John C. Linnell

Cobalamins in fibroblasts cultured from normal control subjects and patients with methylmalonic aciduria.

Extract: The intracellular content and proportional distribution of B12 (cobalamin) derivatives in fibroblasts cultured from patients with various forms of methylmalonic aciduria, as well as from normal control subjects, has been determined by a two-dimensional chromatobioautographic technique. Each line of fibroblasts was grown in the presence of four concentrations of cobalamin, ranging from the 0.04–0.07 pmol/ml contained in the basal medium to 74 pmol/ml (100 ng/ml), added in form of hydroxocobalamin (OHCBI). Control cells grown in the basal medium contained substantial proportions of both methylcobalamin (MeCbl) and adenosyl-cobalamin (AdoCbl), with the former predominating. As increasing concentrations of OH-CBI were added to the growth medium, the total cellular cobalamin content increased without marked changes in the relative proportions of MeCbl, AdoCbl, and OHCbl. Three different patterns were discernable in the cobalamin distributions of the cells cultured from patients with methylmalonic aciduria (Table 1 and Fig. 1).Speculation: Information about the cobalamin contents of cultured fibroblasts yields further understanding of cobalamin metabolism. Such information, obtained by the technique of two-dimensional chromatobioautography, permits definition of genetically determined abnormalities in the handling of cobalamins, as well as examination of possible correlations between cellular responses to increased concentrations of these compounds and the effectiveness of cobalamins in the clinical treatment of patients.

Tissue distribution of endogenous cobalamins and other corrins in the rat, cat and guinea pig

1. Methylcobalamin, adenosylcobalamin, hydroxocobalamin and cyanocobalamin have been estimated by a chromato-bioautographic techniques in 16 tissues from healthy rats and in five guinea pig tissues. 2. Plasma and erythrocyte cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder hydroxocobalamin predominated or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues but at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those of methylcobalamin in 8 of the 16 tissues. 5. In the rat, cat and guinea pig, levels of methylcobalamin and hydroxobalamin were higher in erythrocytes than in plasma, a pattern almost the complete reverse of that in man.

Effects of low cobalamin diet and chronic cyanide toxicity in baboons

The effects of a low cobalamin (Cbl) diet, together with chronic cyanide or thiocyanate administration in some animals have been investigated in baboons over a period of 42 months. All animals remained healthy throughout the study and gained weight at a similar rate. None became anaemic or showed major haematological changes and there were no major neurological changes. Plasma total Cbl in the animals on the low Cbl diet fell within 9 months to values below the lower limit in man and were lowest at 24 months in baboons not receiving cyanide or thiocyanate. A striking feature in all animals, however, was an apparently seasonal increase in the plasma total Cbl each autumn with a corresponding decrease the following spring. This fluctuation was detected by radioisotopic assay but not by Euglena. Methylmalonic (MMA) excretion after oral valine ranged from 0.1--8.4 mg/24 h and was greatest in animals on the low Cbl diet and not receiving cyanide or thiocyanate. The results suggested an inverse relationship between MMA excretion and plasma total Cbl. Plasma thiocyanate was consistently higher in animals receiving cyanide or thiocyanate and at the end of the study plasma cyanide was highest in animals on the low Cbl diet receiving cyanide. The results support the suggestions that cyanide affects bodily handling of Cbl and that hydroxo-cobalamin plays a part in detoxication of cyanide.

Interference by methylcobalamin analogues with synthesis of cobalamin coenzymes in human lymphocytes in vitro

1. 72 h uptake of cyano[57Co]cobalamin and formation of 57Co-labelled methylcobalamin, adenosylcobalamin and hydroxocobalamin has been estimated with and without the addition of methylcobalamin analogues in phytohaemagglutinin-stimulated lymphocytes from healthy human subjects. 2. Difluorochloromethylcobalamin reduced cell uptake of cyanocobalamin and caused a disproportionate reduction in synthesis of adenosylcobalamin. 3. Methylcobalamin-palladium trichloride reduced cell uptake of cyanobalamin more effectively than did difluorochloromethylcobalamin and reduced the formation of methylcobalamin, adenosylcobalamin and hydroxocobalamin in proportion. 4. The results suggest that in addition to inhibiting uptake of cyanocobalamin, one or both compounds may have interfered directly with the mechanism of synthesis of the cobalamin coenzymes.

Effects of low cobalamin diet and chronic cyanide toxicity on cobalamin distribution in baboons

This paper reports the bodily distribution of total cobalamin and individual cobalamins at the termination of an experiment on the effects of a low cobalamin diet and chronic cyanide or thiocyanate administration in baboons. The results show that the distribution of cobalamins in the tissues of the baboon can be altered by a low cobalamin diet and also by chronic intoxication with cyanide, whether or not the animals are on a low cobalamin diet. All animals on the low cobalamin diet showed a reduction in total and individual cobalamins. In blood plasma and erythrocytes, kidney, spleen, testis and brain, the proportion of methylcobalamin tended to be disproportionately reduced in cobalamin-depleted animals. This reduction was lessened or prevented by the administration of cyanide. Neither cyanide not thiocyanate produced a significant increase in the proportion of cyanocobalamin in plasma, though thiocyanate produced a large increase in cyanocobalamin in erythrocytes. In liver, cyanocobalamin was more than doubled by the administration of cyanide to cobalamin-depleted animals.