214 THE VITAMINS 



of its antiscorbutic properties in 1 hour when exposed to air at room 

 temperature, while a solution of the same reaction kept for 24 hours 

 at room temperature in the absence of air showed no deterioration. 

 Zilva (1923a) reported no loss in activity in slightly acid juice after 

 storage for 3 months in an ordinary conical flask in an evacuated bell 

 jar. 



In later tests (Zilva, 1924a) the decitrated lemon juice was con- 

 centrated to one-tenth its volume, acidified by the addition of 7 grams 

 of citric acid to 1 liter of juice, and placed in 50 cubic centimeters 

 amounts in a series of flasks which were then evacuated, sealed, and 

 stored in a dark cupboard for 3 and 5 months, with no detectable loss 

 in potency. The concentrate was also used after 6 months' storage in 

 the treatment of a severe case of infantile scurvy. The treatment was 

 very intensive, from 2.5 to 6 drams being given every 3 hours during 

 the first 2 days and 1 dram every 6 hours on the following day. The 

 patient was discharged cured in 8 days, during which time the equiva- 

 lent of about 48 lemons had been administered. 



On subjecting decitrated lemon juice to fermentation with yeast in 

 the presence of carbon dioxide, Zilva (1924) was able to bring about 

 further concentration of the fraction containing the antiscorbutic vita- 

 min through destruction of the sugar present. The active fraction thus 

 obtained contained less than half the dry matter of the decitrated juice 

 and had a nitrogen content varying from 0.004 to 0.014 per cent. It 

 was said to give negative biuret, sulfur, and tryptophane tests, and a 

 faint murexide reaction. Precipitates were obtained with mercuric sul- 

 fate, basic lead acetate, and Millon's reagent and occasionally but not 

 invariably with phosphotungstic acid. The solution reduced ammoniacal 

 silver nitrate and gave a marked Pauly reaction. 



Zilva (1924c) next found that much of the inactive material could 

 be removed from decitrated lemon juice by precipitation with alcohol 

 and that the active material was precipitated by basic lead acetate. 

 His method at this time was essentially as follows : 



The lemon juice was first decitrated with excess calcium carbonate and the 

 filtrate concentrated in vacuo at 50° C. to about one-fifth its original volume. A 

 volume of alcohol equal to about one-half the original volume of juice was then 

 added gradually, the inactive precipitate filtered off and washed with a little 

 alcohol and the filtrate and washings distilled in vacuo at 50° C. to remove the 

 alcohol. The residue was then made up to the original volume with distilled water, 

 fermented as previously described, concentrated to small volume, and treated with 

 several volumes of absolute alcohol. After removing the inactive precipitate and 

 excess alcohol as before, basic lead acetate was added to complete precipitation, 

 the active precipitate quickly centrifuged out and dissolved in acetic acid. Mag- 

 nesium sulfate and absolute alcohol were added to remove the lead and other 

 inactive material and the filtrate finally distilled in vacuo at 50° C. 



