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P26. Urinary galactosyl hydroxylysine as a marker of bone resorption
458
Abstracts from the Winter Meeting, December 1993
17.4 f 1.6; p=O.O09 and 56nm/ll f 6 vs 27 f 3; p=O.OOOS respectively. Free androgen index, however, was lower in HPs at 44 + 5 vs 69 + 7; p=O.O08. These results suggest the presence of significant osteopenia in association with haemophilia A. Since chronic liver disease has been shown previously to stimulate SHBG production, liver dysfunction in HI’s may explain the apparent relative hypogonadism found.
P26. Urinary galactosyl hydroxylysine resorption AW Al-Dehami, RGG Russell, R Eastell Department of Human Metabolism and University of Sheffield, Sheffield
as a marker
Clinical
of bone
Biochemistry,
Hydroxylysine is a component of bone collagen that is not reutilised and is less abundant than hydroxyproline. The major form of glycoslated hydroxylysine is bone collagen is galactosyl hydroxylysine (Gal-Hyl). The aim of the study was to identify sources of variability in this measurement. Gal-Hyl was determined by HPLC using the method of Mom (Calcified Tissue International, 1988; 42:87-90). The assay involves HPLC after incubation of urine with dansyl chloride in alkaline conditions. The day-to-day variation was assessed in 12 volunteers over 5 days on 24 hour urine collections and was 10.6%. There was no increase in urinary Gal-Hyl after gelatin loading in 6 healthy adults, although there was a 10 fold increase in hydroxyproline excretion. There was an increase in Cal-Hyl with age in 90 women ages 50-85 years (r= 0.47, pcO.000 1). In this group of women, we also measured deoxypyridinoline by HPLC and fluorescence detection and the excretion of this collagen crosslink correlated with Gal-Hyl (r=0.55, pcO.0001). Thus GalHyl may be a useful marker for measuring bone resorption.
P27. Rabbit osteoblasts constitutively synthesize progelatinase-A (72 kDa) and progelatinaecB (95 kDa) S Bord, RM Hembry, JJ Reynolds, MC Meikle’ Strangeways Research Laboratory, Worts Causezuay, Cambridge, CBI 4RN and *Department of Orthodontics and Paediatric Dentistry, UMDS, (Guys), London Rabbit calvarial osteoblasts were cultured on [14C]-abelled type 1 collagen films and stimulated with either IL-la (1 ng/ml), 1,25(OH)zD3 (10 r&ml) or partially purified porcine mononuclear cell conditioned medium (MCM, 2% v/v). The culture media were changed every 24 h for 6 days. During this time 1,25(OH)zD3 and MCM induced 60% and IL-la 90% collagenolysis, compared to control levels of 35%. High levels of coIlagenase activity were detected after MCM (9.5 u/ml) and ILla (11.0 u/ml) treatment compared to unstimulated controls (co.1 u/ml). However, collagenase levels in the media of 1,25(OH)zD3 treated cultures were. low (approx 0.1 u/ml). Zymography on gelatin substrate SDS gels showed that rabbit osteoblasts constitutively produced progelatinase-A (72 kDa and progelatinase-B (95 kDa). Activation of gelatinase-A occurred following MCM and IL-la treatments at 48h, and at 72h after 1,25(OH)zD3 treatment; unstimulated samples showed only slight activation after % h culture. Progelatinase-B (95 kDa) was present in all media samples, but active gelatinase-B could only be detected in IL-la and MCM-treated culture media. These data suggest that gelatinases as well as collagenase play an important role in preparing bone surfaces for osteoclastic attachment. Supported by the Arthritis and Rheumatism Council and the Medical Research Council.
P28. Phylloquinone (vitamin K1) content of bone in young adults SJ Hodges, S Petursson, D Harrington’, C Hopper, B Henderson, M Harris Joint Departments of Oral Surgery, Eastman Dental and University College Hospitals and Institute of Dental Surgery, London and ‘Haematology Research, Guy’s Hospital, London Recently it has been reported that bone samples from a limited series of elderly osteoarthritic patients undergoing elective hip replacement surgery is rich in vitamin K. This observation, if generally applicable, contrasts with the reported undercarboxylation of serum osteocalcin in postmenopausal women. The phylloquinone content in bone samples from 19 young adults (age range 16-37 yrs, mean 24.8 f 5.8 y’s; 8 male, 11 female) admitted for wisdom tooth extraction has been determined. The bone samples were powdered under liquid nitrogen before being extracted with EM’A (0.5M). An internal standard of tritiated phylloquinone epoxide was added. The mixture was then extracted twice with chloroform/methanol (3:l v/v) and the combined organic extracts reduced to dryness. The residue was treated with lipase in 0.8M phosphate buffer (pH 8.0) for lhr at 37°C. The reaction mixture was further extracted with hexane before purification on silica Sep-Pak cartridges, normal phase HPLC followed by reverse-phase HPLC coupled to electrochemical detection. All procedures were done under conditions of diffus’e light. After taking into account losses during the extraction process it was found that bone samples from young adults contains less vitamin Kl than previously found in the bone of the elderly. The reasons for the lower levels in the bone of young subjects remains unclear, but may reflect the changing lipid content of ageing bone. Further research is needed to examine the relationship between vitamin K, the lipid content of bone and the synthesis of functional vitamin Kdependent bone proteins.
