Vitamin E activity in foods and feedstuffs is derived from a series of compounds of plant origin. Natural vitamin E is the mixture of two classes of compounds, tocopherols and tocotrienols. The term vitamin E, according to the International Union of Pure and Applied Chemistry-International Union of Biochemistry (IUPAC-IUB) Commission on Biochemical Nomenclature, is used as a generic descriptor for all tocol and tocotrienol derivatives that qualitatively exhibit the biologic activity of alpha-tocopherol (IUPAC-IUB, 1973). Both the tocols (tocopherols) and tocotrienols consist of a hydroquinone nucleus and an isoprenoid side chain. Characteristically, tocols have a saturated side chain, whereas the tocotrienols have an unsaturated side chain containing three double bonds. There are four principal compounds of each of these two sources of vitamin E activity (alpha, beta, gamma, delta), differentiated by the presence of methyl (-CH3) groups at positions 5, 7 or 8 of the chroman ring (Figure 4-1). Alpha-tocopherol, the most biologically active of these compounds, is the predominant vitamin E active compound in feedstuffs and the form used commercially for supplementation of animal diets. The biological activity of the other tocols is limited (Table 4-1), but some new functions have recently been found for non alpha-tocopherol forms of vitamin E (Schaffer et al., 2005; Freiser and Jiang, 2009).
There are three asymmetric carbon atoms in the tocopherol molecule located at the 2’, 4′, and 8′ positions. The d-form of alpha-tocopherol has all of the methyl groups in these positions facing in one direction and is referred to as the RRR-form. This is the form found in plants. The dl- or chemically synthesized form of alpha-tocopherol has an equal mixture of the R and S configurations at each of the three positions, (i.e., it contains eight stereoisomers) and is referred to as the all-rac (for racemic) form of the compound.
Commercially there is no truly “natural” tocopherol product available since the d-alpha-tocopherol commercial products are obtained from the original raw material only after several chemical processing steps. Hence, it should be referred to as “natural-derived” and not natural. In addition, the international unit (IU) is the standard of vitamin E activity, and consequently it is the same regardless of the source. However in some studies in several species comparing the natural derived RRR to the synthetic dl-alpha-tocopherol, has been shown to be more effective in elevating plasma and tissue concentrations when administered on an equal IU basis (Jensen et al., 2006). Recent studies with humans, sheep, pigs, guinea pigs, fish and horses in which the elevation of plasma concentrations was measured, indicated that the biopotency of RRR-α-tocopherol compared to all-rac-α-tocopherol was closer to 2:1 than 1.36:1 (Lauridsen et al., 2002; Hidiroglou et al., 2003).
Typically, deodorizer distillates produced during the purification and manufacture of vegetable oils are utilized in the production of d-alpha-tocopherol or tocopheryl acetate. These deodorizer distillates contain a mixture of alpha-, beta-, gamma- and delta-tocopherols, and this mixture is extracted and purified. In the final process, ultra-vacuum molecular distillation is performed and the end material methylated to produce an alpha-tocopherol concentrate. This material may then be acetylated. The acetate ester is used because it is more stable in processing and storage of foods and feeds than the alcohol (tocopherol) form.
All-rac-alpha-tocopheryl acetate is the most common vitamin E form used to supplement animal feeds. This form of vitamin E is manufactured by condensing trimethyl hydroquinone and isophytol and conducting ultra-vacuum molecular distillation, producing a highly purified form of alpha-tocopherol. This material may then be acetylated. As previously stated, all-rac-alpha-tocopherol is a mixture of eight stereoisomers (four enantiomeric pairs) of alpha-tocopheryl acetate. The enantiomeric pairs, racemates, have been shown to be present in equimolar amounts (Cohen et al., 1981; Weiser and Vecchi, 1981, 1982; Scott et al., 1982). This finding indicates that the manufacturing processes employed lead to all-rac-alpha-tocopheryl acetate with similar proportions of all eight stereoisomers (Weiser and Vecchi, 1982).
Alpha-tocopherol is a yellow oil that is insoluble in water but soluble in organic solvents (Illus. 4-1). Tocopherols are extremely resistant to heat, but readily oxidized. Natural vitamin E is subject to destruction by oxidation, which is accelerated by heat, moisture, rancid fat, copper, and iron. Alpha-tocopherol is an excellent naturally occurring antioxidant that protects carotene and other oxidizable materials in the feed and in the body. However, in the process of acting as an anti-oxidant, it is oxidized and becomes biologically inactive.
The naturally occurring tocopherol form is subject to destruction in the digestive tract to some extent, while the acetate ester is not. Much of the acetate is readily split off in the intestinal wall and the alcohol is absorbed, thereby permitting the vitamin to function as a biological antioxidant. Any acetate form absorbed into the body is converted to the alcohol form.
Vitamin E absorption is related to fat digestion and is facilitated by bile and pancreatic lipase (Sitrin et al., 1987). The efficiency of vitamin E adsorption is low and variable (35 to 50%); the absorption efficiency is much lower than that of vitamin A. Absorption of vitamin E is enhanced by the simultaneous digestion and absorption of dietary fats. Whether presented as free alcohol or as esters, most vitamin E is absorbed as the alcohol. Esters are largely hydrolyzed in the intestinal wall, and the free alcohol enters the intestinal lacteals and is transported via the lymph to the general circulation. An alpha-tocopherol transfer protein has been identified (Traber, 2006).
The animal appears to have preference for tocopherol versus other tocols. Rates and amounts of absorption of the various tocopherols and tocotrienols are in the same general order of magnitude as their biological potencies. Alpha-tocopherol is absorbed best, with gamma-tocopherol absorption slightly less than that of alpha-forms but with a more rapid excretion. It can be generally assumed that most of the vitamin E activity within plasma and other animal tissues is alpha-tocopherol (Ullrey, 1981). Vitamin E in plasma is attached mainly to lipoproteins in the globulin fraction within cells and occurs mainly in mitochondria and microsomes. The vitamin is taken up by the liver and is released in combination with low-density lipoprotein (LDL) cholesterol.
Vitamin E does not cross the placenta in any appreciable amounts; however, it is concentrated in colostrum (Van Saunet al., 1989). With respect to neonatal ruminants (Hidiroglou et al., 1969; Van Saun et al., 1989; Njeru et al., 1994) and baby pigs (Mahan, 1991), several investigators have reported limited placental transport of alpha-tocopherol, making neonates highly susceptible to vitamin E deficiency. This may be related to either a decreasing efficiency in placental vitamin E transfer as gestation proceeds, a dilution effect as a result of rapid fetal growth or possibly a decrease in available maternal vitamin E. With limited placental transfer of vitamin E, newborns must rely heavily on ingestion of colostrum as a source of vitamin E. Van Saun et al. (1989) reported decreased fetal serum vitamin E concentrations with increasing fetal age. Additionally, these authors reported less of a decline in fetal serum vitamin E concentration during gestation in fetuses from vitamin E-adequate dams.
There is inefficient placental transfer of vitamin E but high levels of the vitamin have been shown in calves (Nockels, 1991) and lambs (Njeru et al., 1994) after consumption of colostrum. Nockels (1991) reported alpha-tocopherol levels in plasma from beef calves prior to colostrum consumption, and for several days thereafter. Precolostral plasma vitamin E levels averaged 0.2 µg per ml and increased to 3.3 µg per ml at five to eight days of age.
Njeru et al.(1994) fed ewes dl-alpha-tocopherol acetate at graded levels (0, 15, 30 and 60 IU per head daily) to study placental and mammary gland transfer. Supplemental vitamin E had no effect on serum alpha-tocopherol of lambs prior to nursing, averaging 0.35 µg per ml. By day 3, lamb serum tocopherol increased to 1.41, 1.84, 2.43 and 4.46 µg per ml, respectively, for the four supplemental dietary levels of vitamin E (Table 4-2).
Vitamin E at the given levels of supplementation increased colostral alpha-tocopherol at a linear rate of 3.3, 6.8, 8.0, and 9.6 µg per ml, respectively. The importance of providing colostrum rich in vitamin E is quite apparent, as both calves and lambs are born with low levels of the vitamin (Nockels, 1991; Njeru et al., 1994). Low blood vitamin E may lead to diminished disease resistance and immune response in the neonate (Nockels, 1991).
Less than 1% of the dam’s tocopherol intake is secreted in the milk (Millar et al., 1973), however, colostrum tocopherol concentration is directly affected by maternal intake (Whitting and Loosli, 1948). Levels of vitamin E in the colostrum are considerably higher than in milk.
