THE LECITHINS 431 



rather than ciioHne, was forming the insoluble reineckate. Glick'^' and 

 Entenman et al.^^^ have reported modifications of the reineckate procedure 

 for determining the choline content of plasma and tissue extracts. Accord- 

 ing to Entenman and Chaikoff^^^ both of these methods give identical values 

 for blood choline, although the Glick technic gives lower values for choline 

 in liver extract than does the procedure of Entenman and associates. ^^"-^^^ 



In the absence of interfering substances, any of several procedures may be 

 satisfactorily employed for the determination of choline as the reineckate. 

 This is true of the Beattie method, ^^^ in which the choline reineckate is dis- 

 solved in acetone and is determined by coiorimetric comparisons with a 

 standard. The procedure of Jacobi, Baumann, and Meek^*^ is considerably 

 more specific. This involves extraction of the tissues with a 1 : 1 alcohol- 

 ether mixture by boiling for 3 minutes; the extract is then evaporated to 

 dryness, and the residue is saponified for 2 hours with barj^a, followed by 

 neutralization with acetic acid and precipitation with reineckate. It is 

 stated ^^^ that the results obtained by this method agree with those deter 

 mined by the biological procedure using the acetylation technic of Fletcher, 

 Best, and Solandt. ^^^ Other modifications of the reineckate procedure have 

 been proposed by Engel,^^^ Shaw,^^^ and by Winzler and Meserve.^^'' 



Another group of methods involves the degradation of choline to tri- 

 methj'lamine, which may be determined by aeration into sulfuric acid. 

 However, such procedures as those suggested by Lintzeland associates, ^^•*^' 

 as well as by lOein and Linser,^^* are open to the same criticism as are the 

 earlier ones, in that they are nonspecific. 



The biological methods have long been regarded as standard. One of 

 these is based upon the effect of acetylcholine in producing contraction of 

 smooth muscle. The method was proposed in 1906 by Hunt and Taveau,^^" 

 who found that acetylcholine was 1000 to 100,000 times as active as choline 

 in causing such a response. Probably the most satisfactory procedure is 

 that of Chang and Gaddum^" as modified by Fletcher et al.^^* 



An entirely new biological procedure has been made available by the use 

 of the ascomycete, the red bread mold Neurospora crassa.'^^^ One of the two 

 mutants which do not produce choline can grow only if choline itself is spe- 



"' D. Glick, J. Biol. Chem., 156, 643-651 (1944). 



»»« C. Entenman, A. Taurog, and I. L. Chaikoff, /. Biol. Chem., 155, 13-18 (1944). 



'" C. Entenman and I. L. Chaikoff, ./. Biol. Chem., 160, 377-385 (1945). 



i»2 F. J. R. Beattie, Biochem. J., 30, 1.554-1559 (1936). 



»" H. P. Jacobi, C. A. Baumann, and W. J. Meek, /. Biol. Chem., 138, 571-582 (1941). 



"* J. P. Fletcher, C. H. Best, and O. M. Solandt, Biochem. J., 29, 2278-2284 (1935). 



w R. W. Engel, J. Biol. Chem., 144, 701-710 (1942). 



iw F. H. Shaw, Biochem. J., 32, 1002-1007 (1938). 



'«^ R. J. Winzler and E. R. Meserve, J. Biol. Chem., 159, .395-397 (1945). 



"» W. Lintzel and S. Foimin, Biochem. Z., '238, 438-451 (1931). 



•89 W. Lintzel and G. Monasterio, Biochem. Z., 241, 273-279 (1931). 



'»» R. Hunt and R. de M. Taveau, BriL Med. J., 1906, II, 1788-1791. 



»'» N. H. Horowitz and G. W. Beadle, J. Biol. Chem., 150. 325-333 (1943). 



