Properties and Metabolism

Riboflavin exists in three forms in nature. The forms are free dinucleotide riboflavin and the two coenzyme derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Riboflavin is an odorless, bitter, orange-yellow compound that melts at about 280°C. The molecular structure of riboflavin is shown in Illus. 8-1. Riboflavin is only slightly soluble in water, but readily soluble in dilute basic or strong acidic solutions. It is heat stable in neutral and acidic solutions but not alkaline solutions. Very little is lost in cooking. Aqueous solutions are unstable to visible and ultraviolet light. Instability is increased by heat and alkalinity. Riboflavin plays a key role in problems related to light sensitivity and photodegradation of milk and dairy products. Both light and oxygen have been found to induce riboflavin degradation (Becker et al., 2003). When dry, riboflavin is appreciably less affected by light.

Illustration 8-1

Riboflavin covalently bound to protein is released by proteolytic digestion. Phosphorylated forms (FAD, FMN) of riboflavin are hydrolyzed by phosphatases in the upper gastrointestinal tract to free the vitamin for absorption. Free riboflavin is absorbed by mucosal cells via an active saturable transport system in all parts of the small intestine. Transport of riboflavin by blood plasma is known to involve both loose association with albumin and tight associations with some globulins (McCormick, 1990). A genetically controlled riboflavin binding protein is present in serum and eggs. There is a hereditary recessive disorder in chickens, renal riboflavinuria, in which the riboflavin-binding protein is absent (White, 1996). Eggs become riboflavin-deficient and embryos generally do not survive beyond the fourteenth day of incubation (Clagett, 1971; Rivlin, 2006). Also, if riboflavin-binding protein is in excess it can diminish riboflavin availability to the chicken embryo (Lee and White, 1996). Presumably the lack of the specific vitamin transport protein prevents adequate transfer of dietary riboflavin to the developing fetus and riboflavin losses occur via maternal urine. In addition to poultry, specific binding proteins have been found in serum from pregnant cows and rats, human fetal blood, and uterine secretions in the pig. Hepatic cells from deficient animals have a relatively greater maximal absorption uptake of riboflavin (Rose et al., 1986). Hepatic cell riboflavin absorption occurs via facilitated diffusion. In mucosal cells, riboflavin is phosphorylated to FMN by the enzyme flavokinase (Rivlin, 2006, 2007). The FMN then enters the portal system, where it is bound to plasma albumin, transported to the liver, and there converted to FAD. Rivlin (2006) suggested there may be physiological mechanisms in pregnancy that facilitate transfer of riboflavin from maternal stores to the fetus in a manner that is fundamentally similar to that in the laying hen. Riboflavin is efficiently transferred to the fetus.

Animals do not appear to have the ability to store appreciable amounts of riboflavin, with the liver, kidneys and heart having the greatest concentrations. The liver, the major site of storage, and contains about one-third of the total body riboflavin. Intakes of riboflavin above current needs are rapidly excreted in urine, primarily as free riboflavin. Minor quantities of absorbed riboflavin are excreted in feces, bile and sweat.


Riboflavin is required as part of many enzymes involved with metabolism of carbohydrate, fat and protein. Riboflavin in these coenzyme forms (FMN and FAD) are called flavoproteins and act as intermediaries in the transfer of electrons in biological oxidation-reduction reactions. If levels of riboflavin are low, the respiration process becomes less efficient, and 10% to 15% more feed is required to meet energy needs (Christensen, 1983). The enzymes that function aerobically are called oxidases, and those that function anaerobically are called dehydrogenases. The general function is in oxidation of substrate and generation of energy (i.e., ATP). By involvement in the hydrogen transport system (cytochrome system), flavoproteins function by accepting and passing on hydrogen. Flavoproteins assist in the generation of ATP.Collectively, the flavoproteins show great versatility in accepting and transferring one or two electrons with a range of potentials. Many flavoproteins contain a metal (e.g., iron, molybdenum, copper, zinc), and the combination of flavin and metal ion is often involved in the adjustments of these enzymes in transfers between single- and double-electron donors. Xanthine oxidase contains the metals molybdenum and iron. It converts hypoxanthine to xanthine and the latter to uric acid. It also reacts with aldehydes to form acids, including the conversion of retinal (vitamin A aldehyde) to retinoic acid.

Flavoproteins may accept hydrogen ion (H+) directly from the substrate, thus catalyzing oxidation of the substrate, or it may catalyze the oxidation of some other enzyme by accepting hydrogen ion from it, for example, from the niacin-containing coenzymes, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). More than 100 flavoprotein enzymes may be arbitrarily classified into three groups:

  1. NADH2 dehydrogenases–reduced pyridine nucleotide is a substrate, and the electron acceptor is either a member of the cytochrome system or some other acceptor besides oxygen.
  2. Dehydrogenases–accept electrons directly from the substrate and can pass them to one of the cytochromes.
  3. Oxidases (true)–accept electrons from the substrate and pass them directly to oxygen (O2 is reduced to H2O2); they cannot reduce cytochromes.

