Bone alkaline phosphatase, heat inactivation

Our data support the statement that the heat sensitivity of each enzyme source remains characteristic and independent of the influence of the others in the mixture, and that the resultant heat inactivation is an additive function of the heat-sensitivities of members of the mixture. Bone enzyme from different sources is very consistently heat-sensitive (85-90%), unlike intestinal (50-65%), and liver enzyme (50-75%). However, the heat sensitivity of the LPSAP of normal serum can vary from 33 to 85% and of the non-LPSAP fraction from 50 to 95%. Therefore one cannot determine the identity of the organ sources of serum alkaline phosphatase with a knowledge of only the heat sensitivity and the total alkaline phosphatase. However, by correcting the heat-inactivation of serum by that contributed by intestinal component, one obtains the heat-inactivation of non-intestinal sources of alkaline phosphatase. If this value is 90% or more, the non-intestinal component could be presumed to be of osseous origin if 60% or less, of hepatic origin. 

Measurements op Bone and Liver Alkaline Phosphatase Based on Heat Inactivation"... 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

The application of the heat-sensitive measurement by Posen et al. (P19, P20) has been restricted to sera of patients with uncomplicated liver or bone disease. Here, sera with high inactivation of alkaline phosphatase are interpreted as bone and those that are relatively heat-stable as liver. Particularly disturbing to us were sera from patients with cirrhosis of the liver (patients 5, 6, and 12, Table 13) exhibiting heat sensitivity of non-LPSAP in the bone range. The measurement of heat sensitivity of serum alkaline phosphatase alone cannot be used for the certain identification of liver or bone sources or their mixtures. 

In the present state of knowledge and in the absence of information of the patient s diagnosis, one cannot yet expect to correctly identify a serum as mostly bone or mostly liver in origin with respect to its alkaline phosphatase by heat inactivation or L-phenylalanine inhibition. With the inclusion of additional studies, such as starch-gel electrophoresis, neuraminidase treatment, and continued study over a period of time, one can increase the certainty of the interpretation of the origin of the serum alkaline phosphatase in patients. 

The other approach is the selective inactivation of specific isoenzymes. Placental alkaline phosphatase, for instance, is remarkably stable to heat inactivation. Incubation of the enzyme at 65 °C has no effect on its activity, unlike the other isoenzymes which are inactivated. Other isoenzymes can be differentiated by their stability in other conditions. For instance phenylalanine inhibits placental and intestinal isoenzymes but has little effect on the bone and liver isoenzymes. 

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