Vitamin D designates a group of closely related compounds that possess antirachitic activity. It may be supplied through the diet or by irradiation of the body. The two most prominent forms of vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Ergocalciferol is derived from a common plant steroid, ergosterol, whereas cholecalciferol (Illus. 3-1) is produced from the precursor 7-dehydrocholesterol, exclusively from animal products. The provitamin 7-dehydrocholesterol, derived from cholesterol or squalene, is synthesized in the body and present in large amounts in skin, the intestinal wall and other tissues. Vitamin D precursors have no antirachitic activity.
Vitamin D, in the pure form, occurs as colorless crystals that are insoluble in water but readily soluble in alcohol and other organic solvents. Vitamin D can be destroyed by over-treatment with ultraviolet (UV) light and by peroxidation in the presence of rancidifying polyunsaturated fatty acids (PUFA). Like vitamins A and E, unless vitamin D3 is stabilized, it is destroyed by oxidation. Its oxidative destruction is increased by heat, moisture and trace minerals. There is less destruction of vitamin D3in freeze-dried fish meals during drying, possibly because of decreased atmospheric oxygen. There is negligible loss of crystalline cholecalciferol during storage for one year or of crystalline ergocalciferol for nine months in amber-evacuated capsules at refrigerator temperatures.Vitamin D from the diet is absorbed from the intestinal tract, and is more likely to be absorbed from the ileal portion in greatest amounts due to the longer retention time of food in the distal portion of the intestine (Norman and Henry, 2007). Vitamin D is absorbed from the intestinal tract in association with fats, as are all the fat-soluble vitamins. Like the others, it requires the presence of bile salts for absorption (Braun, 1986), and is absorbed with other neutral lipids via chylomicron into the lymphatic system of animals. It has been reported that only 50% of an oral dose of vitamin D is absorbed. However, considering that sufficient vitamin D is usually produced by daily exposure to sunlight, it is not surprising that the body has not evolved a more efficient mechanism for dietary vitamin D absorption (Norman and Henry, 2007). Effective treatment of rickets by rubbing cod liver oil on the skin indicates that vitamin D can be absorbed through the skin. There are four important variables that selectively determine the amount of vitamin D3 that will be photochemically produced by an exposure of skin to sunlight (Norman and Henry, 2007). The two principal determinants are the quantity and intensity of ultraviolet light (UV) and the appropriate wavelength of the UV light. The third important variable determining skin vitamin D synthesis is the actual concentration of 7-dehydrocholesterol present in the skin. The fourth determinant of vitamin D3 production is the concentration of melanin in the skin (skin color). The darker the skin the longer time required to convert 7-dehydrocholesterol to vitamin D3.Presence of the provitamin 7-dehydrocholesterol in the epidermis of the skin and sebaceous secretions is well recognized. Vitamin D is synthesized in the skin of many herbivores and omnivores, including humans, rats, pigs, horses, poultry, sheep and cattle. However, little 7-dehydrocholesterol is found in the skin of cats and dogs (and likely other carnivores), and therefore little vitamin D is produced in the skin (How et al., 1995). For poultry, Tian et al. (1994) reported that skin of the legs and feet of chickens contains about 30 times as much 7-dehydrocholesterol (provitamin D3) as the body skin. The cholecalciferol formed by the UV irradiation of 7-dehydrocholesterol is removed from the skin into the circulatory system by the blood transport protein for vitamin D, the vitamin D-binding protein (DBP) (Norman and Henry, 2007). Heuser and Norris (1929) showed that 11 to 45 minutes of sunshine daily were sufficient to prevent rickets in growing chicks and that no other further improvements in growth were obtained under these conditions by adding cod liver oil (a rich source of vitamin D).
Production and metabolism of vitamin D necessary to activate the target organs is illustrated in Figure 3-1. By itself vitamin D is biologically inactive and must be converted to a hormonal form in a two-step process before it can function as the hormone 1,25-(OH)2D3. Once in the liver, the first transformation occurs in which a microsomal system hydroxylates the 25-position carbon in the side chain to produce 25-hydroxy-vitamin D [25-(OH)D]. This metabolite is the major circulating form of vitamin D under normal conditions and during vitamin D excess (Littledike and Horst, 1982). The 25-(OHD is then transported to the kidney on the vitamin D transport globulin, where it can be converted in the proximal convoluted cells to a variety of compounds, of which the most important appears to be 1,25 dihydroxy-vitamin D [1,25-(OH)2D] (DeLuca, 2008). Although the kidney is the main site of 1-hydroxylation, other organs can also form 1,25-(OH)2D including the placenta (Johnson, 2006; DeLuca, 2008). The 1,25-(OH)2D is also referred to as calcitriol. Once formed in the kidney, 1,25-(OH)2D is then transported to the intestine, bones or elsewhere in the body, where it is involved in the metabolism of calcium and phosphorus. From studies of vitamin D metabolism, it has been found that the vitamin functions as a hormone. The hormonal form, 1,25-(OH)2D3, is the metabolically active form of the vitamin that functions in intestine and bone, whereas 25-(OH)D and vitamin D do not function at these specific sites under physiological conditions (DeLuca, 2008).
