V. ESTIMATION 247 



unable to confirm the presence of this complex*' ■ le. 27. 47-50 ^^^^ have at- 

 tributed the apparent increase in both instances either to failure to inacti- 

 vate the enzyme (thereby causing loss by oxidation) or to incomplete ex- 

 traction of ascorbic acid or to lack of representative sampling. 



It is now generally accepted that either metaphosphoric acid or oxalic 

 acid is suitable for extraction in the assay of ascorbic acid, provided that 

 precautions are taken along the lines already discussed and that, in the 

 presence of interfering substances, the extract is suitably treated before 

 titration (see pp. 249-250). 



Having ground or l^lended the sample with acid, the slurry is either 

 filtered or centrifuged, the latter procedure being necessary Avhen quan- 

 tities of colloidal substances are present, as, for example, vnth. cooked 

 potatoes. Complete extraction of ascorbic acid is essential. Pressing of the 

 residue through muslin before filtration has therefore been advised,^ and 

 Avashing of the residues is similarly to be emphasized.^^ The ascorbic acid 

 present in the extract is then determined by one or other methods to be 

 described. 



For animal tissues in general, hand grinding with sand and concentrated 

 metaphosphoric acid has been found satisfactory. Special treatment, how- 

 ever, is necessary with some samples, and with blood, for instance, lithium 

 oxalate is added to prevent clotting. The plasma obtained from the oxalated 

 blood is at once deproteinized A\dth metaphosphoric acid and assayed.'^ 

 In collecting urine for estimation of vitamin C, a small amount of glacial 

 acetic acid or metaphosphoric acid is added to retard loss by oxidation, 

 and the subsequent titration is then generally carried out without further 

 treatment, except for such dilution or addition of acid as may be necessary. 



2. Estimation of Ascorbic Acid in Sample Extract 



Numerous chemical methods have been suggested for the determina- 

 tion of ascorbic acid, the majority being based upon colorimetric changes 



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" B. C. Guha and J. C. Pal, Nature 137, 946 (1936). 



« B. C. Guha and P. N. Sen-Gupta, Nature 141, 974 (1938). 



« E. J. Reedman and E. W. McHenry, Biochem. J. 32, 85 (1938). 



" H. Scarborough and C. P. Stewart, Nature 142, 40 (1938). 



« F. De Eds, Food Research 8, 275 (1943). 



*^ W. K. Schwarze and K. Gunther, Pharmazie 4, 161 (1947). 



^fi" W. N. Sumerwell and R. R. Soalock, J. Biol. Chcm. 196, 753 (1952). 



'""' Z. Prochazka and S. Koristek, Collection Czechoslov. Chem. Communs. 16, 65 



(1951). 

 « G. L. Mack, Nature 138, 505 (1936). 



« G. L. Mack and D. K. Tressler, /. Biol. Chem. 118, 735 (1937). 

 « M. Hochberg, D. Mehiick, and B. L. Oser, Ind. Eng. Chem. Anal. Ed. 15, 182 (1943). 

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