Alkaline Phosphatase Isoforms Research Articles

Bone turnover prediction in patients with chronic kidney disease (CKD) undergoing hemodialysis using shortened dynamic 18F-NaF PET/CT Ki–Patlak

This study investigated whether Ki–Patlak derived from a shortened scan time for dynamic 18F-NaF PET/CT in chronic kidney disease (CKD) patients undergoing hemodialysis can provide predictive accuracy comparable to that obtained from a longer scan. Twenty-seven patients on chronic hemodialysis, involving a total of 42 scans between December 2021 and August 2023 were recruited. Dynamic 18F-NaF PET/CT scans, lasting 60–90 min, were immediately acquired post-injection, covering the mid-twelfth thoracic vertebra to the pelvis region. Ki–Patlak analysis was performed on bone time–activity curves at 15, 30, 45, 60, and 90 min in the lumbar spine (L1–L4) and both anterior iliac crests. Spearman’s rank correlation (rs) and interclass correlation coefficient were used to assess the correlation and agreement of Ki–Patlak between shortened and standard scan times. Bone-specific alkaline phosphatase (BsAP) and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) were tested for their correlation with individual Ki–Patlak. Strong correlations and good agreement were observed between Ki–Patlak values from shortened 30-min scans and longer 60–90-min scans in both lumbar spine (rs = 0.858, p < 0.001) and anterior iliac crest regions (rs = 0.850, p < 0.001). The correlation between BsAP and Ki–Patlak in the anterior iliac crests was weak and statistically insignificant. This finding suggests that a proposed shortened dynamic 18F-NaF PET/CT scan is effective in assessing bone metabolic flux in CKD patients undergoing hemodialysis, offering a non-invasive alternative approach for bone turnover prediction.

Musculoskeletal pain and muscular weakness as the main symptoms of adult hypophosphatasia in a Spanish cohort: clinical characterization and identification of a new ALPL gene variant.

Hypophosphatasia (HPP) is a rare inherited disorder, caused by mutations in the alkaline phosphatase (ALPL) gene, which encodes for the tissue non-specific alkaline phosphatase (TNSALP) isoform of alkaline phosphatase (ALP). Adult HPP is one of the mild forms that presents with unspecific signs such as osteopenia, osteomalacia and muscle involvement. Our purpose was to identify and characterize possibly misdiagnosed adult HPP patients at a clinical and biochemical level. At the laboratory of Miguel Servet University Hospital we retrospectively reviewed serum ALP levels in adults over a 48-month period. The clinical records of individuals with consistently low ALP levels were reviewed to exclude secondary causes. Those with persistent hypophosphatasemia were screened for symptoms of HPP. The study participants were evaluated at biochemical and genetic levels. We identified 705 ALP determinations (out of 384,000 processed) in 589 patients below the reference range (30 U/l). Only 21 patients with clinical signs and symptoms of HPP were selected for genetic testing. Finally, only 12 patients participated in the study, 83.3% of whom (10/12) harbored a pathogenic or likely pathogenic variant in a heterozygous state. The major symptoms of our cohort were the presence of musculoskeletal pain (100% of patients) and muscular weakness (83.3% patients). Mild HPP patients presenting with diffuse symptoms such as musculoskeletal pain may be undiagnosed or misdiagnosed as osteoporosis patients by routine diagnosis. It is important to identify these individuals, to avoid inappropriate treatment with antiresorptive drugs.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

Deglycosylation Differentially Regulates Weaned Porcine Gut Alkaline Phosphatase Isoform Functionality along the Longitudinal Axis

