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The antibiotic roseoflavin is produced by Streptomyces davaonensis in the stationary phase of growth. To support biosynthesis of the secondary metabolite roseoflavin, S. davaonensis underwent several genetic adaptations with regard to metabolism of the roseoflavin precursor and primary metabolite riboflavin. In addition to 17 riboflavin biosynthesis genes at different chromosomal locations, S. davaonensis contains the riboflavin transporter gene ribM being part of the riboflavin biosynthetic operon ribE1MAB5H. Deletion of this operon generated riboflavin auxotrophic S. davaonensis strains. The finding that S. davaonensis ΔribE1MAB5H was able to grow in a culture medium containing low levels of riboflavin indicated that, in addition to RibM, a second riboflavin transporter is present in this bacterium. The S. davaonensis genes ribXY (former rosXY) represented candidate genes for such a second riboflavin transport system and the results of our experiments now show that RibXY from S. davaonensis is a highly efficient riboflavin importer but not a roseoflavin importer.

Cellular senescence is a state of permanent proliferative arrest induced by a variety of stresses, such as DNA damage. The transcriptional activity of p53 has been known to be essential for senescence induction. It remains unknown, however, whether among the downstream genes of p53, there is a gene that has antisenescence function. Our recent studies have indicated that the expression of SLC52A1 (also known as GPR172B/RFVT1), a riboflavin transporter, is up-regulated specifically in senescent cells depending on p53, but the relationship between senescence and SLC52A1 or riboflavin has not been described. Here, we examined the role of SLC52A1 in senescence. We found that knockdown of SLC52A1 promoted senescence phenotypes induced by DNA damage in tumor and normal cells. The senescence suppressive action of SLC52A1 was dependent on its riboflavin transport activity. Furthermore, elevation of intracellular riboflavin led to activation of mitochondrial membrane potential (MMP) mediated by the mitochondrial electron transport chain complex II. Finally, the SLC52A1-dependent activation of MMP inhibited the AMPK-p53 pathway, a central mediator of mitochondria dysfunction–related senescence. These results suggest that SLC52A1 contributes to suppress senescence through the uptake of riboflavin and acts downstream of p53 as a negative feedback mechanism to limit aberrant senescence induction.

Background Brown-Vialetto-Van Laere syndrome (BVVLS) and Fazio-Londe disease (FLD) are rare neurological disorders presenting with pontobulbar palsy, muscle weakness and respiratory insufficiency. Mutations in SLC52A2 (hRFVT-2) or SLC52A3 (hRFVT-3) genes can be responsible for these disorders with an autosomal recessive pattern of inheritance. The aim of this study was to screen for mutations in SLC52A2 and SLC52A3 among Indian families diagnosed with BVVLS and FLD. Methods SLC52A2 and SLC52A3 were screened in one FLD and three BVVLS patients by exon-specific amplification using PCR and sequencing. In silico predictions using bioinformatics tools and confocal imaging using HEK-293 cells were performed to determine the functional impact of identified mutations. Results Genetic analysis of a mother and son with BVVLS was identified with a novel homozygous mutation c.710C>T (p.Ala237Val) in SLC52A3. This variant was found to have an autosomal pseudodominant pattern of inheritance, which was neither listed in the Exome Variant Server or in the 1000 Genomes Project database. In silico analysis and confocal imaging of the p.Ala237Val variant showed higher degree of disorderness in hRFVT-3 that could affect riboflavin transport. Furthermore, a common homozygous mutation c.62A>G (p.Asn21Ser) was identified in other BVVLS and FLD patients. Despite having different clinical phenotypes, both BVVLS and FLD can be attributed to this mutation. Conclusion A rare and peculiar pattern of autosomal pseudodominant inheritance is observed for the first time in two genetically related BVVLS cases with Indian origin and a common mutation c.62A>G (p.Asn21Ser) in SLC52A3 can be responsible for both BVVLS and FLD with variable phenotypes.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD), or glutaric aciduria type II (GAII), is a group of clinically heterogeneous disorders caused by mutations in electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) - the two enzymes responsible for the re-oxidation of enzyme-bound flavin adenine dinucleotide (FADH2) via electron transfer to the respiratory chain at the level of coenzyme Q10. Over the past decade, an increasing body of evidence has further coupled mutations in FAD metabolism (including intercellular riboflavin transport, FAD biosynthesis and FAD transport) to MADD-like phenotypes. In this review we provide a detailed description of the overarching and specific metabolic pathways involved in MADD. We examine the eight associated genes (ETFA, ETFB, ETFDH, FLAD1, SLC25A32 and SLC52A1-3) and clinical phenotypes, and report ∼436 causative mutations following a systematic literature review. Finally, we focus attention on the value and shortcomings of current diagnostic approaches, as well as current and future therapeutic options for MADD and its phenotypic disorders.