Figure 3-1: The Functional Metabolism of Vitamin D3 Necessary to Activate Target Organs of Intestine, Bone and Kidney
One question that is still unanswered is whether the hormone from 1,25-(OH)2D3 acts alone or if there some response from a second vitamin D metabolite or hormone [e.g., 24,25-(OH)2D3] (Feldman et al., 2005). However, this question was probably answered in a study where the 24-position of 25-(OH)D3 was blocked with fluoro groups to prevent 24-hydroxylation (DeLuca, 2008). For two generations all systems were normal, indicating a need for only 1,25-(OH)2D3. Therefore research indicates that 1,25-(OH)2 D3 appears to be the only functional form of vitamin D in biology (DeLuca, 2008).Production of 1,25-(OH)2D3 is very carefully regulated by parathyroid hormone (PTH) in response to serum calcium and phosphate (PO43-) concentrations. Under conditions of calcium stress, PTH activates renal mitochondrial 1 alpha-hydroxylases, which convert 25-(OH)D to 1,25-(OH)2D, and inactivates renal and extrarenal 24- and 23-hydroxylases, which convert the 25-(OH)D and any 1,25-(OH)2D formed to inactive metabolites (Goff et al., 1991b). Under conditions with little calcium stress (when little PTH is secreted), the 1 alpha-hydroxylase can also be directly stimulated by low blood calcium or phosphorus concentration. High plasma 1,25-(OH)2D concentration has an inhibitory effect on renal 1 alpha-hydroxylase and a stimulatory effect on tissue 24- and 23-hydroxylases (Engstrom et al.,1987). Thus, production and catabolism of the hormone 1,25-(OH)2D3 are tightly regulated. It is now known that the most important point of regulation of the vitamin D endocrine system occurs through stringent control of the activity of the renal 1 alpha-hydroxylase. In this way, the production of the hormone 1,25-(OH)2D3 can be modulated according to the calcium needs of the organism (Norman and Henry, 2007).For most mammals, vitamin D, 25-(OH)D, and possibly 24,25-(OH)2D3 and 1,25-(OH)2D are all transported on the same protein, called transcalciferin, or vitamin D-binding protein (DBP). In contrast to aquatic species, which store significant amounts of vitamin D in the liver, land animals, do not store appreciable amounts of the vitamin. The body has some ability to store vitamin D, although to a much lesser extent than vitamin A. Principal stores of vitamin D occur in blood and liver, but it is also found in lungs, kidneys and elsewhere in the body. During times of deprivation, vitamin D in these tissues is released slowly, thus meeting vitamin D needs of the animal over a longer period of time (Norman and Henry, 2007). Excretion of absorbed vitamin D and its metabolites occurs primarily in feces with the aid of bile salts. For mammals, 1,25-(OH)2D is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate (Lester, 1986). A liberal intake of vitamin D during gestation does provide a sufficient store in newborns to help prevent early rickets. For example, newborn lambs can be provided enough to meet their needs for six weeks. Parenteral cholecalciferol treatment of sows before parturition proved an effective means of supplementing young piglets with cholecalciferol (via the sow’s milk) and its more polar metabolites via placental transport (Goff et al., 1984).
The primary function of vitamin D is to elevate plasma calcium and phosphorus to a level that will support normal mineralization of bone as well as other body functions. It is now realized that vitamin D is not only important for mineralization and skeletal growth but has many other roles in regulation of the parathyroid gland, in the immune system, in skin, cancer prevention, in metabolism of foreign chemicals and in cellular development and differentiation. There is a regulatory role of vitamin D 1,25-(OH)2D3 in immune cell functions (Reinhardt and Hustmeyer, 1987), the release of insulin in relation to glucose challenge (DeLuca, 2008), and reproduction in both males and females (DeLuca, 2008). Experiments in mice have illustrated that the autoimmune diseases of multiple sclerosis and rheumatoid arthritis can be successfully treated with the vitamin D hormone and its analogs (DeLuca and Zierold, 1998).Two hormones, thyrocalcitonin (calcitonin) and PTH, function in a delicate relationship with 1,25-(OH)2D3 to control blood calcium and phosphorus levels (Engstrom and Littledike, 1986). Production rate of 1,25-(OH)2D is under physiological control as well as dietary control. Calcitonin, contrary to the other two, regulates high serum calcium levels by (1) depressing gut absorption, (2) halting bone demineralization, and (3) depressing reabsorption in the kidney. Vitamin D elevates plasma calcium and phosphorus by stimulating specific ion pump mechanisms in the intestine, bone and kidney. These three sources of calcium and phosphorus provide reservoirs that enable vitamin D to elevate calcium and phosphorus in blood to levels necessary for normal bone mineralization and for other functions ascribed to calcium. In the target tissue, the hormone enters the cell and binds to a cytosolic receptor or a nuclear receptor. 1,25-(OH)2D3 regulates gene expression through its binding to tissue-specific receptors and subsequent interaction between the bound receptor and the DNA (Norman and Henry, 2006). The receptor-hormone complex moves to the nucleus where it binds to the chromatin and stimulates the transcription of particular genes to produce specific mRNAs, which code for the synthesis of specific proteins. Evidence for transcription regulation of a specific gene typically includes 1,25-(OH)2D-induced modulation in mRNA levels. Additionally, evidence may include measurements of transcription and/or the presence of a vitamin D responsive element within the promoter region of the gene (Hannah and Norman, 1994). Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and a vitamin A receptor (RXR) within the nucleus of the cell as the active complex for mediating positive transcriptional effects of 1,25-(OH)2D. The two receptors (vitamins D and A) selectively interact with specific hormone response elements composed of direct repeats of specific nucleotides located in the promoter of regulated genes. The complex that binds to these elements actually consists of three distinct elements: the 1,25-(OH)2D hormonal ligand, the vitamin D receptor (VDR) and one of the vitamin A (retinoid) X receptors (RXR) (Kliewer et al., 1992; Whitfield et al., 1995).