Vitamin E is stored throughout all body tissues, with highest storage in the liver. However, liver contains only a small fraction of total body stores, in contrast to vitamin A, for which about 95% of the body reserves are in the liver. Small amounts of vitamin E will persist tenaciously in the body for a long time. However, stores are exhausted rapidly by polyunsaturated fatty acids (PUFA) in the tissues, the rate of disappearance being proportional to the intake of PUFA. A major excretion route of absorbed vitamin E is bile in which tocopherol appears mostly in the free form (McDowell, 2000). Tocopherol entering the circulatory system becomes distributed throughout the body with the majority localizing in the fatty tissues. Subcellular fractions from different tissues vary considerably in their tocopherol content, with the highest levels found in membranous organelles, such as microsomes and mitochondria, that contain highly active oxidation-reduction systems (McCay et al., 1981; Taylor et al., 1976)
Vitamin E has been shown to be essential for integrity and optimum function of reproductive, muscular, circulatory, nervous, and immune systems (Hoekstra, 1975; Sheffy and Schultz, 1979; Bendich, 1987; McDowell, 2000). It is well established that some functions of vitamin E, however, can be fulfilled in part or entirely by traces of selenium or by certain synthetic antioxidants. Even sulfur-bearing amino acids, cystine and methionine, affect certain vitamin E functions. Considerable evidence indicates there may be undiscovered metabolic roles for vitamin E, which may be paralleled biologically by roles of selenium and possibly other substances. The most widely accepted functions of vitamin E are discussed in this section.
Vitamin E has several different but related functions. One of the most important functions is its role as an intercellular and intracellular antioxidant (Figure 4-2). Vitamin E is part of the body’s intracellular defense against the adverse effects of reactive oxygen and free radicals that initiate oxidation of unsaturated phospholipids (Chow, 1979) and critical sulfhydryl groups (Brownlee et al., 1977).
Figure 4-2: Placement of Antioxidant Systems Within the Cell
In dogs antioxidants, including vitamin E, can achieve sustained increases in circulating levels of antioxidants that exert a protective effect by decreasing in DNA damage. This leads to improved immunological performance (Heaton et al., 2002). Free-radical damage has been associated with a variety of degenerative disorders and the aging process in general (Jewell et al., 2000; Skoumalova et al., 2003). Disorders where vitamin E has been beneficial to dogs and or cats include chronic hepatitis (Honeckman, 2003), renal failure (Yu and Paetau-Robinson, 2006; Varzi et al., 2007), congestive heart failure (Freeman et al., 2005) and immune-mediated hemolytic anemia (Pesillo et al., 2004).
Free-radical reactions are ubiquitous in biological systems and are associated with energy metabolism, biosynthetic reactions, natural defense mechanisms, detoxification, and intra- and intercellular signaling pathways. Support for the antioxidant role of vitamin E in vivo also comes from observations that synthetic antioxidants can either prevent or alleviate certain clinical signs of vitamin E deficiency diseases.
In order to improve the oxidative stability, and thus increase the shelf life of meat, antioxidants have been successfully added to animal feeds. In the last few years, different compounds such as carotenoids, vitamin C, selenium and plant extracts have been tested in different experiments in order to verify their potential antioxidant effect on meat. Of all of them, alpha-tocopherol has demonstrated the highest biological efficiency in preventing the lipid oxidation in vivo (Barroeta, 2007).
Vitamin E functions as a membrane-bound antioxidant, trapping lipid peroxyl free radicals produced from unsaturated fatty acids under conditions of “oxidative stress.” Orientation of vitamin E within cell membranes appears to be critical to its functionality (Dunnett, 2003). Lipids, especially phospholipids, present in cell membranes are particularly susceptible to oxidative damage. This damage is positively correlated with the degree of unsaturation of its fatty acids.
Vitamin E functions as a quenching agent for free radical molecules with single, highly reactive electrons in their outer shells. Free radicals attract a hydrogen atom, along with its electron, away from the chain structure, satisfying the electron needs of the original free radical, but leaving the PUFA short one electron. Thus, a fatty acid free radical is formed that joins with molecular oxygen to form a peroxyl radical that steals a hydrogen-electron unit from yet another PUFA. This reaction can continue in a chain, resulting in the destruction of thousands of PUFA molecules (Gardner, 1989; Herdt and Stowe, 1991). Free radicals can be extremely damaging to biological systems (Padh, 1991).
Highly reactive oxygen species such as the superoxide anion radical (O2-), hydroxyl radical (·OH), hydrogen peroxide (H2O2) and singlet oxygen (O2-) are continuously produced in the course of normal aerobic cellular metabolism. Also, phagocytic granulocytes undergo respiratory bursts to produce oxygen radicals to destroy the intracellular pathogens. However, these oxidative products can, in turn, damage healthy cells if they are not eliminated. Antioxidants serve to stabilize these highly reactive free radicals, thereby maintaining the structural and functional integrity of cells (Chew, 1995). Therefore, antioxidants are very important to immune defense and health of humans and animals.
The antioxidant function of vitamin E is closely related to and synergistic with the role of selenium. Selenium has been found to be part of 25 selenoproteins with most of the functions unknown, although these selenoproteins generally participate in antioxidant and anabolic processes (Hatfield and Gladyshev, 2002). Selenium has been shown to act in aqueous cell media (cytosol and mitochondrial matrix) by destroying hydrogen peroxide and hydroperoxides via the enzyme glutathione peroxidase (GSH-Px), of which it is a co-factor. In this capacity, it prevents oxidation of unsaturated lipid materials within cells, thus protecting fats within the cell membrane from breaking down. The various GSH-Px enzymes are characterized by different tissue specificites and are expressed from different genes. In general, different forms of GSH-Px perform their protective functions in concert, with each providing antioxidant protection at different sites of the body. It is the oxidation of vitamin E that prevents oxidation of other lipid materials to free radicals and peroxides within cells, thus protecting the cell membrane from damage (Drouchner, 1976). If lipid hydroperoxides are allowed to form in the absence of adequate tocopherols, direct cellular tissue damage can result, in which peroxidation of lipids destroys structural integrity of the cell and causes metabolic derangement.
Lipid membranes have an abundance of unsaturated fatty acids and thus are very susceptible peroxide formation. The peroxidation of membrane lipids can, in turn, cause oxidation of membrane proteins (Leeson and Summers, 2001). These reactions are enhanced by the presence of metal ions, especially iron. Broilers fed additional alpha-tocopherol of between 30 and 50 mg per kg of diet (13.6 and 22.7 mg per lb) had reduction in lipid peroxidation as evidence by the decrease in malondialdehyde (Zhang et al., 2009).
Vitamin E reacts or functions as a chain-breaking antioxidant, thereby neutralizing free radicals and preventing oxidation of lipids within membranes. Free radicals may damage not only their cell of origin, but migrate and damage adjacent cells in which more free radicals are produced in a chain reaction leading to tissue destruction (Nockels, 1991). At least one important function of vitamin E is to interrupt production of free radicals at the initial stage.
Myodystrophic tissue is common in cases of vitamin E-selenium deficiency, with leakage of cellular compounds such as creatinine and various transaminases through affected membranes into plasma. The more active the cell (e.g., the cells of skeletal and involuntary muscles), the greater is the inflow of lipids for energy supply and the greater is the risk of tissue damage if vitamin E is limiting. This antioxidant property also ensures erythrocyte stability and maintenance of capillary blood vessel integrity.
Interruption of fat peroxidation by tocopherol explains the well-established observation that dietary tocopherols protect or spare body supplies of such oxidizable materials as vitamin A, vitamin C and the carotenes. Certain deficiency signs of vitamin E (i.e., muscular dystrophy) can be prevented by diet supplementation with other antioxidant nutrients, which helps validate the antioxidant role of tocopherols. Semen quality of boars was improved with selenium and vitamin E supplementation, in which vitamin E helped maintain sperm integrity in combination with selenium (Marin-Guzman et al., 1989). Chemical antioxidants are stored at very low levels, and thus are not as effective as tocopherol. It is clear that highly unsaturated fatty acids in the diet increase vitamin E requirements (McDowell, 2000). When acting as an antioxidant, vitamin E supplies become depleted, which explains the frequent observation that the presence of dietary unsaturated fats (susceptible to peroxidation) increases or precipitates a vitamin E deficiency.
Alpha-tocopherol may be involved in the formation of structural components of biological membranes, thus exerting a unique influence on architecture of membrane phospholipids (Ullrey, 1981). It is reported that alpha-tocopherol stimulated the incorporation of 14C from linoleic acid into arachidonic acid in fibroblast phospholipids. Also, it was found that alpha-tocopherol exerted a pronounced stimulatory influence on formation of prostaglandin E from arachidonic acid, while a chemical antioxidant had no effect.
Vitamin E is an inhibitor of platelet aggregation in pigs (McIntosh et al., 1985), and may play a role by inhibiting peroxidation of arachidonic acid, which is required for formation of prostaglandins involved in platelet aggregation (Panganamala and Cornwell, 1982; Machlin, 1991).
