DSM in Animal Nutrition & Health
Properties and Metabolism
Vitamin B6 refers to a group of three compounds: pyridoxol (pyridoxine), pyridoxal and pyridoxamine. Pyridoxol is the predominant form in plants, whereas pyridoxal and pyridoxamine are vitamin forms generally found in animal products. These three forms have equal activity when administered to animals, but are not equivalent when administered to various microorganisms. Two additional vitamin B6 forms found in foods are the coenzyme forms of pyridoxal phosphate (PLP) and pyridoxamine phosphate. Various forms of vitamin B6 found in animal tissues are interconvertible, with vitamin B6metabolically active mainly as PLP and to a lesser degree as pyridoxamine phosphate. Vitamin B6 has been shown to be stable to heat, acid and alkali; however, exposure to light, especially in neutral or alkaline media, is highly destructive. The free base and the commonly available hydrochloride salt are soluble in water and alcohol (Illus. 9-1).
There are several vitamin B6 antagonists, which either compete for reactive sites of apoenzymes or react with PLP to form inactive compounds. The presence of a vitamin B6 antagonist in linseed meal is of particular interest to animal nutritionists. This substance was identified in 1967 as hydrazic acid and was found to have antibiotic properties (Parsonsand Klostermann, 1967). Pesticides (e.g., carbaryl, propoxur or thiram) can be antagonistic to vitamin B6. Feeding a diet enriched with vitamin B6 prevented disturbances in the active transport of methionine in rats intoxicated with pesticides (Witkowska et al., 1992). Digestion of vitamin B6 would first involve splitting the vitamin, as it is bound to the protein portion of foods. Vitamin B6 is absorbed mainly in the jejunum but also in the ileum by passive diffusion. Absorption from the colon is insignificant, even though colon microflora synthesize the vitamin. However, Durst et al. (1989) administered vitamin B6 in the cecum of sows and concluded that the vitamin was absorbed at this location. Little information is available on digestion and absorption of vitamin B6 in ruminants. However, large quantities of the vitamin are synthesized in the rumen.
Vitamin B6 compounds are all absorbed from the diet in the dephosphorylated forms. The small intestine is rich in alkaline phosphatases for the dephosphorylation reaction. Sakurai et al. (1992) reported that a physiological dose of pyridoxamine was rapidly transformed to pyridoxal in the intestinal tissues and then released in the form of pyridoxal into the portal blood. Under normal conditions, most of the vitamin B6 in the blood is present as PLP that is linked to proteins, largely albumin in the plasma and hemoglobin in the red blood cells (McCormick, 2006). After absorption, B6 compounds rapidly appear in the liver, where they are mostly converted into PLP, considered to be the most active vitamin form in metabolism. Pyridoxal phosphate is the major B6 form in goat milk, accounting for 75% of the vitamin B6 activity (Coburn et al., 1992). Both niacin (as nicotinamide adenine dinucleotide phosphate [NADP]-dependent enzyme) and riboflavin (as the flavoprotein pyridoxamine phosphate oxidase) are important for conversion of vitamin B6 forms and phosphorylation reactions (Kodentsova et al., 1993).
Although other tissues also contribute to vitamin B6 metabolism, the liver is thought to be responsible for forming PLP found in plasma. Pyridoxal and PLP found in circulation are associated primarily with plasma albumin and red blood cell hemoglobin (Mehansho and Henderson, 1980). Pyridoxal phosphate accounts for 60% of plasma vitamin B6. Researchers do not agree on whether pyridoxal or PLP is the transport form of B6 (Driskell, 1984).
Only small quantities of vitamin B6 are stored in the body. The vitamin is widely distributed in various tissues, mainly as PLP or pyridoxamine phosphate. Vitamin B6 readily passes through the placenta. Pyridoxal crosses the human placenta readily in both directions (Delport et al., 1991; Schenker et al., 1992). Pyridoxic acid is the major excretory metabolite of the vitamin, eliminated via the urine. Also, small quantities of pyridoxol, pyridoxal and pyridoxamine, as well as their phosphorylated derivatives, are excreted into the urine (Henderson, 1984). Vitamin B6metabolism is altered in renal failure, as observed in rats which exhibited plasma pyridoxal phosphate levels 43% lower than controls (Wei and Young, 1994).
