Ácido fólico em excesso: efeitos sobre o metabolismo das vitaminas B2 e B6, o catabolismo do triptofano e a resposta imune / Folic acid excess: effects on vitamin B2 and B6 metabolism, tryptophan catabolism and immune response

Introdução: A suplementação com ácido fólico (AF) é recomendada em algumas condições para evitar a deficiência de folato, como para mulheres no período periconcepcional e durante a gestação. Atualmente, existe uma preocupação quanto ao consumo excessivo de AF pela população pelo uso de suplementos com altas doses dessa vitamina. As vitaminas B6 e B2 agem como cofatores no metabolismo de um carbono, e o uso de altas doses de AF pode influenciar o metabolismo de ambas vitaminas e, consequentemente, interferir em metabolismos importantes das quais elas participam, como a via das quinureninas, e no sistema imune. Objetivo: Avaliar os efeitos da intervenção diária com uma alta dose de AF (5 mg) por 90 dias sobre marcadores do estado das vitaminas do complexo B, e as consequências sobre os metabólitos da via das quinureninas e o sistema imune em adultos saudáveis. Material e Métodos: 64 indivíduos saudáveis foram submetidos à intervenção diária com 5 mg de AF por 90 dias. Coletas de sangue foram realizadas antes (baseline) e após 45 e 90 dias de intervenção. As concentrações séricas de folato e vitamina B12 foram avaliadas por métodos microbiológicos. As concentrações séricas das vitaminas B6 (piridoxal 5'-fosfato (PLP), piridoxal (PL) e ácido 4-piridóxico (PA)), B2 (riboflavina e flavina mononucleotídeo (FMN)), B1 (tiamina e tiamina monofosfato (TMP)) e B3 (ácido nicotínico, nicotinamida e N1-metilnicotinamida), bem como de triptofano, quinurenina e metabólitos, foram avaliadas por LC-MS/MS. A proteína C-reativa ultrassensível (PCRus) foi determinada por imunoturbidimetria, e as concentrações séricas de interleucina (IL)-6, IL-8, IL-10, interferon gama (IFN-γ) e fator de necrose tumoral alfa (TNF-α) foram avaliadas por ensaio multiplex. A expressão de RNAm de DHFR (dihidrofolato redutase), MTHFR (metilenotetrahidrofolato redutase), IL8, TNFA e IFNG em leucócitos mononucleares (PBMC) foram avaliadas por PCR em tempo real. O número de células T regulatórias (Treg) (CD3+, CD4+, CD25high, FoxP3+, CD127-) foi avaliado após incubação dos PBMC com PMA e ionomicina ou veículo por 18h, por imunofenotipagem. Resultados: Houve um grande aumento das concentrações de folato sérico após 45 e 90 dias de intervenção com AF. Não houve diferença nas concentrações de vitamina B12 antes e após a intervenção. As concentrações séricas de PLP foram semelhantes antes e após a intervenção, entretanto, um aumento de PL sérico foi observado após 45 e 90 dias, e de PA após 45 dias, quando comparado ao baseline. Riboflavina e FMN foram maiores após 45 e 90 dias em relação ao baseline. A tiamina sérica foi menor após 45 dias, e as concentrações de TMP foram maiores após 90 dias quando comparados aos períodos anteriores. Não houve diferença nas concentrações de vitamina B3 antes e após a intervenção. Dentre os metabólitos da via das quinureninas, apenas o ácido antranílico apresentou aumento após 45 e 90 dias, enquanto o ácido picolínico diminuiu após 90 dias. PCRus, IL-6, IL-8, IL-10, IFN-γ e TNF-α séricos foram semelhantes no baseline e após a intervenção. Um aumento da expressão de RNAm de DHFR e TNFA foi observado após, respectivamente, 90 dias e 45 e 90 dias de intervenção. Após 90 dias de intervenção com AF, foi observada diminuição do número de células Treg após estímulo com PMA e ionomicina. Conclusão: O uso diário de 5 mg de AF foi associado a alterações nas concentrações séricas de marcadores do estado de vitaminas do complexo B e da via das quinureninas, bem como a diminuição do número de células Treg

