Variation of pH of clinical samples as a source of error in enzyme determinations.

IN THE DETERMINATION of enzymes in serum, blood or urine, the sample is added to a buffer designed to bring the pH after admixture to the peak of the enzyme activity curve. Body fluids themselves, however, are buffered and the pH of each fluid, even when fresh, can vary within certain limits, e.g., blood can vary from about pH 7 to 7.8 and urine from about pH 4.6 to 8. In. addition, samples of blood, serum and urine in contact with air lose C02, resulting in an increase in pH. Figure 1 shows the change in. pH with time of two of many sera studied at room temperature (25#{176}), in the refrigerator (4#{176}) and in the frozen state (-10#{176}). The rapidity with which the pH increase occurs is dependent on several factors, including the volume of fluid, the surface area and the amount of agitation of the fluid during storage. It is seen that a pH increase of 1 unit can occur in serum. To illustrate the possible magnitude of errors in certain tests due to variation in sample pH, determinations were performed for alkaline phosphatase by the method of Shinowara, Jones and Reinhart (1). The pH activity curves for serum alkaline phosphatase, run on sera with normal as well as elevated enzyme concentrations, consistently showed an optimum at approximately pH 9.7, which is significantly higher than the optimum of pH 9.3 reported by Shinowara, et at. (1). The pH readings were taken on a Beckman model G pH meter at 37#{176}, the temperature employed in the incubations.