Vitamin E is perhaps the most studied nutrient related to the immune response (Meydani and Han, 2006). Evidence accumulated over the years and in many species indicates that vitamin E is an essential nutrient for the normal function of the immune system. Furthermore, studies suggest that through disease reducing beneficial effects of certain nutrients, such as vitamin E, can be through their effect on the immune response.
Considerable attention is presently being directed to the roles that vitamin E and selenium play in protecting leukocytes and macrophages during phagocytosis, the mechanism whereby animals immunologically kill invading bacteria. Both vitamin E and selenium may help these cells survive the toxic products that are produced in order to effectively kill ingested bacteria (Badwey and Karnovsky, 1980). Macrophages and neutrophils from vitamin E-deficient animals have decreased phagocytic activity (Burkholder and Swecker, 1990).
Since vitamin E acts as a tissue antioxidant and aids in quenching free radicals produced in the body, any infection or other stress factor may exacerbate depletion of the limited vitamin E stores from various tissues. With respect to immunocompetency, dietary requirements may be adequate for normal growth and production; however, higher levels have been shown to influence positively both cellular and humoral immune status of ruminant species. The former two responses are generally used as criteria for determining the requirement of a nutrient. During stress and disease, there is an increase in production of glucocorticoids, epinephrine, eicosanoids, and phagocytic activity. Eicosanoid and corticoid synthesis and phagocytic respiratory bursts are prominent producers of free radicals, which challenge the animal’s antioxidant systems. Vitamin E has been implicated in stimulation of serum antibody synthesis, particularly IgG antibodies (Tengerdy, 1980). The protective effects of vitamin E on animal health may be involved with its role in reduction of glucocorticoids, which are known to be immunosuppressive (Golub and Gershwin, 1985). In rats an in vivo inflammatory challenge decreased vitamin E blood and liver concentrations (Fritsche and McGuire, 1996). Vitamin E also most likely has an immunoenhancing effect by virtue of altering arachidonic acid metabolism and subsequent synthesis of prostaglandins, thromboxanes and leukotrienes. Under increased stress conditions, levels of these compounds by endogenous synthesis or exogenous entry may adversely affect immune cell function (Hadden, 1987).
The effects of vitamin E and selenium supplementation on protection against infection by several types of pathogenic organisms, as well as antibody titers and phagocytosis of the pathogens, have been reported for calves (Cipriano et al., 1982; Reddy et al., 1985; 1987b) and lambs (Reffett et al., 1988; Finch and Turner, 1989; Turner and Finch, 1990). As an example, calves receiving 125 IU of vitamin E daily were able to maximize their immune responses compared to calves receiving low dietary vitamin E (Reddy et al., 1987a). In sows, vitamin E restriction depressed lymphocytes and polymorphonuclear cells for immune function (Wuryastuti et al., 1993). Dogs with vitamin E deficiency had a depressed proliferative lymphocyte responsiveness (Langweiler et al., 1983).
Antioxidants, including vitamin E, play a role in resistance to viral infection. Vitamin E deficiency allows a normally benign virus to cause disease (Beck et al., 1994). In mice, enhanced virulence of a virus resulted in myocardial injury that was prevented with vitamin E adequacy. A selenium or vitamin E deficiency leads to a change in viral phenotype, such that an avirulent strain of a virus becomes virulent and a virulent strain becomes more virulent (Beck, 1997; 2007; Sheridan and Beck, 2008). Thus, host nutritional status should be considered a driving force for the emergence of new viral strains or newly pathogenic strains of known viruses.
Vitamin E and selenium appear to enhance host defenses against infections by improving phagocytic cell function. Both vitamin E and GSH-Px are antioxidants that protect phagocytic cells and surrounding tissues from oxidative attack by free radicals produced by the respiratory burst of neutrophils and macrophages during phagocytosis (Baboir, 1984; Baker and Cohen, 1983). Hogan et al. (1990, 1992) reported that vitamin E supplementation of diets increased intracellular kill of Staphylococcus aureus and Esherichia coli by neutrophils.
There is limited evidence that vitamin E is involved in biological oxidation-reduction reactions (Hoffmann-La Roche, 1972). Vitamin E also appears to regulate the biosynthesis of DNA within cells. Vitamin E appears to be of particular importance in cellular respiration of heart and skeletal muscles (Leeson and Summers, 2001).
Both vitamin E and selenium provide protection against toxicity of various heavy metals (Whanger, 1981). Vitamin E is highly effective in reducing toxicity of metals such as silver, arsenic and lead, and shows slight effects against cadmium and mercury toxicity.
Vitamin E can be effective against other toxic substances. For example, treatment with vitamin E gave protection to weanling pigs against monensin-induced skeletal muscle damage (Van Vleet et al., 1987). Recent research has shown a beneficial response for vitamin E supplementation on male reproduction for bulls fed high concentrations of gossypol. Velasquez-Pereira et al. (1998) reported that bulls which received 14 mg free gossypol per kg body weight had a lower (P < 0.05) percentage of normal sperm than those which also received supplemental vitamin E, 30% versus 55%, respectively (Table 4-3). Likewise, sperm production per gram of parenchyma and total daily sperm production were higher (P < 0.05) when gossypol-treated animals also received vitamin E. Bulls receiving gossypol exhibited more sexual inactivity (P < 0.05) than bulls in other treatments (Table 4-4). Vitamin E supplementation to bulls receiving gossypol improved number of mounts in the first test and time of first service in the second test. The final conclusion of the Florida data is that vitamin E is effective in reducing or eliminating important gossypol toxicity effects for male cattle.
There is a close working relationship between vitamin E and selenium within tissues. Selenium has a sparing effect on vitamin E and delays onset of deficiency signs. Likewise, vitamin E and sulfur amino acids partially protect against or delay onset of several forms of selenium deficiency syndromes. Tissue breakdown occurs in most species receiving diets deficient in both vitamin E and selenium, mainly through peroxidation. Peroxides and hydroperoxides are highly destructive to tissue integrity and lead to disease development. It now appears that vitamin E in cellular and subcellular membranes is the first line of defense against peroxidation of vital phospholipids (Figure 4-2), but even with adequate vitamin E, some peroxides are formed. Selenium, as part of the enzyme GSH-Px, is a second line of defense that destroys these peroxides before they have an opportunity to cause damage to membranes. Therefore, selenium, vitamin E and sulfur-containing amino acids, through different biochemical mechanisms, are capable of preventing some of the same nutritional diseases. Vitamin E prevents fatty acid hydroperoxide formation, sulfur amino acids are precursors of GSH-Px, and selenium is a component of GSH-Px (Smith et al., 1974).
Although alpha-tocopherol has been the most widely studied form of vitamin E other tocopherols and tocotrienols have recently been shown to have biological significance (Qureshi et al., 2001; Eder et al., 2002; McCormick and Parker, 2004; Schaffer et al., 2005; Nakagawa et al., 2007; Sun et al., 2008; Freiser and Jiang, 2009). The greater emphasis on the alpha-tocopherol undoubtedly arises from observations that gamma-tocopherol and delta-tocopherol are only 10% and 1% as effective as alpha-tocopherol, respectively, in experimental animal models of vitamin E deficiency.
Tocotrienols have been shown to possess excellent antioxidant activity in vitro and have been suggested to suppress reactive oxygen substances more efficiently than tocopherols (Schaffer et al., 2005). Studies have shown that tocotrienols exert more significant neuroprotective, anti-cancer and cholesterol-lowering properties than do tocopherols (Qureshi et al., 2001; Sun et al., 2008). Gamma-tocopherol has beneficial properties as an anti-inflammatory and possibly anti-atherogenic and anti-cancer agent (Wolf, 2006). Research with tocotrienols and non-alpha tocopherols has been carried out with laboratory animals and in vitro studies; the significance for companion animals is unknown.
Additional functions of vitamin E that have been reported (Scott et al., 1982) include: (1) normal phosphorylation reactions, especially of high-energy phosphate compounds, such as creatine phosphate and adenosine triphosphate (ATP); (2) a role in synthesis of vitamin C (ascorbic acid); (3) a role in synthesis of ubiquinone; and (4) a role in sulfur amino acid metabolism. Pappu et al. (1978) have reported vitamin E to play a role in vitamin B12 metabolism. A deficiency of vitamin E interfered with conversion of vitamin B12 to its coenzyme 5′ deoxyadenosylcobalamin and concomitantly, metabolism of methylmalonyl-CoA to succinyl-CoA. For humans, Turley and Brewster (1993) suggest that cellular deficiency of adenosylcobalamin may be one mechanism by which vitamin E deficiency leads to neurologic injury.
In rats, vitamin E deficiency has been reported to inhibit vitamin D metabolism in the liver and kidneys by interfering with the formation of active metabolites and decreasing the concentration of the hormone-receptor complexes in the target tissue. Liver vitamin D hydroxylase activity decreased by 39%, 25-(OH)D3 1-alpha hydroxylase activity in the kidneys decreased by 22% and 24-hydroxylase activity by 52% (Sergeev et al., 1990).