P29. Measurement of peri-pmathetic bone mineral density pre and poet total hip arthroplaety B Lees, PR AIlen*, JC Stevenson Lynn Institute for Metabolic Research, London hW8 9SQ and *Lewisham Hospital, London SE13 6LH Dual energy X-ray absorptiometry (DXA) allows the quantification of changes in bone mineral density (BMD) following total hip arthroplasty (THA) which cannot be accurately determined by conventional radiography. This has important implications, since bone loss due to stress shielding may be an important factor in the subsequent failure of the implant. In thii prospective study we compared the BMD of the proximal femur of 7 individuals (4 males and 3 females) preTHA and then 6,12,24 and 52 weeks following uncemented THA in the same hip. BMD measurements were made by DXA (Lunar DPX). Four regions of interest 20 mm in height were selected, A and B (lateral sites) which approximate to Gruen zone I, and C and D (medial sites) which approximate to Gruen zone 7. Bone loss compared to pre THA was significant in all regions 24 weeks post THA (regions A, C and D -12.8%, -15.7% and -8.9% respectively ~~0.05, and region B -15.6% p-3.01) and remained reduced at 52 weeks post THA (regions A and B -14.1% and -19.5% respectively, p
P30. Analysis of a truncated mRNA for alkaline phosphatase in two human oateoaarcoma cell lines NN Ali, J Rowe, NM Teich Laboratory of Viral-mediated Cell Differentiaiton, Imperial Cancer Research Fund, London
Abstracts from the Winter Meeting, December 1993
17.4 f 1.6; p=O.O09 and 56nm/ll f 6 vs 27 f 3; p=O.OOOS respectively. Free androgen index, however, was lower in HPs at 44 + 5 vs 69 + 7; p=O.O08. These results suggest the presence of significant osteopenia in association with haemophilia A. Since chronic liver disease has been shown previously to stimulate SHBG production, liver dysfunction in HI’s may explain the apparent relative hypogonadism found.
P26. Urinary galactosyl hydroxylysine resorption AW Al-Dehami, RGG Russell, R Eastell Department of Human Metabolism and University of Sheffield, Sheffield
as a marker
Clinical
of bone
Biochemistry,
Hydroxylysine is a component of bone collagen that is not reutilised and is less abundant than hydroxyproline. The major form of glycoslated hydroxylysine is bone collagen is galactosyl hydroxylysine (Gal-Hyl). The aim of the study was to identify sources of variability in this measurement. Gal-Hyl was determined by HPLC using the method of Mom (Calcified Tissue International, 1988; 42:87-90). The assay involves HPLC after incubation of urine with dansyl chloride in alkaline conditions. The day-to-day variation was assessed in 12 volunteers over 5 days on 24 hour urine collections and was 10.6%. There was no increase in urinary Gal-Hyl after gelatin loading in 6 healthy adults, although there was a 10 fold increase in hydroxyproline excretion. There was an increase in Cal-Hyl with age in 90 women ages 50-85 years (r= 0.47, pcO.000 1). In this group of women, we also measured deoxypyridinoline by HPLC and fluorescence detection and the excretion of this collagen crosslink correlated with Gal-Hyl (r=0.55, pcO.0001). Thus GalHyl may be a useful marker for measuring bone resorption.
