Riboflavin Depletion Research Articles

Riboflavin depletion promotes longevity and metabolic hormesis in Caenorhabditis elegans.

Riboflavin is an essential cofactor in many enzymatic processes and in the production of flavin adenine dinucleotide (FAD). Here, we report that the partial depletion of riboflavin through knockdown of the C.elegans riboflavin transporter 1 (rft-1) promotes metabolic health by reducing intracellular flavin concentrations. Knockdown of rft-1 significantly increases lifespan in a manner dependent upon AMP-activated protein kinase (AMPK)/aak-2, the mitochondrial unfolded protein response, and FOXO/daf-16. Riboflavin depletion promotes altered energetic and redox states and increases adiposity, independent of lifespan genetic dependencies. Riboflavin-depleted animals also exhibit the activation of caloric restriction reporters without any reduction in caloric intake. Our findings indicate that riboflavin depletion activates an integrated hormetic response that promotes lifespan and healthspan in C.elegans.

A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells.

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H2O2 was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport.IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle–Related Gene Transcription

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.

Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

Riboflavin depletion of intestinal cells in vitro leads to impaired energy generation and enhanced oxidative stress

Riboflavin is an essential component of the human diet, with an established role for its derivative cofactors in oxidative metabolism. Our previous in vivo data suggest that riboflavin may act as a signalling molecule in the intestinal lumen, regulating crypt development and cell turnover. Our in vitro studies in riboflavin-depleted intestinal cells in culture indicate that riboflavin depletion impairs normal mitosis. The aim of the study was to establish an improved intestinal cell model of riboflavin depletion using the structural analogue of riboflavin, lumiflavin (7,8,10-trimethyl-isoalloxazine) and to determine effects on cell function. The study was conducted using three intestinal cell lines, Caco-2, HCT116 and HT29 cells. Cell growth was inhibited in all three cell lines, in a lumiflavin concentration-dependent manner. Riboflavin depletion was confirmed through a significant decrease in intracellular riboflavin concentrations in Caco-2 and HT29 cell lines and a significant increase in the activation coefficient for the flavin adenine dinucleotide-dependent enzyme glutathione reductase. Riboflavin depletion led to a significant reduction in intracellular ATP concentration, and an enhanced generation of reactive oxygen species was also observed in response to riboflavin depletion, in all cell lines; effects were at least fivefold greater in Caco-2 cells than other cells. Riboflavin-depleted Caco-2 and HCT116 cells also showed an irreversible loss of proliferative potential. A model system of intracellular riboflavin depletion in intestinal epithelial cells has been developed. Riboflavin depletion induced by lumiflavin results in oxidative stress and a disruption of energy generation, which may contribute to observed effects on cell proliferation.

Riboflavin Depletion Impairs Cell Proliferation in Adult Human Duodenum: Identification of Potential Effectors

Riboflavin (vitamin B2) is an essential dietary component with a known function in oxidative metabolism. Our previous data using a rat model of riboflavin deficiency suggested that riboflavin also functions as a luminal signaling molecule regulating crypt development and cell turnover. Riboflavin deficiency is prevalent in both high- and low-income countries across the globe. This study aims to establish whether riboflavin deficiency has consequences for gastrointestinal (GI) morphology in adults and what the effects and effectors of any such alteration may be. Duodenal biopsies and blood samples were collected from a cross-section of gastroscopy patients. Crypt morphology and cell division were studied by immunohistochemistry, and biochemical riboflavin status was determined. Additionally a cell culture model of riboflavin deficiency was developed and analyzed using a combination of flow cytometry, and microarray and clonogenic assays. Duodenal crypts from subjects in the lowest quartile of riboflavin status were significantly shorter (P=0.023), less cellular (P=0.007), and had fewer cell divisions (P=0.034) than the crypts of subjects in the top quartile of riboflavin status. Following riboflavin depletion of colon cells in culture, cell cycle slowed. Microscopy revealed impaired mitosis and accumulation of aneuploid cells. Alterations in gene expression profiles reflected this alteration, with several mitosis-related genes altered, including AspM, cyclin B1, and Birc5 downregulated and Kif23 upregulated. Riboflavin depletion in vitro caused irreversible loss of proliferative potential of cells. Riboflavin depletion in adult humans impairs proliferation and proliferative potential of intestinal cells, which may have implications for gastrointestinal function.

