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Leukocyte alkaline phosphatase in cancer patient.

Leukocyte alkaline phosphatase (LAP) activity in peripheral blood was determined in 113 patients with non-metastatic malignancies, 36 patients with metastatic spread, and 22 individuals who served as controls. The LAP score of the non-metastatic cancer patients was significantly higher (p less than 0.001) than the controls, and the score of the metastatic group was further increased significantly as compared to both control (p less than 0.001) and non-metastatic groups (p less than 0.001). The results of this survey suggest that the level of LAP score in peripheral blood might be used to differentiate between non-metastatic and metastatic disease and might serve as an aid in prognosis of the patients.

Alkaline Phosphatase↗

Plasma alkaline phosphatase isoenzymes in diabetes mellitus.

Alkaline phosphatase (ALP) isoenzymes were measured in type 1 diabetics, type 2 diabetics and in a non-diabetic control group. Within the diabetics and the control group, intestinal ALP activity was significantly higher in BO secretors than A secretors or ABO non-secretors. There was no difference in intestinal ALP activity between type 1 and type 2 diabetics but the diabetics had a significantly higher activity of this isoenzyme than the corresponding blood group/secretor status category of the control group. Liver ALP was significantly higher in the diabetics compared with the control group. Bone ALP showed no significant difference between the diabetics and the control group.

Adult↗

The prognostic value of serum alkaline phosphatase in osteosarcoma of the limbs.

The values for serum alkaline phosphatase were evaluated in 340 patients affected with osteosarcoma of the limbs. The percentage of patients with high values for the enzyme at the onset of disease was significantly higher in the 69 cases who presented with metastases at the time of diagnosis, as compared to the 271 cases in which the neoplasm still appeared to be localized (81% vs 62%: p less than 0.001). In this last group of patients, treated with neoadjuvant chemotherapy, an evident relationship between values for alkaline phosphatase and prognosis was demonstrated. The percentage of patients who developed metastases was in fact 22% in the group with normal serum values for the enzyme at the onset of the disease and 40% for the patients with high values of the same (P less than 0.001). These data confirm the prognostic value for serum alkaline phosphatase in osteosarcoma, which should be taken into consideration when planning chemotherapy and in classifying patients for randomized therapeutic studies.

Adolescent↗

Utilizing ultrafiltration to remove alkaline phosphatase from clinical analyzer water.

Alkaline phosphatase (ALP) conjugated to antibodies is often used in enzyme immunoassays (EIAs). These assays are notably sensitive to experimental conditions. A possible source of interference is bacterial ALP, which is released when bacterial contamination occurs in clinical analyzers. Preliminary experiments led to the selection of a detection kit, ALP source, and specific types of tubes for collecting water samples and performing assays. The release of ALP from various strains of bacteria identified in pure water was demonstrated (10-30 x 10(6) cfu/mL released 6-10 microU/microL). It was shown that ultrafiltration is totally efficient in removing ALP from water, while residual ALP activity (2.21 microU/microL after filtration of an ALP solution of 6.22 microU/microL) was observed after filtration using a 0.22-mum filter.

Alkaline Phosphatase↗

Alkaline phosphatase. Laboratory and clinical implications.

Alkaline phosphatase (ALP), which was discovered in 1907, is extensively reviewed. The first section deals with biochemical aspects of ALP, e.g. the anchorage of ALP to cell membranes via a phosphatidylinositol linkage, the charge and molecular mass heterogeneities and their causes, and methods for the separation and analysis of ALP isoforms using the newest electrophoresis techniques, such as affinity electrophoresis with wheatgerm lectin and isoelectric focusing in immobilized pH gradients. The second section deals exclusively with the clinical implications of ALP analysis in diseased states, e.g. cholestasis, hyper- and hypophosphatasemias and neutrophil ALP. Extensively discussed is the involvement of ALP in numerous bone diseases. The role of ALP and its isoforms in tumours and their applicability as potential tumour markers is critically evaluated.

Alkaline Phosphatase↗

Factors predictive of liver histopathological appearance in chronic alcoholic pancreatitis with common bile duct stenosis and increased serum alkaline phosphatase.

In the course of alcoholic chronic pancreatitis, increased serum alkaline phosphatase level is usually caused by common bile duct stenosis but may also be due to alcoholic liver disease. The aims of this prospective study were to investigate whether clinical, biochemical and radiological factors could predict liver histopathological appearance. The study comprised 48 patients with chronic alcoholic pancreatitis, common bile duct stenosis and increased serum alkaline phosphatase levels; clinical, biochemical, radiological and histological data were recorded in all cases. Liver biopsy examination (surgical [n = 45] or intercostal [n = 3]) showed (a) biliary obstructive liver abnormalities (n = 33), which were severe in 20 cases (biliary fibrosis in 15, secondary biliary cirrhosis in 3, secondary sclerosing cholangitis in 2) and moderate in 13 cases; (b) alcoholic liver disease in 9; and (c) normal liver in 6. Clinical, biochemical and radiological data were not statistically different between patients with biliary obstructive liver disease and those with alcoholic liver disease. Forty-five patients underwent surgery; two patients with alcoholic hepatitis died after surgery, at the beginning of this study. We conclude that in chronic alcoholic pancreatitis with common bile duct stenosis and increased serum alkaline phosphatase levels, clinical, biochemical and radiological data cannot be used to predict the type of liver lesions. Therefore liver biopsy is warranted to identify (a) alcoholic hepatitis, which increases operative risk; and (b) biliary obstructive liver disease, frequent and often severe, in which surgical biliary decompression should be considered.