Vitamin E requirements are exceedingly difficult to determine because of the interrelationships with other dietary factors. Therefore, its requirement is dependent on dietary levels of PUFA, antioxidants, sulfur amino acids, and selenium. The requirement may be increased with higher levels of PUFA, oxidizing agents, vitamin A, carotenoids, gossypol and trace minerals, and decreased with increasing levels of fat-soluble antioxidants, sulfur amino acids or selenium (Dove and Ewan, 1990; Nockels, 1990; McDowell, 2000; Hendriks et al., 2002).
Vitamin E requirements vary between strains and breeds of animals. Surai et al., (2002) compared antioxidant status in two lines of breeder broiler genotypes: fast growing and slow growing. A difference in antioxidant requirement was found; the fast growing genotype had a higher vitamin E requirement. A comparative study of vitamin E deficiency compared domestic shorthaired and Siamese breeds of cats (Fytianou et al., 2006). There was a delay of Siamese cats to develop symptomatic steatitis, which was presumably attributed to an inherent resistance as a result of the long-standing evolution of more efficient antioxidant mechanisms.
The levels of PUFA found in unsaturated oils such as cod liver oil, corn oil, soybean oil, sunflower seed oil and linseed oil all increase vitamin E requirements. This is especially true if these oils are allowed to undergo oxidative rancidity in the diet or are in the process of peroxidation when consumed by the animal. If they become completely rancid before ingestion, the only damage is the destruction of the vitamin E present in the oil and in the feed containing the rancidifying oil. But if they are undergoing active oxidative rancidity at the time of consumption, they apparently cause destruction of body stores of vitamin E as well (Scott et al., 1982). A combination of the stress of infection and presence of oxidized fats in swine diets was reported to exaggerate vitamin E needs still further (Tiege et al., 1978). These researchers reported that supplements of 100 IU vitamin E per kg (45 IU per lb) of diet and 0.1 ppm selenium did not entirely prevent deficiency lesions in weanling pigs afflicted with dysentery and fed 3% cod liver oil.
Of all factors, the most important determinant of vitamin E requirements is the dietary concentration of unsaturated fatty acids. A diet that contains high levels of fish oil may cause a three- to four-fold increase in a cat’s daily requirement for alpha-tocopherol. Early cases of pansteatitis (“yellow fat disease”) occurred almost exclusively in cats that were fed a canned, commercial fish-based cat food, of which red tuna was the principal type of fish.
Since the PUFA content of membranes can be altered by dietary fats, it is not surprising that the dietary requirement for vitamin E is closely related to the dietary concentration of PUFA. When a large amount of polyunsaturated fat is fed after it has been stripped of tocopherols, as much as 100 mg alpha-tocopherol per kg (45.5 mg per lb) of diet may be insufficient to protect against lipofuscin formation (Hayes et al., 1969).
Harris and Embree (1963) have proposed a dietary alpha-tocopherol:PUFA ratio (mg/g) of 0.6:1 as a minimum to protect against PUFA peroxidation. This is a step towards more accuracy in determining vitamin E requirements for pets. It should be realized, however, that some PUFA require much more vitamin E to counteract detrimental effects than others. Fish oil PUFA that contain arachidonic acid (20:4), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6) require much more vitamin E for stabilization than do typical plant PUFA such as linoleic acid (18:2) and linolenic acid (18:3) (McDowell, 2000). The longer the carbon chain of a fatty acid and the more double bonds, the greater the vitamin E requirement. As an example, when 5% tuna oil (long carbon chain and greater unsaturation) was substituted for 5% of lard (shorter carbon chain and less unsaturation), steatitis was severe in cats unsupplemented with vitamin E. The severity of steatitis was diminished by supplemental vitamin E, but was not entirely prevented by 34 IU per kg (15.5 IU per lb) of diet. When 136 IU per kg (61.8 IU per lb) of diet was provided, no lesions were seen (Gershoff and Norkin, 1962). By-product organs (e.g., liver) in the diet would also have more of the longer-chained, more unsaturated fatty acids.
Exact requirements for vitamin E in dogs and cats is difficult to establish, but they are definitely a function of the peroxidizable PUFA consumed, and probably vary from as little as 5 mg per kg (2.3 mg per lb) of diet to as much as 100 mg per kg (45.5 mg per lb) when a high quantity of PUFA is fed. The requirement would range from approximately 0.25 to 5 IU per day for a cat and 1 to 20 IU per day for the average-sized dog.
The amount of vitamin E needed to maintain adequate growth and reproduction would not necessarily be enough to ensure optimal immune function as noted previously. For example, in the rat, Bendich et al. (1986) reports that 15 IU per kg vitamin E (7 IU per lb) of diet prevented muscle abnormalities and 50 IU per kg (23 IU per lb) of diet was necessary to prevent red blood cell breakdown, but for maximum lymphocyte stimulation 200 IU vitamin E per kg (91 IU per lb) of diet was required. For pigs, 150 IU per kg (68 IU per lb) of supplemental vitamin E resulted in a higher lysozyme activity and a higher rate of yeast lysis (Riedel-Caspari et al., 1986).
Stress, exercise, infection and tissue trauma all increase vitamin E requirements (Nockels, 1991). Handling and bleeding heifers periodically in a 10-day period resulted in a large decrease in the vitamin E content of red blood cells and a 62% decrease in neutrophil vitamin E levels (Nockels, 1991). Vitamin E supplementation to stressed cattle reduced neutrophil vitamin E (Hogan et al., 1990) and increased immune response (Golub and Gershwin, 1985) in stressed calves. Nockels et al. (1996) noted that for most sampled tissues, stress did not affect alpha-tocopherol concentration, although other indicators confirmed a deficiency.
Requirements of both vitamin E and selenium are greatly dependent on the dietary concentrations of each other and they are mutually replaceable above certain limits. The relationship has been quantified to a certain degree for poultry. Chicks consuming a diet containing 100 IU per kg (45.5 IU per lb) vitamin E required 0.01 ppm selenium, while those receiving no added vitamin E required 0.05 ppm selenium (Thompson and Scott, 1969). Determination of vitamin E requirements is further complicated because the body has the ability to store both vitamin E and selenium. A number of studies to establish requirements for both nutrients have underestimated the requirements by failing to account for their augmentation from body stores as well as experimental dietary concentrations.
Van Vleet (1975) fed beagle puppies a diet containing 5% stripped lard and Torula yeast and varying levels of vitamin E or selenium. Feeding alpha-tocopherol at 30 IU per kg (13.6 IU per lb) of diet alone or 1 ppm selenium alone as selenite prevented deficiency both clinically and histopathologically, whereas 0.5 ppm selenium only prevented clinical signs of deficiency.
Assuming a dry diet containing not more than 1% linoleic acid and at least 0.1 ppm selenium, the NRC (2006) recommended allowance for Vitamin E is 30 mg per kg (13.6 mg per lb) of diet for all life stages of dogs. If dietary PUFA levels are increased, it is suggested that an alpha-tocopherol:PUFA ratio (mg/g) of at least 0.5 be maintained. As noted previously, vitamin E should be increased further if the PUFA are derived from fish or animal organs (e.g., liver) versus vegetable oils. AAFCO (2007) recommends considerably higher dietary levels of vitamin E than does the NRC (2006). For all classes of dogs, 50 IU of vitamin E is recommended per kg (22.7 IU per lb) of diet.
The need for vitamin E in the diet of cats is markedly influenced by dietary composition. Because of the nature of the diets consumed by cats, they are more susceptible than dogs to vitamin E deficiency. Cat diets, especially canned, are typically higher in fat than dog diets and are frequently based on fish products, especially tuna, containing long-chain PUFAs that are prone to lipid peroxidation.
Cordy (1954), Coffin and Holzworth (1954), Munson et al. (1958), and Griffiths et al. (1960) have noted an association of steatitis (yellow fat disease, vitamin E deficiency) with consumption of fish-based diets, particularly red tuna.
Cordy (1954) fed kittens diets based on fish and additional fish-liver oil. Death and signs of vitamin E deficiency were noted in controls or in those receiving only 10 mg alpha-tocopherol daily. The kittens receiving 20 to 40 mg per day of alpha-tocopherol were normal. Gershoff and Norkin (1962) reported that 34 or 68 IU per kg (15.5 or 30.9 IU per lb) vitamin E prevented vitamin deficiency for cats receiving a 20% vitamin E-free lard diet. However, when 5% of the lard was replaced by 5% tuna oil, 34 IU vitamin E per kg (15.5 IU per lb) did not prevent steatitis, but 136 IU vitamin E per kg (61.8 IU per lb) was effective.
