原文

A specific method for determination of total ascorbic acids in urine by the α,α´- dipyridyl method

Summary

Application of theα,α´-dipyridyl method for determination of ascorbic acid in urine is described.
The urine sample was acidified with trichloroacetic acid and shaken with activated carbon to remove interfering substances.
The acid filtrate was first neutralized (pH7.0) by adding Na2HPO4.
The dehydroascorbic acid was then reduced back to ascorbic acid by incubation with dithiothreitol.
After removal of the excess dithiothreitol with N-ethylmaleimide, ascorbic acid was determined by measuring the reduction of ferric ion.
the ferrous ion produced was coupled toα,α´-dipyridyl in the presence of H3PO4.
Ferrous ion in urine samples, which theoretically interferes with the method, was removed by a combination of Na2HPO4 and H3PO4.

Introduction

theα,α´-dinitrophenylhydrazine (DNP) method [1-4] which is most widely used for the determination of ascorbic acid (AsA) has some disadvantages.
It is complex and time consuming. For example, coupling DNP with dehydroascorbic acid (DAsA) requires a 3-h incubation, and dropwise addition of concentrated sulfuric acid is necessary to dissolve osazones.
in 1955, Sullivan and Clarke [5] described a method for assaying AsA which depends on the reduction of Fe3+ and estimation of the Fe2+ as a red-orangeα,α´-dipyridyl (DP) complex.
Other reducing materials are inhibited by the presence of H3PO4 and a high degree of acidity, pH 1-2. They applied the method to the determination of AsA in urine, orange juice and honey.
The method was improved by Maikel [6] and Zannoni et al. [7] who assayed AsA in animal tissues.
The author has reinvestigated these methods and found that they are comparatively sensitive and simple enough for many analyses but not specific for the estimation of urinary AsA because some urinary constituents cross-react.
The values obtained by the DP method were always higher than those obtained by the DNP method.
The urinary constituents capable of affecting the DP method can be removed by adsorption with activated carbon in the presence of trichloroacetic acid (TCA).
At this time AsA is oxidized to DAsA.
this oxidation means that the DP method cannot be used to estimate urinary AsA, but a simple reducing system of DAsA to AsA using dithiothreitol (DTT) and N-ethylmaleimide (NEM) was recently established by the author [8].
application of this system brought about as specific a method as the DNP method for determination of total urinary ascorbic acids (AsA + DAsA).