DSM in Animal Nutrition & Health
Properties and Metabolism
Folacin is the generic descriptor not only for the original vitamin, folic acid, but also for related compounds that qualitatively show folic acid activity; the pure substance being designated pterylomonoglutamic acid. Its chemical structure contains three distinct parts, consisting of glutamic acid, a para-aminobenzoic acid (PABA) residue and a pteridine nucleus (Illus. 12-1). The PABA portion of the vitamin structure was once thought to be a vitamin. Research has shown that if the folic acid requirement of the organism is met, there is no need to add PABA to the diet.
Much of the naturally occurring folic acid in feedstuffs is conjugated with varying numbers of extra glutamic acid molecules. Polyglutamate forms of folic acid, usually containing three to seven glutamyl residues linked by peptide bonds, are the natural coenzymes being abundant in every tissue examined (Wagner, 1984). Synthesized folic acid, however, is the monoglutamate form. It has been concluded that there are more biologically active forms of folic acid than any other known vitamin. Folic acid is a yellowish-orange crystalline powder, tasteless and odorless, and insoluble in alcohol, ether and other organic solvents. It is slightly soluble in hot water in the acid form but quite soluble in the salt form. It is fairly stable to air and heat in neutral and alkaline solution, but unstable in acid solution. From 70% to 100% of folic acid activity is destroyed on autoclaving at pH 1 (O’Dell and Hogan, 1943). It is readily degraded by light and ultraviolet radiation. Cooking can considerably reduce the folic acid activity of food. Large losses in food can occur during food preparation such as heating, particularly under oxidative conditions (Gregory, 1989). Sulfonamides are analogs of the folic acid biosynthetic intermediate PABA and are widely used as antibacterial agents (Brown, 1962). By competing with PABA, sulfonamides prevent folic acid synthesis so that microorganisms cannot multiply, with the result that an important source of folic acid to the animal is reduced or eliminated. Polyglutamate forms of folic acid are digested via hydrolysis to pteroylmonoglutamate prior to transport across the intestinal mucosa. The enzyme responsible for the hydrolysis of pteroylpolyglutamate is a carboxypeptidase known as folate conjugase (Baugh and Krumdieck, 1971). Most likely, several conjugase enzymes are responsible for hydrolysis of the long-chain folate polyglutamates to the monoglutamates, which then enter the mucosal cell (Rosenberg and Newmann, 1974). Pteroylmonoglutamate is absorbed predominantly in the duodenum and jejunum, apparently by an active process involving sodium. Using an everted intestinal sac model, Tactacan et al. (2011) reported the presence of a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folic acid. Kesavan and Noronha (1983) suggested from rat results that luminal conjugase is a secretion of pancreatic origin and that the hydrolysis of polyglutamate forms of folic acid occur in the lumen rather than at the mucosal surface or within the mucosal cell.
Dietary folates, after hydrolysis and absorption from the intestine, are transported in plasma as monoglutamate derivatives, predominantly as 5-methyl-tetrahydrofolate. The monoglutamate derivatives then enter cells by specific transport systems. There the pteroylpolyglutamates, the major folic acid form in cells, are built up again stepwise by an enzyme, folate polyglutamate synthetase. Polyglutamates serve to keep folic acid within the cells since only the monoglutamate forms are transported across membranes and only monoglutamates are found in plasma and urine (Wagner, 1995). Tactacan et al. (2010b) reported that there is a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folic acid.
Specific folate-binding proteins (FBPs) that bind folic acid mono- and polyglutamates are known to exist in many tissues and body fluids, including liver, kidney, small intestinal brush border membranes, leukemic granulocytes, blood serum and milk (Tani and Iwai, 1984). Physiological roles of these FBPs are unknown, although they have been suggested to play a role in folic acid transport analogous to the intrinsic factor in the absorption of vitamin B12. Studies showed that about 79% to 88% of labeled folic acid is absorbed, and that absorption is rapid since serum concentrations usually peak about two hours after ingestion. The mean availability of folic acid in seven separate food items was found to be close to 50%, ranging from 37% to 72% (Babu and Skrikantia, 1976). Folic acid is widely distributed in tissues largely in the conjugated polyglutamate forms. Urinary excretion of folic acid represents a small fraction of total excretion. Fecal folic acid concentrations are quite high, often higher than intake, representing not only undigested folic acid but more importantly the considerable bacterial synthesis of the vitamin in the intestine. Bile contains high levels of folic acid due to enterohepatic circulation, with most biliary folic acid reabsorbed in the intestine.
