19.1: Riboflavin (20b.1)

Riboflavin (7,8-dimethyl-10-ribityl-isoalloxazine) was first synthesized in 1935. Its structure consists of an isoalloxazine ring attached to a ribityl side chain Figure 20b.1. A detailed review of ribo­flavin and health is provided elsewhere (Powers, 2003; Thakur et al., 2017; Suwannasom et al., 2020).

20b.1.1 Functions of ribo­flavin

Ribo­flavin is a component of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which act as coenzymes of different flavo­proteins involved in oxidation-reduction reactions that are central to energy production, intermediary metab­olism, drug metab­olism as well as the maintenance of anti­oxidant status (Figure 20b.1). Of the two coenzymes, FMN is formed first, from free ribo­flavin, by ATP-dependent phos­phoryla­tion; a reaction catalyzed by cytosolic flavo­kinase (EC 2.7.1.26). Next, most of this FMN is combined with a molecule of ATP to form FAD; this step is catalyzed by the FAD-dependent FAD syn­thetase (EC 2.7.7.2). In turn, FAD can be converted into forms covalently bound to tissue proteins. Thyroid hormones regulate the synthesis of FMN and FAD as well as the formation of the covalently bound flavins (Rivlin, 1970; Pinto and Rivlin, 1979).

Figure 20b.1: Structure of riboflavin and the two coenzymes derived from flavin, flavin mononucleotide, and flavin adenine dinucleotide.

Both FMN and FAD are also cofactors for several enzymes, including glutathione reductase, xanthine oxidase, L‑amino oxidase, and nico­tinamide adenine dinucleo­tide (NAD) dehy­drogenase. Results from animal studies also suggest that flavins are cofactors in the cyclical β‑oxi­dation of fatty acids (Olpin and Bates, 1982a; Olpin and Bates, 1982b). Around 84% of human flavo­proteins are FAD‑depen­dent and only 16% use FMN as a cofactor (Leinhart et al., 2013).

Ribo­flavin coenzymes are also involved in the metab­olism of four other vitamins — folate, vitamin B₁₂, vitamin B₆, and niacin. Flavin adenine dinucleo­tide is a cofactor for 5,10‑methyl­enetetra­hydro­folate reductase (MTHFR; EC 1.7.99.5), a key enzyme in folate metab­olism involved in the conversion of 5,10‑methyl­enetetra­hydro­folate to 5-methyl­tetra­hydro­folate, which is required for the remethylation of homo­cysteine to meth­ionine. Both FMN and FAD act as cofactors for the enzyme meth­ionine synthase reductase (EC 1.16.1.8) which is responsible for the regeneration of methyl­cobalamin, the bio­logically active form of vitamin B₁₂ that is also involved in the remethyl­ation of homo­cysteine to meth­ionine. In addition, FMN acts as a cofactor for pyri­dox­ine (pyridox­amine) phos­phate oxidase (PPO; EC 1.4.3.5), an important enzyme in vitamin B₆ metab­olism that converts the 5‑phos­phates of both pyri­dox­ine and pyri­dox­amine to the coenzyme pyri­dox­al‑5´‑phos­phate. Finally, the FAD‑dependent kynurenine hydroxylase is involved in the conversion of tryptophan to niacin. As a result, severe ribo­flavin deficiency can cause functional deficiency with disturb­ances in the meta­bolic pathways of these vitamins.

Absorption of ribo­flavin occurs mainly in the proximal small intestine by a saturable carrier-mediated mechanism; the maximal amount of ribo­flavin that can be absorbed from a single dose is about 27mg (Zempleni et al., 1996). Intestinal micro­flora is able to synthesize ribo­flavin, some of which is absorbed in the colon by a carrier-mediated process (Said et al., 2000; Said, 2013 ). The vitamin is transported in plasma as free ribo­flavin complexed with albumin and other proteins, mainly immuno­globulins, which also bind flavin coenzymes (Innis et al., 1985).

20b.1.2 Deficiency of ribo­flavin in humans

The classical signs of ribo­flavin deficiency, termed aribo­flavinosis, are angular stomatitis, cheilosis, glossitis and anemia. Corneal vascular­ization, dermato­logical changes, and neuro­logical alterations may also occur but are not specific for aribo­flavinosis (Northrop-Clewes and Thurnham, 2012). Some environ­mental factors may also influence the clinical signs and symptoms of ribo­flavin deficiency.

Aribo­flavinosis is rarely encountered in isolation, usually occurring in association with other vitamin deficiency states. In a study of Irish elderly, 49% had suboptimal ribo­flavin status, 39% had suboptimal vitamin B₆status, and 21% had con­current ribo­flavin and vitamin B₆ deficiencies. After supple­mentation with ribo­flavin alone, both the ribo­flavin deficiency and the low plasma pyri­dox­al‑5´‑phos­phate levels were corrected (Madigan et al., 1998).

Some evidence suggests that ribo­flavin deficiency is linked with impaired mobilization of iron from intra­cellular stores (i.e. hepatic ferritin), increased rate of iron loss from the gastro­intestinal tract, as well as decreased absorption of iron (Powers, 2003). This could explain the low hemoglobin con­cen­trations and hypo­chromic anemia which often are observed in ribo­flavin deficiency (Aljaadi et al., 2019). Studies conducted in population groups with a com­promised ribo­flavin status such as school children (Buzina et al., 1979; Charoenlarp et al., 1980), men (Fairweather-Tait et al., 1992), pregnant (Decker et al., 1977; Suprapto et al., 2002; Ma et al., 2008), lactating (Powers et al., 1985) and women of reproductive age (Powers et al., 2011), have shown that ribo­flavin supple­mentation leads to improve­ment of the hemat­ological status.

