IBMS/ECTS 2001 - PROGRAM and ABSTRACTS
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Diseases of Bone
HUMORAL HYPERCALCEMIA OF MALIGNANCY: SCID/BEIGE MOUSE MODEL OF ADULT T-CELL LYMPHOMA (ATL) INDEPENDENT OF HTLV-1 TAX EXPRESSION
V. Richard1, M. D. Lairmore1, P. L. Green1, G. Feuer2, R. S. Erbe1, B. Albrecht1, C. D'Souza1, E. T. Keller3, J. Dai3, T. F.Rosol1*
1Ohio State University, Columbus, OH, USA
2SUNY Upstate Med. Univ., Syracuse, NY, USA
3University of Michigan, Ann Arbor, MI, USA
The majority of patients with adult T-cell leukemia/lymphoma (ATL) resulting from HTLV-1 infection develop humoral hypercalcemia of malignancy (HHM). We utilized an animal model using SCID/beige mice to study the pathogenesis of HHM. SCID/beige mice were inoculated intraperitoneally with a human ATL line (RV-ATL) and were euthanatized 20-32 days after inoculation. SCID/beige mice with engrafted RV-ATL cells developed lymphoma in the mesentery, liver, thymus, lungs, and spleen. The lymphomas stained positively for human CD45RO surface receptor and normal mouse lymphocytes stained negatively confirming the human origin of the tumors. The ATL cells were immunohistochemically positive for parathyroid hormone-related protein (PTHrP). In addition, PTHrP mRNA was highly expressed in lymphomas when compared to MT-2 cells (HTLV-1 positive cell line). Mice with lymphoma developed severe hypercalcemia (3.7±0.8 vs. 2.2±0.1 mM in controls). Plasma PTHrP (1-84) concentrations were markedly increased in mice with hypercalcemia (12 to 140 pM compared to <0.1 pm in controls), and correlated with the increase in plasma calcium concentrations. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss due to a marked increase osteoclastic bone resorption. RV-ATL cells contained 1.5 HTLV-1 copies of the tax gene as determined by quantitative real-time PCR. However, tax expression was not detected by western blot or RT-PCR in RV-ATL cells, which suggests that factors other than Tax are modulators of PTHrP gene expression. The SCID/bg mouse model mimics HHM as it occurs in ATL patients, and will be useful to investigate the regulation of PTHrP expression by ATL cells in vivo.
FELINE HEAD AND NECK SQUAMOUS CELL CARCINOMA LINE: CHARACTERIZATION, PRODUCTION OF PARATHYROID HORMONE-RELATED PROTEIN, AND REGULATION BY TRANSFORMING GROWTH FACTOR-BETA
S. H. Tannehill-Gregg, E. Ahaus, T. J. Rosol*
The Ohio State University, Columbus, OH, USA
Despite the development of several models for human head and neck squamous cell carcinoma (H&N SCC), a useful natural animal model does not exist for this disfiguring, incurable disease. The cat develops a high rate of H&N SCC, which is similar to the human disease. We have developed a cell line from a laryngeal SCC of a cat. Squamous cell carcinoma is the cancer type most commonly associated with humoral hypercalcemia of malignancy, which is induced by the secretion of parathyroid hormone-related protein (PTHrP). PTHrP production can be upregulated by transforming growth factor-beta (TGF-beta) produced by the tumor cells or surrounding stromal cells. Neoplastic keratinocytes from an advanced laryngeal SCC were maintained in culture for >25 passages. Cells were characterized immunohistochemically and had strong cytokeratin and involucrin staining and absent vimentin and p53 staining. Electron microscopy demonstrated the presence of cytokeratin filaments and small desmosomes, as well as features of anaplasia (irregular cytoplasmic margins with invaginations, surface ruffling and microvilli, abnormal intermediate filament production and arrangement, and irregular nuclear membrane). Karyotype analysis of the SCCF1 cells demonstrated aneuploidy with a stemline chromosome number of 34, loss of chromosomes, and the presence of abnormal "marker" chromosomes. The growth curve of the cells demonstrated logarithmic growth for six days. The cytoplasm stained strongly positive for PTHrP, and secretion was demonstrated by measuring PTHrP (1-84) in culture medium with a two-site immunoradiometric assay. Treatment of SCCF1 cells with TGF-beta resulted in >2-fold increase in PTHrP mRNA, but TGF-beta had no effect on PTHrP mRNA stability. RT-PCR was used to amplify a 321-base pair region of feline PTHrP mRNA encoding the prepro and part of the coding region. The cDNA was cloned into pCR 3.1 (Invitrogen) and sequenced. The comparative nucleotide sequence homology with canine PTHrP was 98%, human 93%, and rat and mouse 87%. The predicted amino acid sequence of PTHrP –22 to 72 was identical to that of dog, with 96% homology to human, and 98% homology to mouse and rat PTHrP. This cell line will permit mechanistic experiments on genetic dysregulation in neoplastic keratinocytes of the feline oral cavity, and development of an important natural model of human head and neck cancer.
33 KD BINDING PROTEIN MAY REGULATE PARATHYROID HORMONE-RELATED PROTEIN MESSENGER RNA STABILITY AFTER TRANSFORMING GROWTH FACTOR-BETA TREATMENT IN SQUAMOUS CARCINOMA CELLS
R. S. Sellers, C. C. Capen, T. J. Rosol*
The Ohio State University, Columbus, OH, USA
Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, is due to expression of parathyroid hormone-related protein (PTHrP). PTHrP mRNA transcription rate and stability are increased in response to TGF-beta, which is often produced by malignant neoplasms. Utilizing a cell-free mRNA degradation assay, the effect of cytoplasmic proteins from vehicle and TGF-beta-treated squamous cell carcinoma cells on the rate of PTHrP mRNA decay was compared between full-length PTHrP mRNA and mRNA with deletion of the 3'-untranslated region (UTR) (truncated PTHrP mRNA). The average half-life of full-length PTHrP mRNA was mildly increased (1.6 fold) with TGF-ß-treated cell extracts. However, when truncated PTHrP mRNA was assayed with TGF-beta-treated cytoplasmic extracts there was a greater than 5-fold increase in PTHrP mRNA half-life and no change in vehicle-treated samples. In addition, the 3'-UTR of PTHrP was not able to destabilize ß-globin mRNA, a normally stable message. These studies revealed that stabilization of PTHrP mRNA in response to TGF-beta may be dependent on cis-acting sequences in the coding region of the mRNA. UV cross-linking of mRNA and cytoplasmic proteins from squamous carcinoma cells treated with either vehicle or TGF-beta and PTHrP mRNA from coding and 3'UTR regions, revealed 33 and 66 kD proteins that bound within a 176 nucleotide mRNA comprised predominately of the terminal coding region with a small region (30 nucleotides) of the 3'UTR. The protein-mRNA binding was present in vehicle-treated cytoplasmic extracts. Protein binding to the mRNA constructs was inhibited by TGF-beta. Therefore TGF-beta expression by cancers may play an important role in the pathogenesis of HHM by modulating PTHrP mRNA stability.
FEMORAL AND LUMBAR SPINE BMD CHANGES IN PATIENTS WITH PRIMARY HYPERPARATHYROIDISM: DIFFERENT IMPROVEMENTS FOLLOWING SUCCESSFUL PARATHYROIDECTOMY IN MALE AND FEMALE PATIENTS
F. Lumachi1*, G. Luisetto2, A. Nardi3, V. Camozzi2, V. Masiero1, G. Favia1
1Endocrine Surgery Unit, Dept Surg & Gastroent Sciences, University of Padua, School of Medicine, 35128 Padova, Italy
2Division of Endocrinology, Dept Med & Surg Sciences, University of Padua, School of Medicine, 35128 Padova, Italy
3Bone Metabolism Unit, City Hospital, Rovigo, Italy
Introduction. The aim of this study was to analyze the femoral neck and lumbar spine BMD changes in patients with PHPT following successful PTx, and to correlate the results with the age and sex of the patients.
Patients and Methods. Thirty-nine patients (median age 57 years, range 23-76) with confirmed PHPT underwent both femoral and lumbar (L2-L4 region) spine osteodensitometry using a dual-energy X-ray absorptiometry. Patients were divided into three groups: Group A (12 premenopausal women, mean age 44.1±9.0 years), Group B (16 postmenopausal women, mean age 64.2±6.9 years; p=0.00, Student's t-test), and Group C (11 men, mean age 54.1±14.8 years; p=NS in respect of women's age). None of the Group B patients had received estrogen replacement therapy at any time, and no Group A patient had used oral contraceptives. Preoperative s-alkaline phosphatase (ALP), s-bone-ALP, s-osteocalcin, and s-PTH 1-88 levels did not differ significantly (p=NS) among the three groups. In Group A patients s-creatinine levels (A=86.50±15.89, B=85.06±22.49, C=83.82±17.85 mmol/L; p=0.016) were lower, whereas s-calcium levels (A=2.94±0.24, B=2.80±0.17, C=2.92±0.25 mmol/L; p=0.012), L2-L4 BMD values (A=0.8588±0.0781, B=0.7301±0.1363, C=0.8002±0.1465 g/cm2; p=0.007), and trochanteric BMD values (A=0.5871±0.0964, B=0.5213±0.2117, C=0.6865±0.9703 g/cm2; p=0.023) were higher.
Results. At short-term (mean 13 months, range 9-15 months) follow-up following successful PTx all biochemical parameters were normal with significant (p<0.05) differences in respect of the preoperative values. Overall postoperative BMD values improved between 4.77% and 11.48% (median 6.37%). Significant differences were found only in Group A (L2-L4=0.8588±0.0781 vs 0.9575±0.1063, p=0.016; femoral neck=0.6056±0.1217 vs 0.722±0.1382, p=0.039; total hip: 0.7415±0.1424 vs 0.8890±0.1267, p=0-013) and in Group C patients (L2-L4=0.8002±0.1465 vs 0.9411±0.137, p=0.023). No difference (p=NS) was found in Group B patients.
Conclusions: At short-term follow-up PTx did not result in a significant increase of BMD in postmenopausal patients with PHPT, suggesting the need of estrogen hormones in complete bone remodeling.
REGULATION OF PTHrP GENE EXPRESSION: A PATTERNS OF DNA METHYLATION IN BREAST CANCER CELL LINES
M. Rousière1, C. Cataisson1, J. Foley2, J. Taboulet1, Z. Bouizar1*
1U.349, Centre Viggo Petersen, Hôpital Lariboisière, 2 rue Ambroise Paré, Paris, France.
2Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA
Parathyroid hormone-related peptide (PTHrP) is expressed in a large fraction of breast cancers that do not produce the syndrome of humoral hypercalcemia of malignancy. In a previous study, we have reported that transcripts of the 1-139 isoform derived from P2 and P3 promoters were more abundant in tumors that ultimately metastasize, and that P3 transcripts were the most abundant in tumors that metastasized to bone. However, mechanisms underlying increased PTHrP expression are largely unknown. We investigated DNA methylation state and PTHrP gene expression in 4 cell lines derived from normal human breast epithelium immortalized with the SV40 large T antigen (S1T6, S1T3, S2T2 and NS2T2A1) that have differential tumorigenic potential as well as the established breast cancer cell lines T47D, MCF7 and MDA 231. The methylation status of PTHrP promoter region was investigated by methylation-sensitive restriction enzyme digestion, and then correlated with PTHrP mRNA expression and PTHrP promoter usage determined by semiquantitative RT-PCR. In cell lines that failed to express or expressed low levels of PTHrP (S1T6, S1T3, T47D and MCF7, MDA 231), the 5' sequences 1.2 to 0.7kb upstream of the P2 promoter were hypermethylated, whereas high PTHrP-expressing lines (S2T2 and NS2T2A1) were hypomethylated at these sites. The high PTHrP-expressing lines had abundant P2-derived transcripts while the low expressing contained low levels of these transcripts. Treatment with 5-azaC increased PTHrP gene mRNA levels in the low PTHrP-expressing lines. In transient transfection experiments, a reporter construct with 2kb of upstream PTHrP sequences containing the P2 and P3 promoters was equally active in both the high and low-PTHrP expressing cell lines. Methylation of this construct in vitro using HpaII amethylase significantly reduced the levels of reporter gene activity in both high and low PTHrP expressing cell lines. Our data provide evidence that methylation of CpG residues upstream of the P2 promoter may regulate PTHrP gene expression in human breast epithelial and breast cancer cell lines.
PTHrP EXPRESSION IN TUMORIGENIC BREAST EPITHELIAL CELL LINES: ROLE OF P3 PROMOTER AND THE ETS-1 SITE
C. Cataisson1, A. Abdalkhani2, M. Rousière1, R. K. Lindemann3, J. Dittmer3, J. Foley2, Z. Bouizar1*
1U.349, Centre Viggo Petersen, Hôpital Lariboisière, 2 rue Ambroise Paré, Paris, France
2Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA
3Abteilung Molekularbiologie, Institut fuer Zellbiologie, Universitaet Tuebingen, 72076 Tuebingen, Germany
Parathyroid hormone-related protein (PTHrP) appears to be the major mediator of humoral hypercalcemia of malignancy and is produced by many tumors, including breast cancers. The human PTHrP gene is a complex transcriptional unit, using at least three individual promoter elements (P1-P3), and alternative splicing of 3’ exons occurs producing mRNAs that are translated into PTHrP proteins of 139, 173, and 141 amino acids. A previous retrospective study suggested that primary tumors that express increased levels of P3-initiated transcripts or mRNAs that encode the 1-139 isoform exhibit an increased probability to metastasize to bone. In this study, we used three human immortalized mammary epithelial cell lines that differ in tumorigenicity and PTHrP expression. Using RT-PCR we investigated 5' and 3' alternative splicing of PTHrP transcripts and PTHrP promoter usage in the lines. The tumorigenic cell lines express higher levels of P3 and P2-initiated transcripts than the non-tumorigenic cell line. In the tumorigenic cell lines, 1-139 is the predominant PTHrP RNA isoform. RNA stability analyses suggest that increased transcriptional activity of the P2 and P3 promoters accounted for the differential mRNA expression among the three cell lines. To identify regulatory elements responsible for the progressively increased expression of PTHrP transcripts in the tumorigenic cell lines, we transfected these cells with a series of human 5' PTHrP reporter gene constructs. Normalized reporter gene activity from constructs that contained P3 in isolation corresponded to the endogenous transcriptional level of this promoter. Using site-directed mutagenesis, we found that a previously described Ets binding site upstream of the P3 promoter was crucial for PTHrP P3 promoter activity in the tumorigenic cell lines. When we determined the expression profiles of the Ets transcription factors Ets1, Ets2 and Esx in the cell lines, we observed that Esx and Ets2 are equally expressed in all three cell lines, while Ets1 levels are significantly higher in the tumorigenic cell lines. These findings suggest that Ets transcription factors such as Ets1 may be involved in the upregulation of PTHrP expression during mammary tumor progression.
USEFULNESS OF 99MTC-SESTAMIBI SUBTRACTION SCINTIGRAPHY AND SPIRAL-CT IN PARATHYROID ADENOMAS LOCALIZATION. ANALYSIS OF RESULTS IN PATIENTS WHO UNDERWENT SUCCESSFUL PARATHYROIDECTOMY
F. Lumachi1*, P. Zucchetta2, G. Luisetto3, F. Angelini4, R. Behboo1, M. C. Marzola2, G. Favia1
1Endocrine Surgery Unit, Dept Surg & Gastroent Sciences
2Nuclear Medicine Service
3Division of Endocrinology, University of Padua, School of Medicine, 35128 Padova, Italy
4Radiology Service, City Hospital, 35128 Padova, Italy
Introduction. In the minimally-invasive era sensitivity of noninvasive localization procedures in patients with primary hyperparathyroidism (PHPT) has improved, but few studies considered the usefulness of a combination of two or more techniques. The aim of this retrospective study was to analyze the results obtained using 99mTc-sestamibi subtraction scintigraphy (SS) and spiral-CT scanning (CT) together in patients who underwent successful parathyroidectomy (PTx).
Patients and Methods. Thirty-nine patients with PHPT due to a single parathyroid (PT) adenoma underwent both SS and CT before successful PTx. There were 12 (30.8%) men and 27 (69.2%) women with an overall median age of 59 years (range 23-78 years). Main preoperative biochemical data was the following: s-calcium = 2.88±0.29 mmol/L, s-alkaline phosphatase = 146.59±92.08 U/L, s-creatinine = 71.774±16.07 micromol/L, s-PTH 1-84 = 183.51±109.05 ng/L. In each patient the final pathology showed a PT adenoma (median maximum size = 18 mm, range 8-30 mm) that was in an ectopic site in 13 (33.3%) patients.
Results. Sensitivity and positive predictive value (PPV) were 84.2% and 82.1% (SS), and 87.2% and 87.2% (CT), respectively (p=NS, chi-squared test). When SS and CT were used together sensitivity reached 100%. There was no difference (p=NS) in SS and CT sensitivity between patients with PT adenomas in typical and ectopic site. Preoperative s-calcium and s-PTH levels of patients with false negative (FN) CT were significantly higher (2.91±0.27 vs 2.59±0.33 mmol/L, P=0.045; 192.41±113.87 vs 123.0±27.77 ng/L, p=0.004, respectively), and the mean size of the PT tumor was smaller (14.12±4.56 vs 18.86±6.22 mm, p=0.046). No differences (p=NS) were found between patients with true positive and FN SS, both in the biochemical parameters and in the mean size of the removed PT gland.
Conclusion. In patients with PHPT the starting preoperative localization procedure should be SS because its sensitivity is independent both from the site (typical or ectopic) and the size of the PT tumor. A combination of SS + CT may improve significantly both sensitivity and PPV in such patients.
RECOMBINANT HUMAN RANK LIGAND (RANKL) AND MACROPHAGE COLONY-STIMULATING FACTOR (M-CSF) FAIL TO RESCUE HUMAN INFANTILE OSTEOCLAST-POOR OSTEOPETROSIS (OP) IN VITRO
H. M. Massey1, C. G. Steward2, C. Wilson3, A. Vellodi3, M. A. Horton4, A. M. Flanagan1 *
1Royal Free and University College London Medical School, London, UK
2Royal Hospital for Sick Children, Bristol, UK
3Great Ormond Street Hospital, London, UK
4Bone and Mineral Centre, University College, London, UK
Osteopetrosis (op) is a disease phenotype characterised by a marked reduction in osteoclastic bone resorption. Normal or increased numbers of osteoclasts compared with healthy individuals are generated in the common variant of this disease. In such cases the defect is most likely to be inherent to the mature osteoclast and can be cured by bone marrow transplantation (BMT). However, op also results from failure of osteoclast formation (osteoclast-poor op). In mice, this phenotype occurs in the absence of M-CSF or RANKL. As these membrane-bound factors are produced by bone marrow stromal osteoclast-supportive cells, neither M-CSF– deficient nor RANKL-deficient variants of op are rescued by BMT. It is not known whether the rare forms of human osteoclast-poor op are caused by genetic mutations encoding M-CSF or RANKL. If this were the case, individuals with the disease could theoretically be rescued by treatment with the appropriate recombinant molecule.
