Riboflavin (Vitamin B2)

Riboflavin, also called vitamin B-2-, was the second B-complex vitamin to be discovered, with essentiality in humans being shown in 1939.^[1]

According to the USDA Food Composition Database, the best sources of dietary riboflavin per 100 grams of food, excluding fortified foods, are:

• 2 to 5 mg: liver (beef, lamb, chicken, etc.)
• 1 to 2 mg: kidney, heart, almonds, muscadine grapes
• 0.5 to 1 mg: goat cheese, game meats, roe and caviar, egg yolk, wheat germ and bran
• 0.3 to 0.5 mg: red meats and pork, shellfish, fatty fish (mackerel, salmon, trout, etc.), sesame seeds and tahini, mushrooms

Riboflavin is relatively heat-stable, with a melting point of 278-282°C and able to withstand heating processes like hot air convection, infrared, high-pressure steam, and microwave during cooking, as well as milk pasteurization.^[2] When dissolved in water, heating the riboflavin solution for 40 minutes at 100, 120 and 150 °C destroys 4, 7, and 28% of the riboflavin, respectively.^[3]

While eventually being degraded in response to heat, riboflavin is relatively heat resistant and should not significantly degrade under most practical situations (cooking foods with riboflavin in them).

Although riboflavin is somewhat heat-resistant, it is highly sensitive to light. Aqueous riboflavin is strongly absorbs and is photodegraded by various ultraviolet and blue wavelengths (223, 267, 373, and 444 nm).^[2] Photodegradation also occurs when riboflavin is in powdered form or tablets,^[4][5] with the powder turning greenish depending on humidity and moisture.^[5]

There may be slightly more photostability when riboflavin is in a liposome,^[6] especially when in the liposome alongside something else that can absorb light and degrade in its place,^[7] but the degradation still occurs in a linear fashion when light is present.^[6]

Riboflavin is unstable in light both as a solid and even more so when in aqueous solution. Riboflavin should be stored in a dry, dark place.

Riboflavin is an essential vitamin used to produce the two flavocoenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).^[1] These coenzymes are involved primarily in reduction-oxidation (redox) reactions in metabolism, with most flavoproteins using FAD (84%) over FMN (16%).^[8]

In energy metabolism, FMN is involved in complex I of the respiratory chain, while FAD is involved in complex II of the respiratory chain, amino acid catabolism, fatty acid beta-oxidation, and proper function of the TCA cycle.

FAD also plays an important role in the body’s antioxidant system, being a cofactor for glutathione reductase. It serves as the link between energy metabolism and the antioxidant system as electrons move from NADPH to FAD to glutathione.

Four riboflavin-dependent enzymes are involved in folate metabolism, vitamin B12 metabolism, and the methionine-homocysteine cycle: methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), dimethylglycine dehydrogenase (DMGDH), and sarcosine dehydrogenase (SARDH).^[8]

Other biosynthetic roles of riboflavin include:

• The biosynthesis of heme requires the FAD-dependent protoporphyrinogen IX oxidase (PPOX).
• The activation of pyridoxal 5-phosphate (PLP), a form of vitamin B6, requires the FMN-dependent pyridoxine 5-phosphate oxidase (PNPO).^[9]
• Synthesis of niacin from tryptophan requires FAD for kynurenine 3-monooxygenase (
• The biosynthesis of coenzyme A from pantothenic acid requires the FMN-dependent 4-phosphopantothenoylcysteine decarboxylase (PPCDC).
• The biosynthesis of cholesterol depends on two FAD-dependent enzymes: squalene monooxygenase (SQLE) and 3β-hydroxysterol 24-reductase (DHCR24).
• Protein folding in the endoplasmic reticulum is FAD-dependent.^[10]

Riboflavin is used to create the coenzymes FAD and FMN, which are used by many enzymes in redox reactions to facilitate energy metabolism, the antioxidant system, and biosynthesis.

