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 (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 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. 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 B6activity (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 vitamin 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 into the urine (Henderson, 1984). Vitamin B6 metabolism is altered in renal failure, as observed in rats exhibiting plasma pyridoxal phosphate levels 43% lower than controls (Wei and Young, 1994).
Vitamin B6in the form of pyridoxal phosphate (PLP) is a co-factor for over 60 enzymatic reactions involved in amino acid, carbohydrate and fatty acid metabolism. Pyridoxal phosphate functions in practically all reactions involved in amino acid metabolism, including transamination, decarboxylation, deamination and desulfhydration, as well as the hydrolysis and synthesis of amino acids. Vitamin B6 participates in enzymatic functions (Merrill and Burnham, 1990; LeKlem, 1991; McCormick, 2006) that include:
- Deaminases: serine, threonine and cystathionine.
- Desulfhydrases and transulfurases.
- Synthesis of niacin from tryptophan.
- Formation of alpha-aminolevulinic acid from succinyl CoA and glycine — the first step in porphyrin synthesis.
- Conversion of linoleic to arachidonic acid in the metabolism of essential fatty acids (a function also requiring biotin); this function is also related to brain sphingolipid production.
- Glycogen phosphorylase: catalyzes glycogen breakdown to glucose-1-phosphate. Pyridoxal phosphate appears not to be a coenzyme for this enzyme but rather to affect the enzyme conformation.
- Synthesis of epinephrine and norepinephrine from either phenylalanine or tyrosine; and therefore effects on neural function. With a vitamin B6 deficiency dopamine release is delayed, which may contribute to motor abnormalities (Tang and Wei, 2004).
- Racemases: PLP-dependent racemases enable certain microorganisms to utilize D-amino acids. Alanine racemase has been detected in mammalian tissues.
- Transmethylation reactions involving methionine.
- Incorporation of iron in hemoglobin synthesis, and therefore effects on erythrocyte function.
- Antibody response to antigens and lymphocyte formation.
- Inflammation - higher vitamin B6 levels were linked to protection against inflammation (Morris et al., 2010).
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). Additional human studies indicate that vitamin B6 status may influence tumor growth and disease processes. Deficiency of the vitamin has been associated with immunodeficiency virus (HIV) and those with uremia or rheumatoid arthritis (Rall and Meydani, 1993).
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 vector for long chain fatty acids for mitochondrial β-oxidation and of certain bases for phospholipid biosynthesis (McCormick, 2006).
Vitamin B6 is required by numerous microorganisms and appears to play a role in rumen metabolism. Vitamin B6enhanced the in vitro production of phenylalanine from its precursors phenylpyruvic acid and phenylacetic acid in mixed rumen bacteria and protozoa (Ruhul and Onodera, 1998). The saccharolytic rumen bacterium, Butyrivibrio fibrisolvens, requires vitamin B6, along with biotin and folic acid (Baldwin and Allison, 1983).
Requirements of vitamin B6 have been found generally to depend on species, age, physiological function, dietary components, intestinal flora and other factors that are not yet fully understood. Quantity of dietary protein affects the requirement for vitamin B6, and nutrients other than protein influence the B6 requirement. Niacin and riboflavin are needed for interconversions of different forms of vitamin B6. Excessive thiamin intake was reported to produce vitamin B6 deficiency in rats (Driskell, 1984). Conversely, vitamin B6is required for synthesis of niacin from tryptophan. Microorganisms of the intestinal tracts of animals produce vitamin B6, but whether significant quantities are absorbed and utilized is in doubt. For ruminants, far more important than large intestinal synthesis is the considerable quantities of vitamin B6 synthesized by ruminal microorganisms. The amounts of vitamin B6 normally synthesized by bacteria in the rumen are sufficient to prevent outward signs of deficiency. In vitro studies conducted with microbial isolates collected from the rumen of goats also demonstrated that vitamin B6 additions enhanced production of lysine by ruminal microorganisms (NRC, 2007b). In addition to lysine being one of the most limiting amino acids for milk production (NRC, 2001), lysine supply may be a major limitation to wool, and perhaps mohair growth (Reis and Sahlu, 1994). Pyridoxal kinase activity was greater in goat mammary tissue than in the liver (Coburn et al., 1992), suggesting that requirements for vitamin B6 may increase during lactation.
Prior to full rumen development, young ruminants require dietary vitamin B6 to prevent deficiency. An exact vitamin B6requirement has not been determined, but calves fed 65 µg of vitamin B6 per kg (29.5 µg per lb) of body weight did not develop deficiency signs (Johnson et al., 1950). This requirement is similar to that determined in humans and swine of similar body weight. Whether ruminal synthesis provides optimal levels of vitamin B6 for high-producing dairy cows is unknown.
