Abstract Alkaline Phosphatase Research Articles

Alkaline Phosphatase Electrochemical Micro-Sensor Based on 3D Graphene Networks for the Monitoring of Osteoblast Activity.

Alkaline phosphatase (ALP) is a significant biomarker that indicates osteoblast activity and skeletal growth. Efficient ALP detection methods are essential in drug development and clinical diagnosis. In this work, we developed an in-situ synthesized three-dimensional graphene networks (3DGNs)-based electrochemical sensor to determine ALP activity. The sensor employs an ALP enzymatic conversion of non-electroactive substrate to electroactive product and presents the ALP activity as an electrochemical signal. With 3DGNs as the catalyst and signal amplifier, a sample consumption of 5 μL and an incubation time of 2 min are enough for the sensor to detect a wide ALP activity range from 10 to 10,000 U/L, with a limit of detection of 5.70 U/L. This facile fabricated sensor provides a quick response, cost-effective and non-destructive approach for monitoring living adherent osteoblast cell activity and holds promise for ALP quantification in other biological systems and clinical samples.

Alternative ELISA Using a RNA Aptamer against Calf Intestinal Alkaline Phosphatase

Alkaline Phosphatase (AP) is extensively used in enzyme-linked immunosorbent assays(ELISA), most requiring expensive and time-intensive reagents to generate (such as AP-conjugated antibodies). In a...

Hela Cells Consist of Two Cell Types, as Evidenced by Cytochemical Staining for Alkaline Phosphatase Activity: A Possible Model for Cancer Stem Cell Study

Alkaline phosphatase (ALP) is known to be expressed in the several somatic stem cells and cancer cells. To investigate whether ALP may be a promising marker for cancer stem cells (CSCs), we examined the expression of ALP in human squamous cell carcinoma HeLa cells using a cytochemical

Ingestion of potato starch containing esterified phosphorus increases alkaline phosphatase activity in the small intestine in rats

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters and plays an important role in phosphorus (P) metabolism. Several nutrients in food have been reported to affect intestinal ALP activity in animal models. Previous reports indicated that high levels of P or phosphate in diets decreased intestinal ALP activity in rats. Because potato starch contains considerable amounts of esterified P, unlike other starch-derived plants, we hypothesized that the feeding of potato starch would decrease ALP activity in the intestinal tract. Male Sprague-Dawley rats (7 weeks old) were fed 3 different types of diet containing 60% corn starch or 1 of 2 types of potato starch with different esterified P content for 1 or 5 weeks. Body weight and food intake of each rat were measured every day throughout the experimental periods. At the end of the feeding periods, the small intestine was removed to determine ALP activity in the mucosal tissues. Significant differences were observed in ALP activity in the small intestine between the 2 feeding periods, among the 4 segments of the small intestine, and among the 3 diet groups. Significant positive linear correlations between the amount of P derived from the starch and mucosal ALP activity were obtained in the jejunum and jejunoileum in rats after feeding for 5 weeks. We concluded, contrary to our hypotheses, that the ingestion of potato starch adaptively increases ALP activity in the upper part of the small intestine of growing rats in an esterified P content-dependent manner.

Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis

Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.

Effect of the apterous 56f mutation on N-acetyltransferase and alkaline phosphatase activities in Drosophila melanogaster females

Alkaline phosphatase (AP) and N-acetyltransferase (NAT) activities were studied in 1-day-old Drosophila melanogaster females of the apterous56f (ap56f) strain, having an elevated level of the juvenile hormone (JH) and a decreased level of dopamine as a result of the mutation, and in the Canton S ancestral wild-type strain in the normal conditions and upon an experimental increase in JH titer. The AP and NAT activities in ap56f females were significantly lower than in Canton S females in the norm. JH application increased the AP activity of mutant females to the level characteristic to JH-treated wild-type females.

Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue

Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.

Purification and properties of alkaline phosphatase of silkworm Bombyx mori

Alkaline phosphatase (AKP), from the succus entericus of silkworm, was purified using 10%–50% ammonium sulfate fractions, ion exchange chromatography of DEAE-Sepharose, and size exclusion chromatography of Sephacryl S-200. The purification fold was 464 times and specified activity was 3 936 U/mg. Optimum pH value of the phosphatase was 10.5, and was stable between pH 7.5 and 11. The optimum temperature of the phosphatase was 40°C and it was unstable over 50°C. K m value of the phosphatase was 1.25 mmol/L. In a given condition, the phosphatase was selectively modified by PCMB, NBS, PMSF, TNBS, SUAN, DTT, BrAc, and IAc, the results indicate that PMSF, SUA, BrAc, IAc, and TNBS could obviously inhibit the activity of the phosphatase, and the degree of inhibition depended on the concentration of these reagents. There was little effect on the activity of phosphatase after treatment by PMSF, DTT, and NBT. We primarily conclude that mercapto and imidazole are essential for AKP from silkworm. Also, Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.

