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P14. Complete androgen insensitivity and osteoporosis
134 PlO. Restoration of circadian phosphate and prrathyroid hormone rhythms in patients with osteoporosis using timed calcium and phosphate supplements J Robinson, C Charlwood, B Durham, WD Fraser Department of Clinical Chemistry, Royal Liverpool University Hospital, Liwtpwl L7 8XP 16 patients (F = 15, h4 = 1) with established osteoporosis who had previously shown an unsatisfactory response to oral bisphosphonate/hormone replacement therapy, received timed calcium and phosphate (PO4) supplements given as Calcichew 500 mg at 0900,120O and Phosphate Sandoz 750 mg at 2200. The presence of circadian PO4 and parathyroid hormone rhythms were established by short overnight and morning sampling, the ratio of Peak PO4 levels and PTH secretion compared between 0300-0430 and 0900-1030. Patients were sampled prior to starting treatment and at 1, 3, 6 and 12 months. PO4 ratios were significantly increased at 1 and 3 months, 1.28, SD 0.15; p=O.O4, and 1.42, SD 0.23; p=O.O05 respectively from the pretreatment mean pf 1.10 SD 0.13. In 6 patients who received treatment for 12 months the ratio increased to 1.31 SD 0.17 but this was not significant. PTH ratios were also significantly increased after 1, 3 and 6 months at 1.38 p=O.O06, 1.70 p=O.O4 and 1.39 p=O.O32 from the pretreatment mean of 1.06 SD 0.18. At 12 months the ratio increased to 1.57 SD 0.37 but was not significant. Calcium and PO4 supplementation increased significantly the circadian rhythm of PO4 and PTH.
p11. Metaphyseal fractures have a differential diagnmis CR Paterson Department of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DDl 9SY Metaphyseal,fractures have often been regarded as typical or even ‘pathognomonic’ of non-accidental injury (D S Ablin et al AJR 1990; 154: 1035-1046). Metaphyseal fractures have been described in osteogenesis imperfecta but controversy exists as to whether they can occur in the absence of overtly abnormal bone. The paper will demonstrate that metaphyseal fractures are frequently seen, particularly in the first year of life, in known cases of osteogenesis imperfecta. Such fractures are found both with and without other radiological abnormalities. In addition metaphyseal fractures and fracture-like abnormalities in the metaphyses are seen in a wide variety of metabolic disorders in the first year of life, including scurvy, copper deficiency, Menkes’ syndrome and hyperparathyroidism. They could be expected in any disorder to collagen maturation at this age. It is important to recognise that this type of skeletal abnormality has a wide differential diagnosis.
Pl2. Effects of VIP and substance P on human bone marrow stromal cells IK Ashton, BA Ashton, S Jennings, SM Eisenstein Centre for Spinal Studies and Department of Rheumatology, Robert jones and Agnes Hunt Orthopaedic Hospital, Oswesty Nerve fibres immunoreactive to the neuropeptides vasoactive intestinal peptide (VIP) and substance P (SP) are found throughout bone and bone marrow. VIP, but not SF, increases cyclic AMP in human osteoblast-like cells, mouse calvaria and various osteoblastic cell lines. VIP also increases colony forming efficiency in rabbit bone marrow stromal cells. The effects of VIP and SP on human bone marrow has now been examined. Stromal cells derived from bone marrow (5 patients, 63-73 years) were grown to confluence in MEM + 10% FCS then replated at 12.5 x 103cells/ml f 2 x IO-Tdexamethasone (DEX) f VIP or SP (10-6.lO-IlM). VIP, but not SP increased cell proliferation with or without DEX. In the absence of DEX both peptides decreased protein synthesis despite the apparent increase in cell number
Abstracts
from the Spring Meeting, March 1994
with VIP. Alkaline phosphatase activity was increased by both VIP and SP in the presence or absence of DEX in 4 of the 5 cell preparations, although the effects were small (max 160% of control) compared with the effects of DEX itself. While these observations suggest that VIP and SP could play a role in the control of bone modelling a more detailed study of which cells in the bone/marrow stroma respond to the neuropeptides is needed.