Vitamin B6 in the form of PLP and, to a lesser degree, pyridoxamine phosphate plays an essential role in the interaction of amino acid, carbohydrate and fatty acid metabolism in the energy-producing TCA cycle. Over 60 enzymes are already known to depend on vitamin B6coenzymes. Pyridoxal phosphate functions in practically all reactions involved in amino acid metabolism, including transamination, decarboxylation, deamination, and desulfhydration, as well as the cleavage or synthesis of amino acids.Vitamin B6 participates in functions that include (Bräunlich, 1974; Marks, 1975; Driskell, 1984; McCormick, 2006):
- (a) Deaminases–for serine, theonine and cystathionine.
- (b) Desulfydrases and transulfurases–interconversion.
- (c) Synthesis of niacin from tryptophan–hydroxykynurenine is not converted to hydroxyanthranilic acid, but rather to xanthurenic acid due to lack of the B6- dependent enzyme, kynureninase.
- (d) Formation of alpha-aminolevulinic acid from succinyl CoA and glycine, the first step in porphyrin synthesis.
- (e) Conversion of linoleic to arachidonic acid in the metabolism of essential fatty acids (this function is controversial).
- (f) Glycogen phosphorylase catalyzes glycogen breakdown to glucose-1-phosphate. Pyridoxal phosphate does not appear to be a coenzyme for the enzyme but rather to affect the enzyme conformation.
- (g) Synthesis of epinephrine and norepinephrine from either phenylalanine or tyrosin–both norepinephrine and epinephrine are involved in carbohydrate metabolism as well as in other body reactions.
- (h) Racemases–PLP-dependent racemases enable certain microorganisms to utilize D-amino acids. Racemases have not yet been detected in mammalian tissues.
- (i) Transmethylation involving methionine.
- (j) Incorporation of iron in hemoglobin synthesis.
- (k) Formation of antibodies–B6 deficiency results in inhibition of the synthesis of globulins that carry antibodies.
- (l) Inflammation-higher vitamin B6 levels were linked to protection against inflammation.
Neurological disorders, including states of agitation and convulsions, result from reduction of B6 enzymes in the brain, including glutamate decarboxylase and gamma-aminobutyric acid transaminase. With a vitamin B6 deficiency dopamine release is delayed, which may contribute to motor abnormalities (Tang and Wei, 2004). Maternal restriction of B6 in rats adversely affected synaptogenesis, neurogenesis and neuron longevity, and differentiation of the progeny (Groziak and Kirksey, 1990; 1997). Recent work in animal models suggests that vitamin B6 deficiency during gestation and lactation alters the function of N-methyl-D-aspartate receptors, a subtype of receptors of the glutamatergic neurotransmitter system thought to play an important role in learning and memory (Guilarte, 1993).
Animal and human studies suggest that a vitamin B6 deficiency affects both humoral and cell-mediated immune responses. In humans, vitamin B6 depletion significantly decreased percentage and total number of lymphocytes, mitogenic responses of peripheral blood lymphocytes to T- and B-cell mitogens and interleukin 2 production (Meydaniet al., 1991). The role of PLP in effecting one-carbon metabolism is important in nucleic acid biosynthesis and immune system function. The PLP is also needed for gluconeogenesis by way of trasnsaminases active on glucogenic amino acids and for lipid metabolism that involves several aspects of PLP function. For example, for production of carnitine needed to act as a vector for long chain fatty acids for mitochondrial β-oxidation and of certain bases for phospholipid biosynthesis (McCormick, 2006). Additional human studies indicate that vitamin B6 status may influence tumor growth and disease processes. Deficiency of the vitamin has been associated with immunological changes observed in the elderly, persons infected with human immunodeficiency virus (HIV) and those with uremia or rheumatoid arthritis (Rall and Meydani, 1993).
