The Association Between Serum Riboflavin and Flavin Mononucleotide With Pancreatic Cancer: Findings From a Prospective Cohort Study.

OBJECTIVES: Vitamin B2 (riboflavin) has a prime role in metabolic reactions imperative to cell cycle and proliferation. We investigated the associations between serum concentrations of riboflavin flavin mononucleotide with the risk of pancreatic cancer in a nested case-control study involving 58 cases and 104 matched controls. METHODS: The Singapore Chinese Health Study, an ongoing prospective cohort study of 63,257 Chinese Singaporeans. Conditional logistic regression method was used to evaluate these associations with adjustment for potential confounders including the level of education, body mass index, smoking status, alcohol consumption, history of diabetes, serum cotinine and pyridoxal 5'-phosphate, estimated glomerular filtration rate, and total methyl donors (ie, the sum of serum choline, betaine, and methionine). RESULTS: The risk of pancreatic cancer increased with increasing level of serum riboflavin in a dose-dependent manner, especially in men (Ptrend = 0.003). The odds ratio (95% confidence intervals) of pancreatic cancer for the second and third tertiles of serum riboflavin, compared with the lowest tertile, were 9.92 (1.65-59.77) and 25.59 (3.09-212.00), respectively. This positive association was stronger in individuals with a longer follow-up period (≥7 years). CONCLUSIONS: The findings suggest a potential role of riboflavin in the development of pancreatic cancer, especially in men.

Prospective study of serum B vitamins levels and oesophageal and gastric cancers in China.

B vitamins play an essential role in DNA synthesis and methylation, and may protect against oesophageal and gastric cancers. In this case-cohort study, subjects were enrolled from the General Population Nutrition Intervention Trial in Linxian, China. Subjects included 498 oesophageal squamous cell carcinomas (OSCCs), 255 gastric cardia adenocarcinomas (GCAs), and an age- and sex-matched sub-cohort of 947 individuals. Baseline serum riboflavin, pyridoxal phosphate (PLP), folate, vitamin B12, and flavin mononucleotide (FMN) were measured for all subjects. We estimated the associations with Cox proportional hazard models, with adjustment for potential confounders. Compared to those in the lowest quartile of serum riboflavin, those in the highest had a 44% lower risk of OSCC (HR: 0.56, 95% CI: 0.41 to 0.75). Serum vitamin B12 as a continuous variable was observed to be significantly inversely associated with OSCC (HR: 0.95, 95% CI: 0.89 to 1.01, P for score test = 0.041). Higher serum FMN levels were significantly associated with increased risk of OSCC (HR: 1.08, 95% CI: 1.01 to 1.16) and GCA (HR: 1.09, 95% CI: 1.00 to 1.20). Our study prompted that B vitamins have the potential role as chemopreventive agents for upper gastrointestinal cancers.

A U-shaped relationship between plasma folate and pancreatic cancer risk in the European Prospective Investigation into Cancer and Nutrition.

Folate intake has shown an inverse association with pancreatic cancer; nevertheless, results from plasma measurements were inconsistent. The aim of this study is to examine the association between plasma total homocysteine, methionine, folate, cobalamin, pyridoxal 5'-phosphate, riboflavin, flavin mononucleotide and pancreatic cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). We conducted a nested case-control study in the EPIC cohort, which has an average of 9.6 years of follow-up (1992-2006), using 463 incident pancreatic cancer cases. Controls were matched to each case by center, sex, age (± 1 year), date (± 1 year) and time (± 3 h) at blood collection and fasting status. Conditional logistic regression was used to calculate the odds ratios (OR) and 95% confidence intervals (CI), adjusting for education, smoking status, plasma cotinine concentration, alcohol drinking, body mass index and diabetes status. We observed a U-shaped association between plasma folate and pancreatic cancer risk. The ORs for plasma folate ≤ 5, 5-10, 10-15 (reference), 15-20, and > 20 nmol/L were 1.58 (95% CI=0.72-3.46), 1.39 (0.93-2.08), 1.0 (reference), 0.79 (0.52-1.21), and 1.34 (0.89-2.02), respectively. Methionine was associated with an increased risk in men (per quintile increment: OR=1.17, 95% CI=1.00-1.38) but not in women (OR=0.91, 95% CI=0.78-1.07; p for heterogeneity <0.01). Our results suggest a U-shaped association between plasma folate and pancreatic cancer risk in both men and women. The positive association that we observed between methionine and pancreatic cancer may be sex dependent and may differ by time of follow-up. However, the mechanisms behind the observed associations warrant further investigation.