P13. Calcium channels in transformed human osteoblasts (U-2 0s) B Somasundaram’, WT Mason’, R Evans, MC Meikle, F McDonald Department of Orthodontics, UMDS, University of London, SE1 9RT and *Department of Neuroendocrinology, Babraham, Cambridgeshire Calcium channels have been examined in transformed human osteoblast cells. In addition the electrophysiology of cultured transformed osteoblast cells was also investigated. U-2 OS cells were grown in culture flasks and then split onto cover slips one day prior to patch clamping the cells. The cells were grown in minimum essential medium (2mM Ca, 4 mM K) at 37OC in 4% CO?. Whole cell patch clamp recordings were used. The solutions used external to the cells allowed examination of both potassium and sodium currents. The cells were held at a membrane potential of -80mV and stepped to various potentials for 500ms. Specific channels identified included sodium and potassium voltage gated channels. Voltage gated calcium channels were not identified. Further to this the cells were deformed by a patch clamp attached on a single cell. intracellular calcium [CaZ+]i fluxes were examined, following loading with 5 PM Fura- AM, at 100 ms intervals. Waves of [CaZ+]i were seen spreading along the length of the individual cell. Following 300 ms of raised [Ca2+]i the level returned to values comparable to those prior to loading. Cells cultured in solutions which were free of calcium demonstrated no change in [Ca2+],.
Pl4. Complete androgen insensitivity and osteoporosis PA Mole, CR Paterson Department of Biochemical Medicine, Ninewells Hxspital Medical School, Dundee DDI 9SY
and
We report identical twins with complete androgen insensitivity (testicular feminisation syndrome). Born prematurely in 1951, one was noted incidentally to have ‘bilateral inguinal hernias’. They were not seen until the age of 18 when both were examined under anaesthesia for primary amenorrhoea. No uterus or ovaries were found but no further investigations were carried out. One twin had a hernia repair with removal of inguinal testes at the age of 22, and was treated intermittently with oestrogens. She was referred at age 38 because of concern about osteoporosis. Her bone density (SPA) was more than 2SD below the mean for females of her age. She has since been on continuous oestrogens and her bone density has increased slightly. The second twin presented with backache at age 33 and was reported to be ‘osteoporotic’ with wedging of two lumbar vertebrae. She was shown to have an XY chromosome content and had a laparotomy with removal of two intra-abdominal testes. She was prescribed oestrogens and, when SPA became available in 1988, had figures about -2SD for her age. On HRT for the subsequent five years her bone density has declined very slowly. We draw attention to the importance of oestrogen replacement from the age of puberty to prevent osteoporosis in these patients.
p11. Metaphyseal fractures have a differential diagnmis CR Paterson Department of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DDl 9SY Metaphyseal,fractures have often been regarded as typical or even ‘pathognomonic’ of non-accidental injury (D S Ablin et al AJR 1990; 154: 1035-1046). Metaphyseal fractures have been described in osteogenesis imperfecta but controversy exists as to whether they can occur in the absence of overtly abnormal bone. The paper will demonstrate that metaphyseal fractures are frequently seen, particularly in the first year of life, in known cases of osteogenesis imperfecta. Such fractures are found both with and without other radiological abnormalities. In addition metaphyseal fractures and fracture-like abnormalities in the metaphyses are seen in a wide variety of metabolic disorders in the first year of life, including scurvy, copper deficiency, Menkes’ syndrome and hyperparathyroidism. They could be expected in any disorder to collagen maturation at this age. It is important to recognise that this type of skeletal abnormality has a wide differential diagnosis.