Riboflavin functions in flavoprotein-enzyme systems to help regulate cellular metabolism. They are specifically involved in metabolism of carbohydrates. Riboflavin is also an essential factor in amino acid metabolism as part of amino acid oxidases. These enzymes oxidize amino acids, which result in the decomposition of the amino acids, yielding ammonia and a keto acid. Distinct oxidized D-amino acids (prosthetic group FAD) and L-amino acids (prosthetic group FMN) are produced. In addition, riboflavin plays a role in fat metabolism (Rivlin, 2006), and an FAD flavoprotein is an important link in fatty acid oxidation. This includes the acyl-coenzyme A dehydrogenases, which are necessary for the stepwise degradation of fatty acids. An FMN flavoprotein is required for synthesis of fatty acids from acetate. Thus flavoproteins are necessary for both degradation and synthesis of fatty acids.

Riboflavin and other vitamins play an important role in skin development, tensile strength and healing rates (Ward, 1993). A deficiency in riboflavin can slow epithelialization of wounds by 4-5 days (Lakshmi et al., 1989), reduce collagen content by 25%, and decrease tensile strength of wounds by 45%. Riboflavin deficiency can increase skin homocysteine two- to four-fold, which ultimately impairs the cross-link formation of collagen (Lakshmi et al., 1990). Marginal field deficiencies of riboflavin could increase skin tears and cause longer healing times.

Among the enzymes that require riboflavin is the FMN-dependent oxidase responsible for conversion of phosphorylated pyridoxine (vitamin B6) to a functional coenzyme. Riboflavin deficiency also results in a decrease in the conversion of the vitamin B6 coenzyme pyridoxal phosphate to the main vitamin B6 urinary excretory product of 4-pyridoxic acid (Kodentsova et al., 1993). Riboflavin deficiency has an effect on iron metabolism, with less iron absorbed and an increased rate of iron loss due to an accelerated rate of small intestinal epithelial turnover (Powers et al., 1991, 1993).

Riboflavin, co-enzyme Q10, and niacin are associated with poly (ADP-ribose) which functions in post-translational modification of nuclear proteins. The poly ADP-ribosylated proteins function in DNA repair, replication and cell differentiation (Premkumar et al., 2008).


Riboflavin requirements vary with heredity, growth, environment, age, activity, health, other dietary components and synthesis by the host. Microbial synthesis of riboflavin has been shown to occur in the gastrointestinal tract of a number of animal species and thus affects requirements. Depending on the species, utilization depends on the composition of the diet (Christensen, 1973) and incidence of coprophagy. Young rats fed a riboflavin-free, purified diet with sucrose as the carbohydrate source will cease to grow. However, when sucrose is replaced by starch, sorbitol, or lactose, growth is comparable to that of rats supplied with riboflavin (Fridericia et al., 1927; Haenel et al., 1959). Excretion of riboflavin in urine and feces is also dependent on the carbohydrate in the diet and is suppressed by inclusion of sulfa drugs in the diet (De and Roy, 1951). Antibiotics, such as tetracycline, penicillin and streptomycin, reduce the requirements of several animal species for riboflavin via stimulation of microorganisms that synthesize riboflavin, or inhibition of microorganisms in the gut that compete for riboflavin.For the dog and cat, the contribution of microbial riboflavin synthesis is not known. However, Gershoff et al. (1959a) fed varying levels of fat and carbohydrates to kittens and concluded that a high-carbohydrate, low-fat diet favored synthesis of riboflavin by intestinal microorganisms as indicated by greater urinary and fecal excretion. The high-carbohydrate diet may also have favored utilization or retention of riboflavin. The high-fat diets, 46% vs. 11% of metabolizable energy (ME) from fat, increased the riboflavin requirement in kittens from 0.15 to 0.20 mg per day (Gershoff et al., 1959a).

A. Requirements for Dogs

According to the NRC (2006) the riboflavin requirements for all life stages of dogs is 5.3 mg per kg (2.4 mg per lb) of diets. These levels will provide adequate amounts of the vitamin for reasonable tissue storage. Using an accurate indicator of riboflavin body status (erythrocyte glutathione reductase), Cline et al. (1996) reported adult dog requirements to be 66.8 µg riboflavin per kg (30.4 µg per lb) body weight.On a feed basis, Axelrod et al. (1941) reported a minimal requirement of 2 mg per kg (0.91 mg per lb) of diet, but tissue storage was low, which suggested that 4 mg per kg (1.8 mg per lb) of diet was a satisfactory level. The Association of American Feed Control Officials (AAFCO, 2007) recommends 2.2 mg riboflavin per kg (1 mg per lb) of diet for all classes of dogs.

