26 Samuel Gurin and Roscoe O. Brady 



methyl carbons of acetate, change from 18:12 to 15:12. We have 

 shown by the use of doubly-labelled acetate ^^CHg^^coOH, that the 

 ratio of these two carbon atoms in cholesterol is equal to 1-25, or 

 15:12. It appears to us that a mechanism which involves the forma- 

 tion of cross-linkages in a folded chain would, on the basis of present 

 evidence, not be unreasonable. I may further mention that squalene 

 has recently been shown to be much more widely distributed than was 

 originally assumed. It has now been isolated from human sebum, and 

 therefore the possibility that the function of this hydrocarbon is that of 

 a precursor for the steroids would appear to be worth considering. 



Dauben: It is very interesting to hear about the role of squalene in 

 cholesterol synthesis. Dr. Chaikoff and I in Berkeley have arrived at 

 the same conclusion from experiments using dimethylacrylic acid in the 

 slice and in the intact animal. We have gone one step farther than Dr. 

 Bloch. We have synthesized radioactive squalene and now have it in 

 the animal to find out if the specifically labelled squalene will come out 

 with the right distribution of marked carbons. For example, if you 

 label it properly and it folds around you should either get 4-C or 6-C 

 labelled cholesterol. 



Rittenberg: We have been interested in problems quite similar to 

 those Dr. Gurin has just reported on. We have attempted to investigate 

 the role of a more highly reduced Cg unit than acetate, and have chosen 

 ethyl alcohol as a possible substrate (Curran, G. L., and Rittenberg, 

 D., 1951, J. biol. Chem., 190, 17). We tried ethyl alcohol because we 

 thought it was a much more pleasant compound to incubate with a 

 tissue slice than acetaldehyde. We wanted to know whether acetalde- 

 hyde is utilized in cholesterol synthesis by some direct route or whether 

 it is first converted to acetate. Of course, such problems cannot be 

 attacked with ^^C labelling and instead we labelled the a carbon of 

 ethyl alcohol with deuterium. We found that while the carbon atoms 

 are utilized, neither in the in vitro nor the in vivo systems does the 

 deuterium of ethyl alcohol yield deuterio cholesterol. We therefore 

 consider it unlikely that ethyl alcohol or acetaldehyde can be inter- 

 mediates. 



Curran in my laboratory has been working with carbonyl labelled 

 acetoacetate and he finds results similar to those reported by Dr. Gurin, 

 that is, that acetoacetate is utilized for cholesterol formation. However, 

 by a procedure very similar to that which Dr. Gurin reports he finds a 

 very considerably higher conversion of acetoacetate to acetate, of the 

 order of 5 per cent conversion. Of course this raises the question whether 

 the utilization goes by way of acetate, because a 5 per cent conversion 

 is more than adequate to supply all the Cg units required for cholesterol 

 synthesis. He has therefore carried out a dilution experiment to test 

 whether acetate is the intermediate, by incubating i*C-labelled acetone 

 and normal acetate. For comparison, let us consider a similar experi- 

 ment using carbonyl labelled acetone, which goes very effectively to 

 acetate. If you incubate one equivalent of acetone with one equivalent 

 of normal acetate you dilute the ^^C activity of the cholesterol by a 

 factor of about 10 or 20, indicating very strongly, as Dr. Bloch's work 