Two infants were diagnosed with a malignant form of human op and a histological diagnosis of the osteoclast-poor variant was made. Peripheral blood mononuclear cells (PBMCs) from both children were cultured in the presence of recombinant soluble M-CSF (25 ng/ml) and RANKL (30 –100 ng/ml) for 2 and 3 weeks. Control cultures included PBMCs from age-matched children, one of whom had an osteoclast-rich form of op. Formation of osteoclasts (alphavbeta3VNR+, TRAP+, cathepsin K+ cells co-localising with F-actin ring+ cells, upto 100µm in diameter) occurred in all the control cultures, and 20-80% of the surface of the bone slices were resorbed in the healthy individuals. No bone resorption occurred in the osteoclast-rich op cultures. In contrast, PBMC cultures from the osteoclast-poor op children, formed very occasional small mono- and binucleate (10µm diameter) osteoclasts identified as F-actin ring-positive cells which also expressed alphavbeta3 VNR and TRAP.
Since neither M-CSF or RANKL rescued the two cases of osteoclast-poor op in vitro there would be little benefit in treating the two children with either of these soluble recombinant proteins. The findings also indicate that the genetic mutation is unlikely to lie in the genes encoding these molecules. Finally, these results demonstrate that this experimental model replicates the human op phenotype in vivo and should prove useful in analysing the pathogenesis of the various forms of human osteopetrosis.
COMPARISON OF AN INJURY STRESS-RESPONSE ON SKELETAL GROWTH IN TWO INBRED STRAINS OF MICE.
B. M. Bowman*, C. C. Hooper, J. Shelby, S. C. Miller
University of Utah, Salt Lake City, UT, USA
Genetically distinct inbred mice differ in their immune and endocrine responses to stress. The Balb/c mouse, a stress-responsive strain, responds to stress trauma with increased and sustained levels of corticosterone and increased production of inflammatory cyctokines. Additionally, we have demonstrated profound bone loss following burn trauma in the Balb/c mouse (J. Burn Care Rehab, 1997). Osteopenia following burn trauma is also observed in humans. To explore the genetic predisposition for osteopenia, we studied the effects of burn trauma on cancellous and cortical bone in the Balb/c and a more stress-resistant strain, C3H/HeN.
Eight-week-old male Balb/c and C3H/HeN mice were divided into sham and injury groups. The mice received an 18% total body surface area scald injury under deep surgical anesthesia. The size of the thermal injury was calculated using Meeh's formula for total body surface area in mice. The sham group was treated identically without receiving the thermal injury. Both the sham and injury groups were injected with calcein and tetracycline on day 3 and day 8 following the burn as fluorescent bone markers and euthanized on day 10 post-injury.
The bone formation on the cortical bone surfaces decreases to almost zero following the injury in both strains of mice. The cancellous bone in the proximal tibia metaphysis also showed significant decreases in the percent labeled-surface, percent mineralizing surface and bone formation rates in both strains of mice. Cancellous bone formation indices of the sham groups were significantly different in the two strains of mice. The Balb/c sham group had a lower percent labeled-surface, percent mineralizing surface and bone formation rate than the sham CeH/ HeN mice. Balb/c mice exhibited a greater change of percent bone between the sham group and the injury stressed group than the C3H/HeN mice.
Both strains of mice had decreases in bone mass and formation in response to thermal injury. The responses, however, were more severe in the Balb/c strain compared with the C3H strain. These results indicate genetic predispositions of the skeleton to burn trauma and may relate to differences in immune and endocrine responses to trauma in these strains.
THE EFFECT OF ALENDRONATE ON LONG BONE GROWTH AND GROWTH PLATE IN THE OIM MOUSE MODEL FOR OSTEOGENESIS IMPERFECTA
K. D. Evans1*, R. B. Martin2, S. T. Lau2, A. M. Oberbauer1
1Department of Animal Science, University of California Davis, Davis, CA, USA
2Orthopaedic Research Laboratories, UC Davis Medical Center, Sacramento, CA, USA
The bisphosphonate, alendronate, reduces remodeling in post-menopausal osteoporosis by inhibiting osteoclast function. This has led to the use of bisphosphonates as a treatment for osteogenesis imperfecta (OI). Though alendronate treatment of OI enhances bone strength, thereby reducing fractures, the consequences of alendronate on linear bone growth are not well defined. This study investigated the effect of alendronate on growth plate area and long bone length using a mouse model for OI. Vehicle (0 mg/kg/mo), low (0.05 mg/kg/mo) and high (10 mg/kg/mo) alendronate doses were injected weekly between 4-12 wks of age. Humerus and ulna from both sexes of oim/oim, oim/wt, and wt/wt genotypes were evaluated. Humerus length in both sexes of the oim/wt and wt/wt genotypes was inhibited by the high dose, but only males of the oim/oim genotype displayed high dose inhibition (P<0.003). For the ulna (with growth plates that close at an earlier age), the high dose inhibited length in the oim/wt genotype for both sexes (P<0.05), but did not affect the length in the wt/wt genotype for either sex. The high dose in males, but not females, shortened the oim/oim ulnas. The sex differences within the oim/oim genotype may be attributable to differences in the hormonal milieu between males and females, which plays a major role in bone growth and development. A strong genotype effect with animals showing a significant decremental gradation in bone length correlating to the expression of mutant alleles was also observed across all analyses. The low dose and the vehicle dose were equivalent (P>0.05). Alendronate significantly increased growth plate area with increasing dose. Additionally, the high dose produced taller growth plates in all genotypes by increasing hypertrophic cell number, indicating a failure of vascular invasion-induced apoptosis at the chondro-osseous junction. Growth plates in the oim/oim genotype were taller showing a disrupted cell structure similar to human OI patients, while no differences were observed between oim/wt and wt/wt growth plates. These results indicate that while alendronate may increase the strength of growing bones exhibiting OI, high doses may impair growth plate function through alteration of growth plate organization and mineralization processes.
HYPOPHOSPHATEMIC HYPOPARATHYROIDISM AND RICKETS WITH CALCIUM SUPPLEMENTATION AND HYPOCALCITRIOLIC HYPERPARATHYROIDISM AND OSTEOCHONDROSIS WITH CALCIUM AND PHOSPHORUS SUPPLEMENTATION IN YOUNG DOGS
I. Schoenmakers1*, G. Voorhout1, C. S. Carlson2, J. A. Mol1, D. Richardson3, H. A. W. Hazewinkel1
1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
2Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA
3Hill's Science and Technology Center, Topeka, Kansas, USA
The consequences of calcium (Ca) and/or vitamin D (Vit D) deficiency at young age are well defined, whereas relatively little is known of the effects Ca over-supplementation, as may be introduced by the fortification of artificial milk and solid foods. Since at a young age, Ca absorption is a function of Ca intake and availability, due to the passive transfer of Ca across the intestinal epithelium, high Ca uptake may be expected.
We investigated the consequences of Ca supplementation, with or without a proportional high phosphorus (P) intake on the hormonal Ca regulation, Ca and P balance and skeletal development in 3 groups of rapidly growing dogs. Dogs were either fed a diet according to the requirements (controls; 1.04% Ca on dry matter base (dmb)), or with a tripled Ca content (HCaNP; 3.11 Ca % on dmb), from the earliest moment of solid food intake, until half way their rapid growth phase. Since alterations in dietary Ca intake influences P availability, absorption and metabolism, the third group was fed a diet with both an tripled Ca and P content (HCaHP; 3.10 % Ca, 2.77% P on dmb).
HCaNP feeding led to hypercalcemia, hypophosphatemia and hypoparathyroidism, together with similar plasma calcitonin, 1,25(OH)2 Vit D and 24,25(OH)2 Vit D concentrations and elevated 25(OH) Vit D concentrations when compared to controls, and radiological and histological characteristics of rickets. This may in part be attributed to the discrepancy between the balance of Ca and P. Food intake was below the energy requirements, and may have contributed to the decrease in the plasma IGF-1 concentration in this group.
Dogs fed the HCaHP-diet, gradually developed hyperparathyroidism and hypercalcitoninemia, together with decreasing 1,25(OH)2 Vit D, but normal 25(OH) Vit D and 24,25(OH)2 Vit D concentrations. The absorbed Ca:P ratio was appropriate to allow for the calcification of newly formed osteoid, but endochondral ossification was disturbed as reflected by focal areas of delayed ossification, i.e. as in osteochondrosis.
In conclusion, excessive dietary Ca supplementation during growth leads to hypophosphatemic rickets when dietary Ca and P are not proportionally elevated, and to osteochondrosis when dietary P is equally available.
PHEX GENE-EXPRESSION IN HUMAN LUNG AND A549 CELLS
E. Zoidis1*, C. Ghirlanda1, K. Tisa1, M. Gosteli-Peter1, W. Weder2, J. Zapf1, C. Schmid1
1Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital of Zurich, CH-8091 Zurich, Switzerland
2Division of Thoracic Surgery, Department of Surgery, University Hospital of Zurich, CH-8091 Zurich, Switzerland
X-linked hypophosphatemia, a renal phosphate (Pi) wasting disorder, is caused by inactivating mutations in the PHEX gene. PHEX is a membrane-bound endopeptidase encoded on the X chromosome and highly homologous to other type II integral membrane endopeptidases such as endothelin-converting enzyme-1 and neprilysin. Its substrates and products are unknown. In the rat, Phex mRNA (6 kb) is expressed predominantly in bones and lungs. Since few studies have addressed PHEX/Phex expression in the lung and since species differences may exist, we studied PHEX mRNA expression in adult human lung and in the human alveolar epithelial cell-derived A549 (non-small lung cancer) cell line. Considering the possibility that PHEX may not only be a Pi regulating but also a Pi-regulated gene, we also studied Pi transport and Pi transporter (PiT-1) mRNA expression in these cells. PiT-1 mRNA (4 kb) is expressed in adult human lung and in A549 cells. Pi transport in A 549 cells is a carrier-mediated process dependent on the presence of Na+ (Nad) in the extracellular space. The Km for Pi was 0.089±0.011 mM. NadPi transport rate was barely (1.11±0.03 -fold, 4 experiments) increased in response to 24 h Pi deprivation. Total RNA from A549 cells was reverse-transcribed (RT): PCR using human PHEX primers resulted in the expected 700 bp product. It was sequenced and PHEX-identical. Northern blot analysis revealed also neprilysin mRNA expression in A549 cells. A549 cells were kept for 48 h in test media. Semiquantitative RT-PCR experiments showed that PHEX expression levels were 5-fold higher in cells exposed to 5% FCS, as compared to serum-free medium with or without 5 nM IGF I. PHEX and neprilysin expression was also detected in normal human lung tissue (obtained from patients undergoing pneumectomy for lung cancer) by RT-PCR.
Our data demonstrate expression of PHEX and neprilysin transcripts in A549 cells and in human lung. Similarly to neprilysin PHEX may control the activity of bioactive peptides targeting blood vessels and the kidney.
THE BRITTLE MOUSE MODEL FOR NON-LETHAL OSTEOGENESIS IMPERFECTA HAS DECREASED BONE DENSITY, BONE FORMATION RATE AND FEMUR STRENGTH
J. C. Marini1*, C. Bergwitz1, A. Forlino1, E. P. Frankenburg2, K. M. Kozloff2, S. A. Goldstein2, M. Nahounou3, G. Gronowicz3
1Heritable Disorders Branch, NICHD, NIH, USA
2Orthopedic Research Labs, U Michigan School of Medicine, USA
3Dept of Orthopedics, U Conn Health Center, USA
We have developed a knock-in murine model (Brtl IV) for non-lethal osteogenesis imperfecta which reproduces the skeletal, biochemical and molecular features of type IV OI. This mouse carries a point mutation in one col1a1 allele, resulting in a G349C substitution in a1(I) collagen chains. Brittle mice are 2/3 to 3/ 4 the size of normal littermates. There is generalized undermineralization of the skeleton. Their rib cage has a flared base and narrow apex; multiple rib fractures are evident at birth. There are also fractures and bowing of the long bones. Histologically, long bone are undermineralized with disorganized trabeculae.
We examined the BMD of Brtl mice using the Piximus densitometer. Two month old Brtl males and females have significantly decreased total body, lumbar spine and femoral BMD, averaging 84%, 73-74% and 76-78% of age and gender matched normal littermates at these sites, respectively. By 6 months of age, Brtl males still have significantly decreased total body (88% of control) and lumbar spine (82% of control) BMD. However, the femoral BMD in 6 month old Brtl males is not significatly decreased compared to wild type. This finding results from a relative increase in Brtl femoral BD with age (p=0.0001), in comparison to a stable femoral BMD in normal males. The femurs of Brtl mice were subjected to biomechanical evaluation, including whole bone 4-point bending to failure. Geometric properties including cortical thickness, area and moment of inertia were evaluated utilizing micro-CT. Preliminary data indicates that Brtl bone is weaker at 1 and 2 months of age, with reduction in energy to failure on the bending test. Pre-and post-yield displacements are similar, suggesting that the bone does not have a "brittle" failure pattern. Microspecimens from the cortices of bone are being evaluated for changes in the material properties of the bone ECM. Preliminary histomorphometric analysis indicates that 2 month old male Brtl mice have decreased total bone volume and femoral cortical width, as compared to normal littermates. At 8 months of age, Brtl mice continue to demonstrate decreased bone volume and trabecular bone area with a decreased bone formation rate.
FOOT ALGO-DISTROPHIC, POST-TRAUMATIC SYNDROME. ISSUES AND POSTURE MODIFICATIONS
F. M. Donelli
No affiliations
Algo-neurodystrophia defines a distinct, poly-tissue syndrome, marked by pain, hyperalgia, vasomotorial disorders due to a hypertonic sympathetic nervous system; analgetic function inability, skin and appendage trophic disorders, subsequent bone atrophy.
Its etiology is unknown while predisposing factors are: trauma and surgery. It usually occurs three to four weeks after trauma or, even later, clinically symptoms can be classified in three stages:
Stage 1: - initial stage: pain is localized and the region concerned is hot
Stage 2: - status: pain is more acute and the region concerned is colder and cyanotic
Stage 3: - larger stage: pain ceases and dystrophic alterations become chronic.
It seems that the algo-dystrophic syndrome is more frequent in adult patients aged 40 to 60. While its incidence looks similar in both sexes.
Diagnostic Course
Instrumental tests: alterations of hormonal bio-chemical parametres of the phosphor-calcium metabolism. X-ray and scintillography, CAT and magnetic resonance, total-body bone densitometric test.
Therapy and Posture Significance in Functional Recovery:
A pharmacological treatment is obtainable by systemic inhibition of the sympathetic nervous system - sympathetic blocks, as well as ganglion blocks at the star-shaped ganglion and, finally, through localized venous infusion of sympathetico-lytic agents. Treatments by clodronate phleboclysis should also be of choice.
A physical rehabilitating treatment should also be associated to hydro-kinesitherapy and shock-wave therapy (Swiss-dolor clast).
Posture maintaining, e.g. body and limbs position in respect to each other and as a function of external space is a task of the locomotive system which should show a perfect functional aesthetic morphology of the trunk and the limbs, of receptors and efferent canals, that collect information from outside, and from which we learn the posture relationship to the environment.
Postural Pathology: Frequent and difficult to be understood. In our opinion the approach should aim to:
a) evaluate the walking;
b) evaluate various contributory causes.
In conclusion the state of the art and vision of myo-articular mechanics aknowledge the importance of postural alterations, as a post-traumatic exitus of foot algo-dystrophy, of upper systems reaching as far as the upper limb.
LONG-TERM TREATMENT WITH RECOMBINANT HUMAN GROWTH HORMONE ATTENUATES BONE LOSS FOLLOWING BURNS IN CHILDREN
G. L. Klein1,2*, D. W. Hart1, S. Mohan3, S. E. Wolf1,2, D. N. Herndon1,2
1University of Texas Medical Branch, Galveston, TX, USA
2Shriners Burns Hospital, Galveston, TX, USA
3Pettis VA Hospital and Loma Linda University Medical School, Loma Linda, CA, USA
Burn injury greater than 40 percent body surface area results in both acute and chronic changes to bone. Acutely, patients are hypocalcemic and magnesium-depleted possibly due to increased parathyroid calcium receptor expression and resultant hypoparathyroidism. Bone formation is reduced, possibly related to increased endogenous production of glucocorticoids and pro-inflammatory cytokines, or to immobilization. Chronically, pediatric patients have low bone mineral density (BMD) z-scores and increased extrapolated fracture incidence a mean of 5 years post-burn. Late-onset vitamin D depletion secondary to lack of sun exposure may play a role. The long-term BMD reduction may reduce peak bone mass. Acutely, these children are functionally growth hormone deficient due to low levels of insulin-like growth factor-1 (IGF-1). Our aim was to determine if one year's therapy with recombinant human growth hormone (rHGH) could improve bone mass. We performed a preliminary study in which 12 children with burns more than 40 percent body surface area, ages 4-19 yr were randomized to either rHGH 0.05 mg/kg/d subcutaneously or placebo in double blind fashion. Treatment began at discharge (baseline) and continued till 1 year post-burn. We measured whole body BMD and bone mineral content (BMC) at baseline and 1 year and serum IGF-1 and IGF binding proteins -4 and -5 (IGFBP4 and -5) at 1 year. At 1 year there was an increase from baseline in BMC in the rHGH group of 138±51(SD)% (n=4) compared to placebo, 84±20% (n=8) from baseline, p=0.016 by rank-sum test. There were no differences between groups in BMD, IGF-1 or IGFBP4 or 5, though IGFBP4, a putative inhibitor of IGF-1 access to tissue receptors remained elevated versus age-matched normals, combined mean 552±261 ng/ml with normal range of 177-368. These data suggest that rHGH may increase BMC and bone size compared to placebo despite high circulating levels of IGFBP4. A larger scale study is now underway to confirm these results.
LONG-TERM FOLLOW UP OF BONE MASS IN TREATED ADDISON’S DISEASE
E. Jodar*, G. Martínez, S. Azriel, A. Serraclara, E. De Juan, M. Ruiz Valdepeñas, F. Hawkins
Servicio Endocrinología, Hospital Universitario 12 de Octubre, Madrid, Spain
The potential deleterious effects on bone mineral density (BMD) of usual prescribed doses of glucocorticoids in patients with Addison’s disease is controversial.
Our aim was to evaluate the long-term changes in BMD of patients with treated Addison´s disease.
Patients and methods: We studied 25 patients (6 men, 19 women; aged 59±11 years) with treated Addison’s disease (Hidrocortisone 30mg/d or Prednisone 7.5 mg/day). The mean duration of the disease was 22.7±11.7 years and the mean fasting cortisol and ACTH levels were 2.3±1.3 mcg/dl and 501±389 pg/ml. BMD was measured in the lumbar spine (LS), femoral neck (FN) and radius (third distal (TDR), ultra distal (UDR) and mid distal (MDR)) by DXA (Hologic QDR1000w, expressed as gr/cm2 and z-score). Rates of bone loss were calculated according previous DXA measurements in LS (48 and 60 months before) and in TDR, UDR and MDR (48 months before). Serum calcium, phosphorus, ALP, bone ALP, BGP, iPTH and 25(OH) vitamin D also urine PYR were measured.
