Bone turnover biomarkers reflect radiation-induced bone injuries in women with non-metastatic rectal cancer.

Preoperative radiotherapy (RT) for non-metastatic rectal cancer reduces local recurrence rates but can cause pelvic insufficiency fractures. Despite the high morbidity from RT-induced skeletal injuries, predictive and preventive measures are lacking. How these injuries are reflected by bone biomarkers are largely unknown. The aim was to assess longitudinal changes in bone biomarkers and their relation to RT-related bone injuries in women with rectal cancer. This longitudinal cohort study includes 47 women with non-metastatic rectal cancer treated with surgery ± preoperative RT with or without chemotherapy. Sclerostin, bioactive sclerostin, C-terminal telopeptide cross-links of collagen type I (CTX), bone-specific alkaline phosphatase (BALP), and type I procollagen intact N-terminal propeptide (PINP) were measured at baseline, after RT, and 1 yr postoperatively. Pelvic magnetic resonance imaging was used for detection of skeletal injury. Sixteen of 36 (44%) irradiated women had radiation-induced bone injuries and were compared to 11 women (RT-) and 20 women (RT+) without bone injuries. Serum CTX, BALP, and PINP increased during the first year after RT in women with radiation-induced bone injuries. The difference in mean change of CTX (p=.037) and BALP (p=.042) was conferred by longitudinal regression analyses adjusted for serum estradiol. Serum sclerostin and bioactive sclerostin remained stable over time. Taken together, bone markers may be of interest for future research on fracture prediction or preventive measures in women susceptible to radiation-induced bone injury. Due to few measure points, the full pattern cannot be captured regarding the relation over time between bone biomarkers and skeletal injury from irradiation.

Demographic and disease-related factors impact bone turnover and vitamin D in children with hemato-oncological diseases.

Children with hemato-oncological diseases may have significant skeletal morbidity, not only during and after treatment but also at the time of diagnosis before cancer treatment. This study was designed to evaluate the vitamin D status and circulating bone metabolic markers and their determinants in children at the time of diagnostic evaluation for hemato-oncological disease. This cross-sectional study included 165 children (91 males, median age 6.9 yr range 0.2-17.7 yr). Of them, 76 patients were diagnosed with extracranial or intracranial solid tumors, 83 with leukemia, and 6 with bone marrow failure. Bone metabolism was assessed by measuring serum 25OHD, PTH, bone alkaline phosphatase, intact N-terminal propeptide of type I procollagen, and C-terminal cross-linked telopeptide of type I collagen. Vitamin D deficiency was found in 30.9% of children. Lower 25OHD levels were associated with older age, lack of vitamin D supplementation, season outside summer, and a country of parental origin located between latitudes -45° and 45°. Children diagnosed with leukemia had lower levels of markers of bone formation and bone resorption than those who had solid tumors or bone marrow failure. In conclusion, vitamin D deficiency was observed in one-third of children with newly diagnosed cancer. Bone turnover markers were decreased in children with leukemia, possibly because of the suppression of osteoblasts and osteoclasts by leukemic cells. The identification of patients with suboptimal vitamin D status and compromised bone remodeling at cancer diagnosis may aid in the development of supportive treatment to reduce the adverse effects of cancer and its treatment.

Low bone mineral density and reduced bone-specific alkaline phosphatase in 5q spinal muscular atrophy type 2 and type 3: A 2-year prospective study of bone health.

AIM: Individuals with spinal muscular atrophy (SMA) are at risk of developing skeletal problems. This study investigated bone mineral density (BMD), bone turnover markers and motor function in children and adolescents with SMA type 2 and type 3 over a two-year period. The effect of nusinersen was studied in a subgroup. METHODS: Single-centre study, including 20 patients, 2-18 years, of whom ten patients received nusinersen treatment. BMD was measured by dual-energy X-ray absorptiometry. RESULTS: All patients had low BMD levels at baseline; mean Z-score -2.3 for total body less head (TBLH) and -2.9 for total hip left (THL). Significant correlations were found both at baseline and for the follow-up change for motor function and Z-scores (TBLH and THL). For the whole study group, reduced bone formation and unchanged bone resorption, assessed by bone-specific alkaline phosphatase (BALP) (p = 0.0006, ES = -0.83) and C-terminal cross-linking telopeptide of type I collagen (CTX), respectively, were found over the study period. However, BALP decreased less in the nusinersen treatment group, which suggests a positive development on bone mass in these patients. CONCLUSION: Bone health evaluation is important in follow-up programmes for SMA patients. Further investigations are warranted for individuals on survival motor neuron-targeted treatments.