The requirement for vitamin B6 has been found generally to depend on species, age, physiological function, dietary components, the intestinal flora and other factors that are not yet fully understood. Vitamin B6 is produced by microorganisms in intestinal tracts of animals, but whether significant quantities are absorbed and utilized is in doubt. Animals practicing coprophagy (e.g., the dog) would obviously be receiving vitamin B6from this source.Breed of animal and environmental temperature have been shown to influence vitamin B6 requirements for some species (e.g., poultry and rats). Regarding ambient temperature, when rats are housed at 33°C they needed twice as much vitamin B6 as when they were housed at 19°C (Bräunlich, 1974).
Quantity of dietary protein affects the requirement for vitamin B6 in both animals and humans. The requirement is increased when high-protein diets are fed. For example, when feed contained 60% casein instead of 20%, the level of pyridoxine required by mice was three times as high (Miller and Baumann, 1945). Research findings indicate that the requirement of growing cats is positively related to the level of protein in the diet (Axelrod et al., 1945; Bai et al., 1991). For a 30% casein diet, the B6 requirement was between 1 and 2 mg per kg (0.45 and 0.91 mg per lb) of diet, but for a 60% casein diet the requirement was 2.0 or more mg per kg (0.91 mg per lb) of diet (Morris and Rogers, 1994).
A number of studies have suggested that amino acid imbalance has an adverse effect on vitamin B6 status, in that weight gain was depressed and survival was decreased when large amounts of a single amino acid were added to rat diets limited in the vitamin. High tryptophan, methionine and other amino acids increase the need for vitamin B6 (Scott et al., 1982).
Certain feed antagonists as well as bioavailability of B6 in feeds and nutrients other than protein influence the B6requirement. Niacin and riboflavin are needed for interconversions of different forms of vitamin B6, with an overdose of thiamin reported to produce vitamin B6 deficiency in rats (LeKlem, 1991). Roth-Maier and Kirchgessner (1993) suggested that adult sows are able to maintain optimal metabolic functions over eight weeks by utilizing bacterially synthesized vitamin B6. Cellulose supplemented to these pigs increased total vitamin B6 excretion from 3.4 to 5.2 mg and 25% to 50% was excreted in urine. In rats administered sulfasalazine, a vitamin B6 deficiency was aggravated, suggesting that the intestinal synthesis of the vitamin was affected (Trumbo and Raidi, 1991). A large amount of literature for humans (McDowell, 2000) has shown that vitamin B6 requirements are elevated as a result of drugs and inborn errors of metabolism. It is likely that the vitamin B6 requirements of dogs and cats are likewise altered by unrecognized inborn errors of metabolism and by drugs.
Flesh-eaters, such as cats, derive considerable energy from dietary protein. Because these animals have high transaminase activity, it is logical to expect that their vitamin B6 turnover, and therefore requirement, would be higher than that of omnivores. The vitamin B6 requirement of the cat is about 60% higher than that of the dog (NRC, 2006; AAFCO, 2007).
A. Requirements for Dogs
According to the NRC (2006) the vitamin B6 requirement for all classes of dogs is 1.5 mg per kg (0.68 mg per lb) of diet. This recommendation is in agreement with the AAFCO (2007).
In early research (Michaud and Elvehjem, 1944) with growing dogs, those given 5 µg vitamin B6 per kg (2.3 µg per lb) body weight died before evidence of anemia appeared, whereas 10 µg per kg (4.5 µg per lb) gave fairly good growth, but not equal to dogs with 60 µg per kg (27.3 µg per lb).
B. Requirements for Cats
Gershoff et al. (1959b), using three- to six-month-old kittens fed a semi-purified diet, reported that 1 mg of pyridoxine HCl per kg (0.45 mg per lb) of diet was not adequate for all cats, but twice that amount permitted normal growth and hematology. However, urinary excretion of oxalate was greater in cats given a diet containing 2 mg pyridoxine per kg (0.91 mg per lb) than in those receiving a diet containing 4 mg pyridoxine per kg (1. 8 mg per lb). A minimal requirement of 2.5 mg pyridoxine per kg (1.1 mg per lb) of diet is recommended for all classes of cats (NRC, 2006). Bai et al. (1989) suggest that 1 mg of vitamin B6 per kg (0.45 mg per lb) of diet is insufficient for growing kittens, but that 2 mg per kg (0.91 mg per lb) is adequate when kittens received a 35% casein diet. Blanchard et al.(1991) reported kidney lesions in kittens fed only 1 mg vitamin B6 per kg (0.45 mg per lb) of diet and suggest that 2.0 mg per kg (0.91 mg per lb) of diet was sufficient for growing kittens. As previously noted, Morris and Rogers (1991) suggested 2 or more mg vitamin B6 per kg (0.91 mg per lb) for diets containing high levels of protein (e.g., 60% casein). AAFCO (2007) recommended 2.4 mg per kg (1.1 mg per lb) of diet for all classes of cats, in agreement with the NRC (2006).
