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DSM in Animal Nutrition & Health

Vitamin C

Properties and Metabolism

Vitamin C occurs in two forms, namely L-ascorbic acid (reduced form) and dehydro-L-ascorbic acid (oxidized form). Although in nature the vitamin is primarily present as ascorbic acid, both forms are biologically active. The L-isomer of ascorbic acid is biologically active; the D-isomer is not. In nature, the reduced form of ascorbic acid may reversibly oxidize to the dehydroxidized form, the dehydroascorbic acid is irreversibly oxidized to the inactive diketogulonic acid. Since this change takes place readily, vitamin C is very susceptible to destruction through oxidation, which is accelerated by heat and light. Reversible oxidation-reduction of ascorbic acid with dehydroascorbic acid is the most important chemical property of vitamin C and the basis for its known physiological activities and stabilities (Moser and Bendich, 1991). Vitamin C is the least stable and, therefore, most easily destroyed of all the vitamins. Metabolic need for ascorbic acid is a general one among species, but a dietary need is limited to humans, subhuman primates, guinea pigs, fruit-eating bats, some birds (including the red-vented bulbul and related Passeriformes species), insects, fish (such as coho salmon, rainbow trout, and carp), and perhaps certain reptiles (McDowell, 2000). Under normal conditions, dogs and cats can synthesize vitamin C within their body. Because of de novo synthesis, vitamin C is not technically a dietary required vitamin for healthy dogs and cats.

Vitamin C is a white to yellow-tinged crystalline powder. It crystallizes out of water as square or oblong crystals (Illus. 6-1), slightly soluble in acetone and lower alcohols. A 0.5% solution of ascorbic acid in water is strongly acid with a pH of 3. The vitamin is more stable in an acid than an alkaline medium. A number of chemical substances, such as air pollutants, industrial toxins, heavy metals and tobacco smoke, as well as several pharmacologically active compounds, among them some antidepressants and diuretics, are antagonistic to vitamin C and can lead to increased requirements for the vitamin.

 

Illustration 6-1

6Illustration_6-1_VitaminC

Vitamin C is absorbed in a manner similar to carbohydrates (monosaccharides). Intestinal absorption in vitamin C-dependent animals appears to require a sodium-dependent active transport system (Johnston, 2006). It is assumed that those species that are not scurvy prone do have an absorption mechanism by diffusion (Spencer et al., 1963). Ascorbic acid is readily absorbed when quantities ingested are small, but limited intestinal absorption occurs when excess amounts of ascorbic acid are ingested. Bioavailability of vitamin C in feeds is limited, but apparently 80% to 90% appears to be absorbed (Kallner et al., 1977). Site of absorption in the guinea pig is in the duodenal and proximal small intestine, whereas the rat showed highest absorption in the ileum (Hornig et al., 1984). In its metabolism, ascorbic acid is first converted to dehydroascorbate by several enzymes or nonenzymatic processes and can then be reduced back to ascorbic acid in cells (Johnston et al., 2007). Absorbed vitamin C readily equilibrates with the body pool of the vitamin. No specific binding proteins for ascorbic acid have been reported, and it is suggested that the vitamin is retained by binding to subcellular structures.

Ascorbic acid is widely distributed throughout the tissues, both in animals capable of synthesizing vitamin C as well as in those dependent on an adequate dietary amount of the vitamin. In experimental animals, highest concentrations of vitamin C are found in the pituitary and adrenal glands, and high levels are also found in the liver, spleen, brain and pancreas. Vitamin C also tends to localize around healing wounds. Tissue levels are decreased by virtually all forms of stress, which also stimulates the biosynthesis of the vitamin in those animals capable of synthesis.

Ascorbic acid is excreted mainly in urine, with small amounts in sweat and feces. In guinea pigs, rats, and rabbits, CO2is the major excretory mechanism for vitamin C. Primates do not normally utilize the CO2 catabolic pathway, with the main loss occurring in the urine. Urinary excretion of vitamin C depends on body stores, intake and renal function.

