Determination of Phenol Red in Gastric Contents. ∗

Abstract

A dilution indicator in gastric analysis is a substance employed for determining quantitatively the proportion of residual test meal present in a sample of gastric contents. Of the numerous substances so employed, phenolphthalein and phenol red (P.R.) are the most common. The former, however, is not suitable for the purpose because of its low solubility in water (Hollander, Penner, and Saltzman 1 ), but the latter possesses most of the characteristics requisite for an ideal dilution indicator (Gorham, 2 Bulger, et al. 3 Wilhelmj, et al. 4 ). A method for determining P.R. in the presence of bile and protein suspensions is described by Wilhelmj. His procedure is unsatisfactory because: (1) it involves a subjective color correction previous to a colorimetric determination, (2) in the presence of dark green bile even this correction is impossible, (3) it necessitates the preparation of a different standard of comparison for each bile-containing specimen, (4) it does not include a standardized technique for adding the (variable) volume of color-correcting solution to the colorimetric standard without introducing an indeterminate change in the P.R. concentration. Furthermore, no data are cited to indicate its quantitative reliability.

In order to eliminate these faults, we have developed a simple and reliable method for the simultaneous removal of bile pigment and protein without loss of P.R. by means of freshly precipitated Zn(OH)₂. Following preliminary centrifuging, 5 ml. of the P.R. solution are treated with finely powdered CaO, sufficient to neutralize the gastric HC1 and make the solution distinctly alkaline; then one ml. each of NaOH (0.5 N) and ZnSO₄ (0.55 N) are added (the latter dropwise), mixed thoroughly, and centrifuged after standing for IS minutes; finally, 5 ml. of the supernatant fluid are treated with 2 ml of Na₃PO₄ (0.5 N) to remove excess Zn and simultaneously to adjust the pH to a value suitable for colorimetry. After centrifuging, the supernatant liquid is filtered with suction and compared in the colorimeter with a standard P.R, solution (the test meal itself) similarly treated. Color differences between unknown and standard were practically non-existent. Comparison of treated with untreated standard solutions showed that the Zn treatment causes no significant loss of P.R. We have already run several dozen determinations on P.R. solutions in egg albumin, gastric pouch secretion from dogs, and human stomach contents; the concentration of gastric fluid varied from 25% to 95% and of P.R. from 0.2 mg. to 4.0 mg. per 100 ml. of mixture. Each solution was prepared from a different specimen of gastric contents; at least half of these were thick with dark green bile. In practically every case observed and calculated results agreed to within 0.04 mg. per 100 ml.; this corresponds to 1% of the initial concentration of the indicator in the test meal, which is accurate enough for all clinical and experimental purposes.

Concerning the pH to which the P.R. must be adjusted for color-imetry: Wilhelmj uses one ml. of saturated NaOH to 6.5 ml. of P.R. solution, which is equivalent to a final concentration of about 1.8 N NaOH. It is generally known, however, that P.R. will lose its color very rapidly in the presence of strong alkali (Thiel 5 ) and we have found that this color loss may be appreciable even in one N NaOH (pH about 14) after one-half hour. In Wilhelmj's procedure, therefore, unless the solutions are alkalinized only a short time before being read and fresh portions of standard solution are prepared at short intervals, there is a great likelihood of error due to this progressive loss of color. On the other hand, we have found that at pH 11-12 no appreciable loss of color occurs, even after 4 hours; also, by colorimetric comparison of P.R. solutions at pH's 11 and 12 and by examination of the pH-dissociation curve (Clark, 6 p. 58), we have shown that the indicator attains its maximum degree of color in this same pH range. Since the pH of Na₃PO₄ solutions is likewise around 12, it follows that our P.R. solutions require no further alkali after the addition of excess phosphate for removal of residual Zn.