Determination of Reduced Ascorbic Acid in Small Amounts of Blood

Abstract

We previously presented data as to the quantity of reduced ascorbic acid in blood plasma and showed its dependence upon the dietary supply of vitamin C, or of ascorbic acid administered as a medicament. The data were obtained by deproteinizing plasma by means of tungstic acid and titrating immediately the reduced ascorbic acid present with 2:6 dichlorobenzenoneindophenol. In working with infants, and when making repeated observations on the same individual, it is desirable to have a method which requires a minimum of blood. We have, therefore, developed a micro-method requiring but 0.3 ml. blood, which may readily be secured from the finger of an adult, or the heel of an infant. In the guinea pig, blood is obtained by cardiac puncture, or sometimes by incision of the marginal vein of the ear. In the latter case, a special blood pipette is required. Collection of Blood. Collect approximately 0.3 ml. blood from a lancet wound, into a small phial (Fig. 1, B) containing sufficient powdered potassium oxalate to prevent coagulation. Stir immediately with a toothpick or small glass rod. The phial is made by sealing the end of a piece of glass tubing of approximately 10 mm. outside diameter, and, while hot, pressing it against a piece of iron to form a flat base. The tubing is cut off at about 15 mm. from the closed end. The cut end is then ground flat on a fine stone wheel and the outer wall ground to a taper for a distance of 5 mm. from the top. The grinding produces a thin edge which greatly facilitates the collection of blood from the heel of an infant. It is unnecessary to grind the side wall if the phial is used for finger blood only (which is permitted to drop directly into the phial as the finger is milked), or if Mood is transferred from a syringe. The phial is stoppered (Stopper A), then placed in a suitable holder (a cork with a recess cut in it) and lowered into the metal tube of a centrifuge. It is centrifuged for a few minutes in the usual manner.