Vitamin B12

What it is
In the exciting period of nutritional research in the 1920s and 1930s it was found that liver extracts and other concentrations of animal origin stimulated the growth of farm animals and could be used as a treatment for human pernicious anaemia. For many years the active principle was known as the animal protein factor and its chemical nature eluded scientists. Then, in 1948, a crystal-line material with the same characteristics was isolated from liver and called vitamin B12. Its chemical structure was clarified in 1955 but it was not synthesised until 1973. Until then supplies of vitamin B12 were prepared by extraction from fermentation products and this is still the most economical method of commercial production.

The chemical structure of vitamin B12 is the most complex of all the vitamins. The basic unit is a corrin nucleus which consists of a ring structure of four 5-membered rings joined by corner nitrogen atoms. In the active centre of the corrin nucleus is a cobalt atom. Thus, vitamin B12 is the only vitamin to contain a mineral element. The main material with vitamin B12 activity is believed to be cyanocobalamin which has a -CN group attached to the cobalt atom. There seems to be some doubt if this is a true feature of the vitamin or whether it is an artefact. Other groups such as -OH or -NO3 can also be attached to the molecule without affecting its vitamin status. However, there are many other corrinoids which appear analogous with cyanocobalamin but have very little, or no, vitamin B12 activity.

What it does
Vitamin B12 is an essential part of several enzyme systems. Most of these involve the transfer or synthesis of single-carbon units. Thus, vitamin B12 is responsible for a number of basic metabolic functions in association with other vitamins such as folic acid. The most important tasks relate to the metabolism of proteins but it also features in the metabolism of fats and of carbohydrates.

Under normal feed conditions vitamin B12 is probably linked to peptides or even to protein. This link is broken during digestion. The released vitamin B12 molecule cannot be absorbed through the intestinal wall without a carrier. Various products, collectively known as the intrinsic factor, have been shown to carry vitamin B12. These vary between species but most appear to be glycoproteins. Only cats appear to be able to absorb vitamin B12 without the intervention of the intrinsic factor.

The physiological activities of vitamin B12 are very closely inter-related with those of folic acid but the actual mechanisms are poorly understood. It is known that one activity is the formation of labile methyl groups which play a significant part in the biosynthesis of methionine which, in turn, affects the synthesis of body proteins. There is good reason to believe that the impairment of protein synthesis is the principal cause of the growth depression which is frequently observed in animals deficient of vitamin B12. The cobalt atom appears to be responsible for the transmethylating capacity of cobalamin because the methyl- cobalt derivative is formed.

One interesting and important function of vitamin B12 is the metabolism of propionate products of dietary or metabolic origin. Propionate is converted into succinate in the Kreb’s cycle. Propionate contains three, and succinate four, carbon atoms. The extra methyl group is supplied through methylmalonyl-CoA which is activated by methylmalonyl-CoA isomerase, a vitamin B12-dependent enzyme.

Vitamin B12 appears to exert a calming influence on horses, particularly those which are fresh or frisky, or generally excitable. This hyperactive condition may be due to a generous supply of biotin (vitamin H) and 1 mg/day of vitamin B12 can act as an ”antidote”.

If too much is given
Although excess vitamin B12 is not excreted rapidly, no toxicity or unpleasant reactions have been reported in any species of animal even at extremely high dosage levels. Many organs are capable of storing it for short periods; 30-60% of the body’s reserves are held in the liver. There is no destruction of vitamin B12 during metabolism but a regular excretion via the kidneys and through the bile into faeces.

Many bacteria are capable of synthesising vitamin B12 when sufficient cobalt is available. Bacteria inhabiting the lower parts of the digestive tracts of horses can synthesise corrinoids when the diet contains sufficient cobalt. However it has been shown that most of these corrinoids are vitamin B12 analogues with no biological activity. As the cobalt content in the diet is increased more vitamin B12 analogues are produced and true vitamin B12 production may actually fall. This suggests that, even in a cobalt-deficient area, there is a limit to the amount of cobalt which should be supplied. However, it is doubtful whether horses benefit to any major extent from the vitamin production in the lower gut because it is beyond the area where absorption into the system takes place.

For reasons which are still not understood, the action of vitamin B12 is adversely affected by the presence of high levels of niacin or of oxidising agents. If these are present in the feed additional vitamin B12 is required. Oestrogens also antagonise the action of vitamin B12.

