DSM in Animal Nutrition & Health
Properties and Metabolism
Folic acid (pteroylmonoglutamic acid) consists of one pteridine nucleus, one molecule of para-amino benzoic acid and one glutamic acid moiety (Illus. 12-1). Para-amino benzoic acid (PABA) is required by some microorganisms for the synthesis of folic acid. The vitamin is referred to by the terms folic acid, folacin and folate.
Naturally occurring folic acid in feedstuffs is conjugated with varying numbers of glutamic acid molecules. Polyglutamate forms of folic acid, usually containing three to seven glutamyl residues linked by peptide bonds, are the most abundant coenzyme form in living tissues (Wagner, 1984). Synthetic folic acid is the monoglutamate form. Folic acid has the largest number of biologically active forms of any vitamin. Over 100 active forms have been identified, varying according to the reduction state of the pteridine nucleus (di- or tetrahydrofolate), the presence or absence of methyl or other single-carbon groups on the N5 and N10 atoms, and the number of glutamic acid residues present (Girard, 1998a). Folic acid is a yellow-orange crystalline powder, tasteless and odorless, and insoluble in alcohol, ether and other organic solvents. The acid form is slightly soluble in hot water, but folate salts are quite soluble. Folic acid is fairly stable to oxygen 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). Light and ultraviolet radiation readily degrade folic acid. Cooking reduces folic acid activity. Polyglutamate forms of folic acid are hydrolyzed to pteroylmonoglutamate in the small intestine prior to absorption. 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 polyglutumates 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 and also in a passive process. In fact, the relative importance of passive absorption changes according to the folic acid supply, increasing with the amounts of folic acid available (Soliset al., 2008). Kesavan and Noronha (1983) suggest, based on data from rats, that luminal conjugase is a pancreatic secretion and that the hydrolysis of polyglutamate forms of folic acid occur in the lumen rather than at the mucosal surface or within the mucosal cells.
After hydrolysis and absorption from the intestine, folates are transported in plasma as monoglutamate derivatives, predominantly as 5-methyl-tetrahydrofolate. These monoglutamate derivatives are absorbed from circulation by specific tissue transport systems. Then the pteroylpolyglutamates, the major folic acid form in cells, are synthesized in stepwise fashion by the enzyme folate polyglutamate synthetase.
Specific folate-binding proteins (FBP) have been identified in the liver, kidney, small intestinal brush border membranes, leukocytes, reticulocytes, blood serum and milk (Tani and Iwai, 1984). The physiological roles of these FBPs are unknown, although it has been suggested they play a role in folic acid transport analogous to the intrinsic factor in the absorption of vitamin B12.
Folate is both actively and passively transported across the placenta during late gestation, and the vitamin is stored primarily in the liver of the developing fetus (Narkewicz et al., 2002). Folate status of the newborn kid is relatively low. Content of folic acid is greatest in the colostrum and declines precipitously with the onset of copious milk production (Girard et al., 1996). Starting at parturition, folate concentrations in milk decrease until 4 weeks after parturition when folate concentrations reach a plateau which is stable until the end of lactation (Girard et al., 1995). Folate balance of the kid may be more precarious than that of lambs because concentration of folic acid in goat’s milk is at least five times less than that of ewe’s milk.
Studies have shown that 79% to 88% of labeled folic acid is absorbed, and that absorption is rapid, with serum concentrations usually peaking about two hours after ingestion. The mean availability of folic acid in seven separate foods was close to 50%, varying 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. However, fecal folic acid concentrations are quite high, often higher than intake, representing not only undigested folic acid, but more importantly, considerable bacterial synthesis in the large intestine. Bile contains high levels of folic acid due to enterohepatic circulation, with most biliary folic acid reabsorbed from the small intestine. This suggests that a physiologic mechanism exists to regulate folate re-uptake based on tissue requirements.
Folic acid, in the form 5, 6, 7, 8-tetrahydrofolic acid (THF), is essential for the transfer and interconversion of single-carbon units, including formyl, methyl and methylene groups (Bailey and Gregory, 1999, 2006). Some biosynthetic relationships of one-carbon units are shown in Figure 12-1. Single-carbon units are generated primarily during amino acid metabolism and are used in the metabolic interconversions of amino acids and the biosynthesis of the purine and pyrimidine components of nucleic acids. Therefore, folic acid requirement is closely linked to the rate of cell division.
