Vitamin B12 KEY POINTS BIOCHEMICAL FUNCTIONS INTRODUCTION. A Stewart TRUSWELL

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1 Nutrition & Dietetics 2007; 64 (Suppl. 4): S120 S125 DOI: /j x Vitamin B12 A Stewart TRUSWELL University of Sydney, Sydney, New South Wales, Australia KEY POINTS Subclinical vitamin B12 deficiency (low serum vitamin B12, and/or raised methylmalonate) occurs in 10% or more of older people, in Australia and other developed countries. Causes include inadequate liberation of vitamin B12 from its natural binding to the protein in foods because of poor gastric acid production. There is evidence that in some cases, the diet has been inadequate in animal foods. At present, it is not clear whether these low biochemical values lead to any serious consequences. The recent recommended dietary allowance reports for vitamin B12 in the USA and Canada recommend that older people should obtain most of their vitamin B12 from fortified foods or from supplements of free (crystalline) vitamin B12. In Australia and New Zealand, advice in the Nutrient Reference Values report is that older people with low stomach acid secretion May require higher intakes of vitamin B12 rich foods (e.g. meat) vitamin B12 fortified foods (not generally available here) or supplements. Dietary deficiency of vitamin B12 occurs in strict vegans. It is usually subclinical. But very severe clinical deficiency has been reported in infants breastfed by a vegan mother. Now that folate intakes have been increased by fortification of foods mandatory in North America, at present voluntary in Australia and New Zealand some experts and some evidence warn that it may be advisable for people to increase B12 intake. Meat and meat products are the major source of vitamin B12 in Britain. Presumably this would be similar in Australia. Liver and kidney are the foods richest in this vitamin. INTRODUCTION Vitamin B12, or cobalamin, was the last vitamin to be isolated. 1,2 It has the most complex structure and largest molecular weight (1335 Da) of all the vitamins. Dorothy Hodgkin was awarded the Nobel Prize (Chemistry, 1964) for elucidating its structure by X-ray crystallography. The vitamin is the only active substance in the human body to contain an atom of cobalt, which gives vitamin B12 its red colour. The human requirement for vitamin B12 is the lowest for any of the known essential nutrients (2 mg/day), 3 and body stores last longer, when there is no intake, than for any other (essential) nutrient. Vitamin B12 is synthesised by some anaerobic microorganisms in particular in the rumen of cows and S120 sheep, which require traces of cobalt in the pasture. Humans eat this vitamin preformed in animal foods: meat, milk, eggs and fish. No plant food has ever been shown to contain vitamin B12 consistently unless it is contaminated, for example by manure. In McCance and Widdowson s food tables, 4 all the vitamin B12 columns for vegetables and fruits and cereals have an 0 unless the line is for a mixed dish or a fortified (British) breakfast cereal. This review discusses the biochemical functions of vitamin B12, clinical deficiency diseases associated with vitamin B12, causes of deficiency, vegan infants and the elderly, nutritional intakes in Australia and New Zealand and meeting dietary requirements. BIOCHEMICAL FUNCTIONS Methionine synthase (in the cytosol) requires methylcobalamin as cofactor, and methylmalonyl CoA mutase (in mitochondria) requires 5 deoxyadenosylcobalamin as coenzyme. In bacteria, vitamin B12 participates in several other enzyme reactions. Methionine synthase is also called N-5 methyltetrahydrofolate: homocysteine methyltransferase. It sits at the junction between two important metabolic processes: synthesis of DNA and the methylation reactions via S-adenosylmethionine. If vitamin B12 is lacking, folate is trapped as methyl tetrahydrofolate. The metabolite 5, 10 methylene tetrahydrofolate is not formed, and this is specifically required for conversion of deoxyuridylate to thymidylate, one of the four essential bases in DNA synthesis. In this situation, cell nuclei cannot divide, and there is megaloblastic change which affects rapidly dividing cells of the bone marrow (making blood cells), the gastrointestinal epithelium and germinal epithelium. Effects include anaemia and increased plasma homocysteine. Methylmalonyl CoA mutase (MMCoA mutase) is the second and less central enzyme that requires vitamin B12. It deals with products of oxidation of odd-chain fatty acids and of the carbon skeletons of four amino acids as well as propionate itself. Propionyl CoA is converted to L-methylmalonyl CoA and the mutase (with vitamin B12 as coenzyme) converts this to succinyl CoA, which is part of the tricarboxylic acid (Krebs) cycle of metabolism. It was formerly thought that the neuropathic disease in vitamin B12 deficiency (which can occur without megaloblastic anaemia) would be due to failure of this second enzyme. But in inborn errors with absence of MMCoA mutase or of synthesis of the adenocobalamin coenzyme (without dietary B12 deficiency), the main feature is acidosis and accumulation of methylmalonic acid (MMA) in plasma and urine. 5 Neuropathy is not a feature. On the other hand, exposure of monkeys to nitrous oxide which inhibits