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we discovered that SLC22A14 is a riboflavin transporter localized at the mitochondrial inner membranes of spermatozoa midpiece, and showed via genetic, biochemical, multi-omic and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we found that fatty acid β-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from TCA cycle, but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrated that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wildtype mice treated with riboflavin deficient diet mimics those in Slc22a14 knockout mice, confirming that altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for development of male contraceptives.

Background The waterborne diarrheagenic bacterium Vibrio cholerae, cause of the pandemic cholera disease, thrives in a variety of environments ranging from estuarine waters to the human intestinal tract. This species has two ways to obtain the essential micronutrient riboflavin, de novo biosynthesis and environmental uptake through the RibN importer. The way these functions interrelate to fulfill riboflavin needs in different conditions in this species is unknown. Results This study analyzed the contributions of riboflavin biosynthesis and transport to the culturability of Vibrio cholerae in river and seawater in vitro and in the Caenorhabditis elegans nematode host model. Elimination of the ribD riboflavin biosynthetic gene renders the bacteria riboflavin-auxotrophic, while a ribN mutant strain has no growth defect in minimal media. When growing in river water, deletion of ribD causes an impairment in culturability. In this condition, the ∆ribN strain has a defect to compete against a wild type strain but outcompetes the ∆ribD strain. The latter effect is inverted by the addition of riboflavin to the water. In contrast, growth in seawater causes a loss in culturability independent of riboflavin biosynthesis or transport. In the C. elegans model, only the ∆ribD strain is attenuated. Conclusion Results indicate that while riboflavin biosynthesis seems to outweigh riboflavin uptake, the latter may still provide a selective advantage to V. cholerae in some environments.

Deficiency or defective transport of riboflavin (RF) is known to cause neurological disorders, cataract, cardiovascular anomalies, and various cancers by altering the biochemical pathways. Mechanisms and regulation of RF uptake process is well characterized in the cells of intestine, liver, kidney, and brain origin, while very little is known in the heart. Hence, we aimed to understand the expression and regulation of RF transporters (rRFVT-1 and rRFVT-2) in cardiomyocytes during RF deficiency and also investigated the role of RF in ischemic cardiomyopathy and mitochondrial dysfunction in vivo. Riboflavin uptake assay revealed that RF transport in H9C2 is (1) significantly higher at pH 7.5, (2) independent of Na+ and (3) saturable with a Km of 3.746 µM. For in vivo studies, male Wistar rats (110–130 g) were provided riboflavin deficient food containing 0.3 ± 0.05 mg/kg riboflavin for 7 weeks, which resulted in over expression of both RFVTs in mRNA and protein level. RF deprivation resulted in the accumulation of cardiac biomarkers, histopathological abnormalities, and reduced mitochondrial membrane potential which evidenced the key role of RF in the development of cardiovascular pathogenesis. Besides, adaptive regulation of RF transporters upon RF deficiency signifies that RFVTs can be considered as an effective delivery system for drugs against cardiac diseases.

Riboflavin (vitamin B2) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [3H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [3H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l -ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [3H]riboflavin uptake with a Km of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [3H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na+- and Cl−-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na+- and Cl−-independent. [3H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 &plusmn, 0.07 &micro, M and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B2) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [3H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [3H]riboflavin with a Km of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [3H]riboflavin uptake was affected under a K+-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [3H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the “RfnT-like” CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb3-Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

Summary Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid β-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives., Graphical abstract, Highlights • Slc22a14 deficiency results in decreased sperm motility and male infertility • Slc22a14 ablation disrupts fatty acid β-oxidation and flavoenzyme activity • Slc22a14 is a riboflavin transporter located at inner mitochondrial membrane in sperm, Long-chain fatty acid β-oxidation is an important energy source during epididymal maturation of spermatozoa. Kuang et al. show that Slc22a14 is a riboflavin transporter located at the inner mitochondrial membrane. Slc22a14 deficiency in the spermatozoa mid-piece disrupts the riboflavin transport and subsequently alters flavoenzyme-mediated bioenergetic metabolism, resulting in male infertility.

