528 BIOTIN 



anced except for a large proportion of egg white as the sole source of pro- 

 tein developed peculiar and impressive skin changes. These were accom- 

 panied by progressive emaciation and led finally to a fatal outcome. Diets 

 rich in raw or slightly cooked fresh whole egg, egg white, or commercial 

 Chinese dried egg white have been found equally detrimental. The egg 

 white was found to be deprived to its toxic properties by continued and 

 intensive treatment with heat, as well as by digestion with pepsin or in- 

 cubation with hydrochloric acid, probably owing to hydrolysis.^^ An or- 

 ganic protective substance, present in different foodstuffs, was found hke- 

 wise to be able to neutralize the toxic effect of egg white when the protective 

 substance was given as a supplement to the otherwise disease-producing 

 diet rich in egg white. This substance has been called the "protective factor 

 X" by Boas,^ vitamin H by Gyorgy,^" or "the factor protective against 

 egg-white injury" by Lease and Parsons.^* 



Isolation of the active substance was first achieved even before identifi- 

 cation of biotinwith vitamin H (the anti-egg white injury factor) from egg 

 yolk.^ The yolks of 1000 fresh eggs were treated with acetone, and the fil- 

 trate after concentration was precipitated with four volumes of alcohol. 

 The active principle was found in the precipitate, which was dissolved in 

 water and the impurities removed by precipitation with lead acetate. The 

 filtrate was freed from lead and treated with phosphotungstic acid. The 

 precipitate, containing the active factor, was decomposed with baryta, 

 and the solution was shaken with charcoal. The adsorbate was first washed 

 with 50% alcohol, followed by elution with 60% acetone containing 2.5% 

 of ammonia, leading to an active fraction. A second precipitation with 

 phosphotungstic acid and decomposition with baryta gave an active frac- 

 tion soluble in alcohol. Treatment with mercuric chloride removed impuri- 

 ties. After esterification with methanolic hydrogen chloride further impuri- 

 ties were removed by precipitation with picrolonic acid and then with 

 rufianic acid. Finally, a very potent preparation was obtained by decom- 

 position of the reineckate. From a second preparation, using dried egg 

 yolk as starting material, the methyl ester was obtained in crystalline 

 form by high vacuum distillation and crystallization from a mixture of 

 chloroform and light petroleum. The substance had a m.p. of 146 to 147° 

 and was active on Saccharomyces in a dilution of 1 in 10,000,000,000. 



Later, Kogl and Pons^^ succeeded by the use of molecular distillation 

 to purify further the crude product and even to increase the yield. The 

 biotin methyl ester was crystallized from mesityl oxide which gave a 

 purer product, m.p. 161 to 165°. 



Before the identity of biotin with vitamin H (the anti-egg white injury 



" J. G. Lease and H. T. Parsons, Biochem. J. 28, 2109 (1934). 



" F. Kogl and L. Pons, Hoppe-Seyler's Z. physiol. Chem. 269, 61 (19-11). 



