COMBINED FORMS— EXTRACTION 39 



from its various combined forms. Piatt and Glock 101 have shown that 

 when fresh rat tissues are carefully dried in the frozen and finely divided 

 condition the water extract of the powder contains partly free and partly 

 combined (requiring acid hydrolysis) inositol. 



Choline 



The principal bound forms in which choline occurs are the lecithins 

 and sphingomyelins. Since the chemistry of naturally occurring phos- 

 pholipides and related compounds is not satisfactory, it being extremely 

 difficult or impossible to obtain such compounds in the pure state, one 

 would not dare be dogmatic as to the existence of other lipides in which 

 choline also is bound. 



From the standpoint of physiology, an extremely important bound form 

 of choline is acetylcholine. From the quantitative standpoint, however, 

 the amount of natural choline which is bound in this manner is extremely 

 small. 



There are certain close relatives of choline (dimethylaminoethanol, 

 monomethylaminoethanol, and ethanolamine) all of which are possible 

 components of lipides. These should be considered in connection with 

 choline studies. 



The extraction of total choline, both combined and uncombined, from 

 tissues is accomplished by the use of exhaustive absolute methanol ex- 

 traction. 102 Mixtures of ethanol and ether and other solvents have been 

 used, but the yield in every case is smaller. 102 - 103 



Lecithins which contain most of the bound choline can be precipitated 

 from an aqueous medium with acetone, leaving free choline in solution. 



Acid digestion of tissues has been used to free choline from its combina- 

 tions. 104 Autoclaving the entire tissue with SN HC1 for 2 hours yields a 

 solution which contains choline in the free form. 105 



Folic Acid (Pteroylglutamic Acid, P.G.A.) 



This vitamin has not been known long enough for its more complex 

 combined forms to be recognized. There is every reason to suppose that it 

 acts catalytically as a coenzyme and is therefore bound (perhaps loosely) 

 to proteins. These protein combinations are unknown. 



Of the three combined forms, probably the most revealing is the sim- 

 plest, i.e., formylfolic acid. 100 This has been made synthetically and 

 evidence for its natural occurrence and biological functioning has accu- 

 mulated. The next combined form, listed in order of simplicity, has two 

 extra (three total) glutamic acid residues joined to the glutamic acid 

 portion by peptide linkage. This was designated "fermentation L. casei 

 factor," since it was obtained from a fermentation residue. 107 A third 