Figure 12-1: Folic Acid Metabolism Requiring Single-Carbon Units
Adapted from Scott et al., (1982) and McDowell (1989)
The biochemical function of tetrahydrofolate is the binding and activation of single-carbon units, rendering them interconvertible by oxidation or reduction and transferable to appropriate acceptor molecules. The polyglutamic acid form of folic acid is required for nucleotide synthesis, while pteroylmonoglutamate is the transport form of folic acid (Brody, 1991; Bailey and Gregory, 2006). Numerous reactions involving single-carbon transfer require folic acid. These include:
- Purine and pyrimidine synthesis (adenine, guanine, thymine, cystosine and uracil);
- Interconversion of serine and glycine;
- Utilization of glycine as a carbon source for synthetic pathways (glycine cleavage system);
- Histidine degradation;
- Methionine synthetase, a vitamin B12 enzyme using 5-methyl-THF and homocysteine as substrates;
- Methionyl-tRNA transformylase, a required step in initiation of all protein synthesis.
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, and embryonic and fetal tissues. 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). Without adequate folate, the normal maturation of primordial red blood cells in bone marrow is arrested at the megaloblast stage. As a result, a characteristic macrocytic anemia develops. White blood cell formation is also affected, resulting in abnormalities such as thrombopenia, leukopenia and multilobed neutrophils.
Vitamin B12 is necessary for the synthesis of labile methyl groups and in this way interacts with folic acid, which is also essential for these reactions. 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 the choline requirement. Vitamin B6 is required for the serine and glycine metabolizing pathways and can therefore interact with folic acid status.
Folic acid is required for normal immune function. In rats, the immune system was severely inhibited with folic acid deficiency, due to disruption of leukocyte production (Kumar and Axelrod, 1978).
Various species differ markedly in their requirements for folic acid. Also folic acid requirements within species have been shown to differ between genotypes (Solis et al., 2008). Young ruminants without a fully functional rumen require folic acid supplementation of milk replacer. There was no indication of folic acid deficiency in calves fed a synthetic milk containing 52 µg of folic acid per kg (23.6 µg per lb) of liquid fed at 10% of body weight (Wiese et al., 1947). In lambs, 0.39 mg of folic acid per liter in milk replacer prevented deficiency (NRC, 1985). The NRC (1989) for dairy cattle recommends that milk replacer for calves should contain 0.5 mg of folic acid per kg (0.23 mg per lb). Weekly injections of 160 mg of folic acid given to dairy cows starting 45 days after breeding increased both the placental and colostral transfer of folic acid to calves, although calf performance was not affected (Girard et al., 1995). Supplemental folic acid fed at either 2 or 4 mg per kg (.9 or 1.8 mg per lb) body weight significantly increased serum folic acid concentration, but did not alter serum levels of either vitamin B12 or vitamin B6 (Girard and Matte, 1999). The primary source of folic acid for ruminants is rumen microbial synthesis, although intestinal microbial synthesis also occurs. Certain antibiotics inhibit microbial synthesis of folic acid in both the rumen and intestine. Sulfonamides are folic acid antagonists. Mycotoxins can also inhibit microbial intestinal synthesis of folic acid in swine (Purser, 1981). Rumen folic acid synthesis is greater with high-concentrate rations than with high-forage rations (Girard et al., 1994). Fluctuations in the concentration of serum folates observed during the first months of life of young ruminants may be an indication that synthesis of folates by ruminal microflora is not sufficient to meet requirements during weaning (Girard et al., 1989a). Girard et al. (1989a) observed that the serum folic acid concentration in calves at two weeks of age is half that of four-month-old heifers. An age effect on development of the rumen was observed, in which parenteral folic acid markedly increased serum folic acid in two-week-old heifers, while in four-month-old heifers the increase was less marked. Similarly, Dumoulin et al. (1991) observed that supplemental folic acid increased calf growth rate during the first five weeks after weaning but not thereafter, suggesting that folic acid status was marginal during the postweaning phase. In the same experiment, there was no effect of supplemental folic acid on mammary gland development of heifers (Petitclerc et al., 1999).
For adult dairy cows, Girard et al. (1989b) reported that serum folates can be increased by an intramuscular injection of folic acid, but the effect on serum and milk folates is reduced in lactating and pregnant cows. Serum folates are higher in non-gravid cows than in pregnant cows (Girard, 1998a, b). Serum folates decrease by 40% from two months postpartum until the next calving. A significant time effect was shown as the gain in serum folate concentration due to dietary supplementation was greater in the first eight weeks of lactation than later in lactation (Girard and Matte, 1999; Girard et al., 2005). It seems that increasing serum folate concentrations during early lactation could result from a decreased ability of the cells to retain and use folates. A reason for this may be generally lower serum vitamin B12 levels of 181 µg/ml at early lactation compared with 252 pg/ml in the later lactation; therefore folates can get into the methyl-trap where the folate form is not functional. Tremblay et al. (1991) reported that serum folates of dairy cows are lower one month prior to calving than two months after calving. These results suggest a higher folate requirement during pregnancy in dairy cows. Girard et al. (1995) also reported that apparent tissue utilization of serum folates was greater in pregnant, nonlactating dairy cows than in nonpregnant, lactating cows in early lactation. Likewise, in sheep serum, folates were significantly lower during pregnancy in breeds that give birth to larger numbers of lambs, for example, Romanov and Finnsheep more than Suffolk (Girard et al., 1996).