Most feedstuffs, except fruits, are good sources of vitamin B6. In general, muscle, liver, vegetables, whole grain cereals and their by-products, and nuts are among the best sources. The bioavailability of two common feed ingredients is 65% for soybean meal with corn varying from 45% to 56% (McDowell and Ward, 2008). The vitamin B6 present in cereal grains is concentrated mainly in bran; the rest contains only small amounts. The richest source is royal jelly produced by bees (5,000 µg per kg or 2,273 µg per lb). Most vitamin B6 in animal products is in the form of pyridoxal and pryridoxamine phosphates. In plants and seeds the usual form is pyridoxol (McDowell, 2000). The principal source of vitamin B6 to ruminants is that obtained by microbial synthesis in the rumen.
The level of vitamin B6 contained in all feeds is affected by processing and subsequent storage. Of the several forms, pyridoxol (pyridoxine) is far more stable than either pyridoxal or pyridoxamine. Therefore, the processing losses of vitamin B6 tend to be highly variable (9% to 40%), with plant-derived foods (which contain mostly pyridoxine) losing little if any of the vitamin, and animal products (mostly pyridoxal and pyridoxamine) losing large quantities (Hand et al., 2010). Vitamin B6 loss during cooking, processing, refining and storage has been reported to be as high as 70% (Shideler, 1983) or in the range of 0% to 40% (Birdsall, 1975). Losses may be caused by heat, light, and various agents that can promote oxidation. Two-hour sunlight exposure may destroy half of the vitamin B6 in milk. Blanching of rehydrated lima beans resulted in a loss of 20% of the vitamin B6, but, more significantly, the availability of the vitamin was reduced by almost 50% (Ekanayake and Nelson, 1990). Irradiation as a potential method for microbial control of poultry feed results in a loss of 15% of vitamin B6 potency (Leeson and Marcotte, 1993).
Pyridoxine-5’–beta-D-glucoside (PNG), a conjugated form of vitamin B6, has been shown to be abundant in various plant-derived foods. This form of B6 may account for up to 50% of the total vitamin B6 content of oilseeds such as soybeans and sunflower seeds. The utilization of dietary PNG relative to pyridoxine has been shown to be 30% in rats and 50% in humans (Gregory et al., 1991). In suckling rats, the availability of vitamin B6 derived from PNG is only 25% compared with pyridoxine (Trumbo and Gregory, 1989). The glycosylated PNG can quantitatively alter the metabolism of pyridoxine in vivo; hence, it partially impairs the metabolic utilization of co-ingested non-glycosylated forms of vitamin B6 (Nakano and Gregory, 1995; Nakano et al., 1997).
Commercially, vitamin B6 is available as crystalline pyridoxine and various dilutions. Pyridoxine hydrochloride contains 82.3% of vitamin B6 activity. Dry premixes are used in feeds, while the crystalline product is used in parenteral and oral pharmaceuticals as well as in feeds.