2 Vitamin B12 methionine synthase produces spinal cord demyelination, similar to human neuropathy. The most likely mechanism for neuropathy is impaired methylation of myelin basic protein. 6 CLINICAL DEFICIENCY DISEASES There are two major clinical syndromes of vitamin B12 deficiency: megaloblastic anaemia and/or disease of the nervous system, neuropathy. A patient can have anaemia alone or neuropathy alone, or one preceding the other. With megaloblastic anaemia there is anaemia, enlarged red cells (macrocytosis), or hypersegmented neutrophil leucocytes, and in the bone marrow, the nucleated red cell precursors show megaloblastic change. The white cell count and the platelet count can be low. Elsewhere the tongue may be sore (glossitis) and there is likely to be infertility. B12-deficient neuropathy can affect different parts of the nervous system. The most usual presentation is subacute combined degeneration of the spinal cord, in which there is loss of proprioceptive sensation from demyelination of the posterior columns and spastic weakness of the lower limbs from involvement of the lateral (pyramidal) tracts. Peripheral neuropathy may predominate or accompany this spinal cord disease. Less commonly, neuropsychiatric disorders have been recognised in association with vitamin B12 deficiency, for example memory loss or depression. In infants, the neurological dysfunction can be severe and progress to coma. Most cases of vitamin B12 deficiency are due to interference with one stage of its complicated mechanism of absorption. Pure dietary deficiency is less common. In the stomach, vitamin B12 is split from its binding to dietary protein by acid and pepsin. At the same time, intrinsic factor (IF) is secreted by the (normal) stomach s parietal cells. In the more alkaline ph of the duodenum, IF attaches to the vitamin B12. The vitamin B12/IF complex passes down the small intestine (not digested) and is absorbed only at a specific site in the terminal ileum. After three to four hours, the vitamin B12 appears in the blood carried on transcobalamin II. Vitamin B12 is excreted in the bile into the duodenum. Most of this then combines with IF and is absorbed at the terminal ileum. This entero-hepatic cycle helps conserve the vitamin. The primary method for measurement of serum vitamin B12 is microbiological or, more usually, radioassay. Different authorities set the lower reference level between 100 and 225 pmol/l. 7 9 (To convert pg/ml to pmol/l, multiply by 0.74.) One reason for differences between these numbers depends on the methods used for what is a very minute concentration of substance. The lower levels of serum B12 are likely to coincide with clinical deficiency disease. 10 More common is biochemical, subclinical deficiency, usually in elderly people in whom serum B12 above 200 pmol/l can be accompanied by raised serum methylmalonate (MMA) and homocysteine. Raised serum methylmalonate is apparently a more sensitive indicator of low vitamin B12 status than serum B12. It is not raised in folate deficiency. Serum homocysteine is increased with folate and vitamin B12 deficiency, so it is less useful for diagnosing vitamin B12 status. Cut-off values for serum MMA differ between methods and laboratories. That a raised serum MMA is due to vitamin B12 deficiency can be confirmed by response to supplementation with crystalline vitamin B12. CAUSES OF VITAMIN B12 DEFICIENCY Because of the relatively large stores in the liver enough for several years the onset of B12 deficiency is gradual. Biochemical, subclinical deficiency low serum B12, and/or raised methylmalonate occurs first and is more common than clinical deficiency megaloblastic anaemia and/or neuropathy. There are four groups of causes: 1 Inadequate dietary intake can affect vegans and is most severe and dangerous in breastfed infants of vegan mothers (see below). It is also seen in poorly nourished (vegetarian) adults, for example, in some populations in India. 2 Several (but rare) varieties of inborn errors of cobalamin metabolism. 3 Interference with absorption Pernicious anaemia. There are auto-immune antibodies to IF, so cobalamin cannot be absorbed. This is the most common cause of severe deficiency. It is more frequent in people of northern European descent and usually affects older people. Gastric atrophy or post-gastrectomy. Some IF may be produced but there is failure to free vitamin B-12 from food proteins. Drugs that suppress gastric acid (cimetidine, omeprazole) or interfere with B-12/IF absorption (slow K, metformin). Disease of the terminal ileum (the only place B-12/IF complex can be absorbed) e.g. in Crohn s disease or after resection of that part of the bowel. 