The NRC (2006) recommends a minimum vitamin E requirement of 38 mg per kg (17.3 mg per lb) of diet for kittens after weaning and adults at maintenance. For cats in late gestation and peak lactation the vitamin E requirement is 31 mg per kg (14.1 mg per lb) of diet. It is suggested that a relatively low-fat diet (< 10% dry weight) containing antioxidants and adequate selenium would greatly reduce this requirement. High-PUFA diets, especially those containing fish oil, will increase the suggested minimum three- to four-fold. AAFCO (2006) recommends 30 IU per kg (13.6 IU per lb) of diet for all classes of cats. AAFCO (2006) recommends the addition of 10 IU vitamin E above minimum level per g of fish oil per kg (4.5 IU per g per lb) of diet.
Many vitamin E analyses of foods and feedstuffs have been reported using a variety of analytical techniques. However, there is a lack of characterization of individual tocopherols in the majority of analyses. Total tocopherol analysis of a food or feedstuffs is of limited value in providing a reliable estimate of the biological vitamin E values.
Because alpha-tocopherol is the most active form of vitamin E, many nutritionists prefer listing this form in feeds versus the unreliable total tocopherol values. Some of the less active tocopherols, particularly gamma-tocopherol, are present in mixed diets in amounts of two to four times greater than that of alpha-tocopherol. Cort et al. (1983) utilizing HPLC (high pressure liquid chromatography) assay procedures, which allow separation of alpha and non-alpha forms of both tocopherol and tocotrienols, determined that corn, corn gluten meal, oats, barely and wheat contained significant amounts of alpha-tocopherol. If only alpha-tocopherol in a mixed diet is reported, the value in mg can be increased by 20% to account for other tocopherols that are present, thus giving an approximation of total vitamin E activity as mg of alpha-tocopherol equivalents.
Vitamin E is widespread in nature, with the richest sources being vegetable oils, cereal products containing these oils, eggs, liver, legumes and, in general, green plants. In nature, the synthesis of vitamin E is a function of plants and thus their products are by far the principal sources. It is abundant in whole cereal grains, particularly in germ, and thus in by-products containing the germ (McDowell, 2000; Traber, 2006). Feed table averages are often of little value in predicting individual content of feedstuffs or bioavailability of vitamins.
Vitamin E content of 42 varieties of corn varied from 11.1 to 36.4 IU per kg, (5.0 to 16.5 IU per lb), a 3.3-fold difference (McDowell and Ward, 2008). There is wide variation in vitamin content of particular feeds with many feeds having a three- to ten-fold range in reported alpha-tocopherol values. Naturally occurring vitamin E activity of feedstuffs cannot be accurately estimated from earlier published vitamin E or tocopherol values. Alpha-tocopherol is especially high in wheatgerm oil, safflower oil and sunflower oil. Corn and soybean oil contains predominantly gamma-tocopherol, as well as some tocotrienols (McDowell, 2000; Traber, 2006). Cottonseed oil contains both alpha- and gamma-tocopherols in equal proportions. Table 4-5 presents the alpha-tocopherol content of various feedstuffs compared to previously published assay values.
Milk is highly variable in vitamin E content. As a source of vitamin E, there can be a five-fold seasonal difference in the alpha-tocopherol content of cow’s milk (McDowell, 2000). The vitamin E content of colostrum is of special importance for the newborn, because at birth many species have very small amounts of vitamin E in their tissues. Colostrum from dairy cows has been reported to have a mean value of 1.9 µg alpha-tocopherol per ml, declining at 30 days to 0.3 µg alpha-tocopherol per ml (Hidiroglou, 1989). In this study, milk tocopherol was raised from 0.3 mg per ml to 1.6 mg per ml 12 hours after an intraperitoneal injection of dl-alpha-tocopherol acetate. Supplemental vitamin E to ewes at graded levels of 0, 15, 30 and 60 IU (as dl-alpha-tocopherol acetate) increased colostral alpha-tocopherol at a linear rate of 3.3, 6.8, 8.1, and 9.6 µg per ml, respectively (Njeru et al., 1994). In sows, colostrum alpha-tocopherol concentration can be elevated by increasing the gestation dietary level of vitamin E or via injection during the last four days of pregnancy. Colostrum alpha-tocopherol concentration was approximately five-fold higher than later milks (Mahan and Vallet, 1997).
Animal by-products supply only small amounts of vitamin E, and milk and dairy products are poor sources. Eggs, particularly the yolks, make a significant contribution, depending on the diet of the hen. Wheat germ oil is the most concentrated natural source, and various other oils, such as soybean and peanut, and particularly cottonseed are also rich sources of vitamin E. Unfortunately, most of the oilseed meals now marketed are almost devoid of these oils because of their removal by solvent extraction (Maynard et al., 1979). Green forage and other leafy materials, including good quality leaf meals, are very good sources, with alfalfa being especially rich. Concentration of tocopherols per unit of dry matter in fresh herbage is between five and 10 times greater than that in some cereals of their by-products (Hardy and Frape, 1983). A more available form of vitamin E is present in pastures and green forages, containing ample quantities of alpha-tocopherol versus lower bioavailable forms in grains (McDowell, 2004). Variability in forage vitamin E content is so great, both between and within farms, that one must have current results of representative samples to ensure proper vitamin E fortification programs (Harvey and Bieber-Wlaschny, 1988). These authors indicated that preciously published values on vitamin E content of forages are unacceptable for use in feed formulation.
Stability of all naturally occurring tocopherols is poor and substantial losses of vitamin E activity occur in feedstuffs when processed and stored, as well as in manufacturing and storage of finished feeds (McDowell et al., 1996; Gadient, 1986; Dove and Ewan, 1991). Vitamin E sources in these ingredients are unstable under conditions that promote oxidation of feedstuffs– heat, oxygen, moisture, oxidizing fats and trace minerals. Vegetable oils that normally are excellent sources of vitamin E can be extremely low in the vitamin if oxidation has been promoted. Not only does oxidized oil have little or no vitamin E, but it will destroy the vitamin E in other feed ingredients and deplete animal tissue stores of vitamin E. Rats fed a diet containing 15% oxidized frying oils had significantly lower alpha-tocopherol in plasma and tissues (Liu and Huang, 1995; 1996).
For concentrates, oxidation increases following grinding, mixing with minerals, the addition of fat, and pelleting. When feeds are pelleted, destruction of both vitamins E and A may occur if the diet does not contain sufficient anitoxidants to prevent their accelerated oxidation under conditions of moisture and high temperature. Iron salts (i.e., ferric chloride) can completely destroy vitamin E. Artificial dehydration or processing of forages and grains will reduce availability of tocopherol as well as that of selenium. For example, in one study, 80% of the vitamin E was lost in hay making (Kinget al., 1967), whereas ensiling or rapid dehydration of forages retains most of the vitamin. Vitamin E content in forage is affected by stage of maturity at time of forage cutting and the period of time from cutting to dehydration. Storage losses can reach 50% in one month and losses during drying in the swath can amount to as much as 50% within four days. Vitamin E losses of 54% to 73% have been observed in alfalfa stored at 33°C for 12 weeks, and 5% to 33% losses have been obtained with commercial dehydration of alfalfa.
In a study testing vitamin E stability, Orstadius et al. (1963) reported that vitamin E content of corn was reduced from 30 to 50 mg per kg (14 to 23 mg per lb) to about 5 mg per kg (2.2 mg per lb) of dry weight as a result of artificial drying at 100°C for 24 hours under continuous flow of air. Similarly, it has been reported that artificial drying of corn for 40 minutes at 87°C produced an average 10% loss of alpha-tocopherol and 12% loss of other tocopherols (Adams, 1973). When corn was dried for 54 minutes at 93°C, the alpha-tocopherol loss averaged 41%. Artificial drying of corn results in a much lower vitamin E content. Young et al. (1975) reported a concentration of 9.3 or 20 mg per kg (4 or 9 mg per lb) alpha-tocopherol in artificially dried corn versus undried, respectively. Preservation of moist grains by ensiling caused almost complete loss of vitamin E activity. Corn stored as acid-treated (propionic or acetic-propionic mixture) high moisture corn contained approximately 1 mg per kg (0.45 mg per lb) dry matter of alpha-tocopherol, whereas similar corn artificially dried following harvesting contained approximately 5.7 mg per kg (2.6 mg per lb) alpha-tocopherol (Young et al., 1978). Apparently damage is not due to moisture alone, but to combined propionic acid/moisture effect (McMurray et al., 1980). Further decomposition of alpha-tocopherol occurs over a more extended period of time until the grain eventually has alpha-tocopherol levels of less than 1 mg per kg (0.45 mg per lb), which is commonly found in propionic acid-treated barley.
The most active form of vitamin E, as previously stated, found in feed ingredients is d-alpha-tocopherol. For many years, the primary source of vitamin E in animal feed was the natural tocopherols found in green plant materials and seeds. Commercially available sources of vitamin E activity are shown in Table 4-6. Differences in biopotency of the stereoisomers of alpha-tocopherol can been seen from the definition of International Unit (IU).