Circulating microRNAs in young individuals with long-duration type 1 diabetes in comparison with healthy controls.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional control of gene expression and might be used as biomarkers for diabetes-related complications. The aim of this case-control study was to explore potential differences in circulating miRNAs in young individuals with long-duration type 1 diabetes (T1D) compared to healthy controls, and how identified miRNAs are expressed across different tissues. Twelve adolescents, age 15.0-17.9 years, with T1D duration of more than 8 years (mean 11.1 years), were enrolled from the Swedish diabetes quality registry. An age-matched control group was recruited. Circulating miRNAs (n = 187) were analyzed by quantitative PCR. We observed that 27 miRNAs were upregulated and one was downregulated in T1D. Six of these miRNAs were tissue-enriched (blood cells, gastrointestinal, nerve, and thyroid tissues). Six miRNAs with the largest difference in plasma, five up-regulated (hsa-miR-101-3p, hsa-miR-135a-5p, hsa-miR-143-3p, hsa-miR-223-3p and hsa-miR-410-3p (novel for T1D)) and one down-regulated (hsa-miR-495-3p), with P-values below 0.01, were selected for further in-silico analyses. AKT1, VEGFA and IGF-1 were identified as common targets. In conclusion, 28 of the investigated miRNAs were differently regulated in long-duration T1D in comparison with controls. Several associations with cancer were found for the six miRNAs with the largest difference in plasma.

Lipocalin-2 is associated with FGF23 in WNT1 and PLS3 osteoporosis.

Background: The pathogenic mechanisms of early-onset osteoporosis caused by WNT1 and PLS3 mutations are incompletely understood and diagnostic biomarkers of these disorders are limited. Recently, lipocalin-2 has been recognized as an osteokine involved in bone development and homeostasis. However, the role of lipocalin-2 in WNT1 and PLS3 osteoporosis is unknown. Objective: We aimed to investigate if plasma lipocalin-2 could be utilized as a biomarker for WNT1 and PLS3 osteoporosis and to evaluate the association between lipocalin-2 and other parameters of bone metabolism. Methods: We measured plasma lipocalin-2 in 17 WNT1 and 14 PLS3 mutation-positive patients and compared them to those of 34 mutation-negative (MN) healthy subjects. We investigated possible associations between lipocalin-2 and several bone biomarkers including collagen type I cross-linked C-telopeptide (CTX), alkaline phosphatase (ALP), type I procollagen intact N-terminal propeptide (PINP), intact and C-terminal fibroblast growth factor 23 (FGF23), dickkopf-1 (DKK1) and sclerostin as well as parameters of iron metabolism (iron, transferrin, transferrin saturation, soluble transferrin receptor and ferritin). Results: We found no differences in plasma lipocalin-2 levels in WNT1 or PLS3 patients compared with MN subjects. However, lipocalin-2 was associated with C-terminal FGF23 in WNT1 patients (r=0.62; p=0.008) and PLS3 patients (r=0.63, p=0.017), and with intact FGF23 in PLS3 patients (r=0.80; p<0.001). In addition, lipocalin-2 correlated with serum transferrin in WNT1 patients (r=0.72; p=0.001). Conclusion: We conclude that plasma lipocalin-2 is not altered in WNT1 or PLS3 mutation-positive subjects but is associated with FGF23 in abnormal WNT1 or PLS3 signaling and with iron status in abnormal WNT1 signaling.

Impact of radiotherapy on bone health in women with rectal cancer - A prospective cohort study.