Introduction: Folic acid (FA) supplementation is recommended in some conditions to avoid folate deficiency, as women during periconceptional period and pregnancy. Currently, there is a concern about the excessive consumption of FA by population by using supplements with high doses of this vitamin. Vitamins B6 and B2 are cofactors of enzymes of one carbon metabolism and, consequently, may disturb key metabolism in which they participate, as kynurenine pathway, and the immune system. Aim: To assess the effects of a daily intervention with high dose of FA (5 mg) for 90 days on biomarkers of complex B vitamins status and its outcomes in kynurenine pathway metabolites and immune system in healthy adults. Material and Methods: 64 healthy individuals were submitted to a daily intervention with 5 mg of FA for 90 days. Blood samples were collected before (baseline) and after 45 and 90 days of intervention. Serum folate and vitamin B12 were assessed by microbiological assays. Serum vitamin B6 (pyridoxal 5'-phosphate (PLP), pyridoxal (PL) and 4-pyridoxic acid (PA)), vitamin B2 (riboflavin and flavin mononucleotide (FMN)), vitamin B1 (thiamin and thiamin monophosphate)) and vitamin B3 (nicotinic acid, nicotinamide and N1-methylnicotinamide), as well as tryptophan, kynurenine and metabolites, were assessed by LC-MS/MS. C-reactive protein (hs-CPR) was assessed by immunoturbidimetry, and serum interleukin (IL)-6, IL-8, IL-10, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were assessed by multiplex assay. Mononuclear leukocytes mRNA expression of DHFR (dihydrofolate reductase), MTHFR (methylenetetrahydrofolate reductase), IL8, TNFA and IFNG were assessed by real time PCR. Regulatory T Cell (Treg) number (CD3+, CD4+, CD25high, FoxP3+, CD127-) was determined after mononuclear leukocytes incubation with PMA and ionomycin or vehicle for 18h, by immunophenotyping. Results: A great increase on serum folate was observed after 45 and 90 days of FA intervention. No differences in serum vitamin B12 were observed before and after intervention. Serum PLP was similar before and after intervention, however, an increase in serum PL was observed after 45 and 90 days, and in PA after 45 days, when compared to baseline. Riboflavin and FMN were increased after 45 and 90 days than in baseline. Serum thiamine was decreased after 45 days than in baseline. Serum TMP was increased after 90 days when compared with previous timepoints. No differences in vitamin B3 were observed after and before FA intervention. Among kynurenine pathway metabolites, anthranilic acid was increased after 45 and 90 days, while picolinic acid was decreased after 90 days. hs-CPR, serum IL-6, IL-8, IL-10, IFN-γ and TNF-α were similar at baseline and after intervention. An increase on mRNA expression of DHFR and TNFA was observed after, respectively, 90 days and 45 and 90 days of intervention. After 90 days of FA intervention, it was observed a decrease on Treg cell number after PMA and ionomycin stimulation. Conclusion: Daily use of 5 mg of FA was associated with changes in serum markers of B-complex vitamins status and kynurenine pathway, as well as decreased number of Treg cells

Severe neurological complication following adjustable gastric banding.

AIM: In the last years with the increase of bariatric surgery, first of all as a result of new indications, a rise in the incidence of nutrient-related complications has been observed. Currently little is known about the impact of post-bariatric malnutrition and neurological complications. Wernicke's encephalopathy is a severe neurological syndrome which occurs as a result of thiamine deficiency. Wernicke-Korsakoff syndrome must be considered a serious neurological complication of bariatric surgery with significant morbidity and mortality, with rapidly progressing neurological symptoms, and must be treated immediately. CASE REPORT: We report the case of a 35 years-old male patient, affected by morbid obesity, anxious-depressive syndrome and alcohol use disorder, who after adjustable gastric banding implanted in another hospital developed a severe malnutrition and neurological syndrome. The patient showed poor adherence to the follow-up and to the dietary indications and after all, we needed to place a PEG for enteral nutrition in order to resolve the malnutrition condition and the neurological syndrome. Our experience emphasizes that preoperative selection and assessment of a patient's nutritional status according to guidelines, is required to identify potential problems, and that bariatric surgeons or physicians caring for patient who have undergone bariatric surgery should be familiar with the constellation of nutritional and neurological disorder that may occur after surgery. CONCLUSION: We want to remark the importance of preoperative selection of the patients, the follow-up and the cooperation between patient and physician in order to obtain the best result and avoid severe complications.

