DSM in Animal Nutrition & Health
Properties and Metabolism
Riboflavin exists in three forms in nature: as free dinucleotide riboflavin and as 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 structure of riboflavin is depicted in Illus. 8-1. Riboflavin is only slightly soluble in water but readily soluble in dilute basic or strong acidic solutions. It is quite stable to heat in neutral and acidic, but not alkaline solutions. Very little is lost in cooking. Aqueous solutions are unstable to visible and ultraviolet light. This instability is increased by heat and alkalinity. Riboflavin plays a key role in problems related to light sensitivity and photo degradation of milk and dairy products. Both light and oxygen have been found to induce riboflavin degradation (Beckeret al., 2003). When dry, riboflavin is not affected appreciably by light, but in solution it is quickly destroyed. Mash feeds left exposed to direct sunlight for several days and frequently stirred are subject to some loss (Maynard et al., 1979).
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 enters mucosal cells of the small intestine after apparently being absorbed in all parts of the small intestine. Cells from deficient animals have a greater maximal absorption uptake of riboflavin (Rose et al., 1986). At low concentrations, riboflavin absorption is an active carrier-mediated process. At high concentrations, however, riboflavin is absorbed by passive diffusion, proportional to concentration. Liver cells from deficient animals have a relatively greater maximal absorption uptake of riboflavin (Rose et al.,1986). Liver 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, and is transported to the liver, where it is converted to FAD. Serum riboflavin-binding proteins appear to influence placental transfer and fetal or maternal distribution of riboflavin (Rivlin, 2007). Riboflavin-binding proteins have also been reported to be present in the serum and uterine secretions in the pig. Animals do not appear to have the ability to store appreciable amounts of riboflavin, with the liver, kidney and heart having the greatest concentrations. The 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 part of many enzymes essential to utilization of carbohydrate, fat and protein. These coenzyme forms (FMN and FAD) are called flavoproteins and act as intermediaries in the energy transactions of electrons in biological oxidation-reduction reactions. If levels are low, the energy from an inefficient respiration process is diminished and 10% to 15% more feed is required (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, flavoproteins function by accepting and passing on hydrogen, undergoing alternate oxidation and reduction.Flavoproteins may either accept reduced hydrogen directly from the substrate (the material being oxidized) or catalyze the oxidation of some other enzyme by accepting hydrogen from it, for example, from the niacin-containing coenzymes, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). About 40 flavoprotein enzymes may be arbitrarily classified into three groups:
- NADH2 dehydrogenases—enzymes whose substrate is a reduced pyridine nucleotide and the electron acceptor is either a member of the cytochrome system or some other acceptor besides oxygen.
- Dehydrogenases—enzymes that accept electrons directly from substrate and can pass them to one of the cytochromes.
- Oxidases (true)—enzymes that accept electrons from 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, although it is also 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 corresponding amino acids that decompose to give ammonia and a keto acid. There are distinct oxidized D-amino acids (prosthetic group FAD) and L-amino acids (prosthetic group FMN). 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 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, and ultimately increase costly downgrades.
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 (Rivlin, 2007). Riboflavin deficiency has an effect on iron metabolism, with less iron absorbed and an increased rate or 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. Hughes (1940) proposed that the daily minimum requirement of riboflavin for young growing pigs ranged between 2.2 and 6.6 mg per 100 kg (1 and 3 mg per 100 lbs) body weight. Krider et al. (1949) stated that 3.1 mg of riboflavin per kg (1.4 mg per lb) of ration seemed to represent the practical minimum requirement for weanling pigs fed in drylot. Mitchell et al. (1950) found the riboflavin requirement of growing pigs to be approximately 1.2 and 2.3 ppm when pigs were fed at environmental temperatures of 85°F and 42°F, respectively. These values were reported to be equivalent to 1.4 and 4.2 mg riboflavin per 45.5 kg of body weight, respectively. Forbes and Haines (1952) determined that baby pigs fed a “synthetic” milk diet and kept at an environmental temperature of 85°C with a relative humidity of 70% had a riboflavin requirement of between 1.5 and 2.0 mg per gram of dry matter. Miller et al. (1954) reported that for optimum growth and feed efficiency the riboflavin requirement for the baby pig is approximately 3.0 mg per kg (1.4 mg per lb) of solids. Terrill et al. (1955) indicated that for the growing pig kept at an environmental temperature of approximately 53°F, the riboflavin requirement is between 