INTRODUCTION: Pelvic radiotherapy (RT) increases the risk of pelvic insufficiency fractures. The aim was to investigate if RT is associated with changes in serum bone biomarkers in women with rectal cancer, and to examine the incidence of radiation-induced bone injuries and the association with bone biomarkers. MATERIAL AND METHODS: Women diagnosed with rectal cancer stage I-III, planned for abdominal surgery ± preoperative (chemo) RT, were prospectively included and followed one year. Serum bone biomarkers comprised sclerostin (regulatory of bone formation), CTX (resorption), BALP and PINP (formation). A subgroup was investigated with annual pelvic magnetic resonance imaging (MRI). The association between RT and bone biomarkers was explored in regression models. RESULTS: Of 134 included women, 104 had surgery with preoperative RT. The formation markers BALP and PINP increased from baseline to one year in the RT-exposed group (p < 0.001, longitudinal comparison). In the adjusted regression analysis, the mean increase in PINP was higher in the RT-exposed than the unexposed group (17.6 (3.6-31.5) µg/L, p = 0.013). Sclerostin and CTX did not change within groups nor differed between groups. Radiation-induced injuries were detected in 16 (42%) of 38 women with available MRI. At one year, BALP was higher among women with than without bone injuries (p = 0.018, cross-sectional comparison). CONCLUSIONS: Preoperative RT was associated with an increase in the formation marker PINP, which could represent bone recovery following RT-induced injuries, commonly observed in participants evaluated with MRI. These findings should be further explored in larger prospective studies on bone health in rectal cancer patients.

Impaired renal clearance among Swedish adolescents born preterm.

AIM: To determine whether adolescents born before 28 gestational weeks have an increased risk for renal impairment. METHODS: Swedish infants, born before 28 gestational weeks in 2001 and 2002, were identified from a local register. A total of 16 children, 12 females and 4 males, were examined at 16-17 years of age with 51 Cr-EDTA clearance. A comparison group (n = 26) was used. RESULTS: Most study participants (n = 13) had normal blood pressure; one individual had hypertension stage 1. All study participants had results within the reference interval for ionised calcium, parathyroid hormone, intact fibroblast growth factor-23 and for urinary albumin-to-creatinine ratio. Four out of 16 participants (25%) had a 51 Cr-EDTA clearance less than 90 ml/min/1.73 m2 , indicating a reduced kidney function. Measured 51 Cr-EDTA clearance values were significantly lower in the study group than in the comparison group (p = 0.0012). Five study participants (31%) were referred for further investigations. CONCLUSION: Swedish children born before 28 gestational weeks have an increased risk of renal impairment later in life, suggesting that the kidney function in these individuals should be assessed, at least once, during adolescence.

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.

[Appropriate use of vitamin D assessments in primary health care: Impact of new strategies for the introduction and follow-up of analyses in Östergötland]. / D-vitaminanalyser i primärvård måste användas ändamålsenligt - Ny strategi i Östergötland för införande och uppföljning av analyser gav bra resultat.

Systematic reviews and meta-analyses have shown that there is little justification for vitamin D supplements to prevent infections, cancer and cardiovascular disease. Despite the limited evidence of effectiveness, the total number of ordered serum 25-hydroxyvitamin D (25(OH)D) tests has increased considerably in recent years. There seems to be an overuse of this test that does not provide meaningful benefit for patients. A passive introduction of new tests leads generally to a slow initiation of value-based diagnostics, as well as overuse and underuse of diagnostic tests. In this study, in Region Östergötland, we applied a ¼Choosing wisely« model that reversed a rising trend of 25(OH)D tests and reduced the number of unnecessary tests. The findings point to the need for strategic plans for introducing new analyses and approaches to counteract misuse of laboratory diagnostics. We recommend ¼Choosing wisely« models for the introduction of new analyses to facilitate appropriate laboratory diagnostics and to counteract long-term overuse.

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.

Bone alkaline phosphatase isoforms in hemodialysis patients with low versus non-low bone turnover: a diagnostic test study.

