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| thiamine(1+) diphosphate |
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| CHEBI:9532 |
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| A thiamine phosphate that is thiamine(1+) in which the hydroxy group has been replaced by a diphosphate group. It is the active form of vitamin B1 and an essential cofactor for enzymes in key metabolic pathways. |
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This entity has been manually annotated by the ChEBI Team.
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| eMolecules:2726636, ZINC000008215517 |
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more structures >>
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Ideal conformer Mrv1927 07192113343D starting HoverWatcher_5 Time for openFile(TPP - Ideal conformer Mrv1927 07192113343D 45 46 0 0 0 0 999 V2000 -6.0960 -2.2730 -0.0220 N 0 0 0 0 0 0 0 0 0 0 0 0 -7.0120 -1.6120 0.6560 C 0 0 0 0 0 0 0 0 0 0 0 0 -8.0350 -2.3820 1.4510 C 0 0 0 0 0 0 0 0 0 0 0 0 -7.0580 -0.2950 0.6600 N 0 0 0 0 0 0 0 0 0 0 0 0 -6.1720 0.4170 -0.0300 C 0 0 0 0 0 0 0 0 0 0 0 0 -6.2230 1.8010 -0.0240 N 0 0 0 0 0 0 0 0 0 0 0 0 -5.1880 -0.2540 -0.7600 C 0 0 0 0 0 0 0 0 0 0 0 0 -5.1780 -1.6340 -0.7330 C 0 0 0 0 0 0 0 0 0 0 0 0 -4.1620 0.5110 -1.5570 C 0 0 1 0 0 0 0 0 0 0 0 0 -3.0860 0.9560 -0.6680 N 0 3 0 0 0 4 0 0 0 0 0 0 -3.1220 2.1120 -0.0950 C 0 0 0 0 0 0 0 0 0 0 0 0 -1.7300 2.3340 0.8730 S 0 0 0 0 0 0 0 0 0 0 0 0 -1.1430 0.7380 0.4100 C 0 0 0 0 0 0 0 0 0 0 0 0 -2.0430 0.1920 -0.4140 C 0 0 0 0 0 0 0 0 0 0 0 0 -1.8750 -1.1820 -1.0090 C 0 0 0 0 0 0 0 0 0 0 0 0 0.1420 0.0990 0.8690 C 0 0 1 0 0 0 0 0 0 0 0 0 1.2670 0.4630 -0.1020 C 0 0 1 0 0 0 0 0 0 0 0 0 2.4860 -0.1440 0.3320 O 0 0 0 0 0 0 0 0 0 0 0 0 3.8800 0.0270 -0.4550 P 0 0 2 0 0 5 0 0 0 0 0 0 4.1810 1.4670 -0.6170 O 0 0 0 0 0 0 0 0 0 0 0 0 3.7650 -0.6630 -1.9050 O 0 0 0 0 0 0 0 0 0 0 0 0 5.0600 -0.6770 0.3830 O 0 0 0 0 0 0 0 0 0 0 0 0 6.6640 -0.5360 0.3920 P 0 0 1 0 0 5 0 0 0 0 0 0 7.1540 -0.3930 -0.9970 O 0 0 0 0 0 0 0 0 0 0 0 0 7.0830 0.7630 1.2460 O 0 0 0 0 0 0 0 0 0 0 0 0 7.3140 -1.8490 1.0590 O 0 0 0 0 0 0 0 0 0 0 0 0 -7.6600 -2.5480 2.4610 H 0 0 0 0 0 0 0 0 0 0 0 0 -8.9640 -1.8140 1.4960 H 0 0 0 0 0 0 0 0 0 0 0 0 -8.2200 -3.3430 0.9700 H 0 0 0 0 0 0 0 0 0 0 0 0 -6.9090 2.2590 0.4860 H 0 0 0 0 0 0 0 0 0 0 0 0 -5.5720 2.3130 -0.5300 H 0 0 0 0 0 0 0 0 0 0 0 0 -4.4320 -2.1860 -1.2850 H 0 0 0 0 0 0 0 0 0 0 0 0 -3.7480 -0.1360 -2.3310 H 0 0 0 0 0 0 0 0 0 0 0 0 -4.6330 1.3770 -2.0200 H 0 0 0 0 0 0 0 0 0 0 0 0 -3.9160 2.8350 -0.2100 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.2560 -1.1170 -1.9040 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.3940 -1.8360 -0.2820 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.8520 -1.5860 -1.2720 H 0 0 0 0 0 0 0 0 0 0 0 0 0.3920 0.4600 1.8660 H 0 0 0 0 0 0 0 0 0 0 0 0 0.0200 -0.9840 0.8950 H 0 0 0 0 0 0 0 0 0 0 0 0 1.0170 0.1010 -1.1000 H 0 0 0 0 0 0 0 0 0 0 0 0 1.3890 1.5450 -0.1280 H 0 0 0 0 0 0 0 0 0 0 0 0 3.5680 -1.6090 -1.8760 H 0 0 0 0 0 0 0 0 0 0 0 0 6.7930 0.7380 2.1680 H 0 0 0 0 0 0 0 0 0 0 0 0 8.2800 -1.8330 1.0980 H 0 0 0 0 0 0 0 0 0 0 0 0 1 2 2 0 0 0 0 1 8 1 0 0 0 0 2 3 1 0 0 0 0 2 4 1 0 0 0 0 3 27 1 0 0 0 0 3 28 1 0 0 0 0 3 29 1 0 0 0 0 4 5 2 0 0 0 0 5 6 1 0 0 0 0 5 7 1 0 0 0 0 6 30 1 0 0 0 0 6 31 1 0 0 0 0 7 8 2 0 0 0 0 7 9 1 0 0 0 0 8 32 1 0 0 0 0 9 10 1 0 0 0 0 9 33 1 0 0 0 0 9 34 1 0 0 0 0 10 11 2 0 0 0 0 10 14 1 0 0 0 0 11 12 1 0 0 0 0 11 35 1 0 0 0 0 12 13 1 0 0 0 0 13 14 2 0 0 0 0 13 16 1 0 0 0 0 14 15 1 0 0 0 0 15 36 1 0 0 0 0 15 37 1 0 0 0 0 15 38 1 0 0 0 0 16 17 1 0 0 0 0 16 39 1 0 0 0 0 16 40 1 0 0 0 0 17 18 1 0 0 0 0 17 41 1 0 0 0 0 17 42 1 0 0 0 0 19 18 1 1 0 0 0 19 20 2 0 0 0 0 19 21 1 0 0 0 0 19 22 1 0 0 0 0 21 43 1 0 0 0 0 22 23 1 0 0 0 0 23 24 2 0 0 0 0 23 25 1 0 0 0 0 23 26 1 0 0 0 0 25 44 1 0 0 0 0 26 45 1 0 0 0 0 M CHG 1 10 1 M END): 15 ms reading 45 atoms ModelSet: haveSymmetry:false haveUnitcells:false haveFractionalCoord:false 1 model in this collection. 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| Thiamine pyrophosphate (TPP or ThPP), or thiamine diphosphate (ThDP), or cocarboxylase is a thiamine (vitamin B1) derivative which is produced by the enzyme thiamine diphosphokinase. Thiamine pyrophosphate is a cofactor that is present in all living systems, in which it catalyzes several biochemical reactions.
