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Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P<0.0001). At 11 wks, only WT+S EDL stress in vitro was greater than all other groups at 50-150 Hz. The in vitro relative stress-frequency relationship of mdx+S EDL was left shifted from the other treatment groups. Inflammatory genetic markers were increased in mdx+S mice. These data suggest treatment of mdx mice with 0.1% SM improves ankle flexor torque in vivo, causes a left shift of the stress-frequency relationship in vitro, and modulates the inflammatory gene signature. / Master of Science

This study was designed to examine if a sphingolipid-enriched lipid fraction from wheat gluten could affect the incidence of type I diabetes in BioBreeding diabetes prone (BBdp) rats. Wheat gluten was extracted with a chloroform-methanol (CM) mixture to isolate most of the lipids. Isolated lipids were subjected to silica gel column chromatography and saponification to remove most of neutral lipids and phospholipids, leaving behind a lipid fraction enriched in sphingolipids. This sphingolipid-enriched lipid fraction was used in a BBdp rat feeding study. BBdp rats were fed with one of five diets from weaning at 23 days of age until 125 days of age: a hydrolyzed casein based diet (HC), a NTP-2000 standard rodent diet (NTP-2000), a wheat gluten based diet (WG), a sphingolipid-free wheat gluten based diet (WGSLF), and a hydrolyzed casein plus sphingolipid-enriched lipid fraction diet (HC+SL). The yield of sphingolipid-enriched lipid fraction was about 0.62% of wheat gluten. The content of glycosylceramide in sphingolipid-enriched lipid fraction was increased more than five fold compared to that in total isolated lipids. Rats fed the NTP-2000 diet had the highest incidence of diabetes; while rats on the HC diet had the lowest diabetes incidence. There was no significant difference with regard to the onset age of diabetes among rats in the five diet groups. The addition of sphingolipid-enriched fraction to the HC diet caused a significant increase in the incidence of diabetes in BBdp rats in the first 80 days of the study. However, the ultimate diabetes incidence at day 125 was not changed. The removal of lipids from wheat gluten did not change the diabetes incidence in BBdp rats at any stages of the feeding study. These findings suggest that the sphingolipid-enriched fraction from wheat gluten acted as a possible promoter but not as a trigger of the development of type I diabetes in BBdp rats. There must be something that remains in wheat gluten after chloroform-methanol extraction that serves as a trigger for type I diabetes in these rodents. Type I diabetes in this animal model for the human disease seems to be caused by multiple factors, most likely, by the interaction of sphingolipids and some other unknown substances in wheat gluten. / Master of Science

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is being tested in clinical trials as a relatively non-toxic anti-cancer agent. 4-HPR induces de novo sphingolipid biosynthesis and production of ceramide has been suggested to contribute to the growth arrest and apoptosis. To characterize the types of ceramides that might be involved, we used liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) to analyze the sphingolipids, and found that 4-HPR increased total sphingolipid amounts, but unexpectedly, ceramides (i.e., N-acylsphingosines) changed very little, and in some cases decreased. Instead, dihydroceramides (i.e., N-acylsphinganines) increased as much as 10-fold, both as the free species and as the backbones of dihydrosphingomyelins and dihydrohexosylceramides. To determine if 4-HPR inhibits dihydroceramide desaturase, we synthesized NBD-dihydroceramide and treated Hek293 cells with 4-HPR and analyzed the metabolites by HPLC. These analyses showed that NBD-dihydroceramide was taken up by the cells and converted to NBD-ceramides and more complex NBD-sphingolipids in control cells, however, within one hour of treatment with 10 ~{ and L~}M 4-HPR, the production of NBD-ceramide was blocked. In vitro assays of the desaturase using NBD-dihydroceramide also showed rapid and complete inhibition by 4-HPR. Interestingly, when Hek cells were treated with 4-HPR for one hour then the medium was changed, the recovery of dihydroceramide desaturase activity was very slow (i.e., t1/2 > 66 h); therefore, either 4-HPR is difficult to remove from cells or the inhibition is essentially irreversible. These findings establish that 4-HPR not only induces de novo sphingolipid biosynthesis but also inhibits dihydroceramide desaturase, resulting in production of abnormally high proportions of sphingolipids with dihydroceramide as the backbone. This raises the possibility that some of the effects of 4-HPR on cell behavior may be due to the presence of these abnormal species.