4 Nitrous oxide, when used as an anaesthetic agent (or in someone addicted to it), can cause irreversible oxidation of the cobalt atom of vitamin B12 and render the vitamin inactive. VEGAN INFANTS Severe vitamin B12 deficiency has usually occurred in infants exclusively breastfed by their vegan mothers in Western countries. Typically, the baby is normal for the first few months and then stops developing, becomes lethargic, stops growing, is weak and pale, may have abnormal twitching and writhing movements, and can no longer sit or move the head. There is megaloblastic anaemia, very low serum vitamin B12 and excretion of MMA and homocysteine in the urine. CAT scan shows cerebral atrophy; the electroencephalogram is abnormal. Some of the babies of vegan mothers deteriorated to the stage of coma before they were diagnosed and treated their mothers did not trust conventional medical practice. Treatment with B12 injections produced rapid improvement of all abnormalities, but in some cases, the child s IQ had not reached normal a year or more later. It is remarkable that the vegan mothers were not obviously ill while their baby became comatose; yet both depended on the same inadequate supply of vitamin B12. The vitamin B12 S121

3 A Stewart Truswell concentration was lower in breast milk than in the mother s serum. In developing countries, similar cases have been reported in breastfed infants of poor mothers subsisting on diets low in animal foods. OLDER ADULTS In older people, biochemical signs of vitamin B12 deficiency become increasingly prevalent among those in their 70s and 80s of life. 15,16 As many as 10 15% of people aged over 75 years may show this when tested. The great majority of these people do not have pernicious anaemia (lack of IF). Nor do they have megaloblastic anaemia or obvious neuropathy. However, they have a low, or low normal, serum B12 with raised serum MMA and homocysteine, and evidence has accumulated that the raised homocysteine is associated with cognitive decline. The most likely explanation for about half of these cases (after excluding renal failure as a cause of raised MMA) is impaired gastric acid and pepsin secretion, so that vitamin B12 is not liberated from its binding to dietary proteins. But not all such people have features of atrophic gastritis when they have been investigated for this. 17 A Lancet editorial 18 set out the possibilities: Inadequate intake Inadequate release of protein-bound cobalamin because of impaired gastric acid and pepsin secretion Alterations in the binding site of cobalamin to transcobalamin II Alteration in the metabolism of cobalamin A minority of cases have early pernicious anaemia There is evidence in studies that, in some of the older people with subclinical vitamin B12 deficiency, the cause is inadequate B12 intake. Blundell et al. 19 reported that 15 of 200 geriatric patients had low serum levels on admission to hospital, then after a period in hospital, serum B12s had risen moderately, presumably on a more nutritious diet. A nutritionally depleted diet may have been responsible for the low vitamin values. In the Framingham Offspring Study, 20 subjects with low plasma B12 had significantly lower intakes of supplements and fortified breakfast cereals. They also had significantly lower intakes of dairy products. Their meat intakes had been lower too, although the difference here was not statistically significant. Two possibilities could explain these findings: either the range of meat intakes was too small to yield significant differences, or in dairy foods, the B12 is more bioavailable in these older people. Raised plasma homocysteine has been linked epidemiologically with cardiovascular disease and with cognitive decline and dementia. Only the latter will be considered here, although one mechanism of this relationship could be via cerebral vascular disease. People with senile dementia have been found to have raised serum homocysteine, low vitamin B12 and folate. 21 The low B-vitamin levels could be secondary to poor nutrition, due to the dementia. However, several prospective studies have reported that hyperhomocysteinaemia tends to predict cognitive decline and dementia S122 This then raises the question whether treatment with all or some of the three B vitamins that lower plasma homocysteine folate, vitamin B12 and vitamin B6 could delay cognitive decline or help prevent dementia. Eight trials have been reported in which folic acid and other B vitamins were given. Three of these lasted one 29 or two years, 30,31 and they included vitamin B12: plasma homocysteine was lowered, but there were no improvements in various tests of cognitive function. Among randomised controlled trials with folic acid alone, one 32 out of three found improvement in cognitive tests. It was a large trial with 818 participants who were relatively young (50 70 years). The treatment group were given 800 mg/day folic acid; the trial lasted three years, and the main benefits were in memory and speed of information-processing functions (i.e. short of dementia). 