Thiamine pyrophosphate is synthesized in the cytosol and is required in the cytosol for the activity of transketolase and in the mitochondria for the activity of pyruvate-, oxoglutarate- and branched chain keto acid dehydrogenases. To date, the yeast ThPP carrier (Tpc1p) the human Tpc and the Drosophila melanogaster have been identified as being responsible for the mitochondrial transport of ThPP and ThMP. It was first discovered as an essential nutrient (vitamin) in humans through its link with the peripheral nervous system disease beriberi, which results from a deficiency of thiamine in the diet.
TPP works as a coenzyme in many enzymatic reactions, such as:
Pyruvate dehydrogenase complex
Pyruvate decarboxylase in ethanol fermentation
Alpha-ketoglutarate dehydrogenase complex
Branched-chain amino acid dehydrogenase complex
2-hydroxyphytanoyl-CoA lyase
Transketolase |
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Read full article at Wikipedia
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InChI=1S/C12H18N4O7P2S/c1- 8-11(3-4-22-25(20,21)23-24(17,18)19)26-7-16(8)6-10-5-14-9(2)15-12(10)13/h5,7H,3-4,6H2,1-2H3,(H4-,13,14,15,17,18,19,20,21)/p+1 |
| AYEKOFBPNLCAJY-UHFFFAOYSA-O |
| CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=C(N)N=C(C)N=C1 |
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Mus musculus
(NCBI:txid10090)
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See:
PubMed
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Mus musculus
(NCBI:txid10090)
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Source: BioModels - MODEL1507180067
See:
PubMed
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Mus musculus
(NCBI:txid10090)
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From MetaboLights
See:
MetaboLights Study
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Mus musculus
(NCBI:txid10090)
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From MetaboLights
See:
MetaboLights Study
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Arabidopsis thaliana
(NCBI:txid3702)
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See:
PubMed
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Saccharomyces cerevisiae
(NCBI:txid4932)
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See:
PubMed
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Escherichia coli
(NCBI:txid562)
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See:
PubMed
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Escherichia coli
(NCBI:txid562)
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See:
PubMed
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Homo sapiens
(NCBI:txid9606)
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See:
PubMed
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Homo sapiens
(NCBI:txid9606)
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Found in
cerebrospinal fluid
(UBERON:0001359).
See:
PubMed
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Homo sapiens
(NCBI:txid9606)
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Found in
blood
(UBERON:0000178).
See:
PubMed
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Bronsted base
A molecular entity capable of accepting a hydron from a donor (Bronsted acid).
(via organic amino compound )
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fundamental metabolite
Any metabolite produced by all living cells.
cofactor
An organic molecule or ion (usually a metal ion) that is required by an enzyme for its activity. It may be attached either loosely (coenzyme) or tightly (prosthetic group).
water-soluble vitamin (role)
Any vitamin that dissolves in water and readily absorbed into tissues for immediate use. Unlike the fat-soluble vitamins, they are not stored in the body and need to be replenished regularly in the diet and will rarely accumulate to toxic levels since they are quickly excreted from the body via urine.
(via B vitamin )
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nutraceutical
A product in capsule, tablet or liquid form that provide essential nutrients, such as a vitamin, an essential mineral, a protein, an herb, or similar nutritional substance.
(via B vitamin )
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View more via ChEBI Ontology
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3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-[2-(diphosphooxy)ethyl]-4-methyl-1,3-thiazol-3-ium
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3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-(4,6,6-trihydroxy-4,6-dioxido-3,5-dioxa-4,6-diphosphahex-1-yl)thiazolium
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ChEBI
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3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-{[hydroxy(phosphonooxy)phosphoryl]oxy}ethyl)-4-methyl-1,3-thiazol-3-ium
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IUPAC
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ThDP
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ChEBI
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thiamin diphosphate
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KEGG COMPOUND
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thiamin pyrophosphate
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KEGG COMPOUND
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thiamine diphosphate
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KEGG COMPOUND
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thiamine pyrophosphate
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PDBeChem
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thiamine-pyrophosphate
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HMDB
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ThPP
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KEGG COMPOUND
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TPP
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KEGG COMPOUND
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136-08-3
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CAS Registry Number
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ChemIDplus
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3577792
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Reaxys Registry Number
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Reaxys
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Sabui S, Romero JM, Said HM (2021)
Developmental maturation of the colonic uptake process of the microbiota-generated thiamin pyrophosphate.
American journal of physiology. Gastrointestinal and liver physiology 320, G829-G835 [PubMed:33759569]
[show Abstract]
The water-soluble vitamin B1 is essential for normal human health and physiology. In its main biologically active form, i.e., thiamin pyrophosphate (TPP), the vitamin plays many critical roles in cell metabolism; thus, its deficiency leads to a variety of adverse effects. Humans/mammals obtain vitamin B1 from two exogenous sources: diet and gut microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP, and colonocytes can efficiently absorb this TPP via a high-affinity and specific carrier-mediated mechanism that involves the recently cloned colonic TPP transporter (cTPPT; product of SLC44A4 gene). There is nothing currently known about colonic uptake of TPP during early stages of life and whether the process undergoes developmental regulation. We addressed this issue using the mouse as animal model. Our results showed that colonic uptake of TPP undergoes developmental upregulation as the animal moves from the suckling period to weanling and adulthood. This upregulation in uptake was found to be associated with a parallel induction in level of expression of the cTPPT protein, mRNA, and heterogeneous nuclear RNA, suggesting possible involvement of transcriptional mechanism(s). We also found a parallel upregulation in the level of expression of the two nuclear factors that drive activity of the SLC44A4 promoter (i.e., CREB-1 and Elf-3) with maturation. These results demonstrate, for the first time, to our knowledge, that colonic TPP uptake process and cTPPT expression are developmentally upregulated and that this upregulation is likely driven via transcriptional mechanism(s).NEW & NOTEWORTHY The colonic carrier-mediated uptake process of the microbiota-generated and phosphorylated form of vitamin B1, i.e., thiamin pyrophosphate, undergoes ontogenic changes that parallel the development of the gut microbiota (and their ability to generate vitamins) during early stages of life. | Nauton L, Hecquet L, Théry V (2021)
QM/MM Study of Human Transketolase: Thiamine Diphosphate Activation Mechanism and Complete Catalytic Cycle.