It is well known that vitamin D stimulates active transport of calcium and phosphorus across intestinal epithelium. This stimulation does not involve PTH directly but involves the active form of vitamin D. Parathyroid hormone indirectly stimulates intestinal calcium absorption by stimulating production of 1,25-(OH)2D3under conditions of hypocalcemia. The mechanism whereby vitamin D stimulates calcium and phosphorus absorption is still not completely understood. Current evidence (Wasserman, 1981) indicates that 1,25-(OH)2D is transferred to the nucleus of the intestinal cell, where it interacts with the chromatin material. In response to the 1,25-(OH)2D, specific RNAs are elaborated by the nucleus, and when these are translated into specific proteins by ribosomes, the events leading to enhancement of calcium and phosphorus absorption occur (Scott et al., 1982).In the intestine, 1,25-(OH)2D3 promotes synthesis of calbindin (calcium-binding protein, CaBP) and other proteins and stimulates calcium and phosphorus absorption. Vitamin D has also been reported to influence magnesium absorption as well as calcium and phosphorus balance (Miller et al., 1965). This calbindin is not present in the intestine of rachitic chicks but appears following vitamin D supplementation. Originally, it was felt that vitamin D did not regulate phosphorus absorption and transport, but in 1963 it was demonstrated, through the use of an in vitro inverted sac technique, that vitamin D does in fact play such a role (Harrison and Harrison, 1963).
During bone formation in young animals, minerals are deposited on the protein matrix. This is accompanied by an invasion of blood vessels that gives rise to trabecular bone. This process causes bones to elongate. During a vitamin D deficiency, this organic matrix fails to mineralize, causing rickets in the young and osteomalacia in adults. The active metabolite, 1,25-(OH)2D3, brings about mineralization of the bone matrix. Vitamin D has another function in bone, namely, in mobilization of calcium from bone to the extracellular fluid compartment. This function is shared by PTH (Garabedian et al., 1974), requires metabolic energy, and presumably transports calcium and phosphorus across the bone membrane by acting on osteocytes and osteoclasts. Rapid, acute plasma calcium regulation is due to the interaction of plasma calcium with calcium-binding sites in bone material as blood comes in contact with bone. Changes in plasma calcium are brought about by a change in the proportion of high- and low-affinity calcium-binding sites, access to which is regulated by osteoclasts and osteoblasts, respectively (Bronner and Stein, 1995). Another role of vitamin D has been proposed in addition to its involvement in bone, namely, in the biosynthesis of collagen in preparation for mineralization (Gonnerman et al., 1976).
There is evidence that vitamin D functions in the distal renal tubules to improve calcium reabsorption and is mediated by the calcium-binding protein, calbindin (Bronner and Stein, 1995). It is known that 99% of the renal-filtered calcium is reabsorbed in the absence of vitamin D and PTH. The remaining 1% is under control of these two hormonal agents, although it is not known whether they work in concert. It has been shown that 1,25-(OH)2D3 functions in improving renal reabsorption of calcium (Sutton and Dirks, 1978).
The well-known effects of vitamin D relate to biochemical changes occurring in the intestine, bone and kidney. More recent research indicated that vitamin D has important functions in addition to mineralization and skeletal growth. The first evidence of non-calcium and phosphorous related activities of the vitamin D hormone was the demonstration of its receptor in tissue not related to bone metabolism. Many roles have been identified in regulation of the parathyroid gland, in the immune system, in skin, in cancer prevention and in cellular development and differentiation.A receptor for the active metabolite 1,25-(OH)2D3 has been isolated in the pancreas, parathyroid glands, bone marrow, certain cells of the ovary and brain, endocrine cells of the stomach, breast epithelial cells, skin fibroblasts and keratinocytes, suggesting that 1,25-(OH)2D has additional functions in a wide variety of cells, glands and organs (Machlin and Sauberlich, 1994). To date, more than 50 genes have been reported to be transcriptionally regulated by 1,25-(OH)2D (Hannah and Norman, 1994).Vitamin D has also been shown to be required for embryonic development of the chick. Vitamin D treatment stimulated yolk calcium mobilization and the vitamin D-dependent Ca2+ binding protein, calbindin, is present in the yolk sac (Tuan and Suyama, 1996). These findings strongly suggest that the hormonal action of 1,25-(OH)2D3on yolk sac calcium transport is mediated by the regulated expression and activity of calbindin, analogous to the response of the adult intestine. 1,25-(OH)2D is also essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (Elaroussi et al., 1994).In the dog Calbindin has been found to play an important role in modulating the activity of neurons in the dentate gyrus (associated with the hippocampus part of the brain). Choi et al.(2009) compared calbindin immunoreactivity in the dentate gyrus of dogs of various ages (German shepherds). The number of calbindin neurons decreased in the aged dog brain and this may be associated with reduction of function in the dentate gyrus.
The actions of 1,25-(OH)2D are recognized as being involved in regulation of the growth and differentiation of a variety of cell types, including those of the hematopoietic and immune systems (Lemire, 1992). Recent studies have suggested 1,25-(OH)2D3 as an immunoregulatory hormone. Aslam et al. (1998) reported that vitamin D deficiency depresses the cellular immune responses in young broiler chicks. Turkey osteomyelitis, a disease that affects commercially produced turkeys and disease incidence in E. coli-challenged birds was also decreased with vitamin D metabolites (Huff et al., 2002). Elevated 1,25-(OH)2D also was associated with a significant 70% enhancement of lymphocyte proliferation in cells treated with pokeweed mitogen (Hustmeyer et al., 1994). 1,25-(OH)2D also inhibits growth of certain malignant cell types and promotes their differentiation (Colston et al., 1981; DeLuca, 2008). 1,25-(OH)2D3 has been reported to inhibit proliferation of leukemic cells (Pakkla et al., 1995), breast cancer cells (Vink van Wijngaarden et al., 1995) colorectal cells (Cross et al., 1995). and to produce cells that suppress inflammation (Cantorna, 2006). A deficiency of vitamin D may promote prostate cancer (Skowronski et al., 1995). Also, 1,25-(OH)2D3 and its analogs may be effective in treating some forms of psoriasis (Kragballe et al., 1991). The therapeutic value of vitamin D and its analogs has been under rigorous evaluation in numerous laboratories around the world.