Results: BMD (z-score; 95% confidence interval (95%CI)) was normal in LS: (– 1.15; 0.07); FN: (-0.90; 0.22) and UDR (-0.77; 0.36) and was slightly reduced in TDR: (-1.51; -0.37) and in MDR (-1.56; -0.30). No significant bone loss (g/cm2; 95%CI) was detected in LS (during 60 months): (-0.021; 0.023); and radius (during 48 months); TDR: (0.002; 0.021); UDR (-0.001; 0.025); MDR (-0.002; 0.021). All bone markers were normal: ALP: 145±55 UI/l, bone ALP: 15.9±5.1 ng/ml, BGP: 6.1±1.8 ng/ml, iPTH: 34.4±10.5; 25(OH) vitamin D: 33.1±14.3; urine PYR: 34.1±7.9 nmol/mmol Cr.
Conclusions: Patients with treated Addison’s disease show normal bone mineral density and normal bone turnover. No significant changes in bone mineral density were detected during long-term (48 and 60 months) of follow-up.
THE CHANGES OF SERUM FGF-2 LEVELS FOLLOWING BONE MARROW TRANSPLANTATION: IMPACT ON BONE MINERAL DENSITY
M. I. Kang*, W. Y. Lee, E. S. Oh, K. W. Oh, J. H. Han, B. Y. Cha, K. W. Lee, H. Y. Son, S. K. Kang, C. C.Kim
The Catholic University of Korea, College of Medicine, Seoul, Korea
FGF-2 plays a critical role in bone growth and osteogenesis. Loss of bone mass is usually detected after BMT and the rapid impairment of bone formation and the increase in bone resorption might play a role in post-BMT bone loss. Among the mechanisms of impaired bone formation following BMT, it is suggested that marrow stromal microenvironment is damaged by myeloablative chemo-radiotherapy. There has been no report in which the relationship between serum FGF-2 levels and bone mineral density (BMD) in human was studied. The purpose of this study was to identify the change of serum FGF-2 levels following BMT and its association with the change of BMD and bone turnover. We have prospectively investigated 10 patients undergoing BMT. BMD was measured with DEXA before BMT and 1 yr after BMT. Serum calcium, phosphorus, creatinine, and biochemical markers of bone turnover were measured before BMT and 1, 2, 3, 4 and 12 wks, 6 months, and 1 yr after BMT. Serum FGF-2 was measured in all patients before BMT and 1, 3, and 12 wks after BMT. The mean bone loss in the lumbar spine and total proximal femur, calculated as the percent change from the baseline to the level at 1 yr was 7.9%(p<0.05) and 14.4% (p<0.01), respectively. The serum ICTP, bone resorption marker, increased progressively until 4 weeks after BMT. Thereafter, it decreased gradually to reach basal values after 1 year. Serum osteocalcin, bone formation marker, decreased progressively until 3 wks after BMT. Thereafter, returned to basal level at 1 yr. Serum FGF-2 decreased to nadir at 1 wk and gradually recovered to basal values at 3 Ms. There was a strong negative correlation between the actual change of lumbar spine BMD during post-BMT 1 yr and FGF-2 serum level at 3 wk (r=-0.82, p<0.01), but not proximal femur. There was no correlation between serum FGF-2 levels and bone turnover markers during the entire period. In conclusion, there was a significant decline in the serum FGF-2 levels at immediate post-BMT period and it was related with the decrease of lumbar spine BMD during 1 year following BMT.
RELATIONSHIP BETWEEN BONE TURNOVER BIOCHEMICAL MARKERS AND ACTIVITY OF CROHN´S DISEASE IN THE PATHOGENESIS OF SECONDARY OSTEOPOROSIS.
M. S. Parisi*, A. C. Bagur, C. A. Mautalen
Sección Osteopatías Médicas, Hospital de Clínicas, Universidad de Buenos Aires, Argentina
Osteoporosis is a known extraintestinal complication of Crohn´s Disease (CD). The pathogenesis of decrease bone mass is multifactorial, and the relative contribution of the primary disease process, as compared with secondary factors like corticosteroid use, has not been well defined.
We report 8 months follow up of bone turnover biochemical markers in a 27 year old woman first seen by us in February 2000, with diagnosis of CD made 3 years before. She has only received one month of corticosteroid treatment 2 years before our first visit.
At presentation she reported 3 years of amenorrhea, and a resection of the jejune 2 years before. BMI was 19kg/m2. Laboratory test: serum calcium 7.1mg/dl (NV:8.9-10.4), phosphate 1.3mg/dl(NV:2.6-4.4), 25OH-vitamin D 13pg/ml(NV:18-62), PTHmm 20pg/ml(NV:20-100), BAP 240UI/l(NV:31-95). Urinary calcium 98mg/d(NV:80-200), CTX 1730ug/mmolCreat(NV:10-400). Bone mineral density(DEXA): L2-L4: 0.748g/cm2, Zscore –3.8 and total skeleton: 0.808g/cm2, Zscore –4.0. X ray of the lumbar spine revealed a fracture of L5. Treatment with prednisone 40mg/d, Mezalazina 1g/d, vitamin D 60 000UI/d, calcitriol 0.5ug/d and calcium 1000mg/d was started. Dose of prednisone was progressively diminished. During the 8 months follow up, peak levels of BAP and CTX coincided with clinical relapse of CD. An important decrease of values was seen after a new administration of high doses of prednisone. On the 7 month of follow up treatment with Mezalazina was changed by Aziatoprina 50mg/d.
Pathogenesis of secondary osteoporosis in this patient is multifactorial: amenorrhea, resection of the jejune, low BMI. At the time of BMD determination corticosteroid administration was not enough to produce this severe decrease of bone mass. The coincidence of peak levels of BAP and CTX with clinical relapse of CD support the hypothesis that the underlining inflammatory process contribute to increase bone turnover, and it is probably related to the pathogenesis of secondary osteoporosis.
PRELIMINARY REPORT: STUDY OF BONE MASS AND MINERAL METABOLISM IN CHRONIC HEPATITIS C MALE PATIENTS TREATED WITH ALPHA-INTERFERON AND RIBAVIRIN.
G. Martínez1*, E. Jodar1, A. Soto1, F. Hawkins1, J. A. Solis-Herruzo2
1Service of Endocrinology, Hospital 12 de Octubre, Madrid
2Service of Gastroenterology, Hospital 12 de Octubre, Madrid
Few studies have analyzed the effect of antiviral therapy on bone mass and mineral metabolism. We have conducted a cross-sectional study in 30 chronic hepatitis C caucasian male patients (mean age 41±5.2 SE years, range 31-48) ramdonly selected from our outpatient Unit, and treated with alpha-interferon (IFN) alone or in combination with ribavirin (RBV).
Patients and Methods: Thirteen patients (Group 1) were treated with IFN (3 MU tiw) for 12 months and 17 patients (Group 2) were treated with IFN (3 MU tiw) plus RBV (1000-1200 mg/day). Lumbar (L2-L4) BMD was measured with DXA (Hologic QDR 4500, Waltham, MA, USA) at the end of treatment. As control group 218 Caucasian Spanish normal males were used. T and Z-scores were obtained from sex and age-matched BMD. Bone formation markers (serum osteocalcin & bone-specific alkaline phosphatase), bone resorption (urinary pyridinoline & 24 hrs. calcium excretion) and calcitropic hormones (serum iPTH and 25-OH vitamin D) were measured using well standardized methods.
Results: No differences were found between groups in age, known duration of disease, route of transmision of HCV, serum ALT levels, HCV viral load or Knodell's histologic activity index score. Bone mass was significantly lower in group 2 than in group 1 patients (BMD 0.889±0.066 vs 1.108±0.076; T-score -1.25±0.55 vs 0.57±0.63; Z score -0.89±0.43 vs 0.67±0.54, p<0.001). 9 patients (52.9%) in group 2 had osteopenia (-2.5< Tscore<-1.0), but none had T-score<-2.5. No significant differences were found between groups in bone alkaline phosphatase, osteocalcin, pyridinoline or calcitropic hormones. Mean values of bone markers were in the normal range in both groups. Twenty-four hour urinary calcium excretion was significantly disminished in group 2 (79±36 mg/day vs 218±97 mg/day, p<0.001).
Conclusion: These preliminary results show a decreased bone mass and low bone turnover after combination therapy of chronic hepatitis C that include RBV. Longitudinal studies are needed to ascertain the effect of RBV on bone mass.
BONE MINERAL DENSITY (BMD), BIOCHEMICAL MARKERS AND FRACTURES IN PERITONEAL DIALYSIS (PD) PATIENTS
A. L. Negri1*, M. Alvarez Quiroga2, M. Bravo2, A. Marino1, E. E. Fradinger1, C. E. Bogado1, J. R. Zanchetta1
1Instituto de Investigaciones Metablicas, Buenos Aires, AR
2RTC Argentina, Suc. Caballito, Buenos Aires, AR
End stage renal disease is associated with skeletal abnormalities known as renal osteodystrophy. We studied the severity of skeletal changes in PD patients by measurement of BMD with a Lunar DPX densitometer at the femoral neck (FN) lumbar spine (LS) and total body (TB) and by biochemical markers of bone turnover (iPTH, bAP, CTX and PINCP). 43 patients were studied: 32 females (7 pre and 25 postmenopausal) and 11 men. Patients were 52±14 years of age (mean±SD) and had been in PD for a mean of 29 months (range 3 to 77); 82% of them had been previously on hemodialysis for a mean of 56.2 months (range 1 to 316). Calcium concentration in the PD fluid was 3.5 meq/l in 82.5% of the patients and 2.5 meq/l in the rest. BMD results are expressed as a function of age and gender (Z score) and in relation to the young adult (T score). Mean BMD Z and T scores were normal at all measurement sites. However, severe osteopenia-osteoporosis (T score less or equal -2) was seen in 36% of females and 50% of males at the FN, while at the LS it was seen in 33% of females and in no men. By biochemical markers (iPTH less than 150 pg/ml or bAP less than 27 U/l) 43% of the patients were classified as having low turnover. There was no correlation between biochemical markers and BMD at any site. Five of the 43 patients (4 females and 1 male) had 9 fractures (3 ribs, 3 pelvis, 1 metatarsal, 1 metacarpal and 1 ankle). There were no differences in Z scores or T scores at any site between patients with or without fractures. We conclude that skeletal changes and fractures in PD are less severe than those observed in hemodialysis patients and this could be probably related to the high prevalence of low bone turnover seen in these patients.
OSTEOBLASTIC STIMULATION AND TUMOR NECROSIS FACTOR-RELATED ACTIVATION-INDUCED CYTOKINE (TRANCE) EXPRESSION IN IDIOPATHIC HYPERCALCIURIA (IH)
E. Alonzo*, E. Hernandez, R. Carlini, E. Bellorin-Font, J. R. Weisinger
Division of Nephrology, Hospital Universitario de Caracas, Venezuela
Decreased bone mass has been reported in patients with IH. Bone histomorphometric studies in these patients have shown increased osteoclastic bone resorption, reduced bone formation and defective mineralization. Previous studies from our laboratory have demonstrated increased production of bone resorting cytokines by peripheral blood mononuclear cells obtained from IH patients. Since osteoclast formation requires an interaction with cells of the osteoblastic lineage, we cultured a normal human osteoblast cell line (NHost) in the presence of sera obtained from 20 IH (urinary calcium 272±85 mg/day), 20 normocalciuric stone formers (urinary calcium 80.2±37.7 mg/day), and 10 healthy controls. After confluence, osteoblasts were incubated for 24 and 48 hours in 24 well plates in the presence of increasing concentrations of sera (0, 10 and 20%). Alkaline phosphatase (ALP) activity in cell lyzates using nitrophenylphosphate as the substrate and TRANCE expression by immunoblotting were determined. Results were expressed as units of enzymatic activity per L per mg protein. ALP activity obtained from osteoblast cultures in the presence of IH sera was significantly increased when compared to those obtained after incubation with normocalciuric sera (2,456.9±1,274.8 vs. 1,519.6±1,224.9 U/L/mg protein, p< 0.02 and 2,959.7±1,807 vs. 1,435.9±1,023.8 U/L/mg protein, p<0.01 for incubations with 10% and 20% serum, respectively). Similarly, results obtained from the IH patients were significantly higher when compared to those from healthy controls at all serum concentrations. No difference was achieved between the results obtained from normocalciurics or healthy controls. TRANCE expression was significantly higher in the presence of IH serum as compared to the effect of control or normocalciuric sera. Our results demonstrate that normal human osteoblasts increased their activity in the presence of IH sera, suggesting that osteoblastic activation may play a role in the pathogenesis of the bone alterations observed in IH.
THE PATHOGENESIS OF POST-TRANSPLANT (TX) OSTEODYSTROPHY AS DETECTED BY EARLY ALTERATIONS IN BONE REMODELING.
E. Rojas1*, R. Carlini1, P. Clesca1, A. Arminio1, J. R. Weisinger1, K. A. Hruska2, E. Bellorin-Font1
1Division of Nephrology, Hospital Universitario de Caracas, Venezuela
2Renal Division, Washington University Medical School, St. Louis, MO, USA
Although pre-existing bone disease and the effects of steroids and cyclosporine (CsA) have been postulated as causes of osteoporosis after renal Tx, the cellular mechanisms remain unclear. Recent studies suggest that steroids inhibit osteoblastogenesis and promote osteoblast (OB) apoptosis. To examine the possible role of these mechanisms after Tx, we performed bone biopsies in 7 patients (pts) 3 weeks after a successful Tx. In 4 pts receiving kidneys from living related donors, a control biopsy was performed the same day of Tx. Apoptosis was determined by the method of TUNEL. Immunosuppression consisted of steroids, CsA and MMF. None of the biopsies pre-Tx revealed apoptosis. In 3 pts with pre-Tx PTH levels less than twice normal, adynamic bone disorder was observed in 2 and osteomalacia in 1. The post-Tx biopsies of these pts revealed markedly reduced OB surfaces and number compared to pts with high PTH. In addition, almost all OB were flattened and resting. Apoptotic cells were observed in the proximity of osteoid seams in 2 of the pts. In 4 pts, pre-Tx PTH levels were elevated (1093±160 pg/ml), and 3 of them had evidence of osteitis fibrosa and 1 of mixed bone disorder. Following Tx, they exhibited a significant change in OB morphology, demonstrating a marked shift towards quiescence from the cuboidal morphology of active OB. OB surfaces were not reduced, and OB apoptosis was not observed in the pre-Tx nor post-Tx biopsies. The present study demonstrates a major decrease in OB activity early after Tx as assessed morphologically and evidence of OB apoptosis in pts with low PTH. In pts with high PTH, decreased OB activity but no loss of number or apoptosis was observed at this early time point. The data are compatible with PTH induced osteoblastogenesis and suggests that impaired osteoblastogenesis and apoptosis may play important roles in the pathogenesis of post-Tx osteoporosis.
CIRCULATING OSTEOPROTEGERIN (OPG) IS AN UREMIC TOXIN TO CAUSE BONE AND PARATHYROID DISORDERS IN DIALYSIS PATIENTS
J. J. Kazama
Niigata University, Niigata, Japan, Kobe University, Kobe, Japan
National Sakura Hospital, Chiba, Japan
Tokyo Jikeikai Medical School, Tokyo, Japan
Tokai University, Kanagawa, Japan
The calcemic action of PTH is decreased in uremic patients. This phenomenon is referred as the increased skeletal resistance to PTH in uremia and has been considered to be a major cause of uremic bone and parathyroid diseases. Since the calcemic action of PTH mainly appears through osteoclastic bone resorption in uremic patients, we investigate the role of circulating osteoprotegerin (OPG) using an ELISA system (Cosmo-Bio, Japan). Serum OPG levels in pre-dialysis patients inversely correlated with glomerular filtration rate (p<0.001). Serum OPG levels in dialysis patients were significantly higher than that of age and gender matched healthy controls (2.67±1.39 ng/dl vs 0.91±0.28 ng/dl, p<0.001). The OPG molecules in uremic serum preserved the binding affinity to RANKL/OPGL. No significant correlations were noted between serum OPG levels and intPTH, calcium or inorganic phosphate levels in dialysis patients. In 20 non-diabetic dialysis patients underwent iliac bone biopsy examination, serum OPG levels showed a negative correlation with ES/BS (p=0.064). Patients with serum OPG levels greater than 2.2 ng/ml had less ES/BS (10.83±6.03%) than those with <2.2 ng/ml (21.16±11.74%, p=0.044) while no significant difference in intPTH levels was noted between these groups (341.14±427.09 pg/ml vs 198.69±158.83 pg/ml, ns). In summary, circulating OPG levels increased in uremic patients. Bone biopsy suggested that serum OPG level was an independent factor to modify bone metabolism in uremic patients. Thus circulating OPG may be an uremic toxin to develop uremic bone and parathyroid diseases through increasing skeletal resistance to PTH.
THE CHANGE OF SERUM TNF-ALPHA CONCENTRATION IN PATIENTS WITH OSTEODYSTROPHY AFTER TREATMENT OF 1-ALPHA(OH)D3
Z. M. Ding*, S. D. Zhu, R. Y. Yuan
Department of Internal Medicine, Beijing Ji Shui Tan Hospital
Objectives: To observe change of the serum TNF-alpha concentration when patients with osteodystrophy have been treated by 1-alpha(OH)D3.
Methods: Twelve patients with chronic renal failure and renal osteodystrophy treated with 1-alpha(OH)D3, 0.5µg /day for 2 weeks and 4 weeks individually.
Results: The serum TNF-alpha concentration descended obviously after 1-alpha(OH)D3 treatment.
Conclusions: 1-alpha(OH)D3 may regulate TNF-alpha producting and secreting in mononuclear leukocyte.
DETERMINATION OF BIOCHEMICAL MARKERS OF BONE METABOLISM IN TYPE 2 DIABETES MELLITUS PATIENTS
G. Y. Zhang*, B. G. Duan, H. Song, Y. Xue
Department of Endocrinology, Beijing Ji Shui Tan Hospital
Objective To investigate the characteristic of bone metabolism in type 2 diabetes mellitus. Methods The levels of serum 25OHD, IBGP and urine HOP, Crosslaps were determined in 64 type 2 diabetes mellitus patient. Results (1) The levels of serum 25OHD in type 2 diabetes mellitus group were lower than that in normal controls (Male and premenopause women vs controls P<0.01; postmenopause women vs controls P<0.05). (2) The levels of serum IBGP in the whole type 2 diabetes mellitus group were lower than that in normal controls (P<0.01). (3) The levels of urine HOP in men group were higher than that in normal (P<0.05). The levels of urine Crosslaps in the whole diabetic group were higher than that in normal (premenopause women vs controls P<0.05; Male and postmenopause women vs controls P<0.01). Conclusion The data suggest that the abnormal bone metabolism in type 2 diabetes mellitus are the lack of Vitamin D, low bone formation and turnover, heighten bone resorption. The serum IBGP and the urine Crosslaps are sensitive biochemical markers of bone metabolism.