0.9 and 1.4 mg per kg (0.4 and 0.65 mg per lb) of diet. Seymour et al. (1968) did not observe consistent interactions between riboflavin and temperatures ranging from -4° to 32°C and indicated that between 3.0 and 4.0 mg of riboflavin per kg (1.4 and 1.8 per lb) of diet was required for baby pigs.Swine have a riboflavin requirement between 2 and 4 mg per kg (0.91 and 1.8 mg per lb) of diet (NRC, 1998). The NRC (1998) requirement declines as the pig grows, from 4 mg per kg (1.8 mg per lb) of feed for pigs 1 to 5 kg (2.2 to 11 lb) in body weight to 2 mg per kg (0.91 mg per lb) for growing-finishing hogs weighing 50 to 100 kg (110 to 220 lbs). Based upon sow farrowing performance and erythrocyte glutathione reductase (an indicator of riboflavin status), Frank et al. (1984) estimated the available riboflavin requirement for pregnancy to be about 6.5 mg daily. Using the same criteria, the suggested lactational requirement was about 16 mg daily (Frank et al., 1985; 1988). Frank et al. (1988) suggested that first-litter gilts have a higher requirement for riboflavin than the second-litter sow based on needs for both maternal growth and reproduction. With regard to erythrocyte glutathione reductase, Pettigrew et al. (1996) confirmed that its activity coefficient (EGRAC) is a sensitive biochemical indicator of riboflavin deficiency. After activity of erthrocyte glutathione reductase is measured with and without flavin adenine dinucleotide in the assay medium, the ratio of the activities is expressed as EGRAC. A high coefficient is indicative of a riboflavin deficiency. As expected, sows fed a low-riboflavin diet had higher EGRAC values than those consuming diets supplemented with higher levels of riboflavin. However, these authors determined that EGRAC values cannot be used to distinguish the period when sows are most likely to be deficient in riboflavin. In their experiment, there were only minor fluctuations in EGRAC values based on the stage of the reproductive cycle.
Early work (Warkany and Schraffenberger, 1944) indicated that riboflavin-deficient pregnant female rats produced offspring with congenital malformations, especially defects associated with skeletal development as embryonic tissue differentiation was abnormal. Subsequent studies (Miller et al., 1962) noted that female rats that were riboflavin deficient had embryos with depressed total riboflavin and FAD as well as abnormal skeletal development. Additionally, these authors observed that a 60% reduction in total riboflavin and FAD in the rat embryos resulted in sterility and embryonic and fetal death.
Murray et al. (1980) and Moffatt et al. (1980) reported that free riboflavin increases markedly in uterine secretions of pigs between days six and nine of both the estrus cycle and pregnancy; during this period, uterine flushing appears to have a distinct yellow color. These concentrations of riboflavin are much greater than those found in blood or colostrum of sows. The significance of these increased concentrations of riboflavin in uterine secretions of sows is unknown; however, they occur when blastocyst development is occurring, a stage critical to embryonic development and survival.
Bazer and Zavy (1988) reported that 100 mg supplemental riboflavin per day provided on days four to 10 after the onset of estrus resulted in higher litter size, embryonic survival and allantoic fluid volume at day 30 of gestation in gilts and increased rates of conception and more live pigs at birth, day 21 and day 42 of lactation in primiparous sows. This is in contrast to Luce et al. (1990) who reported no benefit at 100 mg per day riboflavin supplementation provided on days four to 10 postbreeding in sows consuming 1.8 kg (4.0 lbs) of feed supplemented with 6.6 mg riboflavin per kg (3.0 mg per lb). Pettigrew et al. (1996) fed 10 (control), 60, 110 or 160 mg of riboflavin per day from breeding through 21 days after breeding. Although the riboflavin supplementation tended to increase the percentage of sows farrowing (66.7%, 85.7%, 93.3% and 86.7% for 10, 60, 110 and 160 mg riboflavin per day, respectively), no effect on litter size was detected.
Increased dietary fat or protein increases requirements for riboflavin in rats and chickens. It was assumed that high urinary riboflavin excretion during periods of negative nitrogen balance for a number of species was a reflection of impaired riboflavin utilization or retention. However, Turkki and Holtzapple (1982) suggested, in studies with rats, that the effect of protein on riboflavin requirement is related to rate of growth and not to protein intake per se. Lutz and Stahly (1998) evaluated the dietary riboflavin requirement for protein versus fat accretion in pigs. These authors indicated that protein accretion, but not fat accretion, substantially increased the required dietary riboflavin levels. It was calculated that riboflavin needs for body protein accretion are six times higher than the requirement for body fat accretion. In addition, for high-lean, high-health status pigs, the requirement for riboflavin was found to be greater than the 1998 NRC estimate. Lutz and Stahly (1998) reported that as dietary riboflavin concentration increased (0, 3.7 or 7.4 mg per kg), efficiency of feed utilization and protein accretion increased linearly in both moderate- and high-lean gain strains of pigs.
