68 THE BIOCHEMISTRY OF B VITAMINS 



and the pathogenic nature of the organism used are deterrents to its use 

 as an assay method. 



Physiological Assay. An interesting and important method for choline 

 determination involves acetylating it quantitatively and determining the 

 acetylcholine by its stimulation of the contraction of isolated muscle from 

 rabbit intestine. The amount of acetylcholine necessary to elicit a con- 

 traction about 75 per cent of the maximum may be from 0.01 to 0.03 /xg 

 per ml, depending upon the muscle preparation and the exact conditions ; 

 so the method has high sensitivity. 200 



There are interfering substances and the method possesses many dan- 

 gerous pitfalls for the chemist, 201 but inherently it has great advantages 

 and it seems likely that cooperative studies by biochemists (who are more 

 expert in preparing suitable extracts and eliminating by adsorption and 

 otherwise interfering substances) and physiologists (who are more expert 

 with respect to dealing with muscle preparations) might evolve an assay 

 method more sensitive, more specific, and more satisfactory generally 

 than any yet devised. In its present form it has been applied to many 

 materials. 200 Duplicate assays occasionally differed by almost 30 per cent. 



Animal Assays. The development of an animal assay procedure for 

 choline is complicated by the fact that choline is not an absolute require- 

 ment; methionine and other naturally occurring substances possess 

 choline-like activity when fed to animals. 202 Engel, 1S3 nevertheless, has 

 developed an assay method for choline and choline-like substances which 

 depends upon its ability to prevent kidney hemorrhages in rats receiving 

 a choline-deficient diet. The rats had to be carefully matched and used 

 in considerable numbers (more often 11 or 12 for each test), but the 

 results were such that a 10 per cent increase or decrease of the material 

 tested could be detected. Liver was found to be about 25 per cent more 

 effective than could be accounted for on the basis of its choline content 

 as determined chemically by the reineckate method, and this was inter- 

 preted to mean the presence in it of other substances which are physiologi- 

 cally like choline. Otherwise, the bioassays agreed with the chemical 

 method rather closely. 



Folic Acid (Pteroyl Glutamic Acid, P.G.A.) 



Determination of folic acid may be performed (under delimiting con- 

 ditions) chemically, microbiologically or by animal assays. None of the 

 methods has been fully standardized, particularly so as to be readily 

 applied to tissues of all types. 



Chemical Determination. Hutchings, et al. 203 have developed a chemi- 

 cal method for the determination of folic acid and related compounds, 

 taking advantage of the fact that by reduction in acid solution with zinc 