CLINICAL STUDY ABOUT THE RELATIONSHIP BETWEEN DIABETES AND OSTEOPOROSIS
P. S. Li*, H. Qian, H. Jiang
Department of Gerontology, 304 Hospital, Beijing 100037, China
Objective: To investigate the relationship between type 2 diabetes and osteoporosis(op). Methods: Bone mineral density (BMD) of lumbar 2-4 and Ward's triangle was measured between diabetic group and two control groups (healthy young and elder using DXA, Norland-X236). The correlation between the parameters of glucose metabolism and bone metabolism parameters was observed.
Results: The BMD(L2-4) of young group was obviously higher than that of the elder groups (p<0.05). In the diabetic group, the BMD of L2-4 and Ward's were higher than that in elder group (p>0.05). The relevant analysis suggests there was no significant relativity among age | duration of diabetes and the BMD | serum BGP | ALK-B | Pyr-D | calcium | phosphorus and urine calcium in diabetic group.
There were obvious positive relations between body-weight index and serum phosphorus and BMD (P<0.05 and P<0.01).
Conclusions: Low body weight is a risk for diabetic patients to get OP. BMD in diabetic patients is a bit higher than in normal elder, but no significant difference.
IDENTIFICATION OF OSTEOPROTEGERIN AND OSTEOPROTEGERIN-LIGAND IN CALCIFIED ATHEROSCLEROSIC LESIONS
C. Dhore1*, J. Cleutjens1, S. van der Linden2, M. Daemen1, P. Geusens2,3
1Dept. of Pathology, CARIM, University of Maastricht, The Nethelands
2Dept. of Rheumathology, University Hospital, Maastricht, The Netherlands
3Biomedical Research Institute, Limburg University Centre, Diepenbeek, Belgium
The nature of ectopic bone and depositions of amorpheous calcifications in advanced atherosclerosic lesions is not well understood. Accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. We studied the presence of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in ectopic bone and amorpheous calcifications of type Vb human atherosclerotic lesions derived from the abdominal aorta.
The localization of OPG and OPGL was determined by immunohistochemistry in 5 patients with ectopic bone formation (Group 1) and in 7 patients with amorpheous calcified depositions without bone structure (Group 2).
In group 1, areas of lamellar bone were found containing lining cells, osteoblasts, osteoid, trabecular bone with cement lines, osteocytes and osteoclasts. In some areas cartilage structures were found containing matrix, lacunas and chondrocytes. In group 2, amorphous calcium deposits were found.
OPGL was absent in and around the bone matrix in group 1. Faint OPGL depositions were close to areas of inflammation, which included T cells. In the lesions with cartilage structure, no OPG or OPGL could be identified. In group 2, deposition of OPGL was found in areas immediately around amorphous calcium depositions. OPG was found in the lining cells and in some of the smooth muscle cells in group 1 but could not be detected in group 2.
These results indicate that OPG and OPGL are involved in ectopic bone formation and in the deposition of amorpheous calcium in atherosclerosis. This could provide a base for the study of the effect of bone-specific agents on the progression of atherosclerosis.
BONE HISTOMORPHOMETRY IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE
G. Lehmann*, T. Eidner, G. Stein, G. Hein
Dept. of Internal Medicine IV, Rheumatology & Osteology, University of Jena, Germany
Inflammatory Bowel Disease (IBD) is often followed by a loss of bone mass and bone quality. The resulting metabolic osteopathy should be characterized using laboratory and bone histomorphometric parameters.
We investigated 19 patients (9 men, 10 women, age 23-68 years) suffering from Crohn's disease (16) or ulcerative colitis (3). The mean duration of the disease was 5,2 years (0,5-23). At the time of investigation, 16 patients were treated with corticosteroids (for a mean of 1,9 ys.). As serologic parameters we determined ESR, CRP, calcium, 25-OH-vitamin-D3, PTH and osteocalcin. After tetracycline labelling, bone biopsies were taken from the right anterior iliac crest applying a vertical technique. The samples were embedded in methylmetacrylate, sectioned on a heavy-duty microtome and stained (Masson-Goldner, Gomori, Giemsa). The following histomorphometric parameters were obtained using a Merz grid: bone volume (BV, in % of total bone volume), osteoid surface (OS), eroded surface (ES), osteoblast- (ObS) and osteoclast-covered surface (OcS), mineralized surface (MS, each in % of bone surface) and mineral apposition rate (MAR in microm).
Patients with IBD showed increased CRP-levels (30,2±39,1 mg/l) and normal levels of calcium (2,32±0,21 mmol/l), 25-OH-vitamin-D3 (20,0±10,3 ng/ml) and PTH (25,6±10,9 ng/l), with a tendency towards low osteocalcin-levels (5,7±3,1 ng/ ml, reference 5-12). Histomorphometric parameters of IBD-patients were as follows (reference value of healthy young adults in parenthesis): BV 16,2±6,8% (20,8), OS 8,5±8,6% (16,5), ObS 4,1±6,8% (3,2), ES 21,8±11,1% (5,8), OcS 3,4±3,5% (2,1), MS 6,6±5,0% (8,0), MAR 0,73±0,29microm (0,75).
Osteopathy in IBD may be caused by a reduced calcium and vitamin-D resorption, the systemic action of inflammatory cytokines and the effect of corticosteriod treatment on bone. The normal levels of calcium, vitamin D and PTH in our patients do not support the hypothesis of a calcium deficiency as a main pathogenetic aspect. Small osteoid surface and low osteocalcin levels reflect a reduced bone formation, which is likely to be caused by the effects of corticosteriods on bone. In contrast, both the elevation of eroded surface and osteoclast-covered bone surface point to an increased bone resorption.
According to our histomorphometric parameters, reduced bone formation and increased bone resorption seem to be characteristic features of osteopathy in patients with IBD.
HIGH PREVALENCE AND INCIDENCE OF VERTEBRAL DEFORMITIES IN ANKYLOSING SPONDYLITIS PATIENTS WITH HYPERKYPHOSIS
P. P. M. M. Geusens1,2*, D. Vosse1, D. van der Heijde1, J. Vanhoof2, J. Raus2, S. van der Linden1
1Department of Rheumatology, University Hospital, Maastricht, The Netherlands
2Biomedical Research Institute, Limburg University Centre, Diepenbeek, Belgium
Objective. To study the prevalence and incidence of deformities of vertebrae in patients with ankylosing spondylitis (AS) in relation to hyperkyphosis of the spine.
Methods. Altogether 50 patients with AS were studied. Hyperkyphosis was measured by the occiput-wall distance (OXD). Anterior (Ha), mid- (Hm) and posterior height (Hp) of the vertebrae and intervertebral discs were measured on lateral x-rays of the thoracic (Th5-Th12) and lumbar spine (L1-L5). Vertebral shapes were analysed according to McCloskey et al (Osteoporosis Int, 1993, 138). A new deformity was defined as a decrease in Ha/Hp of >15% for vertebrae and >50% for discs. Hyperkyphosis was defined as an OWD of >1 cm.
Results. In the thoracic spine, the prevalence of vertebral deformities was higher in patients with kyperkyphosis (n=38) compared to patients without hyperkyphosis (n=12) (45% versus 8%, p=0.01). Patients with one or more deformed thoracic vertebrae had a higher mean OWD than patients without deformed vertebrae (12±7 cm versus 7±6 cm, p<0.01). Deformities of lumbar vertebrae and discs did not contribute to hyperkyphosis.
In 11 patients follow-up X-rays were available during a follow-up of 2 to 18 years. Fourteen new vertebral deformities were found in 7 patients. The mean increase of OWD (5 cm, range: 0-14 cm) was significantly correlated with the combination of progression of wedging of the vertebrae and of the discs (r=0.813, p<0.01).
Conclusions. In patients with AS and hyperkyphosis, deformities of the thoracic vertebrae occur frequently and, together with wedging of the thoracic discs, contribute significantly to the presence and progression of hyperkyphosis.
ASSESSMENT OF BONE MINERAL DENSITY AT DISTAL FOREARM IN THE COURSE OF ANKYLOSING SPONDYLITIS
H. Przepiera-Bêdzak*, M. Brzosko
Department of Rheumatology, PAM, Szczecin, Poland
Objective: To examine the changes of bone mineral density at forearm in the course of ankylosing spondylitis (AS).
Materials and methods: We studied 46 males with AS. The patients were assessed clinically and bone mineral density was measured at distal forearm by DEXA method three times during follow up of the disease. We assessed activity of the disease in each subject according to the following indexes: BASFI, BASDAI, BASG, BASMI. After one year follow up 38 subjects had performed the second measurement of BMD. After next two years follow up 27 patients had assessed the third BMD at forearm.
Results: Mean age of patients at the first bone mineral density measurement was 49 yrs. Mean disease duration was 20,8 yrs. Mean value of BMD at forearm at the first measurement was -0,07. After one year follow up mean BMD decreased to -0,26, and after three years follow up from the first measurement it decreased to -1,71. We found significant decreasing of BMD at distal forearm during three years in the course of AS (p=0,0001).
We found significant negative correlation between BMD at distal forearm and disease duration (respectively: -0,43 for the first, -0,40 for the second and -0,44 for the third). There was also negative correlation between BMD and age of patients for all three measurements (respectively: -0,49 for the first; -0,31 for the second and –0,36 for the third).
There was also negative correlation between BMD after three years follow up and BASMI index assessed at the start of the observation (-0,27).
Conclusion: There is a decreasing of BMD at peripheral skeleton in the course of AS.
WADDLING GAIT AND INVALIDATING RICKETS AS PRESENTING SIGNS OF HEREDITARY TYROSINEMIA TYPE 1
D. Rigante1, E. Holme3, G. Segni1, A. Stabile1, P. Caradonna2*
1Dept. of Pediatrics, UCSC, Rome, Italy
2Dept. of Int. Medicine and Geriatrics, UCSC, Rome, Italy
3Dept. of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
Hereditary tyrosinemia type 1 (HT-1) is caused by deficiency of fumarylacetoacetase, the last enzyme for tyrosine degradation, leading to the accumulation of highly reactive compounds with electrophilic properties which are thought to cause hepatic and renal damages.
Rarely, as in the case reported, HT-1 may present with severe rickets-like expression of renal tubular failure-and minimal signs of hepatic involvement.
RR, female child, full-term born in Italy, came to the first observation in our Policlinic at the age of 3 years with waddling gait and progressive inability in walking. Clinical examination showed proximal muscle weakness, "bracelet-like" enlargement of wrists and overgrowth of the external malleoli. Blood tests revealed glycaemiae variable from 50 to 70 mg/dl, inorganic phosphor 1.5 mg/dl and alkaline phosphatase 6625 UI/l (osseous isoenzyme: 97%). Radiography of wrists and ankles showed signs evocative for active hypophosphatemic rickets. Diagnosis of Fanconi syndrome was firstly evidenced, but because of liver palpable at 3.5 cm from the costal arch, confirmed by ultrasound-scan, a liver biopsy was decided: it revealed diffuse cyrrhosis. Furthermore the relentless muscular weakness combined with complete loss of deambulation required a metabolic counseling. An increased tyrosine with grossly elevated a-fetoprotein were found in serum; the detection of serum organic acids revealed the presence of succinylaceton suggesting the diagnosis of hereditary tyrosinaemia type 1 (global incidence 1: 100,000). Specific treatment with NTBC (1 mg/kg of body weight/day, divided in two doses) was started. Since then, the child has showed considerable progress with good hepatic function and bone age corresponding to anagraphic one. Actually she is 7 years old and her weight and height are respectively 22.1 kg (40 centile) and 116.5 cm (60 centile).
Traditionally the only feasible treatment for HT-1 has been a phenylalanine/ tyrosine restricted diet, but NTBC, a triketone with herbicidal activity which inhibits competitively a higher enzyme in the tyrosine catabolic pathway, is available since 1991, having the capability to prevent synthesis of toxic metabolites. We believe that diagnosis of HT-1 must be considered promptly in order to begin as soon as possible NTBC treatment which appears to correct effectively the metabolic abnormalities of this disease and to avoid severe disturbances in skeletal development.
EXPRESSION OF BONE INDUCING FACTORS IN RATS WITH OR WITHOUT A TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY
U. Scherbel1,2*, J. Khurana3, C. T. Born1, P. Riess4,2, W. G. DeLong1
1University of Pennsylvania, Department of Orthopaedic Surgery
2University of Cologne, Department of Surgery II
3University of Pennsylvania, Department of Pathology
4University of Pennsylvania, Department of Neurosurgery
Background: There is a well-established relationship between brain injury and heterotopic ossification (HO). Patients with brain injury are known to have an increased tendency to form ectopic bone and are given corticosteroids, nonsteroidal anti-inflammatory drugs, disphosphonates, and radiotherapy as a preventative measure. The mechanism is not known but may involve humoral or other circulating factors. One of the possible links between brain injury and bone formation are the bone morphogenic proteins (BMP’s), which play a major role after brain injury in terms of neuronal plasticity but can also induce bone formation at orthotopic and ectopic sides.
We utilized a well established rat head-injury model and fracture model to study this phenomenon at the histologic and molecular level.
Design: Forty Sprague-Dawley rats were used. Ten rats were head-injured using a fluid percussion brain injury device (with a known and reproducible amount of force). Ten had head injury and a mid-tibial fracture. Ten animals underwent fracture but no head injury and 10 animals were controls (neither head-injury nor fracture). The animals were sacrificed at 3 days. Bone and soft-tissue from the femur was snap-frozen and a part of it was formalin fixed for histology and immunohistochernistry. Immunostains with antibodies to c-fos, c-jun, BMP-2 and BMP-4 were utilized on the organs, soft- tissues and bone. RT-PCR was performed on tissue from the soft-tissue and bone with primers for BMP-2, 4 and 6. Differential displays were also performed on tissues from these animals.
Results: 75 bands were differentially expressed in the head-injured animals as, compared to the controls. Additionally, RT-PCR showed the presence of increased BMP-4 expression in the head injured (with and without femoral fractures) as compared to the control group. By immunostainng BMPs, c-fos and e-jun were increased in the region of the fracture.
Conclusions: Our study revealed differences at the tissue level in animals with head- injury. There was differential expression of several genes including BMP-4 that got activated by head injury, perhaps by a circulating factor or a direct nerval pathway. This might be a possible mechanism by which head injury induces ectopic ossification.
SERUM PROPEPTIDES OF TYPE I PROCOLLAGEN FOR THE ASSESSMENT OF RENAL OSTEODYSTROPHY IN DIALYSED PATIENTS
Z. Nowak*, M. Konieczna, Z. Wankowicz
Postgraduate Military Medical Centre, Warsaw, Poland
The evaluation of renal osteodystrophy in everyday practise is based on non-invasive measurements. We compared clinical usefulness of some non-invasive biochemical bone turnover markers in monitoring renal osteodystrophy with special interest to the modality of dialysis: hemodialysis vs. continuous ambulatory peritoneal dialysis (HD/CAPD).
Was studied 58 patients: 29 on CAPD for 47±28 months (15M. 14F, aged 53± 23 yr.), vs. 29 on HD for 43±25 months (16M, 13F aged 59±25 yr.). The following parameters were determined in serum: iPTH, Ca, P., totalAP, aminoterminal propeptide of type I procollagen – PINP, carboxyterminal propeptide of type I procollagen – PICP as a markers of bone formation and type I collagen crosslinked telopeptide - ICTP as a marker of bone resorption. These measurements were repeated every 3rd month during 18mths study.
In CAPD pts. significantly lower values of iPTH were found in comparison with HD patients (188±103 vs. 428±243 pg/ml; p<0.01). Similar results were found with respect to PINP. There were not significant differences between values of PICP, ICTP, P, Ca between both groups (CAPD/HD). As a result we recognised secondary hyperparathyroidismus (sHPT) in 20/29pts. (70%) on HD and only in 6/29 (21%) on CAPD. Low turnover renal osteodystrophy was recognised in 18/29pts, (62%) on CAPD and only in 4/29 (14%) on HD.
The study showed that in patients treated with CAPD predominant bone lesion seems to be low turnover renal osteodystrophy, opposite to HD patients in whom sHPT might be more frequent. Only monitoring of serum levels of iPTH and PINP might be useful in assessment of bone turnover in dialysed patients.
| Marker | Group | 0mnth | 3mnth | 6mnth | 9mnth | 12mnth | 18mnth |
| iPTHpg./ml. | HD | 428±243 | 383±218 | 337±189 | 322±207 | 313±152 | 302±132 |
| iPTHpg./ml. | CAPD | 188±103 | 199±96 | 184±98 | 216±102 | 248±115 | 227±109 |
| PINPug./ml. | HD | 158±40 | 162±39 | 165±46 | 168±47 | 180±54 | 187±72 |
| PINPug./ml. | CAPD | 124±43 | 122±45 | 138±56 | 121±41 | 135±40 | 154±58 |
| PICPug./ml. | HD | 294±122 | 282±104 | 358±125 | 330±131 | 381±137 | 326±127 |
| PICPug./ml. | CAPD | 315±138 | 338±122 | 329±139 | 384±136 | 375±118 | 359±115 |
IL-6 IS INVOLVED IN BONE REMODELING AFTER KIDNEY TRANSPLANTATION
V. Kusec*, R. Smalcelj, D. Rogic, Z. Puretic
Central Laboratory, Dialysis Unit, University Hospital Zagreb, Zagreb, Croatia
Disorder of bone metabolism occurring during chronic dialysis treatment frequently persists after successful kidney transplantation. Increased parathyroid hormone (PTH) secretion and bone turnover take years to normalise. PTH stimulates osteoblasts to release factors which induce osteoclast action. IL-6 is regulated by PTH, secreted by osteoblasts, and involved in bone resorption. Aim of this study was to analyse levels and relationships of PTH, IL-6 and markers of bone remodeling in patients with successful kidney graft function. Nineteen patients received a kidney transplant within 12 months (2-8) and 45 within 16-175 months. None of the patients had rejection crisis at least three months before the commencement of the study. Intact PTH, bone alkaline phosphatase (BALP), osteocalcin (OC), procollagen type I propeptide (P1CP) and IL-6 were measured in serum and deoxypyridinoline crosslinks (DPD) in urine by ELISA kits. No difference existed between sexes, or patient groups regarding age and dialysis duration. Patients in the first post-transplant year had significantly higher BALP, OC, DPD, and IL-6, and proportions of increased BALP and OC values. Post-transplantation period correlated significantly and negatively with BALP, OC, P1CP and DPD. Statistically significant and positive correlation was found between PTH and BALP, OC, DPD, IL-6 and also between IL-6 and DPD. These results indicate that IL-6 mediates bone resorption in PTH-regulated higher bone turnover within the first post-transplant year.
ANTHROPOMETRIC DIAGNOSIS OF "TRUE" OSTEOPENIA IN CAPD PATIENTS BASED ON DEXA-ASSESSED MUSCLE-BONE RELATIONSHIPS
A. Negri*, J. L. Ferretti, G. Cointry, J. R. Zanchetta
Instituto de Investigaciones Metabolicas, Buenos Aires, Argentina
We have shown that, in agreement with the mechanostat theory, the DEXA-assessed, whole-body muscle (lean) and bone masses (BMC, both as raw values and adjusted to fat mass) correlated linearly keeping similar slopes but different intercepts in children, men, and pre- and post-menopausal women [Ferretti, Bone 22:693, 1998]. Similitude between slopes would have reflected the natural muscle-bone influence as predicted by the mechanostat theory [Frost, CTI 62:1, 1998], which should be a constant for Homo sapiens. Differences in intercepts would correspond to the interaction of the endocrine-metabolic environment with the muscle-bone relationships (shifts in the mechanostat setpoint). Reference, percentile charts were performed based in the corresponding 1,450 data for the 4 different conditions, in order to allow an anthropometric differentiation between "physiological" and "true" osteopenias (proportionate or disproportionate muscle-bone masses, respectively) according to Frost's criteria [Frost, Bone 20:385, 1997].
