BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 33 



in the stearic-lactobacillic acid fraction derived from chro- 

 matography. Acetone-water mixtures of increasing acetone 

 content serve as eluant for the various fatty acids. The 

 initial hydroxylation step converts the monoethenoid fatty 

 acids, mainly cis-vaccenic acid, into the corresponding di- 

 hydroxy derivatives. In contrast to the unsaturated acids, 

 which cannot be cleanly separated from the saturates by 

 rubber chromatography, the dihydroxy acids evolve from the 

 column with the eluants of the lowest acetone content and 

 are thus readily separable from the saturated acids and 

 lactobacillic acid. This "dihydroxy" fraction is a measure 

 of the proportion of unsaturated fatty acids both Ciq and 

 Cis in the bacterial lipids. A typical chromatogram ob- 

 tained with a synthetic mixture of fatty acids relating frac- 

 tion number to the volume of 0.0 LV sodium hydroxide 

 required to titrate each fraction to the phenolphthalein 

 endpoint is illustrated in Fig. 2.1. In this experiment the 

 recovery of individual fatty acids ranged from 81 to 110% 

 of theory. Lactobacillic and stearic acids evolve from the 

 column as a single inseparable peak. The lactobacillic acid 

 content of this fraction is readily determined by microbio- 

 logical assay with L. delbrueckii. A typical curve relating 

 lactobacillic acid content of the medium and cell growth 

 is illustrated on Fig. 2.2. The studies which are summarized 

 on Table 2.1 demonstrate conclusively that addition of 

 stearic acid to the lactobacillic acid samples does not inter- 

 fere with the biological assay. Verification of this point 

 was of considerable importance since saturated fatty acids 

 may increase synergistically the growth promoting potency 

 of certain unsaturated fatty acids (3). 



Gas-liquid chromatography of the methyl esters of bac- 

 terial fatty acids (4, 5, 6) has largely replaced the older 

 procedure, since it offers significant advantages in speed. 



