Riboflavin deficiency reduces bone mineral density in rats by compromising osteoblast function.

Insufficient riboflavin intake has been associated with poor bone health. This study aimed to investigate the effect of riboflavin deficiency on bone health in vivo and in vitro. Riboflavin deficiency was successfully developed in rats and osteoblasts. The results indicated that bone mineral density, serum bone alkaline phosphatase, bone phosphorus, and bone calcium were significantly decreased while serum ionized calcium and osteocalcin were significantly increased in the riboflavin-deficient rats. Riboflavin deficiency also induced the reduction of Runx2, Osterix, and BMP-2/Smad1/5/9 cascade in the femur. These results were further verified in cellular experiments. Our findings demonstrated that alkaline phosphatase activities and calcified nodules were significantly decreased while intracellular osteocalcin and pro-collagen I c-terminal propeptide were significantly increased in the riboflavin-deficient osteoblasts. Additionally, the protein expression of Osterix, Runx2, and BMP-2/Smad1/5/9 cascade were significantly decreased while the protein expression of p-p38 MAPK were significantly increased in the riboflavin-deficient cells compared to the control cells. Blockage of p38 MAPK signaling pathway with SB203580 reversed these effects in riboflavin-deficient osteoblastic cells. Our data suggest that riboflavin deficiency causes osteoblast malfunction and retards bone matrix mineralization via p38 MAPK/BMP-2/Smad1/5/9 signaling pathway.

Causes and Clinical Sequelae of Riboflavin Deficiency.

Riboflavin, in its cofactor forms flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), plays fundamental roles in energy metabolism, cellular antioxidant potential, and metabolic interactions with other micronutrients, including iron, vitamin B6, and folate. Severe riboflavin deficiency, largely confined to low-income countries, clinically manifests as cheilosis, angular stomatitis, glossitis, seborrheic dermatitis, and severe anemia with erythroid hypoplasia. Subclinical deficiency may be much more widespread, including in high-income countries, but typically goes undetected because riboflavin biomarkers are rarely measured in human studies. There are adverse health consequences of low and deficient riboflavin status throughout the life cycle, including anemia and hypertension, that could contribute substantially to the global burden of disease. This review considers the available evidence on causes, detection, and consequences of riboflavin deficiency, ranging from clinical deficiency signs to manifestations associated with less severe deficiency, and the related research, public health, and policy priorities.

Riboflavin (Vitamin B2) Deficiency Induces Apoptosis Mediated by Endoplasmic Reticulum Stress and the CHOP Pathway in HepG2 Cells.

Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway.

Riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Riboflavin deficiency led to lower blood cholesterol level and higher content of hepatic cholesterol in rats and the mechanisms are not clarified yet. We hypothesized that riboflavin deficiency might alter cholesterol homeostasis via apolipoprotein B100, one of the important proteins in cholesterol transport. To test this hypothesis, HepG2 cells were cultured in riboflavin-deficient media for 4 days to develop riboflavin deficiency. Compared to riboflavin-sufficient cells, the mRNA (0. 37 ± 0.04 vs 1.03 ± 0.29 relative expression level, n = 3) and protein expressions of apolipoprotein B100 (intracellular: 173.7 ± 14.4 vs 254.8 ± 47.2 µg/mg protein; extracellular: 93.8 ± 31.1 vs 161.6 ± 23.9 µg/mg protein; n = 3) were significantly reduced in riboflavin-deficient cells (P < 0.05). Endoplasmic reticulum oxidoreductin 1 and protein disulfide isomerase, two enzymes involved in the oxidative folding of apolipoprotein B100, were also lower remarkably in expression at both mRNA and protein levels. Meanwhile, intracellular cholesterol was increased (256.3 ± 17.1 µM/g protein vs 181.4 ± 23.9 µM/g protein, n = 4) and extracellular cholesterol decreased (110.0 ± 23.2 µM/g protein vs 166.2 ± 34.6 µM/g protein, n = 4) significantly in riboflavin-deficient cells (P < 0.05). Very low-density lipoprotein was also diminished (29.0 ± 6.1 µM/g protein vs 67.0 ± 11.0 µM/g protein, n = 4) in the culture media (P < 0.05). These findings suggest that riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Dietary riboflavin deficiency induces genomic instability of esophageal squamous cells that is associated with gut microbiota dysbiosis in rats.

