Bone Alkaline Phosphatase Isoforms Research Articles

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.

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.

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 <0.0001), bALP (r 0.89, p <0.0001) and iPTH (r 0.87, p<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.

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.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

歯科用インプラント体埋入後におけるインプラント体周囲骨吸収

Purpose: The objective of this study was to clarify the relationship between the bone resorption around dental implants and bone metabolism. Patients and Methods: Eleven post-menopausal female patients were enrolled in the clinical study. Twenty-three dental implants were inserted in the posterior mandibular edentulous region. Dental radiograms evaluated the alveolar bone absorption ratio （ABAR） of post-surgical peri-implant bone resorption. Bone alkaline phosphatase isoform （ALP） and bone metabolism marker （PICP and ICTP） in serum were measured before surgery as markers of bone synthesis and degradation. A semi-quantitative ratio of the bone uptake of a scintigraphic study at one month post-surgery was calculated. Results: ABAR was not correlated with serum PICP, ICTP, or ALP. However, the correlation between ABAR and the ICTP to PICP ratio representing bone formation was higher than that of each relation. ABAR increased with the magnitude of the bone uptake ratio of the scintigraphic study. There was a relation between PICP and bone uptake: uptake decreased with increasing PICP. Conclusion: Peri-implant bone resorption may be related with enhanced bone metabolism in the region of implant placement. Bone formation capacity is likely to be important for inhibiting post-surgical peri-implant bone resorption.

Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

Calcifying Human Aortic Smooth Muscle Cells Express Different Bone Alkaline Phosphatase Isoforms, Including the Novel B1x Isoform

Background: Vascular calcification, causing cardiovascular morbidity and mortality, is associated with hyperphosphatemia in chronic kidney disease (CKD). In vitro, phosphate induces transdifferentiation of vascular smooth muscle cells to osteoblast-like cells that express alkaline phosphatase (ALP). In vivo, raised serum ALP activities are associated with increased mortality. A new bone ALP isoform (B1x) has been identified in serum from CKD patients. The present study investigated the different ALP isoforms in calcifying human aortic smooth muscle cells (HAoSMCs). Methods: HAoSMCs were cultured for 30 days in medium containing 5 or 10 mmol/l β-glycerophosphate in the presence or absence of the ALP-specific inhibitor tetramisole. Results: All known bone-specific ALP (BALP) isoforms (B/I, B1x, B1 and B2) were identified in HAoSMCs. β-Glycerophosphate stimulated calcification of HAoSMCs, which was associated with increased BALP isoforms B/I, B1x and B2. Tetramisole inhibited the β-glycerophosphate-induced HAoSMC calcification, which was paralleled by the inhibition of the B1x and B/I, but not the other isoforms. Conclusions: HAoSMCs express the four known BALP isoforms. B/I, B1x and B2 could be essential for soft tissue calcification. B/I and B1x were more affected by tetramisole than the other isoforms, which suggests different biological functions during calcification of HAoSMCs.

Biochemical bone markers in the assessment and pamidronate treatment of children and adolescents with osteogenesis imperfecta

To assess the role of biochemical bone markers in classification of children with osteogenesis imperfecta (OI), their possible association with vertebral compression fractures in milder forms of OI and their role in monitoring of intravenous pamidronate (APD) treatment. Serum total alkaline phosphatase (ALP), bone ALP isoforms (in a subgroup), osteocalcin, type I procollagen carboxy-terminal propeptide, carboxy-terminal telopeptide of type I collagen, and urine deoxypyridinoline (DPD) were measured in a cross-sectional study of 130 untreated individuals, 0.25-20.9years (median 6.7), with OI types I, III and IV. Of those, sixty-nine were also assessed longitudinally during monthly APD treatment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. Significant differences in bone markers, however not sufficient for individual clinical use, were found in the larger untreated group but not between subgroups with or without vertebral compressions. All bone markers decreased during treatment for 1.0-12.5years, but with different relative amounts. Changes were not correlated to the improvement in BMD, mobility or pain. Bone markers are, despite significant differences, not useful for the classification of OI type in the individual child and are not associated with vertebral compressions. Serum ALP and urinary DPD are sensitive in monitoring bisphosphonate treatment.

Circulating and tissue-derived isoforms of bone alkaline phosphatase in Paget's disease of bone

Background. Alkaline phosphatase (ALP) is routinely used in the assessment of Paget's disease of bone (PDB); however, the individual bone ALP isoforms (B/I, B1, and B2) have not been investigated in this disorder. Methods. Subjects comprised 37 patients (mean age 74 years) with symptomatic PDB confirmed by radiograph and stratified into high and low total ALP activity groups and 66 healthy individuals (mean age 64 years). Extracts of human cancellous and cortical bone tissues were also investigated. The bone ALP isoforms, measured by HPLC, were compared with two bone ALP immunoassays (Metra® and Ostase®), and the bone formation marker intact amino-terminal procollagen type I propeptide (iPINP). Results. All bone ALP isoforms were increased in high ALP activity PDB compared with the low ALP activity and control groups (p < 0.0001). The B2 isoform had the greater relative activity representing 36%, 50%, and 71%, of the total ALP activity in the control, low and high ALP activity groups, respectively. Compared with controls, B2 was increased in the low ALP activity PDB group (p < 0.05). ROC analysis showed a validity of approximately 80% for B2 to discriminate patients with PDB. Conclusion. All bone ALP isoforms were increased in patients with high ALP activity PDB and the B2 isoform was even elevated in the low ALP activity PDB group. The bone ALP isoform B2 may be of use in the management of PDB but that has to be further elucidated in subsequent studies.

