244 ASCORBIC ACID 



A further quantitative bioassay method has been worked out, based 

 upon the observation that the level of serum "alkaline" phosphatase, i.e., 

 the enzyme Avith an optimum activity at pH 8.6 to 9.0, falls considerably in 

 scurvy and increases again when ascorbic acid is administered.'^ A curative 

 rather than preventive procedure is recommended, owing to the more con- 

 sistent behavior of the animals. Consequently, the procedure adopted is 

 first to reduce the level of serum "alkaline" phosphatase in the experimental 

 animals to about 4 to 5 units, and then to administer doses of the test 

 sample at varying ascorbic acid levels. The serum phosphatase is deter- 

 mined at various intervals from 3 to 15 days, and the ascorbic acid content 

 is determined on the basis that 0.225 mg. of ascorbic acid per day is the 

 critical dose for a 5-day response, and 0.20 mg. for a 10-day response. 



Apart from the precautions which normally have to be observed in the 

 biological assay of any vitamin, special care has to be taken when es- 

 timating the ascorbic acid content of fresh fruit and vegetables, in order 

 to ensure uniformity of the sample dosed. For instance, a constant source of 

 supply of the material under test is necessary in order to minimize the 

 effect of varietal, soil, and other conditions on the vitamin (see p. 261), 

 and in order to avoid possible storage effect only freshly harvested tissue 

 should be used. Uneven distribution of the vitamin in one individual fruit 

 or vegetable (see p. 261) can usually be overcome by cutting a wedge 

 from each of several units; and, to avoid enzymic oxidation of the vitamin, 

 each wedge should be fed immediately after cutting. Cutting and mixing 

 the full weight of sample before dosing should be avoided. When the effect 

 of household cooking is being studied, the greatest care is needed to repro- 

 duce exactly the conditions of cooking from one day to another. If this 

 precaution is not taken the composition of the feed may not be constant. 

 However, with processed foods prepared on an industrial scale, it is usually 

 relatively easy to ensure uniformity of both the raw material and the 

 treatment to which it is subjected. 



B. CHEMICAL METHODS 



1. Preparation of Sample Extract 



One of the most important features in the chemical estimation of as- 

 corbic acid lies in the satisfactory preparation of the sample extract, and 

 this is especially true of plant tissue. The main aim of the analyst is to effect 

 complete extraction of the ascorbic acid in an unchanged form, i.e., without 

 incurring loss by oxidation, at the same time reducing to a minimum the 

 extraction of other substances which might interfere in the test reaction. 

 In addition, care has to be taken that the material extracted is representa- 

 tive of the sample under test — an important consideration in xiew of the 



' B. S. Gould and H. Schwachman, J. Biol. Chem. 151, 439 (1943). 