Now we analyzed the same variables determined in 16 men, 16 pre-MP and 36 post-MP women with chronic renal insufficiency who underwent COPD. The aim was to test the hypothesis that this systemic disturb would shift the mechanostat setpoint so that the intercepts of the whole-body BMC / lean mass curves for each studied group would be lower than those of the corresponding reference charts (i.e., that a diagnosis of a "true" osteopenia could be established).
The BMC / lean mass curves were linear, positive and significant for all groups. The statistical adjustment of the data improved when the BMC was adjusted to a common, 18-kg fat mass. The slopes of the curves did not differ significantly from those of the reference charts. However, the intercepts were significantly lower than normal in all cases, ranging approximately at the 5 percentile level of the corresponding, reference correlations. The condition would correspond to a "true" osteopenia according to Frost's criteria.
These results may be interpreted as supporting the hypothesis that the systemic disturbance affecting the studied patients altered the natural muscle-bone interrelationships as described by the employed procedure.
BONE GEOMETRY AND BIOMECHANICS IN SPONTANEOUSLY DIABETIC, ESS RATS
S. Daniele*, G. R. Cointry, M. Meta, S. Montenegro, M. Tarres, S. Martinez, J. L. Ferretti, L. Morisoli
Rosario University Biochemistry and Medicine Schools, Rosario, Argentina
This study aims to compare the biomechanical properties of the femur shafts of 9 adult, spontaneously (type-II) diabetic, eSS rats (Univ. of Rosario) with those of 10 unaffected controls. Tomographic scans (pQCT, XCT-960A, Stratec, Germany) of mid-diaphyses allowed determining the cortical cross-section area, volumetric mineral content and density (CSA, vBMC, vBMD), periosteal and endosteal perimeters and moment of inertia (CSMI, architectural indicator for bending strength). Three-point bending tests provided the diaphyseal stiffness and the fracture load. The elastic modulus of cortical bone (E, approaching the stiffness of the "solid" bone matrix) was indirectly calculated from mechanical and geometric indicators.
There were no significant body weight differences between groups. The eSS bones were 43% stiffer (p<0.001) but 17% weaker (p<0.007) than controls, showing a curious dissociation between stiffness and strength. They also had smaller periosteal and endosteal perimeters, CSA, vBMC and CSMI (-51%, p<0.001), and overnormal values of vBMD and E (+3.8% and +154%, p<0.02, p<0.001).
The higher bone stiffnes of eSS bones can be explained by the higher E of the tissue, and this on time can be attributed to the small but meaningful increase in vBMD. Nevertheless, the assayed model is unable to describe bone mineralization qualitatively (porosity, crystallization, etc.) and to rule out other, mineralization-unrelated, microstructural determinants of the tissue stiffness. The diaphyseal weakness may have derived from a combination of the strikingly high tissue stiffness (E, inductor of both, strength and brittleness) and the highly deteriorated cortical design (CSMIs). This condition could only come from a severe delay in skeletal development that would have impeded the optimization of the diaphyseal architecture, as much as to overcome any strengthening effect eventually derived from the bone material stiffening.
These striking results are congruent with those observed by others in streptozotocin-induced diabetes [Einhorn, 1988] but differ from those produced by other kind of spontaneous diabetes [Verhaeghe, 2000]. We are currently unable to extrapolate this analysis to the interpretation of the human diabetic bone disease.
ACQUIRED HYPOPHOSPHATEMIC ONCOGENIC OSTEOMALACIA: PROBLEMS OF DIAGNOSIS, TREATMENT AND LONG-TERM MANAGEMENT
G. P. R. Clunie*, P. E. Fox, T. C. B. Stamp
Division of Bone and Mineral Metabolism, Royal National Orthopaedic Hospital, Stanmore Middlesex HA7 4LP, UK
Oncogenic hypophosphatemic osteomalacia (OHO) is a rare acquired condition though one associated with considerable morbidity. Reports have often emphasised the ability of surgical excision of an underlying tumour to effect a cure. However, reports have infrequently addressed issues such as the risk of relapse after surgery, optimal drug treatment and it's risks in the long-term.
The delay in diagnosis, morbidity, difficulty in searching for tumours, clinical relapse after surgery, long-term treatment and treatment-related morbidity are reported in four patients who were followed for 3-22 years after diagnosis.
Patients had had features suggestive of OHO for 5-12 years before diagnosis. Multiple pseudofractures or fractures were present in all patients at diagnosis. Tumours identified after extensive imaging procedures included a periscapular sclerosing haemangioma, an ethmoid sinus haemangiopericytoma and a vulval haemangiopericytoma. In the fourth patient no tumour was found. All tumours were difficult to remove and histopathology was unable to confirm complete excision. All patients responded to combination treatment with phosphate and either alphacalcidol or calcitriol in the short-term. In the 3 patients who had surgery, treatment was withdrawn when clinical and the major radiological indices reflecting osteomalacia had resolved. The patients relapsed (clinically and biochemically) respectively after 1, 3 and within 36 months after treatment withdrawal. Further imaging did not disclose an identifiable tumour in 2 patients; the third, who had had a vulval lesion, declined further investigation. Treatment was re-established successfully in all these 3 patients. Hypercalcaemia, nephrocalcinosis and renal failure have complicated long-term management in one patient. One patient remained well for 20 years on treatment then died of a colonic carcinoma. The other 2 patients remain well on phosphate/calcitriol after 3 and 6 years respectively.
Though emphasised in the literature, a delay in diagnosis of OHO is still prevalent. Morbidity is considerable. Extensive imaging is recommended but is not always successful at identifying tumours. Relapse after surgery, especially where tumours are inaccessible, may be common. Long-term treatment with phosphate and calcitriol/alfacalcidol may be necessary and can be complicated by serious metabolic and renal abnormalities.
HYPERCALCEMIA AND OSTEOMALACIA COMPLICATED BY OSTEOPETROSIS CAUSED BY HIGH CONCENTRATION OF FLUORIDE AND ALUMINIUM IN CHILDREN
F. C. Li
The Hygiene Anti-epidemic Station of Liu Panshui City, Guizhou, 553000, PR China
27 2-11year old patients with fluoride and aluminum intoxication were observed. We found that there were 22 (81%) patients suffering from osteomalacia complicated with osteopetrosis. The results also indicated that serum levels of Calcium and phosphorus increased in sick children. The serum Ca2+ was in the normal range, the serum P3+ decreased. Considering the condition of the adult patients, we think that the appearances of bone damage caused by fluoride and aluminum koinpnia have the relationship with the rate of bone metabolism, cell biochemistry, and the change of deflection of physicochemical osteodesmosis.
X-LINKED HYPOPHOSPHATEMIA IN A SOUTH AFRICAN POPULATION
D. Basu1,2*, J. M. Pettifor1, J. G. R. Kromberg2
1MRC Mineral Metabolism Research Unit, University of Witwatersrand
2Department of Human Genetics, South African Institute for Medical Research, University of Witwatersrand
X-linked Hypophosphatemia (XLH) is inherited as an X-linked dominant trait with the mutant gene (PHEX gene) being located in Xp22.1-22.2 region of the X chromosome. The most striking clinical features of this disorder are growth retardation, lower limb deformities and hypophosphatemia, and a defect in renal tubular phosphate reabsorption. Females are usually less severely affected than males. Affected children usually have short stature, and bowing of lower extremities, whereas adults develop dental disease and osteoarthritis. Although generally inherited as an X-linked condition, some 30% of cases appear to be the result of sporadic mutations.
The aims of this study were to investigate a series of South African XLH patients, to describe the pattern of their clinical presentation with particular attention to ethnic differences in presentation, inheritance and response to therapy and lastly, to determine the perceptions and psychosocial problems associated with XLH.
So far fifty patients (15 males and 35 females) have been investigated at our clinic. Thirty-two of them were black, sixteen white and two Indian. The mean age of clinical onset was 3 1/2 years (range 1 1/2 -5 years). 54% of the cases were apparently sporadic. The prevalence of sporadic mutations is 65% among the black patients and 37.5% among the white patients. Corrective surgery has been performed in 87.5% of cases and thereafter deformities were found to be mild. All the patients were taking phosphate and one-alpha-hydroxy-cholecalciferol supplementation. Their blood biochemistry results were better while they were on this treatment. The Psychosocial study involved interviews with 20 parents and 6 patients (16 years or more). The finding showed that this disorder had not only affected the family life but also the lives of the patients and the interpersonal relationship. Hereditary nature of the condition was not clear to the parent even after attending clinics for many years.
The findings show that South African patients with XLH have similar features to the other studies but a higher prevalence of sporadic mutations in the black patients. Better counselling services are needed to improve the understanding of this condition among the parents of the affected children.
HYPOVITAMINOSIS D MYOPATHY CHARACTERISED BY 31P MR SPECTROSCOPY
H. Glerup1*, H. Stødkilde-Jørgensen2, E. F. Eriksen1
1Dept of Endocrinol and Metab C, Aarhus Amtssygehus, Aarhus University Hospital, Aarhus, Denmark
2MR-Center, Skejby Sygehus, Aarhus University Hospital, Aarhus, Denmark
Myopathy with reduced muscle power, fatigue and muscle pain is a well known symptom complex in hypovitaminosis D. However, the nature of hypovitaminosis D myopathy is only partly understood. In order to investigate the intracellular energy and phosphate metabolism in skeletal muscle 13 patients with hypovitaminosis D (D-) (s-25-hydroxyvitamin D 7.8±2.0 nmol/l) and 8 age-matched controls with normal (D+) vitamin D status were included. A MR-scanner (1.5 Tesla) was used for recording of 31P-MR-spectra in the calf muscle at rest and during work (56 J) and recovery (32 recordings in 29 minutes). 6 patients with hypovitaminosis D had another 31P-MR-spectroscopy performed after 3 months of vitamin D treatment.
Results: Rest values of Phosphocreatine (Pcr), Phosphate (Pi) and Pcr/(Pcr+Pi) were lower in D- compared to D+ (6.9±0.6 vs 9.3±1.7, p=0.10, 8.3±0.7 vs 9.2±0.6, NS, 0.889±0.008 vs 0.909±0.004, p<0.05) whereas Pi/Pcr was higher in D- (1.26±0.09 vs 1.02±0.06, p<0.05). During work Pcr/(Pcr+Pi) decreased more in D- compared to D+, whereas Pi/Pcr increased more in D-. At recovery T 1/2 of Pi/Pcr in D- was 56s compared to 36s in D+ (p<0.05). T 1/2 of Pi was D- 64s vs D+ 45 s (p<0.05). Vitamin D treatment tended to normalise all indices, but only reached significance in Pi/Pcr recovery.
Discussion: Our study revealed significant perturbations in intracellular skeletal muscle energy and phosphate metabolism. Energy production seems to be compromised in vitamin D deficiency. Vitamin D is known to by important in transmembranous transport of calcium and phosphate, and this could possibly explain the disturbance in the energy production. Atrophy of fast twitch muscle fibres has been described in vitamin D deficiency. This could possibly explain the increased levels of Pi/Pcr. Normalisation of Pi/Pcr during vitamin D treatment could represent normalisation of the fast twitch fibres.
CHARACTERISATION AND PARTIAL PURIFICATION OF PHOSPHATE UPTAKE INHIBITORY ACTIVITY OF CONDITIONED MEDIUM FROM A SECOND ONCOGENIC OSTEOMALACIA TUMOUR CELL LINE
A. E. Nelson1,2*, M. Mirams1,2, O. Ljunggren3, E. A. Martin1,2, R. S. Mason2, B. G. Robinson1
1Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia
2Physiology Department, Institute of Biomedical Research, University of Sydney, Sydney, Australia
3Department of Internal Medicine, University Hospital, Uppsala, Sweden
There is evidence that oncogenic osteomalacia (OOM) is caused by a circulating factor that regulates renal phosphate reabsorption and metabolism of 1,25-dihydroxyvitamin D, however, this factor is yet to be identified. Although some candidate genes have been proposed, the factor responsible for renal phosphate wasting in OOM has not been identified. The condition is characterised by renal phosphate wasting and impaired mineralisation. A second cell line has been established from a tumour that caused oncogenic osteomalacia. After the tumour, classified histologically as angiodysplasia, was removed from the patient, there was rapid reversal of his symptoms and normalisation of abnormal biochemistry. The cell cultures exhibit an epithelial morphology rather than showing the mesenchymal appearance of the cell cultures from an earlier tumour, which we have previously shown to produce renal phosphate uptake inhibitory bioactivity (1). Similarly, serum-free conditioned medium from the new cell line has been demonstrated to consistently inhibit phosphate uptake by renal OK 3B2 cells, up to 50%, following preincubation with the OK cells for 20 hours. The phosphate uptake inhibitory activity was dose-dependent and enhanced by heat treatment at 95 deg C. We have previously reported that the phosphate transport regulator was stable to trypsin treatment, though papain-sensitive. We now report that after heat treatment, the activity of the medium from the new cell line was suppressed by trypsin. This clearly characterises the material as a protein. Partial concentration has been carried out using hydrophobic chromatography. A peak of phosphate uptake inhibitory activity has been identified following purification by HPLC.
In conclusion, the phosphate uptake inhibitory bioactivity produced by a second cell line established from an oncogenic osteomalacia-causing tumour has been characterised and partially purified.
(1) Nelson et al, 1996. Mol Cell Endo, 123:17-23.
BIOCHEMICAL RESPONSE FOLLOWING CALCIUM TREATMENT OF RICKETS IN NIGERIAN CHILDREN
M. Worsfold1*, C. A. Sharp1, D. E. Powell1, L. M. Oginni2, M. W. J. Davie1
1Charles Salt Research Centre, Robert Jones and Agnes Hunt Hospital, Oswestry, Shropshire, UK
2Dept. of Orthopaedic Surgery, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
13 boys and 13 girls aged 2-5 years with radiographically confirmed rickets received 2.7g calcium lactate per day for 6 months. There was relief of leg pain within one month of treatment and they were more active. The mean X-ray score [Lancet (1999) pp296-297] improved from 3.3 at baseline to 1.7 at 3 months and 0.9 at 6 months. 12 cases (46%) were healed radiologically after 6 months of treatment. 11 others (42%) were markedly improved while 2 (8%) showed no significant improvement. One patient was worse after 6 months.
At presentation the mean plasma concentrations of alkaline phosphatase (ALP), 1,25dihydroxyvitamin D, parathyroid hormone (PTH), type I collagen carboxy-terminal and amino-terminal propeptides (PICP, PINP), and carboxy-terminal telopeptide (ICTP) were elevated while the plasma and urinary calcium were reduced compared with our local normal ranges. Pre-treatment plasma 25hydroxyvitamin D was low (<25pmol/L) in 4 patients (15%) but the 1,25dihydroxyvitamin D level was low (220pmol/L) in only 1 (4%) of them.
During calcium treatment mean ALP fell from 536IU/L (SD 262) to 312 (SD 185) (p=0.036) in 4 months and 1,25dihydroxyvitaminD fell from 511pmol/L (SD=176) to 298 (SD=115) (p=0.036) in 4 months. ICTP fell from 30microg/L (SD=24) to 16 (SD=5.0) (p=0.03). PICP and PINP fell progressively by 22% and 29% at 4 months, but this was not statistically significant. Mean plasma calcium increased from 2.22mmol/L (SD 0.21) to 2.28 (SD 0.15) (p=0.06) at 2 months while mean urinary calcium/creatinine ratio increased from 0.06 (SD 0.04) to 0.17 (SD 0.09) (p=0.05) after 2 months.
At baseline, (and during treatment, data not shown) ALP correlated with ICTP (r=0.64, p<0.01), PINP (r=0.69, p<0.01) and PTH(r=0.63, p<0.01) and PTH correlated with ICTP (r=0.51, p=0.01). Baseline X-ray scores correlated with PTH (r=0.58, p=0.008); ALP (r=0.82, p<0.001); PINP (r=0.60, p=0.002); PICP/PINP (r=0.63, p=0.001); ICTP (r=0.50); p=0.01); INTP (r=0.55, p=0.05); 1,25 D (r=0.51, p=0.01). Changes in X-ray scores at 6 months correlated with changes in PTH (r= 0.54), ALP (r= 0.63) and PINP (r= 0.33) at 4 months.
In conclusion, calcium supplements alone produced clinical, radiological and biochemical improvement in these Nigerian children with rickets within 2 months.
SEVERE OSTEOMALACIA WITH NORMAL BONE BIOCHEMISTRY PRESENTING AS OSTEOARTHRITIS
G. A. Campbell
Addenbrookes Hospital, Cambridge, UK
Vitamin D deficiency severe enough to present with an insufficiency fracture(Loosers Zone)rarely presents with normal bone biochemistry. A patient with normal bone biochemistry but possessing a Looseres Zone and biopsy proven osteomalacia is described.
Miss A.H., a 95 year old lady with long standing generalised osteoarthritis, was referred for outpatient rehabilitation. She had lived alone in sheltered accomodation for many years. For the last year she had not left her flat on account of increasing pain in her joints. She was referred on account of suffering increasing pain in her right knee for the previous two months that had not responded to simple analgesics. This had resulted in her becoming reluctant to mobilise and she was becoming bedridden.
On examination she had a painful deformed right knee and she was felt initially to have an acute exacerbation of her osteoarthritis. An Xray, however, showed a medial tibial insufficiency fracture and a subsequent bone scan, evidence of metabolic bone disease in spite of normal bone biochemistry. An iliac crest bone biopsy confirmed histological osteomalacia.
A diagnosis of privational osteomalacia secondary to lack of exposure to ultraviolet light was made. She was commenced on calcium and ergocalciferol(800 International Units)supplements daily. The pain in her knee subsided sufficiently within a month for her to begin to remobilise around her flat.
Osteomalacia with normal bone biochemistry has been previously described, but such a presentation, which would most likely have been missed without the Xray, is extremely rare.