Folic acid, in the form 5, 6, 7, 8-tetrahydrofolic acid (THF), is indispensable in the transfer of single-carbon units in various reactions, a role analogous to that of pantothenic acid in the transfer of two-carbon units (Bailey and Gregory, 2006). The one-carbon units can be formyl, methylene or methyl groups. Some biosynthetic relationships of one-carbon units are shown in Figure 12-1. The major in vivo pathway providing methyl groups involves transfer of a one-carbon unit from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate, which is subsequently reduced to 5-methyltetrahydrofolate. Methyltetrahydrofolate then supplies methyl groups to remethylate homocysteine in the activated methyl cycle, providing methionine for synthesis of the important methyl donor agent S-adenosylmethionine (Krumdieck, 1990; Jacob et al., 1994). Some biosynthetic relationships of one-carbon units are generated primarily during amino acid metabolism and are used in the metabolic interconversions of amino acids. They are also used in the biosynthesis of the purine and pyrimidine components of nucleic acids, which are needed for cell division. The important physiological function of THF consists of binding the single-carbon (C1) units to the vitamin molecule, thus transforming them to “active formic acid” or “active formaldehyde.” These are interconvertible by reduction or oxidation and transferable to appropriate acceptors. Folic acid polyglutamates work at least as well as or better than the corresponding monoglutamate forms in every enzyme system examined (Wagner, 1995). It is now accepted that the pteroylpolyglutamates are the acceptors and donors of one-carbon units in amino acid and nucleotide metabolism, while the monoglutamate is merely a transport form.
Figure 12-1: Folic Acid metabolism requiring single carbon units
Specific reactions involving single-carbon transfer by folic acid compounds are (a) purine and pyrimidine synthesis, (b) interconversion of serine and glycine, (c) glycine–carbon as a source of C1 units for many syntheses, (d) histidine degradation and (e) synthesis of methyl groups for such compounds as methionine, choline and thymine (a pyrimidine base). In rats an adequate supply of folic acid and related methyl donors can benefit fetal development directly by improving lipid metabolism in fetal as well as maternal tissues (McNeil et al.,2009).Purine bases (adenine and guanine), as well as thymine, are constituents of nucleic acids, and with a folic acid deficiency, there is a reduction in the biosynthesis of nucleic acids essential for cell formation and function. Hence, deficiency of the vitamin leads to impaired cell division and alterations of protein synthesis. These effects are most noticeable in rapidly growing tissues such as red blood cells, leukocytes, intestinal mucosa, embryos and fetuses. In absence of adequate nucleoproteins, normal maturation of primordial red blood cells does not take place and hematopoiesis is inhibited at the megaloblast stage. As a result of this megaloblastic arrest for normal red blood cell maturation in bone marrow, a characteristic macrocytic anemia develops. White blood cell formation is also affected, resulting in thrombopenia, leukopenia and multi-lobed neutrophils. Vitamin B12 is necessary in reduction of one-carbon compounds of the oxidation stage of formate and formaldehyde, and in this way it participates with folic acid in biosynthesis of labile methyl groups (Savage and Lindenbaum, 1995). Folic acid is also essentially involved in all these reactions of labile methyl groups. The metabolism of labile methyl groups plays an important role in the biosynthesis of methionine from homocysteine and of choline from ethanolamine. Folic acid has a sparing effect on requirements of choline. The critical role of both folic acid and vitamin B12in synthesis of choline is discussed in the choline chapter. Folic acid is needed to maintain the immune system; the blastogenic response of T lymphocytes to certain mitogens is decreased in folic acid-deficient humans and animals, and the thymus is preferentially altered (Dhur et al., 1991). The effects of folic acid deficiency upon humoral immunity have been more thoroughly investigated in animals than in humans, and the antibody responses to several antigens have been shown to decrease. As de novo synthesis of methyl groups requires the participation of folic acid coenzymes, the effect of folic acid deficiency on pancreatic exocrine function was examined in rats (Balaghi and Wagner, 1992; Balaghiet al., 1993). Pancreatic secretion was significantly reduced in the deficient group compared with the pair-fed control groups after five weeks.
