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 most 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% 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. Kesavan and Noronha (1983) suggest from rat results that luminal conjugase is a secretion of pancreatic origin and that the hydrolysis of polyglutamate forms of folic acid occurs 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 intestine 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 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 B12 in 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 (Balachi and Wagner, 1992; Balaghi et al., 1993). Pancreatic secretion was significantly reduced in the deficient group compared with the pair-fed control groups after five weeks.
Folic acid requirements for monogastric species would be dependent on degree of intestinal folic acid synthesis and utilization by the animal. The levels of antibacterials added to the feed will affect microbial synthesis of folic acid. Sulfa drugs are folic acid antagonists. In the chicken, sulfa drugs have been shown to increase the requirement (Scott et al., 1982) for this vitamin. Moldy feeds have also been shown to contain antagonists (e.g., mycotoxins) that inhibit microbial intestinal synthesis of folic acid in swine (Purser, 1981).In dogs and cats, sulfonamides have been shown to severely reduce intestinal microbial synthesis of folic acid. Deficiency signs can occur in dogs after extended oral administration of sulfonamides (NRC, 2006). Carvalho da Silva et al. (1955) were not able to induce folic acid deficiency in two- to three-month-old kittens with a purified diet containing no added folic acid or vitamin B12 unless 0.6% to 2.0% of sulfaguanidine or sulfathalidine was included. Dogs and cats being treated with sulfonamides would have a need for dietary sources of folic acid.
Self-synthesis of folic acid is dependent 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). Keagy and Oace (1984) reported that dietary fiber had an effect on folic acid utilization. Xylan, wheat bran and beans stimulated folic acid synthesis in the rat, reflected as higher fecal and liver folic acid. Animals that practice coprophagy would have a lower dietary need for folic acid, as feces are a rich source of the vitamin (Abad and Gregory, 1987). Dogs would therefore receive part of their folic acid requirement via coprophagy, while cats would not have this source of the vitamin.
Folic acid requirements are dependent 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 have an effect on folic acid needs. Although most folic acid in feedstuffs for pets is present in the conjugated form, apparently dogs and cats are capable of utilizing it. Folic acid requirements are related to type and level of production. The more rapid the growth or production rates, the greater the need for folic acid due to its role in nucleic acid synthesis.
Folic acid requirements are related to type and level of production. Growth rate, age and pregnancy influence folic acid requirements; however, this information is not available for dogs and cats. 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 an enhanced folic acid catabolism that was a feature of pregnancy per se and not simply due to increased weight. All dietary forms of folic acid are available to the dog since it has the conjugase enzyme to break the polyglutamates down to the monoglutamate form that is required for absorption (Baugh et al., 1975).
Some investigators have assumed that dogs do not need preformed folic acid in their diets. However, folic acid deficiency occurred in laboratory dogs fed a semi-purified diet (Afonsky, 1954). The dogs had weight loss and a decline in hemoglobin concentration. Subcutaneous injections of 15 µg of folic acid per kg (6.82 µg per lb) of body weight restored hemoglobin concentration.The folic acid requirement of dogs fed an adequate diet of nonpurified ingredients that does not contain bacteriostatic agents is probably met by microbial synthesis in the intestine (NRC, 2006). While intestinal synthesis in dogs has been demonstrated by Bernstein et al. (1972; 1975), the extent to which it contributes to meeting body needs has not been quantified. Diets inadequate in choline, methionine, and vitamin B12 may induce deficiencies because of their interaction with folic acid. According to the NRC (2006) the folic acid requirements for all classes of dogs is 270 µg per kg (123 µg per lb) of diet. The Association of American Feed Control Officials (AAFCO, 2007) recommends 180 µg folic acid per kg (82 µg per lb) of feed for all classes of dogs.
Carvalho da Silva et al. (1955) reported that a growth response was obtained in sulfa-treated, deficient kittens with either two oral doses of 0.8 mg of folic acid administered 24 hours apart or with one oral dose of 1.0 mg folic acid. The latter treatment produced a response that persisted for about a month. Data on folic acid requirements of cats are extremely limited; the NRC (2006) recommend 750 µg per kg (341 µg per lb) of diet for all classes of cats, the AAFCO (2007) recommend 800 µg per kg (364 µg per lb) of diet for all cats.