Journal of chemical information and modeling 61, 3502-3515 [PubMed:34161071]
[show Abstract]
A computational model for human transketolase was proposed, showing that thiamine diphosphate activation was based on His110 in place of His481 reported in yeast transketolase. In addition, a complete catalytic reaction pathway was investigated using d-xylulose-5-phosphate and d-ribose-5-phosphate as substrates, showing at every step a perfect superimposition of our model with high-resolution crystallographic structures 3MOS, 4KXV, and 4KXX. This study shows that H2N4' of the active thiamine diphosphate "V form" no longer has a self-activating role but allows self-stabilization of the cofactor and of the Breslow intermediate. These advances in our knowledge of the human transketolase mechanism offer interesting prospects for the design of new drugs, this enzyme being involved in several diseases, and for a better understanding of the reactions catalyzed by transketolases from other sources. | Leandro J, Khamrui S, Wang H, Suebsuwong C, Nemeria NS, Huynh K, Moustakim M, Secor C, Wang M, Dodatko T, Stauffer B, Wilson CG, Yu C, Arkin MR, Jordan F, Sanchez R, DeVita RJ, Lazarus MB, Houten SM (2020)
Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex.
ACS chemical biology 15, 2041-2047 [PubMed:32633484]
[show Abstract]
DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies. | Noordally ZB, Trichtinger C, Dalvit I, Hofmann M, Roux C, Zamboni N, Pourcel L, Gas-Pascual E, Gisler A, Fitzpatrick TB (2020)
The coenzyme thiamine diphosphate displays a daily rhythm in the Arabidopsis nucleus.
Communications biology 3, 209 [PubMed:32372067]
[show Abstract]
In plants, metabolic homeostasis-the driving force of growth and development-is achieved through the dynamic behavior of a network of enzymes, many of which depend on coenzymes for activity. The circadian clock is established to influence coordination of supply and demand of metabolites. Metabolic oscillations independent of the circadian clock, particularly at the subcellular level is unexplored. Here, we reveal a metabolic rhythm of the essential coenzyme thiamine diphosphate (TDP) in the Arabidopsis nucleus. We show there is temporal separation of the clock control of cellular biosynthesis and transport of TDP at the transcriptional level. Taking advantage of the sole reported riboswitch metabolite sensor in plants, we show that TDP oscillates in the nucleus. This oscillation is a function of a light-dark cycle and is independent of circadian clock control. The findings are important to understand plant fitness in terms of metabolite rhythms. | Moraes RCM, Singulani MP, Gonçalves AC, Portari GV, Torrão ADS (2020)
Oral benfotiamine reverts cognitive deficit and increase thiamine diphosphate levels in the brain of a rat model of neurodegeneration.
Experimental gerontology 141, 111097 [PubMed:32987117]
[show Abstract]
It is well known that patients with Alzheimer's disease (AD) have imbalances in blood thiamine concentrations and lower activity of thiamine-dependent enzymes. Benfotiamine, a more bioavailable thiamine analog, has been proposed as an alternative to counteract these changes related to thiamine metabolism. Thus, our study aimed to analyze the effects of benfotiamine supplementation on brain thiamine absorption, as well as on parameters related to neuronal energy metabolism and disease progression in an experimental model of sporadic AD induced by intracerebroventricular injection of streptozotocin (STZ) in rats. The supplementation with 150 mg/kg of benfotiamine for 30 days increased the concentrations of thiamine diphosphate in the hippocampus and entorhinal cortex. This led to an improvement in mitochondria enzymes and insulin signaling pathway, with inactivation of GSK3α/β and ERK1/2, which are two tau-kinases related to the progression of AD, which could decrease tau hyperphosphorylation and apoptosis signaling. Besides, we observed an increased amount of Glun2b subunit of NMDA receptors, decreased inflammation, and improvement of cognitive deficit. Together, these results suggest that benfotiamine could be a potential therapeutic approach in the treatment of sporadic AD. | Marsden SR, McMillan DGG, Hanefeld U (2020)
Assessing the Thiamine Diphosphate Dependent Pyruvate Dehydrogenase E1 Subunit for Carboligation Reactions with Aliphatic Ketoacids.
International journal of molecular sciences 21, E8641 [PubMed:33207817]
[show Abstract]
The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from Escherichia coli (EcPDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, EcPDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that EcPDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones. WT EcPDH E1 shows a 180-250-fold higher catalytic efficiency towards 2-oxobutyrate or pyruvate, respectively, in comparison to engineered transketolase variants from Geobacillus stearothermophilus (TKGST). Its broad active site cleft allows for the efficient conversion of both (R)- and (S)-configured α-hydroxyaldehydes, next to linear and branched aliphatic aldehydes as acceptor substrates under kinetically controlled conditions. The alternate, thermodynamically controlled self-reaction of aliphatic aldehydes was shown to be limited to low levels of conversion, which we propose to be due to their large hydration constants. Additionally, the thermodynamically controlled approach was demonstrated to suffer from a loss of stereoselectivity, which makes it unfeasible for aliphatic substrates. | Taylor AJ, Talwar D, Lee SJ, Cox L, Mayxay M, Newton PN (2020)
Comparison of Thiamin Diphosphate High-Performance Liquid Chromatography and Erythrocyte Transketolase Assays for Evaluating Thiamin Status in Malaria Patients without Beriberi.
The American journal of tropical medicine and hygiene 103, 2600-2604 [PubMed:32996449]
[show Abstract]
Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measure thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) were compared with thiamin status assessed by the measurement of TDP by HPLC. 55% of 230 included patients were male, and the median age was 10 (range 0.5-73) years. Using α ≥ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4-80.5%), with specificity of 60.8 (95% CI: 53.2-68.1%). There was a significant inverse correlation between the results of the two tests (Kendall's tau = -0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall's tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays. | Rosado-Souza L, Proost S, Moulin M, Bergmann S, Bocobza SE, Aharoni A, Fitzpatrick TB, Mutwil M, Fernie AR, Obata T (2019)
Appropriate Thiamin Pyrophosphate Levels Are Required for Acclimation to Changes in Photoperiod.
Plant physiology 180, 185-197 [PubMed:30837347]
[show Abstract]
Thiamin pyrophosphate (TPP) is the active form of vitamin B1 and works as an essential cofactor for enzymes in key metabolic pathways, such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. Although its action as a coenzyme has been well documented, the roles of TPP in plant metabolism are still not fully understood. Here, we investigated the functions of TPP in the regulation of the metabolic networks during photoperiod transition using previously described Arabidopsis (Arabidopsis thaliana) riboswitch mutant plants, which accumulate thiamin vitamers. The results show that photosynthetic and metabolic phenotypes of TPP riboswitch mutants are photoperiod dependent. Additionally, the mutants are more distinct from control plants when plants are transferred from a short-day to a long-day photoperiod, suggesting that TPP also plays a role in metabolic acclimation to the photoperiod. Control plants showed changes in the amplitude of diurnal oscillation in the levels of metabolites, including glycine, maltose, and fumarate, following the photoperiod transition. Interestingly, many of these changes are not present in TPP riboswitch mutant plants, demonstrating their lack of metabolic flexibility. Our results also indicate a close relationship between photorespiration and the TCA cycle, as TPP riboswitch mutants accumulate less photorespiratory intermediates. This study shows the potential role of vitamin B1 in the diurnal regulation of central carbon metabolism in plants and the importance of maintaining appropriate cellular levels of thiamin vitamers for the plant's metabolic flexibility and ability to acclimate to an altered photoperiod. | Iacopetta D, Carrisi C, De Filippis G, Calcagnile VM, Cappello AR, Chimento A, Curcio R, Santoro A, Vozza A, Dolce V, Palmieri F, Capobianco L (2010)
The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster.