Microbial synthesis of riboflavin has been shown to occur in the gastrointestinal tract of a number of animal species and thus affects requirements. However, utilization of this endogenously synthesized riboflavin varies from species to species. Within a single species, utilization depends on diet composition and incidence of coprophagy. 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.
Riboflavin is synthesized by green plants, yeast, fungi and some bacteria. Rapidly growing, green, leafy vegetables and forages, particularly alfalfa are a good source, with the vitamin richest in the leaves. Cereals and their byproducts have a rather low content, in contrast to their supply of thiamin. Oilseed meals are fair sources, whereas, grains and protein meals contain some riboflavin, but should not be relied on as the sole source of riboflavin. Riboflavin is more bioavailable from animal products than plant sources. Krider et al. (1949) determined that crystalline riboflavin produced a similar response to an equal amount of dried fermentation solubles as the riboflavin source when these forms were provided to weaning pigs fed in drylot.
Riboflavin is one of the more stable vitamins, but can be readily destroyed by ultraviolet (UV) rays or sunlight. Appreciable amounts may be lost upon exposure to light. Up to one half of the riboflavin content is lost in cooking eggs and pork chops in light and most of the vitamin in milk stored in clear glass bottles is also lost. (McDowell, 2000). Sun drying of fruits and vegetables is likely to lead to substantial losses of vitamin activity. 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, and whole wheat flour contains about two-thirds more riboflavin than white flour (McDowell; 2000).
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 powder, spray-dried powders and dry dilutions. Riboflavin 5’-phosphate sodium salt is available for applications requiring a water-soluble source of riboflavin. High-potency, USP or feed-grade powders are electrostatic, hydroscopic, and dusty, and thus they 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).
Only a few of the feedstuffs fed to swine contain enough riboflavin to meet their requirements (McDowell, 2000). Typical swine diets based largely on grains would be often borderline to deficient in riboflavin levels. A decreased rate of growth and lower feed efficiency are common signs of riboflavin deficiency in all species affected. Reduced feed intake was demonstrated in gilts given a lactation diet containing 1.3 mg per kg (0.6 mg per lb) riboflavin. These gilts consumed 30% less feed than those gilts that received diets with 2.3 to 5.3 mg per kg (1.0 to 2.4 mg per lb) riboflavin (Frank et al., 1988). Typical clinical signs often involve the eye, skin and nervous system. Signs of riboflavin deficiency in the young growing pig include anorexia, slow growth (Illus. 8-2), rough hair coat, dermatitis, alopecia, abnormal stiffness, unsteady gait, scours, ulcerative colitis, inflammation of anal mucosa, vomiting, cataracts, light sensitivity and eye lens opacities (Hughes, 1940; Lehrer and Wiese, 1952; Cunha, 1977; NRC, 1998). In severe riboflavin deficiency of pigs, researchers have observed increased blood neutrophil granulocytes, decreased immune response, discolored liver and kidney tissue, fatty liver, collapsed follicles, degenerating ova and degenerating myelin of the sciatic and brachial nerves (NRC, 1998). Lehrer and Wiese (1952) indicated that the external deficiency symptoms observed in their study could be reversed by supplementation of 1 to 1.5 mg of riboflavin per day for 16 days. However, the internal tissue changes were not corrected during this supplementation interval.
Illustration 8-2: Riboflavin Deficiency
Photo B shows a pig that received adequate riboflavin.
Courtesey of the R.W. Luecke, Michigan Agricultural Experimenation Station and J. Nutrition
Miller and Ellis (1951) and Miller et al. (1953) reported that riboflavin deficiency seriously impaired reproduction in pigs. Sows receiving 0.55 mg riboflavin per kg (0.25 mg per lb) of feed failed to reproduce or experienced death. Offspring from riboflavin-deficient sows are shown in (Illus. 8-3). Cunha (1977) summarized the clinical signs for gilts fed a riboflavin-deficient diet during reproduction and lactation as follows:
- erratic or, at times, complete loss of appetite;
- poor gains;
- parturition four to 16 days prematurely;
- one case of death of fetus in advanced stage with resorption in evidence;
- all pigs either were dead at birth or died within 48 hours thereafter;
- enlarged front legs in some pigs, due to gelatinous edema in the connective tissue and generalized edema in many others; and
- two hairless litters. The longer the period on riboflavin-deficient diets, the more severe the deficiency signs became. Christensen (1980) likewise reported resorption of fetuses and premature farrowing for riboflavin-deficient sows.