Evaluation of a Chemiluminescence Method for Measuring Alkaline Phosphatase Activity in WholeMilk of Multiple Species and Bovine Dairy Drinks: Interlaboratory Study

Alkaline phosphatase (ALP) is a ubiquitous enzyme in milk with time-temperature destruction similar to that of certain pathogens destroyed in pasteurization. Measurement of ALP to indicate proper pasteurization is a common practice. Recently the public health level for ALP was decreased to 350 mU/L, a level below the sensitivity of older colorimetric ALP methods. This study was conducted within the structure of the International Dairy Federation and the International Organization for Standardization to evaluate the reproducibility of the chemiluminescence method (Charm PasLite) for ALP at 50, 100, 350, and 500 mU/L in whole milk of multiple species to meet new regulations in the United States and proposed regulations in the European Union (EU). Fifteen laboratories from 8 countries evaluated bovine, goat, sheep, and buffalo milk, bovine skim milk, 20% fat cream, and 2% fat chocolate milk. At ALP levels of 350 and 500 mU/L, the average relative standard deviation for repeatability (RSDr) was 7.5%, and the average relative standard deviation of reproducibility was (RSDR) 15%. For ALP at 100 and 50 mU/L, the average RSDr values were 10.5 and 12.6%, respectively, and the average RSDR values were 18 and 25%, respectively. The limit of detection was 20 mU/L. Results are comparable to those obtained with other enzymatic photo-activated system methods such as the fluorometric method. Results indicate that the method is suitable for measuring ALP in the milk of multiple species and in dairy drinks at U.S. and proposed EU levels.

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.

Abnormal alkaline phosphatase isoenzymes detected in the serum of elderly patients

Alkaline phosphatase (ALP) isoenzyme analysis of 101 832 serum samples was performed by electrophoresis using cellulose acetate membrane, and abnormal bands at the α<formula>1</formula> to α<formula>2</formula> globulin positions were detected in 23 samples. The physicochemical properties of these abnormal ALP isoenzyme fractions were examined. In brief, the abnormal fractions were heat-sensitive and inhibited by L-phenylalanine, and neither sialic acid in their polysaccharide chains nor the glycosyl-phosphatidylinositol anchor was detected by the enzyme treatment. The physicochemical properties of abnormal ALP isoenzyme fractions detected in Patients 1-22 were similar to those of the adult small intestine type. However, the molecular weight of the adult small intestine type abnormal fractions was smaller than that of the normal fractions. These adult small intestine type abnormal bands at the α<formula>1</formula>- to α<formula>2</formula>-globulin positions, which were identified in the serum of Patients 1-22, were detected in elderly patients. Most of them had various basal diseases such as renal insufficiency, fracture, interstitial pneumonia, and chronic pancreatitis. Some of them had severe diseases such as rectal cancer, descending colon cancer, and septic shock. In Patient 23, the polysaccharide chain had sialic acid and was heat sensitive physicochemical properties that were similar to those of the Kasahara ALP variant.

Modeling Alkaline Phosphatase Inactivation in Bovine Milk During High-Temperature Short-Time Pasteurization

Alkaline phosphatase (AP) is used as the indicator enzyme for proper pasteurization of bovine milk. Predictive modeling of AP inactivation during high-temperature short-time (HTST) pasteurization would support regulations; thus ensuring the safety of heat treated milk. Activation energy (Ea) of AP in milk was measured experimentally using the capillary tube method, and Ea was found to be 429252 J/mol. The Ea was used to develop a nonlinear model to describe the thermal inactivation of milk in a small-scale HTST pasteurizer with a plate heat exchanger. Integrated pasteurization effect (PE) was obtained at different holding temperatures (62–72°C) and holding times (3–25 s), by converting times at different temperatures in various sections of the pasteurizer to the equivalent time at the reference temperature (72°C). A nonlinear function was developed to relate the log(% residual AP activity) to PE. The r2 varied from 0.7488 to 0.8311. The validation trial indicated that the model could predict AP activity accurately for the% residual AP activity &gt;1%.