Riboflavin mobilization from eleocyte stores in the earthworm Dendrodrilus rubidus inhabiting aerially-contaminated Ni smelter soil

A 6-week reciprocal transfer laboratory exposure experiment was conducted with two populations of the epigeic earthworm Dendrodrilus rubidus; one population inhabited a site approx. 200 m downwind of an active Ni smelter co-contaminated with Ni and Cu (3648 and 977 μg g −1 d.w., respectively), the other inhabited uncontaminated soil. Worms transferred from unpolluted to Ni/Cu-polluted soil lost body mass (62%); they also had reduced (70%) total coelomocyte number, including autofluorescent eleocytes, and had significantly decreased (92%) riboflavin-derived fluorescence emission measured at 525 nm. Coelomocyte counts were low, and 525 nm emission was negligible in worms maintained on their native Ni/Cu soil. Earthworms and their coelomocytes were unaffected when transferred from Ni/Cu-polluted soil to unpolluted soil. In conclusion, exposing worms to stress-inducing factors, including metal pollution, alters the riboflavin status within the immune-competent cells of D. rubidus, but it requires further in vivo studies to establish whether the reduction in the fluorescence signal is predominantly due to depletion of riboflavin-containing eleocytes, or to riboflavin quenching, or to enzymatic conversion (and thus depletion) of stored riboflavin into its functional immune-potentiating flavin derivatives, FMN and FAD. The flavin budget of D. rubidus coelomocytes recovered by a reproducible extrusion procedure is a potentially useful biomarker for assessing sublethal stress in this early colonizer of disturbed soils.

Basics in Clinical Nutrition: Nutritional support in different clinical situations

Severe malnutrition is always a result of inadequate intake of energy and basic nutrients or it can be a result of increased energy expenditure, which is not matched by an adequate intake. It often develops relatively slowly over a long period, as a result of somatic or psychiatric disease (see related chapters). However, it can develop quickly during a short period of stress starvation. Regardless of the aetiology, severe malnutrition is associated with depletion of body protein, fat, glycogen, potassium, phosphate, magnesium, zinc, selenium, vitamin A, vitamin E, vitamin C, thiamine, pyridoxine, riboflavin and other vitamins. As malnutrition is commonly associated with an increased risk of infection, delayed wound healing and surgical complications, the severely malnourished subject must receive nutritional support, given preferably by the oral or enteral route. Only in the presence of gastrointestinal dysfunction is parenteral nutrition indicated. When starting nutritional support in a severely malnourished patient, one must remember:

Mild Depletion of Dietary Folate Combined with Other B Vitamins Alters Multiple Components of the Wnt Pathway in Mouse Colon ,