Relation between riboflavin, flavin mononucleotide and flavin adenine dinucleotide concentrations in plasma and red cells in patients with critical illness.

BACKGROUND: There is some evidence that the relationship between plasma and red cell vitamin B2 concentrations is perturbed in the critically ill patient. The aim of the present study was to examine the longitudinal interrelationships between riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma and red cells in patients with critical illness. METHODS: Riboflavin, FMN and FAD concentrations were measured, by HPLC, in plasma and red cells in healthy subjects (n=119) and in critically ill patients (n=125) on admission and on follow-up. RESULTS: On admission, compared with the controls, critically ill patients had significantly higher plasma riboflavin and FMN concentrations (p<0.001) and lower median plasma FAD concentrations (p<0.001). In the red cell, FAD concentrations were significantly lower in critically ill patients (p<0.001). In healthy subjects, plasma riboflavin was directly associated with both plasma FMN (r(s)=0.55, p<0.001) and plasma FAD (r(s)=0.49, p<0.001). Red cell riboflavin was directly associated with red cell FMN (r(s)=0.52, p<0.001) but not red cell FAD. In the critically ill patients, plasma riboflavin was not significantly associated with either plasma FMN or FAD. Red cell riboflavin was directly associated with red cell FMN (r(s)=0.79, p<0.001) and red cell FAD (r(s)=0.72, p<0.001). Longitudinal measurements (n=60) were similar. CONCLUSIONS: The relationship between plasma riboflavin, FMN and FAD was significantly perturbed in critical illness. This effect was less pronounced in red cells. Therefore, red cell FAD concentrations are more likely to be a reliable measure of status in the critically ill patient.

Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

Conversion of FAD to FMN and riboflavin in plasma: effects of measuring method.

The stability of flavin adenin dinucleotide (FAD) in plasma was studied under a low-intensity light and FAD was found to be converted to flavin mononucleotide (FMN) and riboflavin (RF) in both human and rat plasma. The hydrolysis rates of FAD in plasma at 4 degrees C were lower than those at 37 degrees C. In addition, the hydrolysis rates were markedly inhibited when EDTA, known as an anticoagulant, was added to plasma. These results indicated that plasma samples in pharmacokinetic studies should be pretreated with EDTA, extracted at the earliest convenience and lower temperature like 4 degrees C to keep a high stability. The pharmacokinetic study after intravenous administration of FAD at a dose of 500 nmol/kg as FAD in rats was performed with plasma samples after addition of EDTA under strict light and temperature control. A measurable amount of FAD in plasma together with rapid conversions of FAD to FMN and RF were observed in rat plasma. The AUC values (mean+/-S.D. of 4 rats) for FAD, FMN and RF were 707+/-378, 3643+/-958 and 30095+/-3544 nmol x min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B2 in the in vivo study.

Normal riboflavin status in malaria patients in Gabon.

Previous publications reported commonly the occurrence of riboflavin deficiency and a positive correlation between riboflavin status and parasitemia in patients with Plasmodium falciparum malaria. In these studies, riboflavin status was determined by erythrocyte glutathione reductase activation coefficients (EGRACs). Inherited low erythrocyte glutathione reductase activity is highly prevalent in malarial regions, however. To rule out falsely diagnosed riboflavin deficiency in affected patients, we conducted an investigation using a high-performance liquid chromatography method (HPLC) instead of the EGRAC method. In 29 infants (age range, 1-5 years), 22 schoolchildren (age range, 6-12 years), and 33 adolescents and adults (age range, 13-74 years) from Lambaréné, Gabon, with acute P. falciparum malaria, plasma concentrations of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) were measured by HPLC. Results were correlated with parasite densities. Profiles of plasma concentrations of all 3 flavin compounds were within the normal range in all patients. Concentrations of free riboflavin were not different between the 3 age groups. In adolescents and adults, FMN and FAD concentrations were higher than in infants (P = 0.002 and P = 0.001) and schoolchildren (P = 0.003 and P = 0.002). Comparing children with hyperparasitemic and uncomplicated malaria, no difference in the concentrations of either flavin compound was found. Neither the concentrations of free riboflavin nor the concentrations of one of the flavin nucleotides correlated with parasitemia within subgroups of age or of children with uncomplicated and hyperparasitemic malaria. Our data indicate that nutritional riboflavin deficiency might have been overestimated in previous malaria studies and do not support a relationship between flavin concentrations and parasitemia in P. falciparum malaria.

Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation.

BACKGROUND: Vitamin B(2) exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B(2) status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. METHODS: EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC > or =1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). RESULTS: Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P <0.05). All variables except plasma FAD responded significantly to riboflavin supplementation compared with placebo (P < or =0.04). The strongest increases were for riboflavin in plasma (83%) and for FMN in erythrocytes (87%). CONCLUSIONS: Concentrations of all B(2) vitamers except plasma FAD are potential indicators of vitamin B(2) status, and plasma riboflavin and erythrocyte FMN may be useful for the assessment of vitamin B(2) status in population studies.

Proliferation of peripheral blood mononuclear cells increases riboflavin influx.

Previously we demonstrated that proliferation of peripheral blood mononuclear cells (PBMC) causes a five-fold increase in cellular uptake of biotin; this increase is mediated by an increased number of biotin transporters on the PBMC surface. In the present study, we investigated the specificity of this phenomenon by determining whether the cellular uptake of riboflavin also increases in proliferating PBMC and whether the increase is also mediated by an increased number of transporters per cell. We characterized [3H]riboflavin uptake in both quiescent and proliferating PBMC. In quiescent PBMC, [3H]riboflavin uptake exhibited saturation kinetics and was reduced by addition of unlabeled riboflavin (P < 0.05) or lumichrome (P < 0.01). These observations are consistent with transporter-mediated uptake. [3H]Riboflavin uptake was reduced at 4 degrees C compared with 37 degrees C (P < 0.01) and by 2, 4-dinitrophenol (P < 0.05) but not by ouabain or incubation in sodium-free medium. These data provide evidence for an energy-dependent but sodium-independent transporter. Proliferating PBMC accumulated approximately four times more [3H]riboflavin than quiescent PBMC (P < 0.05). Because both transporter affinity and transporter number per cell (as judged by maximal transport rate) were similar in quiescent and proliferating PBMC, we hypothesize that the increased riboflavin uptake by proliferating PBMC reflects only increased cellular volume. To test this hypothesis, PBMC volume was reduced using hyperosmolar medium; [3H]riboflavin uptake decreased to about 50% of isotonic controls (P < 0.01). Thus we conclude that proliferating PBMC increase cellular content of riboflavin and biotin by two different mechanisms.

Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography.

We describe an assay which determines simultaneously riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma, using galactoflavin (GF) as an internal standard. The flavins were extracted on a C18 Sep-Pack cartridge after protein precipitation with 10% trichloroacetic acid, and were analyzed on a C18 RP-HPLC with 85% phosphate-magnesium acetate buffer (pH 3.4) and 15% acetonitrile. FAD, FMN, GF and RF extraction recoveries were 101.0-5.6, 97.0-6.5, 97.0-2.0 and 95.0-4.1%, and reproducibilities were 5.9, 6.8, 2.1 and 4.3%, respectively. FAD, FMN and RF values in infant and adolescent plasma were in the range 53.5-108.2, 9.0-25.1 and 12.7-53.4 nM, and 36.5-157.20, 7.1-24.6 and 8.2-57.8 nM, respectively. Using GF as an internal standard improved the quantification of these B2 vitamers.

Concentrations of riboflavin and related organic acids in children with protein-energy malnutrition.

BACKGROUND: Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) concentrations have been little studied in cases of malnutrition. OBJECTIVE: Our objective was to investigate the effects of malnutrition on riboflavin status and riboflavin's relation with thyroid hormones and concentrations of urinary organic acids. DESIGN: Malnourished children from the savannah in Benin (group S, n = 30) and the coast in Togo (group C, n = 30), as well as 24 control subjects from both regions, were studied. Blood riboflavin, FMN, and FAD were analyzed by HPLC; urinary organic acids were analyzed by gas chromatography-mass spectrometry. RESULTS: Children in group S were more severely malnourished than children in group C. Triiodothyronine concentrations were lower in group S than in group C or the control group (1.12 +/- 0.24 compared with 1.74 +/- 0.18 and 2.92 +/- 0.19 nmol/L, respectively; P < 0.0001). Plasma riboflavin concentrations in group S were higher than those in group C or the control group (66.90 +/- 12.75 compared with 28.09 +/- 9.12 and 20.08 +/- 3.03 nmol/L, respectively; P < 0.001). Plasma FAD concentrations in group S were lower than those in group C or the control group (31.57 +/- 10.19 compared with 59.02 +/- 5.60 and 65.35 +/- 5.23 nmol/L, respectively; P < 0.0001). Dicarboxylic aciduria was higher in group C than in group S or the control subjects. CONCLUSIONS: Children in group S had low triiodothyronine concentrations and low conversion of plasma riboflavin into its cofactors, leading to a plasma FAD deficiency. Plasma FAD was not correlated with urinary dicarboxylic acid concentrations.

