Alkaline Phosphatase

operative period and in the post-operative period on days 10, 20, 30, 60 and 90 using physical and X-ray examinations. At the same time points, blood samples were taken to evaluate tALP, BALP and TRAP serum activities and also serum mineral concentrations. Additionally, blood samples were also collected in a sample of nine healthy and skeletally mature dogs (range 2 to 6 years old) to constitute a control group. In these animals the same bone biomarkers were measured every ten days and the mean performed. All samples were collected into plain tubes containing a clot activator at the same hour in the morning, in order to minimize the possible effect of circadian rhythm. The specimens were processed within 30 minutes after the blood collection, centrifuged at 2000xg during 10 minutes and the resulting serum decanted into 2mL polypropylene cryovials. Serum samples were frozen and stored at -80ºC until assayed. All samples were analyzed in duplicate and the mean value obtained was used for statistical analysis. Commercial reagent kits were used for the determinations of tALP (Alkaline Phosphatase ® , ref. 7D55-20, Abbott) and TRAP (Roche ACP®, ref. 04375351190) serum activities and serum minerals concentrations (Calcium Abbott, ref. 7d61-20; Phosphorus Abbott, ref. 7d71-2) by spectrophotometry. BALP serum activity was measured with a reagent kit (MicroVue BAP EIA, Quidel Corporation) by ELISA.

exhaustively washed using distilled water and newly dehydrated. Ten to twenty milligrams of rat demineralized diaphyseal bone matrix was introduced through a small incision in the dorsal subcutaneous tissue of anesthetized young male Wistar rats (50-60 g). Fourteen days after implantation (the period required for the development of maximal alkaline phos- phatase activity), the newly formed plate was removed, rinsed in ice-cold 0.9% (w/v) NaCl, and used for microscopical examination and for alkaline phosphatase extraction (15).

Objective: The study aims to evaluate the effect of alendronate (ALD) on bone-specific alkaline phosphatase (BALP) serum levels on periodontal bone loss in Wistar rats. Design: Periodontitis was induced by ligature around the upper second molar in 36 male Wistar rats (200 g). Groups of six animals received 0.9% saline (SAL) or ALD (0.01; 0.05; 0.25 mg kg 1 , s.c.), over 11 days; then they were sacrificed and their maxillae were removed

A constitutive alkaline phosphatase was purified to apparent homoge- neity as determined by polyacrylamide gel electrophoresis from myce- lia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30 o

a wide variety of phytoplankton species in response to the condition of limited phosphorus. Highly stable in seawater, it is a membrane-associated enzyme that catalyzes the hydrolysis of phosphorus-containing organic compounds such as phosphate esters (Fitzgerald & Nelson 1966; Graziano et al. 1996; Dyhrman 2005; Pandey & Parveen 2011), to obtain orthophosphate, a form of inorganic phosphorus directly available to organisms (Pandey & Parveen 2011). In other words, alkaline phosphatase facilitates the use of dissolved organic phosphorus (DOP) by phytoplankton when dissolved inorganic phosphorus (DIP) is limited in the environment (Lin et al. 2011). The activity of alkaline phosphatase is related to different phosphorus concentrations (Hernández et al. 2002), with it being produced when cells are experiencing phosphate deprivation or it being suppressed in conditions of sufficient phosphate (Kuenzler & Perras 1965; Fitzgerald & Nelson 1966; Grainger 1989; Vance et al. 2003).

Introduction: Interpreting laboratory tests requires reference intervals (RI) that may vary between different populations. For the diagnosis of hypophosphatasia (HPP), lower limits of alkaline phosphatase (ALP) levels must be determined. Objective: To transfer the RI findings for ALP obtained by the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER) in children and adolescents, adjusted for the Brazilian population. Methods: The ALP measures from 1690 subjects (aging from 1-18 years) were analyzed. The CALIPER subgroups and the Clinical and Laboratory Standards Institute (CLSI) guideline were used for validation. Inclusion criteria were patients with normal range of hepatic and renal function, bone metabolism, and blood counts. Exclusion criteria were hospitalization, low weight, and use of drugs that could interfere in the ALP measurement and patients in with more than three orders for ALP measuring test. The RI obtained were considered valid if more than 90% of patients were whitin of the CALIPER RI. Results: The ALP RI results (IU/l) obtained were: 149-301 for both sexes aged 1-9 years; 127-326 for both sexes aged 10-12 years; 62-212 for girls and 129-437 for boys aged 13-14 years; 52-120 for girls and 78-268 for boys aged 15-16 years; 45-97 for girls and 40-129 for boys aged 17-18 years. In 92.4% of the patients, the results were comparable with those of the CALIPER study. Conclusion: The results demonstrated that the ALP RI for Brazilian children and adolescents are comparable to the CALIPER study in 92.4% of the patients and can be used for this population. Key words: alkaline phosphatase; child; reference intervals.