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids involved in signaling cascades that overlap significantly with these modulators of ICl,swell, we investigated the role of sphingolipids in the regulation of ICl,swell in cardiac ventricular myocytes. Under isoosmotic conditions that isolate anions currents, addition of exogenous, cell permeant C2-ceramide (C2-Cer) elicited an outwardly-rectifying Cl− current that reversed near the Cl− equilibrium potential (ECl) in both physiological and symmetrical Cl− gradients. This current was inhibited by the ICl,swell-specific blockers DCPIB. Dihydro-C2-ceramide (C2-H2Cer), the inactive analogue of C2-Cer, failed to elicit current. These data strongly suggest that the identity of C2-Cer-induced Cl− current is ICl,swell and indicate that sphingolipid signaling pathways may be involved. Bacterial sphingomyelinase (SMase), which converts endogenous sphingomyelin in the outer leaflet of the sarcolemmal membrane to native chain-length ceramides, elicited a DCPIB-sensitive Cl− current. SMase-induced current is also suppressed by tamoxifen, which under conditions that isolate anion currents is a specific inhibitor of ICl,swell. SMase-induced ICl,swell was abrogated by ebselen, a membrane permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O. This suggests that ROS are required mediators of SMase-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes and both have been implicated in modulating ICl,swell. Blocking NOX with apocynin or the NOX fusion peptide inhibitor gp91ds-tat had no effect on SMase-induced current. However, pretreatment of cardiomyocytes with gp91ds-tat reduced the maximum current amplitude of SMaseinduced ICl,swell, indicating that NOX may play a time-dependent role in this mechanism. By contrast, the mitochondrial Complex I blocker rotenone, which suppresses extramitochondrial ROS release by Complex III, completely suppresses SMase-induced ICl,swell. Additionally, SMase-induced ICl,swell is partially inhibited by blockade of mitochondrial KATP (mitoKATP) channels with 5-hydroxy-decanoic acid (5-HD). MitoKATP channels have been implicated as modulators of mitochondrial ROS release. Thus these data suggest that mitochondrial ROS generation is required for SMaseinduced activation of ICl,swell. Ceramides are metabolized to form several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase, which converts ceramide to sphingosine, with Derythro-MAPP. SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine. These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P elicited an outwardly rectifying Cl− current that was fully inhibited by DCPIB. As seen with SMaseinduced ICl,swell, S1P-induced ICl,swell was fully inhibited by rotenone. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NOX with apocynin. These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, the fact that exogenous C2-ceramide also activates ICl,swell even though C2-ceramide may not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors PD98059 and U0126 on the activation of ICl,swell. Both of these agents partially inhibited SMase-induced activation of ICl,swell, indicating SMase acts through both ERK-dependent and ERK-independent signaling pathways. HL-1 cells are derived from a murine atrial cell line that retains phenotypic characteristics of adult cardiomyocytes. Recently, ICl,swell has been observed in HL-1 cells with similar regulatory mechanisms to those seen in native cells. We showed that SMase elicits an outwardly-rectifying, DCPIB-sensitive Cl− current that reverses near ECl in HL-1 cells. Finally, we confirmed the production of ROS by SMase-induced signaling by flow cytometry in HL-1 cells using the nominally H2O2-selective fluorescent probe CH2DCFDA-AM. Exposure to SMase increased ROS production, as did the positive control H2O2. SMase-induced ROS generation was suppressed by pretreatment with rotenone but was unaffected by pretreatment with gp91ds-tat. These data indicate that exogenous and endogenous sphingolipids elicit ICl,swell in cardiomyocytes by stimulating mitochondrial ROS production. NOX may contribute to the ROS generation, but is not a required step in this mechanism. Sphingolipid signaling is likely to play an important role in stimulating ROS production and activating ICl,swell in a number of cardiovascular diseases.

