- Email: [email protected]
P6. Biochemical bone markers in anorexia nervosa
Bone Vol. 19, No. 6 December 1996:685-701
Abstracts
rhythm was the same for iFDpd-Metra and Dpd-HPLC although the magnitude of the rhythm was much higher for Dpd-HPLC, (peak-trough amplitudes 30.1% and 70.3% respectively). In 4 clinical models the changes seen in Dlxl measured by HPLC were greater than those seen in ~FDpd-Metra which in turn were greater than those seen in iFDpd-Nichols. We conclude that results from these different assays for Dpd are not interchangeable. P4. Transcriptional inhibition of the et3,thropoetin (EPO) gene by raised extracellular calcium: role of the negative calcium response element
CM Irving, A Kumar, GS McHaffie, SH Ralston
Department of Medicine and Therapeutics. University of Aberdeen Medical School, Foresterhill,AberdeenAB9 2ZD Background: The negative calcium response element type 2 (nCARE) is a DNA control sequence which mediates downregulation of PTH transcription in response to hypercalcaemia. We have previously shown that the nCARE lies within an ALU eleme~nt in the PTH 5' flank (Bone 1995, in press). Since ALU elements are widely distributed throughout the genome, we looked for other nCARE in a search of GenBank and found >150 copies, distributed over a wide range of target genes. To determine if some of these might be functional in genes other than PTH, we studied the etfect of extracellular calcium on EPO transcription, since the promoter of this gene contains 3 copies of the nCARE. Methods: A 5.25Kb insert from the 5' flank of the human EPO gene was cloned into the PGL2 luciferase reporter vector and transfected into BFIK cells with pSVISGal as an internal control for transfection efficiency. The cells were cultured for 48hr in varying medium calcium concentrations (1.5 - 4.Smmol/l) and transcription of the reporter gene assessed by measurement of luciforase activity (corrected for ~-galactosidase) in cell extracts. Results: Transcription was supressed in a dose dependent manner by raised extracellular calcium concentrations. to a nadir of 50-75% of control at 4.0raM (p<0.001). To determine if this effect was associated with binding of nuclear proteins, DNA containing one nCARE was amplified by PCR and used as a probe in a gel shift assay. A hand shift was observed which was competed out by synthetic nCARE oligos, consistent with binding of nuclear proteins to an nCARE motif. Conclusions: These data suggest that EPO transcription is modulated by extracelhilar calcium by a mechanism which involves binding of nuclear proteins to the nCARE, This finding has important implications for transcriptional regulation by calcium of a wide range of target genes and raises the possibility that hypercalcaemia may cause anaemia by supressing EPO transcription.
both forearm bone mass (r=-0.68,p--O.O05 proximal site)and spine DXA (r=--0~59, p=0.02). We conclude that osteoporosis is a potential complication of coeliac disease and that it is related to secondary hyperparathyroidism which is present even in patients who are ostensibly treated by diet.