Clinical riboflavin deficiency (ariboflavinosis) is rare in the developed world, except in pockets of low socioeconomic status, and high in the developing world.^[11] The signs of ariboflavinosis are sore throat; hyperemia and edema of the throat and mouth; cheilosis; angular stomatitis; glossitis; seborrheic dermatitis; and normochromic, normocytic anemia associated with pure erythrocyte cytoplasia of the bone marrow.^[11]

The recommended daily allowance (RDA, or the amount needed to meet the nutritional needs of 97–98% of healthy people in a given demographic) for those 19 years and older is 1.3 mg/day for men and 1.1 mg/day for women.^[11] The RDA increases to 1.4 mg/day for pregnant women, and to 1.6 mg/day for lactating women.

Importantly, the value for lactating women assumed that women provide 0.35 mg/L of riboflavin in breast milk. At least one study of healthy women reported that daily secretion of riboflavin in breast milk was 0.010–0.55 mg and strongly correlated with dietary riboflavin intake from diet and supplements over the range of 1–8 mg/d.^[12] Mothers deficient in riboflavin may deliver only around half of a child's requirements through her breast milk.^[13]

In the US, average riboflavin intake from food alone is 1.9–2.3 mg/d and only 2.9% consume less than the estimated average requirement (EAR) of 1.1 mg/d for men and 0.9 mg/d for women.^[14] When dietary supplements are considered, average intake levels are 2.0–5.7 mg/d and just 2.4% of Americans consume less than the EAR.^[14]

Adults require 1–2 mg of riboflavin per day. In the US, average intakes are above this and deficiency is rare.

Fourteen biomarkers of riboflavin status have been identified, but most have been investigated in only 1-2 studies.^[15] The gold-standard is the erythrocyte glutathione reductase activation coefficient (EGRAC), which represents the ratio of FAD-stimulated to unstimulated enzyme activity.

EGRAC values close to 1.0 indicate glutathione reductase is saturated with FAD, while higher values indicate increasing deficiency of FAD and riboflavin. Sufficient riboflavin status is suggested to correspond to an EGRAC of 1.0 to 1.2, insufficient status to an EGRAC of 1.2 to 1.4, and deficiency to an EGRAC >1.4.^[11]

People who are heterozygous for glucose-6-phosphate dehydrogenase deficiency may have low EGRAC status independent of riboflavin, confounding the measurement.^[16] EGRAC is therefore not an appropriate biomarker to measure riboflavin status in these people.

Although full-blown deficiency of riboflavin is rare in developed worlds, suboptimal status may be widespread. In elderly adults who consumed an average of 1.6 mg/d of riboflavin, 45% had suboptimal riboflavin status that was corrected with an additional 1.6 mg/d of supplemental riboflavin; the EGRAC was improved more so with 25 mg/d.^[17] Another study involved women who were deficient in riboflavin despite consuming an average of 1.14–2.3 mg/d.^[18]

These studies, and others,^[19][20][21] raise questions about the adequacy of the RDA for riboflavin.

Riboflavin status is measured by the EGRAC ratio, where less 1.0 to 1.2 is considered optimal (suggesting saturation of riboflavin in tissues) and higher values suggest increasing riboflavin deficiency. A significant portion of the developed world appear to have suboptimal riboflavin status despite consuming the RDA for riboflavin.

More than 90% of dietary riboflavin is in the form of FAD or FMN; the remaining 10% is comprised of the free form and glycosides or esters.^[11] The most common forms of riboflavin available in supplements are riboflavin and riboflavin 5'-monophosphate.

Dietary riboflavin is mostly FAD or FMN, and supplements supply either free riboflavin or riboflavin 5'-monophosphate.

Dietary sources of riboflavin tend to be in the form of its cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are bound to proteins in the food.^[22] These cofactors are liberated from food by the acidic environment in the stomach releasing free FMN and FAD.^[23][22] FMN and FAD are hydrolyzed into free riboflavin by phosphatases in the intestines, a requirement for absorption.^[24][25]

Riboflavin in food exists in a protein-bound state. The acidic environment of the stomach dissociates these riboflavin-protein complexes, releasing free riboflavin for absorption in the intestines.