The FEBS journal 277, 1172-1181 [PubMed:20121944]
[show Abstract]
The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP. | Höltershinken M, Höhling A, Witte B, Scholz H (2007)
[Thiamine and its derivates in cattle blood measured by HPLC in healthy animals, in patients suffering from CCN and in their cohorts].
DTW. Deutsche tierarztliche Wochenschrift 114, 212-218 [PubMed:17642321]
[show Abstract]
The aim of this project was to develop a routine method to measure thiamine and its derivates in blood, to detect the need of vitamin B1-supply in cattle faster and more precise than until now using the transketolase test. For that, thiamine and its derivates (thiaminediphosphate = TDP,-monophosphate = TMP) were analysed in cattle blood by HPLC technique with post-column derivatization. Moreover, the levels in healthy cattle were compared with those in herds with single CCN cases. EDTA-blood-stored up to 10 days by 6 or 20 degrees C- was the most appropriate substrate. Sensitivitiy and specifity were significantly better than (TT, sum of all fractions): 1.95% and 2.10%; thiamine and TMP: 7.02% and 9.17%. The lowest concentration, which could be measured, was 0.5 ng/ml. 72 samples could be analysed daily. In the blood of 280 healthy calves and cows (group A), 201 clinically unsuspicious animals out of stocks with single cases of CCN (group B) and 12 patients with clinical obvious CCN (group C), thiamine and its phosphates were measured and the means calculated (ng/ml). After this, the thiamine status is characterized by (ng/ml): (A) thiamine 13.5 (4.61-28.8),TDP 51.0 (33.1-82.2), TMP 8.79 (2.23-18.1),TT 73.3 (44.6-114); (B) thiamine 8.73 (0.00-20.0), TDP 43.5 (15.5-75.7),TMP 6.15 (0.00-16.6), TT 59.4 (18.7-96.5); (C) thiamine 3.85 (0.00-1 1.5), TDP 14.5 (0.00-28.3), TMP 1.27 (0.00-4.87), TTP 19.6 (4.92-35.9). So, HPLC with post-column derivatization offers a precise, automatically working method for a fast diagnose of thiamine deficiency. The lowest critical value of TT is 40 ng/ml for healthy animals in the blood. The sample (EDTA-blood) can be posted simply at temperatures up to 20 degrees C. In comparison to other measuring methods, this HPLC-technique enables an early recognition of the risk to come down with CCN in endangered cattle herds. | Ajjawi I, Rodriguez Milla MA, Cushman J, Shintani DK (2007)
Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis.
Plant molecular biology 65, 151-162 [PubMed:17611796]
[show Abstract]
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. | Nemeria N, Chakraborty S, Baykal A, Korotchkina LG, Patel MS, Jordan F (2007)
The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes.
Proceedings of the National Academy of Sciences of the United States of America 104, 78-82 [PubMed:17182735]
[show Abstract]
Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis. | Mojzita D, Hohmann S (2006)
Pdc2 coordinates expression of the THI regulon in the yeast Saccharomyces cerevisiae.
Molecular genetics and genomics : MGG 276, 147-161 [PubMed:16850348]
[show Abstract]
Coordination of gene expression in response to different metabolic signals is crucial for cellular homeostasis. In this work, we addressed the role of Pdc2 in the coordinated control of biosynthesis and demand of an essential metabolic cofactor, thiaminediphosphate (ThDP). The DNA binding protein Pdc2 was initially identified as a regulator of the genes PDC1 and PDC5, which encode isoforms of the glycolytic enzyme pyruvate decarboxylase (Pdc). The Pdc2 has also been implicated as a regulator of genes encoding enzymes in ThDP metabolism. The ThDP is the cofactor of Pdc. Using global and gene-specific expression analysis, we show that Pdc2 is required for the upregulation of all genes controlled by thiamine availability. The Pdc2 seems to act together with Thi2, a known transcriptional regulator of THI genes. The requirement for these two factors differs in a gene-specific manner. While the Thi2, in conjunction with Thi3, seems to control expression of THI genes with respect to thiamine availability, the Pdc2 may link the ThDP demand to carbon source availability. Interestingly, the enzymes Pdc1 and Pdc5 are enriched in the nucleus. Both are known to affect gene expression in an autoregulatory mechanism and expression of both is regulated by glucose and Pdc2, further pointing to a role of Pdc2 in coordinating different metabolic signals. Our analysis helps to further define the THI regulon and hence the spectrum of genes/proteins involved in the ThDP homeostasis. In particular, we identify novel proteins putatively involved in thiamine and/or ThDP transport across the plasma and the mitochondrial membrane. In conclusion, the THI regulon is the most interesting system to study principles of genes expression and metabolic coordination and deserves further attention. | Lindhurst MJ, Fiermonte G, Song S, Struys E, De Leonardis F, Schwartzberg PL, Chen A, Castegna A, Verhoeven N, Mathews CK, Palmieri F, Biesecker LG (2006)
Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia.
Proceedings of the National Academy of Sciences of the United States of America 103, 15927-15932 [PubMed:17035501]
[show Abstract]
SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to alpha-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated alpha-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19(-/-) and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the alpha-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development. | Edwards TE, Ferré-D'Amaré AR (2006)
Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.
Structure (London, England : 1993) 14, 1459-1468 [PubMed:16962976]
[show Abstract]
Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate. | Jordan F, Nemeria NS (2005)
Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.
Bioorganic chemistry 33, 190-215 [PubMed:15888311]
[show Abstract]
Thiamin diphosphate (ThDP), the vitamin B1 coenzyme, is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/C2-carbanion of the thiazolium ring and the C2alpha-carbanion (or enamine) once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state (X-ray) methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, and circular dichroism detection of the 1',4'-imino tautomer of ThDP in some of the intermediates. Some of these methods also enable the investigator to determine the rate-limiting step in the complex series of steps. | Foulon V, Sniekers M, Huysmans E, Asselberghs S, Mahieu V, Mannaerts GP, Van Veldhoven PP, Casteels M (2005)
Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids.