P6. Biochemical bone markers in anorexia nervosa C Mackintosh, J Treasure*, A Ward*, C Moniz
Department of Clinical Biochemistry, Kings College Hospital and *Instituteof Psychiatry,Maudsley Hospital, LondonSE5 Premature osteoporosis is a recognised complication of anorexia nervosa, but the mechanisms for bone loss are unclear. To examine systemic changes in bone turnover, we measured novel biochemical bone markers in 36 anorexia nervosa patients (17-46 years) to complement conventional tests. Anorexia nervosa was diagnosed by standard criteria, 19 were outpatients and 17 in-patients, with average duration of disease 5 and 7 years respectively. 17/19 of outpatients showed no significant weight gain since their previous visit whilst 14/17 inpatients showed a mean weight gain of 9.Skg (range 4-16 kg). Serum Pro-collagen l carboxyterminal peptide (PICP) and alkaline phosphatase were measured for bone formation and serum carboxyterminal teleopeptide (ICTP) and urinary deoxypyridinoline (uDpyd) for resorption.. To overcome spurious interpretation of a creatinine correction for urinary cross-link excretion in subjects with low BMI, serum and urine free pyridinolines were also measured by RIA. Both ICTP (x, SD= 6.5 + 4.2 ug/l) and uDpyd (19.3 ± 14.3 nmol/mmol creat) were raised by 70% and 90% compared to 16 age-matched (range 19-44y) normal controls. No significant differences between in- and out-patients were observed. Although BMI in this group was significantly below normal, creatinine correction did not appear to account for raised urinary pyridinolines, since serum cross-links (measured by RIA) were also elevated. There was no significant difference in bone formation markers from controls: PICP 123:1:52 ug/l, and 114 +14 ug/l respectively (P=NS) but mean PICP was significantly higher in the in-patients 225 ± 14 ug/l, P<0.003. Total alkaline phosphatase was not raised and was predominantly of bone isoform. In untreated anorexia nervosa bone uncoupling with enhanced resorption without concomitant formation occurs and appears to be sustained even several years after the onset of amenorrhoea. With weight gain, bone formation (PICP) increased indicating recovery despite evidence of continued enhanced bone resorption. PT. Architecture of the femoral neck in osteoarthritis JA Bayton, E Morrison, M Neilson, D Russell, J McGadey, SW McDonald, PE McGill
Departmentof Anatomy, Universityof Glasgow,Glasgow P5. Osteopomsis in coeliac disease. PL Selby, M Davies, TW Wames, JE Adams, EB Mawer
University of Manchester BoneDiseaseResearchCentreand Departments of Medicine and Diagnostic Radiology, Manchester Royal Infirmary, Manchester M13 9WL Coeliac disease is a major cause of intestinal malabsorption which has important effects on the absorption of calcium and hence is likely to impact upon the skeleton. In order to assess the effect of coeliac diseasp ~- ~,cme we have measured the bone mass in 27 vauents with histologically proven coeliac disease who had been prescribed a gluten free diet. Bone mass was measured using QCT of the spine, DXA (Lunar) in the spine and femoral neck and by SPA in the non-dominant forearm and expressed as Z scores. Serum PTH and vitamin D metabolites were measured after an overnight fast.
Spine
Spine
QCT
DXA
Femoral neck
Distal Proximal forearm forearm
z score -0.62 -0.27 0.09 -1.42 -1.35 se 0.25 0.35 0.20 0.26 0.25 p vs zero 0.02 0.44 0.66 <0.001 <0.001 P Serum 25OHD concentrations were normal (18.6+2.4ng/ml) but there was secondary hyperparathyroidism ([YfH--63~-17pg/ml) with raised 1,25(OH)2D (55.1±4.2pg/ml: normal=34.9±1.2pg/ml, p=0.001). There was a negative correlation between serum PTH and
Computerised image analysis is providing new information about bone architecture in health and disease. This study examines the human femoral neck in osteoarthritis. Osteoarthritic specimens (4 female aged 63, 63, 67, 74) were obtained at hip replacement. Control hips (1 male aged 85; 2 female aged 68, 85) were collected from post mortem subjects with no arthritic history. The small numbers reflect the length and difficulty of processing. A transverse histological section from each proximal neck was examined using computerised image analysis. Compared to controls, osteoarthritic specimens showed evidence of an increase in cortical bone but no difference in the amount of bone in the medulla, in the number of trabecular profiles or their cross-sectional areas. OA CONTROL % cortical bone 18.49 + 2.00 11.09 + 3.87. % trabecular bone 10.52 + 2.08 9.97+ 1.03 no. trabecular profiles/ram2 0.74 + 0.08 0.60+ 0.17 average area of profile(ram2) 0.18 ± 0.03 0.21+ 0.05 • Standard errors not overlapping. We have devised a grid I to facilitate detailed computerised analysis of bone distribution in a histological section. Use of this revealed that the thickened cortex of the osteoarthritic specimens occurred particularly in the inferior and lower posterior parts of the neck, regions important in weight transmission to the shaft. The distribution of bone in the medulla showed no significant differences between arthritic and control hips. These results may, in part, explain why patients with osteoarthritis rarely sustain fractures of the femoral neck. I. Neilson,M. et al. Clin. Anat 8, 1995.