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p - nitrophenol released from p -nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p - nitrophenol·mg protein -1 ·min -1 ): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP

ABSTRACT: Rheumatoid arthritis is a chronic systemic autoimmune disorder causing inflammation of the synovial joints leading to joint destruction as well as a variety of extra articular features. In this present study, serum Adenosine deaminase and Alkaline phosphatase levels were studied in patients of rheumatoid arthritis. Thirty patients in the age group of 30-60 years of both sexes and equal number of age and sex matched healthy controls were included in this study. In rheumatoid arthritis patients, serum Adenosine deaminase levels were high, the mean serum Adenosine deaminase levels were 60 ± 9.55 and in controls the mean Adenosine deaminase levels were 21 ± 3.04. The p value was significant at p < 0.001. We conclude that serum Adenosine deaminase can be used as a marker for cell mediated immunity to monitor disease activity in rheumatoid arthritis. Serum Alkaline phosphatase was found to be raised in rheumatoid arthritis, the mean serum Alkaline phosphatase levels were 291.63 ± 35.84 in patients of rheumatoid arthritis when compared to healthy controls (196.73 ± 32.71).The p value was significant at p<0.001. The present study has focused on investigating the serum total Alkaline phosphatase levels in Rheumatoid Arthritis Patients and The findings have indicated a raised serum Alkaline phosphatase in patients of rheumatoid arthritis when compared to healthy controls thereby suggesting the role of serum Alkaline phosphatase as a marker of disease activity in rheumatoid arthritis.

30. Heemskerk S, Masereeuw R, Moesker O, Bouw MP, van der Hoeven JG, Peters WH, et al. APSEP Study Group. Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients. Crit Care Med. 2009; 37: 417–423. doi: 10.1097/CCM.0b013e31819598af PMID: 19114895 31. Deji N, Kume S, Araki S, Soumura M, Sugimoto T, Isshiki K, et al. Structural and functional changes in the kidneys of high-fat diet-induced obese mice. Am J Physiol Renal Physiol.2009; 296: F118–126. doi: 10.1152/ajprenal.00110.2008 PMID: 18971213

Fig 3. Expressions of Alpl mRNA and alkaline phosphatase (AP) activity in the early pregnant uterus. (A) The levels of Alpl mRNA were determined by qRT-PCR. Results were normalized to the housekeeping gene Rpl7 and presented as the mean ± SD of three separate experiments, with different letters (a, b, c) indicating statistical difference (p < 0.05; one-way ANOVA and Tukey’s test). (B) Cell-type specific localization of Alpl mRNA was determined by in situ hybridization (n = 3). Inserts show higher magnification (200X) of Alpl mRNA accumulations. (C) AP activity patterns in the periimplantation uterus (n = 6). (D) Levamisole and L-phenylalanine were used as inhibitors of tissue-nonspecific AP (TNAP) and tissue-specific AP (TSAP), respectively. (E) TNAP activity localization in cells of the deciduum and deciduomata (n = 5). The embryo-induced decidua (deciduum) was collected on day 6 of pregnancy. The oil-induced decidua (deciduomata) was collected 48 h after intrauterine instillation of oil in day 4 pseudopregnant mouse. bl, blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium; pdz, primary decidual zone; s, stroma; sdz, secondary decidual zone.