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 nature products, and most feed sources contain predominantly polyglutamyl folic acid. Soybeans, other beans, nuts, some animal 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., 1994). Corresponding values for strained rumen fluid ranged from 0.08 to 0.19 mg per kg (0.036 to 0.086 mg per lb) (Hayeset al., 1966).
Folic acid bioavailability of 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 food folic acid may range from 30% to 80% that of the monoglutamate form, being generally less well utilized from plant-derived foods than from animal products. Bioavailability of orally administered 5-methyl folic acid and 5-formyl folic acid compared to folic acid were found to be equal for rats (Bhandari and Gregory, 1992). 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, with reduced folic acid more stable in foods due to relatively anaerobic conditions and because folic acid is protected from light (Brody, 1991).
Crystalline folic acid, produced by chemical synthesis, is available for feed, food 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 feed 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.
Folic acid deficiency has been produced experimentally in many animal species, with a macrocytic anemia (megaloblastic anemia) and leucopenia (a reduced number of white blood cells) consistent findings. Tissues that have a rapid rate of cell growth or tissue regeneration, such as the epithelial lining of the gastrointestinal tract, epidermis and bone marrow, are principally affected (Hoffbrand, 1978). When megaloblastic anemia is observed in dogs and cats, it is more commonly associated with folic acid deficiency caused by intestinal malabsorption syndromes, a defective diet, folic acid antagonists or an increase in folic acid requirement due to blood loss or hemolysis.For some animals, such as the chick, guinea pig, monkey and pig, the presence of adequate amounts of folic acid in the diet is essential. Deficiency signs can readily be induced by feeding a diet deficient in the vitamin. In other animals, including the dog, cat and rat, folic acid produced by intestinal microflora is usually adequate to meet requirements. Consequently, deficiency signs do not develop unless an intestinal antiseptic is also included in the diet to depress bacterial growth. Folic acid deficiency has been described in dogs and cats but usually only when semi-purified diets were fed in the presence of antibiotics. It is likely that most of the daily requirement for folic acid is met by bacterial synthesis in the intestine.
Assessment of nutritional status of folic acid can involve dietary evaluation, clinical signs, response to supplementation, and laboratory analysis. In folic acid deficiency, formiminoglutamic acid (FIGLU), formed as an intermediate in the degradation of histidine, can no longer be transformed completely into glutamate and formiminotetrahydrofolic acid, and is therefore excreted in urine. This excretion is suitable as a biochemical criterion for diagnosis of folic acid deficiency, appearing at an early stage of deficiency. Because the liver contains a high percentage of stored folic acid, concentration in this organ would serve as a folic acid status indicator. On low-folic acid diets, liver concentrations are depleted in a few months. Clinical signs of folic acid deficiency are extremely variable and are less precise than laboratory analysis to confirm a deficiency. A protocol of folic acid depletion-repletion of rats, followed by growth, liver, serum, and erythrocyte folic acid measurements, has been successful in evaluating the bioavailability of folic acid food sources (Clifford et al., 1990; 1991).
Amyes et al. (1975) determined the folic acid concentrations of cat erythrocytes, plasma and liver and found that these values declined from birth to 32 days. Thenen and Rasmussen (1978) reported a marked depletion of plasma and liver folic acid as weanling kittens were fed a folic acid-deficient diet.
Afonsky (1954) reported weight loss and a progressive decline in hemoglobin concentration in a dog given a semi-purified diet. Folic acid deficiency results in erratic appetite, decreased gain, watery exudate from eyes, glossitis, leukopenia, hypochromic anemia, and decreased antibody response to infectious canine hepatitis and canine distemper virus (NRC, 2006). A positive response was obtained with subcutaneous injections of folic acid.Serum folic acid was increased in various breeds of dogs that received dietary folic acid (Davenport et al., 1994). A fox terrier bitch with chronic ehrlichiosis exhibited a regenerative anemia and thrombopenia associated with marrow hypercellularity. Dysterythropoiesis and dysthrombopoiesis were attributed to a folic acid and vitamin B12 deficiency that was thought to have been the result of medullary hyperconsumption during the subclinical phase of the disease (Caprelle et al., 1994).