AN OCHRONOSIS CASE ASSOCIATED WITH SPINAL CHANGES SIMILAR TO ANKYLOSING SPONDYLITIS AND HIP OSTEOPENIA
Y. Sermez1*, G. Fidan1, N. Oruç1, N. Karabulut2
1Pamukkale University, Department of Endocrinology and Metabolism, Denizli, Turkey
2Pamukkale University, Department of Radiology Department, Denizli, Turkey
Ochronosis is a musculoskeletal manifestation of alkaptonuria, which is a hereditary disorder of amino acid metabolism, secondary to lack of homogentisic acid oxydase enzyme activity. There is an accumulation of homogentisic acid in cartilages and the connective tissues. This deposition results in ochronotic pigmentation and arthropathy with some characteristic radiological findings. In addition, ochronotic patients with HLA-B27 positivity develop spinal changes similar to ankylosing spondylitis. This report describes a 42- years old man with ochronosis that had back and hip pain for last ten years. His family history was positive for alkaptanonuria. HLAB27 was found to be negative. Homogentisic acid was detected in urine sample by qualitative method. There was typical ochronotic pigmentation of ear cartilage. We detected spinal changes similar to ankylosing spondylitis by conventional radiography. Degenerative intervertebral discs and bulging in lumbar vertebrae were found in magnetic resonance imaging (MRI). Dual energy X ray absorptiometry (DEXA) revealed osteopenia in hips, although bone mineral density was normal in vertebrae. Summarizing the results of this report, it should be kept in mind that a rare cause of back pain may be ochronosis, and ochronotic patients may have osteopenia and spinal changes similar to ankylosing spondylitis.
CORTICOSTEROID THERAPY INDUCED REMISSION IN A PATIENT WITH MALIGNANT OSTEOPETROSIS
A. Teti1*, M. Iacobini2, M. Roggini2, A. Taranta3,4, B. Werner2, A. Panero2, S. Migliaccio1,3,4
1Department of Experimental Medicine, University of L’Aquila, Italy
2Department of Pediatrics, University La Sapienza, Rome, Italy
3Department of Medical Physiopathology, University La Sapienza, Rome, Italy
4Istituto Dermopatico dell’Immacolata, Rome, Italy
We report a case of a patient diagnosed with malignant osteopetrosis at birth and treated with prednisone, who had remission of the disease. A female neonate was transferred to our intensive care unit with a diagnosis of "haemorrhagic purpura and suspected neonatal sepsis". The infant was lethargic, hypotonic and presented diffused petechia-like skin lesions, hepatosplenomegalia and convergent strabismus. Blood analysis revealed anemia (Hbg 9.2 gr/dl), alterations of red blood cells (teardrop-like), and presence of immature forms of erythroid and myeloid series. X-ray showed generalised increase in bone density, sclerosis of the skull, transverse wavy stripes of sclerotic bone in the tubular segments, sclerosis of vertebrae, "bone in bone" appearance in phalangies of both hands and feet, and decrease of medullary cavity. On the 1st day, the infant was given a blood transfusion to correct anemia, and vitamin K (1mg/day iv) to correct altered coagulation. This therapy was implemented to prevent extramedullary hematopoiesis, maintain levels of Hgb around 16 gr/dl, and prevent haemolysis by prednisone, that was immediately given on a regimen of 2mg/kg/die. On the 26th and 42nd day, the proband received additional blood transfusions. Due to the marked improvement of symptoms, bone marrow transplantation was not performed, and on the 49th day the proband was discharged. Prednisone was continued at a constant regimen of 5mg/day. At 10 months normal dental eruption was noticed and ocular bandage was prescribed leading to a remarkable correction of the convergent strabismus. At 12 months, an agoaspirate showed normal cellular plasticity and white and red cell lineages. X-ray demonstrated attenuation of the bone sclerosis pattern. At 14 months, a green-stick-like fracture occurred with complete restitutio ad integrum. At 18 months, prednisone therapy was discontinued. Follow-up showed normalisation of all radiological and clinical parameters, including neurological, visual and hearing scores, and acceleration in linear growth. The patient is now 34 months old, appears healthy with a regression of the disease. We believe that the key events of this successful intervention were early diagnosis and immediate administration of corticosteroids, which reversed, at least so far, the catastrophic prognosis in this case of sclerosis bone disease.
A CASE OF NEURAPRAXIA SECONDARY TO MYOSITIS OSSIFICANS PROGRESSIVA, TREATED WITH RADIOTHERAPY
M. Druce1*, V. H. Morris2, T. C. B. Stamp3
1Watford General Hospital, Watford, UK
2University College London Hospital, London, UK
3Royal National Orthopaedic Hospital, Stanmore, UK
The subject was a 34 year old female with a diagnosis of myositis ossificans progressiva. Abnormal toes had been noted at birth and aged 19 she developed progressive swellings and stiffness below the jaw and throughout her body. Her current presentation was with a 5-week history of pain in the right thigh but no palpable lump. Four weeks before admission she developed fixed flexion of the right thigh with inability to actively extend the knee, and numbness of the right leg. Examination findings were consistent with femoral nerve compression. Blood tests were normal except for ESR of 22 and CRP of 36. Surface EMG was consistent with femoral neurapraxia. CT scan showed swelling and inflammation of the right ilio-psoas without new calcification, possibly involving the femoral vessels and femoral nerve. Treatment was with high-dose non-steroidals (indomethacin), disodium etidronate 400mg daily, physiotherapy, hydrotherapy and a single fraction of radiotherapy, dose 10 Gy. Seven days after treatment goniometry showed a reduction in the fixed flexion and improvement in knee movement. After two months some fixed flexion persisted but the pain had subsided. The symptoms of neurapraxia had disappeared. A repeat CT scan showed resolution of the muscle oedema albeit with development of calcification.
COMMENT: Myositis ossificans progressiva is a severe, rare, autosomal dominant condition of ectopic ossification, with primary involvement of skeletal muscles and characteristic skeletal abnormalities. Presenting features include pain, stiffness and inflammatory masses. While ectopic ossification has been described in a number of sites, neural compression has not previously been described in myositis ossificans progressiva. While firm conclusions cannot be drawn from a single case, evidence would suggest a beneficial role for radiotherapy in conjunction with other treatment modalities to reduce pain and progression of ossification. The outcome of this case would suggest a potential role for similar management of acute exacerbations in myositis ossificans progressiva.
CELLULAR ORIGIN AND OSTEOLYTIC ACTIVITY OF THE UNICAMERAL BONE CYST
F. Y. Lee*, G. Osgood, M. Parisien, H. M. Dick
Columbia University, USA
Unicameral bone cyst (UBC) or simple bone cyst (SBC) in growing children is a mysterious tumor-like lesion characterized by a well-defined area of bone destruction. UBC contains fluid and fibrous membrane. UBC is detected usually after pathologic fracture. On histopathologic examination, the membrane consists of fibrous tissue, monocytes, inflammatory cells and occasional giant cells. The pathophysiology of UBC regarding why the bone destruction is so severe is not well understood. The response to various forms of percutaneous treatments such as bone marrow injection, steroid injection and bone grafting is unpredictable. The purpose of out study is to identify the cellular components and regulatory factors related with bone destruction in UBC.
Three patients underwent endoscopic examination to explore the inside aspect of UBC. Under direct vision, pathologic specimens were taken. The endoscopy demonstrated lining structure inside the UBC. The lining fibrous membrane contained scattered areas of vascular proliferation. The fracture callus, despite young age, was scanty. The lining structures and additional 3 specimens from the paraffin embedded archive specimens underwent histopathologic examination and immunohistochemistry using antibodies raised against epithelial membrane antigen, endotheliam markers (CD 31), vimentin, collagen IV, S100 protein, histiocytic marker (CD 68), muscle specific actin (HHF-35), osteoclacin, Ki-67, osteoclast or osteoclast precursor cells (tartate resistant acid phosphatase) and matrix metalloproteinase 1 & 9.
Neither epithelial nor endothelial markers were positive while mesenchymal antigens were positive. There were no specific groups of cells which undergo active proliferation. Tartate resistant acid phosphatase was positive in multinucleated cells (tumor giant cells or osteoclasts) or mononuclear cells in the lining structure of UBC. Matrix metalloproteinase activity was minimal. These findings suggests that UBC or SBC is a lesion of mesenchymal origin with monocytes and osteoclasts which have osteolytic activities. The membrane like structure in UBC is a lesion which is better to be removed rather than left alone. Specific treatment should be designed to target the ostoelytic process in UBC to improve the outcome of patients.
BONE CHANGES IN CHILDREN FOLLOWING LIVER TRANSPLANTATION
C. Moniz1*, L. D'Antiga2, H. Abraha1, M. Buxton-Thomas3, N. Heaton4, M. Rela4, G. Mieli-Vergani2, A. Dhawan2
1Clinical Biochemistry, Kings College Hospital, London, UK
2Child Health
3Nuclear Medicine
4Liver Transplantation
Osteodystrophy is a recognised complication of chronic liver disease in children. Whereas osteomalacia is attributable to vitamin D deficiency, low trauma fractures may have other aetiologies. Since the effect of liver transplantation on bone is poorly investigated we performed a prospective study of bone mass density (BMD) and biochemical markers of bone metabolism in children with chronic liver disease before and after liver transplantation. Six children (2 male) median age 8.8 years (range 3.8-16.6 yrs) with chronic liver disease (biliary atresia 3 and progressive intrahepatic cholestasis 3) were studied at the time of listing for transplant and, 3 and 6 months after liver transplant. Fasting serum 25-OH vitamin D levels, PTH, intact osteocalcin, urinary free deoxypyridinolines (DPyr) adjusted for creatinine and BMD by Lunar DEXA of whole body and spine were measured 3 monthly over one year. Healthy age matched controls were used to compare the results. Post transplant immunosuppression comprised of Neoral, Azathioprin and prednisolone in 5; Tacrolimus and prednisolone in 1. Prednisolone was reduced to a maintenance dose of 0.07 mg/kg/day by the end of 1 month post transplant. Mean results are shown in table 1. BMD improved by 6 months after liver transplant despite an apparent initial worsening at 3 months. Pre-pubertal children showed a greater % increase in BMD than post-pubertal and demonstrated catch-up growth. Changes in bone markers and PTH and Oc signalled alterations in bone turnover which resulted in overall increases in BMD by 1 year. Pre-transplant PTH was within reference limits for normocalcaemia (32±21 ng/L) although in 4/6 cases PTH was below 50% of the range. At 3 months post-transplant there were 3-4 fold increases. Increases in Oc and DPyr. Liver transplantation results in alteration in bone homeostasis restores depressed osteoclastic and osteoblastic activity and increases bone density. However, the effects of post-operative care and immunosuppression need to be studied further to reduce low trauma fractures in the post-operative period.
| Time (months) | 0 | 3 | 6 | 12 |
| Total BMC g/cm2 | 0.76 | 0.79 | 0.83 | 0.85 |
| z-score | -1.6 | -1.85 | -1.77 | -0.35 |
| L2-L4 g/cm2 | 0.59 | 0.60 | 0.68 | 0.71 |
| z-score | -1.9 | -2.15 | -1.67 | -0.79 |
| DPyr (nM/mM) | 22.4 | 54.0 | 63.8 | |
| Oc pg/L | 17.8 | 32.4 | 38.8 |
DIFFERENTIAL DIAGNOSTICS OF INHERITED CONNECTIVE TISSUE DISEASES AND CONTRIBUTION OF CLINICAL, RADIOGRAPH, DENSITOMETRIC AND BIOCHEMICAL EXAMINATIONS TO THE DIAGNOSTIC PROCEDURE
V. Vyskocil1*, J. Varvarovska2, J. Vyskocilova4, K. Koudela3, O. Topolcan5
1Department of Bone Disease
2Department of Pediatric
3Department of Orthopaedic Surgery
4Department of Tuberculosis Respiratory Disease
5Department of Immunobiochemistry
The authors followed 200 patients with connective tissue diseases from 1996 to 2000 (Marfan syndrome, Ehlers Danlos syndrome, osteogenesis imperfecta). The cause for that choice is genetic relationship of collagen defect. The studied groups consists of 45 patients with osteogenesis imperfecta OI, 55 children with Ehlers Danlos syndrome ED and 100 patients with Marfan syndrome MS. Authors tried to apply the criteria combination for OI, MS and ED so that the validity of particular criteria could be evaluated and then used in diagnostic difficulties among the studied diseases.
The patients were examined physically first with stress to the single criteria in OI (classification according to Sillence). In Marfan syndrome the authors followed not only metacarpophalangeal index but also the trunk deformity, joint hyperelasticity, echocardiography, ophthalmological examination and the index between arms´ expansion and body height, eventually the ratio between trunk and limbs.
In ED the authors followed joint hyperelasticity, especially of knee and elbow and vascular eventually organ complication such as pneumothorax and intestinal perforation.
All 3 groups were examined by densitometry and also the markers of bone metabolism were evaluated (serum Ca, P, PTH, osteocalcin, PICP, telopeptid, in urine Ca, P, crosslinks). The present chest deformation was the indication for vital lung capacity measurements and FEV.
Conclusions: In patients with OI the best criteria besides clinical findings were lower stature, asymmetry of the body and densitometry with decrease at least of 15% when compared with the other groups. That group has besides fractures the decrease of PICP and the highest levels of osteocalcin and crosslinks.
The group of MS had no typical criterium in bone metabolism and densitometry when compared with OI and ED.
For Marfan syndrome diagnostics the main criteria are metacarpophalangeal index, the index of arms´expansion to body height, positive ophthalmological findings and body dysproportion. At the same time pulmonary functions were worse in Marfan syndrom than in the groups with OI and ED.
Ehlers Danlos syndrome had markedly increased osteocalcin besides basic diagnostic criteria and at the same time lower BMD of the forearm, close to patients with OI.
The authors assume that the combination of different diagnostic methods is useful and could differentiate among those 3 groups of patients. Molecular genetics is the definite method in the unclear cases.
FIFTEEN TO SEVENTEEN YEAR FOLLOW UP OF THE UNCEMENTED SPONGIOSA METAL SURFACE (SMS) TOTAL HIP ARTHROPLASTY
J. Scholz*, D. Hubalek, J. Osel, C. Höptner
Auguste-Victoria Teaching Hospital, Center for Orthopaedic Surgery, Berlin, Germany
To assess the long-term success of total hip arthroplasty inserted without cement 165 consecutive hip arthroplasties using the Spongiosa Metal Surface (SMS) total hip replacement were reviewed.
Material and Method:
The SMS prosthesis has a macroporous surface structure with a pore size of 1-2mm and a pore depth of up to 3mm similar to that of cancellous bone. Surface structure and implant core are molded in one piece. The prosthesis includes a metal hemispheric acetabular component with a polyethylene liner, a stem in form anatomically adapted to the proximal femur with a Morse taper and a 32mm ceramic head. From 1983 to 1985 165 SMS prostheses were implanted into 159 patients. No patient was lost to follow-up. At the time of latest follow-up, 29 patients had died unrelately to surgery and 13 hips had been revised. Of the remaining 87 patients with 90 total hip arthroplasties were clinically examined (minimum follow-up time was 12 years, mean 156 months), and a.p. and lateral radiographs were taken and analyzed for signs of osteolysis and stress shielding. 33 hips were followed by telephone. A survival rate was calculated using the Kaplan-Meyer method.
Results:
13 hips had to be revised. Both components were revised in one case of infection, the femoral stem only in 10 hips because of aseptic loosening, and in one hip for stem fracture. In one case the cup only was revised for aseptic loosening. In no revision case did the surface structure come loose. The survival rate was 88% at 14 years. Clinical follow-up of 87 patients using the Harris Hip Score showed a median of 89 points. Analysis of recent radiographs did show a variable degree of stress shielding in 21%. Periarticular heterotopic ossifications were apparent in 13 hips (10%). In no case did localized endosteal bone loss or stress shielding lead to revision or radiographic signs of loosening.
Discussion:
Bony ingrowth into the macroporous surface structure of the Spongiosa Metal Surface (SMS) hip prosthesis apparently provides a good shield against osteolysis. Although we did see stress shielding in the area of the greater trochanter frequently, this did not have any negative influence on the long-term success of the implant. The long-term survivorship analysis showed 88% survival of the SMS prosthesis at 14 years with good clinical results.
WHOLE BODY - DUAL ENERGY X-RAY ABSORPTIOMETRY TO TEST BODY COMPOSITION AND BONE MINERAL DENSITY IN CHILDREN WITH MUCOPOLYSACCHARIDOSIS TYPE IV
D. Rigante1, G. Segni1, P. Caradonna2*
1Dept. of Pediatrics, UCSC, Rome, Italy
2Dept. of Int. Medicine and Geriatrics, UCSC, Rome, Italy
There is a strong relationship between fat mass (FM), fat free mass (FFM) and bone mineral density (BMD) which can actively influence skeletal development in children. This consideration may be mostly applied to children inflicted with inborn errors of metabolism electively involving the skeleton.
Mucopolysaccharidosis type IV or Morquio-Brailsford syndrome (MPS IV) is a rare autosomal recessive disease (general frequency: 1:40,000-200,000 births) of the connective tissue resulting from deficient keratan sulphate catabolism and accumulation of undegraded substances in multiple organ systems. There is a spondylo-epiphyseal dysplasia with short trunk dwarfism, kyphoscoliosis, severe growth retardation and joint laxity leading to deformities in the skeletal system and formation of faulty postures. Intelligence of these children is completely normal. At a biochemical level MPS IV is characterized by skeletal infiltration with keratan sulphate and osteoporosis of this syndrome may be explained by many contributing factors, especially the very limited deambulation due to odontoid hypoplasia with atlanto-axial subluxation and hypotonia in the lower limbs. Through whole body - dual energy X-ray absorptiometry (DEXA, Hologic QDR 2000) we have measured FM, FFM and BMD in two children with MPS IV:
1: male, age 11 years, BMI 17.1, FM 3.091 kg (18.6% of body weight); FFM 12.630 kg (81.4% of body weight); lumbar BMD 0.416 (Z-score -1.46); femoral BMD 0.356 (Z-score -2.58).
2: female, age 9 years, BMI 18.2, FM 2.462 kg (14.6% of body weight); FFM 13.853 kg (85.4% of body weight); lumbar BMD 0.521 (Z-score -0.64); femoral BMD 0.323 (Z-score -2.07).
We have found a global reduction in the FM and -above all- a severe osteoporosis at the femoral site (calculated as decrease of BMD greater than 2 SD) in both patients as consequence of long-lasting immobilization and insufficient deambulation. We believe that the walking ability must be stimulated in these children in order to achieve a good mineralization state in the skeleton. A careful follow-up of these indicators through DEXA is highly recommended in patients with MPS IV in order to correct their body mineral deficiencies during childhood.
A RARE FORM OF MELORHEOSTOSIS TREATED WITH BISPHOSPHONATE
J. Donáth1*, B. Fornet2, M. Szilágyi3, M. Bély4, Gy Poór5
1National Institute of Rheumatology and Physiotherapy, Budapest, Hungary
2Haynal Imre University of Health, Budapest, Hungary
3National Institute of Rheumatology and Physiotherapy, Budapest, Hungary
4Hospitaller Brothers of St John of God in Budapest, Budapest, Hungary
5National Institute of Rheumatology and Physiotherapy, Budapest, Hungary
Melorheostosis is a very rare bone disease of unknown etiology. Changes commonly involve a single limb; the lower limbs are involved more frequently than the upper limbs.
We present a case of a 38 year-old man with melorheostosis in the left upper limb, the right part of the hip and the right lower limb. He presented with pain and acute swelling of the right foot.
Alkaline phosphatase and urine pyridinoline crosslink levels were increased. The radiological results were atypical, inspite of cortical thickening and sclerotic changes. Soft tissue ossifications were also observed in the region of the hip joint. Bone scintigraphy was positive and revealed moderately increased uptake. Computed tomography (CT) and magnetic resonance imaging (MRI) could further characterize the lesion and the histological results confirmed the diagnosis. 30 mg of pamidronate were administered intravenously for 6 days. After treatment the patient's pain subsided and the laboratory parameters improved.