Riboflavin (vitamin B2), a water-soluble vitamin, is an essential nutrient in higher organisms as it is not endogenously synthesised, with requirements being met principally by dietary intake. Tissue-specific transporter proteins direct riboflavin to the intracellular machinery responsible for the biosynthesis of the flavocoenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These flavocoenzymes play a vital role in ensuring the functionality of a multitude of flavoproteins involved in bioenergetics, redox homeostasis, DNA repair, chromatin remodelling, protein folding, apoptosis, and other physiologically relevant processes. Hence, it is not surprising that the impairment of flavin homeostasis in humans may lead to multisystem dysfunction including neuromuscular disorders, anaemia, abnormal fetal development, and cardiovascular disease. In this review, we provide an overview of riboflavin absorption, transport, and metabolism. We then focus on the clinical and biochemical features associated with biallelic FLAD1 mutations leading to FAD synthase deficiency, the only known primary defect in flavocoenzyme synthesis, in addition to providing an overview of clinical disorders associated with nutritional deficiency of riboflavin and primary defects of riboflavin transport. Finally, we give a brief overview of disorders of the cellular flavoproteome. Because riboflavin therapy may be beneficial in a number of primary or secondary disorders of the cellular flavoproteome, early recognition and prompt management of these disorders is imperative.

BACKGROUND/AIM Riboflavin transport in enterocytes is mediated by three translocators: RFVT3 located on the apical membrane, and RFVT1 and RFVT2 on the basolateral membrane. The aim of this study was to investigate whether the expression levels of RFVTs are altered in human colorectal cancer (CRC). MATERIALS AND METHODS In human colon adenocarcinoma cell lines (CaCo2, DLD-1, HT-29) and in tissues of patients with CRC, gene and protein expression levels were evaluated by real time-polymerase chain reaction and western blotting. Intracellular flavin content was determined by high-performance liquid chromatography. RESULTS RFVT3 and RFVT2 gene and protein expression levels were higher in DLD-1 and HT-29 compared to Caco2 cells. In HT-29 cells, the RFVT1 protein level was drastically lower. These differences are presumably responsible for the higher total flavin content in DLD-1 and HT-29 cells. In tumor tissues of patients with CRC, RFVT1 content was reduced at both protein and mRNA levels compared to normal mucosa. RFVT3 and RFVT2 gene expression levels were increased, while protein expression was reduced, with a small reduction in riboflavin amount. CONCLUSION This study provides first evidence that transcription/translation of RFVTs are profoundly altered in CRC.

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is a clinically heterogeneous disorder affecting fatty acid and amino acid metabolism. Presentations range from a severe neonatal form with hypoglycemia, metabolic acidosis, and hepatomegaly with or without congenital anomalies to later-onset lipid storage myopathy. Genetic testing for MADD traditionally comprises analysis of ETFA, ETFB, and ETFDH. Patients may respond to pharmacological doses of riboflavin, particularly those with late-onset MADD due to variants in ETFDH. Increasingly other genes involved in riboflavin transport and flavoprotein biosynthesis are recognized as causing a MADD phenotype. Flavin adenine dinucleotide synthase (FADS) deficiency caused by biallelic variants in FLAD1 has been identified in nine previous cases of MADD. FLAD1 missense mutations have been associated with a riboflavin-responsive phenotype; however the effect of riboflavin with biallelic loss of function FLAD1 mutations required further investigation. Herein we describe a novel, truncating variant in FLAD1 causing MADD in an 8-year-old boy. Fibroblast studies showed a dramatic reduction in FADS protein with corresponding reduction in the FAD synthesis rate and FAD cellular content, beyond that previously documented in FLAD1-related MADD. There was apparent biochemical and clinical response to riboflavin treatment, beyond that previously reported in cases of biallelic loss of function variants in FLAD1. Early riboflavin treatment may have attenuated an otherwise severe phenotype.