Vitamins B1, B2, B3 and B9 - Occurrence, Biosynthesis Pathways and Functions in Human Nutrition.

BACKGROUND: Vitamins are chemical compounds whose derivatives are involved in vital metabolic pathways of all living organisms. The complete endogenous biosynthesis of vitamins can be performed by many bacteria, yeast and plants, but humans need to acquire most of these essential nutrients with food. In recent years, new types of action of the well-recognized vitamins or their more sophisticated relationships have been reported. CONCLUSION: In this review we present the current knowledge of factors that can influence the yield and regulation of vitamin B1, B2, B3 and B9 biosynthesis in plants which can be important for human nutrition. A summary of modern methods applied for vitamin analysis in biological materials is also provided. Contributions of selected vitamins to the homeostasis of the human organism, as well as their relations to the progress or prevention of some important diseases such as cancer, cardiovascular diseases, diabetes and Alzheimer's disease are discussed in the light of recent investigations. Better understanding of the mechanisms of vitamin uptake by human tissues and possible metabolic or genetic backgrounds of vitamin deficiencies can open new perspectives on the medical strategies and biotechnological processes of food fortification.

The role of thiamine in cancer: possible genetic and cellular signaling mechanisms.

The relationship between supplemental vitamins and various types of cancer has been the focus of recent investigation, and supplemental vitamins have been reported to modulate cancer rates. A significant association has been demonstrated between cancer and low levels of thiamine in the serum. Genetic studies have helped identify a number of factors that link thiamine to cancer, including the solute carrier transporter (SLC19) gene, transketolase, transcription factor p53, poly(ADP-ribose) polymerase-1 gene, and the reduced form of nicotinamide adenine dinucleotide phosphate. Thiamine supplementation may contribute to a high rate of tumor cell survival, proliferation and chemotherapy resistance. Thiamine has also been implicated in cancer through its effects on matrix metalloproteinases, prostaglandins, cyclooxygenase-2, reactive oxygen species, and nitric oxide synthase. However, some studies have suggested that thiamine may exhibit some antitumor effects. The role of thiamine in cancer is controversial. However, thiamine deficiency may occur in patients with cancer and cause serious disorders, including Wernicke's encephalopathy, that require parenteral thiamine supplementation. A very high dose of thiamine produces a growth-inhibitory effect in cancer. Therefore, further investigations of thiamine in cancer are needed to clarify this relationship.

"Syndrome in syndrome": Wernicke syndrome due to afferent loop syndrome. Case report and review of the literature.

Wernicke syndrome is a rare neurological pathology due to a deficit in vitamin B1. The syndrome is common among alcohol abusers, patients with malignant tumor or gastrointestinal diseases, those who undergo hemodialysis or long-term peritoneal dialysis, pregnant women with hyperemesis, women who breast-feed, patients with hyperthyroidism or anorexia nervosa or gastric or jejunal-ileal bypass surgery for obesity, patients submitted to gastric surgery or prolonged total parenteral nutrition or prolonged intravenous therapy. We report a case of Wernicke syndrome due to afferent loop syndrome characterized by incoercible vomiting.

Prodrug thiamine analogs as inhibitors of the enzyme transketolase.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

The antitrypanosomal drug melarsoprol competitively inhibits thiamin uptake in mouse neuroblastoma cells.