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD 10, a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP i), pyridoxal 5′-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k cat/ K M was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD 10 was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD 10 isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.

Clinical significance of bone alkaline phosphatase isoforms, including the novel B1x isoform, in mild to moderate chronic kidney disease

Mineral bone disorder (MBD) is a common complication of chronic kidney disease (CKD) even during the early stages. Bone alkaline phosphatase (BALP) is a marker of bone formation and plays a pivotal role in the mineralization process. Three BALP isoforms (B/I, B1 and B2) have been identified in healthy individuals and a fourth isoform (B1x) has been discovered in serum from dialysis patients. We investigated these BALP isoforms, type I procollagen intact amino-terminal propeptide (PINP), carboxy-terminal telopeptide of type I collagen (CTX) and tartrate-resistant acid phosphatase isoform 5b (TRACP5b), as well as bone mineral density (BMD) in predialysis CKD patients. PINP, CTX, TRACP5b and BALP isoforms were analysed in serum from 46 patients within CKD stages 3-5. BMD was determined by dual-energy x-ray absorptiometry. PINP, TRACP5b and the BALP isoforms, B/I, B1 and B2, were independent predictors of total hip BMD in all patients. Furthermore, B/I predicted osteopaenia in the hip and in the distal 1/3 of the radius in CKD stage 3. The B1x isoform was detected in nine patients (20%), who had lower GFR, higher phosphate and calcium x phosphate product. We found an association of BALP isoforms and other markers of bone turnover with total hip BMD, which predominantly comprises trabecular bone. The association of the new BALP isoform B1x with risk factors for vascular calcification leads us to hypothesize a possible role for B1x in this process. The significance of the BALP isoforms in CKD remains to be further explored in experimental and clinical settings in conjunction with bone histomorphometry.

Analysis of human bone alkaline phosphatase isoforms: Comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography

Background Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). Methods We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5–9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. Results Whilst the isoforms B1x (pI = 4.48), B1 (pI = 4.32) and B2 (pI = 4.12) resolved as well-defined bands, B/I resolved as a complex (pI = 4.85–6.84). Neuraminidase altered the migration of all BALP isoforms to pI = 6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11–20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54–73%. IEF resolved the commercial BALP into 2 major bands (pI = 4.41 and 4.55). Conclusions Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI = 3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.

The novel bone alkaline phosphatase B1x isoform in children with kidney disease

The bone alkaline phosphatase (BALP) B1x isoform has previously only been identified in some adults with chronic kidney disease on dialysis and in human bone tissue. Twenty-nine patients, 3-20 years of age, with reduced renal function due to a variety of kidney diseases were examined. We measured parathyroid hormone (PTH), biointact (whole 1-84) PTH, osteoprotegerin (OPG), CrossLaps (CTX), tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) type I procollagen intact amino-terminal propeptide (PINP), osteocalcin, total alkaline phosphatase (ALP), and BALP isoforms B/I, B1x, B1, and B2. Fifty percent higher levels were detected of PTH vs. biointact PTH, demonstrating non-(1-84) PTH fragments detected by the PTH assay. Increased activities were found in five, four, and three patients for total ALP, B1, and B2, respectively. Sixteen (55%) patients had increased B/I levels. B1x was identified in two (7%) patients, who had OPG levels in the higher range independently of age, glomerular filtration rate (GFR), and biointact PTH. B1x was identified prior to and after 9 days of growth hormone (GH) therapy in one patient but not after 1, 3, 6, and 12 months, however. In conclusion, our study demonstrates that the novel BALP B1x isoform is occasionally found to be present in children with kidney disease but to a lesser degree in comparison with adults with chronic kidney disease on dialysis. It is essential to perform bone histomorphometry for future investigations in order to elucidate the exact nature of circulating B1x in patients with kidney disease for accurate classification of type of renal bone disease.

