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An evaluation of homocysteine in the assessment of vitamin B₁₂ status of pasture-fed sheep

Furlong, J
Date
2005
Type
Thesis
Fields of Research
ANZSRC::0707 Veterinary Sciences
Abstract
Homocysteine (Hcy) is a component of metabolic pathways that are associated with the exchange of methyl groups vital to the animal's survival. Vitamin B₁₂ (cobalamin) has a key role in these methylation reactions and the measurement of Hcy in plasma could theoretically predict the clinical status of cobalamin (Cbl) deficiency in sheep, which is innately difficult to diagnose. Serum Hcy concentrations are evaluated in comparison with Cbl and another metabolic indicator of Cbl deficiency - methylmalonic acid (MMA), in situations of liveweight gain (L WG) responsiveness to cobalt (Co )/Cbl to determine its utility in pasture-fed sheep. Improvement of HPLC methodology was carried out to resolve peak integrity, precision, run time and stability issues encountered previously in the assay of Hcy. Eighty two pregnant ewes, half of which were supplemented with a Co bullet, were grazed together on a Southland property known to be Co deficient. Half of the lambs from each group were supplemented with 3mg microencapsulated Cbl (SMARTShot™) one month after lambing (day 0), providing four groups. Lambs were blood sampled and weighed, at monthly intervals, until approximately 210 days of age and were weaned at day 89. A 40% difference in mean liveweight was recorded between the supplemented and unsupplemented lamb groups. Mean monthly total homocysteine (tHcy) concentration ranged from 1.5 to 4.5 µmol/l. On day 0 tHcy concentrations in lambs from supplemented and unsupplemented ewe groups were 4.0 and 2.2 µmol/l; a difference which was significant (p <0.001). Subsequently there were no differences between unsupplemented and supplemented lamb groups. This lack of effect occurred despite plasma concentrations of MMA and Cbl at which response to supplementation could be expected and L WG responses to Cbl supplementation were observed. Unsupplemented lamb MMA and Cbl concentrations (> 16 µmol/l and <170 pmol/l, respectively, compared to the deficient status thresholds from reference ranges of > 13 µmol/l and <200 pmol/l respectively) were attained on day 57. No correlation between Hcy and MMA or Cbl concentrations was detected even in individual animals whose performance was severely compromised by deficiency. This suggested that the biological importance of the Hcy pathway means that it is highly conserved, even in extreme deficiency. To pursue this further, data 'from a previous supplementation trial in which 34 individual unsupplemented animals had been removed due to welfare issues, due to severe ill-health, were examined. Mean tHcy concentrations ranging from 6 - 13 µmol/l were recorded, in association with Cbl concentrations below 200 pmol/l throughout and steady rise in MMA concentration to very high values (from 10 - 26 µmol/l). The severity of the deficiency at onset was characterised by rapid response to supplementation although tHcy showed no associated change as a consequence. In conclusion, the measurement of tHcy as a metabolic indicator of Cbl deficiency in sheep on typical New Zealand pasture appears to have no value in detecting the incidence or assessment of the severity of the disease. The possibility that diet and other factors such as alternative pathways, including the betaine - methionine metabolic pathways, may regulate and conserve methylation pathways is discussed, but a conclusion that MMA is the first rate limiting pathway and the metabolic indicator of choice in predicting the clinical Cbl status in sheep, seems most plausible.