SCOPE: Epidemiologic evidence suggests that riboflavin (RBF) deficiency is a specific nutritional predisposition for esophageal cancer. The aim of this study is to investigate the potential roles of gut microbiota in esophageal tumorigenesis caused by the RBF deficiency. METHODS: Male F344 rats were subcutaneously injected with the chemical carcinogen N-nitrosomethylbenzylamine (NMBA, 0.35 mg kg-1). Rats were assigned to 4 groups, denoted as R6 (normal RBF, 6 mg kg-1), R6N (normal RBF combined with NMBA), R6N → R0N (normal RBF conversion to RBF-deficiency), and R0N → R6N (RBF-deficiency conversion to normal RBF). Bacterial communities were analyzed based on high-throughput 16S rRNA gene sequencing. Oxidative DNA damage and double-strand break markers were studied by immunohistochemistry. RESULTS: The R6N → R0N diet enhanced the incidence of esophageal intraepithelial neoplasia (EIN, 40 weeks 66.7% vs. 25 weeks 16.7%, P < 0.05). RBF deficiency and replenishment modulated the gut microbiota composition. The gut microbiota (e.g. Caulobacteraceae, Sphingomonas and Bradyrhizobium) affected xenobiotic biodegradation and the genomic instability of the host. Furthermore, the RBF deficiency aggravated oxidative DNA damage and DNA double-strand breaks (immunohistochemistry) in the esophageal epithelium, whereas the RBF replenishment had the opposite effect (P < 0.05, respectively). CONCLUSIONS: RBF deficiency promotes NMBA-induced esophageal tumorigenesis, which is associated with gut microbiota-associated genomic instability, and offers new insights into the role of RBF deficiency in esophageal carcinogenesis.

In Silico Identification of a Key Residue for Substrate Recognition of the Riboflavin Membrane Transporter RFVT3.

Because of its specific physicochemical properties (fluorescence, photosensitizing, and redox reactions), vitamin B2, also called riboflavin (RF), has been generating a lot of interest in the fields of nanotechnology and bioengineering in the last decade. RF, by targeting its riboflavin transporters (RFVTs) overexpressed in some cancers, is particularly used to functionalize nanovectors for anticancer drug delivery. From a physiopathological point of view, an RF deficiency has been implicated in various pathologies, including mendelian diseases. RF deficiency is mainly due to natural variants of its RFVTs that make them inactive and therefore prevent RF transport. The lack of structural data about RFVT is a major drawback for a better understanding of the role of the mutations in the molecular mechanism of these transporters. In this context, this work was aimed at investigating the 3D structure of RFVT3 and its interactions with RF. For this purpose, we used an in silico procedure including protein threading, docking, and molecular dynamics. Our results propose that the natural variant W17R, known to be responsible for the Brown-Vialetto-Van Laere syndrome, prevents the recognition of RF by RFVT3 and thus blocks its transport. This in silico procedure could be used for elucidating the impact of pathogenic mutations of other proteins. Moreover, the identification of RF binding sites will be useful for the design of RF-functionalized nanovectors.

Riboflavin in Human Health: A Review of Current Evidences.

Riboflavin is a water-soluble vitamin, which was initially isolated from milk. There are two coenzyme forms of riboflavin, flavin mononucleotide and flavin adenine dinucleotide, in which riboflavin plays important roles in the enzymatic reactions. Riboflavin is found in a wide variety of animal and plant foods. Meat and dairy products are the major contributors of riboflavin dietary intake. In this chapter, the latest evidence on the relationship between riboflavin status and specific health risks will be reviewed. Also, some of the mechanisms by which riboflavin exerts its roles will be discussed. The evidence accrued suggests that riboflavin is an antioxidant nutrient which may prevent lipid peroxidation and reperfusion oxidative injury. Moreover, riboflavin deficiency may increase the risk of some cancers. Riboflavin may also exert a neuroprotective effects in some neurological disorders (e.g., Parkinson disease, migraine, and multiple sclerosis) through its role in some pathways that are hypothesized to be impaired in neurological disorders such as antioxidation, myelin formation, mitochondrial function, and iron metabolism.

Riboflavin transporter deficiency mimicking mitochondrial myopathy caused by complex II deficiency.