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma by capillary electrophoresis and laser-induced fluorescence detection.

BACKGROUND: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma. METHODS: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source. RESULTS: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4-9% and 6-12%, respectively. The reference values (median, 5-95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7-42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5-13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5-78.1 nmol/L) for FAD. CONCLUSIONS: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.

Reductive dechlorination of DDT to DDD by rat blood.

The present study provides evidence that the reductive dechlorination of DDT (p, p'-dichlorodiphenyltrichloroethane) to DDD (p, p'-dichlorodiphenyldichloroethane) mediated by rat blood proceeds in the presence of both a reduced pyridine nucleotide and a flavin. The reduction appears to proceed in two steps. The first step is reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, either enzymatically or nonenzymatically. The second step is nonenzymatic reductive dechlorination of DDT by the reduced flavin, catalyzed by the heme group of hemoglobin.

The metabolism of riboflavin in female patients with liver cirrhosis.

The metabolism of vitamin B2 was studied in five female patients with liver cirrhosis of varying etiology. Following the oral administration of 40 mg (106.3 mumol) riboflavin, plasma concentrations of riboflavin and flavo-coenzymes as well as urinary riboflavin excretion were analyzed over a period of 48 h. Results were compared to data obtained for healthy controls (Zempleni J. et al, Am. J. Clin. Nutr., 1996 [15]). About 18% of the administered vitamin was recovered from patients' urine, indicating an absorption similar to healthy subjects (p > 0.05). The area under the riboflavin plasma concentration vs time curve was 1.2-fold larger among patients than controls, but the difference was not significant (p > 0.05). Riboflavin peak concentrations in plasma (315.6 nmol/l) and times when those concentrations were achieved (3.0 h) were similar to those found for healthy subjects (p > 0.05). Flavocoenzyme peak plasma concentrations were increased 1.4-fold above their baseline levels in cirrhotics which was equal to controls (p > 0.05). 7 alpha-Hydroxyriboflavin was detected in the plasma of patients. Distribution and elimination kinetics of riboflavin were analyzed by using a two-compartment open model; the riboflavin plasma disposition rate constants of the patients (k alpha = 0.7232 h-1; k beta = 0.0627 h-1) were not different from controls (p > 0.05). No differences between both groups were found regarding renal excretion (renal clearance, first-order rate constants for renal excretion; p > 0.05). In conclusion, patients with liver cirrhosis of varying etiology and varying medical treatment did not show alterations of riboflavin turnover.

Determination of riboflavin and flavocoenzymes in human blood plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for determining riboflavin, flavin adenine dinucleotide, and flavin mononucleotide in human blood plasma is presented. Flavocoenzymes are determined as flavin mononucleotide after acid hydrolysis of flavin adenine dinucleotide. Metabolites are separated by reversed-phase column chromatography and quantified by their native fluorescence. Criteria of quality are (riboflavin/flavocoenzymes): coefficients of variation 2.8/4.6% (intra-assay) and 2.8/4.4% (inter-assay); recovery 82.4/94.4%; detection limit < 3.0/9.0 nmol/l. Because sample preparation requires only few steps, and the retention times are short ( < 5 min), this method is recommended for use in routine analysis.

Periconceptional vitamin profiles are not suitable for identifying women at risk for neural tube defects.

Folic acid and other vitamin deficiencies may play a role in the etiology of neural tube defects. The Medical Research Council Vitamin Study confirmed the beneficial effect of folic acid supplementation on the prevention of neural tube defects. However, the concentrations of vitamins other than folate were not a common feature of any of the former studies. We measured the concentrations of vitamin A, riboflavin, riboflavine-5'-monophosphate, flavine-adenine-dinucleotide, vitamin B-6, vitamin B-12, vitamin C, vitamin E, folate and ferritin in the serum of women who had previously had a child with a neural tube defect and were planning a further pregnancy. Vitamin and folic acid supplements were supplied before conception to 44 high risk women before conception. Eighteen other high risk women not given supplements were the control group. We concluded that vitamin profiles do not form a suitable means for identifying women at risk for neural tube defects before pregnancy. This endorses the hypothesis that the beneficial effect of folic acid supplementation on the prevention of neural tube defects is possibly at least partly due to the fact that it overrides a relative folic acid shortage caused by a metabolic disorder.

