C H A P T E R
V I T A M I N B 1 2
1. I N T R O D U C T I O N
In 1926, G . R . M i n o t and W.P .Murphy1 demonstrated that patients with
pernicious anaemia could be maintained in normal health by ingestion o f
liver. Subsequently it was discovered that the injection o f liver extracts gave
more reliable results with less inconvenience to the patients. Since that time,
the use o f liver extracts has become routine practice in the treatment not only
o f Addisonian pernicious anaemia, but also o f pernicious anaemia due to
tapeworm, pernicious anaemia o f pregnancy, nutritional megaloblastic an-
aemia, megaloblastic anaemia o f infancy and childhood and megaloblastic
anaemia accompanying steatorrhoea. Three types o f liver extracts have
been used"refined" extracts with a relatively low concentration o f total
solids derived from a large amount o f liver, "crude" extracts with a much
higher total solids content and "proteolysed" extracts in which the liver tissue
is partially broken down before extraction in order to liberate more o f the
active principle. Some anaemias respond more readily to proteolysed and
crude liver extracts than to refined extracts and there are many clinicians
who maintain that refined extracts fail to keep the b lood picture normal for
more than a limited period.
Folic acid, as has already been pointed out (page 611), is an anti-anaemic
factor that is only successful in megaloblastic forms o f anaemia; it has no
effect in subacute combined degeneration o f the cord, and may actually in-
crease the severity o f the nervous symptoms in pernicious anaemia. Clearly
folic acid is different from the substance in liver extract that cures pernicious
anaemia, and potent refined liver extracts do , in fact, contain negligible
amounts o f folic acid.
Attempts to fractionate liver extracts with the object o f isolating the anti-
pernicious anaemia factor have always been difficult because no chemical test
for the factor existed, and no animal or micro-organism was known that
would respond specifically to the factor. The isolation o f more or less pure
preparations o f the anti-pernicious anaemia factor was announced in the
same week by E..Lester Smith2 and Rickes et al.
3 The former obtained by
chromatography two red pigments from an ox liver concentrate prepared by
a method previously described by W.B.Emery and L.F.J.Parker.4 Better
VITAMIN B 1 2 683
yields were obtained from proteolysed liver extracts than from non-pro-
teolysed extracts. One o f the pigments appeared to be produced from the other
by proteolysis. In the early stages o f the fractionation, clinical tests with
pernicious anaemia patients in relapse were used to follow the course o f
purification, but in the later stages the colour o f the fractions was used for this
purpose. The most active preparation gave a response with a dose containing
only 0-3 mg o f total solids and, in addition to being anti-anaemic, was
effective in sub-acute combined degeneration o f the cord. The product was
not pure and was not homogeneous when examined in the Tiselius apparatus.
It contained neither folic acid nor xanthopterin, and had a molecular weight
o f about 3000.
Rickes et al.3 reported the isolation o f the anti-pernicious anaemia factor
in the pure state and gave it the name vitamin B 1 2, but gave no information
about its properties or method o f isolation beyond the fact that the substance
crystallised from aqueous acetone in the form o f red needles which did not
melt below 300C and contained cobalt. Vitamin B 1 2 was shown to be
identical with one o f two unidentified growth factors required by Lacto-
bacillus lactis Dorner and this organism was in fact used by Rickes et al.
in the isolation o f vitamin B 1 2. Both o f the growth factors were present in
refined liver extracts.5 Crystalline vitamin B 1 2 gave a positive response in
three cases o f Addisonian pernicious anaemia following single intramuscular
injections o f 3, 6 and 150 (xg respectively.6
Subsequently, E.Lester Smith7 crystallised the anti-pernicious anaemia
factor from liver, and found it to contain 4 % o f cobalt and, assuming this to
represent one atom per molecule, three atoms o f phosphorus per molecule.
Ellis et al.s and Brink et al.9
9 however, stated that the C o : ratio was 1:1 and
not 1:3, the value now generally accepted. Vitamin B 1 2 was also isolated
independently by B.Ellis, V.Petrow and G . F . S n o o k8 and by H . G . W i j -
menga, J.Lens and A . M i d d l e b e c k .10 Several forms o f vitamin B 1 2 are
known, all clinically active. Vitamin B 1 2a was obtained by catalytic hydro-
gnation o f vitamin B 1 2,1 1
and vitamin B 1 2b was isolated, together with
vitamin B 1 2, from liver and a culture o f Streptomyces aureofaciens.12 Both
have absorption spectra similar to but not identical with that o f vitamin B 1 2 and were subsequently shown to be identical with one another .