The effects of weekly injections of 160 mg folic acid, starting 45 days after breeding and continuing through six weeks of the next lactation, were investigated in lactating dairy cows (Girard and Matte, 1995). Parenteral folic acid supplementation tended to increase milk production and milk protein percentage in the latter half of lactation and increased milk protein percentage in multiparous cows during the first six weeks of the next lactation (Girard and Matte, 1995). In a subsequent trial, cows were fed folic acid at 0, 2 or 4 mg per kg (0, 0.90 or 1.8 mg per lb) body weight starting 30 days before calving and continuing through 305 days of lactation (Girard and Matte, 1998). In this trial, milk production was significantly increased in multiparous cows during the first 200 days of lactation, and especially during the first 100 days of lactation. Cows fed 4 mg folic acid per kg (1.8 mg per lb) of live weight produced 6% more milk (4.8 lbs or 2.2 kg per day) than controls during the first 100 days of lactation and 10% more milk (6.6 lbs or 3 kg per day) from day 100 to day 200. Milk production of primiparous heifers fed folic acid was lower than controls during the first 100 days of lactation and not different thereafter. These results suggested a parity x stage of lactation effect on the response to supplemental folic acid. The same researchers tested the effects of feeding 0, 3 or 6 mg folic acid per kg (0, 1.4 or 2.7 mg per lb) body weight to dairy cows fed rations either sufficient or insufficient in calculated methionine status (Girard et al., 1998). This study used only multiparous cows (n = 54), which were fed supplemental folic acid during the first 305 days of lactation. There was no effect of feeding supplemental folic acid on milk production in this trial despite higher average milk yields of cows compared to previous trials—23,800 versus 10,818 versus 8,595 kg (18,910 lbs) over 305 days.
Based on serum folate concentrations and transport kinetics by erythrocytes, the requirement of folic acid for adult sheep is recommended to be 3 µg per kg (1.36 µg per lb) body weight (NRC, 2007b). Folic acid requirements of sheep with functional rumens would be expected to range from a low of 0.3 ug per kg (0.14 µg per lb) body weight to a high of 3.0 µg per kg (1.13 µg per lb) body weight.
Folic acid is widely distributed in nature, almost exclusively as THF acid derivatives. The most stable THF derivatives are 5-methyl or 5-formyl 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 (pteroylpolyglutamic acid). Soybeans, other beans, nuts, some animal products and citrus fruits are good sources. Folic acid is abundant in green, leafy materials and organ meats such as liver. Cereal grains, milk and eggs do not contain high concentrations of folic acid. However, the availability of folate in foods is a modifying factor. The index of folate availability is highest for egg yolk, cow’s liver and orange juice and lowest in yeast (Seyoum and Selhub, 1998). The abundance of folic acid in green forages is confirmed by greater folic acid concentrations in milk from grazing herds than from herds fed dry hay (Dong and Oace, 1975).
Bioavailability of native folates is influenced by different physic-chemical properties and certain feed constituents. For example, polyglutamyl folates have a lower bioavailability than monoglutamyl folates, as polyglutamyl folates must be hydrolyzed to monoglutamates before absorption (Seyoum and Selhum, 1998). Additionally, the actual amount available for each individual animal varies depending on differences in intestinal pH or general living conditions.
Most naturally occurring folates are relatively unstable. Thus folates exhibit a significant loss of activity during harvesting, storage and processing, but measured folate concentrations are also highly influenced by the method used for sample preparation. The synthetic form, folic acid, is more resistant to chemical oxidation (Scott, 1999).
Concentrations of folic acid in ruminal dry matter range from 0.3 to 0.6 mg per kg (0.14 to 0.27 mg per lb) (Lardinoiset al., 1944). Corresponding values for strained rumen fluid ranged from 0.08 to 0.19 mg per kg (0.036 to 0.086 mg per lb) (Hayes et al., 1966).
Rumen microbial synthesis of folic acid begins with the onset of a functional rumen. Some bacterial species are able to synthesize folates, and some others need them. Different amounts of folates can be synthesized and used in the rumen depending on the feed composition. For steers, Hayes et al., 1996 and Girard et al., 1994 described a relationship between the proportion of concentrates in the diet and the amount of folates in the rumen. High-concentrate diets resulted in an increase of folates. The authors hypothesized that this increase is due to an enhanced microbial activity in the rumen, caused by rapidly degradable carbohydrates.
