Role of riboflavin deficiency in malaria pathophysiology.

The emergence of resistance against antimalarials and insecticides poses a significant threat to malaria elimination strategies. It is crucial to explore potential risk factors for malaria to identify new targets and alternative therapies. Malnutrition is a well-established risk factor for malaria. Deficiencies of micronutrients such as vitamin A, zinc, iron, folic acid, and phenotypic measures of malnutrition, such as stunting and wasting, have been studied extensively in the context of malaria. Vitamin B2, also known as riboflavin, is a micronutrient involved in maintaining cellular homeostasis. Riboflavin deficiency has been shown to have an inverse correlation with malarial parasitaemia. This article reviews the role of riboflavin in maintaining redox homeostasis and probes how riboflavin deficiency could alter malaria pathogenesis by disrupting the balance between oxidants and antioxidants. Though riboflavin analogues have been explored as antimalarials, new in vivo and patient-based research is required to target riboflavin-associated pathways for antimalarial therapy.

Strachan's syndrome and riboflavin deficiency.

Strachan's syndrome comprises a triad of optic, auditory and painful sensory peripheral neuropathy. It has been recognised since the late 19th century and is presumed to result from nutritional deficiency. Patients present acute or subacutely after a period of systemic illness, weight loss or, most commonly, dietary restriction, especially veganism, which can cause riboflavin (vitamin B2) and vitamin B12 deficiencies. The syndrome is more common in people who are black British and often of Jamaican descent. We describe the clinical phenotype using a typical case example, review other endemic nutritional peripheral neuropathies and discuss the potential benefit of riboflavin as a treatment.

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.

A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency.

AIFM1 is a mitochondrial flavoprotein involved in caspase-independent cell death and regulation of respiratory chain complex biogenesis. Mutations in the AIFM1 gene have been associated with multiple clinical phenotypes, but the effectiveness of riboflavin treatment remains controversial. Furthermore, few studies explored the reasons underlying this controversy. We reported a 7-year-old boy with ataxia, sensorimotor neuropathy and muscle weakness. Genetic and histopathological analyses were conducted, along with assessments of mitochondrial function and apoptosis level induced by staurosporine. Riboflavin deficiency and supplementation experiments were performed using fibroblasts. A missense c.1019T > C (p. Met340Thr) variant of AIFM1 was detected in the proband, which caused reduced expression of AIFM1 protein and mitochondrial dysfunction as evidenced by downregulation of mitochondrial complex subunits, respiratory deficiency and collapse of ΔΨm. The proportion of apoptotic cells in mutant fibroblasts was lower than controls after induction of apoptosis. Riboflavin deficiency resulted in decreased AIFM1 protein levels, while supplementation with high concentrations of riboflavin partially increased AIFM1 protein levels in variant fibroblasts. In addition, mitochondrial respiratory function of mutant fibroblasts was partly improved after riboflavin supplementation. Our study elucidated the pathogenicity of the AIFM1 c.1019T > C variant and revealed mutant fibroblasts was intolerant to riboflavin deficiency. Riboflavin supplementation is helpful in maintaining the level of AIFM1 protein and mitochondrial respiratory function. Early riboflavin treatment may serve as a valuable attempt for patients with AIFM1 variant.

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.

Avian riboflavin deficiency causes reliably reproducible peripheral nerve demyelination and, with vitamin supplementation, rapid remyelination.

Riboflavin deficiency produces severe peripheral neve demyelination in young, rapidly growing chickens. While this naturally-occurring vitamin B2 deficiency can cause a debilitating peripheral neuropathy, and mortality, in poultry flocks, it can also be a useful experimental animal model to study the pathogenesis of reliably reproducible peripheral nerve demyelination. Moreover, restitution of normal riboflavin levels in deficient birds results in brisk remyelination. It is the only acquired, primary, demyelinating tomaculous neuropathy described to date in animals. The only other substance that causes peripheral nerve demyelination similar to avian riboflavin deficiency is tellurium and the pathologic features of the peripheral neuropathy produced by this developmental neurotoxin in weanling rats are also described.

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 intake and status and relationship to anemia.