Melarsoprol is the main drug used for the treatment of late-stage sleeping sickness, although it causes severe side-effects such as encephalopathy and polyneuropathy leading to death in some patients. Recent data suggest that melarsoprol and its active metabolite melarsenoxide interfere with thiamin transport and metabolism in E. coli and yeast, but there are no data concerning their possible effects on thiamin metabolism in mammalian cells. We tested both drugs on thiamin transport in cultured mouse neuroblastoma cells using (14)C-labeled thiamin. Melarsoprol, competitively inhibits high-affinity thiamin transport in mouse neuroblastoma cells with a K(i) of 44 micromol/L. However, the active compound melarsenoxide has no inhibitory effect. This suggests that the side effects of melarsoprol treatment are unlikely to be due to inhibition of thiamin transport by melarsenoxide, its main metabolite in the brain.

Differentiation-dependent up-regulation of intestinal thiamin uptake: cellular and molecular mechanisms.

Differentiation of intestinal epithelial cells is associated with up-and-down regulation of expression of a variety of genes including those involved in nutrient uptake. Nothing is known about possible differentiation-dependent regulation of the intestinal thiamin uptake process and the cellular and molecular mechanisms involved in such regulation. Using as models human-derived intestinal epithelial Caco-2 cells and crypt/villus epithelial cells isolated from wild-type and transgenic mice carrying promoters for human thiamin transporter-1 and -2 (hTHTR-1 and hTHTR-2), we addressed this issue. Our results showed that differentiation of Caco-2 cells is associated with a significant up-regulation in carrier-mediated thiamin uptake. Up-regulation was associated with a significant increase in the level of expression of hTHTR-1 and hTHTR-2 protein and mRNA as well as in activity of the corresponding transfected human thiamin transporter-1 (SLC19A2) and -2 (SLC19A3) promoters. Deletion analysis identified the differentiation-responsive region to be at position -356 to -275 bp for the SLC19A2 promoter and at position -77 to -13 bp for the SLC19A3 promoter. In addition, a critical and specific role in the differentiation-mediated effects for an NF1 binding site (-348 to -345 bp) in the SLC19A2 promoter and a SP1/GC-box binding site (-48 to -45 bp) in the SLC19A3 promoter were established using mutational analysis. The physiological relevance of in vitro findings with Caco-2 cells was confirmed in wild-type and transgenic mice by demonstrating that thiamin uptake and mRNA levels of the mouse THTR-1 and THTR-2, as well as activity of human SLC19A2 and SLC19A3 promoters expressed in transgenic mice, were all significantly higher in intestinal villus compared with crypt epithelial cells. These studies demonstrate for the first time that differentiation of intestinal epithelial cells is associated with an up-regulation in thiamin uptake process and that this up-regulation appears to be mediated via transcriptional regulatory mechanisms that involve the SLC19A2 and SLC19A3 genes.

Specific association of thiamine-coated gadolinium nanoparticles with human breast cancer cells expressing thiamine transporters.

Thiamine (vitamin B(1)) was investigated as a tumor-specific ligand for gadolinium nanoparticles. Solid nanoparticles containing gadolinium hexanedione (1.5 mg/mL) were engineered from oil-in-water microemulsion templates and coated with thiamine ligands. Thiamine ligands were synthesized by conjugating thiamine to either distearoylphosphatidylethanolamine (DSPE) or fluorescein via a poly(ethylene glycol) (PEG) spacer (Mw 3350). The efficiency of thiamine ligand attachment to nanoparticles was evaluated using gel permeation chromatography (GPC). Cell association studies were carried using a methotrexate-resistant breast cancer cell line, MTX(R)ZR75, transfected with thiamine transporter genes (THTR1 and THTR2). Thiamine-coated nanoparticle association with THTR1 and THTR2 cells was significantly greater than that with control breast cancer cells (MTX(R)ZR75 transfected with the empty expression vector pREP4) (p < 0.01; t-test). The nanoparticle cell association was significantly dependent on the extent of thiamine ligand coating on nanoparticles, expression of thiamine transporters in cells, temperature of incubation, and the concentration of competitive inhibitor (free thiamine). Further studies are warranted to assess the potential of the engineered thiamine-coated gadolinium (Gd) nanoparticles in neutron capture therapy of tumors.