Gut alkaline phosphatases (AP) dephosphorylate the lipid moiety of endotoxin and other pathogen-associated-molecular patterns members, thus maintaining gut eubiosis and preventing metabolic endotoxemia. Early weaned pigs experience gut dysbiosis, enteric diseases and growth retardation in association with decreased intestinal AP functionality. However, the role of glycosylation in modulation of the weaned porcine gut AP functionality is unclear. Herein three different research approaches were taken to investigate how deglycosylation affected weaned porcine gut AP activity kinetics. In the first approach, weaned porcine jejunal AP isoform (IAP) was fractionated by the fast protein-liquid chromatography and purified IAP fractions were kinetically characterized to be the higher-affinity and lower-capacity glycosylated mature IAP (p < 0.05) in comparison with the lower-affinity and higher-capacity non-glycosylated pre-mature IAP. The second approach enzyme activity kinetic analyses showed that N-deglycosylation of AP by the peptide N-glycosidase-F enzyme reduced (p < 0.05) the IAP maximal activity in the jejunum and ileum and decreased AP affinity (p < 0.05) in the large intestine. In the third approach, the porcine IAP isoform-X1 (IAPX1) gene was overexpressed in the prokaryotic ClearColiBL21 (DE3) cell and the recombinant porcine IAPX1 was associated with reduced (p < 0.05) enzyme affinity and maximal enzyme activity. Therefore, levels of glycosylation can modulate plasticity of weaned porcine gut AP functionality towards maintaining gut microbiome and the whole-body physiological status.

Novel insights into plant defensin ingestion induced metabolic responses in the polyphagous insect pest Helicoverpa armigera

Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera.

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae.

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culex mosquito larvae than its closely related toxin—Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed in Spodoptera frugiperda (Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both Cq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP—a Cry4Ba toxin receptor. In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALP expressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain—DII, including Thr512, Tyr513 and Lys514 in the β10-β11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain—DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected β10-β11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatus larvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform—Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

Evaluation of Diagnostic Role of Alkaline Phosphatase in the Patients of Cerebral Stroke

Background: The cerebral stroke is one of the most common disorders in South Asia. The Pakistani adult population suffered from cerebral strokes in more than 10% of cases. The TNAP isoform of Alkaline Phosphatase is also found in the brain. It is possible to diagnose cerebral stroke patients based on their serum ALP level. In this study, the objective is to evaluate the level of ALP in patients suffering from cerebral strokes. Methodology: This cross-sectional study was conducted at the Department of Medicine at the LUMHS Jamshoro. A total of 55 patients (26 males &amp; 29 females) with cerebral stroke between the ages of 40-70 years were selected according to the inclusion &amp; exclusion criteria between the ages of 40-70 years. By using the AMP (2-amino, 2-methyl, 1 propanol) method, we were able to estimate the serum level of ALP. Results: In 29.1% of the patients, the mean serum ALP level was 85.13 ± 10.93 U/L, while in 70.9% of the patients, the mean serum ALP level was 231 .42 ± 5.76 U/L. As a result of this study, it can be concluded that approximately 70% of cerebral stroke patients had elevated levels of serum ALP levels, which can be used as a diagnostic tool for cerebral stroke patients. Conclusion: Cerebral stroke patients had elevated serum ALP levels, which are a cheap and easily quantifiable biomarker. Keywords: Cerebral Stroke, Alkaline Phosphatase, TNAP, Isoenzym

Alkaline Phosphatase: An Old Friend as Treatment Target for Cardiovascular and Mineral Bone Disorders in Chronic Kidney Disease.

Alkaline phosphatase (ALP) is an evolutionary conserved enzyme and widely used biomarker in clinical practice. Tissue-nonspecific alkaline phosphatase (TNALP) is one of four human isozymes that are expressed as distinct TNALP isoforms after posttranslational modifications, mainly in bone, liver, and kidney tissues. Beyond the well-known effects on bone mineralization, the bone ALP (BALP) isoforms (B/I, B1, B1x, and B2) are also involved in the pathogenesis of ectopic calcification. This narrative review summarizes the recent clinical investigations and mechanisms that link ALP and BALP to inflammation, metabolic syndrome, vascular calcification, endothelial dysfunction, fibrosis, cardiovascular disease, and mortality. The association between ALP, vitamin K, bone metabolism, and fracture risk in patients with chronic kidney disease (CKD) is also discussed. Recent advances in different pharmacological strategies are highlighted, with the potential to modulate the expression of ALP directly and indirectly in CKD–mineral and bone disorder (CKD-MBD), e.g., epigenetic modulation, phosphate binders, calcimimetics, vitamin D, and other anti-fracture treatments. We conclude that the significant evidence for ALP as a pathogenic factor and risk marker in CKD-MBD supports the inclusion of concrete treatment targets for ALP in clinical guidelines. While a target value below 120 U/L is associated with improved survival, further experimental and clinical research should explore interventional strategies with optimal risk–benefit profiles. The future holds great promise for novel drug therapies modulating ALP.