Various animal species differ markedly in their requirements for folic acid. Folic acid requirements for various poultry species range from 0.25 to 1 mg per kg (0.11 to 0.45 mg per lb) of diet (NRC, 1994). Whitehead et al. (1995, 1997) noted that growth and feed conversion efficiencies of broilers were optimized with supplementary folic acid at 1.5 mg per kg (0.68 mg per lb) to broilers on most starter diets. They estimated the total folic acid requirement for broilers to be 1.7 to 2 mg per kg (0.77 to 0.91 mg per lb) and suggested that minimum folic acid supplements for pelleted practical diets are 2.5 to 3 mg per kg (1.1 to 1.4 mg per lb). For turkey breeder hens, the NRC (1994) folic acid requirement is 1 mg per kg (0.45 mg per lb). Robel (1993b) provided 2.64 mg folic acid per kg (1.2 mg per lb), and during the production period of 6 to 10 weeks, the higher level of supplemental folic acid increased poult weight 3.28% and egg weight 2.93%. In weeks 11 to 16 of the production period, the increase was 8.9% and 5.37%, respectively. Ryu et al.(1995) suggested that the folic acid requirement of chicks has not been well established for three reasons: (1) until recently, methods to determine the total amount of free and bound folates in feed ingredients were not established (De Souza and Eitenmiller, 1990); (2) it was once believed that chicks fed diets based on soybean meal did not require supplemental folic acid (Saxena et al., 1954); (3) many nutritional factors alter the folic acid requirement of chicks. The nutritional factors that alter the chick’s response to folic acid supplementation include dietary protein level and the amounts of glycine, serine, vitamin B12and choline.
Folic acid requirements for monogastric species depend on degree of intestinal folic synthesis and utilization by the animal (Rong et al., 1991). Animals that practice coprophagy have a lower dietary need for folic acid, as feces are a rich source of the vitamin (Abad and Gregory, 1987). Many species apparently do not require dietary folic acid because they utilize microbial intestinal synthesis of the vitamin (Rong et al., 1991). However, poultry develop deficiencies on diets low in folic acid (Pesti et al., 1991). Self-synthesis of folic acid depends on dietary composition. For poultry, some research has indicated higher folic acid requirements for very high protein diets or when sucrose was the only source of carbohydrates (Scott et al., 1982). Additionally, in growing male chicks, the severity of retrovirus infection by lesion sources was consistently higher when chick diets were less than two times the NRC requirement (Cook et al., 1983). Pesti and Rowland (1989) reported a growth response with 2 mg per kg (0.9 mg per lb) folic acid supplementation in broilers and noted that this response is often obscured if methionine or choline is supplemented. Ryu et al. (1995) concluded that chicks fed practical ingredient-based diets require 1.3 mg folic acid per kg (0.59 mg per lb) of diet with low levels of choline but 1.2 mg folic acid (0.54 mg per lb) when choline is offered near the NRC-recommended level of 1,300 mg per kg (591 mg per lb) of choline.
Pesti et al. (1991) reported that chicks fed standard ingredient-based diets require 1.45 mg folic acid per kg (0.66 mg per lb) of diet with low levels of choline but need only 1.25 mg folic acid per kg (0.57 mg per lb) when choline is offered near the NRC recommendation. With 0.85% methionine plus cystine, total dietary folic acid requirements for maximum growth are estimated to be 1.8 mg per kg (0.82 mg per lb) and with 0.87% methionine plus cystine, the folic acid requirements were 1.47 mg per kg (0.74 mg per lb).
The levels of antibacterials added to the feed affect microbial synthesis of folic acid. Sulfa drugs, which are sometimes added to poultry diets, are folic acid antagonists. In the chicken, sulfa drugs have been shown to increase the requirements (Scott et al., 1982). Moldy feeds have also been shown to contain antagonists (i.e., mycotoxins) that inhibit microbial intestinal synthesis in swine (Purser, 1981).