Illustration 8-3: Riboflavin Deficiency
Courtesy of T.J. Cunha and Washington State University
Riboflavin deficiency has led to anestrus (Esch et al., 1981). Deficiency of riboflavin in post-pubertal gilts has led to a cessation of ovarian cyclicity without overt signs of deficiency. Gilts fed a riboflavin-deficient diet had a progressively longer average time interval between consecutive estrus periods until becoming anestrus 63 days after the beginning of the study. Teratogenic effects have been observed, including skeletal abnormalities, shortened bones and fusions between ribs (Zintzen, 1975).
Riboflavin is one of the vitamins most likely to be deficient for swine. Riboflavin fortification levels should be adjusted, especially to offset the exclusion or reduced amounts of riboflavin-rich ingredients from computerized best-cost swine formulations. Swine diets based on grains and plant protein sources are generally deficient in riboflavin. Only a few feedstuffs fed to swine contain enough riboflavin to meet the requirements of growth and reproduction. As early as the 1950s (Briggs and Beeson, 1951), researchers realized that weanling pigs, when raised in drylot and fed an ideal combination of high-quality protein supplements in a grain ration fortified with essential minerals, vitamins (including B12) and an antibiotic, had improved growth and feed efficiency when three B-vitamins (riboflavin, pantothenic acid and niacin) were provided. Swine in confinement become more dependent on adequate vitamin (including riboflavin) and trace mineral supplementation, because best-cost feed formulation limit the number of vitamin-rich feed ingredients. The greater the variety of feed ingredients, the lower the chance of vitamin and trace element deficiencies for animals and humans alike (McDowell, 2000). Stahly et al (2007) evaluated the B-vitamin needs of strains of pigs with high and moderate lean tissue growth. The test vitamins were riboflavin and four other B-vitamins (folic acid, niacin, pantothenic acid and vitamin B12) and were fed an additional 0, 100, 200, 300% of the NRC requirements for weanling pigs. In both lean strains of pigs, rate and efficiency of growth were improved as dietary B-vitamins were increased. Pigs from a high lean strain consumed less feed, gained body weight faster and more efficiently than pigs of the moderate lean strain. Based on these data, the dietary needs for one or more of the five B-vitamins are greater than the current NRC (1998) estimates, particularly in pigs expressing a high rate of lean tissue growth.
Of the five B-vitamins typically supplemented to growing pigs fed grain and plant protein based diets, the two B-vitamins most likely to be deficient are riboflavin and vitamin B12. Riboflavin would likely be limiting, as Lutz and Stahly (1998) reported dietary riboflavin concentration increased feed efficiency and protein accretion linearly in high-lean gaining pigs.
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 as only the top of concentrate mixtures in automatic feeders would be affected. In dry form, riboflavin is extremely resistant to oxidation even when heated in air for long periods. While it has been shown that 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 (Hoffmann-La Roche, 1969). Riboflavin is quite stable in multivitamin premixes (Frye, 1978). One report demonstrated a 98% retention of riboflavin after six months in a vitamin premix; however retention was only 59% when the premix contained choline and trace minerals (Coelho, 1991).
Some producers have removed vitamin and trace mineral premixes from finishing diets three to six weeks prior to slaughter. This is often not advisable in relation to the risk of reduced performance. Shaw et al. (2002) removed the vitamin premix 28 days prior to slaughter, this withdrawl significantly reduced riboflavin in the longissimus dorsi muscle.
A large body of evidence has accumulated that treatment with riboflavin in excess of nutritional requirements has very little toxicity, either for experimental animals or for humans (Rivlin, 2007). There are no reports of riboflavin toxicity studies in swine. Most data from rats suggest that dietary levels between 10 and 20 times the requirement (possibly 100 times) can be tolerated safely (NRC, 1998). Campbell and Combs (1990) fed diets containing up to 0.7% supplemental riboflavin to growing and finishing pigs and observed no effect on performance. When massive amounts of riboflavin are administered orally, only a small fraction of the dose is absorbed, the remainder being 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 excessive amounts of riboflavin and its coenzyme derivatives appears to be limited.