13 Vitamin B 1 2
has been isolated from S. griseus,1* which is now the commercial source o f
The elucidation o f the chemical structure o f vitamin B 1 2 resulted from the
work o f a number o f research groups in the United Kingdom, United States
and Switzerland, notable contributions being made by the British Drug
Houses Ltd. team led by V.Petrow, by the Glaxo group under E.Lester
Smith, by the Merck group under K.Folkers, and by Lord Todd's group at
the University o f Cambridge. It was the last-named group in association with
Dorothy Crowfoot Hodgkin and E.Lester Smith that in 1955 published the
first complete formula for vitamin B 1 2,1 5
modified slightly in the following
684 THE VITAMIN CO-FACTORS OF ENZYME SYSTEMS
year in the light o f further crystallographic evidence. The various forms o f
vitamin B 1 2 have the structure (I) . Cyanocobalamin, the form used for the
structural investigations has structure ( I ; R = C N ) .
1. G . R . M I N O T and W . P . M U R P H Y , / . Amer. Med. Assoc., 1926 , 8 7 , 4 7 0 . 2 . E . L . S M I T H , Nature, 1 9 4 8 , 1 6 1 , 6 3 8 . 3 . E . L . R I C K E S , N . G . B R I N K , F . R . K O N I U S Z Y , T . R . W O O D and K . F O L K E R S , Science, 1 9 4 8 ,
1 0 7 , 3 9 6 ; 1948 , 1 0 8 , 1 3 5 .
4 . W . B . E M E R Y and L . F . J . P A R K E R , Biochem. J. Proc, 1946 , 4 0 , iv. 5. M . S . S H O R B , Science, 1 9 4 8 , 1 0 7 , 3 9 7 . 6 . R . W E S T , ibid., 3 9 8 . 7. E . L . S M I T H , Nature, 1948 , 1 6 2 , 144 . 8. B . E L L I S , V . P E T R O W and G . F . S N O O K , J. Pharm. Pharmacol, 1949 , 1, 6 0 , 2 8 7 . 9. N . G . B R I N K , D . E . W O L F , E . K A C Z K A , E . L . R I C K E S , F . R . K O N I U S Z Y , T . R . W O O D and
K . F O L K E R S , J. Amer. Chem. Soc, 1949 , 7 1 , 1854 . 10. H . G . W I J M E N G A , J . L E N S and A . M I D D L E B E C K , Chem. Weekbl, 1949 , 4 5 , 3 4 2 . 11 . E . K A C Z K A , D . E . W O L F and K . F O L K E R S , / . Amer. Chem. Soc, 1 9 4 9 , 7 1 , 1 5 1 4 . 12 . J . V . P I E R C E , A . C . P A G E , E . L . R . S T O K S T A D and T . H . J U K E S , ibid., 2 9 5 2 . 13 . E . A . K A C Z K A , D . E . W O L F , F . A . K U E H L and K . F O L K E R S , Science, 1950 , 1 1 2 , 3 5 4 ;
H . G . W I J M E N G A , W . L . C . V E E R and J .LENS, Biochim. Biophys. Acta, 1950 , 6 , 2 2 9 . 14. E . L . R I C K E S , N . G . B R I N K , F . R . K O N I U S Z Y , T . R . W O O D and K . F O L K E R S , Science, 1 9 4 8 ,
1 0 8 , 6 3 4 .
15. D . C . H O D G K I N , J . P I C K W O R T H , J . H . R O B E R T S O N , K . N . T R U E B L O O D , R . J . P R O S E N and
J . G . W H I T E , Nature, 1 9 5 5 , 1 7 6 , 3 2 5 ; R . B O N N E T T , J . R . C A N N O N , A . W . J O H N S O N , I . S U T H E R L A N D , A . R . T O D D and E . L E S T E R S M I T H , ibid., 3 2 8 ; D . C . H O D G K I N , A . W . JOHNSON and A . R . T O D D , Chem. Soc. Special Publ. No. 3, p. 1 0 9 ; D . C . H O D G K I N , J . K A M P E R , M . M A C K A Y , J . P I C K W O R T H , K . N . T R U E B L O O D and J . G . W H I T E , Nature, 1956 , 1 7 8 , 6 4 .
N H ^ O - C H J - C H J Me Me CHjOD-N^
References to Section 1
V I T A M I N B 1 2 685
2. I S O L A T I O N A N D P U R I F I C A T I O N OF V I T A M I N B 1 2
A N D R E L A T E D F A C T O R S
Isolation and Purification
The method used by E.Lester Smith and L.F.J.Parker1 to isolate vita-
min B 1 2 from liver extracts was as follows: Proteolysed liver extract was treated with charcoal, and the adsorbate was eluted with hot 65 % ethanol.
The extract was purified by partition chromatography on damp silica or
starch using aqueous butanol or isopropanol as the moving phase. Activity,
as indicated by clinical trials, was found to be associated with a pink zone
which was further purified by repeated partition chromatography and frac-
tional precipitation with ammonium sulphate. Finally, the material was
crystallised from aqueous acetone in the form o f red needles.
Merck & C o .2 isolated vitamin B 1 2 from either liver extract or a culture
o f S. griseus by chromatographic adsorption on activated alumina or char-
coal. The former was eluted with methanol or aqueous methanol and the
latter with an aqueous solution o f acetone, butan