Biallelic likely pathogenic variants in SLC52A2 and SLC52A3 cause riboflavin transporter deficiency. It is characterized by muscle weakness, ataxia, progressive ponto-bulbar palsy, amyotrophy, and sensorineural hearing loss. Oral riboflavin halts disease progression and may reverse symptoms. We report two new patients whose clinical and biochemical features were mimicking mitochondrial myopathy. Patient 1 is an 8-year-old male with global developmental delay, axial and appendicular hypotonia, ataxia, and sensorineural hearing loss. His muscle biopsy showed complex II deficiency and ragged red fibers consistent with mitochondrial myopathy. Whole exome sequencing revealed a homozygous likely pathogenic variant in SLC52A2 (c.917G>A; p.Gly306Glu). Patient 2 is a 14-month-old boy with global developmental delay, respiratory insufficiency requiring ventilator support within the first year of life. His muscle biopsy revealed combined complex II + III deficiency and ragged red fibers consistent with mitochondrial myopathy. Whole exome sequencing identified a homozygous likely pathogenic variant in SCL52A3 (c.1223G>A; p.Gly408Asp). We report two new patients with riboflavin transporter deficiency, caused by mutations in two different riboflavin transporter genes. Both patients presented with complex II deficiency. This treatable neurometabolic disorder can mimic mitochondrial myopathy. In patients with complex II deficiency, riboflavin transporter deficiency should be included in the differential diagnosis to allow early treatment and improve neurodevelopmental outcome.

Adaptive regulation of riboflavin transport in heart: effect of dietary riboflavin deficiency in cardiovascular pathogenesis.

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 deficiency induces a significant change in proteomic profiles in HepG2 cells.

Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC-MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson's disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency.

Vitamin B2 deficiency enhances the pro-inflammatory activity of adipocyte, consequences for insulin resistance and metabolic syndrome development.

AIMS: Adipose tissue is an endocrine organ important for regulation of such physiological processes as energy metabolism or lipids homeostasis. In an obesity state, it participates in the induction of chronic systemic inflammation accompanied by pro-inflammatory cytokines and fatty acid elevation. For this reasons, adipose tissue is involved in, e.g., insulin resistance, type 2 diabetes or hyperlipidemia development. In our previous study, we have shown that riboflavin deficiency induces a pathological pro-inflammatory response of macrophages, the main component of adipose tissue. Therefore, in the current study, we investigated the alteration of the pro-inflammatory activity of adipocytes. MAIN METHODS: The study was conducted on mouse 3T3 L1 preadipocytes differentiated to adipocyte and culture in the state of riboflavin deficiency (3.1nM) or control condition (10.4nM). The cell viability, adiposity and glucose uptake was assessed. Moreover, mRNA expression, as well as crucial pro-inflammatory cytokines (TNFα, IL-6) and adipokines (adiponectin, leptin, resistin) release and NFκB activation, were evaluated. KEY FINDINGS: Results showed that riboflavin deprivation induced a significant elevation in adipocyte lipolysis and enhance obesity-related apoptosis of adipocytes. The generation of reactive oxygen species was enhanced in riboflavin-deficient adipocytes by 43%. Moreover, NFκB phosphorylation and the expression and release of both TNFα, IL-6 as well as leptin were elevated in a deficient group what was accompanied by a reduction of adiponectin level. CONCLUSION: Our study shows that riboflavin deficiency can promote the intensification of pro-inflammatory activity of adipocyte cells, leading consequently to the severity of chronic inflammation that accompanies obesity state.

Riboflavin Deficiency in Rats Decreases de novo Formate Production but Does Not Affect Plasma Formate Concentration.

Background: The one-carbon metabolism pathway is highly dependent on a number of B vitamins in order to provide one-carbon units for purine and thymidylate biosynthesis as well as homocysteine remethylation. Previous studies have examined folate and vitamin B-12 deficiency and their effects on formate metabolism; as of yet, to our knowledge, no studies on the effects of riboflavin deficiency on formate metabolism have been published.Objective: Our objective was to determine the effects of riboflavin deficiency on formate metabolism.Methods: Weanling male rats were randomly assigned either to control, riboflavin-replete (RR) or to experimental, riboflavin-deficient (RD) versions of the AIN-93G diet for 13 d, at which time a constant infusion of [13C]-formate was carried out to ascertain the effects of deficiency on formate production. Gas chromatography-mass spectrometry was used to measure plasma formate concentration and [13C]-formate enrichment. HPLC, LC-mass spectrometry (MS)/MS, and enzymatic assays were used for the measurement of one-carbon precursors and other metabolites.Results: RD rats had significantly lower rates of formate production (15%) as well as significantly reduced hepatic methylenetetrahydrofolate reductase activity (69%) and protein concentration (54%) compared with RR rats. There was no difference in plasma formate concentrations between the groups. Plasma serine, a potential one-carbon precursor, was significantly higher in RD rats (467 ± 73 µM) than in RR rats (368 ± 52 µM).Conclusions: Although deficiencies in folate and vitamin B-12 lead to major changes in plasma formate concentrations, riboflavin deficiency results in no significant difference; this disagrees with the prediction of a published mathematical model. Our observation of a lower rate of formate production is consistent with a role for flavoproteins in this process.