Folic acid requirements depend on the form in which it is fed and concentrations and interrelationships of other nutrients. Deficiencies of choline, vitamin B12, iron and vitamin C all affect folic acid needs. Although most folic acid in poultry feedstuffs is present in conjugated form, the young chick is expected to be fully capable of utilizing it.
Folic acid requirements are related to type and level of production. Growth rate, age and pregnancy influence folic acid requirements. The requirement decreases with age because diminished growth rate reduces the need for DNA synthesis. Increased catabolism of folic acid is a feature of pregnancy. Studies with both rats (McNulty et al., 1993) and humans (McPartlin et al., 1993) demonstrated enhanced folic acid catabolism; this was a feature of pregnancy per se and not simply due to increased weight. In poultry, the folic acid requirement for egg hatchability is higher than that for production (NRC, 1994). Taylor (1947) reported that 0.12 mg folic acid per kg (0.05 mg per lb) diet was satisfactory for egg production but that higher levels were required for good hatchability.
Folic acid is widely distributed in nature, almost exclusively as THF acid derivatives; the stable ones have a methyl or formyl group in the 5-position and generally possess three or more glutamic acid residues in glutamyl linkages. Only limited amounts of free folic acid occur in natural products, and most feed sources contain predominantly polyglutamyl folic acid. Soybeans, other beans, nuts, some animals products and citrus fruits are good sources. Cereal grains, milk and eggs are generally poor sources of the vitamin. Folic acid is abundant in green leafy materials and organ meats. The abundance of folic acid in green forages is shown by the higher concentrations of folic acid in milk from grazing herds than from herds fed dry hay (Dong and Oace, 1975).
For ruminants, the three naturally occurring sources of folic acid are from ruminal synthesis, intestinal synthesis and dietary sources, and the greatest contribution is from ruminal microbial synthesis. Concentrations of folic acid in ruminal dry matter ranges from 0.3 to 0.6 mg per kg (0.14 to 0.27 mg per lb) (Lardinois et al., 1990). Corresponding values for strained rumen fluid ranges from 0.08 to 0.19 mg per kg (0.036 to 0.086 mg per lb) (Hayes et al., 1966).
Folic acid bioavailability in a variety of foods was found to generally exceed 70% (Clifford et al., 1990). Bioavailability of monoglutamate folic acid is substantially greater than polyglutamyl forms (Gregory et al., 1991a; Clifford et al., 1990). The availability of folic acid may range from 30% to 80% in the monoglutamate form. Bioavailability of orally administered 5-methyl folic and 5-formyl folic acid were found to be equal with folic acid for rats (Bhandari and Gregory, 1992).
Supplementation with both folic acid and 5-methyltetrahydrofolate had equivalent effects in enhancing egg folic acid concentrations and improving folic acid status in laying hens (Tactacan et al., 2010a).
A considerable loss of folic acid (50% to 90%) occurs during cooking or processing of foods. Folic acid is sensitive to light and heating, particularly in acid solution. Under aerobic conditions, destruction of most folic acid forms is significant with heating.
Crystalline folic acid, produced by chemical synthesis, is available for feed, foods and pharmaceuticals. Several lines of evidence indicate higher bioavailability of added folic acid than naturally occurring folates in many foods (Gregory, 2001). Although folacin is only sparingly soluble in water, the sodium salt is quite soluble and is used in injections as well as food supplements (McGinnis, 1986; Tremblay et al., 1986). Spray-dried folic acid and dilutions of USP crystalline folic acid are the most widely used product forms in animal feeds. Synthetic folacin supplements are highly available, natural forms are approximately 50% less available.