B. Requirements for Cats

Using semi-purified diets for kittens, Leahy et al. (1967) concluded that riboflavin requirements for growth did not exceed 100 µg per day or approximately 1 mg per kg (0.45 mg per lb) of diet. Although the riboflavin requirement for cats is relatively low, when there is an increased demand for riboflavin, such as during lactation or for cats with various infectious diseases, the dietary requirement is higher (Hoffmann-La Roche, 1981). The NRC (2006) suggests a minimal requirement of 4 mg riboflavin per kg (1.8 mg per lb) of diet for growth. AAFCO (2007) also recommends 4 mg riboflavin per kg (1.8 mg per lb) of diet for all classes of cats.


Riboflavin is synthesized by green plants, yeast, fungi and some bacteria. Rapidly growing green leafy vegetable and forages, particularly alfalfa, are good sources, and the leaves have the highest content of the vitamin. Cereals and their by-products have a rather low riboflavin content, in contrast to their high thiamin content. Oilseed meals are fair sources, whereas grains should not be relied on as the sole sources of riboflavin.

Riboflavin is one of the more stable vitamins, but can be readily destroyed by ultraviolet (UV) rays of sunlight. Appreciable amounts may be lost upon exposure to light; up to one half the riboflavin content is lost in cooking eggs and pork chops in light and most of the vitamin is lost from milk stored in clear glass bottles (McDowell, 2000). The riboflavin content of the milk of cows or goats is many times higher than that of their feed because of rumen synthesis. Human milk contains about 0.5 mg riboflavin per liter, while the content of cow’s milk is three times as high (i.e., 1.7 mg per liter). Milling of rice and wheat results in considerable loss of riboflavin since most of the vitamin is in the germ and bran, which are removed during this process. About one-half the riboflavin content is lost when rice is milled. Whole wheat flour contains about two-thirds more riboflavin than white flour (McDowell, 2000).

Riboflavin is more bioavailable from animal products than plant sources. Flavin complexes in plants are more stable to digestion and thus less digestible than animal sources. Singh and Deodhar (1992) concluded that milk factor(s) enhanced intestinal uptake of riboflavin in the rat intestine. Fermentation significantly increases the proportions of riboflavin present in the free form, resulting in a greater bioavailability of the vitamin in curd than in milk. Bioavailability of riboflavin was less for chicks fed corn-soybean than for those fed purified amino acid diets; riboflavin bioavailability in the corn-soybean meal diet was 59.1% (Chung and Baker, 1990).

Riboflavin is commercially available as a crystalline compound produced by chemical synthesis or fermentation. It is available to the feed, food and pharmaceutical industries as a high-potency, USP or feed-grade crystalline powder; spray-dried powders; and dry dilutions. Riboflavin 5’-phosphate sodium salt is available for applications requiring a water-dispersible source of riboflavin. High-potency, USP or feed-grade crystalline powders are electrostatic, hygroscopic and dusty, and thus do not flow freely and show poor distribution in feeds. In contrast, dilutions of riboflavin, such as spray-dried powders or other concentrated, dry dilution products, or inclusion in a premix, reduce electrostaticity and hygroscopicity for better flowability and distribution in feeds (Adams, 1978).


Pet foods based largely on grains could be borderline to deficient in riboflavin. A decreased rate of growth and lower feed efficiency are common signs of riboflavin deficiency in all species affected. Typical clinical signs often involve the eye, skin, and nervous system. Riboflavin deficiency would not be expected in young nursing puppies or kittens, as milk is a rich source of the vitamin. Several methods have been used to assess nutritional status of riboflavin. These include clinical signs, blood and urine levels of the vitamin, and measurement of enzymatic coenzyme activity. Riboflavin deficiency is accompanied by a reduction in erythrocyte riboflavin concentration, a reduced urinary excretion of riboflavin, and a low urinary recovery of a riboflavin test load (Axelrod et al., 1941; Potter et al., 1942; Noel et al., 1972). Erythrocyte glutathione reductase assay is currently the preferred test for diagnosis of riboflavin deficiency in a number of species including dogs (Cline et al., 1996).