Characteristics of vitamin B6 deficiency in most species are retarded growth, dermatitis, epileptic-like convulsions, anemia and a partial alopecia. Due to the predominate function of the vitamin in protein metabolism, in vitamin B6deficiency a fall in nitrogen retention is observed, feed protein is not well utilized, nitrogen excretion is excessive and impaired tryptophan metabolism may result.An indication of a vitamin B6 deficiency is elevated urinary levels of xanthurenic acid and kynurenic acid, indicating incomplete conversion of tryptophan. Axelrod et al. (1945) showed that following a tryptophan load, young vitamin B6-deficient dogs excreted xanthurenic acid and kynurenine in their urine. Dogs supplemented with pyridoxine excreted kynurenine and kynurenic acid but no xanthurenic acid. Measuring tryptophan metabolites does not apply to cats because their tryptophan metabolism is different from most other mammalian species that have been studied (Carvalho de Silva, 1959a). Buckmaster et al. (1993) reported that recording brain stem auditory evoked potentials (BAEP) provided a noninvasive means of detecting the effects of vitamin B6 deficiency in cats.
Direct measurement of one or more forms of vitamin B6 in plasma, urine or erythrocytes is a relatively reliable indicator of vitamin B6 status. Urinary 4-pyridoxic acid (4-PA) excretion is considered a short-term indicator of vitamin B6 status, due to the fact that 4-PA reflects recent vitamin B6 intake rather than the underlying state of tissue reserve. Often 4-PA is not detectable in the urine of vitamin B6-deficient subjects. One of the most commonly used measures of vitamin B6 status is the measurement of erythrocyte alanine and aspartic acid transaminase(s) activity (Leklem, 1991; McCormick, 2006).
A. Deficiency in Dogs
A typical consequence of a B6 deficiency in both young and old dogs is a microcytic, hypochromic anemia. The anemia is severe with hemoglobin values as low as 1.4 g per dl (Fouts et al., 1939). It is probably caused by an inability to synthesize amino-levulinic acid, a precursor of heme. In addition to decreased appetite and body weight loss, elevated plasma iron levels, convulsions and death, there are pathological changes, including ataxia, cardiac dilatation and hypertrophy, congestion of various tissues and demyelination of peripheral nerves (NRC, 1985). Acute deficiency of vitamin B6 in growing puppies may lead to sudden death with clinical signs of only anorexia, slow growth or body weight loss.
B. Deficiency in Cats
Clinical signs of vitamin B6 deficiency in cats include growth depression, a mild microcytic, hypochromic anemia with elevated serum iron, convulsive seizures and irreversible kidney lesions consisting of areas of tubular atrophy and dilatation, fibrosis and calcium oxalate nephrosis (Gershoff et al., 1959b). Vitamin B6-induced renal damage due to the deposition of large amounts of calcium-oxalate throughout the kidneys has been reported by a number of researchers (Gershoff et al., 1959b; Carvalho Da Silva et al., 1959a; Blanchard et al., 1991; Kirk et al., 1995). With a vitamin B6 repletion diet there is a decline in urinary oxalates and improvement of hematocrits and weight gains (Bai et al., 1989; Blanchard et al., 1991).
Histologically, the kidneys of vitamin B6-deficient cats resemble those seen in humans suffering from idiopathic oxalosis. Abnormal amounts of hemosiderin are deposited in the spleen and liver, giving the liver a bright orange color. Thus, as in the dog, it is clear that the anemia is not related to an inability to absorb iron (Ralston Purina, 1987).
Vitamin B6 deficiency has been reported to produce behavioral, neurophysiological and neuropathological abnormalities in a variety of species including cats. Buckmaster et al. (1993) used BAEP and determined that vitamin B6 deficiency in cats affected peripheral and brainstem auditory pathways. The finding of prolonged interwave intervals in vitamin B6-deficient cats was consistent with slowed axonal conduction velocity secondary to defective myelination. The growth of mammary tumor cells was inhibited with the supplementation of vitamin B6 in a dose-dependent manner (Shimada et al., 2006).
Vitamin B6 is one of the B vitamins that is least likely to be deficient in livestock and pet diets. Because of its wide distribution in feedstuffs, nutritionists generally expect adequate levels in typical pet diets. Nevertheless, vitamin B6deficiencies have been reported for dogs and cats when diets were prepared and processed in such a way as to destroy the vitamin or the diet used sources where the vitamin B6was biologically unavailable. Concern that a dog or cat is vitamin B6-deficient probably will not arise unless the animal is convulsive and/or has a microcytic hypochromic anemia. However, a nondetectable subclinical deficiency of vitamin B6 may affect performance (e.g., the immune response), but may not exhibit the more traditional signs of deficiency such as microcytic hypochromic anemia. If the clinical diagnosis of a dog or cat is consistent with vitamin B6 deficiency, the diagnosis can be confirmed by an injection of 25 mg of pyridoxine, which will stop convulsive seizures within one to two hours and will initiate a hematologic response (Ralston Purina, 1987).
