94] nM; and hemodialysis patients (2.73 [1.52-5.76] nM). Oxythiamine was converted to the transketolase inhibitor oxythiamine pyrophosphate. The red blood cell oxythiamine pyrophosphate concentration was significantly increased by 4-fold in hemodialysis (healthy individuals, 15.9 nM and hemodialysis patients, 66.1 nM). This accounted for the significant concomitant 41% loss of transketolase activity (mU/mg hemoglobin) from 0.410 in healthy individuals to 0.240 in hemodialysis patients. This may be corrected by displacement with excess thiamine pyrophosphate and explain lifting of decreased transketolase activity by high-dose thiamine supplementation in previous studies. Oxythiamine is likely of dietary origin through cooking of acidic thiamine-containing foods. Experimentally, trace levels of oxythiamine were not formed from thiamine degradation under physiologic conditions but rather under acidic conditions at 100(°)C. Thus, monitoring of the plasma oxythiamine concentration in renal failure and implementation of high-dose thiamine supplements to counter it may help improve the clinical outcome of patients with renal failure.

1,25-Dihydroxyvitamin D increases the gene expression of enzymes protecting from glucolipotoxicity in peripheral blood mononuclear cells and human primary endothelial cells.

Besides its classical function as an orchestrator of calcium and phosphorus homeostasis, vitamin D also affects insulin secretion and tissue efficiency. A number of studies have consistently reported the inverse relationship between vitamin D deficiency and type 2 diabetes. Activation of certain metabolic pathways and down-stream transcription factors may protect from glucolipotoxicity and their targeted activation -e.g. by vitamin D - might explain the detrimental role of vitamin D deficiency in diabetes. The aim of the study was to quantify gene and protein expression of selected enzymes involved in the protection from glucolipotoxicity, specifically glyoxalase 1 (GLO1), and other enzymes with antioxidant activity - hemoxygenase (HMOX), thiamin pyrophosphokinase (TPK1) and transketolase (TKT), under normo- and hyperglycemic conditions and upon addition of vitamin D in peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). The results of our study indicate that the active form of vitamin D regulates gene expression of enzymes opposing the harmful effect of glucolipotoxicity whose activities appear to be suppressed by hyperglycemia. However, we were unable to confirm this effect on protein expression. While we cannot speculate on the effect of vitamin D on diabetes itself our results support its role in the protection against existing glucolipotoxicity therefore possibly translating into the prevention of development of diabetic complications.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 µM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen.

Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization.

Bioinformatic and metabolomic analysis reveals miR-155 regulates thiamine level in breast cancer.

microRNA-155 (miR-155) is one of the well-known oncogenic miRNA implicated in various types of tumors. Thiamine, commonly known as vitamin B1, is one of critical cofactors for energy metabolic enzymes including pyruvate dehydrogenase, alpha ketoglutarate dehydrogenase, and transketolase. Here we report a novel role of miR-155 in cancer metabolism through the up-regulation of thiamine in breast cancer cells. A bioinformatic analysis of miRNA array and metabolite-profiling data from NCI-60 cancer cell panel revealed thiamine as a metabolite positively correlated with the miR-155 expression level. We confirmed it in MCF7, MDA-MB-436 and two human primary breast cancer cells by showing reduced thiamine levels upon a knock-down of miR-155. To understand how the miR-155 controls thiamine level, a set of key molecules for thiamine homeostasis were further analyzed after the knockdown of miR-155. The results showed the expression of two thiamine transporter genes (SLC19A2, SLC25A19) as well as thiamine pyrophosphokinase-1 (TPK1) were decreased in both RNA and protein level in miR-155 dependent manner. Finally, we confirm the finding by showing a positive correlation between miR-155 and thiamine level in 71 triple negative breast tumors. Taken altogether, our study demonstrates a role of miR-155 in thiamine homeostasis and suggests a function of this oncogenic miRNA on breast cancer metabolism.

Expanding the clinical and molecular spectrum of thiamine pyrophosphokinase deficiency: a treatable neurological disorder caused by TPK1 mutations.

Thiamine pyrophosphokinase (TPK) produces thiamine pyrophosphate, a cofactor for a number of enzymes, including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. Episodic encephalopathy type thiamine metabolism dysfunction (OMIM 614458) due to TPK1 mutations is a recently described rare disorder. The mechanism of the disease, its phenotype and treatment are not entirely clear. We present two patients with novel homozygous TPK1 mutations (Patient 1 with p.Ser160Leu and Patient 2 with p.Asp222His). Unlike the previously described phenotype, Patient 2 presented with a Leigh syndrome like non-episodic early-onset global developmental delay, thus extending the phenotypic spectrum of the disorder. We, therefore, propose that TPK deficiency may be a better name for the condition. The two cases help to further refine the neuroradiological features of TPK deficiency and show that MRI changes can be either fleeting or progressive and can affect either white or gray matter. We also show that in some cases lactic acidosis can be absent and 2-ketoglutaric aciduria may be the only biochemical marker. Furthermore, we have established the assays for TPK enzyme activity measurement and thiamine pyrophosphate quantification in frozen muscle and blood. These tests will help to diagnose or confirm the diagnosis of TPK deficiency in a clinical setting. Early thiamine supplementation prevented encephalopathic episodes and improved developmental progression of Patient 1, emphasizing the importance of early diagnosis and treatment of TPK deficiency. We present evidence suggesting that thiamine supplementation may rescue TPK enzyme activity. Lastly, in silico protein structural analysis shows that the p.Ser160Leu mutation is predicted to interfere with TPK dimerization, which may be a novel mechanism for the disease.

[Extracting and study of biochemical properties of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium].