ECF transporters are a family of active transporters for vitamins. They are composed of four subunits: a membrane-embedded substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT) and two ATP-binding-cassette ATPases (EcfA and EcfA'). We have investigated the mechanism of the ECF transporter for riboflavin from the pathogen Listeria monocytogenes, LmECF-RibU. Using structural and biochemical approaches, we found that ATP binding to the EcfAA' ATPases drives a conformational change that dissociates the S subunit from the EcfAA'T ECF module. Upon release from the ECF module, the RibU S subunit then binds the riboflavin transport substrate. We also find that S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Our results explain how ECF transporters capture the transport substrate and reproduce the in vivo observations on S-subunit competition for which the family was named.

Riboflavin transporters (rft-1 and rft-2), orthologous to human riboflavin transporter-3 (hRVFT-3), are identified and characterized in Caenorhabditis elegans. However, studies pertaining to functional contribution of rft-2 in maintaining body homeostatic riboflavin levels and its regulation are very limited. In this study, the expression pattern of rft-2 at different life stages of C. elegans was studied through real-time PCR, and found to be consistent from larval to adult stages that demonstrate its involvement in maintaining the body homeostatic riboflavin levels at whole animal level all through its life. A possible regulation of rft-2 expression at mRNA levels at whole animal was studied after adaptation to low and high concentrations of riboflavin. Abundance of rft-2 transcript was upregulated in riboflavin-deficient conditions (10 nM), while it was downregulated with riboflavin-supplemented conditions (2 mM) as compared with control (10 meu M). Further, the 5'-regulatory region of the rft-2 gene was cloned, and transgenic nematodes expressing transcriptional rft-2 promoter::GFP fusion constructs were generated. The expression of rft-2 was found to be adaptively regulated in vivo when transgenic worms were maintained under different extracellular riboflavin levels, which was also mediated partly via changes in the rft-2 levels that directs towards the possible involvement of transcriptional regulatory events.

The Drosophila eye color gene brown is known to control the transport of pteridine precursors in adult eyes. The Brown protein belongs to the ATP-binding cassette (ABC) transporter G family, which includes proteins encoded by the genes brown, scarlet, and white. These genes are responsible for pigmentation in Drosophila and the domestic silkworm Bombyx mori. Although orthologs of brown are conserved among insects, the function of this gene is only known in Drosophila. Here, we elucidated the function of the B. mori ortholog Bm-brown. We examined the spatial and temporal expression profiles of Bm-brown and found that this gene was specifically and continuously expressed in larval Malpighian tubules (MTs), indicating this gene has a special function in MTs. We then successfully obtained a Bm-brown knockout (KO) strain based on a wild-type (WT) strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system. We found that larval MTs of the KO strain were white, whereas those of WT were yellow. It is known that larval yellow MTs of WT are due to the accumulation of riboflavin. Therefore, we compared the riboflavin contents of MTs of KO and WT strains, and found that the riboflavin level in the KO strain was 20 fold less than that in WT during the 5th instar period. MTs are known to exhibit a similar milky color in w-3 mutant larvae due to a deficiency of riboflavin accumulation. The responsible gene for w-3 mutant is the Bmwh3 gene, which is orthologous to Drosophila white. Thus, we speculate that Bm-brown is heterodimerized with Bmwh3, similar to Brown/White in Drosophila, and acts as a riboflavin transporter in silkworm MTs.

Background Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. Methodology/Principal findings Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. Conclusions/Significance The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target., Author summary In this work, we show that riboflavin plays a key role in the trypanosomatid life cycles and describe a novel family of riboflavin transporters (RibJ) with uptake function. Despite the vital importance of riboflavin for all living cells, RibJ are the first transporters described in protists. We functionally characterized the T. cruzi and T. brucei RibJ members and the effect of riboflavin analogs on parasite physiology. The structural and biochemical differences presented between human transporters and RibJ members make riboflavin transport and downstream metabolism, attractive and potential trypanosomatid targets.

Brown-Vialetto-Van Laere syndrome (BVVLS) represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter (RFVT) genes, SLC52A2 and SLC52A3, have recently been linked to BVVLS. However, the genetic frequency, neuropathology and downstream consequences of RFVT mutations have previously been undefined. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between RFVT mutations and BVVLS, identifying twenty-two pathogenic mutations in SLC52A2 and SLC52A3, fourteen of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain (ETC), we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. ETC complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila RFVT homologue revealed reduced levels of riboflavin, downstream metabolites, and ETC complex I activity. RFVT knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings indicate mitochondrial dysfunction as a downstream consequence of RFVT gene defects in BVVLS and validate riboflavin esters as a potential therapeutic strategy.