Maternal and zygotic expression of the endoderm-specific alkaline phosphatase gene in embryos of the ascidian, Halocynthia roretzi

Alkaline phosphatase (AP) activity is expressed by endodermal cells of ascidian larvae. It was reported previously that the expression of AP activity is resistant to treatment with actinomycin D, a transcription inhibitor that inhibits the appearance of several other tissue-specific molecules and morphological markers of tissue formation in developing ascidians. The resistance of AP expression to actinomycin D treatment suggests that endodermal AP activity does not depend on zygotic transcription and that its appearance is mediated by the translational activation of maternal AP mRNA present in ascidian eggs. However, it was also shown that anucleate merogons do not develop AP activity. To directly examine whether maternal AP transcripts are present in the cytoplasm of eggs, we isolated a cDNA of an endoderm-specific AP in Halocynthia roretzi and examined the temporal and spatial expressions of this gene during embryogenesis using Northern blots and in situ hybridization. Maternal AP transcripts were detected in oocytes, cleaving-stage embryos, and in gastrulae, and endoderm-specific AP transcripts dramatically increased about 14 times from the neurula stage to the larval stage in endoderm precursor cells. These results suggest that the differentiation of endoderm is primarily correlated with the activation of zygotic transcription of the AP gene, presumably by egg cytoplasmic factors, similar to how muscle and epidermis are believed to develop.

The bone isoenzyme of alkaline phosphatase in hypercalcaemic cancer patients.

Alkaline phosphatase (AP) is the classic marker of bone formation, especially in cancer patients, but the interpretation of its measurement is complicated by the existence of various circulating isoenzymes, especially of liver origin. The introduction of a mass measurement of the bone isoenzyme of AP (BAP) by an immunoradiometric assay has markedly improved the sensitivity and the specificity of the determination. We measured BAP and other markers of bone turnover in 46 patients with tumour-induced hypercalcaemia (TIH), which is an interesting model for evaluating markers of bone formation because of the uncoupling between bone formation and bone resorption found by histomorphometric techniques. The extent of bone metastatic involvement was evaluated by planimetry on bone scintigraphy. Mean (+/- S.D.) BAP concentrations were slightly higher in patients with TIH than in healthy subjects, 15.5 +/- 8.5 versus 12.4 +/- 3.5 micrograms/L (P < 0.05). However, the scatter of the data in TIH patients was quite marked. Increased values (10/46 patients, 22%) occurred only in patients with bone metastases. Total AP, gamma GT and BGP levels, as well as markers of bone resorption, were not significantly different between patients with or without bone metastases. BAP levels were significantly correlated with AP (rs = 0.63; P < 0.01) but not with BGP levels nor with markers of bone resorption. BAP levels were also correlated with the extent of bone uptake at scintigraphy (rs = 0.54; P < 0.01), but this was not the case for total AP or BGP. In the 36 patients re-evaluated when normocalcemic after pamidronate therapy, BAP levels increased from 16.3 +/- 9.2 to 22.2 +/- 21.3 micrograms/L (P < 0.05) but there were no significant changes in AP or BGP concentrations. In summary, our data confirm the existence of an uncoupling in bone turnover in TIH and indicate that cancer hypercalcaemia is another pathological condition characterised by a discordance between BAP and BGP concentrations. BAP levels appear to be a better reflection of bone metastatic involvement than total AP or BGP and their short-term increase after pamidronate therapy could reflect the recently described effects of bisphosphonates on osteoblasts.

Characterization of alkaline and acid phosphatases from skeletal muscles of young and old rats.

Alkaline phosphatase (ALP) and acid phosphatase (ACP) specific activities were measured in gastrocnemius muscles of female Wistar rats ranging in age from 2 to 30 months. ALP activity reached a peak at 12 months, with a subsequent slow decline with age. ACP activity increased sharply up to 12 months of age, followed by a slower elevation up to the age of 30 months. Using histochemical staining techniques and electron microscopy, the presence of ALP was demonstrated in the sarcolemma of gastrocnemius muscles, as well as in some capillaries around muscle fibers. ALP and ACP were isolated further from muscles of young (12 months) and old (30 months) animals by applying ion-exchange chromatography and separation on a Sephadex G-200 column. The purity of ALP was shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From a calibrated Sephadex G-200 column and SDS-PAGE, the molecular mass of ALP was determined as 116 kilodaltons (kDa), a dimer of two 56-kDa monomers. The ACP major peak of the Sephadex G-200 column revealed a molecular mass of 40 kDa. No differences in molecular mass or in amino acid analysis were detected between ALP(s) from young and old animals, indicating that most probably the decline of activity with age is due to some post-translational events, as has been shown in the past for many other enzymes and proteins.