In the small intestine, riboflavin is taken up by its transporters designated RFVTs,^[26] including RFVT1 (SLC52A1),^[27] RFVT2 (SLC52A2)^[28] and RFVT3 (SLC52A3),^[29][30] which are all expressed in the apical membranes of both the jejunum and ileum.^[26][30]

Bioavailability of riboflavin (as orally administred FMN) is better when taken with food than when taken in isolation at doses of 10-30mg. No differences have been found at lower doses,however, with 5mg riboflavin having a bioavailability of around 58% with or without food.^[31] Food sources seem similar in riboflavin absorption (400μg riboflavin), with spinach and milk both having 60-67% riboflavin bioavailability.^[32]

Higher doses of riboflavin have been shown to have lower bioavailabilities of 14.5% (150mg) and 8.3% (300mg) as assessed by urinary recovery of riboflavin.^[31] This is consistent with the threshold for intestinal riboflavin saturation, which seems to occur around the 27-50mg range.^[33][31] It should be noted that riboflavin can be absorbed passively through intestinal epithelial cells, suggesting that doses in excess of the threshold for transporter saturation can still be absorbed.^[34]

The small intestines mediate most of the absorption of riboflavin, where it appears to be a simple absorption process. Low supplemental doses (5mg) can be taken with or without food, while higher doses are better absorbed with food.

Some riboflavin uptake can occur in the colon (large intestine) also via a saturable transporter.^[35] Although colonic transporters seem to have less maximal capacity than those in the small intestine,^[36] they seem to be equally potent with low (0.1µM) concentrations of riboflavin.^[35] These transporters may have a role in taking up the riboflavin produced by intestinal bacteria,^[37][38] with Lactobacillus and potentially bifidobacteria being able to produce riboflavin (ex vivo in food products^[39]).

The colon is capable of absorbing some riboflavin at a lesser capacity than the small intestines, which may faciliate the absorption of riboflavin produced by intestinal bacteria. The overall contribution of intestinal bacteria to riboflavin status is not currently known.

Renal (kidney) elimination of riboflavin is mediated by the same transporters expressed in the intestines (RFVTs) which mediate resorption back into the blood. Expression of these transporters can be reduced by chronic administration of ethanol, reducing resorption and resulting in increased elimination.^[40]

One study assessing riboflavin repletion found that supplementation was able to increase riboflavin stores in the body after six weeks (1.6mg daily)^[41] with higher doses (2-4mg) being able to increase stores at four weeks, with levels remaining elevated over eight weeks.^[42] After supplementation is ceased, riboflavin stores have been shown to return to baseline after six months.^[41]

Although the cause of migraine headaches is unknown, a large number of studies have noted impaired mitochondrial function in patients with migraines.^[43] Defects in both brain and mitochondrial energy metabolism have been associated with complicated migraine,^[44][45] migraine with aura,^[46][47] and migraine without aura.^[48] As a precursor to FMN and FAD, riboflavin is an essential component of mitochondrial ATP production. Thus, riboflavin has been investigated as a treatment for migraines, in hopes that it may augment the impaired mitochondrial function that is thought to be the cause of headaches.^[43] Riboflavin treatment has shown some efficacy in this regard, reducing the frequency of migraines and encephalopathy in patients with MELAS (mitochondrial encephalomyopathy and stroke-like episodes).^[43][49][49]

Migraine headaches are associated with impaired mitochondrial metabolism. Because riboflavin is a source of FAD and FMN cofactors important for mitochondrial ATP production, it has been investigated for the treatment of migraine headaches.

In subjects diagnosed with migraine (with or without aura) with a headache frequency of 2-8 headaches a month who were given a high dose of riboflavin (400mg) over three months, there was a reduction in headache frequency and duration without any influence on headache intensity. The therapeutic effect persisted for three months after supplement cessation.^[50] Similar benefits of 400mg riboflavin over placebo have been noted elsewhere, with 59-68.4% of patients reporting a greater than 50% reduction in migraine frequency.^[51][52][53] One study using 25mg riboflavin as the 'placebo' treatment also noted that 44% of participants reported more than a 50% reduction in migraine frequency.^[54] It is uncertain, despite the aforementioned study, if lower doses are equally effective as other studies using 200mg^[55] and 50mg^[56] have failed to find benefits with riboflavin treatment. Due to the fact that some studies have failed to note benefits with riboflavin treatment, it has been suggested that there may be a mitochondrial genetic component to riboflavin therapeutic response.^[53]