Investigations on the intestinal availability of native thiamin in selected foods and feedstuffs.

The aim of the present study was to investigate the precaecal digestibility as a quantitative measure for the intestinal availability of naturally occurring thiamin from selected foods and feedstuffs. Therefore, three experiments were conducted to examine the following foods and feedstuffs: Eggs, bananas, white cabbage, corn, milk, fish, barley, soybeans, rice, wheat bran, brewer's yeast, rye and soybean meal. The foods and food processing procedures were made with regard to their relevance in human and animal nutrition. For all experiments male pigs with an initial live weight between 33 and 40 kg were fitted with an end-to-end ileo-rectal anastomosis with preserved ileo-caeco-colicvalve. Three weeks after surgery, the digestibility trials were carried out from week 4 to week 9 and week 12 to week 17 after surgery. The animals were fed the individual experimental diets for a period of 12 days while digesta were collected twice a day quantitatively during the final 5 days of this period. Precaecal digestibility for thiamin from all tested foods and feedstuffs was within a range from 73% to 94% with the highest values from boiled soybeans, boiled rice and barley, and the lowest value from steamed fish. In comparison with the animal products the plant products show on average a nearly equal precaecal digestibility for thiamin (87.3% versus 83.5%). Moreover, all tested foods and feedstuffs exhibit a relatively good intestinal availability of thiamin.

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

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

Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small intestine.

1. Rat small intestinal basolateral membrane vesicles (BLMVs) were prepared and found to be 31% non-vesiculated and 69% vesiculated, 4.9% right side out and 63.8% inside out. 2. Thiamine uptake by BLMVs followed a hyperbolic time course reaching equilibrium after 60-90 min incubation. Uptake was not affected by the transmembrane potential or by the presence or absence of Na+ or K+ in the incubation medium. 3. At concentrations below 1.25 microM, [3H]thiamine was taken up mainly by a saturable mechanism with an apparent Michaelis-Menten constant (Km) = 1.32 microM and maximal flux (Jmax) = 1.93 pmol (mg protein)-1 (4 s)-1. At higher concentrations, a non-saturable mechanism prevailed. 4. Only 29% of [3H]thiamine taken up by the vesicles was membrane bound, the remaining being translocated into the vesicular space. No thiamine phosphoesters could be detected inside the vesicles. 5. In the absence of ATP, the Na(+)-K(+)-ATPase inhibitors ouabain, frusemide and vanadate reduced thiamine uptake by 35, 30 and 15% respectively. 6. In experiments conducted with K+ inside the vesicles and Na+, Mg2+ and ATP outside, the time course of thiamine uptake by BLMVs displayed an overshoot (80-90% increment) at 30 s incubation as compared to controls. When ATP was replaced with phosphocreatine, or when NaCl was replaced with isosmotic amounts of KCl, the overshoot disappeared. 7. The thiamine analogues pyrithiamine, amprolium and 4'-oxythiamine decreased the ATPase-dependent transport of [3H]thiamine by 100, 86 and 31% respectively. 8. These results provide evidence that the transport of thiamine by BLMVs is coupled directly to the hydrolysis of ATP (primary active transport).

[The role of Na K ATPase in thiamine and lipoic acid interrelations during their absorption in the gastrointestinal tract of mice]. / Rol' Na+, K+ ATFazy vo vzaimootnosheniiakh tiamina i lipoevoi kisloty pri vsasyvanii, proiskhodiashchem v zheludochno-kishechnom trakte myshei.

35S-lipoic acid (20 mumol/kg) with different doses of thiamine or 35S-thiamine (50 mumol/kg) with unlabelled lipoic acid where administrated to the white mice stomach. It was established that absorption and entrance of both lipoic acid and thiamine when they were in 1:5 quantities in organs and tissues were maximal. The activity of Na+, K-ATPase determined in stomach, duodenum and small intestine was the maximal too.