Riboflavin is an essential micronutrient used for flavin mononucleotide and flavin adenine dinucleotide biosynthesis that plays a crucial role in metabolic reactions and is critical for normal cellular functions, growth, and development. The inability of vertebrates to synthesize riboflavin and its pivotal role in metabolism necessitate specialized processes for their transport into a particular cell and efflux for utilization by other cells. In humans, dietary absorption from enterocytes and subsequent intracellular distribution of riboflavin involve primary active transport, which is mediated by three transporters, namely RFVT1 ( SLC52A1 ), RFTV2 ( SLC52A2 ), and RFVT3 ( SLC52A3 ). Studies have extensively characterized the functional properties of riboflavin transport from diverse cells of human origin. Proper transporter functioning is essential for maintaining cells and body homeostasis, whereas the inability to maintain homeostasis has led to death or a disease. To gain insight into riboflavin transporters, this review summarizes their genomic distribution, their structural topologies, and the associated clinical manifestations.

Riboflavin (vitamin B2) acts as an intermediary during various biochemical oxidation-reduction reactions in the liver. Hepatic riboflavin homeostasis is suggested to be maintained through its transporter(s). Riboflavin transporters, RFVT2/Slc52a2 and RFVT3/Slc52a3, have been identified in rodents. However, the role of each RFVT in the hepatic homeostasis of riboflavin has not yet been fully clarified. In this study, we assessed the contribution of each RFVT to riboflavin uptake into the liver using in vitro and in vivo studies. The uptake of riboflavin by mouse primary hepatocytes increased in a time-dependent and a concentration-dependent manner. Riboflavin transport was independent of extracellular Na(+). However, the uptake decreased slightly along with the extracellular pH increases. Real-time PCR analysis revealed that the mRNA level of Slc52a2, or coding for mouse (m)RFVT2, in the mouse liver was 10 times higher than that of Slc52a3 (coding for mRFVT3). The uptake of riboflavin at pH 7.4 by primary hepatocytes was significantly decreased by the transfection of Slc52a2-small interfering RNA (siRNA), but not Slc52a3-siRNA. Furthermore, we also confirmed the contribution of riboflavin transporters in vivo. The riboflavin concentrations in plasma, but not in the liver, were significantly decreased in mice fed on a riboflavin-deficient diet for 8 weeks. The expression of Slc52a2 mRNA was significantly upregulated by riboflavin deprivation. These results strongly suggest that mRFVT2 was involved in hepatic riboflavin homeostasis.

The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus . Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B 2 ), RoF, and AF in the human pathogen Listeria monocytogenes , a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945 , is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes . IMPORTANCE The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus . RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes . We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.

Homeostasis of riboflavin should be maintained by transporters. Previous in vitro studies have elucidated basic information about riboflavin transporter RFVT3 encoded by SLC52A3 gene. However, the contribution of RFVT3 to the maintenance of riboflavin homeostasis and the significance in vivo remain unclear. Here, we investigated the physiological role of RFVT3 using Slc52a3 knockout (Slc52a3−/−) mice. Most Slc52a3−/− mice died with hyperlipidemia and hypoglycemia within 48 hr after birth. The plasma and tissue riboflavin concentrations in Slc52a3−/− mice at postnatal day 0 were dramatically lower than those in wild-type (WT) littermates. Slc52a3−/− fetuses showed a lower capacity of placental riboflavin transport compared with WT fetuses. Riboflavin supplement during pregnancy and after birth reduced neonatal death and metabolic disorders. To our knowledge, this is the first report to indicate that Rfvt3 contributes to placental riboflavin transport, and that disruption of Slc52a3 gene caused neonatal mortality with hyperlipidemia and hypoglycemia owing to riboflavin deficiency.