Riboflavin in its coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, is essential for multiple redox reactions necessary for energy production, antioxidant protection, and metabolism of other B vitamins, such as niacin, pyridoxine, and folate. Erythrocyte glutathione reductase activity coefficient (EGRac) is a biomarker of riboflavin status; ratios ≥1.40 are commonly interpreted as indicating biochemical deficiency. Most research on riboflavin status comes from low-income countries and rural settings, which reported high rates of riboflavin deficiency and inadequate intake. However, some studies suggest that riboflavin deficiency, based on the functional indicator EGRac, is also of concern in middle- and high-income countries. Biochemical riboflavin deficiency that does not cause clinical symptoms may contribute to anemia, particularly among women and children. Riboflavin enhances iron absorption, and riboflavin deficiency decreases iron mobilization from stores. The current knowledge on riboflavin's role in metabolic processes and its biochemical status is summarized in this review, and the available evidence on the role of riboflavin in anemia among different populations is discussed.

Thiamin and riboflavin status with related enzyme activities in pulmonary tuberculosis with diabetes mellitus in Shandong province of China.

BACKGROUND AND OBJECTIVES: Poor nutritional status is a common finding in pulmonary tuberculosis (TB) patients with and without type 2 diabetes mellitus (T2DM), thiamin (VB-1) and riboflavin (VB-2) are coenzymes important for the activation of many enzymes involved in improving nutritional status. We aimed to investigate enzymatic activities and the associations between VB-1 and VB-2, and their relations to nutritional status in TB and TB+T2DM patients. METHODS AND STUDY DESIGN: This was a cross-sectional study that prospectively enrolled TB 40 patients with or without T2DM respectively from the Chest Hospital of Qingdao and 76 healthy controls with similar age and gender distributions were recruited from the medical center of the affiliated hospital of Qingdao Medical College. The erythrocyte transketolase activation coefficient (ETKac, for VB-1 deficiency), the glutathione reductase activation coefficient (EGRac, for VB-2 deficiency), and metabolic enzyme activities were analyzed. RESULTS: VB-1 and VB-2 deficiency rates were higher, and enzyme activities were lower in TB and TB+T2DM relative to control group. ETKac and EGRac were negatively correlated with enzyme activities, either with body mass index (BMI), while enzyme activities were positively associated with BMI. CONCLUSIONS: VB-1 and VB-2 concentrations were lower in TB patients with or without T2DM relative to controls, with concomitant reductions in the activity levels of key metabolic enzymes. Significant correlations were observed between VB-1 and VB-2 concentrations and the activity of these metabolic enzymes, they all correlated with nutrition status. VB-1 and VB-2 concentrations may thus impact metabolic enzyme activity and thereby influence nutritional status.

Riboflavin deficiency leads to irreversible cellular changes in the RPE and disrupts retinal function through alterations in cellular metabolic homeostasis.

Ariboflavinosis is a pathological condition occurring as a result of riboflavin deficiency. This condition is treatable if detected early enough, but it lacks timely diagnosis. Critical symptoms of ariboflavinosis include neurological and visual manifestations, yet the effects of flavin deficiency on the retina are not well investigated. Here, using a diet induced mouse model of riboflavin deficiency, we provide the first evidence of how retinal function and metabolism are closely intertwined with riboflavin homeostasis. We find that diet induced riboflavin deficiency causes severe decreases in retinal function accompanied by structural changes in the neural retina and retinal pigment epithelium (RPE). This is preceded by increased signs of cellular oxidative stress and metabolic disorder, in particular dysregulation in lipid metabolism, which is essential for both photoreceptors and the RPE. Though many of these deleterious phenotypes can be ameliorated by riboflavin supplementation, our data suggests that some patients may continue to suffer from multiple pathologies at later ages. These studies provide an essential cellular and mechanistic foundation linking defects in cellular flavin levels with the manifestation of functional deficiencies in the visual system and paves the way for a more in-depth understanding of the cellular consequences of ariboflavinosis.

The Effect of a Single Bout of Exercise on Vitamin B2 Status Is Not Different between High- and Low-Fit Females.

