Alkaline phosphatase activity assay

Dyhrman, S.T. and Palenik, B.P. (1999) Phosphate stress in cultures and field populations of the dinoflagel late Prorocentrum minimum detected using a single-cell alkaline phosphatase activity assay. Applied and Environmental Microbiology 55, 3205-3212. 

Urinary alkaline phosphatase activity. II. An analytical validation of the assay method. JAMA (1963), 185, 953-957. 

In addition to the classical symptoms of zinc deficiency mentioned above, the following unusual conditions have been reported liver and spleen enlargement, abnormal dark adaptation and abnormalities of taste. Several laboratory procedures for diagnosing zinc deficiency are available. Measurement of zinc levels in plasma is useful in certain cases. Levels of zinc in the red cells and hair may be used for assessment of body zinc status. More accurate and useful parameters are neutrophil zinc determination and quantitative assay of alkaline phosphatase activity in neutrophils. Determination of zinc in 24 h urine may help diagnose deficiency if sickle cell disease, chronic renal disease and liver cirrhosis are ruled out. A metabolic balance study may clearly distinguish zinc-deficient subjects. 

Partially denatured alkaline phosphatase was assayed and compared to control enzyme. With a 15 min incubation of 8x10 M control enzyme, 9.8 3.3 peaks were observed per assay (n=4) with activities of 138 107 s . This is about twice the number observed in the single molecule assays different enzyme lots were used for the two studies. Denatured enzyme produced 4.4 1.5 peaks/assay (n=4) with an average activity of 118 97 s . At the 85% confidence limit, the means of the activities are identical and at the 75% confidence limit the standard deviations are identical. A 50% loss of the activity in bulk assay results in 45 9%... 

The substrate for the indicator reaction, dichloroindophenylbutyrate, is pale yellow. The product of the reaction is deep blue, with an absorbance maximum at 620 nm. The ingenuity of this method lies in the production of an enzyme inhibitor, (oxotrifluor-obutyl)phenol, rather than a species that is directly quantitated. The inhibitor affects the activity of the esterase, resulting in catalytic amplification of the initial alkaline phosphatase activity. The detection limit of the amplified assay has been reported as 3.2 x 10 14M, a 100-fold improvement over the detection limit of the standard alkaline phosphatase assay. 

Fig. 2 Brush border enzyme activity in primary rat proximal tubular cells (RPTC) as a function of age. RPTC were cultured in 35 mm culture dishes for 4,6,8,10, and 12 days. Cell homogenates were assayed for alkaline phosphatase activity (ALP) and gamma glutamyl transpeptidase activity (GGT). Values were correlated to homogenate protein content and are expressed as a fraction of isolated proximal tubular fragment activity/mg protein (day 0). Results are the mean of three culture dishes from two different primary preparations. Statistical significance from day 0 as analyzed by one way ANOVA with a Dunnett s post test is denoted by where p<0.05 and by where p<0.01...

The cytochemical assay for alkaline phosphatase activity is semiquanti-tative, and the absolute results were not expected to correspond to the direct measurement of enzyme activity in supernatants surrounding sheared... 

The attachment of the ferrocene groups to small molecules has been used in two main classes of biosensor in the first, the conjugate is an enzyme substrate such that a shift in its redox potential occurs following enzymatic modification. An example is ferrocenylethyl phosphate (Figure 9) that facilitates the electrochemical assay of alkaline phosphatase, a common enzyme label for immunoassays. The redox potential of the alcohol product is lower than the phosphate ester and hence by poising the potential, the product can be selectively measured and so the alkaline phosphatase activity measured. The sensitivity of this method was further enhanced by the use of stripping voltammetry to detect the product. ... 

Similar resnlts can be obtained by the Ishikawa-Var-I assay. This second bioassay, developed by Littlefield et dl.f is based on the ability of componnds with estrogenic activity to stimnlate alkaline phosphatase activity in a human endometrial... 

After seven days of culture on titanium and nanotubulartita-nia surfaces, the MSCs were provided with complete media to initiate differentiation and matrix deposition. Alkaline phosphatase activity (ALP) activity was measured for up to three weeks of culture after providing complete media. The ALP levels are age dependent however the levels are elevated during the period of active bone growth, and thus were measured. A colorimetric assay was used to measure the ALP levels. Figure 16.16(top) shows the ALP activity normalized with total protein content to account for difference in number of cells present on each surface. The cells present on nanotubular surfaces show higher ALP levels compare to those on titanium surfaces. There is approximately 50% increase in ALP levels on nanotubular surfaces after three weeks of culture (p < 0.05). 

A procedure for measuring alkaline phosphatase activity via a colorimetric assay follows (Holtorf et al., 2005) ... 

Biocompatibile hydroxyapatite/coUagen bionanocomposites were also studied by Santos et al. [232] using osteoblast cell culture assay. Their results showed that, after 72 h, their bionanocomposites did adversely affect ceU morphology, and had similar cell viability and alkaline phosphatase activity related to the control. This demonstrated that these bionanocomposites can potentially be used in bone tissue replacement applications. 

After bone marrow stromal cells were seeded and cultured on PHBHHx, their proliferation was investigated by MTT. Differentiation of the cells was assessed by measuring alkaline phosphatase activity and by histochemical assay. The wettability and thermal properties of PHBHHx films were also studied. The... 

Belfield, A. and Golderg, D. M. (1971). Revised assay for serum alkaline phosphatase activity using 4-amino antipyrine. Enzyme., 12 561. 

Enzyme Reference Serums. Several companies sell lyophilized or stabilized reference serums for the calibration of instruments and for quality control. The label values given for the enzymatic activity of these serums should never be taken at face value, as at times they may be quite erroneous (19,33). Also, these values should only be used for the assay with which they were standardized, as interconversion of activity from one method to another for the same enzyme may often lead to marked errors. For instance, it is not recommended that alkaline phosphatase expressed in Bodansky units be multiplied by a factor to convert it to the units of the Ring-Armstrong method, or any other method for that matter. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

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