Poultry are more susceptible to lack of folic acid than other farm livestock, as a deficiency can readily be produced by feeding a folic acid-deficient diet. Folic acid deficiency, as indicated by retarded growth and feed efficiency, could be produced in 15-day-old chicks fed corn-soybean meal diets (Pesti et al., 1991). In folic acid deficiency, megaloblastic arrest of erythrocyte formation in bone marrow causes severe macrocytic anemia as one of the first signs. Folic acid deficiency in chicks is also characterized by poor growth, very poor feathering, an anemic appearance, and perosis (Illus. 12-3 and 12-4). The chicks become lethargic, and feed intake declines. As anemia develops, the comb becomes waxy white and the mucous membrane of the mouth becomes pale (Siddons, 1978). Turkey poults fed a folic acid-deficient diet show reduced growth rate and increased mortality (Illus. 12-5). The birds develop a spastic type of cervical paralysis in which the neck is stiff and extended but with only a moderate degree of anemia. Poults with cervical paralysis die within 2 days after the onset of these signs unless folic acid is administered immediately (Scott et al., 1982). Erythrocytes of deficient birds tend to be large in diameter, and their nuclei are less dense than those of birds receiving supplementary folic acid (Schweigert et al., 1948).
Illustration 12-3: Folic Acid Deficiency in the Chicken
Reduced growth, poor feathering.
Courtesy of M.L. Sunde, University of Wisconsin
Illustration 12-4: Folic Acid Deficiency, Perosis
Note the weakened condition of the legs and the way this folic acid-deficient bird holds its left wing. This five-weel-old chick is suffering from cervical paralysis. A deficient bird will shake the end of the wing and quiver at times.
Courtesy of M.L. Sunde, University of Wisconsin
Illustration 12-5: Folic Acid Deficiency
This poult was hatched from a hen fed a diet low in folic acid.
Courtesy of M.L. Sunde, University of Wisconsin
Folic acid deficiency also results in poor feather development for chicks and turkeys, with the shafts weak and brittle. Folic acid, lysine, and iron are required for feather pigmentation, as depigmentation occurs in colored feathers during a deficiency of the vitamin. It appears that egg production is less affected by folic acid deficiency than the development of the chick or poult. Egg and poult weights were significantly increased when turkey hens received higher dietary folic acid and when eggs were injected with folic acid (Robel, 1993b). Inadequate folic acid provided to the hen impairs the oviduct’s response to estrogen and ability to form albumen (NRC, 1994). An inadequate intake of folic acid by breeding hens results in poor hatchability and a marked increase in embryonic mortality (Illus. 12-6), which occurs during the last days of incubation. A deformed beak and bending of the tibiotarsus are signs of the embryonic deficiency. Chicks that successfully emerge are stunted and have feathers that are poorly developed and abnormally pigmented (NRC, 1994).
Illustration 12-6: Folic Acid Deficiency, Embryonic Mortality
This abnormal embryo was from an egg laid by a hen fed a diet low in folic acid.
Courtesy of M.L. Sunde, University of Wisconsin
Folic acid deficiency has sometimes been associated with perosis, or slipped tendon. Pollard and Creek (1964) demonstrated histologically that the lesions of folic acid-deficient bones and cartilage are different from those produced by choline or manganese deficiencies. Abnormal structure of the hyaline cartilage is found in folic acid-deficient chicks, and ossification is retarded. These disorders are not found in chicks deficient in choline or manganese, although bone deformities and slipped tendons are found in both types of disorders. However, Bechtel (1964) claimed that choline is effective in preventing perosis only when sufficient folic acid is present in the diet. Dietary choline content has been shown to affect the chicks’ requirement for folic acid. When the diet contained adequate choline, the folic acid requirement was 0.47 mg per kg (0.21 mg per lb) of diet, but this increased to 0.96 mg per kg (0.44 mg per lb) diet when the diet was choline deficient (Young et al., 1955). Increasing the protein content of the diet has also been shown to increase the incidence and severity of perosis in chicks receiving low levels of dietary folic acid. It is suggested that this increased requirement for folic acid in high-protein diets for poultry is a consequence of greater demand for folic acid in uric acid formation (Creek and Vasaitis, 1963). Folic acid appears to be necessary for cell mitosis. In the absence of folic acid, oviduct growth is not increased in estrogen-treated chicks. The production of water-soluble proteins (particularly the albumen fraction) in the hormone-stimulated oviduct is also greatly reduced, and there is an alteration in the amino acid composition of these proteins. The percentages of arginine, leucine, serine and tryptophan are decreased and those of glycine and methionine, increased (Siddons, 1978).