High-fitness individuals have been suggested to be at risk of a poor vitamin B2 (riboflavin) status due to a potentially higher vitamin B2 demand, as measured by the erythrocyte glutathione reductase (EGR) activation coefficient (EGRAC). Longer-term exercise interventions have been shown to result in a lower vitamin B2 status, but studies are contradictory. Short-term exercise effects potentially contribute to discrepancies between studies but have only been tested in limited study populations. This study investigated if vitamin B2 status, measured by EGRAC, is affected by a single exercise bout in females who differ in fitness levels, and that represents long-term physical activity. At baseline and overnight after a 60-min cycling bout at 70% V·O2peak, EGR activity and EGRAC were measured in 31 young female adults, divided into a high-fit (V·O2peak ≥ 47 mL/kg/min, N = 15) and low-fit (V·O2peak ≤ 37 mL/kg/min, N = 16) group. A single exercise bout significantly increased EGR activity in high-fit and low-fit females (Ptime = 0.006). This response was not affected by fitness level (Ptime*group = 0.256). The effect of exercise on EGRAC was not significant (Ptime = 0.079) and not influenced by EGR activity. The exercise response of EGRAC was not significantly different between high-fit and low-fit females (Ptime*group = 0.141). Thus, a single exercise bout increased EGR activity, but did not affect EGRAC, indicating that vitamin B2 status was not affected. The exercise response on EGRAC and EGR did not differ between high-fit and low-fit females.

Effects of riboflavin deficiency on the lipid metabolism of duck breeders and duck embryos.

This study aimed to evaluate the effects of dietary riboflavin deficiency (RD) on the lipid metabolism of duck breeders and duck embryos. A total of 40 female 40-wk-old white Pekin duck breeders were randomly divided into 2 groups, received either RD diet (1.48 mg riboflavin/kg) or control diet (16.48 mg riboflavin/kg, CON) for 14 wk. Each group consisted of 20 duck breeders (10 replicates per group, 2 birds per replicate), and all experiment birds were single-caged. At the end of the experiment, reproductive performance, hepatic riboflavin, hepatic flavin mononucleotide (FMN), hepatic flavin adenine dinucleotide (FAD), hepatic morphology, hepatic lipid contents, and hepatic protein expression of duck breeders and duck embryos were measured. The results showed that the RD had no effect on egg production and egg fertility but reduced egg hatchability, duck embryo weight, hepatic riboflavin, FMN, and FAD status compared to results obtained in the CON group (all P < 0.05). Livers from RD ducks presented enlarged lipid droplets, excessive accumulation of total lipids, triglycerides, and free fatty acids (all P < 0.05). In addition to excessive lipids accumulation, medium-chain specific acyl-CoA dehydrogenase expression was downregulated (P < 0.05), and short-chain specific acyl-CoA dehydrogenase expression was upregulated in maternal and embryonic livers (P < 0.05). RD did not affect maternal hepatic acyl-CoA dehydrogenase family member 9 (ACAD9) expression, but duck embryonic hepatic ACAD9 expression was reduced in the RD group (P < 0.05). Collectively, dietary RD conditioned lower egg hatchability and inhibited the development of duck embryos. Increased accumulation of lipids, both maternal and embryo, was impaired due to the reduced flavin protein expression, which caused inhibition of hepatic lipids utilization. These findings suggest that abnormal duck embryonic growth and low hatchability caused by RD might be associated with disorders of lipid metabolism in maternal as well as embryos.

Effect of Vitamin B2-Deficient Diet on Hydroxyproline- or Obesity-Induced Hyperoxaluria in Mice.