Perry (1978) listed reasons for needed supplementation of vitamin B6 for animal diets as follows: (a) great variations in amounts of B6 in individual ingredients, (b) variable bioavailability of this vitamin in ingredients, (c) losses reported during processing of ingredients, (d) discrepancies between activity for test organisms vs. those for animals, (e) a higher vitamin B6 requirement due to a marginal level of methionine in the diet and (f) high protein diets.
Variability of vitamin B6 in feeds depends on the sample origin, conditions of growth, climate, weather conditions and other local factors. Yen et al. (1976) determined available vitamin B6 in corn and soybean meal using a chick growth assay. Corn was found to be 38% to 45% available and B6 in soybean meal 58% to 62% available. It is probably equally available for the dog and cat. There was little difference in availability between corn samples not heated and the heated to 120°C. However, corn heated to 160°C contained significantly less available B6. Level of vitamin B6contained in feedstuffs is also affected by processing and subsequent storage. Bioavailability can be as low as 40% to 50% after heat processing of feedstuffs.
Predominant losses of vitamin B6 activity in feedstuffs occur in the pyridoxal and pyridoxamine forms, with pyridoxine the more stable form. Supplemental vitamin B6 is reported to have a higher bioavailability and stability than the naturally occurring vitamin. Naturally occurring vitamin B6 in retorted milk products exhibited only 50% of the bioavailability of synthesized B6 or B6 in formulas that were fortified with the vitamin prior to thermal processing (Tomarelli et al., 1955).
The recovery of vitamin B6, as pyridoxine hydrochloride, in a multivitamin premix not containing trace minerals, was 100% even after three months of storage at 37°C. However, stability in a premix containing trace minerals was poor, with only 45% recovery after three months at 37°C (Adams, 1982). Verbeeck (1975) found vitamin B6 to be stable in premixes with minerals as sulfates. However, if minerals in the form of carbonates and oxides are used, 25% of the vitamin can be lost over a three-month period. Stress agents such as choline chloride help catalyze this destruction. Gadient (1986) considers pyridoxine to be very sensitive to heat, slightly sensitive to moisture and light, and insensitive to oxygen. Retention of B6 activity in pelleted feeds after three months at room temperature should be 80% to 100% as a general rule.
Insufficient data are available to support estimates of the maximum dietary tolerance of vitamin B6 for cats. It is suggested, primarily from dog and rat data, that dietary levels of more than 1,000 times the nutritional requirements are needed to produce signs of toxicity in these species (NRC, 1987). Dogs given oral doses of 20 mg of pyridoxine per kg (9.1 mg per lb) of body weight reported no signs of toxicity (Unna and Antopol, 1940). Higher doses of the vitamin have been found to produce signs of toxicity. Phillips et al. (1978) reported that ataxia, muscle weakness, and loss of balance developed between 40 and 75 days in dogs that received 200 mg of pyridoxine per kg (90.9 mg per lb) of body weight. Histological examination of the tissues revealed bilateral loss of myelin and axons in the dorsal funiculi and loss of myelin in individual fibers of the dorsal nerve roots. A lesser amount of pathological damage was observed in dogs receiving 50 mg per kg (22.7 mg per lb) of body weight daily. Dogs fed daily doses of 250 mg per kg (113.6 mg per lb) began to develop incoordination and ataxia within the first week of treatment.
Krinke et al. (1980) administered daily oral doses of 300 mg of pyridoxine per kg (136.4 mg per lb) of body weight to pairs of seven- to 11-month-old beagle dogs for 78 days. Treated dogs developed a swaying gait and became unable to walk. It was concluded that excess vitamin B6 produced a toxic, peripheral, sensory neuronopathy involving degeneration of the dorsal root ganglia, gasserian ganglia, and sensory nerve fibers.