Unlike man, rats and our common poultry and livestock, dogs and cats have a nutritional requirement for vitamin D even when sufficient sunlight is available. This is because vitamin D3 is not produced in skin through action of UV irradiation on 7-dehydrocholesterol in sufficient quantities to prevent rickets (How et al., 1994a, b; 1995). Hazewinkel et al. (1987) indicated that rickets in dogs could not be prevented or treated by ultraviolet radiation; these dogs developed clinical, biochemical and histological signs of rickets. In the skin of the dog and cat the concentrations of the precursor 7-dehydrocholesterol are low and the precursor is inadequately converted to vitamin D. It is suggested that carnivores do not need to provide their own vitamin D, since fat, liver and blood of their prey will fulfill this need (How et al.,1995).Vitamin D requirements of cats and dogs are suggested to be sufficiently high to produce normal growth, calcification, production and reproduction, provided that diets contain recommended levels of calcium and available phosphorus. Species differences can be illustrated by the fact that adequate intakes of calcium and phosphorus in a diet that contains only enough vitamin D to produce normal bone in the rat or pig will quickly cause the development of rickets in chicks. Cats and dogs have low vitamin D requirements when calcium, phosphorus and the ratio of the minerals are correct. The need for vitamin D depends to a large extent on the ratio of calcium to phosphorus. As this ratio becomes either wider or narrower than the optimum, the requirement for vitamin D increases, but no amount will compensate for severe deficiencies of either calcium or phosphorus. A calcium to phosphorus ratio of 1.2:1 is suggested for dogs with no optimum ratio yet established for cats (NRC, 2006).Amounts of dietary calcium and phosphorus and the physical and chemical forms in which they are presented must be considered when determining requirements for vitamin D. High dietary calcium concentrations can precipitate phosphates as insoluble calcium phosphate. Soluble calcium salts are more readily absorbed, and oxalates tend to interfere with absorption, but some of this interference can be overcome by dietary vitamin D. Correspondingly, while the phosphorus of inorganic orthophosphate tends to be well absorbed, other factors being favorable, that of phytic acid, which is the predominant phosphorus compound of unprocessed cereal grains and oilseeds, seems to be poorly available to monogastric species. Phosphorus absorption is mostly independent of vitamin D intake, with the inefficient absorption in rickets being secondary to failure of calcium absorption, and the improvement upon vitamin administration being a result of improved calcium absorption.
Requirements for vitamin D are dependent on dietary concentrations of calcium and phosphorus, the dietary calcium:phosphorus ratio, physiological stage of development and perhaps sex and breed (NRC, 2006). Kozelka et al. (1933) found that collie puppies were protected from rickets by 1 to 1.3 IU vitamin D (irradiated ergosterol) per kg (0.45 to 0.59 IU per lb) of body weight per day. When a diet containing low levels of calcium (0.08%) and phosphorus (0.1%) was fed to pups, they developed rickets. Three of five pups fed the low calcium and phosphorus diet with 100 IU vitamin D per kg (45.5 IU per lb) of body weight daily did not develop rickets while a fourth had very slight rachitic changes. Part-great dane puppies were fed with a calcium:phosphorus ratio of 1.2:1 or 2.0:1 and provided 12 IU vitamin D per kg (5.5 IU per lb) of body weight per day. The puppy receiving a calcium:phosphorus ratio of 2.0:1 became severely rachitic (Arnold and Elvehjem, 1939). Michaud and Elvehjem (1944) concluded that, with a dietary calcium:phosphorus ratio of 1.2:1, daily intakes of 10 to 20 IU vitamin D per kg (4.5 to 9.1 IU per lb) of body weight were adequate, even for large breeds.The current NRC (2006) vitamin D recommendations for dogs is 13.8 µg of vitamin D3 per kg of diet (6.3 µg per lb) for all classes of dogs. The AAFCO (2007) recommendation is 500 IU per kg (227 IU per lb) of diet.
Cats have a low requirement for vitamin D. In kittens, rickets is generally due to calcium deficiency or imbalanced calcium:phosphorus ratio rather than to vitamin D deficiency. Rivers et al. (1979) reported that the incidence of rickets in cats is almost totally independent of a dietary source of vitamin D, even during growth, assuming they are fed a diet with adequate concentrations (and a correct ratio) of calcium and phosphorus.Gershoff et al. (1957b) found that 250 IU of cholecalciferol given orally, twice a week, prevented the development of rickets in kittens fed a semi-purified diet from three to six months of age to 21 months of age. Diets containing 1,111 IU of vitamin D per kg (505 IU per lb) of dry weight have protected kittens from rickets (Gershoff, 1972). The current dietary vitamin D recommendation of theNRC (2006) for kittens is that they be provided 5.6 µg vitamin D3 per kg (2.5 µg vitamin D3 per lb) of diet. For adults at maintenance and for cats in late gestation or peak lactation the requirement is 7.0 µg vitamin D3 per kg (3.2 µg vitamin D3 per lb) of diet. On a feed basis, AAFO (2007) recommends 750 IU per kg (341 IU per lb) for cats in growth and reproduction and 500 IU per kg (227 IU per lb) for maintenance.