SCOPE: Hyperoxaluria is a major cause of kidney stone disease. Around half of the oxalate in mammals is supplied from the diet and the other half is endogenously synthesized from glyoxylate. Reduction of hepatic glycolate oxidase (GO) activity is one approach to reduce endogenous production of oxalate. However, there are currently few effective dietary approaches to reduce hepatic GO activity. METHODS AND RESULTS: In the present study, it is investigated whether restriction of dietary vitamin B2 (VB2) can reduce hepatic GO activity and oxalate excretion in mice with hyperoxaluria induce by hydroxyproline (Hyp) or obesity. It is found that VB2 restriction significantly reduces hepatic GO activity in both the Hyp- and obesity-induced model of hyperoxaluria in mice. However, VB2 restriction reduces urinary oxalate excretion only in the Hyp-treated mice and not the obese mice. This difference could be due to the contribution of endogenous oxalate production that manifests as increased hepatic GO activity in Hyp-treated mice but not obese mice. CONCLUSION: Together these results suggest that VB2 restriction could be a new dietary approach to improve hyperoxaluria when endogenous production of oxalate is increased.

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 ariboflavinosis and esophageal epithelial atrophy in association with modification of gut microbiota in rats.

PURPOSE: Riboflavin deficiency causes ariboflavinosis, a common nutritional deficiency disease. The purpose of this study is to investigate the effects of riboflavin deficiency on the important internal organs and its potential mechanisms. METHODS: Experiment 1, male F344 rats were randomly assigned to R6 (normal riboflavin, 6 mg/kg) and R0 (riboflavin-deficient, 0 mg/kg) groups. Experiment 2 rats were assigned to R6, R0.6 (0.6 mg/kg) and R0.06 (0.06 mg/kg) groups. Experiment 3 rats were assigned to R6 and R0 → R6 (riboflavin replenishment) groups. Bacterial communities were analyzed based on 16S rRNA gene sequencing. RESULTS: Riboflavin deficiency induced ariboflavinosis (R0.06 46.7%; R0 72%) and esophageal epithelial atrophy (R0.06 40%; R0 44%) in rats, while the R6 group did not display symptoms (P < 0.001, respectively). Esophageal epithelial atrophy occurred simultaneously (R0.06 66.7%; R0 63.6%) with ariboflavinosis or appeared alone (R0.06 33.3%; R0 36.4%). Esophagus is the most vulnerable internal organ. Riboflavin deficiency followed by replenishment (R0 → R6) was effective in treating ariboflavinosis (83.3% vs. 0%, P < 0.001) and esophageal epithelial atrophy (66.7% vs. 20%, P = 0.17). Riboflavin deficiency modulated gut microbiota composition. The several key genera (Romboutsia, Turicibacter and Clostridium sensu stricto 1) were strongly correlated with ariboflavinosis and esophageal epithelial atrophy (P < 0.01 or P < 0.05). The potential mechanism is that gut microbiota affects body's xenobiotic biodegradation and metabolism, and genomic instability. CONCLUSIONS: Riboflavin deficiency induces ariboflavinosis and esophageal epithelial atrophy by modulating the gut microbiota, and offers new Queryinsight into riboflavin deficiency and esophageal lesions.

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.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice.

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3-/-) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3-/- embryos. Slc52a3-/- mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Hematologic presentation and the role of untargeted metabolomics analysis in monitoring treatment for riboflavin transporter deficiency.

Riboflavin transporter deficiency (RTD) (MIM #614707) is a neurogenetic disorder with its most common manifestations including sensorineural hearing loss, peripheral neuropathy, respiratory insufficiency, and bulbar palsy. Here, we present a 2-year-old boy whose initial presentation was severe macrocytic anemia necessitating multiple blood transfusions and intermittent neutropenia; he subsequently developed ataxia and dysarthria. Trio-exome sequencing detected compound heterozygous variants in SLC52A2 that were classified as pathogenic and a variant of uncertain significance. Bone marrow evaluation demonstrated megaloblastic changes. Notably, his anemia and neutropenia resolved after treatment with oral riboflavin, thus expanding the clinical phenotype of this disorder. We reiterate the importance of starting riboflavin supplementation in a young child who presents with macrocytic anemia and neurological features while awaiting biochemical and genetic work up. We detected multiple biochemical abnormalities with the help of untargeted metabolomics analysis associated with abnormal flavin adenine nucleotide function which normalized after treatment, emphasizing the reversible pathomechanisms involved in this disorder. The utility of untargeted metabolomics analysis to monitor the effects of riboflavin supplementation in RTD has not been previously reported.