COMBINED FORMS— EXTRACTION 37 



acid treatment. 78 This makes possible the utilization of one treatment 

 after another, if necessary. 79 The present evidence is against the idea that 

 other acid labile forms of vitamin B G exist, as once appeared to be the 

 case. 80 



Biotin 



Though biotin itself is readily water-soluble, only a minute por- 

 tion of that present in tissues, sometimes as little as 0.1 per cent, is 

 extracted by hot water. 81 Unquestionably biotin exists naturally primarily 

 in combined forms, but little information is as yet available regarding 

 these forms. 



Avidin, 82, 83> 84 a protein constituent of egg white, appears to combine 

 stoichiometrically with biotin to form a heat-labile complex. Avidin- 

 biotin constitutes one of the combined forms of biotin, but the distribution 

 of avidin is limited, so far as is known, to eggs and oviduct tissues, 85 and 

 so from the standpoint of general occurrence this form can be of only 

 minor importance. The more abundant forms of combined biotin differ 

 from avidin-biotin in that they are not dissociated in hot water. 



Biotin unquestionably is associated with protein enzymes as a coenzyme 

 or portion thereof, as will be discussed in a later section, but knowledge 

 regarding these biotin-containing enzymes is almost nonexistent. 



Extraction of biotin from various tissues indicates that it may not 

 always be combined in the same way. For example, exhaustive dialysis 

 of egg yolk (the original source of biotin used by Kogl and co-workers) 

 followed by cold-water extraction yields a nondialyzable form which is 

 active for yeast. Dialysis of liver tissue, on the other hand, does not render 

 the biotin extractable by cold water, though it could be released by 

 enzymes. 81 Probably the most widely used extraction method is to auto- 

 clave the material with 6iV sulfuric acid for 2 hours at 15 lbs. pres- 

 sure si, so, 87. j n mos t cases this yields the maximum amount of biotin. 

 Prolonged autoclaving under these conditions causes appreciable destruc- 

 tion. Autoclaving with 18./V sulfuric acid for 2 hours caused 20-40 per 

 cent destruction. 



This method of extraction cannot be accepted as universally the best 

 since in some cases, particularly plant materials, there is considerable 

 destruction. 8, 8S This difference in behavior may not be due to differences 

 in combination but to the presence of substances in certain extracts which 

 may, under the conditions used, interact with biotin. Incidentally, it may 

 be remarked that the term "heat lability" as applied to vitamins is a very 

 uncertain and indefinite one, inasmuch as the substances with which a 

 vitamin is heated may be fully as important in determining rate of 

 destruction as is the temperature of heating. 