32 Subjects for this trial had to have raised plasma homocysteine and B12s above 200 pmol/l. Raised homocysteine is neurotoxic through overstimulation of N-methyl-D-aspartate receptors and has been associated with thinning of the hippocampus. Vitamin B12 deficiency can affect cerebral function. An adequate trial of vitamin B12 in asymptomatic elderly people with biochemical evidence of B12 deficiency and careful cognitive tests has yet to be conducted. 33 In the meantime, as Clarke concluded, it may be prudent to exclude vitamin B12 deficiency in patients with suspected dementia or cognitive impairment. This section can best be summed up by quoting Carmel, 7 one of America s leading experts on vitamin B-12. Alzheimer disease is often accompanied by unexplained low cobalamin levels. However, unlike the milder cognitive dysfunction sometimes seen in cobalamin deficiency, the symptoms of Alzheimer disease do not respond to cobalamin therapy, and a causal connection has not been found. An even stronger association of Alzheimer disease with homocysteine and folate status has been suggested, but is similarly murky. It has been aptly pointed out that attribution of neurologic problems to cobalamin deficiency requires more than just finding low serum cobalamin levels. 7 Another question for subclinical vitamin B12 deficiency is whether it is aggravated by increased folate intake or status. This was considered unlikely in the National Health & Medical Research Council (NHMRC) Folate Fortification report. 34 But a recent analysis by the Tufts group of data from the US NHANES found that, for senior adults with low vitamin B12 status in (serum cobalamin <148 pmol/l), high serum folate (>59 nmol/l) was associated with anaemia and cognitive impairment. However, high serum folate was associated with protection against cognitive impairment when vitamin B12 status was normal. 35 This is a new situation that has arisen since mandatory folic acid fortification in North America. 36 VITAMIN B12 INTAKE IN AUSTRALIAN AND NEW ZEALAND POPULATIONS There are no data on the vitamin B12 content in Australian foods. Table 1 is from the British food tables. 4 Vitamin B12

4 Vitamin B12 Table 1 Food sources of vitamin B12 (mg/100 g edible portion) Breakfast cereals (a) Whole & skim milk 0.4 (more if condensed or dried) Human milk 0.1 Cheeses 1 2 Yoghurt 0.2 Egg 2.5 Duck egg 5.6 Beef steak 2 Lamb, pork 1 2 Veal 1 Chicken <1 Duck 3 Turkey 2 Rabbit 10 Ox heart 14 Lamb kidney 55 Ox kidney 31 Ox liver 110 Chicken liver 56 Lamb liver 84 Pig liver 25 Tripe Tr Most fish 1 2 Oily fish Anchovies 11 Herring 6 Mackerel 10 Pilchards 12 Salmon 5 Sardines 28 Tuna 5 Mussels 22 Marmite 0.5 (a) If present, this is from fortification; zero in Australia. From McCance and Widdowson. 4 was not included in the 1985 Australian National Nutrition Survey. Vitamin B12 intakes were, however, estimated in samples of Australians investigated with postal food frequency questionnaires by CSIRO Adelaide. For vitamin B12 content of foods, overseas data were used. In the 1988 and 1993 national survey (2315 and 1733 people), mean B12 intakes were 5.5 mg in men and 4.7 mg in women. Around 5% of men and 10% of women reported intakes below the RDI (then 2.0 mg/day). 37 In surveys in Victoria (1985 and 1990) with similar methodology but more subjects (nearly 3000), the percentage with intakes below the RDI were 15% in men aged over 60 years and 25% in women. 38 The most informative study of different representative groups in Australia was conducted by Mann et al. in Melbourne. 39 The results in men in their 30s show that serum B12s were lower in lacto-ovo-vegetarians (mean 211 pmol/l) than in omnivores (334 pmol/l) and, in vegans, the mean value (145 pmol/l) bordered on the deficient range. High meat eaters averaged 402 pmol/l. The same low serum B12s in vegans have been seen consistently in other studies performed in Israel, Finland, Slovakia and the UK. A recent survey as part of the Blue Mountains (NSW) Eye Study (BMES) found that older people in Australia have a similar proportion with low serum vitamin B12 to studies in developed countries overseas. 