Made available in DSpace on 2016-04-28T20:17:26Z (GMT). No. of bitstreams: 1 2006 - Rodrigo Alcantara de Carvalho.pdf: 803403 bytes, checksum: 7a35f34784a3f9cb58b545f52959a6a0 (MD5) Previous issue date: 2006-09-22 / The world wide contamination of foods and byproducts with mycotoxins is a problem of extreme importance. Mycotoxins are secondary metabolites produced by fungi that have adverse effects to human and animal health. The fumonisins and aflatoxins are the mycotoxins of greatest agro-economic importance due to the damage brought to animal production. Important steps in the synthesis of these mycotoxins are described, as such as their metabolism and adverse effects. This work evaluates the determination of the biomarkers sphinganin (Sa) and sphingosin (So) in poultry serum as a diagnose method for fumonisin exposition, and compares the obtained data with ration analysis. Of the evaluated animals only 49% exhibited 2 sphingolipids (Sa and So), 53,04% exhibited only So and 2,04% exhibited only Sa. It was detected at ration the presence of fumonisin B1 at concentration of 374,92&#956;g.kg-1 and aflatoxins B1, B2, and G1 at concentrations of 440,99&#956;g.kg-1, 169,92&#956;g.kg-1 and 494,58&#956;g.kg-1, respectively. It was possible to partially establish the grade of intoxication of the poultry and to verify that the sensibility of the biomarker can be affected by the presence of fumonisin such as aflatoxins. / A contamina??o mundial de alimentos e subprodutos com micotoxinas ? um problema de extrema import?ncia. As micotoxinas s?o metab?litos secund?rios produzidos por fungos, que possuem efeitos adversos ? sa?de humana e animal. As fumonisinas e aflatoxinas s?o as micotoxinas de maior import?ncia agro-econ?mica pelos preju?zos acarretados ? produ??o animal. Passos importantes na s?ntese destas micotoxinas s?o descritos, assim como seu metabolismo e efeitos adversos. Este trabalho avalia a determina??o dos biomarcadores esfinganina (Sa) e esfingosina (So) no soro de frangos de corte como m?todo de diagn?stico para exposi??o ?s fumonisinas e compara os dados obtidos com a an?lise da ra??o. Dos animais avaliados apenas 49% exibiram quantidades detect?veis de esfingolip?deos no soro, dentre estes, apenas 44,9% exibiram os 2 esfingolip?deos (Sa e So), 53,06% exibiram somente So e 2,04% exibiram somente Sa. Foi detectada na ra??o a presen?a de fumonisina B1 na concentra??o de 374,92&#956;g.kg-1 e de aflatoxinas B1, B2 e G1 nas concentra??es de 440,99&#956;g.kg-1, 169,92&#956;g.kg-1 e 494,58&#956;g.kg-1, respectivamente. Foi poss?vel estabelecer parcialmente o grau de intoxica??o dos frangos e verificar que a sensibilidade do biomarcador pode ser afetada tanto pela presen?a de fumonisina quanto de aflatoxina.