BACKGROUND: Renal osteodystrophy encompasses the bone histologic abnormalities seen in patients with chronic kidney disease (CKD). The bone-specific alkaline phosphatase (bALP) isoform B1x is exclusively found in serum of some patients with CKD. STUDY DESIGN: The aim of this cross-sectional diagnostic test study was to examine the relationship between serum bALP isoform activity and histomorphometric parameters of bone in patients with CKD receiving maintenance hemodialysis. SETTINGS & PARTICIPANTS: Anterior iliac crest bone biopsy samples from 40 patients with CKD were selected on the basis of bone turnover for histomorphometric analysis. There were samples from 20 patients with low and 20 with non-low bone turnover. INDEX TEST: In serum, bALP, bALP isoforms (B/I, B1x, B1, and B2), and parathyroid hormone (PTH) were measured. REFERENCE TEST: Low bone turnover was defined by mineral apposition rate < 0.36µm/d. Non-low bone turnover was defined by mineral apposition rate ≥ 0.36µm/d. OTHER MEASUREMENTS: PTH. RESULTS: B1x was found in 21 patients (53%) who had lower median levels of bALP, 18.6 versus 46.9U/L; B/I, 0.10 versus 0.22 µkat/L; B1, 0.40 versus 0.88 µkat/L; B2, 1.21 versus 2.66 µkat/L; and PTH, 49 versus 287pg/mL, compared with patients without B1x (P<0.001). 13 patients (65%) with low bone turnover and 8 patients (40%) with non-low bone turnover (P<0.2) had detectable B1x. B1x correlated inversely with histomorphometric parameters of bone turnover. Receiver operating characteristic curves showed that B1x can be used for the diagnosis of low bone turnover (area under the curve [AUC], 0.83), whereas bALP (AUC, 0.89) and PTH (AUC, 0.85) are useful for the diagnosis of non-low bone turnover. LIMITATIONS: Small number of study participants. Requirement of high-performance liquid chromatography methods for measurement of B1x. CONCLUSIONS: B1x, PTH, and bALP have similar diagnostic accuracy in distinguishing low from non-low bone turnover. The presence of B1x is diagnostic of low bone turnover, whereas elevated bALP and PTH levels are useful for the diagnosis of non-low bone turnover.

Bone alkaline phosphatase in CKD-mineral bone disorder.

Overall and cardiovascular mortality in patients with chronic kidney disease (CKD) is greatly increased, without obvious current effective treatments. Mineral and bone disorder (MBD) is a common manifestation of CKD and contributes to the high risk of fracture and cardiovascular mortality in these patients. Traditionally, clinical management of CKD-MBD focused on attenuation of secondary hyperparathyroidism due to impaired renal activation of vitamin D and phosphate retention, although recently, adynamic forms of renal bone disease have become more prevalent. Definitive diagnosis was based on histologic (histomorphometric) analysis of bone biopsy material supported by radiologic changes and changes in levels of surrogate laboratory markers. Of these various markers, parathyroid hormone (PTH) has been considered to be the most sensitive and currently is the most frequently used; however, the many pitfalls of measuring PTH in patients with CKD increasingly are appreciated. We propose an alternative or complementary approach using bone alkaline phosphatase (ALP), which is directly related to bone turnover, reflects bone histomorphometry, and predicts outcomes in hemodialysis patients. Here, we consider the overall merits of bone ALP as a marker of bone turnover in adults with CKD-MBD, examine published bone histomorphometric data comparing bone ALP to PTH, and discuss possible pathogenic mechanisms by which bone ALP may be linked to outcomes in patients with CKD.

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 ß-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. ß-Glycerophosphate stimulated calcification of HAoSMCs, which was associated with increased BALP isoforms B/I, B1x and B2. Tetramisole inhibited the ß-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.

Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis.

Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-ß in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

Targeting RANKL for reduction of bone loss around unstable implants: OPG-Fc compared to alendronate in a model for mechanically induced loosening.

