4-nitrophenyl phosphate--characterization of high-purity materials for measuring alkaline phosphatase activity in human serum.

1981 ◽  
Vol 27 (1) ◽  
pp. 135-143 ◽  
Author(s):  
G N Bowers ◽  
R B McComb ◽  
A Upretti

Abstract We studied 53 lots of 4-nitrophenyl phosphate (I), obtained from 20 different commercial suppliers, and used this information to set specifications for it. Using these well-defined specifications, we classified 21 lots of I as "unacceptable," 26 lots as "borderline," and six as "acceptable." All lots were shown to contain some 4-nitrophenol and inorganic phosphate. However, "acceptable" I had < 0.3 mmol of 4-nitrophenol and < 10 mmol of inorganic phosphate per mole of I. The mole concentration of I (based on disodium hexahydrate, formula weight 371) was determined by enzymic conversion to 4-nitrophenol in five lots of "acceptable" materials. The mole fraction of I ranged from 0.982 to 0.998. From these measurements and from estimates of impurities that absorb at 311 nm, as determined by liquid chromatography and spectrophotometry at other wavelengths, our best estimate of the molar absorptivity of I at 311 nm in 10 mmol/L NaOH at 25 degrees C was 9867 L x mol-1 x cm-1, with a total uncertainty of 76 L x mol-1 x cm-1. We recommend that I used in clinical laboratories for measurement of alkaline phosphatase activity in serum meet the specifications given in this paper: I content > 98%, maximum activity > 98% in comparative testing with other "acceptable" lots of I, and impurities not to exceed the values cited above.

1983 ◽  
Vol 29 (5) ◽  
pp. 751-761 ◽  
Author(s):  
N W Tietz ◽  
C A Burtis ◽  
P Duncan ◽  
K Ervin ◽  
C J Petitclerc ◽  
...  

Abstract We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.


1980 ◽  
Vol 26 (6) ◽  
pp. 724-729 ◽  
Author(s):  
G N Bowers ◽  
R B McComb ◽  
R G Christensen ◽  
R Schaffer

Abstract We describe specifications for high-purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase activity in human serum. Some lots of 4-nitrophenol available commercially met these specifications, but several did not. The latter can be purified to meet our specifications by recrystallization or sublimation. The molar absorptivity of 4-nitrophenol (35 μmol/L) IN 10 mmol/L NaOH at 25 °C at 401 nm is 18380 +/− 90 L.mol−1.cm−1.


1977 ◽  
Vol 23 (10) ◽  
pp. 1873-1877 ◽  
Author(s):  
K Tanishima ◽  
Y Minamikawa ◽  
N Yokogawa ◽  
M Takeshita

Abstract We studied the effectiveness of glycerol or ethylene glycol in preventing the increase in alkaline phosphatase activity of lyophilized or refrigerated quality-control serum after reconstitution or repeated freezing and thawing. Control serum reconstituted completely from the lyophilized state with subsequent storage at -20 degrees C showed a considerable decrease in alkaline phosphatase activity immediately after thawing, and a gradual increase in activity on allowing it to stand at room temperature. Adding glycerol or ethylene glycol to the reconstituted serum obviated these changes in activity, glycerol being more effective than ethylene glycol. Reconstituted serum with added glycerol maintained maximum activity before refrigeration during either storage for 30 days or on repeated freezing and thawing. Practical applications of this glycerol-supplemented control serum are discussed.


1981 ◽  
Vol 27 (10) ◽  
pp. 1729-1732 ◽  
Author(s):  
V Chromý ◽  
L Zahradnícek ◽  
J Voznícek

Abstract We describe a method for determining serum alkaline phosphatase activity with use of N-methyl-D-glucamine buffer, Na+ is a definite activator, whereas NH4+ and Li+ inhibit enzyme activity. Optimum reaction conditions are: methylglucamine buffer, 0.35 mol/L, pH 10.2 +/- 0.1 (30 degrees C); NaCl, 70 mmol/L; MgCl2, 0.5 mmol/L; disodium 4-nitrophenyl phosphate, 15 mmol/L; reaction temperature, 30 degrees C; reaction time, 2 min. The assay conditions are optimum for all human serum isoenzymes.


1965 ◽  
Vol 22 (3) ◽  
pp. 793-799 ◽  
Author(s):  
N. J. Antia ◽  
A. Watt

Evidence has been obtained for acid phosphatase activity (on p-nitrophenyl phosphate as substrate) at pH 4.8 in cell-free extracts of Phaeodactylum tricornutum, Skeletonema costatum, Cyclotella nana, Monochrysis lutheri, Isochrysis galbana, and Dunaliella tertiolecta grown photo-autotrophically in pure culture. No alkaline phosphatase activity at pH 10.5 was observed.


1976 ◽  
Vol 22 (10) ◽  
pp. 1737-1740 ◽  
Author(s):  
R C Elser

Abstract Thymol blue monophosphate is evaluated as a substrate for use in kinetic bichromatic analysis of serum alkaline phosphatase activity. The effect of abnormally high concentrations of hemoglobin, bilirubin, or chylomicrons is negligible. The expected range for ambulatory adults established by assaying sera from 206 blood donors, was 12-44 U/lliter at 37 degrees C. Comparison of the method with a commonly used p-nitrophenyl phosphate method yielded a correlation coefficient, r, of 0.9949. Advantages of the method are the reduced cost per test and the exceptionally stable substrate.


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