Alkaline phosphatase analysis

C16. Comfort, D., and Campbell, D. J., One-step automated alkaline phosphatase analysis. Clin. Chim. Acta 14, 263-269 (1966). 

A complete physical examination and laboratory analysis are needed to rule out secondary causes and to assess kyphosis and back pain. Laboratory testing may include complete blood count, liver function tests, creatinine, urea nitrogen, calcium, phosphorus, alkaline phosphatase, albumin, thyroid-stimulating hormone, free testosterone, 25-hydroxyvitamin D, and 24-hour urine concentrations of calcium and phosphorus. Urine or serum biomarkers (e.g., cross-linked N-telopeptides of type 1 collagen, osteocalcin) are sometimes used. 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

The ABC detection system has been shown to be more sensitive than most other detection system (5,6), primarily because of the large size of the preformed ABC complexes, which result in amplification of the signals. Alternative detection systems for immunohistochemical analysis include the peroxidase-antiperoxidase (PAP) (1) and the alkaline phosphatase-antialkaline phosphatase (APAAP) systems (7) (see Chapter 24). These approaches are conceptually and technically similar, and will not be discussed here. 

Serum alkaline phosphatase elevations have been reported following administration of salt-poor albumin (B5). Placenta is very rich in a heat-stable alkaline phosphatase, and albumin prepared from placental blood has a high activity of this enzyme. In one cirrhotic patient who received 1-6 units per day of albumin obtained from pooled human blood and/or human placenta, the alkaline phosphatase before infusion was 5 Bodansky units and by the thirteenth day of administration had reached a value of 160 units. The physician administering the albumin at first thought the patient was having a severe toxic liver reaction and stopped the therapy. The alkaline phosphatase then started to go down and within 10 days returned to normal levels. Analysis of the albumin indicated that it contained 470 units of alkaline phosphatase activity and was probably responsible for the observed elevations in the serum enzyme activity. Albumin prepared from venous blood did not cause an alkaline phosphatase elevation, but placenta-albumin caused elevations with a half-life of about 8 days (Ml). 

Enzyme activity measurements are greatly affected by buffer systems used in analysis. In measuring alkaline phosphatase activity under optimal conditions, ethylaminoethanol buffer yielded activity 3.8 times... 

A detailed analysis of solid-state structures of several unrelated systems that catalyze the hydrolysis of diphosphate esters (alkaline phosphatase and the Klenow fragment , among others) revealed that their active sites invariably contain conserved carboxylate... 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

By analysis of the products with glucose oxidase, it was shown that the anomeric composition of the glucose liberated from glucose-6-phos-phate by the enzymes acid or alkaline phosphatase or by glucose-6-phosphatase from rat liver was essentially the same as that of the substrate, thus indicating a lack of anomeric specificity for these enzymes also (106). 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Figure 36.7. Example of Immusoft window appearing in the Analysis menu during the processing of the fluidic steps of an assay. The figure shows the electrochemical monitoring of the fluid flux within the microchannels obtained during the 30 loading steps of the microchannel coating procedure chosen for alkaline phosphatase assays.

Methods Adapted. At the present time, six methods of interest to clinical chemists are available for analysis with the Autoanalyzer. They are methods for glucose, urea nitrogen, chloride, calcium, inorganic phosphate, and alkaline phosphatase. Micromodifications are available... 

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