262 THE VITAMINS 



laboratories in this respect. It is probably because of the complete 

 deprivation and consequent early death of our animals that the lung 

 trouble did not show itself in them. Where the diet is deficient in, 

 rather than devoid of, this vitamin, we fully confirm the findings of 

 Steenbock and Nelson that vitamin A deficiency leads to marked in- 

 crease in the incidence of lung disease. 



For quantitative estimates of relative amounts of vitamin A in 

 which the highest degree of accuracy is desired it is necessary to con- 

 duct the comparative studies in strict parallel with enough experiments 

 to eliminate the effect of individual variability, and with rigid control 

 of all factors such as the previous nutritional history, the weight and 

 sex of experimental animal, litter variability, and rate of growth during 

 the test period. So far as possible, rats of the same litter should be 

 used for those tests the results of which are to be most directly com- 

 pared with one another. Rats from one litter behave more uniformly 

 than a miscellaneous selection even though the latter appear to be in a 

 similar condition. 



There is clearly a tendency toward an inverse relationship between 

 the body weight of the animal and its rate of gain upon a given limited 

 allowance of vitamin A. Undoubtedly, too, this may also be stated 

 conversely; i.e., that among animals of the same species and age, the 

 amount of vitamin A required to support a given rate of gain tends 

 to vary with the size of the individual. This corresponds with the 

 similar relationships previously established in the case of vitamin B. 

 Therefore, only such animals as do not differ greatly among themselves 

 in size at the end of the depletion period should be used. For condi- 

 tions such as those of our laboratory, a minimum limit of 70 or 75 

 grams and a maximum limit of 100 grams at the end of the depletion 

 period would seem to be desirable. 



The early experience of several workers, which indicated that male 

 and female rats may be used interchangeably in determinations of vita- 

 min A by the method here employed, with gains of about 3 grams per 

 week in the test period, has now been confirmed by the data of large 

 numbers of experiments made in such manner as to permit of direct 

 comparison of the quantitative results yielded by the two sexes. Thus the 

 average gain shown during an 8-week test period by 148 males was 

 24.6 grams, and by 148 directly comparable females was 22.3 grams. 

 Statistically, the average difference of 2.3 grams (or slightly less than 

 0.3 gram per week) is probably, though not certainly, significant. Prac- 

 tically, it seems so small as to be safely negligible, although for investi- 

 gations in which an extraordinary degree of refinement is sought it 