 

Functions

Ascorbic acid has been found to be involved in a number of biochemical processes. Function of vitamin C is related to its reversible oxidation and reduction characteristics; however, the exact role of this vitamin in the living system is not completely understood, since a coenzyme form has not yet been reported. In addition to the relationship of ascorbic acid to hydroxylase enzymes, Franceschi (1992) suggests that vitamin C is required for differentiation of connective tissue such as muscle, cartilage and bone derived from mesenchyme (embryonic cells capable of developing into connective tissue). It is proposed that the collagen matrix produced by ascorbic acid-treated cells provides a permissive environment for tissue-specific gene expression. A common finding in all studies is that vitamin C can alter the expression of multiple genes as cells progress through specific differentiation programs (Ikeda et al., 1997). The most clearly established functional role for vitamin C involves collagen biosynthesis. Beneficial effects result from ascorbic acid in the synthesis of “repair” collagen. Alteration of basement membrane collagen synthesis and its integrity in mucosal epithelium during vitamin C restriction might explain the mechanism by which the capillary fragility is induced in scurvy as well as the increased incidences of periodontal disease under vitamin C deprivation. Failure of wounds to heal and gum and bone changes, resulting from vitamin C undernutrition, are direct consequences of reduction of insoluble collagen fibers.

Biochemical and physiological functions of vitamin C have been reviewed (Sauberlich, 1990; Moser and Bendich, 1991; Padh, 1991; Gershoff, 1993; Johnston, 2006; Johnston et al., 2007). The functional importance of vitamin C, other than the previously mentioned role in collagen synthesis, includes the following:

  • (a) Due to the ease with which ascorbic acid can be oxidized and reversibly reduced, it is probable that it plays an important role in reactions involving electron transfer in the cell. Almost all terminal oxidases in plant and animal tissues are capable of directly or indirectly catalyzing the oxidation of L-ascorbic acid. Such enzymes include ascorbic acid oxidase, cytochrome oxidase, phenolase, and peroxidase. In addition, its oxidation is readily induced under aerobic conditions by many metal ions and quinones.
  • (b) Metabolic oxidation of certain amino acids, including tyrosine.
  • (c) Ascorbic acid has a role in metal ion metabolism due to its reducing and chelating properties. It can result in enhanced absorption of minerals from the diet and their mobilization and distribution throughout the body. Ascorbic acid promotes non-heme iron absorption from food (Olivares et al., 1997) and acts by reducing the ferric iron at the acid pH in the stomach and by forming complexes with iron ions that stay in solution at alkaline conditions in the duodenum. Also, a sufficient vitamin C status is the prerequisite for the C-1 hydroxylation of vitamin D3 and its storage form 25-(OH)D3 to the active form 1,25-(OH)2D3 (Suter, 1990).
  • (d) Carnitine is synthesized from lysine and methionine and is dependent on two hydroxylases, both containing ferrous iron and L-ascorbic acid. Vitamin C deficiency can reduce the formation of carnitine, which can result in accumulation of triglycerides in blood and physical fatigue and lassitude associated with scurvy (Ha et al., 1994). About 98% of total body carnitine is in muscle; skeletal and heart muscle carnitine concentrations are reduced by 50% in vitamin C-deficient guinea pigs compared with controls (Johnston, 2006).
  • (e) Interrelationships of vitamin C to B vitamins are known, as tissue levels and urinary excretion of vitamin C are affected in animals with deficiencies of thiamin, riboflavin, pantothenic acid, folic acid and biotin.
  • (f) Vitamin C has been demonstrated to inhibit nitrosamines, which are potent carcinogens. The vitamin is effective in detoxifying high nitrate diets for ruminants (Aseltine, 1990).
  • (g) Vitamin C is involved in controlling synthesis of glucocorticoids (corticosteroids) in the adrenal gland. The protective effects of vitamin C (also vitamin E) on health may partially be a result of reducing circulating levels of glucocorticoids (Nockels, 1990). During stress, glucocorticoids, which suppress the immune response, are elevated. Vitamin C reduces adrenal glucocorticoid synthesis, helping to maintain immunocompetence. In addition, ascorbate can regenerate the reduced form of alpha-tocopherol, perhaps accounting for observed sparing effects of these vitamins (Jacob, 1995). In the process of sparing fatty acid oxidation, tocopherol is oxidized to the tocopheryl free radical. Ascorbic acid can donate an electron to the tocopheryl free radical, regenerating the reduced antioxidant form of tocopherol.
  • (h) Ascorbic acid is found in up to a ten-fold concentration in seminal fluid as compared to serum levels. Decreasing levels have caused nonspecific sperm agglutination. In a review of ascorbic acid and fertility, Luck et al. (1995) suggest how three of ascorbic acid’s principal functions, namely its promotion of collagen synthesis, its role in hormone production, and its ability to protect cells from free radicals, may explain its reproductive actions.
  • (i) Vitamin C has a biological role in keratinocytes. Because skin must provide the first line of defense against environmental free radical attack (e.g., sunburn, skin aging and skin cancer), it has developed a complex antioxidant network that includes enzymatic and non-enzymatic components. The epidermis is composed of several layers of keratinocytes supplied with enzymes (superoxide dismutase, catalase, thioredoxin reductase, and glutathione reductase) and low-molecular-weight antioxidant molecules (e.g., tocopherol, glutathione, and ascorbic acid) (Podda and Grundmann-Kollmann, 2001).
  • In keratinocytes, vitamin C contributes to counteract oxidative stress via transcriptional and post-translational mechanisms. Vitamin C can: 1) act directly by scavenging reactive oxygen species (ROS) generated by stressors; 2) prevent ROS-mediated cell damage by modulating gene expression; 3) regulate keratinocyte differentiation maintaining a balanced redox state; and 4) promote cell cycle arrest and apoptosis in response to DNA damage (Catani et al., 2005).
  • (j) Ascorbic acid is reported to have a stimulating effect on the phagocytic activity of leukocytes, the function of the reticuloendothelial system and formation of antibodies. As an effective scavenger of reactive oxygen species, ascorbic acid minimizes the oxidative stress associated with the respiratory burst of activated phagocytic leukocytes, thereby functioning to control the inflammation and tissue damage associated with immune responses (Chien et al., 2004). Ascorbic acid levels are very high in phagocytic cells, with these cells using free radicals and other highly reactive oxygen containing molecules to help kill pathogens that invade the body. In the process, however, cells and tissues may be damaged by these reactive species. Ascorbic acid helps to protect these cells from oxidative damage (McDowell, 2006).