P27. Rabbit osteoblasts constitutively synthesize progelatinase-A (72 kDa) and progelatinaecB (95 kDa) S Bord, RM Hembry, JJ Reynolds, MC Meikle’ Strangeways Research Laboratory, Worts Causezuay, Cambridge, CBI 4RN and *Department of Orthodontics and Paediatric Dentistry, UMDS, (Guys), London Rabbit calvarial osteoblasts were cultured on [14C]-abelled type 1 collagen films and stimulated with either IL-la (1 ng/ml), 1,25(OH)zD3 (10 r&ml) or partially purified porcine mononuclear cell conditioned medium (MCM, 2% v/v). The culture media were changed every 24 h for 6 days. During this time 1,25(OH)zD3 and MCM induced 60% and IL-la 90% collagenolysis, compared to control levels of 35%. High levels of coIlagenase activity were detected after MCM (9.5 u/ml) and ILla (11.0 u/ml) treatment compared to unstimulated controls (co.1 u/ml). However, collagenase levels in the media of 1,25(OH)zD3 treated cultures were. low (approx 0.1 u/ml). Zymography on gelatin substrate SDS gels showed that rabbit osteoblasts constitutively produced progelatinase-A (72 kDa and progelatinase-B (95 kDa). Activation of gelatinase-A occurred following MCM and IL-la treatments at 48h, and at 72h after 1,25(OH)zD3 treatment; unstimulated samples showed only slight activation after % h culture. Progelatinase-B (95 kDa) was present in all media samples, but active gelatinase-B could only be detected in IL-la and MCM-treated culture media. These data suggest that gelatinases as well as collagenase play an important role in preparing bone surfaces for osteoclastic attachment. Supported by the Arthritis and Rheumatism Council and the Medical Research Council.
P28. Phylloquinone (vitamin K1) content of bone in young adults SJ Hodges, S Petursson, D Harrington’, C Hopper, B Henderson, M Harris Joint Departments of Oral Surgery, Eastman Dental and University College Hospitals and Institute of Dental Surgery, London and ‘Haematology Research, Guy’s Hospital, London Recently it has been reported that bone samples from a limited series of elderly osteoarthritic patients undergoing elective hip replacement surgery is rich in vitamin K. This observation, if generally applicable, contrasts with the reported undercarboxylation of serum osteocalcin in postmenopausal women. The phylloquinone content in bone samples from 19 young adults (age range 16-37 yrs, mean 24.8 f 5.8 y’s; 8 male, 11 female) admitted for wisdom tooth extraction has been determined. The bone samples were powdered under liquid nitrogen before being extracted with EM’A (0.5M). An internal standard of tritiated phylloquinone epoxide was added. The mixture was then extracted twice with chloroform/methanol (3:l v/v) and the combined organic extracts reduced to dryness. The residue was treated with lipase in 0.8M phosphate buffer (pH 8.0) for lhr at 37°C. The reaction mixture was further extracted with hexane before purification on silica Sep-Pak cartridges, normal phase HPLC followed by reverse-phase HPLC coupled to electrochemical detection. All procedures were done under conditions of diffus’e light. After taking into account losses during the extraction process it was found that bone samples from young adults contains less vitamin Kl than previously found in the bone of the elderly. The reasons for the lower levels in the bone of young subjects remains unclear, but may reflect the changing lipid content of ageing bone. Further research is needed to examine the relationship between vitamin K, the lipid content of bone and the synthesis of functional vitamin Kdependent bone proteins.
P29. Measurement of peri-pmathetic bone mineral density pre and poet total hip arthroplaety B Lees, PR AIlen*, JC Stevenson Lynn Institute for Metabolic Research, London hW8 9SQ and *Lewisham Hospital, London SE13 6LH Dual energy X-ray absorptiometry (DXA) allows the quantification of changes in bone mineral density (BMD) following total hip arthroplasty (THA) which cannot be accurately determined by conventional radiography. This has important implications, since bone loss due to stress shielding may be an important factor in the subsequent failure of the implant. In thii prospective study we compared the BMD of the proximal femur of 7 individuals (4 males and 3 females) preTHA and then 6,12,24 and 52 weeks following uncemented THA in the same hip. BMD measurements were made by DXA (Lunar DPX). Four regions of interest 20 mm in height were selected, A and B (lateral sites) which approximate to Gruen zone I, and C and D (medial sites) which approximate to Gruen zone 7. Bone loss compared to pre THA was significant in all regions 24 weeks post THA (regions A, C and D -12.8%, -15.7% and -8.9% respectively ~~0.05, and region B -15.6% p-3.01) and remained reduced at 52 weeks post THA (regions A and B -14.1% and -19.5% respectively, p
P30. Analysis of a truncated mRNA for alkaline phosphatase in two human oateoaarcoma cell lines NN Ali, J Rowe, NM Teich Laboratory of Viral-mediated Cell Differentiaiton, Imperial Cancer Research Fund, London