Made available in DSpace on 2016-04-28T20:17:27Z (GMT). No. of bitstreams: 1 2005 - Jose Marcio Pimentel Martins.pdf: 659991 bytes, checksum: 0bc398add38956a83884eac082b821df (MD5) Previous issue date: 2005-07-22 / Fumonisins are a group of mycotoxins produced by species of the gender Fusarium, fungi of world wide distribution and with high prevalence in grains, mainly in corn, that frequently transmit these toxins to by-products or to foods destined to the human and animal consumption. The fumonisins are specific inhibitors of the synthesis of sphingolipids, causing sphinganine (Sa) and sphingosine (So) accumulations in the cells. The proportion among Sa and So has been told as being an effective intoxication biomarker for fumonisins in animals and humans. Could be used as a biomarker, in epidemic risings about the exhibition of a certain population to the fumonisins. In the equine it causes leukoencephalomalacia (ELEM), pulmonary edema in swine, liver toxicity, liver carcinogenicity and kidney disease in rats, poultry and rabbits, besides several effects in the immune system. The main ingredient of the rations used for swine is the corn and their derived and for that the mycotoxins research, especially fumonisins in the rations used in pigs farms is of vital importance due to the carted economical losses. There were used samples of blood and urine of four swine farms that provided swine to the slaughterhouse, in the municipal district of Rio de Janeiro. The samples of blood were obtained during the sangria and urine from vesical puncture. All samples were maintained under cooling and freezing until the analysis. The number of collected samples was regarding 10 % of the animals sloughed of each property / day, making the total of 76 samples, for 60 days. For the sphingolipids determination the methods used were proposed by CASTEGNARO et al. (1996, 1998), for analysis in liquid chromatograph of high efficiency. The results demonstrated that 17,77 % of the serum samples had the typical profile of intoxication for fumonisins, in 11,11 % it showed suggestive profile of the aflatoxins influence. In one of the properties 100 % of the appraised animals presented typical alterations of fumonisin action. For the urine samples, 26 % indicated the typical action of the fumonisins and in 39,13 % the alterations indicated the influence of the aflatoxins besides the fumonisins. Therefore the use of this biomarker for detecting fumonisin exposition in natural conditions was shown effective, however it should be considered the behavior as much of the Sa as So, allowing a deeper evaluation. / Fumonisinas s?o m grupo de micotoxinas produzidas por esp?cies do g?nero Fusarium, fungo de ampla distribui??o mundial e com alta preval?ncia em gr?os, principalmente no milho, que freq?entemente veiculam estas toxinas para subprodutos ou para alimentos destinados ao consumo humano e animal. As fumonisinas s?o inibidores espec?ficos da s?ntese dos esfingolip?dios, causando ac?mulos de esfinganina (Sa) e esfingosina (So) nas c?lulas. A propor??o entre Sa e So tem sido relatada como sendo eficaz biomarcador de intoxica??o por fumonisinas em animais e humanos, podendo ser utilizada em levantamentos epidemiol?gicos sobre a exposi??o de uma determinada popula??o ?s fumonisinas. Nos eq??deos causam leucoencefalomal?cia eq?ina (LEME), edema pulmonar e hidrot?rax em su?nos, hepatotoxidez, hepatocarcinogenicidade e nefropatias em ratos, aves e coelhos, al?m de diversos efeitos no sistema imune. O principal ingrediente das ra??es utilizadas para su?nos ? o milho e seus derivados e por isso a pesquisa de micotoxinas, em especial fumonisinas nas ra??es utilizadas em suinoculturas ? de vital import?ncia devido ?s perdas econ?micas acarretadas. Foram utilizadas amostras de sangue e urina de quatro granjas fornecedoras de um abatedouro de su?nos, localizado no munic?pio do Rio de Janeiro. As amostras de sangue foram obtidas durante a sangria e as de urina por pun??o vesical, no momento da eviscera??o.Todas mantidas sob refrigera??o e congelamento at? a an?lise. O n?mero de amostras coletadas foi referente a 10 % dos animais abatidos de cada propriedade / dia, perfazendo o total de 76 amostras, durante 60 dias. Para determina??o de esfingolip?dios foram utilizados os m?todos propostos por CASTEGNARO et al. (1996, 1998), para an?lise por cromatografia l?quida de alta efici?ncia. Os resultados demonstraram que 17,77 % das amostras de soro tinham o perfil t?pico de intoxica??o para fumonisinas, em 11,11 % apresentaram perfil sugestivo da influ?ncia de aflatoxinas. Em uma das propriedades 100 % dos animais avaliados apresentaram altera??es t?picas da a??o das fumonisinas. Nas amostras de urina, 26 % indicaram a a??o t?pica das fumonisinas e em 39,13 % as altera??es indicaram a influ?ncia das aflatoxinas al?m das fumonisinas. A utiliza??o deste biomarcador em condi??es naturais mostrou-se eficaz na detec??o da exposi??o ?s fumonisinas, por?m deve-se considerar o comportamento tanto da So quanto de Sa, que permitem uma avalia??o mais abrangente.