Riboflavin and health: A review of recent human research.

There has lately been a renewed interest in Riboflavin owing to insight into its recognition as an essential component of cellular biochemistry. The knowledge of the mechanisms and regulation of intestinal absorption of riboflavin and its health implications has significantly been expanded in recent years. The purpose of this review is to provide an overview of the importance of riboflavin, its absorption and metabolism in health and diseased conditions, its deficiency and its association with various health diseases, and metabolic disorders. Efforts have been made to review the available information in literature on the relationship between riboflavin and various clinical abnormalities. The role of riboflavin has also been dealt in the prevention of a wide array of health diseases like migraine, anemia, cancer, hyperglycemia, hypertension, diabetes mellitus, and oxidative stress directly or indirectly. The riboflavin deficiency has profound effect on iron absorption, metabolism of tryptophan, mitochondrial dysfunction, gastrointestinal tract, brain dysfunction, and metabolism of other vitamins as well as is associated with skin disorders. Toxicological and photosensitizing properties of riboflavin make it suitable for biological use, such as virus inactivation, excellent photosensitizer, and promising adjuvant in chemo radiotherapy in cancer treatment. A number of recent studies have indicated and highlighted the cellular processes and biological effects associated with riboflavin supplementation in metabolic diseases. Overall, a deeper understanding of these emerging roles of riboflavin intake is essential to design better therapies for future.

Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet.

This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish (P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity (P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor ß1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1ß, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase ß, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased (P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet.

Dietary riboflavin deficiency decreases immunity and antioxidant capacity, and changes tight junction proteins and related signaling molecules mRNA expression in the gills of young grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary riboflavin on the growth, gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). Fish were fed six diets containing graded levels of riboflavin (0.63-10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme and acid phosphatase activities, and complement component 3 content in the gills of fish (P < 0.05). Moreover, riboflavin deficiency caused oxidative damage, which might be partly due to decrease copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in the gills of fish (P < 0.05). Furthermore, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor ß1), tight junction proteins (Occludin, zonula occludens 1, Claudin-c and Claudin-3), signaling molecules (inhibitor of κBα, target of rapamycin and NF-E2-related factor 2) and antioxidant enzymes (copper, zinc superoxide dismutase and glutathione reductase) were significantly decreased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1ß), signaling molecules (nuclear factor kappa B p65, IκB kinase ß, IκB kinase γ, Kelch-like-ECH-associated protein 1b and myosin light chain kinase) and tight junction protein Claudin-12 were significantly increased (P < 0.05) in the gills of fish fed riboflavin-deficient diet. In addition, this study indicated for the first time that young fish fed a riboflavin-deficient diet exhibited anorexia and poor growth. In conclusion, riboflavin deficiency decreased growth and gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish. Based on percent weight gain, gill lysozyme activity and reduced glutathione content, the dietary riboflavin requirements for young grass carp (275-722 g) were estimated to be 5.85, 7.39 and 6.34 mg/kg diet, respectively.

Immunomodulatory effect of riboflavin deficiency and enrichment - reversible pathological response versus silencing of inflammatory activation.

Ariboflavinosis, that is, vitamin B2 deficiency, is a common problem affecting the populations of both developing and affluent countries. Teenagers, elderly people, pregnant women, and alcohol abusers represent groups that are particularly susceptible to this condition. This study was aimed to determine the effect of different riboflavin concentrations (deficiency and supplementation) on macrophages response induced by bacteria or yeast-derived factors i.e. lipopolysaccharide (LPS) and zymosan, respectively. Mouse macrophage RAW 264.7 cells were cultured for 5 days in a medium with a riboflavin concentration corresponding to moderate riboflavin deficiency (3.1 nM), physiological state (10.4 nM), or vitamin pill supplementation (300 nM). On the third or fourth day of deprivation, the medium in some groups was supplemented with riboflavin (300 nM). Macrophages activation were assessed after LPS or zymosan stimulation. Short-term (5 days) riboflavin deprivation resulted in the pathological macrophages activation, manifested especially in a reduction of cell viability and excess release of tumor necrosis factor-α (TNF-α) and high-mobility group box 1 (HMGB1) protein. Moreover, the levels of inducible nitric oxide synthase (iNOS), nitric oxide (NO), heat shock protein (Hsp72), interleukin 1ß (IL-1ß), monocyte chemoattractant protein-1 (MCP-1), and interleukin 10 (IL-10) decreased after riboflavin deprivation, but medium enrichment with riboflavin (300 nM) on the third or fourth day reversed this effect. In the riboflavin-supplemented group, LPS-stimulated macrophages showed lower mortality accompanied by higher Hsp72 expression, reduction of Toll-like receptor 4 (TLR4) and TNF-α, and elevation of NO, IL-6, and IL-10. Moreover, the TLR6, NO, iNOS, IL-1ß, MCP-1, and the keratinocyte chemoattractant (KC) levels significantly decreased in the zymosan-stimulated groups maintained in riboflavin-enriched medium. We conclude that short-term riboflavin deficiency significantly impairs the ability of macrophages to induce proper immune response, while riboflavin enrichment decreases the proinflammatory activation of macrophages.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Riboflavin deprivation inhibits macrophage viability and activity - a study on the RAW 264.7 cell line.