Recent studies elucidated how riboflavin transporters and FAD forming enzymes work in humans and create a coordinated flavin network ensuring the maintenance of cellular flavoproteome. Alteration of this network may be causative of severe metabolic disorders such as multiple acyl-CoA dehydrogenase deficiency (MADD) or Brown-Vialetto-van Laere syndrome. A crucial step in the maintenance of FAD homeostasis is riboflavin uptake by plasma and mitochondrial membranes. Therefore, studies on recently identified human plasma membrane riboflavin transporters are presented, together with those in which still unidentified mitochondrial riboflavin transporter(s) have been described. A main goal of future research is to fill the gaps still existing as for some transcriptional, functional and structural details of human FAD synthases (FADS) encoded by FLAD1 gene, a novel "redox sensing" enzyme. In the frame of the hypothesis that FADS, acting as a "FAD chaperone", could play a crucial role in the biogenesis of mitochondrial flavo-proteome, several basic functional aspects of flavin cofactor delivery to cognate apo-flavoenzyme are also briefly dealt with. The establishment of model organisms performing altered FAD homeostasis will improve the molecular description of human pathologies. The molecular and functional studies of transporters and enzymes herereported, provide guidelines for improving therapies which may have beneficial effects on the altered metabolism.

Brown-Vialetto-Van Laere syndrome was first described in 1894 as a rare neurodegenerative disorder characterized by progressive sensorineural deafness in combination with childhood amyotrophic lateral sclerosis. Mutations in the gene, SLC52A3 (formerly C20orf54), one of three known riboflavin transporter genes, have recently been shown to underlie a number of severe cases of Brown-Vialetto-Van Laere syndrome; however, cases and families with this disease exist that do not appear to be caused by SLC52A3 mutations. We used a combination of linkage and exome sequencing to identify the disease causing mutation in an extended Lebanese Brown-Vialetto-Van Laere kindred, whose affected members were negative for SLC52A3 mutations. We identified a novel mutation in a second member of the riboflavin transporter gene family (gene symbol: SLC52A2) as the cause of disease in this family. The same mutation was identified in one additional subject, from 44 screened. Within this group of 44 patients, we also identified two additional cases with SLC52A3 mutations, but none with mutations in the remaining member of this gene family, SLC52A1. We believe this strongly supports the notion that defective riboflavin transport plays an important role in Brown-Vialetto-Van Laere syndrome. Initial work has indicated that patients with SLC52A3 defects respond to riboflavin treatment clinically and biochemically. Clearly, this makes an excellent candidate therapy for the SLC52A2 mutation-positive patients identified here. Initial riboflavin treatment of one of these patients shows promising results.

Riboflavin and ubiquinone (Coenzyme Q(10), CoQ(10)) deficiencies are heterogeneous groups of autosomal recessive conditions affecting both children and adults. Riboflavin (vitamin B(2))-derived cofactors are essential for the function of numerous dehydrogenases. Genetic defects of the riboflavin transport have been detected in Brown-Vialetto-Van Laere and Fazio-Londe syndromes (C20orf54), and haploinsufficiency of GPR172B has been proposed in one patient to cause persistent riboflavin deficiency. Mutations in the electron tranferring fravoprotein genes (ETFA/ETFB) and its dehydrogenase (ETFDH) are causative for multiple acyl-CoA dehydrogenase deficiency. Mutations in ACAD9, encoding the acyl-CoA dehydrogenase 9 protein were recently reported in mitochondrial disease with respiratory chain complex I deficiency. All these conditions may respond to riboflavin therapy. CoQ(10) is a lipid-soluble component of the cell membranes, where it functions as a mobile electron and proton carrier, but also participates in other cellular processes as a potent antioxidant, and by influencing pyrimidine metabolism. The increasing number of molecular defects in enzymes of the CoQ(10) biosynthetic pathways (PDSS1, PDSS2, COQ2, COQ6, COQ9, CABC1/ADCK3) underlies the importance of these conditions. The clinical heterogeneity may reflect blocks at different levels in the complex biosynthetic pathway. Despite the identification of several primary CoQ(10) deficiency genes, the number of reported patients is still low, and no true genotype-phenotype correlations are known which makes the genetic diagnosis still difficult. Additionally to primary CoQ(10) deficiencies, where the mutation impairs a protein directly involved in CoQ(10) biosynthesis, we can differentiate secondary deficiencies. CoQ(10) supplementation may be beneficial in both primary and secondary deficiencies and therefore the early recognition of these diseases is of utmost importance.

Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment.

