ESTIMATION 



blank. 31 The method was still farther improved by adsorbing the 

 nicotinic acid on Lloyd's reagent and eluting with dilute alkali as in 

 the method of Dann and Handler ; the values thus obtained were in 

 very close agreement with microbiological assay results obtained 

 using L. arabinosus.^^ 



Estimation of Nicotinic Acid in Blood 



The Konig reaction was used for the estimation of nicotinic acid in 

 blood by E. Kodicek/^ by Klein et al.^^ and by M. J. C. Allenson.^* 

 The last-named used an enzyme from certain soil bacteria to eliminate 

 interference from other chromogens, the difference between the colours 

 produced with cyanogen bromide and metol before and after incuba- 

 tion with the enzyme preparation being assumed to be proportional 

 to the nicotinic acid content of the blood. B. D. Kochhar ^^ de- 

 proteinised the blood with trichloroacetic acid and hydrolysed the 

 filtrate with hydrochloric acid. 



Estimation of Nicotinic Acid in Urine 



The estunation of nicotinic acid in urine is complicated for two 

 reasons. In the first place, urine contains pigments and chromogens 

 that interfere with the measurement of the coloured compound pro- 

 duced from nicotinic acid. The problem of eliminating interference 

 from this source was solved by Y. L. Wang and E. Kodicek,^^ who 

 extracted the urine with isobutanol, after digestion with acid, and 

 then treated the extract with potassium permanganate solution to 

 oxidise residual impurities. Kodicek's original method ^^ was found 

 to be unsatisfactory for the assay of urine, but almost theoretical 

 recoveries of nicotinic acid added to urine were obtained when a lower 

 temperature was used, together with a shorter time for the reaction 

 between cyanogen bromide and nicotinic acid, and larger amounts of 

 _^-aminoacetophenone . 



The other reason why nicotinic acid assays of urine are compli- 

 cated is that, as already stated (page 220), several forms of nicotinic 

 acid exist in urine, most, but not all, of which are derived from in- 

 gested nicotinic acid or nicotinamide. 



Urine assays are usually carried out with the object of assessing 

 nutritional status (see page 259), that is, the extent to which ingested 

 nicotinic acid is utilised by the body. In this event, not only must 

 the amount of nicotinic acid and nicotinamide in the urine be esti- 

 mated, but also the nicotinuric acid, trigonelline, N^-methylnicotin- 

 amide, N^-methyl-6-pyridone-3-carboxyl amide and other meta- 

 bolites into which they may have been converted diu-ing their passage 



223 



