Alkaline phosphatase determination

Apart from isoenzymes, hyperphosphatasemia in diseases of liver and bone has made the serum alkaline phosphatase determination the most frequently requisitioned enzyme assay for over 30 years. Obviously, it has also fixed for the great majority (C17) the expectation that liver and bone normally constitute the major sources of serum alkaline phosphatase. Uneasiness rarely is expressed in the literature regarding the patients with bone and liver disease whose enzyme values are not elevated, or in the cases of hyperphosphatasemia where neither liver nor bone disorders exist. 

Fig. 5. Relation between serum alkaline phosphatase determined in the Auto-Analyzer and by the manual method. AutoAnalyzer units are King-Armstrong, and manual units are Bodansky-Shinowara (F14).

C35. Cowan, R. J., and Young, K. A., Evaluation of serum alkaline phosphatase determination in patients with positive bone scans. Cancer 32, 887-889 (1973). 

G14. Goldberg, D. M., and Ellis, G., An assessment of acid and alkaline phosphatase determinations in the diagnosis of prostatic cancer.. Clin. Pathol. 26, 387-388... 

Phosphatase Test. The phosphatase [9001-78-9] test is a chemical method for measuring the efficiency of pasteurization. AH raw milk contains phosphatase and the thermal resistance of this enzyme is greater than that of pathogens over the range of time and temperature of heat treatments recognized for proper pasteurization. Phosphatase tests are based on the principle that alkaline phosphatase is able, under proper conditions of temperature and pH, to Hberate phenol [108-95-2] from a disodium phenyl phosphate substrate. The amount of Hberated phenol, which is proportional to the amount of enzyme present, is determined by the reaction of Hberated phenol with 2,6-dichloroquinone chloroimide and colorimetric measurement of the indophenol blue formed. Under-pasteurization as well as contamination of a properly pasteurized product with raw milk can be detected by this test. 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

The dosage of flucytosine is 150—200 mg/kg orally in four portions every six hours. A 1% flucytosine solution has been developed for intravenous adrninistration. In some countries, a 10% ointment is also available. In patients with normal renal function, flucytosine is seldom toxic, but occasionally severe toxicity may be observed (leukopenia and thrombocytopenia). Plasma levels should be determined and the dose in patients with impaired renal function should be checked. Liver function tests (transaininases and alkaline phosphatase) should be performed regularly. In some patients with high flucytosine plasma levels, hepatic disorders have been observed (24). 

Along with an effective electrolyte and screening program for genetic disease, the laboratory of Neonatology needs to have the capability of analyzing for other components in blood serum, which aid in the diagnosis of disease. These include such determinations as alkaline phosphatase, and various other enzymes, creatinine, uric acid and a host of other components which are normally assayed by the main clinical laboratory. 

Bessey, 0 A Lowry, 0. H and Brock, M J A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum ... 

Winkelman, J. Nadler, S. Demetrio, J. and Pileggi, V. J. Clinical usefulness of alkaline phosphatase isoenzyme determinations. Am. J. Clin. 

Roos, R. A simplified method for the determination of serum alkaline phosphatase activity. Scand. J. Clin. 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

Now look at rows 21-24. Notice how ALKALINE PHOSPHATASE is truncated to ALKALINE PHOSP. This happens because the default behavior of the Import Wizard, PROC IMPORT, and the External File Interface (EFI) is that they scan only 20 rows deep into the file to determine variable attributes. Text field truncation is a common problem here. Another problem is that if a field appears to be numeric in the first 20 rows but later has character text beyond the scanning depth of PROC IMPORT, the procedure will terminate with an error message. There are two workarounds for this scanning depth problem. 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

---