689
Abstracts
rhythm was the same for iFDpd-Metra and Dpd-HPLC although the magnitude of the rhythm was much higher for Dpd-HPLC, (peak-trough amplitudes 30.1% and 70.3% respectively). In 4 clinical models the changes seen in Dlxl measured by HPLC were greater than those seen in ~FDpd-Metra which in turn were greater than those seen in iFDpd-Nichols. We conclude that results from these different assays for Dpd are not interchangeable. P4. Transcriptional inhibition of the et3,thropoetin (EPO) gene by raised extracellular calcium: role of the negative calcium response element
CM Irving, A Kumar, GS McHaffie, SH Ralston
Department of Medicine and Therapeutics. University of Aberdeen Medical School, Foresterhill,AberdeenAB9 2ZD Background: The negative calcium response element type 2 (nCARE) is a DNA control sequence which mediates downregulation of PTH transcription in response to hypercalcaemia. We have previously shown that the nCARE lies within an ALU eleme~nt in the PTH 5' flank (Bone 1995, in press). Since ALU elements are widely distributed throughout the genome, we looked for other nCARE in a search of GenBank and found >150 copies, distributed over a wide range of target genes. To determine if some of these might be functional in genes other than PTH, we studied the etfect of extracellular calcium on EPO transcription, since the promoter of this gene contains 3 copies of the nCARE. Methods: A 5.25Kb insert from the 5' flank of the human EPO gene was cloned into the PGL2 luciferase reporter vector and transfected into BFIK cells with pSVISGal as an internal control for transfection efficiency. The cells were cultured for 48hr in varying medium calcium concentrations (1.5 - 4.Smmol/l) and transcription of the reporter gene assessed by measurement of luciforase activity (corrected for ~-galactosidase) in cell extracts. Results: Transcription was supressed in a dose dependent manner by raised extracellular calcium concentrations. to a nadir of 50-75% of control at 4.0raM (p<0.001). To determine if this effect was associated with binding of nuclear proteins, DNA containing one nCARE was amplified by PCR and used as a probe in a gel shift assay. A hand shift was observed which was competed out by synthetic nCARE oligos, consistent with binding of nuclear proteins to an nCARE motif. Conclusions: These data suggest that EPO transcription is modulated by extracelhilar calcium by a mechanism which involves binding of nuclear proteins to the nCARE, This finding has important implications for transcriptional regulation by calcium of a wide range of target genes and raises the possibility that hypercalcaemia may cause anaemia by supressing EPO transcription.
both forearm bone mass (r=-0.68,p--O.O05 proximal site)and spine DXA (r=--0~59, p=0.02). We conclude that osteoporosis is a potential complication of coeliac disease and that it is related to secondary hyperparathyroidism which is present even in patients who are ostensibly treated by diet.
P6. Biochemical bone markers in anorexia nervosa C Mackintosh, J Treasure*, A Ward*, C Moniz
Department of Clinical Biochemistry, Kings College Hospital and *Instituteof Psychiatry,Maudsley Hospital, LondonSE5 Premature osteoporosis is a recognised complication of anorexia nervosa, but the mechanisms for bone loss are unclear. To examine systemic changes in bone turnover, we measured novel biochemical bone markers in 36 anorexia nervosa patients (17-46 years) to complement conventional tests. Anorexia nervosa was diagnosed by standard criteria, 19 were outpatients and 17 in-patients, with average duration of disease 5 and 7 years respectively. 