The ability of riboflavin to reduce migraine intensity is either not present, as noted in one study,^[50] or relatively modest, with a 21% reduction in intensity noted in another study.^[52] Moreover, other work comparing 25mg riboflavin to 400mg riboflavin alongside other migraine treatments (300mg magnesium and 100mg feverfew) failed to note any differences in migraine intensity between treatments.^[54]

A few studies have found that riboflavin is effective for reducing migraine frequency and duration. The effect of riboflavin on migraine intensity has been inconsistent, with at least one study noting a reduction in headache intensity while others have failed to show any effect. Most studies showing efficacy used 400mg riboflavin daily. The idea that lower (25mg) doses are equally effective is plausible, but requires further testing.

Supplementation of riboflavin (2mg or 4mg) in women who were somewhat deficient in riboflavin (as assessed by EGRAC) over the course of 8 weeks was able to improve hemoglobin status of the blood, with the degree of improvement correlating with the extent of underlying deficiency.^[42] This has also been noted elsewhere with 2mg riboflavin (amongst other nutraceuticals) over 12 weeks, which also slightly improved hemoglobin content in moderately deficient subjects.^[57] Riboflavin taken alongside 5mg Vitamin C has shown similar positive effects in riboflavin- deficient young adults.^[58] Riboflavin is also included in combination supplements to address iron deficiency in third world countries at doses around 2mg;^[59][60][61] while absorption of iron is unaffected by riboflavin, being riboflavin-deficient seems to be associated with less iron utilization.^[61]

One study has noted that removal of supplementation caused the beneficial changes in hemoglobin and hematocrit counts to reverse in adults whose diet was deficient in riboflavin.^[58]

In elderly subjects who appeared to have suboptimal riboflavin status, supplementation of riboflavin (10mg) for four weeks did not appear to significantly alter ferritin concentrations.^[62] Also, neither iron absorption rates nor ferritin levels changed in younger women whose hemoglobin levels increased due to replenishing riboflavin stores via supplementation.^[42]

It seems that proper hemoglobin levels require adequate riboflavin intake (amongst other red blood cell nutrients such as iron). There is no evidence to suggest that supplementing higher than normal riboflavin can enhance red blood cell function, but fixing a riboflavin deficiency can increase hemoglobin levels.

An enzyme known as methylenetetrahydrofolate reductase (MTHFR) which reduces 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, is needed to convert homocysteine (a risk factor for coronary heart disease,^[63][64] into L-methionine.^[65] The activity of the MTHFR enzyme is generally thought to be protective against coronary heart disease, since reduced activity predicts coronary heart disease,^[66] particularly in those deficient in folic acid (from which 5,10-methylenetetrahydrofolate is made).^[67]

One specific gene mutation which reduces activity of the enzyme is MTHFR 677C->T, which causes alanine 222 in the MTHFR protein to be substituted with valine, resulting in reduced biding of the MTHFR enzyme to its riboflavin cofactor (flavin adenine dinucleotide or FAD).^[68] This genetic polymorphism has been identified as a risk factor for coronary heart disease.^[69][66][70] Rates of being homozygous for this polymorphism (known as having the MTHFR 677TT genotype, since each of the two copies of the gene are T's instead of C's) tend to vary between 3-32% across the world.^[71] This polymorphism does not raise the risk of all populations equally, however; the risk for coronary heart disease which this mutation confers is higher in European countries relative to North America.^[70] This difference has been hypothesized to be due to the fact that riboflavin has been fortified in foods in North America for several decades.^[72]

The action of the enzyme MTHFR plays a role in reducing homocysteine levels, an intermediate in metabolism that is a risk factor for coronary heart disease. Mutations that decrease the activity of MTHFR thus increase the risk for heart disease. FAD (which is made in part from riboflavin) is a cofactor for this enzyme.

