Riboflavin exists in nature in three forms: free riboflavin and the coenzyme derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Riboflavin is an odorless, bitter, orange-yellow compound with a melting point of 536°F (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 base or strong acid solutions. It is quite stable to heat at neutral and acidic pH but not in alkaline solutions. Very little is lost in cooking. Aqueous solutions are unstable to visible and ultraviolet light. Heat and alkalinity reduce stability. In the dry state, riboflavin is appreciably more stable to light. 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; Domingos et al., 2011).
Riboflavin covalently bound to protein is released by proteolytic digestion. Phosphorylated forms of riboflavin (FAD, FMN) are hydrolyzed by phosphatases in the upper gastrointestinal tract to free the vitamin for absorption. Free riboflavin is absorbed via an active, saturable transport system in all segments of the small intestine. Hepatic cells from deficient animals have an increased maximal absorption rate of riboflavin (Rose et al., 1986). Hepatic cells also absorb riboflavin via facilitated diffusion. In mucosal cells, riboflavin is phosphorylated to FMN by the enzyme flavokinase (Rivkin, 2006, 2009). The FMN then enters the portal system, where it is bound to plasma albumin and is then transported to the liver, where it is converted to FAD. Riboflavin-binding proteins have been reported to be present in the serum from pregnant cows. Animals do not appear to have the ability to store appreciable amounts of riboflavin; the liver, kidney and heart have the greatest concentrations. Liver, the major site of storage, 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 a cofactor for many enzymatic reactions involved with metabolism of carbohydrate, fat and protein. Riboflavin in coenzyme form (FMN or FAD) is called flavoprotein, and acts as an intermediary 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). Enzymes that function aerobically are called oxidases, and those that function anaerobically are called dehydrogenases. Their general function is in oxidation of substrate and generation of energy (i.e., ATP). By involvement in the electron transport system (cytochrome system), flavoproteins function by transferring electrons. Flavoproteins assist in the generation of ATP. Flavoproteins may accept reduced hydrogen directly from a substrate, thus catalyzing oxidation of the substrate, or may catalyze the oxidation of a second cofactor by accepting a reduced hydrogen from it, for example, from the niacin-containing coenzymes, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). About 40 flavoprotein enzymes have been identified and arbitrarily classified into three groups:
Riboflavin functions in flavoprotein-enzyme systems to help regulate cellular metabolism, and is 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 to ammonia and a keto-acid. Distinct oxidized forms of the 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). FAD flavoprotein plays an important role in fatty acid oxidation, in the acetyl-coenzyme A dehydrogenases, which are necessary for the stepwise degradation of fatty acids. A 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 to 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 by 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 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).
Due to the rumen microbial synthesis of riboflavin, ruminants have no absolute dietary requirement. It has been assumed that riboflavin is synthesized in the rumen because of its relative concentrations in the diet and in rumen contents. The range of values reported for riboflavin range from 4 to 39 mg per kg (1.8 to 17.7 mg per lb) of rumen dry matter, a wide range (Mathison, 1984). Confirmation of a net synthesis in the rumen is illustrated from work ofMcElroy and Goss (1940a) in which the secretion of riboflavin in milk was equivalent to approximately 10 times the intake of the vitamin. Buziassy and Tribe (1960) estimated a net synthesis of 0.9 to 12.0 mg of riboflavin per kg (0.4 to 5.5 mg per lb) of dietary dry matter intake in sheep. Pearson et al. (1953) measured a net synthesis of riboflavin when semi-synthetic diets low in riboflavin were fed. More recently, Miller et al. (1986a) determined a net synthesis equivalent to 78% to 225% of the riboflavin intake (25 to 39 mg per day) in steers fed a diet containing 85% grain. Diet composition influences total microbial synthesis of riboflavin. Data of Miller et al. (1986b) suggest a greater ruminal synthesis of riboflavin with increased dietary concentrate. Buziassy and Tribe (1960) measured increased riboflavin synthesis with increasing dietary protein levels. These authors also noted that ruminal riboflavin synthesis was reduced with higher dietary intakes of the vitamin. Rumen microorganisms can degrade riboflavin, forming hydroxyethylflavine, formylethylflavine and other metabolites (Owen and West, 1970). The importance of ruminal destruction of riboflavin, or of the metabolites produced, is unknown.
