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PubMed1993

Factors associated with serum alkaline phosphatase level.

Serum alkaline phosphatase was measured in a large population of government employees and their dependents, aged 12 years and older. It was found that the concentrations of this enzyme were very high in early adolescence, fell to a low point after the completion of bone growth, and rose thereafter. The concentration was higher in males than females until the age of 50 years; after that it was higher in females. In employees and their spouses (aged 19 years and older), the greater the weight (or weight for height), the higher the concentration. The taller the person, the lower the level of this enzyme. The more cigarettes smoked per day, the higher the enzyme concentration. There was also a weak but statistically significant association of alkaline phosphatase with serum glucose concentrations and blood pressure but not with antihypertensive medications. Other factors were also considered. Some of these factors, particularly age and weight, are associated with substantial difference in the concentration of alkaline phosphatase and could conceivably affect the clinical interpretation of test results.

Adolescent↗
PubMed1976

Molecular properties of rat intestinal alkaline phosphatase.

Rat intestinal alkaline phosphatase is an heterogeneous glycoprotein that contains three protein sub-forms separable by electrophoresis. The molecular weight for the glycoprotein (i.e. the average for the three sub-forms) is 157 000-160 000. Three protein sub-forms are detectable on sodium dodecyl sulphate-polyacrylamide gel electrophoresis that migrate at rates corresponding with molecular weights of 64 000, 79 000 and 92 000. Treatment of native alkaline phosphatase with 6 M guanidine - HC1 or buffer at pH 3.0 results in a product with a molecular weight of 78 000 and 70 000, respectively. Thus it is concluded that each of the three sub-forms is a dimer of identical or closely similar subunits. Limited proteolysis results in the production of new enzymically active sub-forms separable by electrophoresis. Using a bacterial protease it is possible to convert intestinal alkaline phosphatase into a form with a molecular weight of 132 000 without causing any significant change in kinetic properties. Electrophoresis of this new form on sodium dodecyl sulphate polyacrylamide gel suggests that it is composed of 66 000-dalton subunits. The native enzyme contains at least 20% by weight of carbohydrate that probably contributes to microheterogeneity of a second degree superimposed on that stemming from the presence of three protein sub-forms. Treatment with various glycosidases has no effect on electrophoretic behaviour, however. It is suggested that the three sub-forms possibly represent different stages of a maturation process that operates by limited proteolysis of a single parent protein.

Alkaline Phosphatase↗
PubMed1994

Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium.

We assessed the significance of Ca and phosphate (P(i)) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) concentration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan-anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of P(i)-dependent changes. The observed correlation between skeletal ALP-specific activity and P(i) was not unique to osteosarcoma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero-P(i) and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the beta-glycero-P(i)-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by P(i) and that Ca may be involved in ALP release.

Alkaline Phosphatase↗
PubMed1987

Identification of bone and liver metastases from breast cancer by measurement of plasma alkaline phosphatase isoenzyme activity.

Plasma alkaline phosphatase isoenzyme activities were determined in patients with breast cancer to diagnose and monitor bone and liver metastases. Bone alkaline phosphatase activity was increased in 21 of 50 patients (42%) with radiologically confirmed bone metastases, while total alkaline phosphatase activity was increased in only 10 of 50 (20%); liver alkaline phosphatase activity was raised in 12 of 25 patients (48%) with liver metastases. All patients with liver metastases had bone metastases. Bone alkaline phosphatase activity was significantly higher in patients with symptomatic bone disease. Isoenzyme determination provided additional information that would have changed patient management in five of 20 patients who were monitored serially. Measurement of alkaline phosphatase isoenzyme activity, though less sensitive than imaging procedures, can assist in screening for, and in early detection of, a high proportion of bone and liver metastases, and can provide useful objective evidence of their response to treatment.

Adult↗
PubMed1982

Alkaline phosphatase isoenzymes.

The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.

Adolescent↗
PubMed1980

Expression of alkaline phosphatase loci in mammalian tissues.

Alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] have been examined in liver, bone, kidney, intestine, and placenta from nine mammalian species by quantitative inhibition and thermostability studies and compared with alkaline phosphatases in the corresponding human tissues. In humans, three kinds of alkaline phosphatase can be sharply differentiated by these methods, one occurring in liver, bone, and kidney, one in intestine, and one in placenta. They are evidently determined by separate gene loci. In the mammals only two sorts of alkaline phosphatase were found: one, which occurs in liver, bone, kidney, and also placenta, corresponds to the human liver/bone/kidney enzyme and the other corresponds to the human intestinal enzyme. The findings support our earlier proposal that the expression of a distinctive type of alkaline phosphatase in human placenta is the consequence of a late evolutionary event which occurred subsequent to the divergence of the evolutionary lineage leading to humans from the various lineages leading to other mammalian species. The concentrations of the inhibitors, phenylalanine, homoarginine, phenylalanylglycylglycine, and levamisole, required to give 50% inhibition, [I(50)], of the liver/bone/kidney/placental (nonhuman) alkaline phosphatases showed no significant variation among the species. However, the [I(50)] values for the intestinal enzyme varied among species to a much greater extent. This implies that in the liver/bone/kidney/placental (nonhuman) alkaline phosphatase the structures of the binding sites for these inhibitors have been highly conserved during mammalian evolution, but there has been much greater divergence of these structures in the evolution of intestinal alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1967

Neutrophil alkaline phosphatase in pregnancy.

Neutrophil alkaline phosphatase levels have been studied in a group of 194 pregnant women, using a modified technique which permits more rapid counting of the required number of neutrophils with a degree of consistency comparable with standard methods. No correlation was found between neutrophil alkaline phosphatase levels and the period of gestation; in contrast to previous findings, no differences were observed between the means for tests done in early pregnancy and those performed later in pregnancy. There was no demonstrable relationship between neutrophil alkaline phosphatase levels and age, parity, and white cell count. Elevated levels were also observed in women taking oral contraceptives. Thus, although the neutrophil alkaline phosphatase has diagnostic value in certain other disease states, the above findings suggest that it is of little or no value in pregnancy. The possibility of hormonal control of the elevated level of neutrophil alkaline phosphatase seen in pregnancy and simulated pregnancy is discussed, as is the possible relationship to increases in serum alkaline phosphatase in pregnancy.

Adolescent↗
PubMedDate not supplied

[Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis].

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of in vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

Alkaline Phosphatase↗
PubMed1988

Monoclonal alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex: production of antibody, optimization of activity, and use in immunostaining.

A mouse monoclonal antibody, FMC55 (an IgG1), to alkaline phosphatase was prepared and evaluated in immunostaining. Clones producing antibody to alkaline phosphatase were selected using a micro-ELISA which identified antibodies forming active soluble complexes (APAAP) with the enzyme. Conditions that influenced the formation of the complex were investigated by using a quantitative assay in which the complex was captured by a bridging anti-mouse antibody. The ratio of FMC55 to enzyme had a major influence on the activity of the complex. Although all complexes had some activity, those that contained excess antibody had reduced ability to bind to anti-mouse antibody because of competition with excess unlabeled antibody. The optimal complex was formed with 3 micrograms of FMC55 per unit of enzyme. This complex contained neither free enzyme nor free antibody. The molecular weight by gel permeation chromatography was 600,000, giving a composition of two enzyme and two antibody molecules or one enzyme and three antibody molecules. The size of the complex was not altered by adding excess antibody or excess enzyme. Immunoblotting showed that FMC55 bound only to the Mr 140,000 homodimeric form of alkaline phosphatase. The APAAP complex was used in combination with biotin-streptavidin-peroxidase reagent to detect two antigens labeled with two different mouse monoclonal antibodies in the same tissue preparation.

Alkaline Phosphatase↗
PubMed1981

Alkaline phosphatase from human thyroid.

