The estimation and significance of the level of vitamin B12 in serum.

Normal human serum contains a few hundred micro-microgrammes (,u,ug.) of vitamin B12 per ml. A micro-microgramme is one-millionth of a millionth of a gramme, or I/I,000,000,000 of a milligramme. No chemical method is capable of measuring such small amounts. Specialized techniques are necessary and, with one exception, all methods employ microbiological assays. Certain micro-organisms require vitamin B12 for growth in defined media, which must contain all nutrients, other than vitamin B12, needed by the organism. Over a certain range, which varies according to the organism used, the concentration of vitamin B12 is proportional to the amount of growth, which can be assessed either turbidimetrically or by measuring the changes in pH of the medium, or by titrating the amount of acid produced in the medium, after growth. The concentration of vitamin B12 in serum can be computed by comparing the growth responses to known amounts of serum, or suitable extracts of serum, with those produced by known quantities of vitamin B12 The principles underlying microbiological assays are simple, but technically the methods present considerable difficulty. Cleanliness, attention to apparently trifling details of technique and experience are necessary to ensure satisfactory results. These aspects of microbiological assay methods were discussed by Girdwood (ig6oa).