The method of extraction and purification of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium has been elaborated. Kinetic characteristics of T-kinase from non-malignant and tumor tissue of women myometrium have been studied. It has been shown, that malignization of myometrium is accompanied by a decrease in affinity of thiamine pyrophosphokinase from tumor to thiamine and by an increase in sensitivity of the enzyme from tumor to thiochrome.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target.

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

Up-regulation of vitamin B1 homeostasis genes in breast cancer.

An increased carbon flux and exploitation of metabolic pathways for the rapid generation of biosynthetic precursors is a common phenotype observed in breast cancer. To support this metabolic phenotype, cancer cells adaptively regulate the expression of glycolytic enzymes and nutrient transporters. However, activity of several enzymes involved in glucose metabolism requires an adequate supply of cofactors. In particular, vitamin B1 (thiamine) is utilized as an essential cofactor for metabolic enzymes that intersect at critical junctions within the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and thiamine pyrophosphokinase-1 (TPK-1). Therefore, the objective of this study was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. Employing cDNA arrays of breast cancer and normal breast epithelial tissues, SLC19A2, SLC25A19 and TPK-1 were found to be significantly up-regulated. Similarly, up-regulation was also observed in breast cancer cell lines compared to human mammary epithelial cells. Thiamine transport assays and quantitation of intracellular thiamine and thiamine pyrophosphate established a significantly greater extent of thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells. Overall, these findings demonstrate an adaptive response by breast cancer cells to increase cellular availability of thiamine.

Identification of the thiamin pyrophosphokinase gene in rainbow trout: characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development.

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

Construction of a thiamine pyrophosphate high-producing strain of Aspergillus oryzae by overexpression of three genes involved in thiamine biosynthesis.

We have found a gene (thiP) encoding thiamine pyrophosphokinase (TPK) in the Aspergillus oryzae genome. No riboswitch-like region was found in the upstream region of thiP, although it was repressed probably by thiamine pyrophosphate (TPP) as well as thiA and nmtA, which are strictly regulated by TPP-riboswitch sequence. To improve the productivity of TPP in A. oryzae, we constructed the strain in which thiA, nmtA and thiP were overexpressed simultaneously. The resulting strain accumulated intracellular TPP 4-fold higher than did the control strain.

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport.

The renal thiamin reabsorption process plays an important role in regulating thiamin body homeostasis and involves both thiamin transporters-1 and -2 (THTR1 and THTR2). Chronic alcohol use is associated with thiamin deficiency. Although a variety of factors contribute to the development of this deficiency, effects of chronic alcohol use on renal thiamin transport have not been thoroughly examined. We addressed this issue by examining the effect of chronic alcohol feeding of rats with liquid diet on physiological and molecular parameters of renal thiamin transport. Chronic alcohol feeding caused a significant inhibition in carrier-mediated thiamin transport across the renal brush-border membrane and was evident as early as 2 wk after initiation of alcohol feeding. Similarly, thiamin transport across the renal basolateral membrane was significantly inhibited by chronic alcohol feeding. The inhibition in renal thiamin transport was associated with a marked decrease in the level of expression of THTR1 and -2 proteins, mRNAs, and heterogeneous nuclear RNAs. Chronic alcohol feeding also caused a significant reduction in the level of expression of thiamin pyrophosphokinase but not that of the mitochondrial thiamin pyrophosphate transporter. These studies show that chronic alcohol feeding inhibits the entry and exit of thiamin in the polarized renal epithelial cells and that the effect is, at least in part, mediated at the transcriptional level. These findings also suggest that chronic alcohol feeding interferes with the normal homeostasis of thiamin in renal epithelial cells.

Enzymes that control the thiamine diphosphate pool in plant tissues. Properties of thiamine pyrophosphokinase and thiamine-(di)phosphate phosphatase purified from Zea mays seedlings.

The pool of thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The thiamine pyrophosphokinase (TPK) activates thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to thiamine monophosphate (TMP) and then to free thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the K(m) values of 12.4microM and 4.7mM for thiamine and ATP, respectively, and the V(max) value of 360pmol TDPmin(-1)mg(-1) protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The K(m) values for TDP and TMP were 36microM and 49microM, respectively, and the V(max) value for TDP was significantly higher than for TMP (164 versus 60nmolesmin(-1)mg(-1) protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis.

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.

The human malaria parasite Plasmodium falciparum expresses an atypical N-terminally extended pyrophosphokinase with specificity for thiamine.

Vitamin B(1) is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum, are capable of synthesising vitamin B(1)de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B(1) vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min(-1) mg(-1) protein and specificity for thiamine with a K(m) value of 73 microM, while thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of PfTPK with a GFP marker visualised its cytosolic localisation.

Thiamine-responsive congenital lactic acidosis: clinical and biochemical studies.

We studied six infants with thiamine-responsive congenital lactic acidosis and normal pyruvate dehydrogenase complex activity in vitro, through clinical and biochemical analysis. In addition to elevated lactate and pyruvate levels, the data revealed increased urinary excretion of alpha-ketoglutarate, alpha-ketoadipate, and branched chain ketoacids, indicating functional impairment of thiamine-requiring enzymes, such as pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, alpha-ketoadipate dehydrogenase, and branched chain amino acid dehydrogenase. The metabolism of thiamine has not been investigated in patients with thiamine-responsive congenital lactic acidosis. We evaluated two specific transport systems, THTR-1 (SLC19A2) and THTR-2 (SLC19A3), and a pyrophosphorylating enzyme of thiamine, thiamine pyrophosphokinase (hTPK 1), in addition to pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity; no abnormality was found. Although the clinical features of thiamine-responsive congenital lactic acidosis are heterogeneous and clinical responses to thiamine administration vary, we emphasize the importance of early diagnosis and initiation of thiamine therapy before the occurrence of permanent brain damage. Careful monitoring of lactate and pyruvate would be useful in determining thiamine dosage.