Determination of Reduced Ascorbic Acid in Blood

Summary

The ascorbic acid value of blood is materially affected by standing either in the ice-box or at room temperature. Determinations should be carried out within one-half hour after the collection of the blood.

Since the appearance of the clinical methods for determining blood ascorbic acid by Farmer and Abt 1 , 2 considerable interest has been aroused in the study of vitamin C in various diseased conditions. The determination depends on the following proceduure: Deproteinization of the plasma by 10% metaphoric acid 3 and titration of the reduced ascorbic acid present in the filtrate with 2:6 dichlorophenol indophenol. 4

By the use of this method we have obtained high and low ascorbic acid values in presumably normal subjects, or at least in individuals in whom there was no reason to suspect vitamin C deficiency. Furthermore, determinations on the same subject within 2 or 3 hours yielded various blood ascorbic acid values. In all these determinations great care was exercised to avoid the presence of disturbing catalysts. 5

Duplicate and triplicate readings on the sample of plasma by the same investigator or by another, yielded diverse amounts of vitamin C present. Naturally, this difficulty aroused our suspicion as to the accuracy of the method for clinical purposes. Our first thought was that the error lay in observing the true endpoint of the titration due to the oxidation of the dye. Titrations were then carried out by the use of a photometer and the pink endpoint brought to the same intensity in all cases. Even this failed to yield consistent figures where the plasma had been collected some time previous to the experiment. We particularly noticed that samples of blood plasma which gave certain readings at one time, failed to give the same readings 2 or 3 hours later. The common practice of keeping blood, either in the ice-chest or at room-temperature, for various lengths of time was investigated. Two hundred cubic centimeters of blood were withdrawn from a normal individual, oxalated, and plasma separated from the cells. The plasma was divided into 2 portions, one maintained at 26°C. and the other at 0°-5°C. Titrations were carried out hourly, and on a subsequent occasion the experiment was carried out at 10- to 15-minute intervals for 2 hours. In each instance a fresh solution of 10% metaphosphoric acid was carefully made up. The dye-values were repeatedly checked with distilled water and acetic acid for the blank readings. The actual titrations were started within one-half hour from the time the blood was drawn from the patient.