Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The selective removal from 2-amino-2-methyl-1-propanol solutions of impurities that inhibit alkaline phosphatase activity by means of Amberlite chromatography is described. The effects of buffer concentration, column dimensions, adsorbent type, and zinc sulphate addition were investigated. The method was found to improve greatly the quality of commercial 2-amino-2-methyl-1-propanol solutions and to have several advantages over alternative purification methods.
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PMID:A column chromatographic method for the removal from 2-amino-2-methyl-1-propanol of impurities that inhibit alkaline phosphatase activity. 709 45

An optimized assay for alkaline phosphatase (EC 3.1.3.1) is reported. A discrete analyzer, the DuPont Automatic Clinical Analyzer (aca), was used for this study. The assay is based on results of response-surface experimental co-optimization techniques, and response is enhanced over the present aca assay. A key feature is the incorporation of zinc ions, both to fully optimize the assay and to reduce the sensitivity of measured activity to zinc-binding impurities in the buffer, 2-amino-2-methyl-1-propanol. In addition, a simple technique is described for measuring relative concentrations of zinc-binding impurities in this buffer. These features should be considered in the design of any assay for alkaline phosphatase that is based on p-nitrophenyl phosphate as substrate and 2-amino-2-methyl-1-propanol as buffer.
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PMID:Response-surface-optimized, zinc-enhanced assay for serum alkaline phosphatase. 723 80

An inactivator of alkaline phosphatase (EC 3.1.3.1) in 2-amino-2-methyl-1-propanol is demonstrated and characterized. This time-dependent inactivation results from chelation of enzyme-bound Zn2+; it is reversed by addition of Zn2+ and, to a lesser extent, other divalent metal ions. Cu2+ is an effective spectral indicator and can be used to determine the presence and quantity of inactivator. Data obtained from enzyme inactivation, Cu2+ absorbance spectra, "high-performance" liquid chromatography, thin-layer chromatography, Fourier-transform infrared spectroscopy, and mass spectroscopy indicate that the inactivator is 5-amino-3-aza-2,2,5-trimethylhexanol. This compound, even in trace amounts (less than 0.05% on a molar basis), shown to inactivate alkaline phosphatase.
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PMID:Measurement of alkaline phosphatase activity: characterization and identification of an inactivator in 2-amino-2-methyl-1-propanol. 727 99

When 2-amino-2-methyl-1-propanol buffer solutions are extracted with chloroform, impurities inhibiting alkaline phosphatase are selectively removed and this source of variation is thereby greatly decreased. We also studied the effect of adding zinc sulfate to this buffer. A combination of chloroform extraction and zinc sulfate, added to give concentrations from 5 to 10 mumol/L, results in buffer of high and consistent quality. The alkaline phosphatase activities of 12 reference sera of different origin were determined with use of the purified buffers. Those sera, which closely resembled human serum in terms of alkaline phosphatase isoenzyme composition, behaved similarly to human serum. Use of such control sera is strongly recommended.
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PMID:Elimination of inhibitors of alkaline phosphatase from 2-amino-2-methyl-1-propanol. 746 Feb 85

Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.
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PMID:Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis. 822 23

Most UK clinical laboratories use alkaline phosphatase (ALP) methods similar to that proposed by the International Federation of Clinical Chemistry (IFCC), based on the use of 2-amino-2-methyl-1-propanol (AMP) buffer. We present evidence of significant differences in results produced by apparently similar commercial ALP methods using an AMP buffer. We compared Bayer DAX, Dade Dimension and Boehringer Mannheim Hitachi 717 methods. Boehringer and Dade results were higher than Bayer results (Bland and Altman analysis, log transformed data): Boehringer (+23.0%, limits of agreement 1.16-1.31 times Bayer); Dade (+21.9%, limits of agreement 1.13-1.32 times Bayer). Biases were predominantly due to differences in reagents rather than analyser characteristics. Compared to a reagent system prepared exactly as described by the IFCC, Bayer was sub-optimal and Dade and Boehringer methods produced results higher than the IFCC method. Reference ranges and results on patients' samples by the various methods showed large differences but no clinically significant difference was observed in external quality assessment schemes either between Bayer and Boehringer or against method means. Apparently similar methods produce different results in patients' sera: external quality assessment schemes are not useful in highlighting these differences.
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PMID:Alkaline phosphatase activity measurement in the UK by AMP-buffered methods: an appraisal of current practice. 946 50

The effect of feeding, starvation and fibre ingestion on alkaline phosphatase (ALP) activity (E.C. 3.1.3.1) was studied in Wistar rat serum. Using identical assay conditions for total ALP activity determination and for electrophoretic ALP isoenzymes/fractions activity calculation, alpha- and beta-naphthyl phosphates and p-nitrophenyl phosphate were used as substrates and 2-amino-2-methyl-1-propanol/HCI was used as buffer, respectively. Total activity with beta-naphthyl phosphate was significantly higher than with alpha-naphthyl phosphate and p-nitrophenyl phosphate; with alpha-naphthyl phosphate it was significantly higher than with p-nitrophenyl phosphate. With all substrates, fed animals had significantly higher total activity than starving ones. Electrophoresis allowed the separation of two fractions. The second fraction activity was significantly higher in the fed group than in the starving ones, irrespective of the substrate used. Starving animals with fibre showed higher values of this fraction than starving animals without fibre, the difference reaching statistical significance with alpha-naphthyl phosphate. The first fraction predominated in both starved groups and the second in the fed group. The second fraction was identified as intestinal ALP. We conclude that the mechanical stimulation of the digestive tract appears to influence the passage of intestinal ALP to serum. The experimental conditions used enable quantification of electrophoretic fractions based on total activity. Activity depends on the substrate used.
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PMID:Rat serum alkaline phosphatase electrophoretic fractions: variations with feeding, starvation and cellulose fibre ingestion. 1020 51