The Journal of biological chemistry 280, 9802-9812 [PubMed:15644336]
[show Abstract]
2-Hydroxyfatty acids, constituents of brain cerebrosides and sulfatides, were previously reported to be degraded by an alpha-oxidation system, generating fatty acids shortened by one carbon atom. In the current study we used labeled and unlabeled 2-hydroxyoctadecanoic acid to reinvestigate the degradation of this class of lipids. Both in intact and broken cell systems formate was identified as a main reaction product. Furthermore, the generation of an n-1 aldehyde was demonstrated. In permeabilized rat hepatocytes and liver homogenates, studies on cofactor requirements revealed a dependence on ATP, CoA, Mg(2+), thiamine pyrophosphate, and NAD(+). Together with subcellular fractionation data and studies on recombinant enzymes, this led to the following picture. In a first step, the 2-hydroxyfatty acid is activated to an acyl-CoA; subsequently, the 2-hydroxy fatty acyl-CoA is cleaved by 2-hydroxyphytanoyl-CoA lyase, to formyl-CoA and an n-1 aldehyde. The severe inhibition of formate generation by oxythiamin treatment of intact fibroblasts indicates that cleavage through the thiamine pyrophosphate-dependent 2-hydroxyphytanoyl-CoA lyase is the main pathway for the degradation of 2-hydroxyfatty acids. The latter protein was initially characterized as an essential enzyme in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid. Our findings point to a new role for peroxisomes in mammals, i.e. the breakdown of 2-hydroxyfatty acids, at least the long chain 2-hydroxyfatty acids. Most likely, the more abundant very long chain 2-hydroxyfatty acids are degraded in a similar manner. | Casteels M, Foulon V, Mannaerts GP, Van Veldhoven PP (2003)
Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence.
European journal of biochemistry 270, 1619-1627 [PubMed:12694175]
[show Abstract]
3-Methyl-branched fatty acids, as phytanic acid, undergo peroxisomal alpha-oxidation in which they are shortened by 1 carbon atom. This process includes four steps: activation, 2-hydroxylation, thiamine pyrophosphate dependent cleavage and aldehyde dehydrogenation. The thiamine pyrophosphate dependence of the third step is unique in peroxisomal mammalian enzymology. Human pathology due to a deficient alpha-oxidation is mostly linked to mutations in the gene coding for the second enzyme of the sequence, phytanoyl-CoA hydroxylase. | Gerhardt S, Echt S, Busch M, Freigang J, Auerbach G, Bader G, Martin WF, Bacher A, Huber R, Fischer M (2003)
Structure and properties of an engineered transketolase from maize.
Plant physiology 132, 1941-1949 [PubMed:12913150]
[show Abstract]
The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme. | Lévy S, Hervé C, Delacoux E, Erlinger S (2002)
Thiamine deficiency in hepatitis C virus and alcohol-related liver diseases.
Digestive diseases and sciences 47, 543-548 [PubMed:11911339]
[show Abstract]
Thiamine deficiency is a common feature in chronic alcoholic patients, and its pathophysiology remains poorly understood. Until now, thiamine deficiency has been considered to be mainly the result of alcoholism irrespective of the underlying liver disease. The aims of the study were to compare the prevalence of thiamine deficiency in alcohol- and hepatitis C virus-(HCV-) related cirrhosis and in patients with chronic hepatitis C without cirrhosis. Forty patients with alcoholic cirrhosis (group A), 48 patients with HCV-related cirrhosis (group B), and 59 patients with chronic hepatitis C without cirrhosis (group C) were included prospectively. Thiamine status was evaluated by concomitant determination of erythrocyte transketolase activity, thiamine diphosphate (TDP) effect, and direct measurement of erythrocyte thiamine and its phosphate esters by HPLC. Thiamine was mainly present in erythrocytes in its diphosphorylated form. Prevalence of thiamine deficiency and levels of TDP in thiamine-deficient patients were similar in patients of group A (alcoholic cirrhosis) and of group B (viral C cirrhosis). None of the patients with chronic hepatitis (group C) was deficient. Thiamine deficiency was not correlated with the severity of the liver disease or disease activity. No impairment of thiamine phosphorylation was found in the three groups. conclusion, alcoholic or HCV-related cirrhotics have the same range of thiamine deficiency, while no patient without cirrhosis has thiamine deficiency, and impaired phosphorylation does not account for the deficiency observed in cirrhotics. We suggest that thiamine should be given to patients with cirrhosis irrespective of its cause. | Molina JA, Jiménez-Jiménez FJ, Hernánz A, Fernández-Vivancos E, Medina S, de Bustos F, Gómez-Escalonilla C, Sayed Y (2002)
Cerebrospinal fluid levels of thiamine in patients with Alzheimer's disease.
Journal of neural transmission (Vienna, Austria : 1996) 109, 1035-1044 [PubMed:12111441]
[show Abstract]
Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, such as the alpha-ketoglutarate dehydrogenase complex (KGDHC), pyruvate-dehydrogenase complex (PDHC), and transketolase. Some investigators reported decreased thiamine-diphosphate levels and decreased activities of KGDHC, pyruvate-dehydrogenase complex and transketolase in the brain tissue of Alzheimer's disease (AD) patients. We measured cerebrospinal (CSF) levels of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine, using ion-pair reversed phase high performance liquid chromatography, in 33 patients with sporadic AD and 32 matched controls. The mean CSF levels of thiamine-derivatives did not differ significantly from those of controls, while the mean plasma levels of thiamine-diphosphate, free and total thiamine were significantly lower in the AD-patient group. CSF and plasma thiamine levels were not correlated with age, age at onset, duration of the disease, and scores of the MiniMental State Examination, with the exception of plasma thiamine-diphosphate with MiniMental State Examination (r = 0.41, p < 0.05) in the AD-patients group. CSF and plasma values did not predict dementia progression, assessed with the MiniMental State Examination scores. These results suggest that CSF thiamine levels are not related with the risk for and the progression of AD. | Naito E, Ito M, Yokota I, Saijo T, Ogawa Y, Kuroda Y (2002)
Diagnosis and molecular analysis of three male patients with thiamine-responsive pyruvate dehydrogenase complex deficiency.
Journal of the neurological sciences 201, 33-37 [PubMed:12163191]
[show Abstract]
Pyruvate dehydrogenase complex (PDHC) deficiency is a major cause of congenital lactic acidemia in children. PDHC catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment was effective for some patients with PDHC deficiency. We reexamined 30 patients with congenital lactic acidemia of unknown origin who had normal PDHC activity in their cultured fibroblasts using a routine assay with a high (0.4 mM) thiamine pyrophosphate (TPP) concentration. We measured the activity of PDHC in the presence of a low (1x10(-4) mM) TPP concentration, and analyzed for mutations in the E1alpha subunit gene. Three males had low PDHC activity in the presence of 1x10(-4) mM TPP. The DNA sequence of these three patients' X-linked E1alpha subunit revealed a substitution of alanine for valine at position 71 (V71A) in exon 3, phenylalanine for cysteine at position 101 (C101F) in exon 4, and glycine for arginine at position 263 (R263G) in exon 8, respectively. Thiamine treatment was effective in these three patients. Therefore, they had a thiamine-responsive PDHC deficiency due to a point mutation in the E1alpha subunit gene. PDHC activity should be measured at a low TPP concentration to detect thiamine-responsive PDHC deficiency so that thiamine treatment can be initiated as soon as possible. | Marobbio CM, Vozza A, Harding M, Bisaccia F, Palmieri F, Walker JE (2002)
Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate.