Tissue defense mechanisms against free-radical damage generally include vitamin C, vitamin E, and beta-carotene as the major vitamin antioxidant sources. In addition, several metalloenzymes that include glutathione peroxidase (selenium), catalase (iron), and superoxide dismutase (copper, zinc and manganese) are also critical in protecting the internal cellular constituents from oxidative damage. The dietary and tissue balance of all these nutrients is important in protecting tissue against free radical damage. Both in vitro and in vivo studies showed that the antioxidant vitamins generally enhance different aspects of cellular and noncellular immunity. The antioxidant function of these vitamins could, at least in part, enhance immunity by maintaining the functional and structural integrity of important immune cells. A compromised immune system will result in reduced animal production efficiency through increased susceptibility to diseases, thereby leading to increased animal morbidity and mortality.

 

Requirements

A wide variety of plant and animal species can synthesize vitamin C from carbohydrate precursors including glucose and galactose. Ascorbic acid is synthesized by tissues of mammals with the exception of primates (including humans) and the guinea pig. The missing step in the pathway of ascorbic acid biosynthesis in all vitamin C-dependent species has been traced to inability to convert L-gulono-gamma-lactone to 2-keto-L-gulonate, which is transformed by spontaneous isomerization into its tautomeric form, L-ascorbic acid. Vitamin C-dietary dependent species, therefore, lack the enzyme L-gulono-gamma-lactone oxidase.Under normal conditions, dogs and cats have no dietary requirement for vitamin C since they can synthesize the vitamin. Even for species that do synthesize vitamin C, however, it has been shown that the synthesizing capacity of liver microsomal preparations varies widely from animal to animal (Chatterjee, 1978), suggesting possible dietary need for the vitamin for individuals within a species.