Preclinical and clinical studies suggest that diminished folate status increases the risk of colorectal carcinogenesis. However, many biochemical functions of folate are dependent on the adequate availability of other 1-carbon nutrients, including riboflavin, vitamin B-6, and vitamin B-12. Aberrations in the Wnt pathway are thought to play an important role in human colorectal cancers. This study therefore investigated if mild depletion of folate combined with depletion of riboflavin, vitamin B-6, and vitamin B-12 could induce alterations in the Wnt pathway in the colonic mucosa. Ninety-six mice were pair-fed diets with different combinations of B vitamin depletion for 10 wk. Genomic DNA methylation and uracil misincorporation were measured by LC/MS and GC/MS. Gene-specific methylation, strand breaks, and expressions were measured by real-time PCR and immunoblotting. Proliferation and apoptosis were determined by immunohistochemistry. DNA strand breaks within the Apc mutation cluster region were induced by folate depletion combined with inadequacies of riboflavin, vitamin B-6, and vitamin B-12 (P < 0.05), but such effects were not induced by folate depletion alone. Similarly, minor changes in the expression of Apc, β-catenin, and cyclin D1 produced by mild folate depletion were significantly magnified by multiple vitamin depletion. Apoptosis, which can be suppressed by increased Wnt-signaling, was attenuated by the combined deficiency state (P < 0.05) but not by singlet or doublet deficiencies. These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to induce alterations in components of the Wnt pathway may produce such effects when present in conjunction with mild inadequacies of other 1-carbon nutrients.

HepG2 cells develop signs of riboflavin deficiency within 4 days of culture in riboflavin-deficient medium

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential coenzymes in redox reactions. For example, FAD is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/l) medium for up to 6 days; controls were cultured in riboflavin-sufficient (532 nmol/l) medium. The activity of glutathione reductase decreased by 98% within 4 days of riboflavin-deficient culture. Transport rates of riboflavin increased in response to riboflavin depletion, whereas expression of enzymes mediating flavocoenzyme synthesis (flavokinase and FAD synthetase) decreased in response to depletion. The oxidative folding and synthesis of plasminogen and apolipoprotein B-100 was impaired within 4 days of culture in riboflavin-deficient medium; this is consistent with impaired processing of secretory proteins in riboflavin-deficient cells. Riboflavin depletion was associated with increased DNA-binding activities of transcription factors with affinity for endoplasmic reticulum stress elements and nuclear factor κB (NF-κB) consensus elements, suggesting cell stress. Moreover, the abundance of the stress-induced protein GADD153 was greater in riboflavin-deficient cells compared with controls. Riboflavin deficiency was associated with decreased rates of cell proliferation caused by arrest in G1 phase of the cell cycle. These studies are consistent with the hypothesis that HepG2 cells have a great demand for riboflavin and that cell stress develops rapidly if riboflavin supply is marginally low.

Lactococcus lactisis capable of improving the riboflavin status in deficient rats

Lactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegalia that were observed using a riboflavin depletion-repletion model. Riboflavin status and growth rates were greatly improved when the depleted rats were fed with cultures of L. lactis that overproduced this vitamin whereas the native strain did not show the same effect. The present study is the first animal trial with food containing living bacteria that were engineered to overproduce riboflavin. These results pave the way for analysing the effect of similar riboflavin-overproducing LAB in human trials.

Oxidative Folding of Interleukin-2 Is Impaired in Flavin-Deficient Jurkat Cells, Causing Intracellular Accumulation of Interleukin-2 and Increased Expression of Stress Response Genes

Secretory proteins such as interleukin (IL)-2 undergo oxidative folding (disulfide formation) in the endoplasmic reticulum (ER) before secretion. Studies in yeast have suggested that oxidative folding depends on the flavoprotein Ero1p; unfolded proteins accumulate in the ER, triggering cellular stress response. Here, human lymphoid cells (Jurkat cells) were used to model effects of cellular flavin supply on secretion of IL-2 (containing one disulfide bond) and cellular stress response. Cells were cultured in media containing 0.85, 3.1, 12.6 or 300.6 nmol/L riboflavin for 5 wk, representing severely deficient, moderately deficient, physiologic and pharmacologic plasma concentrations in humans, respectively. Transport rates of riboflavin were increased in severely and moderately deficient cells compared with cells cultured in physiologic medium; this increase was not sufficient to prevent intracellular depletion of riboflavin, as judged by glutathione reductase activity and intracellular concentrations of glutathione. Intracellular accumulation of IL-2 was greater in severely deficient cells than in other groups. Nevertheless, severely deficient cells secreted normal amounts of IL-2 into the extracellular space, mediated by increased transcriptional activity of the IL-2 gene. Riboflavin-deficient cells responded to intracellular accumulation of IL-2 with increased expression of genes encoding ubiquitin-activating enzyme E1 and X box-binding protein, consistent with cellular stress. These findings are consistent with the hypothesis that flavin deficiency may cause cellular stress by accumulation of unfolded proteins in human cells.

