PYRIDOXINE 



A modified vitamin Bg-deficient diet for rats was devised by 

 Sarma et al.^ This was based on sucrose and blood-fibrin and, when 

 supplemented with up to 75 /xg. of pyridoxine hydrochloride per 100 g. 

 of diet, gave a linear growth curve. Pyridoxal and pyridoxamine 

 had the same activity as pyridoxine when given separately by mouth 

 or when injected intraperitoneally, but both exhibited reduced activity 

 when given with the diet. Because of this, the results obtained when 

 the method was applied to natural materials were somewhat lower 

 than the results obtained by the yeast growth method (page 311). 



Colorimetric Methods of Estimation 



R. Kuhn and I. Low ^ were the first to describe a colour reaction 

 for pyridoxine. They observed that diazotised sulphanilic acid 

 coupled with the vitamin to give an orange-coloured dyestuff and 

 that a colour was obtained by treating pyridoxine with phosphotung- 

 stomolybic acid reagent and lithium hydroxide. 



M. Swaminathan ^ made use of the colour reaction with diazotised 

 sulphanilic acid to estimate pyridoxine in foodstuffs. The material 

 was first digested with pepsin or papain, and the protein degradation 

 products were removed with phosphotungstic acid, and the purine 

 and other bases by means of silver nitrate and baryta. The pyri- 

 doxine was then adsorbed from the filtrate, acidified to pH i to 2, 

 on to Clarit, from which it was eluted with hot baryta solution. After 

 neutralising the eluate, the colour was developed and compared with 

 that of a standard. The method was used to estimate pyridoxine in 

 urine. 



This colour reaction was also used by Bina et al,^ though they 

 used a somewhat different method of preparing the pyridoxine extract. 

 The food was first hydrolysed by autoclaving with dilute sulphuric 

 acid and was then digested with a mixture of takadiastase and papain 

 after which proteins and other interfering substances were precipi- 

 tated with sodium tungstate. The filtrate was then treated with 

 Saperfiltrol and the adsorbed pyridoxine eluted with alkaline alcohol. 

 These workers subsequently ^° modified the method by using the ion- 

 exchange resin, Amberlite IR-4, to purify the solution, and diazotised 

 ^-aminoacetophenone in place of diazotised sulphanilic acid to develop 

 the colour. 



Another colour reaction was discovered by J. C. Keresztesy and 

 J. R. Stevens,^^ who observed that ferric chloride gave a reddish 

 brown colour with vitamin Bg ; R. D. Greene ^^ rnade use of this 

 reaction to estimate the vitamin in rich concentrates. 



J. V. Scudi and his colleagues ^^ made use of Gibbs' reagent,^* 

 2 : 6-dichloroquinone-chloroimide, to estimate the vitamin in urine. 



310 