Folic acid needs for livestock are often met by good practical diets, and for most species, substantial quantities of folic acid are provided through microbial synthesis. Nevertheless, field observations have been made on folic acid insufficient diets. Green forage is an excellent source of folic acid. Supplementation of folic acid is most needed when animals are in confinement without access to green grazing or preserved green forages. The successful treatment of field cases of folic acid deficiency with supplemental folic acid has demonstrated that commercial feeds do not always supply adequate quantities of the vitamin to poultry (Pesti et al., 1991). Folic acid may be of little benefit when poultry and swine receive only low levels of sulfa drugs, high levels of methionine and choline, and consume grains relatively free of toxin-producing molds. However, since a large percentage of the U.S. corn crop contains some mold contamination, folic acid supplementation should have a positive effect in many commercial poultry and hog operations as well as in other livestock enterprises (Purser, 1981). This is likely to be an even more important consideration in developing tropical countries, where climate conditions favor mold growth. Individual responses to folic acid supplementation to counteract mold effect vary with the class of livestock being fed, species of mold and levels of toxin encountered (Bhavanishankaret al., 1986). Gadient (1986) considers folic acid to be very sensitive to heat and light, slightly sensitive to moisture, and insensitive to oxygen. Folic acid can be lost during storage of premixes, particularly at elevated temperatures (Frye, 1978). After three months of room-temperature storage, 43% of the original folic acid activity was lost. Verbeeck (1975) found folic acid to be stable in premixes without minerals, but there may be as much as 50% loss in a premix with minerals, kept at room temperature for three months. Adams (1982) reported only 38% retention of folic acid activity in a premix without minerals after three weeks at 45°C (113°F). However, he reported 57% retention of activity after three months at room temperature. One suggestion is to almost double the amount of folic acid in a premix at the time of manufacture to ensure that poultry receive the desired amount from that premix, since three to four months is not an unreasonable amount of time from premix manufacture to diet mixing and feeding. Slinger et al. (1979) reported processing and storage losses of folic acid in fish feeds of 5% to 10% for steam-pelleted crumbles and 3% to 7% for extruded crumbles, depending on dietary level. Scott (1966) indicated that an adjustment of 10% to 20% in the folic acid level in poultry feed may be necessary because of pelleting losses.
Research confirms the ability to elevate folic acid in eggs (Table 12-1). Enrichment of eggs with folate is possible when dietary folic acid levels are increased (Herbert et al. 2005; Dickson et al., 2006; Roth-Maier and Böhmer, 2007; Hoey et al., 2009; Ward, 2009). The NRC (1994) requirement for folic acid is 0.25 mg per kg (0.11 mg per lb) of diet. Folic acid in eggs can be increased approximately three-fold by increasing dietary folic acid to 2 to 4 mg per kg (0.9 to 1.8 mg per lb). Roth-Maier and Böhmer (2007) report that one fortified egg can provide up to 76 µg of folic acid. These researchers fed the fortified eggs to pigs and determined the availability of folic acid to be 68%. Coupled with the high bioavailability of yolk folate, the common table egg can be crafted into a good source of natural folate. The small investment to produce folic acid enriched eggs presents an opportunity for egg producers to further elevate the nutritional benefits of egg consumption (Hoey et al., 2009; Ward, 2009). Folic acid in broiler meat can also be enhanced through supplementation of broiler diets with the vitamin (McCann et al., 2004). Providing additional supplemental folic acid, choline and vitamin B12 to laying hen diets positively affected yolk weight and egg phospholipid composition (Krishman and Scheideler, 2010).
Crystalline folic acid produced by chemical synthesis is available for feeds, foods and pharmaceuticals. Although folic acid is only sparingly soluble in water, the sodium salt is quite soluble and is used in injections as well as feed supplements (McGinnis, 1986; Tremblay et al., 1986). Oral supplementation appears to be desirable to maintain maternal stores and to keep pace with the increased folic acid turnover that is seen in rapidly growing tissue. Synthetic folic acid supplements are easy to administer and highly available; natural forms are approximately 50% less available than synthetic forms.
Folic acid generally has been regarded as a nontoxic vitamin (NRC, 1987). Birds are very tolerant of high levels of folic acid, with up to 5,000 times normal intake being needed to induce toxicity. Renal hypertrophy has been described under such conditions (Leeson and Summers, 2001).