The EMBO journal 21, 5653-5661 [PubMed:12411483]
[show Abstract]
The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources. | Singleton CK, Martin PR (2001)
Molecular mechanisms of thiamine utilization.
Current molecular medicine 1, 197-207 [PubMed:11899071]
[show Abstract]
Thiamine is required for all tissues and is found in high concentrations in skeletal muscle, heart, liver, kidneys and brain. A state of severe depletion is seen in patients on a strict thiamine-deficient diet in 18 days, but the most common cause of thiamine deficiency in affluent countries is alcoholism. Thiamine diphosphate is the active form of thiamine, and it serves as a cofactor for several enzymes involved primarily in carbohydrate catabolism. The enzymes are important in the biosynthesis of a number of cell constituents, including neurotransmitters, and for the production of reducing equivalents used in oxidant stress defenses and in biosyntheses and for synthesis of pentoses used as nucleic acid precursors. Because of the latter fact, thiamine utilization is increased in tumor cells. Thiamine uptake by the small intestines and by cells within various organs is mediated by a saturable, high affinity transport system. Alcohol affects thiamine uptake and other aspects of thiamine utilization, and these effects may contribute to the prevalence of thiamine deficiency in alcoholics. The major manifestations of thiamine deficiency in humans involve the cardiovascular (wet beriberi) and nervous (dry beriberi, or neuropathy and/or Wernicke-Korsakoff syndrome) systems. A number of inborn errors of metabolism have been described in which clinical improvements can be documented following administration of pharmacological doses of thiamine, such as thiamine-responsive megaloblastic anemia. Substantial efforts are being made to understand the genetic and biochemical determinants of inter-individual differences in susceptibility to development of thiamine deficiency-related disorders and of the differential vulnerabilities of tissues and cell types to thiamine deficiency. | Talwar D, Davidson H, Cooney J, St JO'Reilly D (2000)
Vitamin B(1) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: comparison with erythrocyte transketolase activation assay.
Clinical chemistry 46, 704-710 [PubMed:10794754]
[show Abstract]
BackgroundThe concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency.MethodsWe used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C(18) column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency.ResultsThe method was linear to at least 200 microgram/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280-590 ng/g hemoglobin and 275-675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP.ConclusionsThe HPLC method is precise and yields results similar to the erythrocyte activation assay. | Frank T, Bitsch R, Maiwald J, Stein G (2000)
High thiamine diphosphate concentrations in erythrocytes can be achieved in dialysis patients by oral administration of benfontiamine.
European journal of clinical pharmacology 56, 251-257 [PubMed:10952481]
[show Abstract]
ObjectiveThe influence of either orally administered S-benzoylthiamine-O-monophosphate (benfotiamine) or thiamine nitrate on the thiamine status was tested in a randomised, two-group comparison study in 20 end-stage renal disease (ESRD) patients. Main outcome measures were the pharmacokinetics of thiamine diphosphate (TDP) in blood, the in vitro erythrocyte transketolase activity, its activation coefficient (alpha-ETK) and the TDP concentration in erythrocytes.MethodsAfter ingestion of a single dose of either 100 mg thiamine nitrate (corresponding to 305 micromol thiamine) or 100 mg benfotiamine (corresponding to 214 micromol thiamine), the blood levels of thiamine phosphate esters were analysed by means of high-performance liquid chromatography for a 24-h period. The TDP concentration in erythrocytes was calculated using the haematocrit and TDP concentration in blood. Erythrocyte transketolase activity and alpha-ETK were measured before and 10 h after administration. The pharmacokinetics of TDP in blood were compared with healthy subjects of other studies retrieved from database query.ResultsRegarding the blood concentrations of TDP, the patients with ESRD had a 4.3 times higher area under the concentration time curve after benfotiamine administration than after thiamine nitrate. After benfotiamine administration, the peak plasma concentration of TDP exceeded that in healthy subjects by 51%. In the ESRD patients, after 24 h, the mean TDP concentration in erythrocytes increased from 158.7+/-30.9 ng/ml initially to 325.8+/-50.9 ng/ml after administration of benfotiamine and from 166.2+/-51.9 ng/ml to 200.5+/-50.0 ng/ml after thiamine nitrate administration. The ratio between the maximum erythrocyte TDP concentration and basal concentration was 2.66+/-0.6 in the benfotiamine group and 1.44+/-0.2 in the group receiving thiamine nitrate (P < 0.001). After 24 h, it was 2.11+/-0.4 and 1.23+/-0.2, respectively. The transketolase activity increased from 3.54+/-0.7 microkat/l initially to 3.84+/-0.6 microkat/l after benfotiamine intake (P = 0.02) and from 3.71+/-0.8 microkat/l to 4.02+/-0.7 microkat/l after thiamine nitrate intake (P = 0.08). Likewise, alpha-ETK decreased from initially 1.10+/-0.07 to 1.04+/-0.04 (P = 0.04) and from 1.12+/-0.05 to 1.08+/-0.06 (P = 0.09). After 24 h, the phosphorylation ratio in whole blood decreased from 12.9+/-6.9 initially to 5.6+/-3.2 after benfotiamine administration (P = 0.02) and from 13.5+/-7.3 to 9.0+/-4.8 (P = 0.03) after administration of thiamine nitrate. No correlation between erythrocyte TDP concentration and transketolase activity and/or alpha-ETK was observed in ESRD patients, either before or 10 h after administration.ConclusionCompared with thiamine nitrate, the oral administration of benfotiamine leads to higher TDP concentrations in erythrocytes accompanied with a significant improvement of the erythrocyte transketolase activity in ESRD patients. | Essama-Tjani JC, Guilland JC, Fuchs F, Lombard M, Richard D (2000)
Changes in thiamin, riboflavin, niacin, beta-carotene, vitamins, C, A, D and E status of French Elderly Subjects during the first year of institutionalization.