Sphingolipids (SLs) are complex lipid-derived structures that are essential components of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives ceramides are known to be involved in multiple processes such as the formation of lipid rafts, cell signalling and membrane trafficking. The first step of SL biosynthesis is universal to all sphingolipid-producing organisms from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase (SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal- 5'-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of structural homology and catalyse the decarboxylative Claisen-like condensation of amino acids with thioester substrates. The SPT enzyme catalyses the formation of the universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis, SPT plays a big role in regulating natural and pathological processes. A lot of research interest has been recently generated by SLs isolated from bacterial members of the human microbiome and their roles in human health. Increasing evidence suggests that some of these SLs possess immunoregulatory effects and can have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal gut-dwelling bacterium that belongs to a few human microbionts known to produce unique iso-branched sphingolipids (isoSLs); these have been shown to influence the human iNKT cell count. The production of SLs in B.fragilis is completely regulated by a gene product BF2461. In this work, BF2461 was expressed and purified; using a combination of UV-vis spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography, it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been assessed with a range of different chain-length substrates, including less common 15 and 17-carbon chain length coenzyme A substrates. The enzyme can produce different types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl- CoA. However, at high levels of PCoA, a substrate inhibition is observed that might point to a natural control mechanism employed by the bacterium in favour of producing iso-branched SLs (isoSLs). The structure of BfSPT has been elucidated in a complex with its amino acid substrate L-serine. Search and analysis of putative SPTs from other microbiome-associated bacteria that produce isoSLs show that they share high similarity with an average amino acid conservation of 74%, suggesting they might be adapted to a particular type of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be structurally characterised, and the first one to have a direct impact on human health. Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine, some common inhibitors of the PLP-dependent enzymes. The structure obtained in the presence of L-penicillamine provides the first direct structural evidence of the inhibitory mechanism by a thiazolidine complex formation in the active site of a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction and inhibition mechanisms of BfSPT as well as opening new prospects into researching this interesting target and its impact on the human microbiome.

A fumonisina B1 (FB1) e uma micotoxina produzida pelo metabolismo secundário de espécies de Fusarium, principalmente F. verticillioides e F. proliferatum, os quais contaminam diversos alimentos antes e apos o processamento, sobretudo o milho e derivados, gerando graves problemas para a Saúde Pública e a qualidade dos alimentos. O objetivo deste trabalho foi avaliar a exposição humana a FB1 presente nos alimentos através da estimativa de ingestão da toxina na dieta e da análise de diferentes biomarcadores presentes em amostras de sangue, urina e cabelo. Além disso, foram investigados os efeitos da toxina através da avaliação de ácido fólico presentes em alimentos e em soro, e os níveis de uréia e creatinina presentes em soro. O estudo foi realizado em dois municípios dos Estados de São Paulo e Santa Catarina, cujos respectivos voluntários foram categorizados como de baixo consumo de derivados de milho (Grupo A, voluntários de Pirassununga/SP) e de alto consumo de derivados de milho (Grupo B, voluntários de Erval Velho/SC). As amostras de alimentos do Grupo A (Pirassununga/SP) foram fornecidas pelos voluntários (n=100) nos meses de Junho/2011, Setembro/2011, Dezembro/2011 e Marco/2012. Os voluntários do Grupo B (Erval