Orthopedic joint prostheses may loosen because of localized bone resorption. Despite initial optimism, there are no reports showing that bisphosphonates can stop the progression of prosthetic loosening once it has begun. This might be due to the strong resorptive stimulus, which continuously recruits new osteoclasts. Therefore, we hypothesized that a treatment targeting osteoclast recruitment would be more efficacious than a treatment reducing osteoclast activity. We used a previously described rat model for instability-induced bone resorption, and compared OPG-Fc with alendronate at a clinically relevant or an extreme dose. A titanium plate was osseointegrated at the rat tibial surface. Instability was simulated by a piston, moving perpendicularly to the bone surface. Piston movement induced bone loss via hydrostatic pressure or fluid flow. Rats were randomized to 5 groups (total n=56), of which 4 were subjected to instability and one was stable. The unstable groups were injected with either high-dose OPG-Fc (10 mg/kg, twice weekly), a high dose of alendronate (20 µg /kg/day), an extreme dose of alendronate (200 µg/kg/day) or saline. Significant protection against resorption could only be shown for OPG-Fc and the extreme alendronate dose. Both alendronate doses reduced serum levels of tartrate-resistant acid phosphatase isoform 5b to a similar extent, demonstrating that the lower dose was able to reduce resorption in the normally remodeling skeleton, although not in the osteolytic lesions caused by instability. Osteoclast numbers in the lesion were increased by the lower bisphosphonate dose and reduced by OPG-Fc. The results suggest the possibility of targeting osteoclast recruitment via the RANKL system in patients with impending prosthetic loosening.

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.

Acidic preparations of platelet concentrates release bone morphogenetic protein-2.

BACKGROUND AND PURPOSE: Growth factors released from platelets have potent effects on fracture and wound healing. The acidic tide of wound healing, i.e. the pH within wounds and fractures, changes from acidic pH to neutral and alkaline pH as the healing process progresses. We investigated the influence of pH on lysed platelet concentrates regarding the release of growth factors. MATERIAL AND METHODS: Platelet concentrates free of leukocyte components were lysed and incubated in buffers with pH between 4.3 and 8.6. Bone morphogenetic protein-2 (BMP-2), platelet-derived growth factor (PDGF), transforming growth factor-beta(TGF-beta), and vascular endothelial growth factor (VEGF) were measured by quantitative enzyme-linked immunosorbent assays. RESULTS: PDGF, TGF-beta, and VEGF were present in all platelet preparations but the levels varied in a pH-dependent fashion. BMP-2 was only detected in the most acidic preparation (pH 4.3), which is interesting since BMP-2 has been reported to be an endogenous mediator of fracture repair and to be responsible for the initiation of fracture healing. INTERPRETATION: Our findings indicate that platelets release substantial amounts of BMP-2 only under conditions of low pH, the milieu associated with the critical initial stage of fracture healing.

Bone mass, biochemical markers and growth in children with chronic kidney disease: a 1-year prospective study.

AIM: This study was designed to investigate bone mineral density (BMD), growth parameters and biochemical markers in children with chronic kidney disease (CKD). METHODS: Sixteen patients, 4-18 years, with CKD were prospectively followed for 1 year. Auxological data, body composition, BMD by dual-energy X-ray absorptiometry, bone age, bone turnover markers, vitamin D, parathyroid hormone (PTH), leptin, osteoprotegerin, insulin-like growth factor-I (IGF-I) and IGF binding protein-3 were measured. A questionnaire regarding bone health and diet was also performed. RESULTS: Delayed bone age was observed (n = 11) and the BMD Z-scores for total body were below zero in seven patients. However, total body BMD (TBBMD) increased in 12 patients. Most patients had increased osteocalcin and carboxy-terminal telopeptide of type I collagen, but normal alkaline phosphatase, type I procollagen intact amino-terminal propeptide and tartrate-resistant acid phosphatase 5b. Ten patients had increased PTH. Most children had normal levels of leptin, osteoprotegerin, IGF-I and IGFBP-3. Leptin, at baseline, correlated with differences in TBBMD over 1 year. CONCLUSIONS: Only seven (44%) had negative Z-scores and TBBMD increased over 1 year. Bone markers at baseline did not predict the longitudinal changes in BMD.

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.

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.