International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition 70, 54-64 [PubMed:10804457]
[show Abstract]
Vitamin status was assessed in 26 recently institutionalized elderly subjects by combining dietary and biochemical measurements of thiamin, riboflavin, niacin, beta-carotene, vitamins C, A, D and E at admission (P1), and 1.5 (P2), 3.0 (P3), 4.5 (P4), 6.0 (P5), 12 (P6) months later. At admission, except for vitamin A, mean vitamin intakes were lower than the 1992 French Recommended Dietary Allowance. Thiamin, vitamins C, A and E status seemed nearly satisfactory as less than one-fourth of the population sample had blood values lower than the cut-off point for thiamin (erythrocyte thiamin pyrophosphate < 0.17 mumol/l), vitamin A (serum retinol < 1.05 mumol/l), vitamin C (serum vitamin C < 11.3 mumol/l) and vitamin E (serum alpha-tocopherol < 9.3 mumol/l) or higher than the cut-off point for thiamin (erythrocyte transketolase activity coefficient > 1.19). Almost half of the subjects for riboflavin, and almost all non supplemented subjects for vitamin D were in risk of vitamin deficiency (46% had an erythrocyte glutathione reductase activity coefficient > 1.19 and 72% had a plasma 25(OH)D3 < 25 nmol/l). During the study, vitamins status remained unchanged for riboflavin, niacin, vitamins A, D and E, improved for vitamin C (P = 0.004) or impaired for thiamin (P = 0.008). Thus, institutionalization seemed to have no effect on riboflavin, niacin, vitamins A, D and E status and a slight effect on thiamin and vitamin C status. | Winston AP, Jamieson CP, Madira W, Gatward NM, Palmer RL (2000)
Prevalence of thiamin deficiency in anorexia nervosa.
The International journal of eating disorders 28, 451-454 [PubMed:11054793]
[show Abstract]
ObjectiveDeficiency of thiamin (vitamin B1) causes a range of neuropsychiatric symptoms that resemble those reported in patients with anorexia nervosa (AN) but the prevalence of thiamin deficiency in AN has not been reliably established. This study was designed to investigate the prevalence of thiamin deficiency in AN.MethodThirty-seven patients attending a specialist eating disorders unit and meeting all or some of the DSM-IV criteria for AN were compared with 50 blood donor controls. All subjects underwent measurement of erythrocyte transketolase activation following the addition of thiamin pyrophosphate, the standard biochemical test for thiamin deficiency. Deficiency was defined as a result more than 2 SD above the mean of the control population.ResultsFourteen patients (38%) had results in the deficient range; 7 (19%) met the most stringent published criterion for deficiency. Deficiency was not related to duration of eating restraint, frequency of vomiting, or alcohol consumption.DiscussionThiamin deficiency may account for some of the neuropsychiatric symptoms of AN and routine screening or supplementation may be indicated. | Schenk G, Duggleby RG, Nixon PF (1998)
Properties and functions of the thiamin diphosphate dependent enzyme transketolase.
The international journal of biochemistry & cell biology 30, 1297-1318 [PubMed:9924800]
[show Abstract]
This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals. | Lynch PL, Trimble ER, Young IS (1997)
High-performance liquid chromatographic determination of thiamine diphosphate in erythrocytes using internal standard methodology.
Journal of chromatography. B, Biomedical sciences and applications 701, 120-123 [PubMed:9389346]
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A high-performance liquid chromatography method for the determination of thiamine diphosphate (vitamin B1) in erythrocytes is presented. The method is robust, accurate and reproducible and due to the use of acetylaneurine as an internal standard, offers advantages over previous methods. | Arjunan P, Umland T, Dyda F, Swaminathan S, Furey W, Sax M, Farrenkopf B, Gao Y, Zhang D, Jordan F (1996)
Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution.
Journal of molecular biology 256, 590-600 [PubMed:8604141]
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The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring. | Lavoie J, Butterworth RF (1995)
Reduced activities of thiamine-dependent enzymes in brains of alcoholics in the absence of Wernicke's encephalopathy.
Alcoholism, clinical and experimental research 19, 1073-1077 [PubMed:7485819]
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The relative roles of alcohol per se, thiamine deficiency, and liver disease in the pathogenesis of alcohol-related brain damage have not been fully elucidated. In particular, the extent to which alterations of brain thiamine metabolism contribute to cognitive dysfunction in alcoholism in the absence of Wernicke's encephalopathy has not been established. In the present study, thiamine diphosphate-dependent enzymes were measured using standard spectrophotometric techniques in homogenates of brain tissue obtained at autopsy from eight alcoholic patients, all of whom died in hepatic coma without clinical or neuropathological evidence of Wernicke's encephalopathy and six nonalcoholic, age-matched controls, matched for autopsy delay time and free, at the time of death, from gross malnutrition or other neurological or psychiatric disorders. Transketolase activities were reduced in cerebellum (by 35%, p < 0.01), thalamus (by 35%, p < 0.01), frontal cortex (by 22%, p < 0.01), temporal cortex (by 20%, p < 0.01), and prefrontal cortex (by 19%, p < 0.01). Activities of the pyruvate dehydrogenase complex were selectively reduced in prefrontal cortex by 25% (p < 0.01). Activities of alpha-ketoglutarate dehydrogenase were within normal limits in all brain regions of alcoholic patients. The generalized reductions of transketolase activity undoubtedly result from thiamine deficiency. Previous studies suggest that the presence of liver disease may exacerbate thiamine deficiency in alcoholics. A sustained loss of transketolase activity in brain could result in disruption of pentose shunt activity and concomitant reductions in reducing equivalents and lipid metabolism within the cell. The selective loss of pyruvate dehydrogenase activity in prefrontal cortex of alcoholic cirrhotics could relate to the phenomenon of hepatic coma.(ABSTRACT TRUNCATED AT 250 WORDS) | Shimon I, Almog S, Vered Z, Seligmann H, Shefi M, Peleg E, Rosenthal T, Motro M, Halkin H, Ezra D (1995)
Improved left ventricular function after thiamine supplementation in patients with congestive heart failure receiving long-term furosemide therapy.
The American journal of medicine 98, 485-490 [PubMed:7733128]
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PurposeWe have previously found thiamine (vitamin B1) deficiency in patients with congestive heart failure (CHF) who had received long-term furosemide therapy. In the present study, we assessed the effect of thiamine repletion on thiamine status, functional capacity, and left ventricular ejection fraction (LVEF) in patients with moderate to severe CHF who had received furosemide in doses of 80 mg/d or more for at least 3 months.Patients and methodsThirty patients were randomized to 1 week of double-blind inpatient therapy with either i.v. thiamine 200 mg/d or placebo (n = 15 each). All previous drugs were continued. Following discharge, all 30 patients received oral thiamine 200 mg/d as outpatients for 6 weeks. Thiamine status was determined by the erythrocyte thiamine-pyrophosphate effect (TPPE). LVEF was determined by echocardiography.ResultsTPPE, diuresis, and LVEF were unchanged with i.v. placebo. After i.v. thiamine, TPPE decreased (11.7% +/- 6.5% to 5.4% +/- 3.2%; P < 0.01). LVEF increased (0.28 +/- 0.11 to 0.32 +/- 0.09; P < 0.05), as did diuresis (1,731 +/- 800 mL/d to 2,389 +/- 752 mL/d; P < 0.02), and sodium excretion (84 +/- 52 mEq/d to 116 +/- 83 mEq/d, P < 0.05). In the 27 patients completing the full 7-week intervention, LVEF rose by 22% (0.27 +/- 0.10 to 0.33 +/- 0.11, P < 0.01).ConclusionsThiamine repletion can improve left ventricular function and biochemical evidence of thiamine deficiency in some patients with moderate-to-severe CHF who are receiving longterm furosemide therapy. | Fidanza F, Simonetti MS, Floridi A, Codini M, Fidanza R (1989)
Comparison of methods for thiamin and riboflavin nutriture in man.