Background: Riboflavin transporter deficiency (RTD), formerly known as Brown-Vialetto-van Laere syndrome, is an early-onset neurodegenerative disorder with distinctive phenotypes. RTD is caused by mutations in either the SLC52A2 or SLC52A3 genes that encode riboflavin transporters RFVT-2 and RFVT-3, respectively. Methods: This was a 3-year retrospective case review from the Cure RTD International Registry. Results: 73 individuals (~60% female, 14 deceased) from 56 families had genetically confirmed RTD Type 2, including 30 novel SLC52A2 mutations (24 missense, 2 nonsense, 4 deletion). The mean ages at symptom onset and at diagnosis were 2.4 years (SD 1.5, range 0.25–8, n=63) and 12.0 years (SD 10.2, range 0.75–52, n=56) respectively. Most common presenting symptoms were sensory ataxia (n=43), sensorineural hearing loss (n=22), nystagmus/visual loss secondary to optic atrophy (n=14), upper limb weakness (n=11), and respiratory insufficiency (n=9). Treatment included high dose riboflavin, other supplements, and supportive care; 7 individuals required transfusions for anemia pre-riboflavin treatment and 17 (25%) received a cochlear implant. The minimum prevalence of RTD was estimated to be 1 per million, with >100 new cases each year. Conclusions: This is the largest case series of RTD to date. Early recognition and prompt riboflavin treatment is essential for survival and optimal outcome.

Transport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting 3H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation.

We report on three patients (two siblings and one unrelated) presenting in infancy with progressive muscle weakness and paralysis of the diaphragm. Metabolic studies revealed a profile of plasma acylcarnitines and urine organic acids suggestive of a mild form of the multiple acyl-CoA dehydrogenation defect (MADD, ethylmalonic/adipic acid syndrome). Subsequently, a profound flavin deficiency in spite of a normal dietary riboflavin intake was established in the plasma of all three children, suggesting a riboflavin transporter defect. Genetic analysis of these patients demonstrated mutations in the C20orf54 gene which encodes the human homolog of a rat riboflavin transporter. This gene was recently implicated in the Brown-Vialetto-Van Laere syndrome, a rare neurological disorder which may either present in infancy with neurological deterioration with hypotonia, respiratory insufficiency and early death, or later in life with deafness and progressive ponto-bulbar palsy. Supplementation of riboflavin rapidly improved the clinical symptoms as well as the biochemical abnormalities in our patients, demonstrating that high dose riboflavin is a potential treatment for the Brown-Vialetto-Van Laere syndrome as well as for the Fazio Londe syndrome which is considered to be the same disease entity without the deafness.

Brown-Vialetto-Van Laere syndrome is a rare neurological disorder with a variable age at onset and clinical course. The key features are progressive ponto-bulbar palsy and bilateral sensorineural deafness. A complex neurological phenotype with a mixed picture of upper and lower motor neuron involvement reminiscent of amyotrophic lateral sclerosis evolves with disease progression. We identified a candidate gene, C20orf54, by studying a consanguineous family with multiple affected individuals and subsequently demonstrated that mutations in this gene were the cause of disease in other, unrelated families.

Intact mitochondria isolated from Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells can take up riboflavin via carrier-mediated systems that operate at different concentration ranges and have different uptake efficiencies. Once inside mitochondria, riboflavin is converted into catalytically active cofactors, FMN and FAD, due to the existence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2). Newly synthesized FAD can be exported from intact mitochondria via a putative FAD exporter. The dependence of FMN synthesis rate on riboflavin concentration shows saturation kinetics with a sigmoidal shape (S0.5, Vmax and Hill coefficient values 0.32 ± 0.12 μm, 1.4 nmol·min−1·mg−1 protein and 3.1, respectively). The FAD-forming enzymes are both activated by MgCl2, and reside in two distinct monofunctional enzymes, which can be physically separated in mitochondrial soluble and membrane-enriched fractions, respectively.