Bovine enamel organ cells express tissue non-specific alkaline phosphatase mRNA.

Alkaline phosphatase (AP) is expressed at high levels in all mineralizing tissues, and the isoform identified in developing enamel has biochemical properties similar to that found in bone. While the bone AP is referred to as the liver/bone/kidney or tissue non-specific (TNS) form, other APs are highly specific for tissue of expression. To determine unequivocally the AP isoform made by enamel organ cells, we constructed a fetal bovine enamel organ cDNA library, which yielded eight AP cDNA clones. In each case, the DNA sequence was homologous to the partial cDNA reported for bovine kidney AP (Garattini et al., 1987). It is concluded that enamel organ cells express the TNS-AP isoform. The extended 3' untranslated region of the cDNA has considerable homology to human TNS-AP, and the conservation of sequence suggests that the 3' end may have a role in post-transcriptional regulation of expression.

Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity in serum

Alkaline phosphatase (ALP) is present in human serum in the form of several isoenzymes. The two major circulating ALP isoenzymes, bone and liver, are difficult to distinguish because they are the products of a single gene and differ only by posttranslational glycosylation. Quantitative measurement of bone ALP (BAP) activity in serum can provide an index for the rate of bone formation. Furthermore, increased BAP activity in serum is indicative of bone disorders. We describe a method in which serum samples are added to a microtiter plate coated with monoclonal anti-BAP antibody and incubated 3 h at room temperature. After the unbound materials are washed off, the bound BAP activity is measured by adding p-nitrophenyl phosphate substrate. The assay demonstrated no cross-reactivity to intestinal or placental ALP and only 3-8% cross-reactivity to liver ALP. The intraassay (n = 21) CVs were 3.9-5.9%, and interassay (n = 8) CVs were 4.4-7.0%. Comparisons of the assay (y) with an IRMA (x) and a wheat germ agglutinin precipitation method (x') gave regression equations of y = 1.32x-6.4, r = 0.99, and y = 1.41x' + 4.8, r = 0.99. The assay detected increased BAP in sera from patients with osteoporosis, Paget disease, osteomalacia, or primary hyperparathyroidism.

Phospholipase Resistance of the Glycosyl-Phosphatidylinositol Membrane Anchor on Human Alkaline Phosphatase

Alkaline phosphatase (ALP) is attached to the cell surface in mammalian tissues via a glycosyl-phosphatidylinositol (GPI) anchor and can be released from the membrane by GPI-specific phospholipases. In a range of cultured human cell lines, however, the sensitivity of ALP to phospholipases was observed to be variable in magnitude (approximately 20-90%). The mechanism of phospholipase resistance was explored with phospholipases of different bond specificities. The results suggest that phospholipase resistance is the result of acylation of the inositol ring in the GPI anchor. The occurrence of phospholipase-resistant forms of ALP may have important implications for the in vivo release and disposition of plasma ALP.

Two alkaline phosphatase genes are expressed during early development in the mouse embryo

Alkaline phosphatase (AP) activity is stage specific in mouse embryos and may be associated with compaction and separation of trophectoderm from inner cell mass in preimplantation development. We previously sequenced a cDNA and two mouse AP genes that could contribute to the AP activity in embryos. Oligonucleotide primers were constructed from the three sequences and used in the reverse transcription-polymerase chain reaction technique to establish that two of the three AP isozymes are transcribed during preimplantation development. The predominant transcript (E-AP) is from a gene highly homologous to the human tissue-specific APs, but different from the mouse intestinal AP. Tissue non-specific (TN) AP also is transcribed, but there is approximately 10 times less TN-AP than E-AP transcript. The TN-AP isozyme is the predominant transcript of 7 to 14 day embryos and primordial germ cells. A switch in predominance from E-AP to TN-AP must occur during early postimplantation development. This study establishes a framework for experiments to determine the functions of the two isozymes during preimplantation development.

Further characterization of serum alkaline phosphatase from male and female beagle dogs.

Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.