Conclusions: Only very few cases of this disorder have been reported in which there was involvement of the long bones in the upper and lower extremities and pelvis. Skeletal scintigraphy can help distinguish melorheostosis from other conditions. CT and MRI reveal the lesion, particularly its clear demarcation from normal bone.
Bisphosphonate treatment may be a new therapeutic approach for the treatment active melorheostosis.
CANDIDATE GENES FOR DEGENERATIVE JOINT DISEASE IN PIGS
D. Jefferies1*, J. Thompson2, W. Smith3, E. Seawright1, C. Farquharson1, C. Whitehead1
1Bone Biology Group, Roslin Institute, Roslin, UK
2SAC Veterinary Services, Penicuik, UK
3SAC Veterinary Science Division, Aberdeen, UK
Degenerative joint disease (DJD) is a widespread problem resulting in lameness and poor welfare in pigs. Although numerous studies have described the various pathologies of DJD, little is known about the aetiology of the condition. As DJD has been shown to have a genetic component we have looked for candidate genes for DJD by studying gene expression levels in normal and affected joints using several approaches. Firstly, we looked at mRNA levels of known cartilage genes using RT-PCR and secondly, we attempted to identify differentially expressed genes using both differential display and suppression PCR subtractive hybridisation. Pigs fed a standard commercial diet or the same diet with 0.1mg/kg 25-hydroxyvitamin D were slaughtered at 4, 8, 12 and 16 weeks of age, in order to follow the progress of the disease. Additional material was obtained from farm reared pigs with lameness. Joints were scored for DJD using a standardized scoring system. Cartilage samples from these animals were digested with collagenase to release the chondrocytes and these cells processed for the extraction of total RNA. These samples were used to screen for genes differentially expressed in DJD. Blood samples from the same pigs were used to determine the serum concentration of vitamin D metabolites, PTH, TNFalpha and IGF-I by immunoassay. Serum concentrations of calcium and inorganic phosphorous were also determined biochemically, as well as the collagen and glycosaminoglycan content of cartilage samples. None of the biochemical parameters measured showed any correlation with the occurrence of DJD. However, the combination of gene expression studies described above has revealed several possible candidate genes for DJD that show altered expression in DJD lesion cartilage. Of 8 genes identified the differential expression of 6 has been confirmed by semi-quantitative RT-PCR.
GNATHO-DIAPHYSEAL DYSPLASIA: A NEW SYNDROME OF FIBRO-OSSEOUS LESIONS IN CRANIO-GNATHIC BONES, UNDERMODELING OF LONG BONES, AND BONE FRAGILITY
A. Corsi1*, M. Riminucci1, M. T. Collins2, S. Wientroub3, P. Gehron Robey2, P. Bianco4
1Università dell'Aquila, Italy
2CSDB, NIDR, NIH, Bethesda, USA
3Souraski Medical Center, Tel Aviv, Israel
4Università "La Sapienza", Roma, Italy
Differential diagnosis of fibro-osseous lesions is a major challenge in the study and clinical management of craniofacial skeletal diseases. We report here the case of a patient previously diagnosed with polyostotic fibrous dyslasia (FD) of bone. At 1 yr, he presented with bilateral, expansile lesions in the maxilla. A radiographic survey also revealed bilateral lytic lesions in the mandible, bowing and cortical thickening of the diaphysis of both tibiae and fibulae, and a healed phalangeal fracture. Over the next 4 years, the child sustained multiple fractures of tubular bones. Upon clinical examination by us at 5 yrs, no other skeletal or extra-skeletal abnormalities were noted, apart from a minimal increase in serum alkaline phosphatase. Activating GNAS1 mutations were not detected in genomic DNA extracted from fresh lesional tissue or lesional stromal cells following PCR amplification of the putative mutant GNAS1 allele using a PNA-based method. Histological examination of all biopsies and surgical samples demonstrated a fibro-osseous lesion in which the cardinal feature was a psammomatoid pattern of mineralization. Based on these features, the diagnosis of psammomatoid ossifying fibroma was made. Undecalcified plastic sections revealed that the psammomatoid bodies represented aberrant calcification of vascular walls. Immunolocalization of alpha-smooth muscle actin demonstrated the presence of myofibroblasts/pericytes within the lesional tissue and disclosed an unusual "disintegration" of lesional blood vessels. MMP-2 expression, known to be expressed in growing or regressing blood vessels, was also observed. This case highlights the need for stringent criteria in the identification of FD, namely, molecular diagnosis. This study also suggests a role for blood vessel abnormalities in the pathogenesis of ossyfing fibroma. Association of craniofacial fibro-osseous lesions with undermodeling of tubular bones and bone fragility has been previously reported, either sporadically, or as an autosomal dominant trait. While it has been speculated that this syndrome represents a variant of osteogenesis imperfecta, the underlying gene defect is still unknown. We believe that our patient represents another example of this undercharacterized syndrome, which we propose should be called gnatho-diaphyseal dysplasia.
ANGIOGENESIS AND EXPRESSION OF ALPHA-SM ACTIN IN FIBROUS DYSPLASIA OF BONE
M. Riminucci1*, A. Corsi1, S. Kuznetsov2, S. Licci3, P. Gehron Robey2, P. Bianco3
1Università dell'Aquila, Italy
2CSDB, NIDR, NIH, Bethesda, USA
3Università "La Sapienza", Roma, Italy
Fibrous dysplasia (FD) is a somatic mosaic disease caused by the expansion of abnormal osteogenic precursors carrying activating mutations of the GNAS1 gene. FD shares certain histological and pathogenetic features with endosteal fibrosis of hyperparathyroidism, noted for proliferation of "myoid" cells expressing the pericytic marker, alpha-smooth muscle actin (SMA). We analyzed the expression of SMA in active FD tissue from six patients and in two biopsies taken from uninvolved sites. SMA expression in normal marrow was restricted to pericytes of capillaries, and was not observed in any other cell type. In contrast, stromal and adventitial cells of sinusoids expressed SMA in lesional tissue. In vitro, clones of mutant stromal cells expressing CBFA1 coexpressed substantially higher levels of SMA mRNA compared to non-mutant clones derived from the same sample. PTH stimulated non-mutant clones to express SMA at similar levels as mutant clones. These data indicated that mutant stromal cells in FD coexpress osteogenic and pericytic markers, and therefore could be related to angiogenesis. Using a point counting technique, vascular density (VD) and the proliferation index (PI) were assessed in FD tissues immunostained for CD34 (a marker of vascular endothelial cells) and Ki67 (a marker of proliferation). VD was increased in all FD samples compared to normal tissues (79.058±18.953 vs 19.215±0.163). The PI was 5.917±2.433 in FD samples; in contrast, stromal cells were not immunolabeled in normal bone marrow. In FD, Ki67 was mainly localized in endothelial cells and pericytes of capillaries and sinusoids, and in the immediately adjacent fibrous tissue, suggesting that newly formed vessels within FD may represent proliferation "hot spots". Interestingly, a direct correlation (n=4) was found between VD in FD sections and colony-forming efficiency (CFE), which estimates the frequency of clonogenic stem cells. Like VD, CFE was remarkably higher in FD than in normal marrow samples. These data suggest that FD stromal cells differ from normal stromal cells by their dual expression of osteogenic and pericytic phenotypic markers, and that angiogenesis may play a significant role in the histogenesis and expansion of FD lesions.
AGE DEPENDENT DEMISE OF MUTATED STEM CELLS AND "NORMALIZATION" OF FIBROUS DYSPLASIA OF BONE
P. Bianco1*, S. A. Kuznetsov2, M. Riminucci3, N. Cherman2, L.W.Fisher2, M. T. Collins2, S. Wientroub4, P. G. Robey2
1Universita "La Sapienza", Rome, Italy
2CSDB, NIDCR, NIH, Bethesda, MD, USA
3Universita dell'Aquila, L'Aquila, Italy
4Tel Aviv Medical Center, Tel Aviv, Israel
Activating GNAS1 mutations underlying fibrous dysplasia of bone are believed to be inherently lethal and to survive through mosaicism. In an in vivo transplantation assay, homogeneous strains of mutant stromal cells are lethal, whereas mosaic strains are effective in generating an FD-like ectopic ossicle. We sought to determine if generation of FD requires a threshold level of pathogenic mutation, and whether mutational load varies across different native lesions and over time. Stromal cell cultures were established from 18 lesions and 4 normal skeletal sites from 18 patients. GNAS1 mutations were determined in stromal monolayers using either a PCR/DNA sequencing method, detecting 1/3 mutant cells, or a PNA-PCR/DNA sequencing method, detecting 1/200 mutant cells. In addition, multiple individual stromal cell clones were analyzed to determine the % proportion of clonogenic (progenitor) stromal cells from a given lesion. 10/10 lesions from patients of age <30, and 1/8 lesions from patients of age >30 years of age contained >1/3 mutant cells, as well as mutant clonogenic cells. All of these lesions had typical FD x-ray and histological features. 7/8 lesions from patients >30 contained <1/3 but >1/200 mutant cells and no clonogenic mutant cells. All of these lesions were typical FD by x-ray, but histology demonstrated either a normal bone/ bone marrow structure (5/7) or normal bone and non-descript marrow changes (2/ 7). We called these lesions "normalized FD." The frequency of clonogenic mutated cells within lesions was inversely correlated with age for the entire patient population. Transplantation of either stromal cell strains or uncultured marrow aspirates (MA) of typical FD into immunocompromised mice generated an FD ossicle, whereas stromal cell strains or MA of normalized FD generated normal bone. Normal skeletal sites of FD patients contained mutant cells with the same frequency as "normalized" sites. Activating GNAS1 mutations are only pathogenic when present in clonogenic stromal (stem) cells, and in >1/3 total stromal cells. Mutant clonogenic cells continuously demise during phases of bone growth and remodeling. This observation unravels an important biological link between the natural history of a lethal gene and the natural history of the human disease it causes.
BONE FORMATION AND STROMA OF BONE MARROW
V. Rugal*, V. Kravets, F. Baranovsky, V. Gonchar
Russian Research Institute of Hematology and Transfusiology, St Petersburg, Russia
AIM. To study bone formation in stromal defects of hemopoiesis.
METHODS. Bone and stromal cells of bone marrow were study in 49 patients with different hypoplasia hemopoiesis connecting with defects of microenvironment using trephinbiopsy. Cultural, morphometric, ultracitochemical methods were used.
RESULTS. The organ culture of bone tissue showed high proliferative activity of cells precursors of bone marrow stroma. The morphometric investigation of the whole bone fragments showed the increase of value of bone trabeculs, in hypoplastic conditions - 1.4 volume increase, in aplasia - 1.7 volume increase. The number of osteogenic cells per unit of square in histological preparation of iliac bone in hypoplasia was 1.5 volume increase, and in aplasia - was 2.0 volume increase. The ultracytochemical analysis demonstrated the increase functional activity of the intramedular and endosteal cells of bone fragments. In osteogenic and stromal cells were found pathological intranuclear bodies.
CONCLUSIONS. Our results indicate close relationship between bone tissue with stromal cells of bone marrow and hemopoietic precursors. Clear that one of the reasons of disorders of bone formations is disturb of the structure and function of stromal and hemopoietic cells of bone marrow. Our results can be used for the establish of reasons of bone formation disorders and improving of treatment of skeleton pathologies. The same scientists have created a new preparation on the basis of this data (See another report).
31P-NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY IN HYPOPHOSPHATASIA: DIAGNOSTIC URINE PROFILE INDICATING MULTIPLE NEW SUBSTRATES FOR BONE ALKALINE PHOSPHATASE
M. P. Whyte1*, M. N. Podgornik1, A. D'Avignon2
1Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, Missouri, USA
2Department of Chemistry, Washington University, St. Louis, Missouri, USA
Hypophosphatasia (HPP) features low serum alkaline phosphatase (ALP) activity and impaired skeletal mineralization (rickets or osteomalacia) due to deactivating mutations in the gene that encodes the tissue non-specific ALP isoenzyme (TNSALP). To date, 3 phosphocompounds are known to accumulate endogenously in HPP patients: phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi) in urine, and pyridoxal 5'-phosphate (PLP) in plasma. PEA, PPi, and PLP seem, therefore, to be natural substrates for TNSALP. Excess PPi, a mineralization inhibitor, may account for the skeletal disease of HPP. Nevertheless, the physiological role of TNSALP is incompletely understood.
To search for additional natural substrates for TNSALP, we performed 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) using urine from 11 children with HPP and contrasted the spectra to 2 healthy controls and 8 patients representing 5 other rachitic disorders. Urine from controls and non-HPP rickets showed the anticipated, single, major 31P-resonance peak consistent with inorganic phosphate (Pi) and only low levels of any other phosphocompounds. In contrast, HPP urine invariably contained 5-6 additional peaks in 10-50 fold excess (Figure). Spiking experiments show a peak for PEA and PPi and a peak tentatively identified as phosphocholine (PC). The identity of these multiple new TNSALP substrates will be presented.
31P-NMRS reveals elevated levels of multiple phosphocompounds in the urine of HPP patients that are diagnostic for this inborn error of metabolism. Importantly, 31P-NMRS of HPP patients indicates that there are several previously unrecognized natural substrates for TNSALP. This discovery should enhance understanding of the physiological role of TNSALP in bone and provide a means to assess potential medical treatments for HPP.
SEQUENCE ANALYSIS OF MEASLES VIRUS NUCLEOCAPSID TRANSCRIPTS IN PATIENTS WITH PAGET'S DISEASE
S. V. Reddy, W. E. Friedrichs, G. D. Roodman*
Univ TX HSC @ SA, San Antonio, TX, USA
It has been debated for almost 30 years whether Paget's disease of bone results from paramyxoviral infection of osteoclasts (OCLs) because of the presence of paramyxoviral-like nuclear inclusions in OCLs from Paget's patients and the detection by in situ hybridization of measles virus (MV) or canine distemper virus (CDV) transcripts in bone cells from pagetic lesions. Furthermore, immunocytochemical studies have demonstrated the presence of several paramyxoviral species in OCLs from Paget's patients. However, others have been unable to detect paramyxoviral transcripts in bone samples from patients with Paget's disease, or marrow cultures from involved sites of patients with Paget's disease. Furthermore, no one has been able to isolate an infectious virus from pagetic bone samples or marrow cells from patients with Paget's disease, nor has a full-length viral gene been cloned from pagetic samples. In the current study, we report the full-length sequence for the MV nucleocapsid (MVNP) gene in bone marrow from an involved site from a patient with Paget's disease and more than 700 base pair (bp) of MVNP sequence in 3 other patients with Paget's disease. These sequences were undetectable in 4 normal marrow samples studied simultaneously. The sequences from the individual patients differed, reflecting the high rate of mutation of MV, and differed from the Edmonston strain MVNP gene used as a positive control. These findings are consistent with the presence of a chronic MV infection in affected sites from these patients with Paget's disease.
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RECENT CHANGES IN PAGET'S DISEASE OF BONE CLINICAL EXPRESSIVENESS
A. A. Morales-Piga1*, F. J. Bachiller-Corral1, V. Abraira2, J. Beltran1, A. Rapado3
1Rheumatology, Ramón y Cajal Hospital, Madrid, Spain
2Investigation Unit, Ramón y Cajal Hospital, Madrid, Spain
3Mineral Metabolism Unit, Fundación Jiménez Díaz; Madrid, Spain
Background. Recent data suggests secular changes implying a current trend towards lesser clinical severity in Paget’s disease of bone (PD).
Objective. To compare the characteristics of two groups of PD patients, as disclosed from a systematically assessed sample.
Design. Hospital-based study of all cases followed up in our Unit since 1980.
Participants. Group I (n=124) represents the cases born before 1926 whereas group II (n= 109) included all those who were born from 1926 on.
Methods. A bone scan performed with 99mTc-EHDP was available for all patients. X-ray of the pelvis and spine, and views of any hot spot observed on the scintigraphy scans were reviewed. The skeletal extent of PD was determined by using the index proposed by Coutris. Alkaline phosphatase and hydroxyproline excretion were determined in blood and urine respectively. Baseline characteristics were recorded on a purpose-designed computerised database.
Results. The proportion of males (47% group I vs. 65% group II; p= 0.007) and the mean age at diagnosis (69.0 vs. 54.3; p< 0.001) differed significantly between groups. The year of birth shows a strong negative correlation with the age at diagnosis (r= – 0.83) and a weak but significant negative correlation with the extent of bone lesion (r= – 0.20; p= 0.002). Likewise, cases who were born prior to 1926 presented a greater percentage of affected skeleton (9.6 vs. 7.06; p=0.001). Multivariate analysis identified extent of skeletal lesions (OR= 0.76; p= 0.01), age at diagnosis (OR= 0.79; p= 0.008), number of involved bones (OR= 1.53; p= 0.03) and occupation (p<0.0001) as the predictive variables linked to year of birth.
Conclusion. Our data are consistent with a temporal tendency toward a smaller number of bone lesions and a lower percent of affected skeleton, both presumably related with changes in the pathogenic mechanisms of the disease. An earlier age of diagnosis in recent times, in absence of any relevant clinical or biochemical differences seems more likely linked to recent changes in referral and sociological patterns.
SEVERITY OF PAGET'S DISEASE IN THE UK: IS IT DECLINING?
G. I. Al-Bahrani1*, R. S. Lawson1, M. C. Prescott1, M. Davies2, P. L. Selby2
1Department of Nuclear Medicine, Manchester Royal Infirmary, Manchester, UK
2Musculoskeletal Research Group, University of Manchester, Manchester, UK
Previous studies have suggested that the prevalence of Paget’s disease of bone is decreasing in the United Kingdom and also that, in New Zealand, patients with Paget’s disease are less severely affected at presentation. The aim of this study was to investigate whether there is also a decline in the severity of Paget’s disease in Britain, and whether there is any change in the age of patients at presentation. Retrospective data were collected from 233 patients (117 females and 116 males) attending the nuclear medicine department for scintigraphy to assess Paget’s disease before treatment.
The patients were divided into two groups; group I comprised 119 patients attending between 1985 and 1994, and group II 114 presenting between 1995 and 1999. The mean age of group I was 68.7 (SD 10.3), while for group II it was 72.0 (SD 9.4). This increase in age at presentation was significant (p=0.01).
Alkaline phosphatase was not normally distributed, and so a logarithmic transformation was used. Mean alkaline phosphatase was significantly higher in group I than group II (705 vs 559 IU/L (normal range 70-330), p=0.03). Severity and distribution of the disease were also assessed from the radionuclide bone scan using a semi-quantitative scale. A high summated score indicates both more lesions and higher severity. The median number of lesions per patient and the median summated score were both significantly lower in patients presenting after 1994 (number of lesions, group I = 4, group II = 3, p=0.04; summated score, group I = 9, group II = 6, p<0.001).
These results suggest that patients with Paget’s disease of bone, not only present at a greater age, but also have less severe disease, as indicated by scintigraphy and biochemical activity. We therefore conclude that the severity of Paget’s disease of bone in this part of the UK is declining in parallel with the previously observed decline in prevalence.