40 In 2963 women and men aged 50 to 80+ years, 6% of the whole sample had serum B12 <125 pmol/l ( very low ), and nearly 17% had levels between 125 and 185 pmol/l ( low ). The lower the B12, the higher the proportion with serum homocysteine >15 mmol/l. Low and very low serum B12 increased progressively from people in their 50, 60s, 70s and 80s of life. So in the octagenarians, 11% had very low and 30% had low serum B12s. The BMES investigators do not report haemoglobin levels in their paper. In a later publication, they note that (as found in much larger numbers in the USA 41 ), older people in the Blue Mountains with higher folate intakes did not have lower serum vitamin B In New Zealand, Green et al. 43 were able to conduct a substudy of the National Nutrition Study (which, unlike its Australian counterpart, included blood analyses). In 446 non-institutionalised men and women aged over 65 years, 12% had serum B12 <148 pmol/l and 28% had pmol/l. They diagnosed atrophic gastritis with serum pepsinogens (ratio of PG1/PG2) and found this in 23% of people with serum B12 below 148 pmol/l. The few B12 supplement users had a reduced risk of low serum B12, but dietary B12 intake above the recommended dietary intake (RDI) or recommended dietary allowance (RDA) (2.0 and 2.4 mg/day) was not associated with lower risk. (There are no other dietary details in the paper.) They note that deficient serum B12 levels were more prevalent than in the USA, and less prevalent in this New Zealand sample than in the UK. Megaloblastic anaemia was absent in the people they sampled. MEETING NUTRITIONAL REQUIREMENTS The 1998 Dietary Reference Intakes for the USA and Canada 44 introduced a new policy which implies that people S123

5 A Stewart Truswell over 51 years of age cannot be relied upon to absorb enough vitamin B12 from natural food. Because 10 to 30 percent of older people are estimated to have atrophic gastritis with low acid secretion they may have decreased bioavailability of B12 from food. So the RDA (of 2.4 mg here) may not meet the needs of 97% of the older age groups. The Institute of Medicine therefore recommends that most of the RDA be obtained by consuming foods fortified with crystalline B12 or with a B12-containing supplement. In the USA, breakfast cereals are commonly fortified with B12. Perhaps because of this or more taking of supplements, only 7% of elderly persons had serum B12 below 185 pmol/l ( Elderly seems to have been used for >60 years, but these percentages are lower than in BMES (above)). The ANZ Nutrient Reference Values report, 3 which usually followed North America, has taken a somewhat different line. Having noted that strict vegans will need supplementation with vitamin B12, the Working Party adds: The natural vitamin B12 in foods may be less bioavailable to the substantial number of older adults who have atrophic gastritis with low stomach acid secretion. People with this condition may require higher intakes of vitamin B12 rich foods, vitamin B12 fortified foods or supplements. Vitamin B12- fortified foods are not generally available in Australian supermarkets, and some multivitamin tablets contain only 1 mg of cyanocobalamin. Blacher et al. 45 in Paris have measured the effect of small doses of pharmaceutical vitamin B12 by mouth in people aged over 70 years who had evidence of gastric disease and low serum B12. With doses from 2.5 up to 80 mg/day for 30 days, there was a dose response effect. A dose of 6 mg would increase serum B12 by an average of 38 pmol/l. What has not been scientifically tested is the effect of that sort of dose from B12-rich food, for example 5 g ox liver (which contains 6 mg vitamin B12) in old people with low serum B12 but no deficiency symptoms (with or without evidence of low gastric acid). CONCLUSION Perhaps we can get the best perspective here by another quote from Carmel: 7 Much remains unknown about subclinical (B-12) deficiency. A few of the affected persons simply have very early pernicious anaemia (PA) and therefore can be expected to progress inexorably to a symptomatic state because of their irreversible malabsorption. However, those without early PA have often remained asymptomatic for at least a decade. Still to be resolved are issues that include the clinical impact of subclinical deficiency, which seems by definition to be small, its course, which appears static in most observations, and whether it can regress spontaneously. The causes of subclinical deficiency are also not well understood in many cases. Malabsorption of free cobalamin such as PA is found in fewer than 5% of cases, whereas food-cobalamin malabsorption may account for 30 40% of cases. No cause has been found in most patients, however. For all these reasons, and because millions of people fall into this category, it is important to answer the outstanding S124 questions and to reach a consensus on what to do abut subclinical (B-12) deficiency. 7,46 REFERENCES 1 Rickes EL, Brink NG, Koniusey FR et al. Crystalline vitamin B12. Science 1948; 107: Smith EL, Parker FJ. Purification of anti-pernicious anaemia factor. Biochem J 1948; 43: viii ix. 3 Australian Government Department of Health & Ageing, National Health & Medical Research Council, New Zealand Minister of Health. Nutrient Reference Values for Australia and New Zealand. Canberra: NHMRC, Food Standards Agency & Institute of Food Research. 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