Cytokinetic and structural responses of the rat small intestine to riboflavin depletion

Abstract:The impaired absorption and metabolism of Fe seen in riboflavin defiaency is attributed, at least in part, to a hyperproliferative response in the small intestine, associated with an altered morphology. Studies were conducted in female weanling Wistar rats to explore further the effect of riboflavin deficiency on the cytokinetics and structure of the small intestine. Feeding a riboflavin-deficient diet for 8 weeks from weaning resulted in a significantly lower villus number, a significant increase in villus length and an increased rate of transit of enterocytes along the villi, compared with weight-matched controls. A second experiment focused on the 3 weeks after weaning and showed that riboflavin deficiency inhibits the increase in villus number observed in control animals over this period. We suggest that riboflavin deficiency induced at weaning impairs the normal increase in villus number and that prolonged deficiency leads to an adaptive increase in length of villi and depth of crypts.

Morphological changes in the rat small intestine in response to riboflavin depletion

Female Wistar rats were weaned onto a diet deficient in riboflavin and compared with weight-matched and ad lib.-fed controls. The effects of riboflavin deficiency on villus morphometry and enterocyte number on the villi in the upper small intestine were studied. Riboflavin depletion was associated with increased villus length and a proportional increase in the number of cell positions along the villi. The total DNA, RNA and protein contents in the intestinal mucosa were not significantly different between any of the groups. Villus hypertrophy in the absence of increased cell number in the small intestine suggests that villus number may be reduced in riboflavin deficiency. Riboflavin deficiency did not influence the number of mucus-producing goblet cells or the amount of mucosal glycoprotein in the small intestine. Impaired production of mucus appeared not to be involved in the structural and functional changes seen in riboflavin deficiency.

Subclinical riboflavin deficiency is associated with outcome of seriously ill patients

The biochemical status of 152 patients in the Intensive Therapy Unit (ITU) was examined in relation to their clinical outcome. Patients who died in ITU or within one week of discharge were found to have significantly poorer riboflavin status than those who survived, but the majority of these measurements were within the normal range. Normal ranges established on a healthy population may therefore not be appropriate in investigation of vitamin nutritional status in seriously ill patients. These results do however suggest that riboflavin status is a risk factor in critically ill patients, and that subclinical riboflavin depletion may affect outcome.

Effects of Combined Riboflavin and Iron Deficiency on the Hematological Status and Tissue Iron Concentrations of the Rat

Interactions of riboflavin and iron deficiencies on utilization and storage of iron in the rat were investigated. Rats were fed diets deficient in iron (ID), riboflavin (RD) or iron and riboflavin (DD) for a period of 7 wk, at which time they were either killed and measurements of hematological indices and tissue iron concentration done or a further group of DD rats was supplemented with either iron, riboflavin or both nutrients for 2 wk. Growth was impaired in all deficient groups but the impairment was greatest in the DD rats. Iron depletion was associated with the greatest reductions in hematological indices and tissue iron concentrations, riboflavin depletion the least, while the combined deficiencies had intermediate effects. Riboflavin deficiency was found to have a “sparing effect” on iron status probably because the lower growth rate associated with riboflavin deficiency reduced iron requirements. Refeeding either iron alone or iron with riboflavin to the DD rats resulted in significant improvement in hematological status, but there was no significant difference in the response to either supplement. Feeding riboflavin alone to DD rats resulted in a further depression of hematological status and tissue iron concentration.

