BIOLOGICAL ESTIMATION 



The salt mixture consists of : NaCl, 23-4 ; MgS04 . 7H2O, 24-6 ; 

 Nas HPO4, 35-8 ; K2HP04,69-6; Ca(H2P04)2, 68-8 ; calcium lactate, 

 15-4 ; iron citrate, 6-o ; KI, 0-2 parts. 



Each rat receives daily, in addition, 3 to 5 drops of cod liver oil to 

 provide vitamins A and D and i ml. of autoclaved yeast extract to 

 provide vitamin Bg complex. The rats are placed in separate cages 

 with wire screens to prevent access to the faeces. Growth continues 

 for two to three weeks and then stops. When the weight has been 

 stationary for not less than five days or has begun to decline, the rats 

 are divided into two groups. Each rat of one group receives daily, 

 for four weeks, 10 mg. of the substance to be tested and each rat of 

 the other group, i unit of the Standard Preparation. This was 

 formerly a fuller's earth adsorbate kept at the National Institute for 

 Medical Research, Hampstead (now Mill Hill), and the Unit of Anti- 

 neuritic Activity (Vitamin BJ for Great Britain and Northern Ireland, 

 which was the same as the international unit, was defined as the specific 

 antineuritic activity contained in 10 mg. of the Standard Preparation. 

 In 1945, after the pure vitamin had become available, the Standard 

 Preparation was changed to a specimen of pure crystalline sjmthetic 

 aneurine hydrochloride now kept at Mill Hill, and the unit was defined 

 as the specific antineuritic activity contained in 3-125 [xg. of the 

 Standard Preparation (Seventh Addendum to the British Pharma- 

 copoeia 1932, Appendix XXIII). 



The average increase in weight of the rats is determined for each 

 group. If the average increase in weight is approximately the same 

 for both groups, the vitamin B^ activity of the test substance is equal 

 to that of the standard. If the increase in weight in the group re- 

 ceiving the test substance is less or greater than in the group receiving 

 the standard, the test is repeated, using a larger or smaller dose of the 

 test substance. Alternatively, for the first trial, two doses of the 

 substance being tested may be given and fourteen rats may be used. 

 In each trial there should be at least two rats receiving no dose ; these 

 should show a gradual decline in weight, ending usually in convulsions 

 caused by vitamin B^ deficiency. In an experiment in which five 

 rats receive the Standard Preparation and five rats receive the test 

 substance, and in which the mean responses are equal, the limits of 

 error (P = 0-99) are 65 and 154 %. 



Another method of assessing the response of vitamin Bj-deficient 

 rats to graded doses of aneurine was suggested by K. H. Coward and 

 B. G. E. Morgan,^^ who observed that the oestrus cycle was inter- 

 rupted in vitamin B^ deficiency, and that the time required to complete 

 three cycles in rats previously deprived of aneurine was inversely 

 proportional to the daily dose of vitamin B^. The method is not of 

 practical importance, however, being less accurate than the growth 



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