Reappraisal of the Role of Alkaline Phosphatase in Hepatocellular Carcinoma.

Background: Alkaline phosphatase (ALP) is a marker of liver function and is associated with biliary tract disease. It was reported as a prognostic factor for hepatocellular carcinoma (HCC). The genetic expression in tumor-tissue microarrays and the perioperative serologic changes in ALP have never been studied for their correlation with HCC prognosis. Methods: The genetic expression of ALP isoforms (placental (ALPP), intestinal (ALPI) and bone/kidney/liver (ALPL)) was analyzed in tumor and non-cancerous areas in 38 patients with HCC after partial hepatectomy. The perioperative change in ALP was further analyzed in a cohort containing 525 patients with HCC to correlate it with oncologic outcomes. A total of 43 HCC patients were enrolled for a volumetry study after major and minor hepatectomy. Results: The genetic expression of the bone/kidney/liver isoform was specifically and significantly higher in non-cancerous areas than in tumors. Patients with HCC with a higher ALP (>81 U/dL) had significantly more major hepatectomies, vascular invasion, and recurrence. Cox regression analysis showed that gender, major hepatectomies, the presence of satellite lesions, higher grades (III or IV) and perioperative changes in liver function tests were independent prognostic factors for recurrence-free survival, and a postoperative increase in the ALP ratio at postoperative day (POD) 7 vs. POD 0 > 1.46 should be emphasized. A liver regeneration rate more than 1.8 and correlation analysis revealed that the ALP level at POD 7 and 30 was significantly higher and correlated with remnant liver growth. Conclusions: This study demonstrated that the perioperative ALP change was an independent prognostic factor for HCC after partial hepatectomies, and the elevation of ALP represented a functional biomarker for the liver but not an HCC biomarker. The higher regeneration capacity was possibly associated with the elevation of ALP after operation.

The Novel Bone Alkaline Phosphatase Isoform B1x Is Associated with Improved 5-Year Survival in Chronic Kidney Disease.

Circulating alkaline phosphatase (ALP) is an independent cardiovascular risk marker. Serum bone ALP (BALP) isoforms indicate bone turnover and comprise approximately 50% of total circulating ALP. In chronic kidney disease (CKD), mortality is highest in patients with increased ALP and BALP and low bone turnover. However, not all low bone turnover states are associated with increased mortality. Chronic inflammation and oxidative stress, features of protein energy wasting syndrome, induce cardiovascular BALP activity and fibro-calcification, while bone turnover is suppressed. Circulating BALP isoform B1x is associated with low ALP and low bone turnover and has been exclusively detected in CKD. We investigated the association of serum B1x with survival, abdominal aortic calcification (AAC) score, and aortic pulse wave velocity (PWV) in CKD. Serum ALP, BALP isoforms, parathyroid hormone (PTH), PWV, and AAC were measured repeatedly over 2 years in 68 prevalent dialysis patients. Mortality was assessed after 5 years. B1x was detected in 53 patients. A competing risk analysis revealed an association of B1x with improved 5-year survival; whereas, baseline PWV, but not AAC score, predicted mortality. However, PWV improved in 26 patients (53%), and B1x was associated with variation of PWV over time (p = 0.03). Patients with B1x had lower PTH and total ALP, suggesting an association with lower bone turnover. In conclusion, B1x is associated with time-varying PWV, lower circulating ALP, and improved survival in CKD, and thus may be an indicator of a reduced cardiovascular risk profile among patients with low bone turnover.

