Tracer studies with the B12 vitamins. 2. Biosynthesis of vitamin B12labelled with 60CO and 32P

Abstract

These attempts failed because the livers contained negligible amounts of vitamin B12. We recognized at the time that it would have been preferable to use ruminants, but such animals were not available to us. Subsequent work suggests that we ought to have extracted the faeces rather than the livers of our animals.About the same time we succeeded in preparing very small amounts of radioactive vitamin B12 concentrates by fermentations with Aerobacter aerogenes isolated from hen faeces. More recently we have returned to this problem, using Streptomyces gri8eu; in the meantime the preparation by this method of [6OCo] vitamin B12 with low or moderately high specific activity has been reported (Chaiet, Rosenblum & Woodbury, 1950;. RESULTS Preparation of vitamin B12 containing 60CoTo avoid diminishing the specific activity of the added radioactive cobalt, we used a semi-synthetic medium (Dulaney, 1948) shown to be nearly free from cobalt. We added only about 0-2 p.p.m. of radio-cobalt, thus minimizing any radiation hazard and securing efficient conversion ofcobalt to vitamin B12. The fermentation liquors were worked up to yield a few mg. of crystalline vitamin B12 by a simple procedure described in the Experimental section. The latest fermentation with 60Co at 3-5 mc./mg. has yielded vitamin B12 with a specific activity of 120 ,uc./mg., which is considerably higher than any previously recorded (Rosenblum & Woodbury, 1951). Preparation of vitamin B12 containing 32pIt was of interest to attempt the preparation of vitamin B12 labelled with radioactive phosphorus, since this would be safer for projected human metabolism experiments than vitamin B12 containing radioactive cobalt with its much longer half-life and penetrating gamma radiation. It was foreseen that it would be difficult to prepare material with adequate specific activity because preliminary experiments had shown that Streptomyce8 gri8eus needs a moderately high level of phosphate in the medium, growth and vitamin B12 production being impaired when the total phosphorus level is reduced to about 0-01 %.It was decided to use 10 mc. Of 32p and to aim at preparing about 1 mg. of the radioactive vitamin. It was then almost immaterial what volume of medium was used for the fermentation; the smaller the volume, the higher the specific activity of the resulting vitamin B12 would be, but the greater the proportion of ordinary vitamin B12 that would have to be added.The radioactive phosphate was added aseptically 24 hr. after fermentation started. This was done in the hope that part of the phosphorus in the medium would already have been taken up by the mould and converted into organic phosphorus compounds that would not exchange with the added 32p. This hope was not realized; the specific activity of the product was the same as that from a pilot run with

Preparation and biodistribution of 32P‐labelled hydroxocobalamin and comparison with 57Co‐labelled cobinamide and cyanocobalamin

“…We produced eight batches of ). The best specific activity achieved was now 1000 times higher than previously achieved by us and a million times than reported in the pioneering work of Smith et al 1 The specific activities of some batches were still low, obviously related to the difficulty of the organism to thrive in low-phosphorus medium. …”

“…Following the isolation of vitamin B 12 , cobalamin (Cbl), a number of radiolabels were introduced into the molecule, 1 but soon radiocobalt isotopes became almost exclusively used as tracers. Using radiocobalt-labelled Cbl, usually in cyano form (CN-Cbl), a large number of observations have been made regarding the absorption, transport, distribution, excretion, and nutritional requirement of this vitamin.…”

Synthesis and nca-Radioiodination of Arylstannyl−Cobalamin Conjugates. Evaluation of Aryliodo−Cobalamin Conjugate Binding to Transcobalamin II and Biodistribution in Mice

“…Early investigators demonstrated that cyanocobalamin could be labeled with cobalt radionuclides (Co-57, -58, -60) (7)(8)(9)(10)(11)(12)(13)(14) and carbon-14 (15). Tritium has also been used to radiolabel cyanocobalamin (16).…”

Preparation and some properties of ferribalamin, the Fe(III)‐analogue of vitamin B12

“…In contrast to heme and chlorophyll the central atom of vitamin B12 can neither be removed nor exchanged with other metals [1 ]. Studies of the function of the cobalt in the B12-coenzyme have been hindered by the limited availability of the relevant substituted corrinoids.…”