According to TheUnited States Pharmacopeia (1980), dl-alpha-tocopheryl acetate (also called dl-rac-tocopheryl acetate) is the International Standard of vitamin E activity with an IU equivalent to 1 milligram of dl-alpha-tocopheryl acetate (Table 4-6). This is the most widely available source of vitamin E activity for the supplementation of animal feeds. The acetate ester of d- or dl-alpha-tocopherol is synthesized to stabilize the compound from oxidation and maintain vitamin E activity. During commercial synthesis of dl-alpha-tocopheryl acetate, all rac-alpha-tocopherol is esterified to the acetate form to stabilize it, with the ester extremely resistant to oxidation. Thus, dl-alpha-tocopherol acetate does not act as an antioxidant in the feed. However, it has antioxidant activity after it is hydrolyzed in the intestine and free dl-alpha-tocopherol is released and absorbed.
The acetate forms of alpha-tocopherol are commercially available from two basic sources: (1) d-alpha-tocopherol vegetable oil refining by-products, molecular distillation to obtain the alpha-form, and then acetylation to form the acetate ester, and (2) dl-alpha-tocopheryl acetate is made by complete chemical synthesis, producing a racemic mixture of equal proportions of eight stereoisomers.
Vitamin E displays the greatest versatility of all vitamins in the range of deficiency signs. Deficiency signs differ among species and even within the same species. Blaxter (1962) reported that muscular dystrophy seemed to be the one syndrome commonly encountered in all species. White muscle disease (WMD), also known as nutritional muscular dystrophy, a serious muscle degeneration disease in young animals, is the major clinical sign of a vitamin E-selenium deficiency in newborns. White muscle disease occurs in all laboratory and farm animals as well as in camels, buffalos, and kangaroos. Muscular dystrophy is reported in a number of wild animals, the condition in antelope, for example, being indistinguishable from WMD in cattle or sheep. Fundamentally, this is Zenker’s degeneration of both skeletal and cardiac muscle fibers. The connective tissue replacement that follows is observed grossly as white striations in the muscle bundles.
Muscular degeneration is reported for dogs with vitamin E deficiency. Steatitis or yellow fat is the most common indication of the vitamin deficiency in cats. Godwin (1975) reported that electrocardiograms of WMD showed a progressive development of a characteristic abnormality and a fall in blood pressure, therefore suggesting that the fundamental change occurring in the vitamin E-selenium deficiency is circulatory failure.
Confirmation of a low vitamin E and/or selenium status in animals is obtained when specific deficiency diseases associated with a lack of these nutrients are present. Likewise, gross lesions and histopathological examinations provide definite evidence of vitamin E and/or selenium deficiency. Unlike dogs, there are few studies which establish the vitamin E status of cats. Diagnosing of steatitis can be made on biopsy or necropsy findings of yellow to orange-brown fat. Histopathologic examination reveals fat necrosis, septal panniculitis, and ceroid within fat vacuoles, macrophages, and giant cells.
Muscular damage as a result of vitamin E and/or selenium deficiencies causes leakage of intercellular contents into the blood. Thus elevated levels of selected enzymes, above normal concentrations for particular species, serve as diagnostic aids in detecting tissue degeneration. Serum enzyme concentrations used to follow incidence of nutritional muscular dystrophy include serum aspartate aminotransferase, lactic dehydrogenase, creatine phosphokinase, and malic dehydrogenase. Enzyme tests are very sensitive, and an elevation of enzyme activity in serum is usually discovered before any pathological changes or clinical signs appear (Tollersrud, 1973). Dogs with muscular dystrophy have had elevated serum creatine phosphokinase (Worden, 1958; Van Vleet, 1975; Sheffy, 1979).
Dogs deficient in vitamin E have an increased erythrocyte fragility as determined by the dialuric acid hemolysis assay (Sheffy and Schultz, 1978; Pillai et al., 1992). In vitamin E-deficient dogs, Pillai et al. (1992) reported that dialuric acid hemolysis (R = -0.89) and spontaneous hemolysis (R = -0.91) increased with declining plasma alpha-tocopherol concentration.
Immune function is altered in vitamin E-deficient dogs and can be used as an additional indicator of vitamin E status. Vitamin E-deficient dogs had significant reduction in antibody titers to canine distemper and infectious canine hepatitis vaccines compared to normal dogs (Sheffy and Schultz, 1978). Dogs deficient in vitamin E had severe impairment of lymphocyte function as assessed by in vitro blastogenic responses to concanavalin A, phytohemagglutinin, pokeweed mitogen and streptolysin O. (Sheffy and Schultz, 1978; Langweiler et al., 1981). The suppression of lymphocyte function was shown to be associated with the presence of a serum immunosuppressive factor, which could be counteracted in vitro by vitamin E, reducing agents, and synthetic antioxidants such as 2-mercaptoethanol, dithrotritol, butylated hydroxytoluene and ethoxyquinone (Langweiler et al., 1983).
Nutritional status with respect to vitamin E is commonly estimated from plasma (or serum) concentration. There is a relatively high correlation between plasma and liver levels of alpha-tocopherol (and also between amount of dietary alpha-tocopherol administered and plasma levels). This has been observed in rats, chicks, pigs, lambs, and calves within rather wide ranges of intake. Plasma tocopherol concentrations of 0.5 to 1 µg per ml are considered low in most animal species, with less than 0.5 µg per ml generally considered a vitamin E deficiency. Thus, plasma alpha-tocopherol concentrations can be used for assay purposes without the necessity of liver biopsy or animal slaughter.
Plasma vitamin E, as true in other species, is an indicator of vitamin E status. Hayes et al. (1970) related plasma tocopherol concentrations of dogs with pathological evidence of vitamin E deficiency. As vitamin E deficiency developed and progressed in dogs, plasma alpha-tocopherol decreased from baseline values of 20.5 to 31.3 µg per ml to 0.07 to 0.1 µg per ml by 90 weeks (Pillai et al., 1992).
A wide range of clinical signs of vitamin E deficiency in dogs has been reported. In general, however, the neuromuscular, vascular and reproductive systems are affected most commonly. Signs of vitamin E deficiency are mostly attributed to membrane dysfunction as a result of the oxidative degradation of polyunsaturated membrane phospholipids and disruption of other critical cellular processes. Definite clinical cases of vitamin E deficiency have been well documented in dogs. Particularly prominent indications of vitamin E deficiency were degeneration of skeletal muscle associated with muscle weakness, degeneration of testicular germinal epithelium and failure of spermatogenesis, failure of gestation, weak and dead pups, and brown pigmentation (lipofuscinosis) of intestinal smooth muscle (NRC, 2006).
In addition to muscular weakness, Van Vleet (1975) reported subcutaneous edema, anorexia, depression, dyspnea, and eventual coma in vitamin E-deficient dogs. Microscopically, these dogs exhibited extensive skeletal muscle degeneration and regeneration, focal endocardial necrosis in the ventricular mycocardium, intestinal lipofuscinosis (brown pigmentation) and renal mineralization.
Van Kruiningen (1967) reported a brown bowel in malabsorbing boxer dogs with granulomatous colitis, and the condition was eventually reproduced experimentally by Hayes et al. (1969), who fed varying levels of polyunsaturated fat to vitamin E-deficient weanling puppies over a 16-week period. The puppies developed an increased susceptibility to hemolysis of red blood cells as well as brown bowel, mild muscle degeneration, and neuralaxonal dystrophy.
Oxidative stress is a risk factor for eye diseases. Vitamin E deficiency has been shown to result in injury to the dog retina. A retinal degeneration was described by Hayes and Rousseau (1970) and reproduced experimentally (Riis et al., 1981) in puppies fed a diet containing stripped corn oil. In as few as three months, ophthalmoscopic lesions were visible, which represented lipid peroxidation and disruption of photoreceptors with accumulation of lipofuscin pigment. Retinal degeneration associated with vitamin E deficiency in hunting dogs was reported by Davidson et al. (1998). Additional reports relating retinal degeneration in dogs with vitamin E include McLellan et al., 2002; and Zapata et al., 2008.
The role of vitamin E deficiency in the development of retinal pigment epithelial dystrophy was investigated in 14 dogs (mostly cocker spaniels) compared to 28 opthalmoscopically normal, healthy control dogs (McLellan et al., 2002). The mean plasma alpha-tocopherol concentration in the normal dogs was 20.2 µg/ml compared with 1.14 µg/ml in the affected dogs. The deficiency of vitamin E in the affected dogs appeared to be the primary cause of retinal pigment epithelial dystrophy, as there was no evidence of any other underlying disease or other dietary deficiency.
