The forms of vitamin B12 in foodstuffs and the occurrence of artefactual sulphitocabalamin

Farquharson, James (1975) The forms of vitamin B12 in foodstuffs and the occurrence of artefactual sulphitocabalamin. PhD thesis, University of Glasgow.

Abstract

Extraction yields of cobalamins were examined with tissues containing both incorporated and added radioactive vitamin B12 (57Co.vit.B12) and pronase used to increase the yields from ca. 65 - 75% by its proteolysis of the vitamin B12 binders. Pronase itself was however found to be a source of vitamin B12 and thus its use was discontinued. The extracted cobalamins were separated by silica gel thin-layer chromatography and the chromatograms developed by the standard bioautography assay, using an Escherichia coli mutant, which was vitamin B12 dependent for growth. An unknown cobalamin which was encountered during these assays was examined by ion-exchange chromautography and light-sensitivity and identified as artefactual sulphite B12, produced from hydroxo B12. To prevent this conversion pH control of the extraction using a bicarbonate buffer was attempted but met with little success, because of the ease of formation of sulphite B12 and the inability to maintain the buffered conditions throughout the extraction. Pre-conversion of the hydroxo B12 ammonia B12 with an ammonia buffer system was successful in eliminating the artefact, and moreover produced a cohalamin which was indistinguishable from hydrozo B12 on chromatography in ammonia containing solvent systems. The light sensitivities of the 'naturally' occurring cohalamins were estimated under controlled light exposure conditions to establish the most suitable method for preparation of tissues and isolation of cobalamins. The forms of vitamin B12 in items of diet were found by the chromatographic and bioautographic analysis of tissue extracts and a degree of quantitation of results introduced by examination of a series of extract diluents. Distinct variations were evident in the forms of vitamin B12 isolated in the three categories of foodstuffs examined, namely dairy produce, meat & poultry (muscle tissue) and fish.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J F Adams
• Keywords: Food science
• Date of Award: 1975
• Depositing User: Enlighten Team
• Unique ID: glathesis:1975-72331
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2019 15:12
• Last Modified: 24 May 2019 15:12
• URI: https://theses.gla.ac.uk/id/eprint/72331