Pl2. Effects of VIP and substance P on human bone marrow stromal cells IK Ashton, BA Ashton, S Jennings, SM Eisenstein Centre for Spinal Studies and Department of Rheumatology, Robert jones and Agnes Hunt Orthopaedic Hospital, Oswesty Nerve fibres immunoreactive to the neuropeptides vasoactive intestinal peptide (VIP) and substance P (SP) are found throughout bone and bone marrow. VIP, but not SF, increases cyclic AMP in human osteoblast-like cells, mouse calvaria and various osteoblastic cell lines. VIP also increases colony forming efficiency in rabbit bone marrow stromal cells. The effects of VIP and SP on human bone marrow has now been examined. Stromal cells derived from bone marrow (5 patients, 63-73 years) were grown to confluence in MEM + 10% FCS then replated at 12.5 x 103cells/ml f 2 x IO-Tdexamethasone (DEX) f VIP or SP (10-6.lO-IlM). VIP, but not SP increased cell proliferation with or without DEX. In the absence of DEX both peptides decreased protein synthesis despite the apparent increase in cell number
Abstracts
from the Spring Meeting, March 1994
with VIP. Alkaline phosphatase activity was increased by both VIP and SP in the presence or absence of DEX in 4 of the 5 cell preparations, although the effects were small (max 160% of control) compared with the effects of DEX itself. While these observations suggest that VIP and SP could play a role in the control of bone modelling a more detailed study of which cells in the bone/marrow stroma respond to the neuropeptides is needed.
P13. Calcium channels in transformed human osteoblasts (U-2 0s) B Somasundaram’, WT Mason’, R Evans, MC Meikle, F McDonald Department of Orthodontics, UMDS, University of London, SE1 9RT and *Department of Neuroendocrinology, Babraham, Cambridgeshire Calcium channels have been examined in transformed human osteoblast cells. In addition the electrophysiology of cultured transformed osteoblast cells was also investigated. U-2 OS cells were grown in culture flasks and then split onto cover slips one day prior to patch clamping the cells. The cells were grown in minimum essential medium (2mM Ca, 4 mM K) at 37OC in 4% CO?. Whole cell patch clamp recordings were used. The solutions used external to the cells allowed examination of both potassium and sodium currents. The cells were held at a membrane potential of -80mV and stepped to various potentials for 500ms. Specific channels identified included sodium and potassium voltage gated channels. Voltage gated calcium channels were not identified. Further to this the cells were deformed by a patch clamp attached on a single cell. intracellular calcium [CaZ+]i fluxes were examined, following loading with 5 PM Fura- AM, at 100 ms intervals. Waves of [CaZ+]i were seen spreading along the length of the individual cell. Following 300 ms of raised [Ca2+]i the level returned to values comparable to those prior to loading. Cells cultured in solutions which were free of calcium demonstrated no change in [Ca2+],.
Pl4. Complete androgen insensitivity and osteoporosis PA Mole, CR Paterson Department of Biochemical Medicine, Ninewells Hxspital Medical School, Dundee DDI 9SY
and
We report identical twins with complete androgen insensitivity (testicular feminisation syndrome). Born prematurely in 1951, one was noted incidentally to have ‘bilateral inguinal hernias’. They were not seen until the age of 18 when both were examined under anaesthesia for primary amenorrhoea. No uterus or ovaries were found but no further investigations were carried out. One twin had a hernia repair with removal of inguinal testes at the age of 22, and was treated intermittently with oestrogens. She was referred at age 38 because of concern about osteoporosis. Her bone density (SPA) was more than 2SD below the mean for females of her age. She has since been on continuous oestrogens and her bone density has increased slightly. The second twin presented with backache at age 33 and was reported to be ‘osteoporotic’ with wedging of two lumbar vertebrae. She was shown to have an XY chromosome content and had a laparotomy with removal of two intra-abdominal testes. She was prescribed oestrogens and, when SPA became available in 1988, had figures about -2SD for her age. On HRT for the subsequent five years her bone density has declined very slowly. We draw attention to the importance of oestrogen replacement from the age of puberty to prevent osteoporosis in these patients.