Gamma Glutamyl Transferase Activity Has Limited Utility in Assessment of Alkaline Phosphatase Elevations.

As part of an ongoing effort to improve healthcare value for patients, laboratories increasingly implement test utilization review. Alkaline phosphatase (ALP) isoenzymes (hereafter: isoenzymes) testing distinguishes the various ALP isoforms to explain elevations in total serum ALP. Gamma glutamyl transferase activity (GGT) has served as a proxy for total ALP elevations attributable to the hepatic isoform given that both are membrane-bound proteins with a shared mechanism of release. We assessed the utility of GGT in evaluating isoenzymes requests. We obtained 8 years of isoenzymes results and identified same-patient GGT measurements obtained within 7 days. We assessed the ability of GGT to predict elevations in hepatic, bone, intestinal, and nonhepatic ALP isoforms overall. We generated ROC curves and calculated sensitivity and specificity using our in-house reference limits for GGT. GGT as a predictor of hepatic isoform elevation had an area under the ROC curve (AUC) of 0.68, and GGT activity above the upper reference limit was 46.6% sensitive and 85.0% specific for hepatic ALP elevation. GGT activity as a predictor of nonhepatic isoform elevation had an AUC of 0.52, and GGT within reference limits was 59.8% sensitive and 46.4% specific for elevation in a nonhepatic ALP isoform. In 133 individuals with hepatic isoform elevations, 93 had a concurrent elevation in a nonhepatic ALP isoform. GGT was reasonably specific but insensitive for hepatic ALP isoform elevation and was a poor predictor of ALP isoform elevation overall, suggesting that its usefulness in evaluating isoenzymes orders is limited.

Clinical Application of the Ratio of Serum Bone Isoform to Total Alkaline Phosphatase in General Practice.

Alkaline phosphatase (ALP) is an enzyme that is expressed in a variety of tissues. Among the isoforms of ALP, bone-specific alkaline phosphatase (BAP) is used as a marker for evaluating bone metabolism. We investigated the clinical usefulness of the ratio of serum BAP to total ALP for the diagnosis of various disorders in general practice. We retrospectively analyzed the cases of 107 Japanese patients whose serum BAP levels were exam-ined, focusing on clinical characteristics. We observed that the BAP/ALP ratios of the patients with fever and those with inflammatory diseases were significantly lower than the ratios of other patient groups. The BAP/ALP ratios of the patients with osteoporosis and those with metabolic bone diseases were higher than those of the patients with other conditions. The BAP/ALP ratio was found to be negatively correlated with age, a cor-relation that has not been found in other ethnicities. The serum BAP/ALP ratio was inversely correlated with serum CRP levels but was positively correlated with serum albumin levels and hemoglobin concentrations. Collectively, our results suggest that the BAP/ALP ratio could be a useful predictor for important geriatric con-ditions seen in general practice.

Altered levels of BMD, PRL, BAP and TRACP-5b in male chronic patients with schizophrenia

Bone mineral density (BMD) has been found to decrease in schizophrenia patients. We examined BMD and the levels of prolactin (PRL), bone alkaline phosphatase (BAP) and tartrate resistant acid phosphatase isoform 5b (TRACP-5b) in male chronic schizophrenia patients and compared them with healthy controls in a Chinese Han population, which has not been reported before. Male patients with chronic schizophrenia (SPs; n = 79) and healthy controls (HCs; n = 56) were recruited. BMD and plasma PRL, BAP and TRACP-5b levels were measured and compared between the two groups. The SPs group was further divided into two subgroups: the elevated PRL group (PRL ≥ 25 ng/ml, EPRL; n = 38) and the normal PRL group (PRL < 25 ng/ml, NPRL; n = 41) in accordance with PRL levels. The levels of BAP and TRACP-5b were measured using sandwich enzyme-linked immunosorbent assay (ELISA) while serum PRL was measured with an Access Immunoassay Analyzer. BMD was determined by quantitative computed tomography. BMD levels significantly decreased and serum PRL and TRACP-5b levels were significantly higher in male chronic schizophrenia patients. The EPRL group had remarkably lower BMD and BAP level and higher TRACP-5b levels compared with the NPRL group and HCs. Moreover, there was a negative correlation between BMD and TRACP-5b in the EPRL group. We found that BMD, BAP and TRACP-5b levels in the EPRL group were significantly different than HCs and the NPRL group. PRL levels in schizophrenia patients may be related to BMD and bone metabolism. Monitoring BMD and markers of bone metabolism in clinical practice may therefore be helpful to understand the bone health status of schizophrenia patients.