Crystalline folic acid, produced by chemical synthesis, is available for feeds, foods and pharmaceuticals. Several lines of evidence indicate higher bioavailability of added folic acid than naturally occurring folates in many foods (Gregory, 2001). 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). Spray-dried folic acid and dilutions of USP crystalline folic acid are the most widely used product forms in animal feeds.
Folic acid deficiency has been produced experimentally in many animal species; macrocytic anemia (megaloblastic anemia) and leukopenia (a reduced number of white blood cells) are consistent findings. Tissues that have a rapid rate of cell growth regeneration, such as the epithelial lining of the gastrointestinal tract, epidermis and bone marrow, are principally affected (Hoffbrand, 1978). A folic acid deficiency has not been demonstrated in the calf, but Drapper and Johnson (1952) reported a deficiency in lambs fed synthetic diets. The disease was characterized by leukopenia, followed by diarrhea, pneumonia and death. Folic acid therapy promoted regeneration of white cells, and 0.39 mg per liter of diet prevented the deficiency (Illus. 12-2). Folic acid deficiency has been reported in sheep that were experiencing a vitamin B12 deficiency. The vitamin B12 deficiency was severe enough to decrease voluntary feed intake to less than 200 g (90.7 lbs) per day (NRC, 2007b).
Illustration 12-2: Folic Acid Deficiency in Lambs
The efficiency of folic acid synthesis by rumen microflora, and whether this is adequate at weaning and later, have not yet been established. For example, in an experiment in which supplemental folic acid was administered to dairy heifers intramuscularly weekly during the first four months of life, average daily gain increased by 8% during the five weeks following weaning (Dumoulin et al., 1991). This supplementation also increased serum and hepatic folates, as well as blood hemoglobin and packed cell volume. These results suggest that marginal folic acid deficiency may develop in calves or lambs during the postweaning period, until full rumen function is achieved. The effects of folic acid supplementation on milk production has been variable. For gestating primiparous and multiparous cows, Girard et al., (2000) found a non-significant increase in milk production of 14% in the last part of lactation due to an intramuscular injection of 160 mg folic acid once per week. However, they could not find an effect on milk production immediately after calving. In contrast, Girard and Matte, (1998) found a 6% increase in milk production during the first 100 days of lactation for multiparous cows receiving 4 mg folic acid per kg (1.82 mg per lb) body weight and a 10% increase from day 100 to day 200. Supplementary folic acid and vitamin B12, increased milk production from 34.7 to 38.9 kg (76.3 to 85.6 lb) per day and increased milk lactose, protein, and total solids yields (Preynat et al., 2009b).
Folic acid fortification is recommended and prudent for milk replacers. As previously discussed, supplementation with folic acid may be warranted during the immediate postweaning period in ruminants. Data from lactating and gestating dairy cows suggest that folic acid status may be less than optimal in multiparous cows, although the responses have not been completely consistent across three experiments. Current studies are examining a possible interaction of folic acid with vitamin B12 (Girard, 1998b; Girard and Matte, 2005). Dietary folic acid supplements and rumen protected methionine modified milk crude protein and casein concentrations, but not yield in mid- and late-lactation. In early lactation, eight weeks after calving, serum concentrations of folates and cysteine were increased whereas vitamin B12 and methionine were decreased. These data, coupled with the slower serum clearance of folates following an intravenous bolus of folic acid suggest that a low supply in vitamin B12during early lactation could interfere with folate use. Gadient (1986) reported that folic acid is 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 storage at room temperature, 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 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 113°F (45°C); however, 57% 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.
For most ruminant species, substantial quantities of folic acid are provided through intestinal microbial synthesis. Ruminant animals receive the majority of their folic acid from rumen microorganisms. Preruminant animals need supplemental folic acid if they are receiving milk substitutes or diets low in the vitamin. Studies (Girard et al., 1989a, b; 1992; 2005; 2007; 2009; Preynat et al., 2009a, b; 2010; Dumoulin et al., 1991) suggest the need for supplemental folic acid under a variety of conditions. Fluctuations in serum concentrations of folic acid during the first months of life of young ruminants may be an indication that synthesis of folic acid by rumen flora is not sufficient to meet requirements during weaning. Similarly, serum folic acid has been shown in dairy cows to decrease 40% from 2 months postpartum to parturition. Supplemental folic acid by intramuscular injection or by diet has been shown to increase serum folic acid concentrations in both pre-ruminant and ruminant animals as well as improve weight gains in calves.
Folic acid has generally been considered a nontoxic vitamin (NRC, 1987). Excess folates are rapidly excreted in urine. No adverse responses to ingestion of folic acid have been documented in any species. 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).