Diagnostic value of bone remodeling markers in the diagnosis of bone metastases in patients with breast cancer

Metastatic spread to bone is common in patients with breast cancer and its early detection is required for the better management of these patients. Several biochemical markers of bone remodeling have been recently developed, in order to assess metastatic bone disease with non radiologic methods. The pyridinolin cross-linked amino-terminal telopeptide of type I collagen (NTx) has been measured in serum and urine as a specific marker of bone collagen breakdown, while the bone-isoform of alkaline phosphatase (BAP) has been used to determine bone formation activity. Thirty-three consecutive ambulatory patients with metastatic breast cancer and bone metastases and 31 with extraskeletal metastases only, matched for age and menopausal status, were studied. Serum levels of NTx and BAP were measured by enzyme-linked immunosorbent assays. The diagnostic accuracy of both markers was evaluated by receiver operating characteristic (ROC) analysis. Patients with bone metastases had significantly higher levels of NTx (37.0 ± 36.9 nM BCE versus 23.5 ± 21.0 nM BCE, P < 0.05) and BAP (57.8 ± 31.7 U/L versus 36.5 ± 28.5 U/L, P < 0.01) compared to those without bone metastases. NTx was positively correlated with BAP ( R = 0.340, P < 0.01). The area under the ROC curve was 0.671 for NTx and 0.755 for BAP. Using a cut-off value of 29.7 nM BCE for NTx, specificity and sensitivity were 87.1% and 45.5%, respectively; in the case of BAP, using a cut-off value of 50.6 U/L, the specificity and sensitivity were 90.3% and 54.5%, respectively. In patients not receiving concomitant hormonal treatment, the area under the ROC curve was 0.724 for NTx and 0.822 for BAP; in this subgroup of patients, using a cut-off value of 30.0 nM BCE for NTx, the specificity and sensitivity were 96.2% and 47.1%, respectively, while using a cut-off value of 50.0 U/L for BAP, the corresponding percentages were 92.3% and 70.6%. Although serum NTx and BAP are quite specific, they are not sensitive enough to diagnose bone metastases in patients with advanced breast cancer. Their diagnostic accuracy, however, is considerably enhanced in patients not receiving hormonal therapy.

Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that B1 and B2 have more (or more reactive) sialic acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts

Background: In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease. Methods: In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD). Results: A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment. Conclusions: The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.

Effect of chronic renal failure on bone turnover and bone alkaline phosphatase isoforms

Biochemical markers of bone turnover are used to monitor metabolic bone disease associated with renal failure. We have applied a comprehensive panel of markers to patients with chronic renal failure (CRF), with particular focus on the isoforms of bone alkaline phosphatase (BALP). Twenty CRF patients undergoing hemodialysis (N = 9) and peritoneal dialysis (N = 11) were measured for serum parathyroid hormone (PTH), osteocalcin, total ALP, and four BALP isoforms (B/I, B1x, B1, and B2) by high-performance liquid chromatography. These BALP isoforms were also compared with BALP measured by three commercial immunoassays (Alkphase-B, Tandem-R Ostase, and Tandem-MP Ostase). Type I collagen turnover was assessed by serum samples using the type I procollagen intact amino- and carboxy-terminal propeptides (PINP and PICP) and two fragments (ICTP and CrossLaps) derived from the carboxy-terminal telopeptide of mature matrix collagen by different degradative pathways. Mean levels of bone turnover markers were elevated in CRF, with marked increases in those markers, osteocalcin, ICTP, and CrossLaps, cleared by the kidney. Total ALP activities were increased corresponding to elevated B/I and B2 isoform levels. The B1 isoform level was not significantly different from healthy controls. B1x was detected in 60% of the patients but was not resolved in healthy individuals. Kendall's tau rank correlation showed that B1x correlated significantly (P < 0.05) with B1 (0.53) and PINP (0.55), and was the only marker to correlate with PTH (0.49). B1x was not significantly correlated with any of the commercial BALP immunoassays. Interestingly, the immunoassay calibrators contained high activities of the B/I peak (39 to 80%) compared with human serum (4%). There are selective differences between the BALP isoforms in CRF compared with healthy adults. The commercial BALP immunoassays are comparable with each other but are unable to distinguish the BALP isoform-specific differences in CRF patients.

Serum isoforms of bone alkaline phosphatase increase during physical exercise in women.

Physical activity is an important factor for maintaining and probably increasing bone mass in humans. However, the mechanism by which this takes place is not completely understood. The purpose of this study was to examine the influence of physical exercise on serum alkaline phosphatase (ALP) and in particular, the bone isoforms of ALP. Six ALP isoforms were quantified by high-performance liquid chromatography: three bone (B/I, B1, and B2), and three liver ALP isoforms. In addition, serum calcium, parathyroid hormone (PTH), and other markers of bone formation and degradation, as measured by osteocalcin and cross-linked carboxyterminal telopeptide of type I collagen (ICTP), were analyzed. The study groups comprised 15 women, 8 postmenopausal (range 51-62 years) and 7 near age of peak bone mass (range 21-27 years). When the postmenopausal women exercised on an ergometer cycle until exhaustion we found significant increases in serum of bone ALP isoforms B1 and B2, and phosphate, even considering the hemoconcentration that occurred during the exercise. When the young women jogged in a moderate tempo for 40-40 minutes the levels of serum B2 and PTH increased. All changes turned towards baseline within 20 minutes after exercise. In conclusion, exercise increased serum ALP bone isoforms B1 and B2, and their responses were differentiated. As B1 and B2 are known to represent specific bone compartments, cortical and trabecular bone, the present findings may indicate different effects on bone of weight- and nonweight-bearing exercise.