Ribo­flavin deficiency has been described in under­nourished pop­ulations in several low income countries, notably among women and children in The Gambia (Bates et al., 1981, 1994) , some elderly persons in Guatemala (Boisvert et al., 1993a), children in Côte d'Ivoire (Rohner et al., 2007), and in some adol­escent refugees from Bhutan living in south­eastern Nepal (Blanck et al., 2002). Infants of mothers with a low ribo­flavin status during gestation are also likely to be born ribo­flavin-deficient (Bates et al., 1982). An accumulating body of evidence based on small as well as pop­ulation-based surveys from developed countries has shown that a sub­optimal ribo­flavin status is more common than previously recognized and those pre­domin­antly affected are adol­escents and young women (Bates et al., 2016; Ward et al., 2020; Aljaadi et al., 2019; Jungert et al., 2020).

Several conditions, including alcoholism, diabetes mellitus, liver disease, thyroid and adrenal insufficiency, and gastro­intestinal and biliary obstruction, may precipitate or exacerbate ribo­flavin deficiency (Rivlin, 2007). Alcohol causes deficiency by interfering with both the digestion and the intestinal absorption of ribo­flavin S (Pinto et al., 1987; Subramanian et al., 2013 ). Anti­epileptic and psycho­tropic drugs such as chlor­promazine, imipramine, and amitriptyline (Apeland et al., 2003; Pinto et al., 1982), as well as some anti­malarial drugs such as quinacrine (Dutta et al., 1985) all inhibit the conversion of ribo­flavin to its active coenzyme derivatives. Drugs, such as tetra­cycline, theo­phylline, and caffeine, as well as metals, such as zinc, copper, and iron, may chelate or form complexes with ribo­flavin and, hence, affect its bio­avail­ability (Sauberlich, 1985).

More vulnerable to ribo­flavin deficiency are individuals with 677C→T poly­morphism in the gene encoding MTHFR enzyme because the variant enzyme has an impaired activity as a result of reduced affinity to the cofactor FAD (Yamada et al., 2001); this typically leads to elevated homo­cysteine con­cen­trations (Frosst et al., 1995). Marked lowering of homo­cysteine has been achieved in people with the impaired variant (TT genotype) with a low dose of ribo­flavin (McNulty et al., 2006). Moreover, genome-wide association studies (Ehret et al., 2011) and clinical studies (Qian et al., 2007; Yang et al., 2014) provide evidence linking the MTHFR 677C→T poly­morphism with blood pressure and increased risk of hyper­tension and hyper­tension in pregnancy by up to 87%. Importantly, emerging evidence from random­ized trials highlights that ribo­flavin supple­mentation can lower blood pressure specifically in adults with TT genotype (Horigan et al., 2010; Wilson et al., 2013). This novel role of ribo­flavin could have important public health implications, considering that the frequency of the variant TT genotype is 10–12% worldwide but it could reach up to 32% in some countries (e.g., Mexico; Wilcken et al., 2003).

Ribo­flavin deficiency has been implicated as a risk factor for cancer in animal studies (Powers, 2003), but the epidemiological evidence based on several meta-analyses is less consistent (Yoon et al., 2016; Yu et al., 2017; Ben et al., 2019; Zeng et al., 2020).

Studies have shown that in malaria endemic regions, individuals with ribo­flavin deficiency are relatively resistant to this infection and have a lower level of parasitemia, but the course of the disease may be more severe than in people with adequate ribo­flavin status (Das et al., 1988).

20b.1.3 Food sources and dietary intakes

Ribo­flavin in foods is present predominantly in the form of FAD and only small quantities are available as FMN. Some flavins bound covalently to protein are also found in certain foods but are largely unavailable as nutritional sources of ribo­flavin; only limited amounts apparently undergo digestion and absorption (Chia et al., 1978).

The major food sources of ribo­flavin are dairy products, especially milk, and meat and fish; most plants contain only small amounts of ribo­flavin. As a result, individuals consuming mainly plant-based diets may be at risk for ribo­flavin deficiency. Ribo­flavin enrichment of flour is mandated in the US and some other countries with the aim of restoring the losses of the vitamin during milling and refining processes. Most of the ready-to-eat breakfast cereals are fortified with ribo­flavin in developed countries. Although it is heat-stable, losses of ribo­flavin do occur if it is exposed to the light and, as it is water soluble, by leaching into the cooking water (Powers, 2003). Bioavailability of ribo­flavin from food is reported to be around 95% (IOM, 2000). There are limited data on the relative bio­availability of ribo­flavin from different food sources but a study using stable isotopes and kinetic modeling did not find a significant difference in ribo­flavin absorption from milk and spinach (Dainty et al., 2007).

Nationally representative surveys from the US, Australia, Ireland, and the UK showed that milk and dairy products, meat and ready-to-eat breakfast cereals are the main dietary contributors to ribo­flavin intake (IOM, 2000; Australian Bureau of Statistics, 2014; National Adult Nutrition Survey, 2011; Bates et al., 2016).

20b.1.4 Effects of high intakes of ribo­flavin

In humans, there is no evidence for ribo­flavin toxicity as a result of excessive intakes (EFSA, 2017). The absorption of orally administered ribo­flavin from both vitamin supple­ments and from natural foodstuffs is limited, and high intakes are rapidly excreted in the urine. Even when 400mg/d of ribo­flavin was given orally with meals for at least 3 months, no short-term side effects were reported (Schoenen et al., 1994). In view of the limited data on adverse effects from high intakes of ribo­flavin, no Tolerable Upper Intake Level for ribo­flavin was set by the U.S. Food and Nutrition Board (IOM, 2000).