Effects of aerobic exercise and weight loss on riboflavin requirements of moderately obese, marginally deficient young women

In a previous study, exercise was shown to increase riboflavin requirements of active, normal weight young women. The present study examined the effect of exercise and weight loss on riboflavin status of moderately overweight women. The experiment was designed as a two-period cross-over with an initial base-line period and two 5-wk metabolic periods. The basic diet contained 1200 kcal with a riboflavin concentration of 0.8 mg/1000 kcal. Exercise consisted of a program of dance exercise. Riboflavin depletion, as measured by increased erythrocyte glutathione reductase activity coefficients and decreased urinary excretion of riboflavin, occurred during both nonexercise and exercise periods. Erythrocyte glutathione reductase activity coefficients increased from a base-line mean of 1.28 ± 0.11 to 1.40 ± 0.12 during nonexercise and to 1.49 ± 0.16 during exercise. Urinary excretion of riboflavin fell from 48 ± 12% of intake during base-line to 30 ± 13% during nonexercise and to 19 ± 6% during exercise. Riboflavin depletion was not related to the rate or composition of weight loss or to change in aerobic capacity.

Accelerated development of riboflavin deficiency by treatment with chlorpromazine

The present study was undertaken to determine whether treatment with chlorpromazine accelerates the depletion of tissue stores of flavin adenine dinucleotide during dietary riboflavin deficiency. These investigations derived their impetus from earlier findings that low doses of chlorpromazine in rats fed abundant riboflavin increase urinary riboflavin excretion and reduce hepatic flavin stores. From 6 to 10 days after beginning to feed on a riboflavin-deficient diet, rats treated with chlorpromazine, 2 mg/kg body weight twice daily, had approximately twice the urinary riboflavin excretion of that of pair-fed saline-treated controls. When the riboflavin-deficient diets and chlorpromazine treatments were extended for 3 weeks and the animals killed, FAD levels in liver, kidney, and heart were markedly lower in drug-treated than in saline-treated animals. When studies were extended for 7 weeks, tissue FAD levels in saline-treated animals declined further and were equal to those of chlorpromazine-treated rats after only 3 weeks of dietary deficiency. Thus, chlorpromazine treatment accelerated urinary riboflavin loss and accelerated tissue depletion of FAD levels during dietary riboflavin deficiency. Brain levels of FAD by contrast were relatively resistant to both dietary riboflavin withdrawal and treatment with chlorpromazine. Subsequent studies showed that urinary riboflavin excretion began to increase within 6 hr of treatment with chlorpromazine. It is concluded that significant riboflavin depletion occurs following treatment with low doses of chlorpromazine, both in animals fed a normal diet and in animals fed a riboflavin-deficient diet, particularly during the early stages of deficiency.

Factors affecting riboflavin requirements of oral contraceptive users and nonusers

Riboflavin depletion has been identified in women on oral contraceptives (OC) but change in riboflavin nutriture has not been consistently demonstrated in all OC user groups studied. Discrepant findings in reports have been attributed to differences of pill formulation or riboflavin intake. Aims of this study were to compare the riboflavin requirements of healthy OC users and nonusers on diets prepared in a metabolic unit. A single daily menu and meal pattern was used. The basic diet providing riboflavin at a level of 0.6 mg/1000 kcal was used in the period of acclimation and period 1. In periods 2 and 3, the riboflavin content of the diet was increased to 0.8 and 1.0 mg/1000 kcal, respectively. The riboflavin status of subjects was monitored by erythrocyte glutathione reductase assay and urinary riboflavin excretion. Eight women on OC and 10 nonusers participated. Erythrocyte glutathione reductase assay values and urinary riboflavin excretion showed intersubject and interperiod differences but no significant group differences (OC versus non-OC) in erythrocyte glutathione reductase values or in urinary riboflavin per g creatinine. It was concluded that when dietary intake is controlled, OC do not significantly influence riboflavin status. Riboflavin needs were related to energy requirements of the subjects.