A. Deficiency in Dogs

Research on riboflavin deficiency in dogs developed when animals were fed semi-purified diets. The first signs of deficiency were seen after three to nine weeks and were characterized by loss of appetite, weight loss, ocular lesions and flaky dermatitis with marked erythema on the hind legs, chest and abdomen. Conjunctivitis with or without corneal vascularization and opacities was also seen (Potter et al., 1942). The ocular lesions are generally bilateral, and progress from a discharge accompanied by conjunctivitis to opacity and vascularization of the cornea (Street et al., 1941a; Potter et al., 1942; Heywood and Partington, 1971; Noel et al., 1972; NRC, 2006).In the final stages of the deficiency disease, muscular weakness develops and progresses within a few days to ataxia, followed by collapse, coma, and death. Dogs also exhibit a unique phenomenon, the collapse syndrome (Street and Cowgill, 1939). Street and Cowgill (1939) used the collapse syndrome as an indicator of deficiency. They reported loss of appetite and muscular weakness before collapse. During the collapse syndrome, the animals were unable to stand and had elevated heart rates (140 to 190 beats per minute), along with vomiting and diarrhea (Potter et al., 1942).

Street et al. (1941a) reported degeneration of the myelin sheath of the posterior spinal cord and the peripheral nerves when adult dogs were fed 8 µg of riboflavin per day for more than 400 days. They suggested this as the basis for the incoordination and loss of deep muscle reflex in the hind limbs observed in the deficient dogs.

B. Deficiency in Cats

Riboflavin deficiency manifests itself in cats after four to eight weeks with anorexia, weight loss and death. Anorexia resulting in weight loss and death is the principal deficiency sign in cats (Gershoff et al., 1959a). Chronic riboflavin deficiency in cats results in hair loss extending to the chest and feet, cataracts, and alopecia with epidermal atrophy. Often with chronic riboflavin deficiency, cats have fatty livers and testicular atrophy (NRC, 2006). Histologically, the cats had fatty livers and testicular hypoplasia. None of the animals exhibited signs of the collapse syndrome associated with riboflavin deficiency in dogs (Gershoff et al., 1959a).

Fortification Considerations

Riboflavin deficiency would be more prevalent in dog diets compared to cat diets. This is because more riboflavin-poor cereal grains form a higher percentage of dog diets. Riboflavin fortification levels should be adjusted, especially to offset the exclusion or reduced amounts of riboflavin-rich ingredients such as milk fermentation and fish and meat by-products. Riboflavin deficiency will not occur if the diet contains meat or dairy products (Watson, 1998). While it is unlikely that a clinical riboflavin deficiency would be seen in cats today, it is not impossible for it to occur in dogs. Most commercial pet foods are supplemented with synthetic riboflavin (Hand et al., 2010). However, there are still a number of hunt clubs, kennels, etc., formulating their own rations rather than purchasing commercial foods. It is in these situations that a deficiency state would most likely occur (Ralston Purina, 1987). If dogs are observed with weight loss, malaise, gastrointestinal disturbances, dermatitis, stomatitis, conjunctivitis and cataracts, a deficiency of riboflavin might be included in the differential diagnosis.

Riboflavin is remarkably stable during heat processing. However, considerable loss may occur if foods are exposed to light during cooking, and some losses occur in feed administered to animals out of doors. Only that portion of riboflavin in the feed exposed to light would be destroyed; therefore, this may be of little significance for pet foods. Up to 26% of riboflavin present in pet food is lost during extrusion (Hoffmann-La Roche, 1981).

In dry form, riboflavin is extremely resistant to oxidation, even when heated in air for long periods. Under typical circumstances, riboflavin has good stability when added to mixed feeds (Hoffmann-La Roche, 1969). Riboflavin is quite stable in multivitamin premixes (Frye, 1978). A recent report demonstrates a 98% retention of riboflavin after six months in a vitamin premix. However, the retention was only 59% when the premix contained choline and trace minerals (Gadient, 1986).

Vitamin Safety

A large body of evidence shows that treatment with riboflavin in excess of nutritional requirements has very little toxicity either for experimental animals or for humans (Rivlin, 2006). There are no reports of riboflavin toxicity occuring in dogs and cats. Most data from rats suggest that dietary levels between 10 and 20 times the requirement (possibly 100 times) can be tolerated safely (NRC, 1987). Dogs are particularly tolerant to large doses of riboflavin. Dogs given a single oral dose of 2 g riboflavin per kg (0.91 g per lb) body weight showed no ill effects (Unna and Greslin, 1942). Similarly, four 10-week-old puppies were fed 25 mg riboflavin per kg (11.36 mg per lb) of body weight for five months; neither toxic signs nor pathological changes in the organs at necropsy were observed. It would appear that feeding levels 42,000 times the NRC requirements for dogs caused no adverse effects (Unna and Greslin, 1942).

When massive amounts of riboflavin are administered orally, only a small fraction of the dose is absorbed, the remainder is excreted in the feces. Lack of toxicity is probably due to the fact that the transport system necessary for the absorption of riboflavin across the gastrointestinal mucosa becomes saturated, limiting riboflavin absorption (Christensen, 1973). Also, capacity of the tissues to store riboflavin and its coenzyme derivatives appears to be limited when excessive amounts are administered.

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