 

A. Requirements for Dogs

Innes (1931) demonstrated that the dog was independent of a dietary supply of vitamin C. Puppies fed a diet devoid of vitamin C for 147 to 154 days showed neither growth impairment or lesions of bones and teeth, although the same diet killed guinea pigs within 25 days with severe signs of scurvy. Furthermore, the livers of dogs on the deficient diet contained the vitamin in sufficient amounts to prevent the onset of scurvy in guinea pigs, indicating that the dog can synthesize vitamin C. Naismith (1958) showed that this ability to synthesize vitamin C is present in puppies during the first weeks of postnatal life. Naismith and Pellet (1960) reported that the concentration of vitamin C in the milk from bitches is approximately four times that of the blood. The comparative rates of liver synthesis of vitamin C in dogs and cats appear to be lower than those in ruminants, rodents, and rabbits (Chatterjee et al., 1975). Dogs synthesized vitamin C in the liver at a rate of 5 µg per mg of protein per hour while cows, rats and rabbits had rates of 68, 39, and 23 µg per mg of protein per hour, respectively.

 

B. Requirements for Cats

No requirement for dietary ascorbic acid has been demonstrated to exist in the cat. Repeated trials have failed to demonstrate a need for dietary vitamin C in cats (Carvalho da Silva, 1950). Successful growth and reproduction are routinely obtained with commercial and purified diets containing no supplemental ascorbic acid (NRC, 2006).

 

Sources

The main sources of vitamin C are fruits and green plants, but some foods of animal origin contain more than traces of the vitamin. Vitamin C occurs in significant quantities in animal organs, such as liver and kidney, but in only small quantities in meat. The milk of bitches contains about four times the concentration of vitamin C as the blood (Naismithand Pellet, 1960). Vitamin C is very low in grains and plant protein supplements. Postharvest storage values vary with time, temperature, damage and enzyme content (Zee et al., 1991).

Of animals raised for food production, sources of vitamin C would be important for fish and next for stressed animals needing the vitamin. L-ascorbic acid is in the most important of the several compounds that have vitamin C activity. Ascorbic acid is commercially available as 100% crystalline, 50% fat-coated, and 97.5% ethylcellulose-coated products and their dilutions. The more soluble sodium salt of ascorbic acid (sodium ascorbate) is also commercially available. Various derivatives of ascorbic acid which are more stable than the parent compound, have been shown to provide antiscorbutic activity. These include L-ascorbate-2-sulfate, L-ascorbyl-2-monophosphate, magnesium-L-ascorbyl-2-phosphate, and L-ascorbyl-2-polyphosphate. When providing supplemental ascorbic acid, it is advisable to use a stabilized form. In storage experiments, ascorbic acid protected in this manner was found to be four times more stable than untreated ascorbic acid crystals (Kolb, 1984).

Adams (1982) reported that coated (ethylcellulose) ascorbic acid showed higher retention after processing than the crystalline form, 84% versus 48%. Retention of ascorbic acid in mash feed was fairly good, but with elevated storage time and temperature, stability was poor in crumbled feeds. Although retention of vitamin C activity in feed containing the ethylcellulose-coated product was low, it was 19% to 32% better than that of the crystalline form.

Recent reports have evaluated polyphosphorylated L-ascorbic acid for fish (Chen and Chang, 1994; Matusiewicz et al., 1995), which has been found to be more stable against oxidation and extrusion. Approximately 50% of the supplemental crystalline ascorbic acid is destroyed during the manufacture of extruded catfish feeds (Lovell and Lim, 1978). An excess of crystalline ascorbic acid is added to commercial formulations to ensure that an adequate concentration of the vitamin is retained during processing. The form of the vitamin selected depends on how the fish feed is to be manufactured and how long it is stored before being fed to the fish. Crystalline L-ascorbic acid or L-ascorbyl-e-polyphosphate were of similar bioavailability for broiler chicks (Pardue et al., 1993). Magnesium-L-ascorbyl-2-phosphate is a stable form of vitamin C that was shown to be available in swine diets (Mahan et al., 1994).