The effect of vitamin E deficiency on reproduction has been reported by Anderson et al. (1939; 1940) and Elvehjemet al. (1944), who described a nutritional deficiency syndrome in puppies from bitches fed evaporated or pasteurized milk diets for extended periods of time. Lactating dams and puppies developed festering skin lesions, and the puppies developed muscle weakness and hemorrhages of body cavities and brain. Other pups were stillborn or died shortly after birth. The syndrome was alleviated by weekly feeding of 40 mg of vitamin E to the bitch during pregnancy.
A deficiency of vitamin E has been implicated in the development of certain dermatological disorders in dogs (Anderson et al., 1939; Worden, 1958; Sheffy, 1979; Scott and Walton, 1985; Scott and Sheffy, 1987; Miller, 1989; Codner and Thatcher, 1993; Jewell and Joshi, 2002) (Illus. 4-2). The occurrence of these skin disorders has been associated with decreased blood levels of vitamin E.
Illustration 4-2: Vitamin E Deficiency, Dermatomyositis
It has been postulated that a subclinical vitamin E deficiency causes suppression of the immune system, which in turn increases a dog’s susceptibility to demodex (skin lesions caused by the demodectic mange mite, Demodex canis). When a group of dogs with demodicosis was treated with supplemental vitamin E, significant improvement was reported. However, other researchers have been unable to reproduce these results.
Scott and Sheffy (1987) experimentally produced vitamin E deficiency skin disorders. They were characterized clinically by an early keratinization defect (seborrhea sicca), a later inflammatory stage (erythroderma) and a tendency to develop secondary pyoderma. The vitamin E anti-inflammatory effect may be related to stabilization of cell and lysosomal membranes against damage induced by free radicals and peroxides. By reducing oxidative damage to cells, vitamin E may inhibit immune-mediated or neoplastic skin diseases associated with ultraviolet irradiation.
Vitamin E therapy has been reported to be effective in dogs with discoid lupus erythematosus (Scott et al., 1983) and has been suggested as therapy for dogs with dermatomyositis (Illus. 4-3) (Muller et al., 1989; Hargis et al., 1985). Vitamin E supplementation has also been evaluated in dogs with acanthosis nigricans. Eight dachshunds with primary acanthosis nigricans showed improvement after 60 days of vitamin E therapy (200 mg twice daily) (Scott and Walton, 1985). All dogs responded with gradual elimination of pruritus, inflammation, lichenification, greasiness, and odor.
Illustration 4-3: Vitamin E Deficiency, Dermatomyositis
Lesions of the leg on a dog with dermatomyositis.
Courtesy of Lynne P. Schmeitzel, University of Tennessee
Steatitis or “yellow fat disease” has been noted when sources of highly unsaturated fatty acids (e.g., tuna fish oil, cod liver oil, and unrefined herring oil) have been fed in the absence of adequate supplemental vitamin E. Known as yellow fat, after the peroxidized ceroid in adipose tissue, it was characterized by extreme hyperesthesia, fever, and a marked rise in leucocytes, primarily as neutrophils and eosinophils. Anorexia, weight loss, listlessness or overt neurological dysfunction and muscle spasms, harsh haircoat, and palpable lumps in the subcutaneous fat were characteristic (Caseet al., 1995). Additional signs were focal interstitial myocarditis and, rarely, muscle fiber degeneration, focal myositis of the skeletal muscle and periportal mononuclear infiltration in the liver (Gershoff and Norkin, 1962). Affected cats are often lethargic, febrile, and exhibit pain on gentle palpation. Subcutaneous and abdominal fat may feel firm or lumpy, and draining tracts may develop. The best way of confirming the condition is histological examination of a fat biopsy (Case et al., 1995). Microscopically, the fat shows focal neutrophilic infiltration with some mononuclear cells. Acid-fast ceroid pigment is present as globules and as peripheral rings in the fat cell vacuoles (NRC, 2006). The white blood cell count is usually elevated (24,000 to 70,000 mg per ml), primarily as a result of neutrophilia, and reflects the degree of fat necrosis.
Vitamin E deficiency in cats became a significant clinical problem when fish products high in PUFA were first used in commercial cat foods that contained inadequate levels of tocopherol (Cordy, 1954; Munson et al., 1958). Later cases of the disease occurred in cats that were fed diets consisting wholly or largely of canned red tuna or fish scraps. Red tuna packed in oil contains high levels of PUFA and low levels of vitamin E. The addition of large amounts of fish products to cat diets appears to be the primary cause of this disease in pet cats (Harvey, 1993; Watson, 1998).
Stephan and Hayes (1978) induced severe vitamin E deficiency with hemolytic anemia and steatitis by feeding a diet containing 15% stripped safflower oil without alpha-tocopherol. Clinical signs of deficiency were prevented by supplementing with alpha-tocopheryl acetate at 100 IU per kg (45.5 IU per lb) diet.
Several tocopherols in plants and certain vegetable oils are the naturally occurring sources of vitamin E activity. Alpha-tocopherol provides the highest vitamin E biological activity. However, the stability of all naturally occurring tocopherols is poor, and substantial losses of vitamin E activity occur in natural ingredients when processed and stored as well as in manufacturing and storage of finished pet foods. To offset losses of vitamin E activity in ingredients, pet foods should be adequately fortified using dl-alpha-tocopheryl acetate, the most stable source of vitamin E activity available for pet food use. Alpha-tocopherol, an alcohol, is stabilized by conversion to the acetate ester namely, dl-alpha-tocopheryl acetate. For pets, the major methods of providing supplemental vitamin E are as part of the complete diet or as an injectable product if a vitamin E-responsive disease condition is expected.
Commercially, the acetate esters of vitamin E are available in purified form or in various dilutions and include: (a) a highly concentrated oily form, for further processing; (b) emulsions incorporated in powders for use in dry premixes or water-dispersible preparations; and (c) adsorbates or absorbates of the oily tocopheryl acetate on selected carriers, in free-flowing, “dry” powder, meal or granules. Vitamin E as the acetate form is highly stable in vitamin premixes, with 98% retention after six months, but in the alcohol form is completely destroyed during the time period (Gadient, 1986).
Injectable vitamin E preparations are also available that contain free d- or dl- alpha-tocopherol. Liquid emulsions of appropriate types can be used for injection. In this form, alpha-tocopherol is more efficiently absorbed from intramuscular injection sites than is a water miscible preparation containing alpha-tocopheryl acetate or either form dissolved in an oily base (Machlin, 1991). Products containing vitamin E and selenium can often be given intramuscularly to dogs exhibiting clinical signs of muscular degeneration. Response to treatment of this condition is extremely variable depending on degree of muscular degeneration.
The need for supplementation of vitamin E is dependent on the requirement of individual species, conditions of production and the amount of available vitamin E in food or feed sources. The most important consideration for pet diets is the feed ingredients. If diets have relatively large quantities of meat and slaughterhouse by-products (e.g., fat and internal organs), good sources of vitamin E will be provided. If, however, grains make up a large proportion of the diet, little available vitamin E will be provided. Adverse conditions, such as poor weather (drought and early frost), molds and insect infestation, will reduce the vitamin E value of feedstuffs. The vitamin E activity in blighted corn was 59% lower than that in sound corn, and the vitamin E activity in lightweight corn averaged 21% below that in sound corn (Adams et al., 1975). Feed spoilage will also promote vitamin E-selenium deficiencies. Therefore, to prevent loss of vitamin E in diets, the producer should use fresh feed at all times because the vitamin is rapidly destroyed under hot, humid conditions. Losses during storage increase as the duration and temperature of storage increase.
For pet foods, the most important feed ingredient that will dictate the need for supplementation is fat source and the stability of the fat source. Large amounts of PUFA can quickly precipitate vitamin E deficiency. Fats from oilseeds are normally not highly detrimental because the oils, in addition to containing high amounts of predominantly linoleic acid (18:2), are likewise sources of vitamin E. Fish oils, however, contain little vitamin E and are highly destructive to vitamin E stores, greatly increasing the requirements of the vitamin. As previously mentioned, a diet that contains high levels of fish oil may cause a three- to four-fold increase in a cat’s daily requirement for vitamin E. Rancid fats should never be fed to pets as they will quickly bring about destruction of fat-soluble vitamins.
It has been suggested that a fixed ratio of vitamin E to PUFA should be maintained in the diet to assure adequate vitamin E intake in the presence of high levels of unsaturated fats, particularly in cat foods containing fish products or added fish oil. In counteracting high levels of PUFAs in pet diets, Harris and Embree (1963) recommended a dietary alpha-tocopherol:PUFA ratio of 0.6:1. This is a good approach but does not differentiate between the major oilseed PUFA of linoleic acid (18:2) and linolenic acid (18:3) from the more highly unsaturated fatty acids of fish oil, including the fatty acids arachidonic (20:4), docosapentaenoic (22:5) and docosahexaenoic (22:6). A more appropriate method to provide sufficient quantities of vitamin E is to follow the AAFCO (2007) recommendation of adding 10 IU vitamin E above the vitamin E requirement per gram of fish oil per kg of diet (4.55 IU per g per lb of diet).