P1430BETA C-TERMINAL TELOPEPTIDE OF TYPE I COLLAGEN: CORRELATIONS WITH MINERAL METABOLISM PARAMETERS AND CARDIAC VALVULAR CALCIFICATIONS IN A COHORT OF HEAMODIALYSIS PATIENTS

Abstract Background and Aims Beta-carboxyterminal telopeptide of type I collagen (beta-CTx) is considered a marker of bone resorption in patients affected by osteoporosis. Nevertheless, its role in mineral bone disorder (MBD) of CKD patients is not clear. The aim of this study was to explore in a group of haemodialysis patients (HDp): 1) The levels of beta-CTx; 2) The correlation between beta-CTx levels and other mineral metabolism (MM) parameters; 3) The association between beta-CTx and CKD-MBD specific therapy 4) The relation between beta-CTx and cardiac valvular calcifications (VC). Method We studied a group of 21 HDp (F/M 9/12, age 68[54-78] years) randomly selected from the HDp actually followed-up in our Department. Dialysis vintage was 52[23-80] months. All HDp were on a three-weekly hemodialysis schedule, with a prescribed dialysate calcium of 1.5 mmol/l. Before the first dialysis session of the week, after the long interdialytic interval, the levels of beta-CTx, total and ionized calcium (tCa and iCa), Phosphorus (P), Magnesium (Mg), intact PTH (iPTH), total and bone isoform of alkaline phosphatase (tALP and bALP), 25-hydroxyvitamin D (25OHVD), 1,25-dihydroxyvitamin D (1,25OH2VD), and other general biochemical parameters were measured (Figure 1). At the same time, data concerning MM therapies were recorded. In all the studied patients, the presence of VC was searched in the the most recent echocardiographic study. Results Beta-CTx levels showed a significant positive correlation with tALP (r 0.92, p &amp;lt;0.0001), bALP (r 0.89, p &amp;lt;0.0001) and iPTH (r 0.87, p&amp;lt;0.0001) and a negative correlation with tCa (r – 0.43, p=0.05) and iCa (r – 0.52, p=0.01). No significant correlation was found with the other MM and general biochemical parameters and with the general features of the studied cohort. 18 patients (85.7%) were taking P binders (72.2% Ca-based and 27.8% Ca-free). In 20 patients (95.2%) vitamin D therapy, either in the natural, active or both natural and active form (35%, 15% and 50%, respectively) was prescribed. 6 patients (28.6%) were in therapy with Cinacalcet. No difference was found in beta-CTx levels according to the presence and the type of each of the above mentioned therapies. Conclusion Taking into account the limitations of this preliminary small size observational study, our data showed that beta-CTx levels are high in HDp. Beta-CTx correlates with iPTH, tALP and bALP. The negative correlation with tCa and iCa probably might be influenced by the negative correlation between iPTH and serum Calcium. Beta-CTx levels are not associated with CKD-MBD specific therapy or with cardiac valvular calcifications. Further prospective and randomized studies, including a higher number of HDp and possibly bone histology are needed to elucidate the possible role of beta-CTx as a marker of bone disease in HDp.

