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

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<ul><li><p>C H A P T E R </p><p>V I T A M I N B 1 2 </p><p>1. I N T R O D U C T I O N </p><p>In 1926, G . R . M i n o t and W.P .Murphy1 demonstrated that patients with </p><p>pernicious anaemia could be maintained in normal health by ingestion o f </p><p>liver. Subsequently it was discovered that the injection o f liver extracts gave </p><p>more reliable results with less inconvenience to the patients. Since that time, </p><p>the use o f liver extracts has become routine practice in the treatment not only </p><p>o f Addisonian pernicious anaemia, but also o f pernicious anaemia due to </p><p>tapeworm, pernicious anaemia o f pregnancy, nutritional megaloblastic an-</p><p>aemia, megaloblastic anaemia o f infancy and childhood and megaloblastic </p><p>anaemia accompanying steatorrhoea. Three types o f liver extracts have </p><p>been used"refined" extracts with a relatively low concentration o f total </p><p>solids derived from a large amount o f liver, "crude" extracts with a much </p><p>higher total solids content and "proteolysed" extracts in which the liver tissue </p><p>is partially broken down before extraction in order to liberate more o f the </p><p>active principle. Some anaemias respond more readily to proteolysed and </p><p>crude liver extracts than to refined extracts and there are many clinicians </p><p>who maintain that refined extracts fail to keep the b lood picture normal for </p><p>more than a limited period. </p><p>Folic acid, as has already been pointed out (page 611), is an anti-anaemic </p><p>factor that is only successful in megaloblastic forms o f anaemia; it has no </p><p>effect in subacute combined degeneration o f the cord, and may actually in-</p><p>crease the severity o f the nervous symptoms in pernicious anaemia. Clearly </p><p>folic acid is different from the substance in liver extract that cures pernicious </p><p>anaemia, and potent refined liver extracts do , in fact, contain negligible </p><p>amounts o f folic acid. </p><p>Attempts to fractionate liver extracts with the object o f isolating the anti-</p><p>pernicious anaemia factor have always been difficult because no chemical test </p><p>for the factor existed, and no animal or micro-organism was known that </p><p>would respond specifically to the factor. The isolation o f more or less pure </p><p>preparations o f the anti-pernicious anaemia factor was announced in the </p><p>same week by E..Lester Smith2 and Rickes et al.</p><p>3 The former obtained by </p><p>chromatography two red pigments from an ox liver concentrate prepared by </p><p>a method previously described by W.B.Emery and L.F.J.Parker.4 Better </p><p>682 </p></li><li><p>VITAMIN B 1 2 683 </p><p>yields were obtained from proteolysed liver extracts than from non-pro-</p><p>teolysed extracts. One o f the pigments appeared to be produced from the other </p><p>by proteolysis. In the early stages o f the fractionation, clinical tests with </p><p>pernicious anaemia patients in relapse were used to follow the course o f </p><p>purification, but in the later stages the colour o f the fractions was used for this </p><p>purpose. The most active preparation gave a response with a dose containing </p><p>only 0-3 mg o f total solids and, in addition to being anti-anaemic, was </p><p>effective in sub-acute combined degeneration o f the cord. The product was </p><p>not pure and was not homogeneous when examined in the Tiselius apparatus. </p><p>It contained neither folic acid nor xanthopterin, and had a molecular weight </p><p>o f about 3000. </p><p>Rickes et al.3 reported the isolation o f the anti-pernicious anaemia factor </p><p>in the pure state and gave it the name vitamin B 1 2, but gave no information </p><p>about its properties or method o f isolation beyond the fact that the substance </p><p>crystallised from aqueous acetone in the form o f red needles which did not </p><p>melt below 300C and contained cobalt. Vitamin B 1 2 was shown to be </p><p>identical with one o f two unidentified growth factors required by Lacto-</p><p>bacillus lactis Dorner and this organism was in fact used by Rickes et al. </p><p>in the isolation o f vitamin B 1 2. Both o f the growth factors were present in </p><p>refined liver extracts.5 Crystalline vitamin B 1 2 gave a positive response in </p><p>three cases o f Addisonian pernicious anaemia following single intramuscular </p><p>injections o f 3, 6 and 150 (xg respectively.6 </p><p>Subsequently, E.Lester Smith7 crystallised the anti-pernicious anaemia </p><p>factor from liver, and found it to contain 4 % o f cobalt and, assuming this to </p><p>represent one atom per molecule, three atoms o f phosphorus per molecule. </p><p>Ellis et al.s and Brink et al.9</p><p>9 however, stated that the C o : ratio was 1:1 and </p><p>not 1:3, the value now generally accepted. Vitamin B 1 2 was also isolated </p><p>independently by B.Ellis, V.Petrow and G . F . S n o o k8 and by H . G . W i j -</p><p>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 </p><p>known, all clinically active. Vitamin B 1 2a was obtained by catalytic hydro-</p><p>gnation o f vitamin B 1 2,1 1</p><p> and vitamin B 1 2b was isolated, together with </p><p>vitamin B 1 2, from liver and a culture o f Streptomyces aureofaciens.12 Both </p><p>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 .