The importance of separation and identification of serum alkaline phosphatase (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. Each serum ALP fraction/isoenzyme quantitation has both practical and theoretical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and serum ALP electrophoretic fractions/isoenzymes activities were quantified. Different results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-methyl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions (9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were used. Higher total serum ALP activity was always observed with beta-naphthyl phosphate, independently of the buffer (or mixture of buffers) and pH used. Electrophoresis allowed the separation of two serum ALP fractions. Activity of both serum ALP electrophoretic fractions was always higher with beta-naphthyl phosphate, except with carbonate/bicarbonate pH 10.4. The effect of a change in pH was buffer- (or mixture of buffers) and substrate-dependent; the addition of a second buffer (to that previously used) was not always accompanied by an increase or decrease (of the same magnitude) in our results. The results obtained with different buffers (or mixture of buffers) were not identical with substrates and pH values. It is concluded that (i) from the same electrophoretic separation of serum ALP fractions/isoenzymes, different values for its activity can be obtained by changing the assay conditions used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoretic fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each case.
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PMID:Importance of assay conditions in visualization and quantitation of serum alkaline phosphatase isoenzymes separated by electrophoresis. 1069 Oct 50

A new reagent carrier, Reflotron ALP, has been developed for the Reflotron system, allowing easy and rapid measurement (in less than 3 minutes) of alkaline phosphatase (ALP) activity in capillary blood, venous blood, heparinized plasma or serum. The evaluation of the analytical performance of the assay was carried out at eight clinical laboratories. The study of the imprecision using the measurements in human samples resulted in coefficients of variation ranging from 1.3% to 4.6% (within-run) and from 3.2% to 4.0% (day-to-day). The analytical specificity of the Reflotron ALP assay agrees well with ALP methods using a N-methyl-D-glucamine buffer solution. The calibration of the Reflotron ALP assay, however, is related to the reference intervals for ALP methods using a diethanolamine buffer solution. Method comparisons were performed with the ALP method on Hitachi instruments using diethanolamine buffer. Reflotron ALP measurements in blood and plasma in 157 randomly selected split samples showed excellent agreement (slope: 0.99; intercept: 0.7 U/l; median bias: 2.3%; median difference from the comparison method: -0.3%). Specimens from pregnant women and adolescents were excluded from this study. Differing values were obtained in a method comparison using 48 samples containing predominantly the ALP bone isoform (slope: 0.81; intercept: 31.5 U/l; median bias: 5.7%; median difference from the comparison method: -12.2%). Regression analysis of the results from 21 sera with prevailing placental ALP gave a slope of 1.51, and an intercept of -41.1 U/l (median bias: 8.6%; median difference from the comparison method: 35.6%). Reflotron ALP was compared with three different wet chemistry procedures using different buffer compounds: N-methyl-D-glucamine or diethanolamine or 2-amino-2-methyl-1-propanol. In samples containing predominantly ALP isoforms not of liver origin, the measurements with N-methyl-D-glucamine buffer gave the best fit with respect to Reflotron. In an interference study with 18 drugs, no effect on the test results could be detected. Total bilirubin up to 750 micromol/l and hemolysis up to 1.7 g/l free hemoglobin did not influence the test. Reflotron ALP proved to be an easy and rapid method with excellent precision. The accuracy related to an ALP method using diethanolamine buffer was good. The systematic differences for ALP in samples from pregnant women and adolescents have to be taken into account. The assay is well suited for differential diagnosis of hepatic diseases in decentralized testing.
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PMID:Alkaline phosphatase activity: new assay for the Reflotron system. Results of the evaluation in eight clinical laboratories. 1125 5

A new assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products. The method proposed is simple, rapid and directly applicable to solid and liquid dairy samples. ALP in the test sample hydrolyzes a non fluorescent substrate, trifluoromethyl-beta-umbelliferone phosphate, to its highly fluorescent phenolate product. The assay is performed in a reverse micellar medium composed of mixed buffer (2-amino-2-methyl-1-propanol buffer pH 9.0 and borate buffer pH 9.0) in AOT/isooctane, at a temperature of 38 degrees C. Total test time is 450 s. Reaction rates are linear (except for butter) up to 8.5 and 11% (v/v) raw milk, for whole milk and chocolate milk, respectively. The detection limits are 0.04, 0.4 and 0.22% (v/v) raw milk, for whole milk, chocolate milk and butter, respectively. The precision of the fluorimetric method was assessed by repeated analysis of a pasteurized milk sample spiked with mixed herd raw milk. The accuracy of the method was evaluated by comparison with an official colorimetric assay using p-nitrophenylphosphate as ALP substrate.
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PMID:Fluorimetric determination of alkaline phosphatase in solid and fluid dairy products. 1896 82


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