SO078APABETALONE, A BROMODOMAIN AND EXTRA-TERMINAL (BET) PROTEIN INHIBITOR, REDUCES ALKALINE PHOSPHATASE IN CVD PATIENTS, IN MICE, AND IN CELL CULTURE SYSTEMS

Abstract Background and Aims Elevated serum alkaline phosphatase (ALP) independently predicts major adverse cardiac events (MACE) by contributing to vascular calcification and endothelial dysfunction arising in chronic kidney disease (CKD) and cardiovascular disease (CVD). Apabetalone is an orally active inhibitor of bromodomain and extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in vascular inflammation and calcification. Here we examined apabetalone’s effects on ALP post-hoc in recent clinical trials, then performed mechanistic studies into apabetalone’s impact on tissue non-specific ALP (TNALP) expression in mice and cell culture. Method Serum ALP was determined in CVD patients in phase 2 trials (3 month ASSERT and 6 month SUSTAIN &amp; ASSURE) and in the phase 3 BETonMACE CVD outcomes trial, including subpopulations with CKD (eGFR&amp;lt;60 mL/min/1.73m2). Apabetalone’s effect on expression of TNALP (gene symbol ALPL) was examined in mice, cultured primary human hepatocytes (PHH), HepaRG, HepG2, vascular smooth muscle cells (VSMCs), and vascular endothelial cells by real-time PCR. TNALP protein levels were assessed by immunoblots and flow cytometry. ALP enzyme activity was measured in enzymatic assays. Results In phase 2 trials, baseline serum ALP independently predicted MACE (hazard ratio [HR] 1.6, 95% CI 1.2-2.2, p=0.001). In the 3 month ASSERT trial, apabetalone dose dependently reduced serum ALP (p&amp;lt;0.001 vs placebo). Prominent reductions in ALP were apparent in patients on apabetalone (n=331) vs placebo (n=166) in combined analysis of the ASSURE &amp; SUSTAIN trials (median % change -11 vs -3.2; p&amp;lt;0.001). In the subset with CKD, patients on apabetalone (n=69) had greater reduction in serum ALP than placebo (n=22; p=0.008). Strikingly, ALP reductions in phase 2 correlated with reduction in MACE (HR 0.58, 95% CI 0.44-0.77, p&amp;lt;0.001). Consistent with phase 2, BETonMACE saw serum ALP reduced by 6.8 U/L with apabetalone (n=1082) vs placebo (n=1070; p&amp;lt;0.001) at 24 weeks. At the conclusion of BETonMACE, fewer MACE occurred in the CKD subgroup with apabetalone (n=124) vs placebo (n=164; HR 0.50 95% CI 0.26-0.96 p=0.032). Neither apabetalone nor statins that control low-density lipoprotein cholesterol inhibited recombinant TNALP enzyme activity, implying that decreased serum ALP activity in patients reflected reduction in TNALP production rather than inhibition of the enzyme. Liver-derived TNALP accounts for ≈50% of circulating ALP. In the liver of mice on high fat diet, apabetalone or JQ1 (BET inhibitors with different chemical scaffolds) reduced Alpl mRNA (p&amp;lt;0.001) with corresponding trends in TNALP activity. In PHH, HepaRG, &amp; HepG2 cells, apabetalone dose dependently suppressed ALPL expression by 60-80%. In HepG2 cells, apabetalone reduced TNALP protein (&amp;gt;55%, p&amp;lt;0.001), enzyme activity (&amp;gt; 40%; p&amp;lt;0.001), and % of TNALP positive cells (15-30%; p&amp;lt;0.001). MZ1, which promotes degradation of BET proteins, downregulated ALPL / TNALP similar to apabetalone. In VSMCs, apabetalone or JQ1 suppressed ALPL gene expression, TNALP protein levels, and enzyme activity, leading to decreases in extracellular calcium deposition. In addition, apabetalone downregulated ALPL expression in human aortic, umbilical vein, and brain microvascular endothelial cells by 50-70%. Conclusion Apabetalone lowers serum ALP in clinical trials, which is consistent with reduced hepatic production of TNALP - the most abundant ALP isoform. Further, apabetalone downregulates ALPL gene expression in vascular cell types while reducing calcification. Together, BET-dependent epigenetic modulation of ALP by apabetalone can affect several pathogenetic processes, and thereby improve cardiovascular outcomes. This study provides insights to the CVD event reductions observed in the CKD subpopulation in the BETonMACE Phase 3 trial.