17/19 of outpatients showed no significant weight gain since their previous visit whilst 14/17 inpatients showed a mean weight gain of 9.Skg (range 4-16 kg). Serum Pro-collagen l carboxyterminal peptide (PICP) and alkaline phosphatase were measured for bone formation and serum carboxyterminal teleopeptide (ICTP) and urinary deoxypyridinoline (uDpyd) for resorption.. To overcome spurious interpretation of a creatinine correction for urinary cross-link excretion in subjects with low BMI, serum and urine free pyridinolines were also measured by RIA. Both ICTP (x, SD= 6.5 + 4.2 ug/l) and uDpyd (19.3 ± 14.3 nmol/mmol creat) were raised by 70% and 90% compared to 16 age-matched (range 19-44y) normal controls. No significant differences between in- and out-patients were observed. Although BMI in this group was significantly below normal, creatinine correction did not appear to account for raised urinary pyridinolines, since serum cross-links (measured by RIA) were also elevated. There was no significant difference in bone formation markers from controls: PICP 123:1:52 ug/l, and 114 +14 ug/l respectively (P=NS) but mean PICP was significantly higher in the in-patients 225 ± 14 ug/l, P<0.003. Total alkaline phosphatase was not raised and was predominantly of bone isoform. In untreated anorexia nervosa bone uncoupling with enhanced resorption without concomitant formation occurs and appears to be sustained even several years after the onset of amenorrhoea. With weight gain, bone formation (PICP) increased indicating recovery despite evidence of continued enhanced bone resorption. PT. Architecture of the femoral neck in osteoarthritis JA Bayton, E Morrison, M Neilson, D Russell, J McGadey, SW McDonald, PE McGill
Departmentof Anatomy, Universityof Glasgow,Glasgow P5. Osteopomsis in coeliac disease. PL Selby, M Davies, TW Wames, JE Adams, EB Mawer
University of Manchester BoneDiseaseResearchCentreand Departments of Medicine and Diagnostic Radiology, Manchester Royal Infirmary, Manchester M13 9WL Coeliac disease is a major cause of intestinal malabsorption which has important effects on the absorption of calcium and hence is likely to impact upon the skeleton. In order to assess the effect of coeliac diseasp ~- ~,cme we have measured the bone mass in 27 vauents with histologically proven coeliac disease who had been prescribed a gluten free diet. Bone mass was measured using QCT of the spine, DXA (Lunar) in the spine and femoral neck and by SPA in the non-dominant forearm and expressed as Z scores. Serum PTH and vitamin D metabolites were measured after an overnight fast.
Spine
Spine
QCT
DXA
Femoral neck
Distal Proximal forearm forearm
z score -0.62 -0.27 0.09 -1.42 -1.35 se 0.25 0.35 0.20 0.26 0.25 p vs zero 0.02 0.44 0.66 <0.001 <0.001 P Serum 25OHD concentrations were normal (18.6+2.4ng/ml) but there was secondary hyperparathyroidism ([YfH--63~-17pg/ml) with raised 1,25(OH)2D (55.1±4.2pg/ml: normal=34.9±1.2pg/ml, p=0.001). There was a negative correlation between serum PTH and
Computerised image analysis is providing new information about bone architecture in health and disease. This study examines the human femoral neck in osteoarthritis. Osteoarthritic specimens (4 female aged 63, 63, 67, 74) were obtained at hip replacement. Control hips (1 male aged 85; 2 female aged 68, 85) were collected from post mortem subjects with no arthritic history. The small numbers reflect the length and difficulty of processing. A transverse histological section from each proximal neck was examined using computerised image analysis. Compared to controls, osteoarthritic specimens showed evidence of an increase in cortical bone but no difference in the amount of bone in the medulla, in the number of trabecular profiles or their cross-sectional areas. OA CONTROL % cortical bone 18.49 + 2.00 11.09 + 3.87. % trabecular bone 10.52 + 2.08 9.97+ 1.03 no. trabecular profiles/ram2 0.74 + 0.08 0.60+ 0.17 average area of profile(ram2) 0.18 ± 0.03 0.21+ 0.05 • Standard errors not overlapping. We have devised a grid I to facilitate detailed computerised analysis of bone distribution in a histological section. Use of this revealed that the thickened cortex of the osteoarthritic specimens occurred particularly in the inferior and lower posterior parts of the neck, regions important in weight transmission to the shaft. The distribution of bone in the medulla showed no significant differences between arthritic and control hips. These results may, in part, explain why patients with osteoarthritis rarely sustain fractures of the femoral neck. I. Neilson,M. et al. Clin. Anat 8, 1995.
689