Some of the gingival fragments, after collection, were placed in PBS (phosphate-buffered saline; 0.01 M, pH 7.3) at 4ºC, wrapped in aluminum foil, and put in the freezer at -82ºC until use. Histological sections with a thickness of 6.0 µm were prepared using a cryostat (Cryostar, UK) at -22ºC. For each fragment, four slides were prepared and stained in duplicate using HE and the histochemical technique. To show ALP activity, an azo dye associated with naphthol was used. The tissue sections were submerged in incubation solution at room temperature for 30 minutes. The incubation solution was made of 10 mL stock solution [25 mg naphthol-AS-BI phosphate; 10 mL N’-N’–dimethylformamide; 1.0 M sodium carbonate (Sigma, USA); distilled water, 0.2 M Tris-HCL buffer; pH 8.3], to which 10 mg of Fast Red TR Salt (Sigma, USA) was added. The tissue sections were then stained with Mayer’s hematoxylin and mounted in glycerine gel (Sigma, USA). Positive and negative controls for the histochemical reaction consisted of specimens of mouse kidney and liver incubated in solutions with and without Fast Red TR Salt, respectively. The tissue sections were evaluated using an immersion objective (1000x). Alkaline phosphatase activity was assessed subjectively by one researcher, considering the location (epithelium and/or connective tissue) and intensity of the reactions: a) strongly positive (dark red), b) weakly positive (light pink), and c) absent.

Total Protein and Alkaline Phosphatase Quantiication Ater 4, 7 and 14 days of plating, the wells were washed three times with slightly warmed PBS and 2 ml of 0.1% Lauryl Sulfate (Sigma) was placed in each well, then let in ambient temperature for 30 minutes. Aliquots of 1 ml of this solution were withdrawn and placed in test tubes along with 500μl of Lowry Solution (Sigma) for 20 minutes at ambient temperature. hen, 250μl of Folin reagent (Sigma) was added, kept for 30 minutes and read using a spectrophotometer (Biomate 3 - hermo Scientiic, Waltham, MA, USA) at 600 nm. he total protein content was calculated, based on a standard curve and expressed as concentration.

Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the São Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C- 8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of São Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.

ABSTRACT - Plant growth promoter microorganisms have been studied as important tools for increasing crop production. Lettuce is the most consumed hardwood crop in the world. Numerous microorganisms are capable of acting in a beneficial way in the growth of this culture. The objective of the present study was to evaluate the efficacy of Trichoderma and Pseudomonas on the microbial biomass, enzymatic activities in sandy soil and lettuce production. The experimental design was completely randomized with ten replicates and treatments: CONT (absolute control); CM (control with cattle manure fertilization); CMB (with fertilization and Pseudomonas sp.); CMF (with fertilization and T. aureoviride) and CMBF (with fertilization and the two microorganisms combined). The fertilizer used was organic with cattle manure in a dose recommended for the culture. This study evaluated the production of lettuce, microbial biomass and the enzymatic activity of acid phosphatase, alkaline phosphatase and urease. The combined application of CMBF was efficient in increasing lettuce production, because it increased 85% of the cv. Veronica cultivated on sandy soil. The combined use of plant growth promoting microorganisms resulted to an increase in microbial biomass. In lettuce crops, it is recommended to use T. aureoviride URM 5158 and Pseudomonas sp. UAGF 14 in lettuce crops, because improved lettuce production, improves the biochemical quality of soils measured by absolute and specific enzymatic activities per unit of microbial biomass.

S. Pullorum are not often present in feces. In these conditions, ELISA can be performed with peroxidase or alkaline phosphatase conjugates. However, the test was more specific when using the alkaline phosphatase conjugate (Table 1). Therefore, the alkaline phosphatase ELISA can be used as a serological test for monitoring poultry flocks to search for Salmonella specific IgG and could be a useful to examine serum samples, egg yolk and dried blood from any kind of poultry flock (Barrow, 1994a).

Abe et al. (8) showed differential expression of alkaline phosphatase (ALP) activity in cell membranes of fibroblasts according to tissue localization in an ultra-structural study of chronically inflamed gingiva. The majority of fibroblasts in the inflamed connective tissue exhibited intense ALP activity, while fibroblasts in the non-inflamed adjacent connective tissue were slightly stained or showed no enzymatic membrane activity. In this study, ALP activity was only observed in adherent fibroblasts. ALP is recognized as a protein that binds to glycosyl-phosphatidyl-inositol. It is also known that this enzyme expressed in gingival fibroblasts is susceptible to cleavage by PI-PLC (1,9). Thus, proteins bound to glycosyl-phosphatidyl-inositol are scarce in the cytoplasm and possess particular physical properties. For example, previous studies showed that ALP exhibits elevated lateral mobility over the cytoplasmic membrane plane (10). Therefore, the polarized localization of ALP membrane activity observed in vitro could describe a fast ALP distribution over adherent cells.