Velho/SC) (n=20) forneceram amostras de alimentos no mês de Abril/2012. Em cada grupo, uma lista com 20 alimentos a base de milho foi entregue aos voluntários, para fornecimento de amostras daqueles disponíveis em suas respectivas residências em cada mês de amostragem, totalizando 122 amostras de derivados de milho no Grupo A e 17 amostras no Grupo B coletadas durante o estudo. Adicionalmente, aplicou-se um Questionário de Frequência Alimentar (QFA) e um Inquérito Recordatório de 24 horas (QIR - 24 h) no momento das coletas de amostras. Em cada mês de amostragem de alimentos, foram coletadas amostras de sangue, urina (somente Grupo A) e cabelo dos voluntários, sendo as amostras armazenadas a -20ºC (urina e cabelo) ou -80ºC (sangue) até o momento das análises. As amostras de alimentos foram submetidas a análise de FB1, sendo que as de farinha de milho foram também analisadas quanto ao teor de ácido fólico. Ambas as análises foram feitas através de cromatografia líquida de alta eficiência (CLAE). Em soro, foram avaliadas a relação esfinganina/esfingosina (Sa/So), resíduos de FB1, ácido fólico, uréia e creatinina. Em urina, foram analisados os níveis de FB1, creatinina para correção do volume urinário e a relação Sa/So. Em cabelo, foram analisados os resíduos de FB1 através de CLAE acoplada a espectrometria de massas. Todos os métodos de análise foram submetidos a procedimento de otimização e validação intra--laboratorial. A incidência de FB1 nos alimentos foi, em média, 72% (n=122) nas amostras do Grupo A (Pirassununga/SP) e 35% (n=17) no Grupo B (Erval Velho/SC). Os maiores níveis foram encontrados em amostras de pipoca provenientes do Grupo B, com uma amostra excedendo o limite de tolerância estabelecido no Brasil (2,500 &micro;g kg-1). A ingestão diária provável média (IDPM) de FB1 no Grupo A foi de 63,3 ng kg-1 peso corpóreo (p.c.) dia-1, que corresponde a 3,1% da ingestão provisória máxima tolerável (IPMT) recomendada para fumonisinas (2.000 ng kg-1 p.c. dia-1). A IDPM do Grupo B apresentou uma média de 190,1 ng kg-1 p.c. dia-1 o que corresponde a 9,5% da IDMT. As concentrações de ácido fólico nas amostras de farinha de milho variaram de < 0,3 &micro;g kg-1 (limite de quantificação do método) a 1.705 &micro;g kg-1, com média de 713 &plusmn; 435 &micro;g kg-1. Somente uma amostra apresentou nível de ácido fólico acima do valor mínimo estabelecido pela ANVISA. Em urina, a incidência de FB1 foi de 33,4% (n=251), com níveis médios de 3,19 &plusmn; 3,15 ng mg-1 de creatinina. Não houve correlação (P&gt;0,05) entre as concentrações de FB1 na urina e nos alimentos. Os níveis de esfinganina foram mais elevados em mulheres, com 25,0% (n=116) de amostras positivas, em comparação à urina de homens, 10,4% (n=96). A relação Sa/So apresentou em média 0,91, 0,77 e 0,89 para urina de mulheres, homens e em combinação, respectivamente. Em soro, os níveis de esfingosina foram em média 2,48 ng mL-1 para o Grupo A e 5,01 ng mL-1 para o Grupo B. A relação Sa/So variou de 0,06 a 3,19 com média de 0,79 para o Grupo A e 0,78 para o Grupo B. Embora tenha havido correlação positiva (r=0,574, P&lt;0,05) entre a relação Sa/So no soro e os dados de consumo de milho e derivados obtidos no QIR-24 h, não foram observadas correlações (P&gt;0,05) entre a ingestão de FB1 e a relação Sa/So na urina ou soro. A concentração de ácido fólico no soro variou de 6,7 a 24,0 ng mL-1 (média de 13,4 &plusmn; 5,4 ng mL-1), com ambos os grupos (A e B) apresentando resultados dentro dos valores de referências. Não foram observados níveis detectáveis de FB1 nas amostras de soro. No entanto, FB1 foi detectada em 4 amostras de cabelo humano (7,2%) dos Grupos A e B, cuja concentração média foi de 21,3 &plusmn; 12,1 ng g-1. Em síntese, os resultados obtidos nas análises de biomarcadores de FB1 no presente trabalho estão de acordo com os valores de IDPM encontrados, indicando que a exposição a FB1 nas populações estudadas não representa um risco a saúde. / Fumonisin B1 (FB1) is a mycotoxin produced by the secondary metabolism of Fusarium species, mainly F. verticillioides and F. proliferatum, which contaminates foods before and after processing and causes serious problems to public health and food quality. The aim of this study was to evaluate the human