 

Deficiency

Clinical signs of scurvy or vitamin C deficiency in those species that cannot synthesize the vitamin include weakness, fatigue, bone pain, loose teeth and hemorrhages of the skin, musculature, adipose tissue and certain organs. Dogs and cats, however, have the capability to synthesize vitamin C and, therefore, would not be expected to show these deficiency signs under normal conditions. However, some studies with dogs have shown that the ability to synthesize vitamin C is compromised under conditions of stress or disease. Starvation or lack of optimum food supplies will affect vitamin C synthesis. Mean plasma ascorbic acid concentration was significantly lower in dogs after they were fasted (Leeet al., 1986). Also, some of the benefits of supplemental vitamin C are with pharmacological doses of ascorbic acid (e.g., 3,000 mg intravenously per day), which may be quite distinct from its nutritional contribution. Completely satisfactory and reliable procedures to assess vitamin C nutritional status have not been developed because of limited knowledge concerning the vitamin’s metabolic functions. However, information concerning adequacy has been determined by an analysis of vitamin C concentrations in serum (plasma), leukocytes, whole blood, or urine. Normal serum ascorbic acid values in dogs should exceed 1.0 mg per 100 ml (Ralston Purina, 1987). Leukocyte vitamin C concentrations provide information concerning body stores of ascorbic acid (Turnbull et al., 1981). Precautions need to be taken to protect the vitamin in solution and to select an assay that measures the vitamin itself and not other substances present.

 

A. Deficiency in Dogs

Scurvy in dogs has been reported (Garlick, 1946; Meier et al., 1957; Holmes, 1962; Hunt, 1962; Vaananen and Wikman, 1979; Kolb, 1984). There is controversy about the therapeutic use of ascorbic acid in canine diseases.Vitamin C deficiency also has been reported to be associated with canine hypertrophic osteodystrophy (HOD) (Grondalen, 1976). This disease affects young, rapidly growing dogs. It is characterized by an enlarged metaphysis of long bones. Hemorrhages are common in the region of bone disorganization. The clinical findings of the acute stage are hyperthermia, anorexia and inability to stand because of great pain in the extremities (Grondalen, 1976). Meier et al.(1957) found that dogs with HOD had low plasma ascorbic acid concentrations and that large doses of vitamin C (100 mg to 200 mg) given orally or IM enhanced healing. Supplementation with ascorbic acid is not always effective. Teareet al. (1980) reported that 600 mg of ascorbic acid twice daily only aggravated the skeletal disease induced by overfeeding protein, energy and calcium to Labrador retriever puppies. The study was weakened by the fact that the pups evaluated did not have clinical signs of acute HOD, which include both lameness and low blood ascorbic acid.

 

B. Deficiency in Cats

Controlled studies evaluating the need for supplementation or describing specific deficiency of vitamin C in cats have not been published. A number of trials have failed to demonstrate a need for dietary ascorbic acid in cats (Carvalho da Silva, 1950; NRC, 2006). Successful growth and reproduction are routinely obtained with commercial and purified diets containing no supplemental ascorbic acid (NRC, 2006). As in dogs, vitamin C synthesis in cats is lower than in other species, including the cow, sheep, rat and rabbit (Rucker et al., 1980). Pietronigro et al. (1983) reports that central nervous system function following spinal cord injury in the cat was associated concentration with large of ascorbic acid in the region of the injury. Treatment of injury with two drugs (naloxone or methyl-prednisolone) preserved neurological function and prevented vitamin C loss.

 

Fortification Considerations

Under practical feeding situations, only humans, nonhuman primates, guinea pigs and fish will develop vitamin C deficiency if diets are lacking in the vitamin. Dogs and cats synthesize ascorbic acid from glucose in either the liver or kidney, and vitamin C deficiency usually does not occur in such animals. In cases of well-balanced nutrition, their tissues receive synthesized vitamin C continuously. Blood and tissue levels of vitamin C are difficult to affect with dietary or injectable vitamin C. However, with nutritionally unbalanced diets, relative vitamin C deficiency may be induced in vitamin C-synthesizing animals as well (Ginter, 1970). Low blood ascorbic acid can be caused by various types of stress, including metabolic disorders, improper nutrition, insufficient vitamin A or beta-carotene intake, and various infectious diseases. Under such conditions, dietary and injectable vitamin C can have a positive effect in vitamin C-synthesizing species. Some researchers have claimed that various diseases of the dog may be associated with insufficient vitamin C. Furthermore, skeletal diseases such as hypertrophic osteodystrophy (HOD), hip dysplasia and a number of others, particularly those common in the large and giant breeds, have been said by some to resemble ascorbic acid deficiency (scurvy). Overall, the data indicate that reduced serum vitamin C is relevant in acute HOD and that supplementary ascorbic acid can be used as a preventive and for treatment. However, such treatment may not be rewarding since there are apparently other interacting mechanisms in the HOD syndrome (Ralston Purina, 1987). A study reported prevention of hip dysplasia (German shepherds) with megadoses of vitamin C given to mothers during pregnancy and after birth to puppies until young adulthood (Strombeck, 1999). The mothers received 2 to 4 g of vitamin C per day and the puppies from 0.5 to 2 g.