Supplementing vitamin E in well-balanced diets has been shown to increase humoral immunity in a number of species (Badwey and Karnovsky 1980; Cipriano et al., 1982; Burkholder and Swecker, 1990). These results suggest that the criteria for establishing requirements based on overt deficiencies or growth do not consider optimal health (Hoffmann-La Roche, 1991). Additional supplemental vitamin E in dog and cat diets would provide the insurance of optimum health for these pets.
Longevity of pets and reduction of “age-related” diseases, such as cancer and heart disease, have a relationship to free radical damage, which can be minimized by antioxidant vitamin supplementation. Vitamin E appears to lengthen life span (Strombeck, 1999). When animals are given vitamin E from a young age, the onset slows for some age-related problems, such as cataracts, cancer, cardiovascular disease and decreased immune function. Dogs with dilated cardiac myopathy have increased oxidative stress compared with normal dogs, and vitamin E concentrations decrease as the disease progresses (Freeman, 1998). Therefore, antioxidant therapy of dogs and cats with cardiac disease is recommended.
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 E supplemental level for a 13.6 kg (30 lb) dog is 90 IU and for a 2.7 kg (6 lb) cat is 18 IU.
Marks et al. (1994) suggest that vitamin E may be beneficial for the management of dogs and cats with copper-associated liver damage because of the antioxidant effects that protect against lipid peroxidation. Vitamin E should be supplemented at levels of 500 mg per day for dogs and 100 mg per day for cats.
The amount of vitamin E required to produce milk is much less than the amount needed to produce milk containing optimum concentrations of the vitamin. Since placental transfer of vitamin E to puppies and kittens is expected to be low, based on studies with other species (Mahan, 1990; Nockels, 1991; Njeru et al., 1994), providing colostrum and later-produced milk high in vitamin E is essential. In sheep, ewes receiving supplemental vitamin E produced colostrum almost three-fold higher in vitamin E than did non-vitamin E-supplemented animals (Njeru et al., 1994). Also, after the nursing period, dogs, and particularly cats, do sometimes receive cow’s milk as part of their diet. Cows receiving higher levels of dietary vitamin E likewise produce milk containing more vitamin E. Feeding supplemental vitamin E as dl-alpha-tocopheryl acetate, providing an equivalent of 500 mg of dl-alpha-tocopherol per cow per day, increased the vitamin E content and oxidative stability of milk (Dunkley et al., 1967).
Vitamin E fed to meat-producing animals will result in a more stable meat product, including stability of the red meat color. Dramatic effects of vitamin E supplementation (500 IU per head daily) to finishing steers on the stability of beef color have been observed (Faustman et al., 1989a). Loin steaks of control steers discolored two to three days sooner than those supplemented with vitamin E. Supplemental dietary vitamin E extended the color shelf-life of loin steaks from 3.7 to 6.3 days. In a subsequent report, Faustman et al. (1989b) observed that vitamin E stabilized the pigments and lipids of meat from the supplemented steers. Except for increased stability of pet foods, the effect of meat color would seem completely unimportant from a nutritional standpoint. However, color is an extremely critical component of fresh red meat appearance and greatly influences the customer’s (pet owner’s) perception of meat quality. This would only be a consideration for pet diets that are predominantly meat (particularly beef).
Oxidative stress, or the imbalance between proxidants and the body’s antioxidant defense system, likely plays a role in the development of skin disease (Jewell and Joshi, 2002). Vitamin E therapy has a role in certain skin disorders in dogs. Apparently, subclinical vitamin E deficiency causes suppression of the immune system, which increases a dog’s susceptibility to certain skin conditions. Vitamin E evidently has an anti-inflammatory effect that may be related to stabilization of cell and lysosomal membranes against damage resulting from free radicals and peroxides (Bissett et al., 1990). Favorable responses to vitamin E supplementation with varying levels of success have resulted from the skin conditions of demodicosis, primary acanthosis nigricans, discoid lupus erythematosus and dermatomyositis (Werner and Harvey, 1995). Vitamin E deficiency in dogs has also been associated with other cutaneous abnormalities (Sheffy, 1979; Worden, 1958). The dermatosis was poorly characterized but included “sores” on the feet and over wear areas as well as generalized scaling and “denudation” (Scott and Sheffy, 1987).
Many dogs with acanthosis nigricans and other skin disorders respond favorably to treatment with systemic glucocorticoids. However, concern with the immediate and long-term side effects of glucocorticoid therapy dictates the need to find alternative treatments, one of which may be supplementation with vitamin E. In many cases, skin disorders are chronic and persistent, and therapy is directed toward control rather than cure.
Quantities of therapeutic vitamin E administered to alleviate skin disorders are experimental and highly variable. For discoid lupus erythematosus, the therapeutic dosage in dogs was 100 to 400 mg of dl-alpha-tocopheryl acetate given orally twice daily (Codner and Thatcher, 1993). For primary acanthosis nigricans, dachshunds showed improvement after 60 days of vitamin E therapy (200 mg twice daily) (Scott and Walton, 1985). A vitamin E supplemented level of 200 mg five times daily was curative for demodicosis for 147 of 149 cases (Fiqueriredo, 1985).
Vitamin E supplementation has been successful in treatment of a number of conditions which are believed to be related to the potential benefits of the antioxidant function of the vitamin. Conditions that respond to supplemental vitamin E in dogs include senile dementia (Skoumalova et al., 2003), chronic hepatitis (Honeckman, 2003), canine immune-mediated hemolytic anemia (Pesillo et al., 2004), congestive heart failure (Freeman et al., 2005) and nephrotoxicity (Varzi et al., 2007). For cats with chronic renal failure, antioxidants, including vitamin E, may be beneficial (Yu and Paetau-Robinson, 2006).
Vitamin E and other antioxidants were tested before and after an oxidant challenge, oral acetaminophen, in cats (Hillet al., 2005). Vitamin E plus cysteine had a protective effect against oxidation. The antioxidant effect of vitamin E has been shown when added to semen extenders to improve seman quality from both dogs (Michael et al., 2009) and cats (Thuwanut et al., 2008).
The effect of vitamin E and selenium supplementation on immune status of dogs vaccinated with antigens against Taenia hydatigena was evaluated (Kandil and Abou-Zeina, 2005). Among other treatments, the best immune response against T. hydatigena was observed in the vitamin E-selenium supplemented groups as evidence by increased production of antibody titer and IgG concentration in comparison with either vaccinated unsupplemented or control groups. Vitamin E and selenium proved to be immune-potentiating to vaccinated dogs and increased the possibility for protection against T. hydatigena infections.
Naturally occurring vitamin E deficiency in cats is usually reported when diets containing excessive amounts of canned red tuna or cod liver oil are fed. The prognosis for steatitis in untreated cats can be grave. Therapy consists of dietary correction, oral vitamin E supplementation (dl-alpha-tocopherol, 25 to 75 mg twice daily), anti-inflammatory doses of corticosteroids [e.g., prednisone, 0.5 to 1.0 mg per kg (0.23 to 0.45 mg per lb) of body weight twice daily] and supportive care (Codner and Thatcher, 1993). The corticosteroid therapy, in addition to decreasing inflammation will reduce pain in the cat. Severely affected cats may fail to improve. However, prognosis for recovery from steatitis is usually very good, but it may be slow in advanced cases.
Vitamin E has a wide margin of safety in animals, and little information is available for toxicity considerations for dogs and cats. Compared with vitamin A and vitamin D, both acute and chronic studies with animals have shown that vitamin E is relatively nontoxic, but not entirely devoid of undesirable effects. However, prolonged excessive consumption of tocopherol may have a depressing effect on the absorption of the other fat-soluble vitamins, and extraordinary parenteral doses (100 to 200 mg per kg per day; 45.5 to 90.0 mg per lb) in kittens have induced hepatosplenomegaly and death (Ralston Purina, 1987). Although alpha-tocopherhol has been the most widely studied, the other three tocopherols and four tocotrienols have recently been shown to have functions apart from alpha-tocopherol. Excess supplementation of diets with alpha-tocopherol reduced serum concentrations of gamma- and delta-tocopherol (Haung and Appel, 2003; Wolf, 2006). For livestock, the effects of high supplemental levels of alpha-tocopherol on other forms of vitamin E are unknown. It has been observed that although vitamin E did not interfere with coagulation in normal dogs, it was able to block oxidation of vitamin K to the epoxide form in warfarin-treated dogs and thereby exacerbated the coagulopathy (Corrigan, 1979). Hypervitaminosis E studies in rats, chicks and humans indicate maximum tolerable levels in the range of 1,000 to 2,000 IU per kg (455 to 910 IU per lb) of diet (NRC, 1987).
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