International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition 59, 40-47 [PubMed:2722424]
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Thiamin and riboflavin nutriture were assessed in 127 apparently healthy individuals aged between 20 and 82 yr, living in small villages near the town of Gubbio. The methods of erythrocyte transketolase activity and HPLC determination of thiamin pyrophosphate (TPP) for thiamin and erythrocyte glutathione reductase and HPLC determination of flavin adenine dinucleotide (FAD) for riboflavin were used. A significant correlation between enzymatic and HPLC methods was found. This was not the case for the correlation between status test and vitamin intake, particularly for thiamin. The vitamer determination in blood by HPLC can be considered a reliable, fast and reasonably accurate method for long-term thiamin and riboflavin nutritional status assessment. | Tate JR, Nixon PF (1987)
Measurement of Michaelis constant for human erythrocyte transketolase and thiamin diphosphate.
Analytical biochemistry 160, 78-87 [PubMed:3565758]
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Human erythrocyte transketolase could be resolved from thiamin diphosphate (TDP) by acidification of the ammonium sulfate precipitate to pH 3.5, but not by other tested procedures. Resolution was 98% by chemical measurement of residual thiamin and 95% by residual enzyme activity. Reconstitution of the resolved preparation by incubation with TDP was dependent upon TDP concentration, duration, temperature, and the presence of dithiothreitol. At low TDP concentrations, 1 h was required for maximum activation; kinetic analysis then yielded an apparent Km value for TDP of 65 nM (SD 14 nM) from 100 erythrocyte lysates and similar values for reconstituted resolved preparations previously purified 400-fold and 10,000-fold. Velocity data obtained by transketolase assays in which the TDP was added to resolved preparations simultaneously with substrates yielded an apparent Km value for TDP of 2.3 microM (SD 1.6 microM) from 114 erythrocyte lysates and similar values for purified preparations. The recovery of activity following resolution and reconstitution ranged from 21 to 60% from lysates and 38 to 70% from purified preparations. Residual ammonium sulfate up to 4.9 mM decreased the apparent Km value for TDP, while a concentration of 11.3 mM increased the value in a manner competitive with TDP and with an apparent Ki value of 2.3 mM. The spectrophotometric assay of transketolase activity was greatly affected by storage of frozen solutions of the substrate ribose 5-phosphate. | Baines M (1985)
Improved high performance liquid chromatographic determination of thiamin diphosphate in erythrocytes.
Clinica chimica acta; international journal of clinical chemistry 153, 43-48 [PubMed:4075519] | Floridi A, Pupita M, Palmerini CA, Fini C, Alberti Fidanza A (1984)
Thiamine pyrophosphate determination in whole blood and erythrocytes by high performance liquid chromatography.
International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition 54, 165-171 [PubMed:6500839]
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A sensitive method for the measurement of thiamine pyrophosphate in whole blood and erythrocytes, using a straight-phase high performance liquid chromatography (HPLC), is described. The method was based on sample extraction with trichloracetic acid, conversion of the vitamer to the corresponding high fluorescent thiochrome ester, and separation and measurement of the derivative by HPLC. Evidence for reproducibility and accuracy of the assay is presented. Data indicating the thiamine pyrophosphate levels to be expected in blood and erythrocytes of healthy humans are included. | Warnock LG (1982)
The measurement of erythrocyte thiamin pyrophosphate by high-performance liquid chromatography.
Analytical biochemistry 126, 394-397 [PubMed:7158773] | Duffy P, Morris H, Neilson G (1981)
Thiamin status of a Melanesian population.
The American journal of clinical nutrition 34, 1584-1592 [PubMed:7270482]
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A semiautomated method is described which uses the Abbott ABA-100 bichromatic analyzer to measure the stimulation of erythrocyte transketolase by thiamin pyrophosphate (the thiamin pyrophosphate effect). The method was accurate with a throughput of 30 samples per hour and is suitable for the study of the thiamin status of large populations. A significant proportion (21%) of a total of 1920 individuals of a predominantly Melanesian population were found to have an elevated thiamin pyrophosphate effect (greater than 15% activation). This same proportion with an elevated thiamin pyrophosphate effect was found in an urban hospital patient group of 346 individuals. Statistical analysis indicated a significant inverse correlation between thiamin pyrophosphate effect and plasma potassium concentration (p less than 0.001). Analysis of human fecal samples for thiaminase I activity demonstrated that this enzyme was present in 23 of a total of 46 samples selected from both the Melanesian and urban hospital patient groups. Preliminary findings indicated that the thiaminase originated from a bacterial source. | Tumanov VN, Trebukhina RV (1981)
[Enzyme activity of thiamine diphosphate biosynthesis and degradation in the mouse liver in the dynamics of B1 avitaminosis development].
Voprosy meditsinskoi khimii 27, 785-788 [PubMed:6121420]
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Activities of thiamin pyrophosphokinase (TPK EC 2.7.6.2) and thiamin pyrophosphatase (TPPase EC 3.6.1.6) were studied in liver homogenate supernatants obtained from mouse (female, body weight of 21-23 g) in dynamics of B1 avitaminosis. As compared with controls distinct deficiency of thiamin led a decrease in coenzyme thiamin diphosphate (TPP) down to 15% as well as of transketolase activity (TPP-dependent enzyme) down to 35%, especially during the terminal stage of the avitaminosis; the process was accompanied by cyclic alterations in activity of TPK and TPPase. Within 9 and 17 days of the B1 avitaminosis the activity of TPK increased by 62% and 38% and the activity of TPPase--decreased by 30% and 20%, respectively. Alterations in the enzymatic activity appear to be characteristic for adaptation in response to thiamin deficiency. | Kjosen B, Seim SH (1977)
The transketolase assay of thiamine in some diseases.
The American journal of clinical nutrition 30, 1591-1596 [PubMed:910736]
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Erythrocyte transketolase activity (ETKA) and the effect of adding thiamine pyrophosphate have been measured in a group of 27 healthy individuals and in 37 patients diagnosed as having diabetes mellitus, anemia, polyneuritis, or malnourishment secondary to vascular disease of the brain. The observed values for the malnourished group did not differ significantly from those for the control group. The low ETKA values in diabetes mellitus seem to be due to a reduced apoenzyme level resulting from the disease itself rather than thiamine deficiency. Polyneuritis patients had low values of ETKA. In the anemic group as a whole the values showed a difference of only marginal significance from those found in the control group, but the patients with pernicious anemia all had a highly significant elevation of the ETKA values. Although the absolute thiamine pyrophosphate effect differ, there are no significant differences in percentage of thiamine pyrophosphate effect between the groups. It appears that differences in the patient groups studied here reflect variations in apoenzyme levels rather than thiamine status. |
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