THE DETERMINATION OF VITAMIN B₁₂‐ACTIVITY IN THE ORGANS AND EXCRETA OF SHEEP

Abstract

The effect of the level of cobalt intake on the vitamin Bi2-active material in the faeces of sheep has already been described (Dawbarn, Hine and Hughes, 1952). The present communication reports an examination of the effect of cobalt status on the vitamin Bi2-activity in rumen contents. METHODS.FOOT microbiological assay methods were employed, each reported to be differently responsive to the varioua vitamin B,g-like factors which are present in rumen conteats (Ford, 1953a, b). The procedures used in this laboratory fur assay of vitamin B,» by Laclohacillus Xeidimannii tube assiiy and Eschcrischia coli mutant plate assay have already been diacuHsed in detail (Dawbarn and Hine, 1954). An Ii. coli tul»e assay, described by Burkbolder (1951), has been used with only minor modifications. In the fourth assay method the toni organism was a chrysomonad, Ochromonas malhamcnsis,^ which has been shown by Ford (195yb) to respond only to vitamin B^ itself (cyanocobalamin), and not to factors A, B and C or pseudovitamin Bij.Tlio estimates of vitamin Bi,-activity made by the epeeific method of Ford (1953b) and the other three methods wore correlated to indicate the presence of the various active subBtanees in the rumen and their changing proportions under different experimental conditions.The methods were first compared by assaying liver, since the vitamin Bij-activity in this or^an is largely attributable to vitamin B,, itself. Estimates obtained by the four methods should therefore be substantially the same.The standard extraction procedure adopted by us involved autoclaving the tissue for 15 minutes in an acetate bufFer at pH 4-6 (extraction method A). Ford (1953b) reported that acetate has an inhibitory effect on the growth of Ochromonas, and recommended that extraction should be carried out by steaming for 30 minutes in water at pH 5 (extraction method B). We therefore compared the two procedures. It has already been shown (Dawbaru and Hine, 1954) that estimates of the vitamin B,, in liver made by the E. coli plate and L. leichmennii tube methods are the same. The results of the other experiments are reported in Table 1.We are indebted to Mr.

Folic acid metabolism in vitamin B12-deficient sheep. Depletion of liver folates

“…Vitamin Bl2 in liver and blood serum was estimated with Lactobacillus leichmanii as described by Hine & Dawbarn (1954).…”

Factors affecting formiminoglutamic acid excretion in vitamin B12 deficiency

“…039 and 438. Values are reproduced from Dawbarn et al (1958b) Measured by the method of Dawbarn & Hine (1954) and Hine & Dawbarn (1954).…”

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism

“…This was assayed with Lactobacillus leichmanii as described by Hine & Dawbarn (1954). Materials L-Methionine was obtained from the Sigma Chemical Co. Enzymes and substrates for determination of adenine nucleotides and nicotinamide nucleotides were obtained from C. F. Boehringer und Soehne G.m.b.H., Mannheim, Germany.…”