Sources of vitamin D are feedstuffs, irradiation, sebaceous material licked from skin or hair or directly absorbed products. For dogs and cats (and presumably other carnivores), vitamin D must be obtained from dietary sources due to the inability of these species to synthesize and utilize vitamin D from precursors in the skin (How et al., 1995).
For grazing livestock in the presence of UV light, no dietary sources of vitamin D are required. The distribution of vitamin D is very limited in nature, although provitamins occur widely. Grains, roots and oilseeds and their numerous by-products for livestock feeds contain insignificant amounts of vitamin D; green fodders are equally poor sources. Ergocalciferol occurs naturally in some mushrooms and cholecalciferol occurs naturally in fish (Johnson and Kimlin, 2006). When various plants, especially pasture species, begin to die and the fading leaves are exposed to UV light, some vitamin D2 is formed, producing vitamin D activity in hay. Its potency depends on local climatic conditions. If produced very quickly in the absence of direct sunlight and baled when still quite green, its potency will be low (Abrams, 1952). The principal source of the antirachitic factor in the diets of farm animals is provided in the action of radiant energy upon ergosterol in forages. Legume hay that is cured to preserve most of its leaves and green color. This contains considerable amounts of vitamin D activity. Alfalfa, for example, will range from 650 to 2,200 IU per kg (295 to 1,000 IU per lb) (Maynard et al., 1979).
Artificially dried and barn-cured hay contains less vitamin D than hay that is properly sun-cured. Even hay dried in the dark immediately after cutting has some of the vitamin present. This is because the dead or injured leaves on the growing plant are responsive to UV irradiation even though the living tissues are not. This is also largely responsible for the vitamin D found in corn silage (Maynard et al., 1979). Under normal conditions, even wilting legume silage furnishes ample vitamin D for livestock. With a few notable exceptions, vitamin D3, is not found in plants. These exceptions include the species Solanum malacoxylon, Cestrum diurnum and Trisetum flavescens (see section on vitamin safety) in which vitamin D occurs as water-soluble beta-glycosides of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3.
For concentrate feed mixtures, the vitamin D that occurs naturally in unfortified feed is generally derived from animal products. Saltwater fish, such as fish liver oils, are extremely rich sources. The probable origin of vitamin D in fish liver is a result of food chains from plankton (Takeuchi et al., 1991). Milk contains a variable amount in it’s fat fraction (5 to 40 IU in cow milk per quart), but neither cow nor human milk contains enough to protect the newborn against rickets (Maynard et al., 1979). Cows milk is reportedly higher in vitamin D when produced during the summer, compared to the winter.
It has generally been assumed that for all but a few species, vitamin D2 and D3 are equally potent. For poultry and other birds and a few of the rare mammalian species that have been studied, including some New World monkeys, vitamin D3 is many times more potent than D2 on a weight basis. Recent evidence indicated that in man, vitamin D2has only 25-30 percent of the biological activity of vitamin D3 (Armas et al., 2004).
The dogma that mammals (other than the New World monkey) do not discriminate between vitamin D2 and D3 has proven incorrect. Data for the pig (Horst and Napoli, 1981) and for ruminants (Sommerfeldt et al., 1981) suggest that these species discriminate in the metabolism of vitamin D2 and D3, with the vitamin D3 being the preferred substrate. Pigs given oral doses of a mixture of vitamin D2 and D3 (1:1 in w/w basis had significantly higher concentrations of plasma vitamin D3, 25-(OH)D3, 24,25-(OH)2D3 and 1,25-(OH)2D3, than corresponding vitamin D2counterparts. Sommerfeldt et al. (1983) reported that the amount of 1,25-(OH)2D in the plasma of ergocalciferol-treated dairy calves was one-half to one-fourth the amount of the cholecalciferol-treated calves. Discrimination against vitamin D2 by ruminants may be, in part, a result of its preferred degradation by rumen microbes or less efficient intestinal absorption. Although recent data suggest a preference for D3 by a number of animals, in practice D2 is still relatively comparable to D3 in antirachitic function except for poultry and certain monkeys. The differential utilization between cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) has not been investigated in dogs, but in cats cholecalciferol is utilized more efficiently than is ergocalciferol to maintain plasma concentrations of 25-(OH)D (Morris, 2002).
Vitamin D3 is the principal source of supplemental vitamin D for livestock and poultry. Vitamin D3 product forms for feed include stabilized gelatin beadlets (with vitamin A), oil dilutions, oil absorbates, emulsions, and spray- and drum-dried powders. Test results have shown that the gelatin beadlet form offers the greatest vitamin D3 stability. Incorporating vitamins D3 and A in the beadlet form provides physical protection from oxidation, and the selected antioxidants included in the beadlets afford chemical protection.
Due to lack of vitamin D in feeds and management systems without direct sunlight, modern livestock and pet operations must provide a supplemental source of the vitamin. Illustrating the diet limitation of vitamin D, a typical corn-soybean meal based diet would contain zero vitamin D. In relation to pets, many animals are kept indoors and dogs are walked at night or in less than sunny places. However, this is of little importance since dogs and cats must rely on dietary sources of vitamin D, as they receive insignificant vitamin D from UV sun irradiation of the skin.