There have been a number of uncontrolled clinical reports purporting to identify either scurvy in dogs or a clinical response to ascorbate supplementation of dogs infected with canine distemper, exhibiting hypertrophic osteodystrophy, hip dysplasia, or other conditions. In a series of controlled experiments that provided vitamin C to puppies infected with either canine herpes virus, kennel cough or canine hepatitis, Sheffy (1972) found no evidence of a beneficial effect of vitamin C on these disease conditions.

Small quantities of vitamin C are sufficient to prevent and cure scurvy. However, larger quantities may be required to maintain good health during adverse environmental and physiological stress as well as certain disease conditions. In recent years, antioxidant vitamins (vitamin C along with vitamin E and beta-carotene) have received a great deal of attention in that they play important roles in animal and human health by inactivating harmful free radicals produced through normal cellular activity and from various stressors. Recommendations for daily antioxidant fortification rates for dogs and cats of vitamin E, vitamin C and beta-carotene have been suggested by Parr (1996). For example, the daily vitamin C recommended supplemental level for a 13.6 kg (30 lb) dog is 60 mg and for a 2.7 kg (6 lb) cat is 12 mg.

Based on the available evidence, no requirement for vitamin C as a nutrient in the diet of dogs of cats is proposed (NRC, 2006). Vitamin C may improve the stability of other nutrients in the diet and may have functions associated with protection against oxidative damage.

Vitamin C is the least stable, and therefore most easily destroyed, of all vitamins. The vitamin is particularly susceptible to destruction through oxidation, a change that is accelerated by heat and light. Choline chloride is particularly destructive to vitamin C, with the unstabilized vitamin almost completely destroyed in a vitamin premix with choline and an average monthly loss of 40% (Gadient, 1986).

When providing supplemental ascorbic acid, it is advisable to use a stabilized form. In feed storage experiments, coated ascorbic acid was found to be four times more stable than crystalline vitamin C (Kolb, 1984). Adams (1978) reported that coated (ethylcellulose) ascorbic acid showed a higher retention after processing than the crystalline form, 84% versus 48%, respectively. Although retention of vitamin C activity in feed containing the ethylcellulose coated product was low, it was 19% to 32% better than retention in feed containing the crystalline form. Phosphorylated vitamin C, ascorbyl mono-phosphate, has the highest stability through pet food processing with retentions of 90 plus percent being typical.

 

Vitamin Safety

In general, high intakes of vitamin C are considered to be of low toxicity. Chronic toxicity studies generally indicate that ascorbic acid is well tolerated in animals. Oral ascorbic acid may be administered to most laboratory animals at doses of several grams per kilogram of body weight without appearance of any obvious general effect on health (NRC, 1987). Male guinea pigs fed 8.7% ascorbic acid for six weeks had decreased bone density and decreased urinary hydroxyproline compared to controls (Bray and Briggs, 1984). Helgebostad (1984) reports that high doses of 100 to 200 mg per kg (45.5 to 91 mg per lb) body weight daily were harmful to mink, with pronounced anemia in pregnant females and reduced number and size of kits.Data are unavailable on tolerance and toxicity of ascorbic acid for dogs and particularly for cats. Intakes of 0.5 and 0.3 g ascorbic acid per day in cats and dogs, respectively, do not appear to affect these animals adversely in short-term studies (Belfield, 1967; Leveque, 1969; Vaananen and Wekman, 1979). Doses of 1,000 mg ascorbic acid per day induced diarrhea in some cats (NRC, 2006). In studies conducted on dogs, Leveque (1969) reported allergic types of reaction in the mouth. These signs disappeared when the level of ascorbic acid intake by the dogs was reduced. However, this observation was incidental, and the study was not controlled.