BIOASSAY OF ESSENTIAL FATTY ACIDS 795 



the bioassay of vitamin A, there are several difficulties in the application of 

 the test to the EFA, In the first place, the period of twenty weeks required 

 for the depletion and assay periods is excessive. The diet, which contains 

 vitamin-test casein, is expensive. Finally, the amount of the test substance 

 which must be administered to ensure that a sufficient dose of EFA will 

 be given may be so great that it constitutes a serious difficulty in the feed- 

 ing. Deuel et alJ^ fed the margarine and butter supplements in daily doses 

 of 250 and 500 mg. The administration of this amount of fat in the test 

 groups was compensated for in the control linoleate tests by giving pro- 

 portionate doses of hydrogenated coconut oil. Since the hydrogenated 

 coconut oil was found to decrease the period of depletion, although the 

 growth effect during the assay period was counteracted by linoleate, Deuel 

 and co-workers-^ consider that the absolute results from the assay may be 

 open to some question. It would now seem preferable to use linoleate- 

 free, partially hydrogenated triolein as the compensating fat in these bio- 

 assays. 



(2) Growth Method of Thomasson Based upon Rationing of Water 



The new weight increase method proposed by Thomasson'^ is based upon 

 the disturbed water metabolism which occurs in EFA deficiency. As 

 early as 1930, Burr and Burr^ reported that rats on fat-free diets drink more 

 water than normal animals do. In the new bioassay procedure, only 

 weanling male rats are used. Animals weighing approximately 40 gm. 

 at weaning, from mothers who had received the Sherman diet, were trans- 

 ferred to a fat-free diet. During the first two weeks on the diet, the water 

 intake of the rats was not limited ; after that the intake of water was limited 

 to 14 ml. per rat per day for the rest of the depletion period and during 

 the period of bioassay. The preparatory depletion period lasted for five 

 weeks (three weeks with water rationing); after this, some groups of ani- 

 mals were dosed with appropriate levels of linoleate, and other groups 

 received the test substance five times per week during the four-week bio- 

 assay period. The linoleate and test substance were given in doses made 

 up to a total volume of 0.2 ml. with hydrogenated coconut oil. The 

 determination of the quantity of essential fatty acids was based upon the 

 slope of the gain-in-weight/log dose curve obtained, compared with that for 

 the standard. Instead of using linoleate directly as a standard, Thomas- 

 son'^ preferred to employ sunflowerseed oil in doses of 10 and 50 mg. One 

 Unit of EFA (also referred to by Thomasson as vitamin F) is defined as the 

 activity of 10 mg. of linoleic acid. Thus, one Unit corresponds almost 



