Reis (1934, 1940, 1951) has demonstrated the presence of alkaline phosphatase in various tissues specifically hydrolysing 5-nucleotidase such as adenosine and inosine-5-phosphoric acids. The enzyme has its optimum action at pH 7.8 and in all human tissues except intestinal mucosa its activity, at the physiological pH range, is much more pronounced than that of the non-specific alkaline phosphatase. Thus ossifying cartilage, one of the classical sites of high alkaline phosphatase activity, although very active against say phenyl phosphoric acids at pH 9, is more active against adenosine-5-phosphoric at pH 7.5. This finding suggested to Reis that the enzyme might play a part in calcification mechanisms. Studies designed to elucidate the physiological significance of phosphatase are frequently based on measurements of enzyme activity in blood serum or plasma, and there is an enormous literature dealing with such variations caused by different pathological conditions. It seemed, therefore, of interest to determine the activity of both serum-5-nucleotidase and non-specific alkaline phosphatase in a number of disease states.Reagents Glycerophosphate Buffer Substrate pH 9.3.-Sodium ,'glycerophosphate, 0.05 g., sodium barbitone 0.424 g. made up to 100 ml. with water, adjusted to pH 9.3 and stored in the cold.Glycerophosphate Buffer Substrate pH 7.5.-As above except that the pH is adjusted to 7.5 by adding approx:-mately 1.2 ml. N HCI.Adenosine-5-phosphate Buffer Substrate pH 7.5. Adenosine-5-phosphate, 0.087 g., and sodium barbitone, 0.424 g., made up to 100 ml. with water and the pH adjusted to 7.5 by adding 1.2 ml. N HCI and stored in the cold.Aminonaphtholsulphonic Acid Reagent.-Fifteen per cent. sodium bisulphite, 195 ml., 1,2,4-aminonaphtholsulphonic acid, 0.5 g., 20% sodium sulphite, 5 ml., stoppered and shaken until dissolved and stored in the cold and used within four weeks.Molybdate Reagent.-Reagent grade ammonium molybdate, 25 g., dissolved in about 200 ml. water. In a I-litre volumetric flask place 300 ml. 10 N H2SO, add the molybdate solution, and dilute with washings to 1 litre with water.Standard Phosphate Solution.-KH2PO4 0.02195 g., and 50 g. trichloracetic acid, made up to 1 litre. This solution contains 0.02 mg. P in 4 ml. MethodsNon-specific alkaline phosphatase activity is high at pH 9 towards phenylphosphate adenosine phosphate and glycerophosphate, in this decreasing order (cf. Reis, 1951). At pH 7.5, however, the total phosphatase activity is equally low towards phenyl-or glycero-phosphate -nd the higher activity towards adenosine-5-phosphate at this reaction is reasonably inferred to be due to the specific 5-nucleotidase. Serum 5-nucleotidase activities were thus measured by subtracting the nonspecific phosphatase activity with glycerophosphate as substrate at pH 7.5 from the total activity with adenosine-5-phosphate at 7.5. Serum non-specific phosphatase activities were determined by incubation with glyxcrophosphate at pH 9.3. Thus for the test solutions ,.2 rpl. serum was mixed with 4.5 ml. o...
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1. A metabolic study in a case of myositis ossificans progressiva is reported. 2. The serum showed an increased power of calcification of rachitic rat cartilage. 3. Estimations of alkaline phosphatase showed slightly raised values. 4. Surgical removal of a bony bar was followed by prolonged administration of ACTH and cortisone, but no effect on the calcium-phosphorus balance or on the re-ossification within the area of operation was observed.
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