DSM in Animal Nutrition & Health
Vitamin B6 refers to a group of three compounds: pyridoxol (pyridoxine), pyridoxal and pyridoxamine. Pyridoxine 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 (Parsons and 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 B6in 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. After absorption, B6 compounds rapidly appear in liver, where they are mostly converted into PLP, considered to be the most active vitamin form in metabolism. Under normal conditions, most of the vitamin B6 in blood is present as PLP that is linked to proteins, largely albumin in the plasma and hemoglobin in the red blood cells (McCormick, 2006). 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 B6forms 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 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 in urine (Henderson, 1984). Vitamin B6 metabolism is altered in renal failure, as observed in rats exhibiting plasma pyridoxal phosphate 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 B6participates 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 (Morris et al., 2010).
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, 1997; 1990). 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 transaminases 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).
Requirements for vitamin B6 have been found to depend on species, age, physiologic function, dietary components, the intestinal flora and other factors that are not yet fully understood. Vitamin B6 is produced by microorganisms of the intestinal tract of poultry, but whether significant quantities are absorbed and utilized is in doubt. Animals practicing coprophagy receive vitamin B6 from this source. Vitamin B6 requirements generally vary from 2.5 to 4.5 mg per kg (1.4 to 2 mg per lb) of diet (NRC, 1994). The requirement for optimum growth of ducklings from day old to three weeks of age was 2.2 mg per kg (1 mg per lb) of vitamin B6 (Yang and Jeng, 1989; Yang et al., 1992). Breed of animal and environmental temperature have been shown to influence vitamin B6 requirements. Lucas et al. (1946) found that crossbred chicks (Rhode Island Red x Barred Plymouth Rock) showed a considerably higher requirement for pyridoxine (B6) than had previously been found for White Leghorn chicks. A Japanese strain of chickens has also been shown to have a higher B6 requirement (Scott et al., 1982). Quantity of dietary protein affects the requirement for vitamin B6 in poultry. The vitamin B6 requirement is increased when high-protein diets are fed. Gries and Scott (1972) found a much higher vitamin B6 requirement in chicks receiving 31% protein than in those receiving a normal 22% protein diet. A number of studies have suggested that amino acid imbalance has an adverse effect on vitamin B6 status. For example, weight gain was depressed and survival was decreased when large amounts of a single amino acid was added to rat diets limited in the vitamin. High amounts of tryptophan, methionine and other amino acids increase the need for vitamin B6 (Scott et al., 1982).
Fisher et al. (1984) reported a consistent deleterious effect of a low-quality protein on vitamin B6 status in rats. Certain feed antagonists, bioavailability of B6 in feed, and nutrients other than protein influence the B6 requirement. Niacin and riboflavin are needed for interconversions of different forms of vitamin B6, with an excess of thiamin reported to produce vitamin B6 deficiency in rats (LeKlem, 1991).
Most feedstuffs 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 bioavalibility of vitamin B6 in soybean meal is 65 percent and in corn it varies from 45 to 56 percent (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 pyridoxamine 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, 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 mostly contain pyridoxine) losing little if any of the vitamin, and animal products (mostly pyridoxal and pyridoxamine) losing large quantities. 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 significant, the availability of the vitamin was reduced almost 50% (Ekanayake and Nelson, 1990). Irradiation as a potential method for microbial control of poultry feed results in a loss of 15% vitamin B6 potency (Lesson and Marcotte, 1993).
Pyridoxing-5’-beta-D-glucoside (PNG), a conjugated form of vitamin B6, had been shown to be abundant in various plant-derived foods (McCormick, 2006). 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, 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 hydrochloride and various dilutions. Pyridoxine hydrochloride contains 82.3% 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 are retarded growth, dermatitis, epileptic-like convulsions, anemia and partial alopecia. Due to the predominant function of the vitamin in protein metabolism, in vitamin B6 deficiency, a reduction in nitrogen retention is observed, feed protein is not well utilized, nitrogen excretion is excessive and impaired tryptophan metabolism may result. Also, lipid metabolism is affected. That is, desaturation and elongation of fatty acids is impaired in growing chickens (An et al., 1995). Chicks fed a vitamin B6-deficient diet have little appetite and grow slowly, with plumage failing to develop fully (Illus. 9-2). Chicks receiving a B6-deficient diet exhibited general weakness after a few days of deprivation. The birds squat in a characteristic posture (Illus. 9-3), with wings slightly spread and head resting on the ground (Bräunlich, 1974). Miller (1963) observed high proportions of pendulous crops in vitamin B6-deficient chicks.
Illustration 9-2: Vitamin B6 Deficiency in Poultry
Picture on left shows inflamed edema of the eyelid in vitamin B6 deficiency. Picture on right shows vitamin B6 deficiency: rough deficient plumage, weaknes and incoordination of movements.
Illustration 9-3: Vitamin B6 Deficiency in the Chicken
Courtesy of L.R. McDowell, University of Florida
A more specific sign of B6 deficiency is the nature of the nervous condition that develops. Deficient chicks are abnormally excitable. As deprivation continues, nervous disorders become increasingly severe (Bräunlich, 1974). There is trembling and vibration of the tip of the tail, with movement stiff and jerky. Chicks run aimlessly about with lowered head and drooping wings (Illus. 9-4). Finally, convulsions develop, during which chicks fall on their side or back, with the legs scrabbling. Violent convulsions cause complete exhaustion and may lead to death (Leeson and Smmers, 2001). These clinical signs may be distinguished from those of encephalomalacia by the greater intensity of activity during a B6deficiency seizure, which results in complete exhaustion and often death (Scott et al., 1982).
Illustration 9-4: Vitamin B6 Deficiency in the Chicken
Neuritis and “squatting position.”