μg of pili in the first well) was added. After wells were washed as previously described, goat pAb to E. coli in gelatin blocking buffer (1:5,000) (or for wells with fimbriae, anti-CS2/CFA/I [2 nd bleed/Ist bleed respectively; 1:1,000) was added for 1 h at RT. Following another washing step, alkaline phosphatase-conjugated rabbit anti- goat immunoglobulin G whole molecule (Sigma) or goat anti- rabbit immunoglobulin G whole molecule (Sigma) in gelatin blocking buffer (1:1,000) was added for 1 h at 37°C. Wells without erythrocytes, or without asialo-GM1 were used as controls. The bound enzyme was detected by the addition of alkaline phosphatase substrate (PNPP, Sigma 71768) diluted in diethanolamine buffer per well for 5 to 30 min at 37°C. The control wells were treated in the same manner except that blank control wells had no bacteria or pili. Absorbance was measured at OD 405 nm with a microplate reader (POLAR star Omega). Each

Osteoporosis is a major health problem. Little is known about the risk factors in premenopause. Sixty 40-50-year old patients with regular menses were studied cross-sectionally. None of the patients were on drugs known to interfere with bone mass. Patients answered a dietary inquiry and had their bone mineral density (BMD) measured. The Z scores were used for the comparisons. A blood sample was taken for the determination of FSH, SHBG, estradiol, testosterone, calcium and alkaline phosphatase. Calcium and creatinine were measured in 24-h urine. A Z score less than -1 was observed for the lumbar spine of 14 patients (23.3%), and for the femur of 24 patients (40%). Patients with a Z score less than -1 for the lumbar spine were older than patients with a Z score ≥ -1 (45.7 vs 43.8 years) and presented higher values of alkaline phosphatase (71.1 ± 18.2 vs 57.1 ± 14.3 IU/l). Multiple regression analysis showed that a lower lumbar spine BMD was associated with higher values of alkaline phosphatase, lower calcium ingestion, a smaller body mass index (BMI), less frequent exercising, and older age. The patients with a Z score less than -1 for the femur were shorter than patients with a Z score ≥-1 (158.2 vs 161.3 cm). Multiple regression analysis showed that a lower femoral BMD was associated with lower BMI, higher alkaline phosphatase and caffeine intake, and less frequent exercising. A lower than expected BMD was observed in a significant proportion of premenopausal women and was associated with lower calcium intake, relatively lower physical activity and lower BMI. We conclude that the classical risk factors for osteoporosis may be present before ovarian failure, and their effect may be partly independent of estrogen levels.

Table 2 - Serum levels of alanine aminotransferase (ALT), alkaline phosphatase (AP), urea (U), creatinine (Cr), hematocrit (Ht) and hemoglobin (Hb) and Urine specific gravity, urinary concentrations of creatinine (UCr) and gamma-glutamyltransferase (GGT), urinary GGT-creatinine ratio, fractional excretion of sodium (FENa), urinary protein-creatinine ratio and urinary albumin- creatinine ratio obtained from twelve cats subjected to hypotension with isoflurane and pretreated (PRE) or treated after hypotension (POST) with 25mgkg -1 of tepoxalin during 5 days. Values of ALT, AP, U, Cr, Ht, Hb, UCr and FENa expressed as

We have demonstrated that the enzyme form lacking a hydrophobic anchor (PLSAP) exhibits surface activity due to the adsorption of the polypeptide moiety of the enzyme at the air/water interface. As expected, the hydrophobic anchor-containing form (DSAP) has a higher surface activity due to the presence of the hydrophobic anchor. The elasticity of alkaline phosphatase at a certain frequency and amplitude depends on the following factors: presence of the GPI anchor, concentration, surface pressure, solubility induced by non-ionic surfactant and surface packing. The anchor-containing form (DSAP) has an elasticity modulus higher than the lacking-anchor form (PLSAP) at the same surface pressure. In the studied frequency, the elasticity modulus is associated to the CAC of the surfactant/enzyme system. Since PLSAP has a high mobility due to its hydrophilic character and low concentration, it displays elasticity values that decrease with concentration. This work will certainly help the understanding of the rheological behavior of enzyme/ phospholipid interfaces and also state the differences in the dilatational elasticity of the liquid interface in the presence of enzymes.