Syncytiotrophoblast extracellular microvesicle profiles in maternal circulation for noninvasive diagnosis of preeclampsia

Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity and mortality incurred on the mother and the fetus. There is a need for improved biomarkers for diagnosis and monitoring of this condition. Placental syncytiotrophoblasts at the maternal-fetal interface release nanoparticles, including extracellular microvesicles, into the maternal blood during pregnancy. Syncytiotrophoblast extracellular microvesicles (STEVs) are being studied for their diagnostic potential and for their potential physiologic role in preeclampsia. We hypothesized that STEV profiles in maternal circulation would be altered under conditions of preeclampsia compared to normal pregnancy. Extracellular vesicles (EVs) released by BeWo cells in vitro showed high expression of syncytin-1, but no plac1 expression, demonstrating that trophoblast cell EVs express syncytin-1 on their surface. Placental alkaline phosphatase also showed high expression on BeWo EVs, but due to concern for cross reactivity to highly prevalent isoforms of intestinal and bone alkaline phosphatase, we utilized syncytin-1 as a marker for STEVs. In vivo, syncytin-1 protein expression was confirmed in maternal plasma EVs from Control and Preeclampsia subjects by Western blot, and overall, lower expression was noted in samples from patients with preeclampsia (n = 8). By nanoparticle analysis, EV profiles from Control and Preeclampsia groups showed similar total plasma EV quantities (p = 0.313) and size distribution (p = 0.415), but STEV quantitative signal, marked by syncytin-1 specific EVs, was significantly decreased in the Preeclampsia group (p = 2.8 × 10−11). Receiver operating characteristic curve demonstrated that STEV signal threshold cut-off of <0.316 was 95.2% sensitive and 95.6% specific for diagnosis of preeclampsia in this cohort (area under curve = 0.975 ± 0.020). In conclusion, we report that the syncytin-1 expressing EV profiles in maternal plasma might serve as a placental tissue specific biomarker for preeclampsia.

A review on biomarkers of bone metabolism in camels (Camelus dromedarius)

Bone metabolism biomarkers of bone formation and bone resorption are released during the bone remodeling processes. In humans as well as in veterinary medicine, these bone biomarkers have attracted much attention in the last decade. However, there are few reports found in the application of bone metabolism biomarkers in camel medicine. In camels, the most commonly used bone formation biomarkers include osteocalcin (OC) and bone-specific alkaline phosphatase (b-ALP). Concerning the bone resorption biomarkers, the most commonly used one is pyridinoline cross-links (PYD). OC is synthesised mostly from by mature osteoblasts. It is believed that OC is associated with mineralisation of newly formed osteoid. Therefore it is considered as a putative biomarker of bone formation and mineralisation. b-ALP is an isoform of alkaline phosphatase and plays an important role in bone formation. With normal liver function in adults, about 50% of total b-ALP is produced from bone in serum. The PYD cross-links, indicators of type I collagen resorption, are found in the mature collagen of bone. It is not only found in mature type I collagen, which is the major type of collagen in bone tissues, but also in collagen types II and III (Eyre et al, 1984). Increased concentrations of PYD in the blood or urine are most commonly considered as indicators of bone resorption. This review article was written to emphasise the importance of commonly used bone formation (OC and b-ALP) and bone resorption (PYD) biomarkers in camels. It is believed that research on bone formation and bone resorption biomarkers in camels will be increasingly used in the future in the diagnosis and prognosis of bone diseases.

Comparison between bone alkaline phosphatase immunoassay and electrophoresis technique in hemodialysis patients.

