Vitamin B12 absorption and metabolism in milk-fed lambs.

AIM: To quantify aspects of absorption and retention of vitamin B12 from milk in pre-ruminant lambs, and the possible effect of activation of dependent metabolic pathways (propionate-succinate) on vitamin B12 concentrations in tissues. METHODS: In Experiment 1, two groups of eight newborn lambs each received either milk substitute alone, or milk substitute with added propionate for 4 weeks. Half of the lambs in each group received vitamin B12 injections twice weekly. Blood and liver samples were taken on Day -1 and at the end of the trial, and blood was also collected twice weekly, for measurement in plasma of vitamin B12, methylmalonic acid (MMA), and blood-binder transcobalamin II/haptocorrin. The lambs were weighed at 0, 2 and 4 weeks. In Experiment 2, 12 milk-fed newborn lambs (n=2 or 3/group) were injected I/M with 0.2, 0.4, 0.8, 1.6 or 3.2 microg vitamin B12 and a further 12 were supplemented orally with 2, 4, 8, 16 or 32 microg vitamin B12, daily for 2 weeks. Three lambs remained as untreated controls. Blood and liver samples were taken at the start and end of the trial, and blood was also collected twice weekly, for measurement of vitamin B12 and MMA concentrations in plasma. The lambs were weighed on Days 0, 7 and 14. In Experiment 3, 14 lambs from Experiment 2 were used to repeat the highest I/M and oral rates of supplementation used in Experiment 2. Blood samples were taken at frequent intervals for 24 hours, and analysed for concentrations of vitamin B12 in plasma. RESULTS: Propionate supplementation was associated with increased concentrations of vitamin B12 in plasma in lambs supplemented with vitamin B12 but not in those not supplemented (p=0.047), but had no detectable effect on concentrations of vitamin B12 in liver (Experiment 1). Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA at a rate of intake of propionate close to that which would depress appetite. Close to 100% of the vitamin B12 administered by I/M injection appeared in plasma in the first hour but 85% was removed from the circulation within 16 hours. The maximum liver concentration was achieved at 900 nmol/kg (=1,200 microg) fresh tissue. There were no significant differences in liveweight gain (LWG) regardless of treatment. CONCLUSIONS: Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA in plasma at a rate of absorption of propionate close to that which would depress appetite. The coefficient of absorption of oral vitamin B12 in milk-fed lambs was low (<10%), and the ability of lambs to retain the vitamin even when tissues had physiologically low concentrations was poor. CLINICAL RELEVANCE: Pre-ruminant lambs had a low requirement for vitamin B12, and even at low tissue concentrations had poor ability to absorb and retain the vitamin. However, if supplementation is required, a method that provides a modest but continual supply may be most effective in protection against deficiency.

Analysis of corrinoids in ovine tissues.

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.

Development of a method for the separation of corrinoids in ovine tissues by HPLC.

A method has been developed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to routinely estimate the distribution of corrinoids (the cobalamins hydroxocobalamin, methylcobalamin and 5'-deoxyadenosylcobalamin, and cobalamin analogues) in liver, plasma, milk, intestinal fluid and faeces. Corrinoids were extracted with a sodium acetate buffer, separated by HPLC and quantified by RIDA. Recoveries of corrinoids were 29% for hydroxocobalamin, 50% for 5'-deoxyadenosylcobalamin and 64% for methylcobalamin. The method allows the routine analysis of many samples and maintains good standards of precision.

Changes in serum concentrations of methylmalonic acid and vitamin B12 in cobalt-supplemented ewes and their lambs on two cobalt-deficient properties.

AIM: To determine concurrent changes in serum methylmalonic acid (MMA) and vitamin B12 concentrations of ewes and their lambs on cobalt-deficient properties, subsequent to cobalt supplementation. METHODS: Three experiments were carried out on two farms. Groups of ewes (n=25-50) were either supplemented with cobalt bullets during late pregnancy, 23-47 days before the mean lambing date, or left unsupplemented. In two experiments, lambs from within each group were supplemented directly by vitamin B12 injection at 3-weekly intervals from birth, and in the third experiment by injection with micro-encapsulated vitamin B12 at tailing and 3 months later. Pasture samples were obtained for analysis of cobalt content at each sampling time. Blood samples were obtained and liveweight recorded from ewes and lambs at approximately monthly intervals. On one farm (two experiments), liver and milk samples were obtained from ewes and liver samples from lambs. RESULTS: Serum vitamin B12 concentrations in unsupplemented ewes fell below 250 pmol/L during early lactation in all experiments and mean concentrations as low as 100 pmol/L were recorded. MMA concentration was maintained below 2 micromol/L in serum from supplemented ewes but increased to mean concentrations ranging from 7 to 14 micromol/L at the nadir of serum vitamin B12 concentration during peak lactation. A significant liveweight response to supplementation was recorded in ewes on one property, and the vitamin B12 concentration in the ewes' milk and in the livers of their lambs more than doubled. No liveweight-gain response to supplementation was observed in lambs on this property. Mean serum MMA concentrations in lambs ranged from <2 in supplemented, to 19.2 micromol/L in unsupplemented lambs, and the latter had concurrent serum vitamin B12 concentrations of >300 pmol/L. Pasture cobalt concentration was lowest at 0.04-0.09 microg/kg dry matter (DM) on the property on which responses in lambs occurred but considerably higher (>0.09 microg/kg DM) on the property on which responses in ewes occurred. On the second property, serum vitamin B12 concentrations in lambs at tailing were extremely low (100 pmol/L), irrespective of supplementation of dams with cobalt. Mean serum MMA concentration was increased to 20 and 42 micromol/L in lambs from supplemented and non-supplemented ewes, respectively. Weight-gain response to direct supplementation of lambs with vitamin B12 occurred during suckling in the latter, but not the former. Lambs from ewes supplemented with vitamin B12 showed a much bigger increase in serum vitamin B12 concentrations a month after supplementation than did lambs from unsupplemented ewes (+1,400 pmol/L vs + 650 pmol/L). CONCLUSIONS: Serum MMA concentration gave a more precise indication of responsiveness to vitamin B12 or cobalt supplementation than serum vitamin B12 concentrations in ewes and lambs. Neither very low serum vitamin B12 nor elevated MMA concentrations were necessarily indicative of responsiveness to supplementation in suckling lambs, but the latter gave an early indication of impending responsiveness. Supplementation of the ewe with a cobalt bullet appeared to protect the growth performance of the lamb for 90 days and influence the subsequent serum vitamin B12 response in the lamb to vitamin B12 supplementation. CLINICAL SIGNIFICANCE: Supplementing ewes with cobalt bullets in late pregnancy can improve the vitamin B12 status of their lambs, and modify their response to vitamin B12 supplementation.