Adult↗

Monitoring skeletal cancer metastases with the bone isoenzyme of tissue unspecific alkaline phosphatase.

The efficacy of bone alkaline phosphatase (ALP) isoenzyme measurement using lectin precipitation in confirming metastatic bone lesions was compared with total ALP and osteocalcin assay in serum. Sixty-five patients with cancer and metastases to bone (n = 44), liver (n = 15) or lymph nodes (n = 6) as well as 33 healthy adults were studied. Assay of bone ALP is as sensitive but more specific than assay of total ALP in the identification of bone metastases. On the other hand, bone ALP did not correlate with osteocalcin, as is the case in other bone diseases. In the serial monitoring of nine patients with skeletal metastases, bone ALP correlated well with the presence of pain and the progression or regression of metastatic spread.

Adult↗

Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma.

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.

Alkaline Phosphatase↗

In situ hybridization: alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections.

Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration of in situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100-200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method. The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.

Alkaline Phosphatase↗

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase.

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.

Alkaline Phosphatase↗

Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimester of gestation.

Human blood plasma contains alkaline phosphatase (EC 3.1.3.1) isoenzymes from bone, liver and intestine. Blood of pregnant women in the third trimester of gestation also contains an isoenzyme of alkaline phosphatase from placenta, placental-AP (PLAP). Serum from pregnant women in the last trimester of gestation shows activity of soluble placental alkaline phosphatase (sol-PLAP). It is also known that a membrane bound high molecular weight placental-AP (high Mr-PLAP) is present in butanol extracts from placental tissue. Nevertheless, up to now, high Mr-PLAP has not been founded in human plasma. A method developed in this laboratory allows detection of membrane-bound alkaline phosphatase in pellet of plasma centrifuged at 100,000x g. By applying this method we have detected high molecular weight placental alkaline phosphatase in plasma of healthy pregnant women in the third trimester of pregnancy.

Alkaline Phosphatase↗

The inhibition and disposition of intestinal alkaline phosphatase.

A primary mechanism of amino acid inhibition of intestinal alkaline phosphatase is postulated to be the formation of a dissociable enzyme-amino acid complex at an allosteric zinc site. The degree of inhibition was highly correlated with the Zn2+ stability constant of each amino acid and the inhibition was reversible by the addition of exogenous Zn2+ or by dialysis. This allosteric amino acid inhibition proved to be a useful probe of the membrane arrangement of the enzyme in the intact tissue. The catalytic site appears to face the lumen based on the poor permeability of the substrate, the accumulation of the coproducts in the luminal bath, and the response of the enzyme to luminal pH. Amino acid inhibition of alkaline phosphatase in the intact tissue was only effective in the presence of sodium; whereas sodium was not required in butanol extracted preparations which lacked the sidedness of the intact tissue. Since amino acid uptake from the intestine is sodium dependent, the allosteric inhibitory site is probably intracellular. The results suggest that the intestinal alkaline phosphatase spans the apical membrane with the catalytic site accessible from the lumen and the allosteric inhibitory site from the cytoplasm.

Alkaline Phosphatase↗

[Alkaline phosphatase in the blood and lymph in diffuse peritonitis].

Activity of alkaline phosphatase was studied in blood and lymph of 52 dogs with experimental peritonitis and in 26 patients with the drained thoracic duct and in 26 patients without lymphodrainage. Pronounced enzyme intoxication was revealed. Under physiological conditions alkaline phosphatase is transported by both blood and lymphatic bed. In peritonitis the lymphatic route of the enzyme transport gets considerably greater. Drainage of the thoracic duct gives good detoxicating effect (decreased alkaline phosphatase activity).

Alkaline Phosphatase↗

Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.

Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 micromol p-nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p-nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60-70 degrees C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N-terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.

Alkaline Phosphatase↗

Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non-equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [S. Orhanović, M. Pavela-Vrancic, Eur. J. Biochem. 270 (2003) 4356-4364]. The significance of interactions at the subunit interface and the involvement of the beta-pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site-directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild-type protein. The T81A mutation, introduced at the subunit interface, significantly affected the protein kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.

Alkaline Phosphatase↗

Heterogeneity of alkaline phosphatases in different HeLa lines.

Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALP structural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the ""derepressed'' placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.

Alkaline Phosphatase↗

The chromatographic and electrophoretic micro-heterogeneity of alkaline phosphatase of rat dental pulp.

Alkaline phosphatase of rat dental pulp was separated into two forms by anion-exchange chromatography, gel-filtration and electrophoresis. The minor activity (AP I) was eluted at 0.1 M of NaCl from a DEAE-cellulose column, and the major activity (AP II) in the broad range of approx. 0.15-0.5 M. These two enzyme activities gave rise to distinct peaks on a TSK-GEL Toyopearl (Fractogel TSK) HW 55-S column and showed different electrophoretic behaviours on both sodium dodecyl sulphate (SDS) and non-SDS polyacrylamide gels. The molecular weights of AP I and AP II were estimated by gel-filtration to be 130,000-150,000 and above 500,000, respectively. On SDS gel electrophoresis, the molecular weight of AP II was changed to 130,000, while AP I had a molecular weight of 150,000. Judging from the sensitivities to heat and some inhibitors, AP I and AP II are biochemically indistinguishable from each other.

Alkaline Phosphatase↗

Structural evidence of functional divergence in human alkaline phosphatases.

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family.

Alkaline Phosphatase↗