</p><p>13 Vitamin B 1 2 </p><p>has been isolated from S. griseus,1* which is now the commercial source o f </p><p>this factor. </p><p>The elucidation o f the chemical structure o f vitamin B 1 2 resulted from the </p><p>work o f a number o f research groups in the United Kingdom, United States </p><p>and Switzerland, notable contributions being made by the British Drug </p><p>Houses Ltd. team led by V.Petrow, by the Glaxo group under E.Lester </p><p>Smith, by the Merck group under K.Folkers, and by Lord Todd's group at </p><p>the University o f Cambridge. It was the last-named group in association with </p><p>Dorothy Crowfoot Hodgkin and E.Lester Smith that in 1955 published the </p><p>first complete formula for vitamin B 1 2,1 5</p><p> modified slightly in the following </p></li><li><p>684 THE VITAMIN CO-FACTORS OF ENZYME SYSTEMS </p><p>year in the light o f further crystallographic evidence. The various forms o f </p><p>vitamin B 1 2 have the structure (I) . Cyanocobalamin, the form used for the </p><p>structural investigations has structure ( I ; R = C N ) . </p><p>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 , </p><p>1 0 7 , 3 9 6 ; 1948 , 1 0 8 , 1 3 5 . </p><p>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 </p><p>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 ; </p><p>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 , </p><p>1 0 8 , 6 3 4 . </p><p>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 </p><p>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 . </p><p>N H ^ O - C H J - C H J Me Me CHjOD-N^ </p><p>References to Section 1 </p></li><li><p>V I T A M I N B 1 2 685 </p><p>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 </p><p>A N D R E L A T E D F A C T O R S </p><p>Isolation and Purification </p><p>The method used by E.Lester Smith and L.F.J.Parker1 to isolate vita-</p><p>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. </p><p>The extract was purified by partition chromatography on damp silica or </p><p>starch using aqueous butanol or isopropanol as the moving phase. Activity, </p><p>as indicated by clinical trials, was found to be associated with a pink zone </p><p>which was further purified by repeated partition chromatography and frac-</p><p>tional precipitation with ammonium sulphate. Finally, the material was </p><p>crystallised from aqueous acetone in the form o f red needles. </p><p>Merck &amp; C o .2 isolated vitamin B 1 2 from either liver extract or a culture </p><p>o f S. griseus by chromatographic adsorption on activated alumina or char-</p><p>coal. The former was eluted with methanol or aqueous methanol and the </p><p>latter with an aqueous solution o f acetone, butanol or benzyl alcohol. In </p><p>either instance, the eluate was evaporated, and the residue dissolved in </p><p>alcohol. After filtration the solution was evaporated and the residue </p><p>dissolved in methanol. Vitamin B 1 2 was precipitated from this solution by the addition o f acetone or ether, the precipitate was dissolved in water and </p><p>the solution treated with acetone. The resulting precipitate was dissolved in </p><p>methanol and several volumes o f acetone were added to precipitate the vita-</p><p>min, which was then dissolved in water and acetone added until a turbidity </p><p>formed. On standing, red crystals o f vitamin B 1 2 separated out. </p><p>Concentrates o f vitamin B 1 2 were also purified prior to crystallisation by counter-current distribution between water and a mixture (3:1) o f toluene and </p><p>tf-cresol. In another Merck process,3 concentrates o f vitamin B 1 2 were ex-</p><p>tracted with an immiscible organic solvent containing an alkyl phenol or </p><p>benzyl alcohol. The concentrate was first washed with mesityl oxide or </p><p>fluorobenzene to remove impurities or the aqueous extract from the solvent </p><p>phase was washed with mesityl oxide or fluorobenzene. Vitamin B 1 2 was also extracted from aqueous concentrates by extraction with a mixture (1:1 </p><p>to 3:1) o f carbon tetrachloride and cresol, and the vitamin recovered by </p><p>adding more carbon tetrachloride, and extracting with