Supplementation of riboflavin (1.6mg) for 12 weeks in subjects positive for the MTHFR 677C->T polymorphism reduced homocysteine levels in only in subjects who were MTHFR 677TT homozygous, where homocysteine levels decreased by 22% in those with normal riboflavin status. Larger decreases (in upwards of 40%) were noted in homozygous individuals with lower riboflavin status, suggesting that riboflavin intake affects homocysteine levels in these individuals.^[72]

In a large assessment of MTHFR mutations, it was found that overall subjects who had the MTHFR 677T allele had higher homocysteine concentrations, but this increase was not found in individuals whose riboflavin status was considered optimal (only in those with lower riboflavin status).^[73] Riboflavin status does not appear to influence homocysteine in the context of another mutation known as MTRR 524T, a flavoprotein that helps refresh methionine synthase, the enzyme that catalyzes the formation of methionine from homocysteine.^[73]

Subjects who are homozygous for the MTHFR 677C->7 polymorphism (aka. MTHFR 677TT) appear to experience greater reductions in homocysteine in response to a relatively low supplemental doses of riboflavin, which is thought to then reduce the risk of coronary heart disease.

Administration of riboflavin at a dose of 1.6mg over 12 weeks failed to significantly reduce total homocysteine concentrations in healthy elderly subjects with suboptimal riboflavin status (11.1% of whom had the MTHFR 677TT genotype).^[74] Although the next phase of the study introducing 400µg folic acid alongside the riboflavin effectively reduced homocysteine levels, riboflavin did not appear to confer additional benefits over folic acid alone.^[74] This was investigated since it was previously noted that after folic acid-induced reductions in homocysteine, Vitamin B12 has a more prominent homocysteine-reducing role^[75] and Vitamin B6 is also effective after folic acid^[76] (although working via a different pathway, reducing homocysteine concentrations by converting it into L-cysteine via cystathionine β-synthase^[77]).

One other study using 10mg riboflavin for a month in elderly subjects with low riboflavin status did find a minor reduction in homocysteine concentrations in serum, although this was not correlated to MTHFR genotype.^[62]

Studies on the effect of riboflavin supplementation on homocysteine levels in elderly populations have yielded mixed results. One study assessing the ability of riboflavin to decrease homocysteine in an elderly population who are predominantly not MTHFR 677TT did not find any benefits of supplementation, even though they had a suboptimal riboflavin status. Another study found the riboflavin caused mild decreases in homocysteine levels, although the MTHFR genotype in the study population was not assessed.

In subjects on medication who had hypertension alongside the MTHFR 677TT genotype, supplementation of riboflavin at 1.6mg daily for 16 weeks was able to promote an additional reduction of blood pressure of 5.6+/-2.6mmHg systolic with no change in diastolic blood pressure.^[78] This dose of riboflavin has elsewhere shown benefits where MTHFR 677TT patients with premature cardiovascular disease were less responsive to medication but normalized (relative to hypertensive subjects without the homogyzous mutation) with riboflavin.^[79] The efficacy of riboflavin supplementation for MTHFR 677TT individuals was further confirmed in the same cohort four years later, despite increases in the number of other blood pressure medications taken; systolic blood pressure in these subjects decreased by -9.2+/-12.8mmHg with a decrease in diastolic blood pressure of -6.0+/-9.9mmHg.^[80]

In nickel-allergic subjects who were irradiated by UVA (385nm) and UVB (300nm) rays under experimental conditions, application of a topical cream containing riboflavin to the site a day prior and again 30 minutes prior to radiation appeared to preserve nickel-induced reddening of the skin, suggesting protection against UV-induced immunosuppression.^[81] Such skin-level immunosuppression can lead to skin cancer.^[82]

Riboflavin is taken up into pancreatic β-cells via transporters similar to those present in the intestines (RFVT1-3, with RFVT3 being more prominent in human pancreatic cells^[83]). Uptake occurs at a similar rate when the concentration is held at 14nM (Km of 0.17+/-0.02μM),^[84] with higher concentrations (10µM) thought to also confer a localized antiinflammatory effect in response to inflammatory cytokines (IL-1β, IFN-γ, and TNF-α).^[85]