The primary vitamin D deficiency disease is a bone disorder called rickets in young animals. It is generally characterized by a decreased concentration of calcium and phosphorus in the organic matrices of cartilage and bone. Vitamin D results in clinical signs similar to those indicating a lack of calcium or phosphorus or both, as all three are concerned with proper bone formation. In the adult, osteomalacia is the counterpart of rickets and, since cartilage growth has ceased, is characterized by a decreased concentration of calcium and phosphorus in the bone matrix. Outward signs of rickets include the following skeletal changes, varying somewhat with species depending on anatomy and severity: (a) weak bones causing curving and bending of bones, (b) enlarged hock and knee joints, (c) tendency to drag hind legs and (d) beaded ribs and deformed thorax. Although there appear to be differences among species in the susceptibility of different bones to such degenerative changes, as well as differences that probably reflect bodily conformation (e.g., dog compared with sheep), there is nevertheless an apparent common pattern (Abrams, 1978). Spongy parts of individual bones and bones relatively rich in such tissues are generally the first and most severely affected. As in simple calcium deficiency, the vertebrae and the bones of the head suffer the greatest degree of resorption. Next come the scapula, sternum and ribs. The most resistant bones are metatarsals and shafts of long bones. Several methods have been used to assess nutritional status of animals deficient in vitamin D. Poor growth rates as well as bone abnormalities in both animals and humans are the chief indications when vitamin D deficiency is substantially advanced. The incomplete calcification of the skeleton is easily detectable with X-rays and reduced bone ash but, like other production-related signs, would not be specific for vitamin D deficiency versus other nutrient inadequacies (e.g., calcium and phosphorus). Deviations from normal in serum calcium, phosphorus and alkaline phosphatase are associated with rickets. For rickets in kittens, serum alkaline phosphatase activity increased markedly in the third month, peaked during the fifth to seventh months, and decreased through the twenty-first month. Serum calcium and inorganic phosphorus concentrations decreased markedly during the acute phase of rickets (Gershoff et al., 1957b).
The dog was one of the first animals in which rickets was produced experimentally. In 1922, Mellanby of Great Britain produced rickets in dogs by feeding them oatmeal (McDowell, 2000). Mellanby (1921), in a summary of his studies on rickets (in which nearly 400 puppies were used), stated that rickets occurred more rapidly in fast-growing than slow-growing dogs. Dogs with rickets became lethargic and had a general loss of muscular tone which did not allow them to run quickly. Rickets in dogs is similar radiographically, histopathologically, and biochemically to the disease in other animals or human beings. An early indication of rickets on radiology was a change in shape of the epiphysis of the distal ulna. This became flattened, with a wavy and indefinite outline, and there was an increase in the width of the epiphyseal cartilage and the adjacent newley calcified bone lost contrast (NRC, 2006). Rickets with typical bone lesions is readily produced in dogs, but clinical signs are frequently confounded by a simultaneous deficiency or imbalance of calcium and phosphorus (NRC, 2006), both resulting in an initial hypocalcemia.Campbell and Douglas (1965) fed a 0.5% calcium and 0.3% phosphorus diet, with no supplemental vitamin D, to puppies for 15 weeks without signs of rickets or osteoporosis. However, when the diet contained 0.08% to 0.10% calcium and 0.13% to 0.15% phosphorus and no supplemental vitamin D, rickets complicated by osteoporosis was observed. Rickets diagnosis in a 12-week-old female St. Bernard was attributed to an inborn error in vitamin D metabolism (Johnson et al., 1988). Physical examination revealed enlargement of the costochondral junctions and the distal metaphyses of the radius, ulna, femur and tibia. When standing, the elbows were slightly abducted and there was mild valgus deviation of the front paws. The dog showed no lameness but was lethargic and inactive. Radiographically, the physes were enlarged radially and axially, and metaphyseal bone adjacent to the physes was widened and cup-shaped. Serum biochemical abnormalities were hypocalcemia, hypomagnesemia, and hyperparathyroidism.
Severe rickets in kittens resulted in enlarged costochondral junctions (“rachitic rosary”) with disorganization in new bone formation and excessive osteoid (NRC, 1986). Classic signs of rickets are rare in kittens and confined to those born in winter, kept permanently in dark quarters, or from queens fed vitamin D-deficient diets.Severe rickets in kittens was produced using vitamin D-deficient diets containing either 1% calcium and 1% phosphorus or 2% calcium and 0.65% phosphorus (Gershoff et al., 1957b). Weight gain was less with the latter diet, and rickets was less severe. Rickets, which developed in about four to five months, was characterized by radiographic and morphologic changes that were similar to bone lesions observed in other species with the disease. The cats that died during acute rickets had a lower percent femur ash than did cats supplemented with vitamin D. Experimental vitamin D deficiency has been produced in cats, resulting in neurologic abnormalities associated with degeneration of the cervical spinal cord (Morris. 1996; Handet al., 2010). Other signs included hypocalcemia, posterior paralysis, ataxia and eventual quadriparesis.
Because natural foods are low in this vitamin, most commercially prepared pet foods are enriched with vitamin D to ensure that dogs and cats receive adequate amounts of this vitamin. This is particularly important since recent studies have shown conclusively that neither dogs or cats receive a significant benefit from synthesis of vitamin D in the skin through exposure to UV irradiation (How, et al., 1994a, b; 1995). Vitamin D deficiency in dogs and cats receiving commercially prepared foods is not common. Rickets is more common with diets that are low in vitamin D and have an accompanying deficiency in dietary calcium and/or phosphorus, or with diets that are imbalanced with respect to calcium and phosphorus (where calcium percentage is less than phosphorus). A number of studies have shown that more vitamin D is required to correct an imbalance of calcium and phosphorus.Morris (1999) found that the time taken for clinical signs of vitamin D deficiency to appear in kittens given a purified vitamin D-free diet (0.8 percent calcium and 0.6 percent phosphorous) depended on the kittens’ initial stores of the vitamin. Kittens from queens receiving a diet containing a high level of vitamin D (400 µg cholecalciferol per kg or 181.8 µg per lb) during pregnancy and lactation were able to complete their growth phase without clinical signs of vitamin D deficiency. However, those from a queen receiving a lower intake of cholecalciferol (about 25 µg per kg or 11.4 µg per lb) in the diet had clinical signs of vitamin D deficiency in six weeks.