exposure to FB1 in food by means of estimated intake of toxin in the diet, and analysis of different biomarkers in serum, urine and hair. In addition, folic acid in food and blood as well urea and creatinin in serum were investigated to evaluate the toxin effects. The study was conducted in two cities of Sao Paulo and Santa Catarina States, where the respective volunteers were categorized as low-consumers of corn products (Group A, volunteers from Pirassununga/SP) and high-consumers of corn products (Group B, volunteers from Erval Velho/SC). Food samples from Group A (Pirassununga/SP) were provided by volunteers (n=100) in June/2011, September/2011, December/2011 and March/2012. The volunteers from Group B (Erval Velho/SC) (n=20) provided food samples in April/2012. In each group, a list of 20 corn products was given to volunteers, to allow them to check and collect the food items available in their homes at each sampling time. The total number of samples of corn products provided by the volunteers were 122 and 17 in Group A and Group B, respectively. Addicionally, a Food Frequency Questionnaire (FFQ) and a 24-Hours Dietary Recall Questionnaire (24h-DRQ) were applied by the time of sample collections. In each month of food samples collection, samples of blood, urine (only Group A) and hair from the volunteers were collected and storage at -20ºC (urine and hair) or -80ºC (blood) until analysis. Food samples were submitted to determination of FB1, and corn meal samples were also evaluated for folic acid levels. Both analysis were performed by high performance liquid chromatography (HPLC). In serum, analyses included sphinganine/sphingosine ratio (Sa/So), FB1 residue, folic acid, urea and creatinine. In urine, the levels of FB1, creatinine to correct urinary volume and Sa/So ratio were evaluated. In hair, FB1 residues were analysed by HPLC coupled to mass spectrometry. All the analytical methods were submitted to optimization and intra-laboratorial validation procedures. The mean incidences of FB1 in corn products were 72% (n=122) in samples of Group A (Pirassununga/SP), and 35% (n=17) of Group B (Erval Velho/SC). The higher levels were found in popcorn from Group B, with one sample exceeding the tolerance limit established in Brazil (2,500 &micro;g kg-1). The mean probable daily intake (PDIM) of FB1 in Group A was 63.3 ng kg-1 body weigh (b.w.) day-1, which corresponds to 3.1% of provisional maximum tolerable intake (PMTDI) recommended for fumonisins (2,000 ng kg-1 b.w. day-1). PDIM of Group B was 190.1 ng kg-1 b.w. day-1, which represents 9.5% of PMTDI. Folic acid levels in corn meal ranged from &LT; 0,3 &micro;g kg-1 (quantification limit) to 1.705 &micro;g kg-1, with a mean of 713 &plusmn; 435 &micro;g kg-1. Only one sample had levels of folic acid above the minimum established by ANVISA. In urine, the incidence of FB1 was 33,4% (n=251), at mean levels of 3,19 &plusmn; 3,15 ng mg-1 of creatinine. There wasn\'t correlation (P&gt;0.05) between concentrations of FB1 in urine and foods. Sphinganine levels were higher in woman, with 25.0% (n=116) of positive samples in comparison to urine of men, 10.4% (n=96). The mean Sa/So ratios were 0.91, 0.77 and 0.89 for urine of women, men and in combination, respectively. In serum, sphingosine presented a mean of 2.48 ng mL-1 to Group A and 5.01 ng mL-1 to Group B. Sa/So ratio ranged from 0.06 to 3.19 with a mean of 0.79 to Group A and 0.78 to Group B. Although a positive correlation (r=0.574, P&lt;0.05) was found between Sa/So ratio in serum and corn consumption data obtained by 24h-DRQ, no correlation was observed (P&gt;0,05) with FB1 intake and Sa/So ratio in urine or serum. Folic acid concentration in serum ranged from 6.7 to 24.0 ng mL-1 (mean of 13.4 &plusmn; 5.4 ng mL-1), with both groups (A and B) presenting levels within the reference valuies. There were no detectable levels of FB1 in serum samples. However, FB1 was detected in 4 human hair samples (7.2%) of Groups A and B, at a mean concentration was 21.3 &plusmn; 12.1 ng g-1. In summary, the results obtained in the analyses of FB1 biomarkers in the present study are in agreement with the PDIM values found, hence indicating that FB1 exposure in the populations studied do not represent a health concern.