The Vitamin Co-Factors of Enzyme Systems || VITAMIN B12

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  • 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

    682

  • 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

    this factor.

    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