Riboflavin deficiencies have been demonstrated in young ruminant animals, and the estimated requirement for calves is 1 to 1.6 mg per kg (0.45 to 0.73 mg per lb) of dry matter (Roy, 1980). The majority of livestock species have requirements of 1 to 4 mg per kg (0.45 to 1.8 mg per lb) of dry matter. Expressed on a body weight basis, the riboflavin requirement of calves fed milk replacer reportedly ranges from 35 to 45 mg per kg (15.9 to 20.5 mg per lb) of body weight. The NRC (1989) suggests that calf milk replacers should contain 6.5 mg per kg (3.0 mg per lb) of dry matter.
Green plants, yeast, fungi and some bacteria synthesize riboflavin. Rapidly growing, green leafy vegetable and forages, particularly alfalfa, are good sources, and the leaves have the highest content of riboflavin. 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 and protein meals contain some riboflavin, but should not be relied on as the sole sources of riboflavin.
Riboflavin is one of the more stable vitamins, but can be rapidly destroyed by ultraviolet (UV) light or 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 in milk stored in clear glass bottles (McDowell, 2000). The riboflavin content of the milk of cows or goats is many times higher than in the diet due to rumen synthesis and probably also to accumulation by the mammary gland. Human milk contains about 0.5 mg riboflavin per liter, while the riboflavin content of cow’s milk is three times higher (i.e., 1.7 mg per liter).
Milling of rice and wheat results in considerable loss of riboflavin because 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 commercially available as a crystalline compound produced either by chemical synthesis or by fermentation. It is available to the feed, food and pharmaceutical industries as a high potency, USP of 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 or show good distribution in feeds. In contrast, dilutions of riboflavin, such as spray-dried powders or other concentrated, dry dilution products, reduce electrostaticity and hygroscopicity for better flowability and distribution in feeds (Adams, 1978).
Riboflavin is not required in the diet of adult ruminants per se because of rumen synthesis. Apparently no response to supplemental riboflavin has been reported in animals with a functional rumen, although tests with present-day levels of milk production and growth may be warranted. Miller et al. (1986a) reported that cattle on a concentrate-silage diet would synthesize approximately 38 mg of riboflavin in the rumen. A dairy cow producing 42 kg (92 lbs) of milk per day loses about 72 mg of riboflavin in milk alone, much more than is consumed in the diet. Riboflavin deficiencies have been demonstrated in young ruminants whose rumen flora is not yet established. Riboflavin deficiency results in redness of the mouth mucosa, lesions in the corner of the mouth and around the edges of the lips and navel, loss of hair, and excessive tear and saliva production (Radostits and Bell, 1970). Non-specific signs are anorexia, chronic diarrhea and reduced growth.
Effects of supplemental riboflavin in adult ruminants have not been well investigated. Osame et al. (1995) reported that intramuscular injection of riboflavin (10 mg per kg or 4.5 mg per lb of body weight in calves; 5 mg per kg or 2.27 mg per lb in cows) increased blood neutrophil count and bactericidal activity one to six days after injection. Milk is an extremely rich source of riboflavin, and therefore young, nursing ruminants should receive ample supplies of the vitamin. Prior to full rumen development, young ruminants require riboflavin. Milk replacer should be formulated to contain at least 6.5 mg per kg (3.0 mg per lb) of dry matter. Riboflavin is remarkably stable during heat processing. However, due to its instability in light, considerable loss may occur in feeds exposed to light during processing. In dry form, riboflavin is extremely resistant to oxidation, even when heated for extended periods. Field-cured alfalfa hay exposed to moisture can lose a significant amount of its riboflavin content in a relatively short time. Under common circumstances, riboflavin has good stability when added to mixed feeds. Riboflavin is quite stable in multivitamin premixes (Frye, 1978). A recent report demonstrates a 99% 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).
Considerable evidence has accumulated that feeding 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 studies in ruminants. Most data from rats suggest that dietary levels between 10 and 20 times the requirement (possibly 100 times) can be tolerated safely (NRC, 1987). When massive amounts of riboflavin are administered orally, only a small fraction of the dose is absorbed; the remainder is excreted in the feces. The lack of toxicity is probably due to saturation of the intestinal transport system (Christensen, 1973). Also, capacity of the tissues to store riboflavin and its coenzyme derivatives appears to be limited.
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