How it is measured
The most accurate assay methods are biochemical, based on test organisms such as Euglena gracilis or Lactobacillus leichmannii. The results are usually quoted in mg/kg feed or in mg/100ml blood. Less commonly used measures of vitamin B12 are the USP (United States Pharmacopoeia) unit, which is the same as 1mcg and the LLD (Lactobacillus lactis Dorner) unit.
Approximately 11,000 LLD units = 1 USP unit
= 1mcg vitamin B12.

Assessment of status
It is extremely difficult to make an accurate assessment of the vitamin B12 status of an animal. The vitamin is largely present in the liver and other organs so that a blood sample is unlikely to be indicative of the overall status unless the animal is almost depleted of vitamin B12. There is also the chemical problem of identifying biologically-active vitamin B12 corrinoids and separating them from inactive corrinoids.

There are considerable doubts regarding the accuracy of some of the existing blood assay methods. Levels of serum vitamin B12 below 200 mg/ml or liver levels below 0.1 mcg/g wet weight indicate deficiency. The excretion of methylmalonic acid (MMA) in the urine can be used to indicate deficiency or adequacy of total supplies since its rate of excretion during vitamin B12 deficiency is 5-12 times normal. Serum MMA can also be used. An alternative urinary metabolite used as an indicator of vitamin B12 deficiency is formiminoglutamic acid (FIGLU) since daily excretion is increased 30 times over normal.

Relationships with other ingredients
Because of its widespread function as a methyl group carrier, vitamin B12 interacts with numerous other micro-ingredients in various metabolic activities. One of its major relationships is with folic acid where there are several combined activities in nucleic acid and methionine synthesis. Various vitamins also affect the rate of absorption of vitamin B12 from the intestine, which is never very efficient at the best of times; folic acid deficiency increases absorption whereas a vitamin B6 deficiency decreases it. Biotin is active with vitamin B12 in methylmalonyl CoA metabolism and so is pantothenic acid which has a vitamin B12-sparing action in this function. Vitamins E and B1 are also synergistic, particularly in blood cell production.

Vitamin B12 improves the uptake and utilisation of carotene from the intestine and the function of vitamin A in maintaining the integrity of mucosal and epithelial cells.

Calcium, copper and ferrous iron also act with vitamin B12, improving absorption from the intestine and the efficiency of its metabolic functions.

Requirements and allowances
Since it is extremely difficult to assess the true vitamin B12 status of animals the accuracy of any measurement of requirement is doubtful. In addition to the determination of vitamin B12 status, the many interactions with other micro-ingredients produce variable requirements depending upon supplies of methionine, choline, folic acid, thiamine and biotin, for example, and the needs increase when the production of propionates during digestion is greater. Most research on the vitamin B12 requirements arrives at a requirement of 10 mg/kg feed dry matter.

In the absence of other information, 10 mcg/kg should be regarded as the minimum to be supplied in the feed. There is evidence of beneficial effects such as the optimization of growth rate when vitamin B12 supplies are supplemented to give a total in the ration of 30 mcg/kg. This should be regarded as the optimum allowance although it should be increased to 120 mcg/kg for hyperactive and performance horses. In the following suggestions for feed supplementation it has been assumed that only minimal amounts of animal protein products are likely to be included in horse feed formulations. Should the animal protein proportion exceed 5% of the mix the optimum recommendations can be reduced by 5 mcg /kg.

    µg / kg   µg / day
Adult performance horses in training  
Adult performance horses in light work   120   800
Mares & stallions   30   120
Young horses 1-2 years   25   75
Foals & young horses less than 1 year   30   30-90

Contents of feed ingredients
Products of vegetable origin contain little or no vitamin B12. The amounts present in animal protein materials are widely variable because of losses during processing. Average figures from tables are liable to be very misleading and any particular sample of a feed may have a widely different vitamin B12 content from that shown in tables.

The very large molecule of vitamin B12 results in a general instability. It is affected by heat, light, acids, alkalis and oxidising agents. It is not seriously affected by moisture. During normal feed mixing and pelleting operations losses of vitamin B12 are of the order of 10%. If the feed is extruded losses may be much greater and can exceed 30%. The pure crystalline cobalamin and powder dilutions are relatively stable to air and heat but are attacked by light, particularly in the UV wavelengths. It is therefore essential to keep vitamin B12 products in light-tight containers, preferably in a cool, dry place. The recommendations for supplementation include an allowance of 20% for losses during processing and storage.