Determination of flavin contents in neutrophils by a sensitive chemiluminescence assay: evidence for no translocation of flavoproteins from the cytosol to the membrane upon cell stimulation.

A sensitive and specific chemiluminescence (CL) method with bacterial luciferase was adapted for accurate measurement of the flavins FAD and FMN in the membrane and cytosolic fractions of neutrophils prepared from pig and human blood. The FAD and FMN contents (FAD/FMN = 100:2) in the membranes were essentially the same in resting (R) and myristate-stimulated (S) cells, although O2(-)-generation was markedly enhanced exclusively in S membranes. The O2(-)-forming activity of S samples remained unchanged or even increased after washing the membranes with buffer, although one-third of the FAD was lost during washing (a decrease from 140 to 95 pmol/10(8) cell-equivalent (CE) during washing). The cytosol is known to contain at least three components that are essential for O2- production (p47-phox, p67-phox, and a G-protein), and that are translocated to membranes upon activation, but its flavin content was one tenth of that of the membranes. The cytosol was treated with fatty acids in the absence of membranes to induce substantial precipitation of p47-phox, p67-phox and a protein of 32 kDa. No difference relative to a solvent-control was noted in the low flavin content of the precipitate indicating that these cytosolic components are not flavoproteins. These results do not support the possibility of translocation of a cytosolic flavoprotein to the membrane upon activation of the respiratory burst.

Flavin levels in the rat retina.

Exposure of riboflavin and its coenzymes adenine dinucleotide (FAD) and riboflavin-5'-phosphate (FMN) to UV and visible light results in the generation of radicals and photodegradative products that can damage surrounding macromolecules. Vertebrates and invertebrates have lost the ability to synthesize riboflavin and must obtain it or its coenzymes from food. The present study evaluated the relationship between FAD, FMN, and riboflavin concentrations in retina and blood of male Sprague-Dawley rats. Rations were provided in the form of purified diets containing 0, 3, 6, 30, and 300 mg riboflavin kg-1 diet. Analysis of flavins by HPLC showed that saturation levels of FAD, FMN and riboflavin in the retina and blood were achieved with diets containing 3 mg riboflavin kg-1. Retinal flavins were not significantly elevated by further increases in dietary riboflavin concentration, but an unidentified flavin appeared in the blood of rats given rations containing concentrations above 3 mg kg-1. The concentration of this unknown flavin varied in proportion to the level of dietary riboflavin.

Analysis of flavins in ocular tissues of the rabbit.

Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes required for the activity of flavoenzymes involved in the transfer of electrons in oxidation-reduction reactions. Flavins are light sensitive and rapidly degrade when exposed to light in the near ultraviolet and visible wavelengths. Some of the byproducts of flavin photodegradation are toxic. A quantitative survey of flavins in rabbit ocular tissues is reported. Adult male Dutch-Belt Rabbits were fed purified diets containing 3, 30, or 300 mg riboflavin/kg for 1 month. A method of aqueous extraction and high-performance liquid chromatography with fluorescence detection was used to measure riboflavin, FMN, and FAD in cornea, lens cortex, lens nucleus, retina, and blood. The retina contained the highest flavin concentration. In all tissues, the primary flavin was FAD followed by FMN and riboflavin. The highest concentration of riboflavin occurred in the cornea followed by the retina, lens cortex, and lens nucleus. A trend toward increasing concentrations of riboflavin occurred in the retina and blood in response to excess dietary riboflavin, but the concentration changes were not statistically significant. The highest concentration of FAD and FMN occurred in the retina followed by the cornea and the lens cortex and nucleus. The relative contribution of riboflavin, FMN, and FAD to the total flavin pool was markedly different in the various tissues of the eye. The proportion of tissue flavins present as riboflavin decreased from anterior to posterior. It was highest in the cornea followed by lens and retina. The pattern of distribution for FMN was: cornea greater than retina greater than lens cortex and nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)