Kealy et al. (1991), in studies with weanling pups, suggested that supplementation of nonpurified, commercially available dog foods with vitamin D may not be necessary. However, this study did not analyze the vitamin D content of the commercial diet, which did contain some animal products potentially rich in the vitamin (e.g., meat and bone meal). Pet foods that contain high protein animal by-products (e.g., blood meal and liver) would likely not need supplemental vitamin D. Cat foods, in particular, that contain fish products would be receiving substantial amounts of vitamin D. Although less than for vitamins A and B12, body storage of vitamin D occurs. During times of low dietary vitamin D concentrations in pet foods, puppies and kittens may be relying on previous stores of the vitamin.
Both synthetic D2 and D3 are quite stable when stored at room temperature. In complete feeds and mineral-vitamin premixes, Schneider (1986) reported activity losses of 10% to 30% after either four or six months of storage at 22°C. However, pure vitamin D3 crystals or vitamin D3 resin is very susceptible to degradation upon exposure to heat or contact with mineral elements. In fact, the resin is stored under refrigeration with nitrogen gas. Dry, stabilized supplements retain potency much longer and can be used in high mineral supplements. It has been shown that vitamin D3 is much more stable than D2 in feeds containing minerals.
Stabilization of the vitamin can be achieved by (a) rapid compression of the mixed feed, for example, into pellets so that air is excluded; (b) storing feed under cool, dry, dark conditions; (c) preventing close contact between the vitamin and potent metallic oxidation catalysts (e.g., manganese); (d) including natural or synthetic antioxidants in the mix. The vitamin can also be protected by enclosing it in durable, gelatin beadlets.
Stability of dry vitamin D supplements is affected most by high temperature, high moisture content and contact with trace minerals such as ferrous sulfate, manganese oxide and others. Hirsch (1982) reports the results of a “conventional” or non-stabilized vitamin D3 product being mixed into a trace mineral premix or into animal feed and stored at ambient room temperature (20°to 25°C) for up to 12 weeks. The feed had lost 31% of its vitamin D activity after 12 weeks, and the trace mineral premix had lost 66% of its activity after only six weeks in storage.
Vitamin D deficiencies may result from (a) errors in vitamin addition to diets, (b) inadequate mixing and distribution in feed, (c) separation of vitamin D particles after mixing, (d) instability of the vitamin content of the supplement, or (e) excessive length of storage of diets under environmental conditions causing vitamin D loss (Hirsch, 1982).
Supplementation considerations are dependent on other dietary ingredients. As previously noted, the requirements for vitamin D are increased several fold by inadequate levels of calcium and/or phosphorus or by improper ratios of these two elements in the diet. A number of reports have indicated that molds in feeds interfere with vitamin D (Cunha, 1977); for example, when corn contains the mold Fusarium roseum, a metabolite of this mold prevents vitamin D3 in the intestinal tract from being absorbed by the chick. A similar deleterious effect on vitamin D metabolism would be expected in dogs and cats.
Other factors that influence vitamin D status are diseases of the endocrine system, intestinal disorders, liver malfunction, kidney disorders and drugs. Liver malfunction limits production of the active forms of the vitamin, while intestinal disorders reduce absorption.
In veterinary medicine, there are clinical conditions for which the judicious employment of vitamin D analogues may be warranted. Evidence suggests that impaired intestinal absorption of calcium due to an acquired defect in vitamin D metabolism plays a significant role in the development of hypocalcemia and bone disorders in chronic renal insufficiency and uremia. Chronic renal disease interferes with the production of 1,25-(OH)2D3 by the kidney, thereby diminishing intestinal calcium transport and resulting in development of hypocalcemia. Gerber et al. (2003) reported both 25-(OH)D3 and 1,25-(OH)2D3 to be significantly lower in dogs with acute renal failure and chronic renal failure. Plasma levels of 25-(OH)D3 were lower following massive resection of the distal small bowel (75%) in adult beagle dogs (Imamura and Yamaguchi, 1992). Concentrations of serum 25-(OH)D were lower in dogs with inflammatory bowel disease than in healthy dogs (Gow et al.., 2011). In dogs with chronic renal disease, 1,25-(OH)2D3 has been advocated for use as a therapeutic agent in the prevention of hypocalcemia and osteodystrophy, with daily dosages as high as 0.1 µg per kg (0.05 µg per lb) body weight (Lewis et al., 1987). Because toxic manifestations of hypervitaminosis D are associated with hypercalcemia, serum calcium levels must be closely monitored when 1,25-(OH)2D3 is given (Lewis et al., 1987; NRC, 2006). For dogs, a high dose of 1,25(OH)2D3 has been shown to exhibit antitumor activity for some animals and is used in cancer chemotherapy (Rassnick et al., 2011).