LONG TERM RESPONSE OF BIOCHEMICAL MARKERS OF BONE TURNOVER AND BONE SCINTIGRAPHIC ACTIVITY AFTER 3 MONTHS OF TILUDRONATE TREATMENT IN PAGET’S DISEASE: PREDICTORS OF RESPONSE TO THERAPY
L. Alvarez1,2*, N. Guañabens1,2, P. Peris1, S. Vidal-Sicart1, I. Ros1, A. Monegal1, J. L. Bedini1, R. Deulofeu1,2, F. Pons1,2, A. M.Ballesta1,2
1Hospital Clínic, Barcelona, Spain
2Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
Aims: 1- To study the relationship between changes of markers of bone turnover and those of bone scintigraphic activity index (SAI) in pagetic patients after tiludronate treatment and 2- to investigate whether or not biochemical markers can predict the response to therapy.
Methods: 15 patients with Paget’s bone disease were studied prospectively. All received 400 mg tiludronate daily for 3 months. Serum total (TAP) and bone (BAP) alkaline phosphatases, propeptide N-terminal of type 1 procollagen (PINP) and urinary hydroxyproline (HYP) and N-terminal telopeptide of collagen type 1 (NTX) were measured at baseline (T-0) and 9 (T-1) and 21 (T-2) months after starting treatment. SAI was performed at the same times. Response to therapy at T-1 (% T-1) and T-2 (% T-2) was calculated and expressed as the mean ± standard error of the mean (SEM) percentages of variation in relation to the baseline. The percentage of variation between T-2 and T-1 (% T-1:T-2) was also calculated. Patients were classified into two groups depending on SAI evolution, i.e.: G-I, patients whose SAI decreased or remained stable between T-1 and T-2 and G-II, patients that showed an increase of SAI between T-1 and T-2.
Results: Expressed as mean±SEM. Bone resorption markers didn’t show significant differences between groups G-I and G-II.
Conclusions: 1- Markers of bone formation, specially BAP, are the first to increase after treatment and those which better reflect the scintigraphic activity. 2- The intensity of the response to therapy at 9 months doesn’t predict the duration of the effect of tiludronate treatment. 3- Increased baseline BAP values (> 81 ng/mL) are associated with a lower duration of response to therapy.
| Time | SAI | TAP (U/L) |
BAP (ng/mL) |
PINP (ng/mL) |
SAI | TAP (U/L) |
BAP (ng/mL) |
PINP (ng/mL) |
| G-I | G-II | |||||||
| T-0 | 11755±4517 | 568±173 | 67±24 | 273±133 | 15499±2610 | 1266±380 | 199±61* | 332±62 |
| T-1 | 5867±2045 | 213±22 | 16±2 | 60±18 | 8056±2101 | 310±60 | 33±9 | 78±15 |
| T-2 | 5556±1970 | 233±27 | 21±4 | 67±17 | 10733±2395 | 569±167 | 73±23* | 122±30 |
| % T-1 | -47±7 | -48±7 | -63±9 | -67±6 | -50±6 | -68±6 | -81±3 | -75±4 |
| % T-2 | -45±7 | -45±7 | -53±11 | -59±7 | -32±6* | -50±6 | -58±8 | -51±5 |
| %T-1:T-2 | -10±5 | 9±4 | 33±12 | 23±12 | 27±3** | 69±20** | 120±18** | 105±10* |
| **p<0.01 vs G-I; *p<0.05 vs G-I | ||||||||
ALTERED TYPE I AND TYPE III COLLAGEN METABOLISM IN THE MINERALISED MATRIX OF BONE AFFECTED BY PAGET'S DISEASE
H. A. Eriksen1*, C. A. Sharp2, M. W. J. Davie2, J. Risteli1
1Department of Clinical Chemistry, University of Oulu, Oulu, Finland
2Charles Salt Centre, Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, UK
Paget's disease of bone (PD) is a skeletal disorder characterised by increased localised bone turnover. Within affected bone matrix, collagen fibrils are irregularly organised resulting in a woven appearance. Type I collagen is the main collagen of normal mineralised bone. To characterise the collagen matrix of mineralised bone with Pagetic involvement we have applied sensitive and specific assays for the cross-linked carboxy- and amino-terminal telopeptides (ICTP, IIINTP) and procollagen propeptide (PIIINP) of types I and III collagen.
Two woven bone samples from very involved (inv), and 2 samples from less involved (less inv) cortical (CB) and trabecular bone (TB) sites were dissected from a femur with extensive PD. For comparison similar CB and TB samples were taken from healthy control femurs. Mineralised collagen fractions (MC) were prepared from all bones. Bone powders were borohydride reduced. Soft tissues were removed by heat denaturation and trypsin digestion prior to EDTA demineralisation. Resulting MCs were heat denatured and trypsin digested. ICTP, PIIINP (Orion Diagnostica, Finland), IIINTP and hydroxyproline were measured in the trypsin digests.
The MC in PD has higher levels of ICTP (2-fold normal) and PIIINP (10-fold normal) than does the MC of normal bone. In PD, involved bone has a greater content of PIIINP antigen than less involved bone (5-fold) or normal bone (about 20-fold). No differences were evident in either the total collagen or IIINTP contents of MC from normal and PD bone.
The PIIINP amounts in MC of PD bone indicate increased type III collagen synthesis, or the presence of type III pN-collagen with the retained amino-terminal propeptide. This may be related to the presence of woven bone, which defines the Pagetic lesion. The unaltered amount of cross-linked type III collagen in the MC of PD bone may suggest increased type III collagen degradation.
| MC (mg/g) | ||||
| Sample | Collagen | ICTP | IIINTP | PIIINP |
| PD CB less inv | 914 | 21.5 | 0.57 | 0.58 |
| PD TB less inv | 776 | 21.0 | 0.54 | 1.24 |
| PD CB inv | 816 | 17.7 | 0.61 | 3.33 |
| PD TB inv | 906 | 18.6 | 0.88 | 5.87 |
| Normal CB 50 | 775 | 10.5 | 0.62 | 0.16 |
| Normal CB 98 | 923 | 12.5 | 0.49 | 0.22 |
| Normal TB 50 | 759 | 11.6 | 0.37 | 0.12 |
| Normal TB 98 | 832 | 12.2 | 0.31 | 0.20 |
BISPHOSPHONATE (OLPADRONATE) RETENTION AND ITS DETERMINANTS IN PAGET'S DISEASE OF BONE
S. C. L. M. Cremers1*, E. Eekhoff2, S. M. van Dam1, J. Hartigh1, N. A. T. Hamdy2, P. Vermeij1, S. E. Papapoulos2
1Leiden University Medical Center, Department of Clinical Pharmacy and Toxicology
2Leiden University Medical Center, Department of Endocrinology
Bisphosphonates (BPs) are the treatment of choice of Paget's disease of bone because they concentrate selectively in the skeleton at active sites and suppress osteoclastic bone resorption. Skeletal involvement and disease activity vary greatly among patients with active disease implying that treatment schedules should be individualized. Understanding the complex pharmacokinetics of BPs can lead to improvements in the management of patients.
We investigated the Whole Body Retention of BP (WBR = dose BP - amount excreted in urine) during 5 days of iv treatment with the N-BP olpadronate 8 mg/ day in 78 patients (47 males, 31 females; mean age 69±11 years) with active disease during admission to a metabolic ward. We further examined the relations between WBR and age, body surface, renal function and pretreatment disease activity.
The mean WBR in the whole group during the 5-day treatment period was 46.5±14.8% with a markedly wide range (from 13.3% to 90.5%). The WBR was significantly related to renal function (r=0.41, p<0.05), assessed as creatinine clearance. Correlation with the activity of the disease was examined after logarithmic transformation of the bone turnover data. All biochemical parameters of bone turnover measured (sAP, uOHP/Cr and uNTx/Cr) were significantly correlated with WBR of olpadronate, even after adjustment for renal function.
Bisphosphonate retention is considerably higher with reduction of renal function (even within the normal range) and with increase in bone turnover.
These results and longitudinally obtained long-term data can help in better understanding the pharmacokinetic/pharmacodynamic properties of BP treatment in Paget's disease of bone and can lead to optimization of therapy in the individual patient.
SEX DIFFERENCES IN PAGET DISEASE OF BONE
F. J. Bachiller-Corral*, A. A. Morales-Piga, J. Beltrán
Rheumatology, Ramón y Cajal Hospital, Madrid, Spain.
Objective: To analyse the differences in age of diagnosis and, number, extension and distribution of lesions, according to sex, in a hospital series of patients with Paget’s Disease of bone (PD).
Patients and methods: A total of 313 patients with PD were evaluated over a period of twenty-two years (1977-1999). In all cases, diagnosis of PD was made by means of usual radiological criteria. A complete body scintigraphy was performed in 93% of patients. Age, sex, skeletal extent and individual affected bones were recorded. We made a prospective follow-up in 233 patients whereas in the remaining 80 cases, the evaluation was retrospective, based on clinical hospital records.
Results: The study sample comprised 168 men (54%) and 145 women, with the mean age of men proving 3 years younger than that of women (64.6 years vs. 67.8, p=0.007). Compared to males, the female group contained a higher percentage of monostotic patients (45.5% vs 35.1%, p=0.06). There were also differences in terms of number of affected sites (3.4 in males vs. 2.5 in females, p=0.007) and overall extension of PD (8.9% vs. 7.2%, p=0.017). No differences in locations of PD lesions were found as regard gender.
Conclusions: In our series, PD is slightly more frequent in men than in women. In addition, males present with the disease at a younger age and with a greater number of sites affected and skeletal extent than do female patients. The mechanical and genetic factors may be determinant for explain these differences.
CANINE PROSTATE INDUCES NEW BONE FORMATION IN MOUSE CALVARIA - A MODEL OF OSTEOINDUCTION BY PROSTATE TISSUE
B. E. LeRoy, R. R. Bahnson, T. J. Rosol*
The Ohio State University, Columbus, OH, USA
Bone metastases of prostate cancer in humans usually results in the formation of ‘osteoblastic’ metastases, which are characterized by localized osteosclerosis and formation of new woven bone on medullary trabeculae. The pathophysiology of the new bone formation at metastatic sites is not currently known, but it is hypothesized that growth factors secreted by the prostate are involved. Unfortunately, most rodent models of prostate cancer that metastasize to bone are osteolytic and not osteoblastic. Significant osteolysis by tumor cells at metastatic sites leads to fractures and bone instability with secondary formation of new periosteal bone for stabilization (callus response). Misinterpretation of new periosteal bone associated with bone stabilization as tumor-cell osteoinduction is another disadvantage of the osteolytic models. To circumvent these problems, we have developed a model system of new bone formation in the calvaria of nude mice stimulated by normal canine prostate tissue. Collagenase-digested normal canine prostate tissue (31 dogs) was implanted adjacent to the calvaria of nude mice (78 mice). Calvaria were examined at 2-6 weeks for changes in the bone microenvironment by histology, calcein uptake at sites of bone mineralization, and tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts in 54 mice with viable prostate tissue at the time of sacrifice. The prostate tissue induced reactive stromal cell proliferation around the prostate acini and abundant new bone formation on the adjacent periosteal surface in 35 of the mice. In some cases new bone formation also was induced on the distant or concave calvarial periosteum. The new bone stained intensely with calcein, which demonstrated regions of bone matrix mineralization. The thickness of control calvaria ranged from 100-130 micrometers, while regions of new bone formation on calvarial surfaces ranged from 250-300 micrometers in thickness. New bone formation was not induced in calvaria of mice implanted with normal kidney, bladder, or muscle tissue. There was a mild (not statistically significant) increase in TRAP (+) osteoclasts in the medullary cavities of calvaria with new bone formation induced by the prostate tissue. In summary, this animal model will be useful for investigating the roles of prostate-derived growth factors on new bone formation, such as endothelin or parathyroid hormone-related protein.
LOW BONE DENSITY IS ASSOCIATED WITH REDUCED LEAN SOFT TISSUE MASS IN PROSTATE CANCER PATIENTS TREATED WITH ANDROGEN ABLATION
Z. Chen*, P. T. Nguyen, M. J. Maricic, F. R. Ahmann, B. L. Dalkin
University of Arizona
Reduced bone mineral density (BMD) and increased risk of osteoporotic fractures among prostate cancer patients after androgen ablation therapy (ABT) have been reported. In this study we investigated whether low bone density is uniformly presented among prostate cancer patients after different modalities of ABT, and whether reduced bone density is associated with changes in body soft tissue composition. Sixty-three patients on ABT for stage C and D prostate cancer during the past 1-5 years and 47 age-matched controls were included in the analysis. Thirty-three of the prostate cancer patients were treated with luteinizing hormone-releasing hormone (LHRH) analogues or total androgen blockade (chemical castration, group A), and 29 had bilateral orchiectomy (surgical castration, group B). All the controls had stable and normal PSA (<4.0 ng/ml) at enrollment. BMD and body composition was measured using the Dual-energy X-ray Absorptiometry (DXA) (Hologic QDR 4500w). In comparison to the normal controls, the prostate cancer patients had significant greater body weight, and greater body fat tissue mass (FTM), while their lean soft tissue mass (LSTM) was reduced. On average, total BMD among the patients was 4.5% lower than the controls. Differences between group A and group B did not appear to be significant for age, weight, height, LSTM and total BMD. However, the average FTM was 22% higher in group B than in group A. After adjusting for age, LSTM was the strongest independent predictor for total BMD. In conclusion, in spite of their significant higher body weight, prostate cancer patients treated with ABT present significant lower BMD and reduced LSTM in comparison to controls. The degree of low bone density is the same among patients who had either chemical castration or surgical castration. The apparent difference in FTM between chemical castration group and surgical castration group should be examined in future studies. Whether low BMD and reduced LSTM are independently attributable to the increased risk of fractures in prostate cancer patients after ABT needs to be investigated.
SERUM MAGNESIUM IS AN INDEPENDENT PREDICTOR OF PARATHYROID HORMONE LEVELS IN DIALYSIS PATIENTS
J. F. Navarro*, C. Mora, M. L. Macía, E. Gallego, J. Chahin, M. L. Méndez, M. L. Rodríguez, A. Rivero, J. García
Hospital Nuestra Señora de Candelaria, Tenerife, Spain
Calcium (Ca), phosphorus (P) and calcitriol are the main factors that control parathyroid hormone (PTH) synthesis and secretion in dialysis patients. It has been suggested that magnesium (Mg) may play a role in the regulation of parathyroid gland function, with acute Mg infusion decreasing PTH secretion. However, the effects of chronic hypermagnesemia (hyperMg) on PTH levels in dialysis population are not known. In order to analyze this aspect, we evaluated 110 hemodialysis (HD) and 51 peritoneal dialysis (PD) patients not previously treated with calcitriol. HyperMg (serum Mg > 2.47 mg/dl) was found in 73% of HD and 58% of PD subjects. Since dialysis patients need higher PTH levels to maintain a normal bone turnover, hypoparathyroidism (hypoPTH) in this population has been defined as a serum intact PTH < 120 pg/ml. Patients with hypoPTH had higher serum Mg compared with subjects with adequate PTH levels, both in HD (3.01±0.33 vs 2.63±0.38 mg/dl) and PD (2.84±0.38 vs 2.22±0.34 mg/dl) (p<0.001). Linear regression analysis showed a negative correlation between Mg and PTH: r=-0.48, p<0.001 (HD) and r=-0.70, p<0.01 (PD). After partial correlation analysis, Mg and PTH were significantly associated: r=-0.58 (HD) and r=-0.57 (PD) (p<0.001). Finally, a forward stepwise multiple regression analysis demonstrated that PTH levels were predicted by Mg independently of Ca and P, both in HD (R=0.57) and PD (R=0.59) (p<0.001).
In conclusion, these data show that Mg is a significant and independent predictor of PTH levels in dialysis patients. These results suggest that hyperMg, a common finding in dialysis subjects, may have a suppresive effect on PTH synthesis and/or secretion in this population.
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EFFECT OF ESTROGEN AND CALCITRIOL REPLACEMENT IN BONE MASS IN RATS WITH RENAL FAILURE IN LONG AND SHORT-TERM ESTROGEN DEPRIVATION.
M. Naves1*, C. Díaz-Corte1, C. Gómez1, S. Braga2, M. T. Fernández-Coto2, M. Serrano1, A. González1, A. Rodríguez1, J. B. Cannata1
1Bone and Mineral Research Unit. Instituto Reina Sofía de Investigación, Oviedo, Spain
2Biochemistry Unit, Hospital Central de Asturias, Oviedo, Spain
Estrogen (E2) deprivation and renal chronic failure (CRF) may affect bone metabolism by different pathways. The aim of this study was to evaluate the effect of E2 and CT on bone in oophorectomized rats with CRF.
In the long-term E2 deprivation (phase I) we used 3-months old female Sprague-Dawley rats divided in 2 groups: CRF or control group (nephrectomy 7/8, n=9), CRF+oophorectomy (one-stage, n=35). Four weeks after surgery, rats were randomly divided in 4 homogeneous groups: placebo (n=8), CT 10 nanog/day/Kg (n=9), 17betaE2 10 microg day/Kg (n=9) and CT+17betaE2 (n=9). In the short-term E2 deprivation (phase II) we used 6-months old female rats divided in 2 groups: CRF (n=4), CRF+oophorectomy (n=27). One week after surgery, rats were randomly divided in the same groups as phase I: placebo (N=7), CT 10 nanog/day/ Kg (n=6), 17betaE2 15 microg day/Kg (n=8) and CT+17betaE2 (n=6). This abstract summarizes the results of biochemical parameters, bone mineral density (BMD) at the lumbar spine and uterus weight after 8 weeks of treatment. However, bone turnover markers and also bone histomorphometry were performed. In the two phases, rats treated either with CT, alone or combined with 17betaE2 showed no differences in BMD with the control group. In these groups BMD increased compared with placebo or 17betaE2 in both phases. Uterus weight was higher in rats receiving 17betaE2.
Biochemical parameters did not show changes after the treatment period. Only serum calcium in groups with CT, alone or combined with 17betaE2, was significantly higher.
In summary, in estrogen deprivation, the treatment with calcitriol alone or in combination with 17betaEstradiol was the most effective treatment to increase bone mass in rats with CRF and estrogen deficiency. The 17betaEstradiol alone group was not capable to increase bone mass despite this treatment preserved the uterus atrophy.
| Phase I | Phase II | ||
| Groups | BMD lumbar (g/cm2) | BMD lumbar (g/cm2) | Uterus weight (mg) |
| Placebo | 0.211±0.016 | 0.239±0.014 | 177±113 |
| CT | 0.234±0.0131 | 0.256 ±0.0111,2 | 210±119 |
| 17betaE2 | 0. 226±0.015 | 0.241±0.015 | 352±981,3/tr |
| 17betaE2+CT | 0.237±0.0101 | 0.268±0.0141,2 | 352±591,3 |
| CRF | 0.249±0.0121 | 0.260±0.0081,2 | 598±2131,3 |
| Mann-Whitney test, 1p<0.05 versus placebo, 2p<0.05 versus 17betaE2, 3p<0.05 versus CT. | |||