Problem of the variability between the different methods using for bone alkaline phosphatase (bALP) determination greately influences the clinical significance of bALP as direct marker of bone metabolism. The aim of this study was to compare immunoassay with electrophoresis technique for bALP determination. We measured bALP in 71 patients on hemo - dialysis with agar gel electrophoresis (ISO-PAL, SEBIA) and immunoassay (OSTASE, Beckman Coulter). The analyzed methods showed significant correlation (Spearman's rho: 0.776, P < 0.01), but we found statistically significant (P < 0.01) positive bias (27%) for the results measured by immunoassay. In support of this, using electrophoresis technique we have detected presence of the intestinal isoenzymes of alkaline phosphatase in 55% of patients with median value of 30% of the total alkaline phoshatase and presence of liver-2 alkaline phosphatase isoform in 42% of patients with median value of 16.6%. The Kendall's W of 0.787 (P<0.0001) revealed significant concordance between two analysed methods. Cusum test showed no significant deviation from linearity (P=0.850). Despite good agreement between immuno - assay methods and electrophoresis technique for bALP determination, interchangeability between these two methods is questionable. Although immunoassays are increasingly used, as fully automated methods, in a large number of laboratories and become routine methods for bALP determination, it should be beared in mind, besides various interferences, also the heterogeneity of the bALP itself, especially in patients on hemodialysis.

Recent advances in intestinal alkaline phosphatase, inflammation, and nutrition.

In recent years, much new data on intestinal alkaline phosphatase (IAP) have been published, and major breakthroughs have been disclosed. The aim of the present review is to critically analyze the publications released over the last 5 years. These breakthroughs include, for example, the direct implication of IAP in intestinal tight junction integrity and barrier function maintenance; chronic intestinal challenge with low concentrations of Salmonella generating long-lasting depletion of IAP and increased susceptibility to inflammation; the suggestion that genetic mutations in the IAP gene in humans contribute to some forms of chronic inflammatory diseases and loss of functional IAP along the gut and in stools; stool IAP as an early biomarker of incipient diabetes in humans; and omega-3 fatty acids as direct inducers of IAP in intestinal tissue. Many recent papers have also explored the prophylactic and therapeutic potential of IAP and other alkaline phosphatase (AP) isoforms in various experimental settings and diseases. Remarkably, nearly all data confirm the potent anti-inflammatory properties of (I)AP and the negative consequences of its inhibition on health. A simplified model of the body AP system integrating the IAP compartment is provided. Finally, the list of nutrients and food components stimulating IAP has continued to grow, thus emphasizing nutrition as a potent lever for limiting inflammation.

Electrochemical sensor for alkaline phosphatase as biomarker for clinical and in vitro applications

Alkaline phosphatase (ALP) is an important biomarker of bone diseases clinically measured in blood as a routine assay by means of optical read-outs, which when compared with electroanalytical methods present lack of miniaturization, slow response and high cost. Thus, electrochemical ALP sensors have arisen as a tool to develop disposable low cost devices with an increased sensitivity for healthcare. However, few of them comply with clinical standards, hence, limiting their application and competitiveness compared to the traditional optical methods. Therefore, we address a rapid test for ALP determination in human samples based on an end-point electrochemical assay using disposable graphite screen-printed electrodes and following international clinical recommendations. Using Square Wave Voltammetry (SWV), we first evaluated the electrochemistry of the enzymatic product phenol both at commercial ALP samples as well as at in vitro samples from osteoblasts cell cultures. ALP present in human serum and saliva from patients under normal and pathological conditions was further quantified by SWV. Additionally, the bone ALP isoform of such human samples was measured via precipitation with lectin wheat germ agglutinin. The resulting sensor showed an operational range between 20 to 1500 U L−1 using 10 μL of sample, a total volume of 100 μL and a reaction time of 10 min at 37 °C in addition to no interference of human samples in the potential window of interest for phenol oxidation. This work demonstrates that the electrochemical ALP sensor, here developed, is a promising alternative against the traditional commercial methods currently used.