After vitamin A, vitamin D is the vitamin next most likely to be consumed in concentrations toxic to animals. Although vitamin D is toxic at high concentrations, short-term administration of as much as 100 times the requirement level may be tolerated. For most species, the presumed maximal safe level of vitamin D3 for long-term feeding conditions (more than 60 days) is four to 10 times the dietary requirement. Studies in a number of species indicate that vitamin D3 is 10 to 20 times more toxic than vitamin D2 when provided in excessive amounts (NRC, 1987). Excessive intake of vitamin D produces a variety of effects, all associated with abnormal elevation of blood calcium. Elevated blood calcium is caused by greatly stimulated bone resorption, as well as increased intestinal calcium absorption. The main pathological effect of ingestion of massive doses of vitamin D is widespread calcification of soft tissues. Pathological changes in these tissues are observed to be inflammation, cellular degeneration, and calcification. Diffuse calcification affects joints, synovial membranes, kidneys, myocardium, pulmonary alveoli, parathyroids, pancreas, lymph glands, arteries, conjunctivae, and corneas. More advanced cases interfere with cartilage growth. As would be expected, the skeletal system undergoes a simultaneous demineralization that results in the thinning of bones. Other common observations of vitamin D toxicity are loss of appetite, extensive weight loss, elevated blood calcium, and lowered blood phosphate. Vitamin D toxicity is enhanced by elevated supplies of dietary calcium and phosphorus and is reduced when the diet is low in calcium. Over-supplementation of vitamin D was a risk factor for chronic heart failure in fast growing commercial broilers (Nain et al., 2007). A diet containing 80,000 IU per kg (36,364 IU per lb) resulted in birds with cardiac arrhythmia and negative QRS axis on lead-ll (an indication of left heart failure), compared to controls. Tryfonidou et al. (2003) reported that excessive vitamin D3 supplementation below the toxic level decreases bone remodeling and causes focal enlargement of the growth plate in growing puppies. Excessive vitamin D concentrations may result in hypercalcemia, soft-tissue calcification and ultimately death (Nakamura et al., 2004).
Toxicity caused by excess vitamin D administration is also associated with plasma 25-(OH)D; concentrations of more than 400 ng per ml are reported (Hughes et al., 1976). Patients suffering from hypervitaminosis D have been shown to exhibit a 15-fold increase in plasma 25-(OH)D concentration as compared with normal individuals.
Morgan et al. (1947) administered a single oral dose of 314,000 to 530,000 IU vitamin D as irradiated ergosterol per kg (142,727 to 240,090 IU per lb) of body weight to 4- to 5-week-old puppies. All exhibited anorexia, polyuria, bloody diarrhea, polydipsia, and prostration. Three were dead within two weeks and a fourth was moribund in five weeks. Extensive calcification was found in the lungs of these dogs, and moderate calcification in the hearts and kidneys. In the dogs that survived, malocclusion, pitting, irregular placement, and poor development of the teeth were seen.Hendricks et al. (1947) fed 10,000 IU vitamin D daily per kg (4,545 IU per lb) of body weight to weaned cocker spaniel puppies. Anorexia developed, growth was retarded, serum calcium was variably increased, jaws and teeth were deformed and soft tissues were calcified–particularly the lungs, kidneys, and stomach. An ill advised high dose injection of vitamin D in a puppy resulted in severe calcification of mucocutaneous and gastrointestinal tissues (Nakamura et al., 2004). After two months, the gastrointestinal and skin disorders disappeared, although calcification of the stomach membranes remained and abnormality of the skeletal system had worsened.
Effects of overdosage of vitamin D were observed at necropsy in a cat that had been given 5 million IU of vitamin D3and 2.5 million IU of vitamin A by mouth over a six-month period. The cat received these vitamins as treatment for a skin ailment, but gradually lost weight and died suddenly. The great vessels, including the aorta and the carotid arteries, and the adrenals were heavily calcified, and calcium was deposited in the stomach wall and parathyroids (Suter, 1957).
Feeding a milk replacer to airedale puppies resulted in poor development and condition, impaired moving capacity, retarded change of teeth and pathological changes in the kidney (renal calcification and sclerosis, fibrosis of glomerula, dilation of the tubuli). The supplement contained excess vitamin D at 3.45 million IU per kg (1.57 million IU per lb).
Historically, vitamin D toxicosis was rarely considered in dogs, and was generally associated with chronic dietary or therapeutic oversupplementation. Reports of hypervitaminosis D in cats have resulted from either accidental ingestion of rodenticides containing cholecalciferol as the active ingredient, consumption of diets based on fish (particularly fish viscera), or errors in diet formulation. Acute vitamin D poisoning has become more common through the ingestion of vitamin D3 rodenticides containing 0.075% cholecalciferol (Livezey and Dorman, 1991; Garlock et al., 1991). Toxicosis due to the ingestion of these products must therefore be included in the differential diagnosis for hypercalcemia in dogs and cats.
Cholecalciferol (vitamin D3) rodenticides have caused significant toxicity in dogs at a fraction of the manufacturer’s reported LD50 for dogs, 88 mg pure cholecalciferol per kg (40 mg per lb) (Garlock et al., 1991). These researchers described a dog estimated to have consumed 1.5 to 3.0 mg per kg (0.68 to 1.36 mg per lb) of body weight of pure cholecalciferol. Clinical signs most commonly associated with the resultant hypercalcemia are polyuria, polydipsia, depression, anorexia, weakness, and vomiting.
Caution for vitamin D toxicity must also be used when formulating home diets for dogs and cats or when enhancing the palatability of commercial diets with higher levels of liver or fish oil, which are all rich sources of vitamin D. Strombeck (1999) hypothesizes that the high incidence of kidney disease in dogs and cats may be related to commercial pet foods that contain excess vitamin D.
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