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 in two common feed ingredients is 65 percent for soybean meal and 45 to 56 percent in corn (McDowell and Ward, 2008). The vitamin B6 present in cereal grains is concentrated mainly in the bran. 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 for ruminants is that obtained by microbial synthesis in the rumen. In an experiment in which sheep were fed a diet containing 1.5 mg of vitamin B6 per kg (0.68 mg per lb), McElroy and Goss (1939) measured 6 to 10 mg per kg (2.7 to 4.5 mg per lb) of vitamin B6 in dried rumen contents. For cattle, McElroy and Goss (1940b) measured 8 to 10 mg of vitamin B6 per kg (3.6 to 4.5 mg per lb) of dried rumen contents in animals fed 1 to 1.5 mg per kg (0.45 to 0.65 mg per lb) of vitamin B6 in the diet. Ewes maintained on a diet containing little vitamin B6 yielded milk that contained as much vitamin B6 as the milk of ewes fed normal diets (McElroy and Goss, 1940b).
For young ruminants, milk is a good source of vitamin B6. Cow’s milk contains approximately 0.30 mg of vitamin B6per liter (0.32 mg per quart) (Gregory, 1975). Therefore, a 50-kg calf fed milk at 10% of body weight would receive approximately 1.75 mg of vitamin B6. The NRC recommends 6.9 ppm of pyridoxine in calf milk replacer powder, which would provide the typical dairy calf with approximately 3 mg of vitamin B6 per day.
Commercially, vitamin B6 is available as crystalline pyridoxine hydrochloride and various dilutions. Pyridoxine hydrochloride contains 82.3% vitamin B6 activity and is a fine, white, crystalline powder. This product form can be used for both water-soluble and dry feed applications.
Characteristics of vitamin B6 deficiency in most species are retarded growth, dermatitis, epileptic-like convulsions, anemia and partial alopecia. Due to the predominant role of vitamin B6in amino acid metabolism, a deficiency is characterized by reduced protein utilization and nitrogen retention and impairment of tryptophan and niacin metabolism. Due to rumen microbial synthesis, vitamin B6 deficiency in ruminants with a functioning rumen has not been reported in the literature. Vitamin B6 has been shown to be essential for the young calf when selected experimental diets are used. Calves that were reared on a “milk substitute” lost appetite within two to four weeks, their growth was impaired, and they progressively showed apathy, diarrhea, anorexia and incoordination. In the last stages, convulsions were soon followed by death (Johnson et al., 1947). The convulsions included thrashing of the legs and head and grinding of the teeth (Johnson et al., 1950).
Postmortem examination of vitamin B6-deficient calves revealed hemorrhages in the epicardium and in the kidneys, demyelination of the peripheral nerves, proliferation of Schwann cells, desquamation of the intestinal mucosa and pneumonia (Johnson et al., 1950). Analyses of the urine from the vitamin B6-deficient calves showed greatly reduced excretion of pyridoxine, pyridoxal, pyridoxamine and pyridoxic acid (the major excretory metabolite). In the early stages of a vitamin B6 deficiency in calves, animals fully recovered after an oral dose of 100 mg of vitamin B6(Johnson et al., 1950). However, if this treatment was delayed until the occurrence of convulsive seizures, it was no longer possible to save the calf, even when the vitamin was administered by injection, indicating that irreparable damage had occurred.
Milk replacers should be fortified with vitamin B6 in accordance with NRC minimum requirements. Calves, such as veal calves, that are raised under accelerated growth programs while on liquid feed may benefit from slightly higher levels of vitamin B6, especially if high rates of protein deposition are achieved. However, this hypothesis has not been tested. Supplemental vitamin B6 is reported to have a higher bioavailability and stability than the naturally occurring vitamin, probably due to the presence of bound pyridoxine in feedstuffs. 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 trace minerals as sulfates. However, carbonate and oxide trace minerals reduced pyridoxine activity by up to 25% after three months. Choline chloride accelerates this reaction. Retention of vitamin B6 in vitamin supplements was 98% after six months, but was only 56% for supplements that also contained choline and trace minerals (Coelho, 1991). Gadient (1986) considers pyridoxine to be very sensitive to heat, slightly sensitive to moisture and light, and insensitive to oxygen. Retention of vitamin B6 activity in pelleted feeds after three months at room temperature is approximately 80% to 100%.
Insufficient data are available to support estimates of the maximum dietary tolerance of vitamin B6 for ruminants. It is suggested, primarily from dog and rat data, that dietary levels of at least 50 times the nutritional requirement are safe for most species (NRC, 1987). Vitamin B6 toxicity causes ataxia, muscle weakness and incoordination at levels approaching 1000 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).