A newborn female presented on the first day of life with clinical and biochemical findings consistent with multiple acyl-CoA dehydrogenase deficiency (MADD). Riboflavin supplementation corrected the biochemical abnormalities 24 h after commencing the vitamin. In vitro acylcarnitine profiling in intact fibroblasts both in normal and riboflavin depleted media showed normal oxidation of fatty acids excluding defects in electron transfer flavoprotein (ETF), or ETF ubiquinone oxidoreductase (ETF:QO), or a genetic abnormality in flavin metabolism. In addition, sequencing of the genes encoding ETF and ETF:QO in the proband did not reveal any pathogenic mutations. Determination of the maternal riboflavin status after delivery showed that the mother was riboflavin deficient. Repeat testing done two years after the infant's birth and while on a normal diet showed that the mother was persistently riboflavin deficient and showed a typical MADD profile on plasma acylcarnitine testing. A possible genetic defect in riboflavin transport of metabolism in the mother is postulated to be the cause of the transient MADD seen in the infant. Sequencing of the SLC16A12, RFK and FLAD1 genes encoding key enzymes in riboflavin transport of metabolism in the mother did not identify any pathogenic mutations. The underlying molecular basis of the mother's defect in riboflavin metabolism remains to be established. Udgivelsesdato: null-null

Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria. Fil: Gutiérrez Preciado, Ana. Universidad Nacional Autónoma de México; México Fil: Torres, Alfredo Gabriel. University of Texas Medical Branch; Estados Unidos Fil: Merino, Enrique. Universidad Nacional Autónoma de México; México Fil: Bonomi, Hernan Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: García Angulo, Víctor Antonio. University of Texas Medical Branch; Estados Unidos

Riboflavin Binding Protein (RBP) is a stable glycophosphoprotein abundant in the eggs of many oviparous species, including chickens. The function of the protein is to transfer riboflavin (Rf) to th...

To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium.rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter.The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl- but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin.Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.

Riboflavin is unique among the water-soluble vitamins in that milk and dairy products make the greatest contribution to its intake in Western diets. Meat and fish are also good sources of riboflavin, and certain fruit and vegetables, especially dark-green vegetables, contain reasonably high concentrations. Biochemical signs of depletion arise within only a few days of dietary deprivation. Poor riboflavin status in Western countries seems to be of most concern for the elderly and adolescents, despite the diversity of riboflavin-rich foods available. However, discrepancies between dietary intake data and biochemical data suggest either that requirements are higher than hitherto thought or that biochemical thresholds for deficiency are inappropriate. This article reviews current evidence that diets low in riboflavin present specific health risks. There is reasonably good evidence that poor riboflavin status interferes with iron handling and contributes to the etiology of anemia when iron intakes are low. Various mechanisms for this have been proposed, including effects on the gastrointestinal tract that might compromise the handling of other nutrients. Riboflavin deficiency has been implicated as a risk factor for cancer, although this has not been satisfactorily established in humans. Current interest is focused on the role that riboflavin plays in determining circulating concentrations of homocysteine, a risk factor for cardiovascular disease. Other mechanisms have been proposed for a protective role of riboflavin in ischemia reperfusion injury; this requires further study. Riboflavin deficiency may exert some of its effects by reducing the metabolism of other B vitamins, notably folate and vitamin B-6.

Previous studies by our laboratory have suggested the potential role of receptor-mediated endocytosis components in the cellular translocation of riboflavin (vitamin B2). To delineate the intracellular compartments and events involved in the internalization of riboflavin, we synthesized a rhodamine-labeled riboflavin conjugate to monitor its movement via fluorescent microscopy. Cellular uptake studies in BeWo cells show that rhodamine-riboflavin conjugate exhibits similar ligand affinity toward the putative riboflavin transport system as [3H]riboflavin, whereas rhodamine does not significantly interfere with its internalization mechanism. Microscope analysis reveals rapid internalization of the rhodamine-riboflavin conjugate via a riboflavin-specific process into acidic vesicular compartments throughout the cells. The intracellular punctate distribution is comparable with that of fluorescein isothiocyanate (FITC)-transferrin, a well characterized receptor-mediated endocytosis substrate. Double-labeling fluorescence microscopy studies further confirm that with 10 min of internalization, rhodamine-riboflavin conjugate substantially concentrates within vesicular structures associated with clathrin, rab5, FITC-transferrin, and the acidotropic marker LysoTracker Blue. In summary, our studies provide, for the first time, direct morphological evidence of the involvement of endocytosis machinery in the intracellular trafficking of riboflavin. The subcellular localization of rhodamine-riboflavin conjugate suggests that, under the experimental conditions in this study, the internalization of riboflavin follows a classical receptor-mediated endocytosis pathway.