In an observational study without controls, Elwood and Colquhoun (1997) reported a decrease in the incidence of cleft palates in Boston terrier puppies from 17.6% to 4.2% following daily supplementation of the bitches with 5 mg folic acid from mating until the pups were 3 weeks of age.
Sheffy (1964) illustrated the importance of folic acid in the immune response of folic acid-deficient puppies inoculated with distemper and hepatitis antigens. Half the dogs were also given 27.5 µg folic acid per kg (12.5 µg per lb) of body weight. Depleted dogs had delayed antibody production responses against both distemper and infectious hepatitis antigens. Antibodies were detected in depleted dogs supplemented with folic acid eight days after challenge with antigen, whereas depleted dogs without folic acid did not show antibodies until 17 days.
Folic acid deficiency has been produced in cats by adding sulfonamides to semipurified diets. Folic acid deficiency was characterized by weight loss, anemia (macrocytic tendencies) and leukopenia. Blood-clotting time was increased and plasma iron concentrations were elevated (Carvalho da Silva et al., 1955). Schalm (1974) has described a megaloblastic anemia in an adult cat that responded to vitamin B12 and folic acid administration. In a survey of 103 cats suffering predominantly from diseases of the alimentary tract (including the liver and pancreas), 33.8% of cats had low folate blood levels. Low blood folate and vitamin B12 concentrations were significantly related to a poor body condition score (Reed et al., 2007).
Typical feeding ingredients for pet foods should contain adequate folic acid. As an example, common, commercial cat foods usually contain from 2 to 25 mg/kg folic acid, with high amounts from the Torula yeast, fish and organ meat components of these diets. Also, for most species, including dogs and cats, substantial quantities of folic acid are provided through microbial synthesis. Thus, the diets normally fed dogs and cats meet the requirements for folic acid. However, when dogs and cats are treated with antibiotics that destroy the intestinal microbes which synthesize folic acid or other disease states exist, concern should be given to having adequate amounts of folic acid in the diet and also adequate amounts of other dietary components such as choline, methionine, and vitamin B12, because of their interactions with this vitamin (Ralston Purina, 1987). Processing of dog and cat foods, which involves high temperatures for prolonged periods in the presence of atmospheric oxygen, may destroy some of the folic acid present. In addition, more folic acid may be lost in the processing fluid if this is not retained in the final product. The presence of ascorbic acid will inhibit the oxidative destruction of folic acid, but this vitamin is not usually present in significant quantities in the feedstuffs used for commercial dog and cat foods.
Gadient (1986) considers folic acid 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 that folic acid is stable in premixes without trace minerals but that there may be as much as a 50% loss in a premix with trace minerals stored at room temperature for three months. Adams (1982) reported only 38% retention of folic acid activity in a premix without trace minerals after three weeks of storage at 45°C. However, 57% retention of activity was retained after three months of storage at room temperature. After six months of storage in a vitamin premix, 97% of folic acid was recovered, but only 43% was recovered in a vitamin premix combined with choline and trace minerals. Commercial pet foods are supplemented with folate to overcome the effects of processing and storage (Hand et al., 2010).
Folic acid has generally been considered a nontoxic vitamin (NRC, 1987). Birds are very tolerant of high levels of folic acid, with up to 5,000 x normal intake being needed to induce toxicity. Renal hypertrophy has been described under such conditions (Leeson and Summers, 2001). Except in poultry, no adverse responses to dietary folic acid have been documented in any species.
Acute intravenous toxicity is very low, with the LD50 in mg per kg body weight being: mice, 600; rat, 500; rabbit, 410; guinea pig, 120. In rats, most of the deaths occurred within 30 minutes of injection (Anonymous, 1961). No information is available on folic acid tolerance for dogs and cats.
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