Alkaline phosphatase activity was found in human thyroid homogenate at a specific activity of about 0.01 units/mg protein. The enzyme responsible appears to be a membrane-bound sialoglycoprotein. The properties of thyroid membrane alkaline phosphatase were examined after extraction with non-ionic detergent or butanol. It was detected mainly as a single electrophoretic form with a mobility similar to that of the liver form of the enzyme. It was readily inhibited by homoarginine, but not by phenylalanine. It reacted in Ouchterlony double diffusion with antiserum raised against liver alkaline phosphatase, but not with antiserum to placental alkaline phosphatase. Heat treatment at 56 degree C for 10 min at pH 7.5 resulted in an approx. 50% loss of enzyme activity. Its relative molecular mass was estimated to be 320000 by gel filtration, and 300000 by gradient gel electrophoresis. It required magnesium for full activity, and had a pH optimum of 10.5 in Tris-borate buffers. It was concluded that thyroid alkaline phosphatase belongs to the liver/bone/kidney isoenzyme group, but may exist in a molecular form distinct from others previously described.

Alkaline Phosphatase↗
PubMed2004

Assessment of pasteurisation of milk and cream produced by on-farm dairies using a fluorimetric method for alkaline phosphatase activity.

The alkaline phosphatase test is used as an indicator of adequate pasteurisation of milk and cream. A proprietary fluorimetric technique (Fluorophos) is a sensitive and quantitative method for the determination of alkaline phosphatase (ALP) activity in milk products. Currently, adequate pasteurisation of milk products is regarded as confirmed in samples that contain a residual bovine ALP activity of < or =500 mU/litre. This is equivalent to the statutory acceptable level of 4ug phenol/ml required by the EC analytical method. The purpose of the present study was to assess the effectiveness of pasteurisation of milk and cream produced by on-farm dairies. In a longitudinal study over a four-year period, 4,999 samples of milk and cream were collected from 130 on-farm dairies and from two large commercial dairies in NW England for comparison. Bovine ALP activity of >500 mU/litre was deemed as a failure and was found in 3.5% of whole milk, 2.4% semiskimmed milk, 5.0% of skimmed milk, and 39% of cream samples from on-farm dairies. Bovine ALP activity of >100 and <500 mU/litre was found in 18.4% of whole milk, 9.3% of semi-skimmed milk, 13.2% skimmed milk and 44.5% of cream samples from on-farm dairies. Results with skimmed milk samples showed significantly lower bovine ALP activity than whole milk. All 409 milk and cream samples from two large commercial dairies passed the fluorimetric test at less than 500 mU/litre of bovine ALP, and 99% of these milk and cream samples had bovine ALP activity of less than 100 mU/litre. The presence of residual bovine phosphatase indicates a failure and may be due to either inadequate pasteurisation or post pasteurisation contamination with raw milk. Residual bovine phosphatase was demonstrated in 108/114 (94.7%) of milk samples with a bovine ALP activity greater than 500 mU/litre, i.e. true failures. Of more concern is that residual bovine phosphatase was found in 395/401 (98.5%) of samples that gave bovine ALP activity greater than 100 mU/litre but equal to or less than 500 mU/litre. Residual bovine phosphatase was demonstrated in 37/108 (30.2%) of cream samples with bovine ALP activity greater than 500 mU/litre. Presence of reactivated bovine phosphatase is not an indication of a failure but can mask the presence of residual bovine phosphatase. Reactivated bovine phosphatase was found in 74/106 (69.8%) of cream samples. Our results confirm that the more sensitive fluorimetric method is suitable for testing pasteurised whole milk and semiskimmed milk, but for statutory purposes the acceptable level of residual bovine phosphatase should be <100 mU/litre. Our findings have highlighted a potential problem when testing skimmed milk and cream samples from on-farm dairies. To ensure public safety we need more stringent standards for the ALP test and new methods that will accurately confirm that pasteurisation of these products has been achieved.

Alkaline Phosphatase↗