Riboflavin is a component of the retina^[86] where it plays roles in aiding function of the photoreceptors in the retina^[87] and structurally protecting the surface;^[88] higher dietary intakes seem to be associated with reduced formation of nuclear cataracts.^[89] Deficiencies of riboflavin, amongst other effects, tend to result in ocular disorders due to damage to the ocular surface.^[88]

While riboflavin concentrations in the retina are responsive to the diet, this only occurs up until 3mg/kg in the rat (riboflavin per kilogram of feed)^[90] and rabbit^[91] as higher feed concentrations do not further increase retinal riboflavin concentration despite increases in serum levels. This may be related to the transporter that mediates riboflavin uptake in the retina, a sodium-independent transporter that appears to be saturated at a concentration of riboflavin between 8nM and 1µM (apparent K_M of 80+/-14nM).^[92]

Riboflavin has a role in the eyes where it aids in photoreceptors (receptors that respond to color) and structurally protects the eyes.

In patients with glaucoma, supplementation of riboflavin (0.8mg) alongside other nutraceutucals including forskolin (from coleus forskohlii) at 15mg, rutin at 200mg, and thiamin at 0.7mg over the course of one month appeared to reduce ocular surface damage as assessed by ocular discomfort (ocular surface disease index) as well as leading to improvments as assessed by three ocular tests (OPI, FBUT, and Schirmer test 1).^[93]

Dietary intake of riboflavin as assessed by food frequency questionnaire (FFQ) in older women appears to be inversely associated with colorectal cancer incidence, with the highest quartile of intake (greater than 3.97mg) having a reduced risk (HR 0.75; 95% CI of 0.62-0.92) when compared to the lowest quartile of total intake (less than 1.8mg).^[94]

Supplementation of 10mg riboflavin daily over the course of six months in subjects with multiple sclerosis failed to lessen the disease severity as assessed by the Expanded Disability Status Scale (EDSS) when compared to placebo as both groups benefitted.^[95] This study failed to find an influence on serum homocysteine concentrations or antioxidant activity (glutathione reductase) in red blood cells.^[95]

Friedreich's ataxia (FRDA) is a genetic disease associated with a mitochondrial mutation that reduces production of a protein called frataxin, increasing iron-mediated oxidative stress which ultimately leads to degeneration of nerve tissue in the spine.^[96][97] Riboflavin is thought to have a role based on studies in S. cerevisiae and C. elegans (fraxatin deficiency models) where the cofactors improved cellular growth and rescued the phenotype resulting from frataxin deficiency.^[98]

One preliminary trial using deferiprone (an iron chelator which may have a role in treating FRDA^[99][100]) with added idebenone (10-20mg/kg) and riboflavin (10-15mg/kg) over the course of numerous months found a possible slowing of disease progression which requires future studies to verify due to the open-label, uncontrolled nature of the study, along with a very high dropout rate.^[101] These two nutraceuticals have been used in conjunction with darbepoetin alfa (a synthetic form of erythropoietin which may increase frataxin expression^[102]) previously for possible benefits in another open-label trial.^[103]

Vitamin B6 needs to be converted in the human body to its active coenzyme form, pyridoxal 5'-phosphate (PLP), in a process which requires riboflavin in its cofactor form of flavin mononucleotide (FMN).^[104]

The absorption of riboflavin from the intestines is influenced by chronic alcohol intake in the rat, as a high alcohol intake for four weeks (36% of calories coming from ethanol) can reduce the expression of riboflavin transporters RFVT1-3.^[40] When tested ex vivo, riboflavin transport appeared to be approximately halved both for intestinal uptake and renal resorption which also saw a decrease in RFVTs.^[40]

Patients with alcoholism tend to have a high prevalence of riboflavin deficiency of between 15-50%,^{[105][106][107]} at times associated with deficiency symptoms such as neuropathies.^[108]

Alcohol exposure seems to reduce the capacity of the intestines to take up riboflavin into the body, resulting in chronic alcoholics being riboflavin-deficient.