Concurrent changes in serum vitamin B12 and methylmalonic acid during cobalt or vitamin B12 supplementation of lambs while suckling and after weaning on properties in the South Island of New Zealand considered to be cobalt-deficient.

AIM: To compare serum analyses of vitamin B12 and methylmalonic acid (MMA) as indices of cobalt/vitamin B12 deficiency in lambs around weaning. METHODS: Lambs on five properties, considered to be cobalt- deficient, were supplemented with either cobalt bullets, or short- or long-acting vitamin B12 preparations. Blood samples, and in some cases liver biopsies, and liveweights were obtained at monthly intervals. Serum samples were assayed for vitamin B12 and MMA and liver for vitamin B12 concentrations. Pasture cobalt concentrations were measured on three of the properties. RESULTS: Pasture cobalt concentrations were generally maintained below 0.07 microg/g dry matter (DM) on the properties sampled. Growth responses to supplementation were observed on only 2/5 properties, despite serum vitamin B12 concentrations being within the currently used 'marginal' reference range (336-499 pmol/L) for at least 3 months on all properties and in the deficient reference range (0-335 pmol/L) for at least 2 months on all farms except one. Serum MMA concentrations in supplemented lambs were <2 micromol/L, except in those animals sampled 1 month after receiving treatment with a short-acting vitamin B12 injection. Serum MMA concentrations in unsupplemented animals on properties on which no growth response to supplementation occurred generally reached peak levels of between 4 and 7 micromol/L at the nadir of serum vitamin B12 concentration. When a growth response was observed, differences in weight gain between supplemented and unsupplemented lambs occurred as mean serum MMA concentrations increased from 9 to 14 micromol/L. On one property where supplementation commenced before weaning, normal growth rates were maintained despite serum vitamin B12 concentrations of 140 pmol/L and serum MMA concentrations in excess of 40 micromol/L serum. CONCLUSIONS: The possibility that current serum vitamin B12 references ranges for diagnosis of cobalt deficiency are set too high and lead to over-diagnosis of responsiveness to cobalt/ vitamin B12 supplementation is discussed. The suggestion is made that serum MMA concentrations in excess of 9-14 micromol/L will provide a more reliable diagnostic test for cobalt deficiency. However, there was sufficient variation between properties in the relationships between cobalt concentrations of pasture and serum vitamin B12 or MMA concentrations to require more rigorous testing of the reliability of using serum MMA concentration for this purpose. The possibility that differences in rumen fermentation and therefore propionate and vitamin B12 production could be involved is discussed. The measurement of serum MMA and vitamin B12 appears to be of little value whilst the lamb is still suckling. CLINICAL SIGNIFICANCE: Serum MMA concentration may offer advantages over serum vitamin B12 concentrations in the diagnosis of a cobalt/vitamin B12 responsiveness in weaned lambs.

A critical evaluation of serum methylmalonic acid and vitamin B12 for the assessment of cobalt deficiency of growing lambs in New Zealand.

AIM: To derive reference ranges for serum methylmalonic acid (MMA) for the diagnosis of cobalt/vitamin B12-responsiveness in lambs and critique existing serum vitamin B12 reference ranges. METHODS: Individual animal data from earlier supplementation trials, involving 225 ewes, 106 suckling lambs, 301 lambs during the suckling and post-weaning periods and 414 weaned lambs, for which weight gain to supplementation was observed, were used to derive relationships between serum vitamin B12 and MMA, and liveweight gain. RESULTS: Serum MMA concentrations were rarely elevated above the norm of <2 micromol/L when serum vitamin B12 concentrations were >375 pmol/L, and not elevated into the range where a liveweight response to supplementation occurred (>10 micromol/L) unless serum vitamin B12 concentrations were below 200 pmol/L. Suckling lambs were able to maintain high growth rates despite elevated serum MMA concentrations (>20 micromol/L). CONCLUSIONS: The current reference ranges used in New Zealand for serum vitamin B12 are set conservatively high. Serum MMA concentrations appear to allow better differentiation of a responsive condition than vitamin B12 concentrations. Serum MMA concentrations >13 micromol/L indicate responsiveness to supplementation whilst concentrations <7 micromol/L indicate unresponsiveness. In the range 7-13 micromol/L, variation in response was observed and predictability of response is less certain, but supplementation is advisable. CLINICAL RELEVANCE: The current reference ranges for vitamin B12 responsiveness are conservatively high and lead to over-diagnosis of vitamin B12 deficiency in ill-thriftiness of sheep.