Riboflavin, or vitamin B2, as a precursor of the coenzymes FAD and FMN, has an indirect influence on many metabolic processes and determines the proper functioning of several systems, including the immune system. In the human population, plasma riboflavin concentration varies from 3·1 nM (in a moderate deficiency, e.g. in pregnant women) to 10·4 nM (in healthy adults) and 300 nM (in cases of riboflavin supplementation). The purpose of the present study was to investigate the effects of riboflavin concentration on the activity and viability of macrophages, i.e. on one of the immunocompetent cell populations. The study was performed on the murine monocyte/macrophage RAW 264.7 cell line cultured in medium with various riboflavin concentrations (3·1, 10·4, 300 and 531 nM). The results show that riboflavin deprivation has negative effects on both the activity and viability of macrophages and reduces their ability to generate an immune response. Signs of riboflavin deficiency developed in RAW 264.7 cells within 4 d of culture in the medium with a low riboflavin concentration (3·1 nM). In particular, the low riboflavin content reduced the proliferation rate and enhanced apoptotic cell death connected with the release of lactate dehydrogenase. The riboflavin deprivation impaired cell adhesion, completely inhibited the respiratory burst and slightly impaired phagocytosis of the zymosan particles. In conclusion, macrophages are sensitive to riboflavin deficiency; thus, a low riboflavin intake in the diet may affect the immune system and may consequently decrease proper host immune defence.

Biosynthesis of flavin cofactors in man: implications in health and disease.

The primary role of the water-soluble vitamin B2, i.e. riboflavin, in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding as well as in diverse regulatory events. Deficiency of riboflavin in men and experimental animal models has been linked to several diseases, including neuromuscular and neurological disorders and cancer. Riboflavin at pharmacological doses has been shown to play unexpected and incompletely understood regulatory roles. Besides a summary on riboflavin uptake and a survey on riboflavin-related diseases, the main focus of this review is on discovery and characterization of FAD synthase (EC 2.7.7.2) and other components of the cellular networks that ensure flavin cofactor homeostasis.Special attention is devoted to the problem of sub-cellular compartmentalization of cofactor synthesis in eukaryotes, made possible by the existence of different FAD synthase isoforms and specific molecular components involved in flavin trafficking across sub-cellular membranes.Another point addressed in this review is the mechanism of cofactor delivery to nascent apo-proteins, especially those localized into mitochondria, where they integrate FAD in a process that involves additional mitochondrial protein(s) still to be identified. Further efforts are necessary to elucidate the role of riboflavin/FAD network in human pathologies and to exploit the structural differences between human and microbial/fungal FAD synthase as the rational basis for developing novel antibiotic/antimycotic drugs.

The discovery and characterization of riboflavin.

The first observation of a pigment in milk with yellow-green fluorescence can be traced to the English chemist Alexander Wynter Blyth in 1872, but it was not until the early 1930s that the substance was characterized as riboflavin. Interest in accessory food factors began in the latter half of the 19th century with the discovery of the first vitamin, thiamin. Thiamin was water soluble and given the name vitamin B(1). However, researchers realized that there were one or more additional water-soluble factors and these were called the vitamin B-2 complex. The search to identify these accessory food factors in milk, whole wheat, yeast, and liver began in the early 1900s. As there is no classical nutritional disease attributable to riboflavin deficiency, it was the growth-stimulating properties of the food extracts given to young rats that provided the tool with which to investigate and eventually extract riboflavin. Riboflavin was the second vitamin to be isolated and the first from the vitamin B-2 complex; the essential nature of the vitamin as a food constituent for man was shown in 1939.