Courtesy of L.R. McDowell, University of Florida
Blood alterations are also typical of vitamin B6 inadequacy in chicks. An extreme deficiency leads to microcytic, polychromatic, hypochromic anemia in conjunction with atrophy of the spleen and thymus (Asmar et al., 1968). Marginal deficiencies provoke microcytic, normochromic polycythemia (Blalock and Thaxton, 1984), and deficient chicks show a decreased immunoglobulin M and immunoglobulin G response to antibody challenge (Blalock et al., 1984). Similar signs of a vitamin B6 deficiency have been observed in turkey poults: loss of appetite, poor growth (Illus. 9-5), oversensitivity, cramps and eventually death. Ducklings not receiving enough vitamin B6 grow slowly, and development of plumage is poor. At five days of age, ducklings showed retarded growth (Yang and Jeng, 1989). Clinical signs, which were first observed at seven days of age, were characterized by decreased appetite, extreme weakness, hyperexcitability, convulsions and death. Hematologic examination at three weeks of age indicated that vitamin B6 deficiency in ducklings resulted in microcytic, hypochromic anemia.
Illustration 9-5: Vitamin B6 Deficiency, Retarded Growth
Note poor growth in a vitamin B6- deficient turkey poult (about four weeks old) compared with a normal poult at right.
Courtesy of T.W. Sullivan, University of Nebraska.
Signs of B6 deficiency in chicks appear very rapidly after introduction of a B6-deficient feed. Fuller and Kifer (1959) reported that signs of a deficiency appeared on the eighth day. Chronic borderline B6 deficiency produces perosis; usually one leg is severely crippled, and one or both of the middle toes may be bent inward at the first joint (Gries and Scott, 1972). Vitamin B6 deficiency in growing chicks affected biomechanical properties of tibial bone, with reduced dry weight and cortical thickness (Masse et al., 1994; 1996). A marked increase in gizzard erosion was found in vitamin B6-deficient chicks (Daghir and Haddad, 1981). For adult poultry, vitamin B6 deficiency results in reduced egg production and hatchability as well as decreased feed consumption, weight loss and death. A severe deficiency [levels of vitamin B6below 0.5 mg per kg (0.23 mg per lb)] of diet causes rapid involution of the ovary, oviduct, comb and wattles in mature laying hens. Involution of testes, comb and wattles occurs in vitamin B6-deficient adult cockerels (Scott et al., 1982).
Vitamin B6 is one of the B vitamins that receives the least amount of attention when poultry rations are formulated. Because of its wide distribution in feedstuffs, nutritionists generally expect adequate levels in typical poultry diets. Evidence to date indicates that corn, soybean meal and other ingredients used to supply energy and protein in practical poultry diets provide the minimum requirement of vitamin B6. However, the bioavailability in corn and soybean meal ranges from only 45% to 65% (Hoffmann-La Roche, 1979). Under certain conditions, vitamin B6 supplementation is warranted for practical growing and breeding diets for poultry and other monogastrics. Fuller et al. (1961) believe that while breeder diets containing corn-soybean meal probably provide the minimum requirement for vitamin B6 to support hatchability, there is little margin of safety. In turkey breeders, there is decreasing deposition of vitamins B6 and B12 in the egg with aging (Robel, 1983). Hatchability of turkey eggs decreased with increasing age of the breeder hen. This maybe related to the decreasing nutrient deposition in the egg that occurs as the hen ages. The amount of supplemental vitamin B6 recommended for monogastric species varies from 1 to 10 mg per kg (0.45 to 4.5 mg per lb) of diet depending on species, age, activity, stress level and field conditions (Bauernfeind, 1974). Reasons for needed supplementation of vitamin B6 include the following (Perry, 1978): (1) great variations in amounts of B6 in individual ingredients; (2) variable bioavailability of this vitamin in feed; (3) losses reported during processing of feed ingredients; (4) discrepancies between activity for test organisms and those for animals; (5) a higher vitamin B6requirement due to a marginal level of methionine in the diet; and (6) high-protein diets. Variability of vitamin B6 in feeds depends on the sample origin, conditions of growth, climate, weather and other local factors. Yen et al. (1976) determined available vitamin B6 in corn and soybean meal using a chick growth assay. Vitamin B6 in corn was found to be 38% to 45% available, and B6 in soybean meal, 58% to 62% available. There was little difference in availability between corn samples not heated and those heated to 120°C (248°F). However, corn heated to 160°C (320°F) contained significantly less available B6. Levels of vitamin B6 contained in feedstuffs are also affected by processing and subsequent storage. In one report, a loss of 30% of B6 was observed in alfalfa meal during the coarse milling and pelleting processes (Bräunlich, 1974). Bioavailability of feedstuffs can be as low as 40% to 50% after heating.
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 higher bioavailability and stability than the naturally occurring vitamin. Naturally occurring vitamin B6 in retorted milk products exhibited only 50% of the bioavailability of synthetic B6 or B6 in formulas fortified with the vitamin prior to thermal processing (Tomarelli et al., 1955).
Insufficient data are available to support estimates of the maximum dietary tolerable levels of vitamin B6 for poultry. It is suggested, primarily from dog and rat data, that dietary levels of at least 50 times the nutritional requirements are safe for most species (NRC, 1987). Vitamin B6 toxicity causes ataxia, muscle weakness and incoordination at levels approaching 1,000 times the requirement (Leeson and Summers, 2001). Large doses of vitamin B6 can produce detrimental effects in experimental animals and humans. Signs of toxicity, which occur most obviously in the peripheral nervous system, include changes in gait and peripheral sensation (Krinke and Fitzgerald, 1988). Changes in central nervous system function were detected in rats fed excessive vitamin B6 using measurement techniques of startle behavior (Schaeffer, 1993).