water. </p><p>N . V . O r g a n o n4 adsorbed impurities from methanolic solutions o f vita-</p><p>min B 1 2 containing not more than 10% o f water on to alumina. They also claimed the isolation o f vitamin B 1 2 from aqueous solutions by extracting with a solution o f phenol in a non-polar solvent, and separating vitamins o f </p><p>the B 1 2 group from one another by partition chromatography on moist silica gel with a mixture o f phenol and a non-polar solvent as the moving </p><p>phase. The Upjohn C o .5 extracted an aqueous concentrate o f vitamin B 1 2 </p></li><li><p>686 THE VITAMIN CO-FACTORS OF ENZYME SYSTEMS </p><p>with a two-phase ternary solvent system consisting o f phenol, water and a </p><p>water-miscible ether; the vitamin was recovered from the upper organic </p><p>solvent phase. Alternatively a water-soluble salt was added to an aqueous </p><p>solution, which was then extracted with a water-miscible alcohol containing </p><p>two to four carbon atoms or a water-miscible ketone containing three to </p><p>four carbon atoms, or a mixture o f two such solvents. Sharp and D o h m e6 </p><p>adsorbed the vitamin on charcoal and eluted the adsorbate with aqueous </p><p>isopropanol or aqueous acetone, treated the eluate with diatomaceous earthy </p><p>eluted the adsorbate with aqueous pyridine, added butanol and recovered </p><p>vitamin B 1 2 from the aqueous phase. The American Cyanamid C o .7 ex-</p><p>tracted the vitamin into a water-insoluble phenol such as /?-chlorophenol, </p><p>and added an organic base e.g. 2,4-lutidine which formed a complex with the </p><p>phenol; this reduced the solubility o f the vitamin in the phenolic phase </p><p>enabling it to be extracted more efficiently into the aqueous phase. </p><p>The isolation o f vitamin B 1 2 in the form o f the readily crystallisable cyano-</p><p>cobalamin by Wijmenga et / .8 was due to their fortunate choice o f cyanide-</p><p>activated papain for the proteolysis o f liver; the cyanide present converted </p><p>the naturally occurring forms o f the vitamin into cyanocobalamin. Treat-</p><p>ment with cyanide has been incorporated into methods for the isolation o f </p><p>vitamin B 1 2 by all subsequent workers. O.Schindler and T.Reichstein9 for </p><p>example digested liver extract with cyanide-activated papain, removed im-</p><p>purities with lead hydroxide, extracted with a mixture (1:3) o f phenol and </p><p>chloroform and precipitated the vitamin from methanol by the addition o f </p><p>acetone. The vitamin was then extracted into phenol-chloroform (1:4) and </p><p>the extract was filtered though a column o f alumina. The vitamin was then </p><p>extracted into phenol-chloroform (1:10) and crystallised several times from </p><p>aqueous acetone. </p><p>The treatment o f vitamin B 1 2 concentrates with cyanides was patented </p><p>by . V. Organon ,10 and the use o f cyanide and thiocyanate solutions for the </p><p>elution o f vitamin B 1 2 from adsorbents and ion-exchange resins by Merck </p><p>&amp; C 0 .1 1</p><p> Pfizer &amp; C o .1 2</p><p> treated vitamin B 1 2 concentrates with cyanide, </p><p>saturated the acidified solution with a salt and extracted the cyanocobalamin </p><p>with an alcoholic solvent, re-extracted into water and then extracted with </p><p>an aliphatic acid such as capric acid, 2-ethylbutyric acid or cyclohexylacetic </p><p>acid. A method o f producing highly pure cyanobalamin by the decomposi-</p><p>tion o f complexes formed with cupric or zinc cuprocyanide was patented </p><p>by U C L A F .1 3</p><p> U C L A F1 4</p><p> also patented the purification o f vitamin B 1 2 by </p><p>forming a crystalline complex with phenol, orcinol, phloroglucinol or hydro-</p><p>quinone, and then decomposing the complex by adding acetone or dioxan </p><p>to the aqueous solution; very pure vitamin B 1 2 crystallised from the solu-</p><p>tion. Complexes from which pure vitamin B 1 2 can similarly be prepared are </p><p>obtained by adding hydroxynaphthoic acid, methylene-bis-(hydroxybenzoic </p><p>acid) or methylene-bis-(hydroxynaphthoic acid) or nuclearly substituted hy-</p><p>droxybenzoic acid to an aqueous solution o f the vitamin at p H 4 to 10 and </p></li><li><p>VITAMIN B 1 2 687 </p><p>then acidifying to p H 1 to 4 .1 5</p><p> The purification o f hydroxocobalamin by </p><p>adsorption on a polysaccharide ion exchanger, such as alginic acid or carboxy-</p><p>methyl-cellulose, and its formation from cyanocobalamin by reduction with </p><p>a titanous compound and re-oxidation after removal o f cyanide, have been </p><p>patented.1 5a </p><p>Partition chromatography has also been used to purify vitamin B 1 2. Strips </p><p>o f filter paper developed with wet n-butanol were used preparatory to micro-</p><p>bi...</p></li></ul>