- Email: [email protected]
A marker for type I collagen degradation identified in urine from patients with Paget’s disease of bone
52S
Clinical aspects: Abstracts
Bone Vol. 24, No. 5, Supplement May 1999:51S–53S
A Marker for Type I Collagen Degradation Identified in Urine From Patients With Paget’s Disease of Bone
A New Radioimmunoassay for C-Terminal Telopeptide of Type I Collagen in the Diagnosis and Follow-Up of Paget’s Disease
S. M. KRANE Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
S. GONNELLI,1 R. PALMIERI,1 C. CEPOLLARO,1 R. MONACO,1 S. PACINI,1 A. MONTAGNANI,1 and C. GENNARI1 1 Institute of Internal Medicine, University of Siena, Siena, Italy
In patients with Paget’s disease, resorption of components of the extracellular matrix may occur at high rates and urinary excretion of hydroxyproline (Hyp)containing peptides may exceed 1 g (7.6 mmol) per day. Analysis of Hypcontaining peptides from Paget’s disease could therefore provide clues as to mechanisms of collagen turnover in bone disease. We reasoned that defined collagen peptides of more than three or four amino acids could be formed and eventually excreted in the urine. We therefore isolated Hyp-containing peptides from urine of patients with severe Paget’s disease (alkaline phosphatase .;1000 IU) by sequential dialysis, Sephadex G-75 chromatography in 1 mol/L CaCl2, and chromatofocusing. Fractions eluting with pI ; 6.2 were resolved by HPLC and peaks collected and amino acid sequencing performed. Several peaks could not be sequenced because of blocked N-termini. One fraction, a major peak isolated by HPLC, was sequenced and comprised a peptide identical to residues 620 – 633 of the collagen a1(I) chain (Ala-Hyp-Gly-Asp-ArgGly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Ala) and was therefore called “peptide/ a1(I)620 – 633.” As described by Ju et al. (J Bone Miner Res 12:S178, 1997), at Metra Biosystems, a KLH-SMCC-Cys N-terminal conjugate of a synthetic peptide with the “peptide/a1(I)620 – 633” sequence was used as immunogen to generate monoclonal antibodies for an ELISA with an alkaline phosphatase-SPDP-Cys conjugate. Intra-assay coefficient of variation was 4%–7%, interassay coefficient of variation was 5%–7%, and mean linearity was 104 6 8% over dilutions of 1:2–1:16. The mean urinary level of peptide/a1(I)620 – 633 in healthy young men and premenopausal women was 30.0 6 17.9 ng/mmol creatinine. The mean level in 30 patients with Paget’s disease was 241.7 with 27 of 30 .60.0 ng/mmol creatinine (vs. normal, p , 0.0001). Measurement by ELISA of the urinary excretion of peptide/ a1(I)620 – 633, first identified in Paget’s disease, is a promising marker for skeletal turnover. Address for correspondence and reprints: Dr. S. M. Krane, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114. PII S8756-3282(99)00054-X
Urinary Crosslinked N-Telopeptide of Type I Collagen and Crosslaps as Markers of Bone Resorption in Patients With Paget’s Disease of Bone S. J. IQBAL, P. WHITTAKER, and T. DAVIES Department of Chemical Pathology, Leicester Royal Infirmary, Leicester, UK Paget’s disease of bone is characterized by focal areas of increased bone remodeling. Total serum alkaline phosphatase (TALP) and urinary hydroxyproline/ creatine (OHP/Cr) have long been used as markers of bone formation and resorption, but these measures lack sensitivity and specificity. We measured urinary cross-linked N-telopeptide of type I collagen (NTx) and the C-terminal peptide of collagen (Crosslaps; CLAPs) as markers of bone resorption. Twenty-one patients, age range 46 – 87 years (13 males, 8 females), with X-ray and biochemical evidence of Paget’s disease of bone were studied. On fasting urine samples, CLAPs (micrograms per milliliter) were measured by a competitive enzyme immunoassay using a microtiter plate (using a CIS kit) and NTx (units as bone collagen equivalents, nanometers BCE) measured by a competitive inhibition enzyme-linked immunosorbent assay (ELISA) using the Osteomark kit. The results for TALP, OHP/Cr, NTx, and CLAPs urine measures were measured per millimole of creatinine (median values and range): TALP, 271, IU/L (56 –1114); OHP/Cr, 32 nmol (7–145); NTx, 423 BCE (22– 4199); and CLAPs, 630 mg (15–2319). Correlation coefficients (r) on log-transformed data were: TALP: vs. NTx, 0.86; vs. CLAPs, 0.69, vs. OHP, 0.84. OHP: vs. NTx, 0.80; vs. CLAPs, 0.72; NTx vs. CLAPs, 0.88. p , 0.001 for all comparisons. NTx and CLAPs appear to reflect bone turnover and require further clinical evaluation in patients with Paget’s disease. Address for correspondence and reprints: Dr. S. J. Iqbal, Department of Chemical Pathology, Leicester Royal Infirmary, Leicester LE1 5WW, UK. PII S8756-3282(99)00055-1
Paget’s disease of bone is a focal disorder characterized by high bone turnover. The activity of Paget’s disease is usually followed by total alkaline phosphatase (TALP). Measurement of new more specific markers of bone turnover would be expected to reflect disease activity more accurately, but it is unclear whether this would affect clinical management. The aim of this study was to evaluate the clinical usefulness of a new radioimmunoassay (RIA) method for the determination of C-terminal telopeptide of type 1 collagen (a-Crosslaps). We studied 34 pagetic patients (25 males and 9 females, age 70.1 6 9.4 years) without treatment and 34 age- and gender-matched controls. A subgroup of 20 patients were treated for 3 months with oral clodronate (800 mg/day). Fasting serum samples, 24 h urine specimens, and second voided 2 h urine specimens were collected for each patient. In all, we measured TALP, bone alkaline phosphatase (BALP; Tandem-R Ostase, Hybritech), osteocalcin (OC; Incstar Corp), hydroxyproline (HOP; Organon), and a-Crosslaps with a new RIA method using a monoclonal antibody (Osteometer BioTech). In our laboratory, the intra- and interassay coefficient of variations for a-Crosslaps were 2.9% and 6.9%, respectively. Receiver operator characteristic (ROC) curve analysis showed that, in Paget’s disease, both serum TALP and BALP had the highest accuracy to a similar extent among markers of bone formation and urinary a-Crosslaps had the most discriminatory power of all markers of bone resorption. No significant difference was found between 24 h and 2 h urine specimens for HOP and a-Crosslaps. Baseline values of a-Crosslaps were markedly higher in pagetic patients than in controls (1894.3 6 909.1 mg/mmol and 281.7 6 119.0 mg/mmol, respectively). All markers, except OC, were significantly correlated (p , 0.01) with the extent of the disease as assessed by bone scan. In patients treated for 3 months with clodronate the decrease of a-Crosslaps was more marked than that of HOP (median 259.5% vs. 238.5%; p , 0.05). Our results indicate that a-Crosslaps measured by this new RIA method may be considered a highly suitable marker of bone resorption to ascertain the extent and activity of Paget’s disease. Address for correspondence and reprints: Dr. S. Gonnelli, Institute of Internal Medicine, University of Siena, 53100 Siena, Italy. PII S8756-3282(99)00056-3
Quantitative Bone Scintigraphy in the Management of Paget’s Disease of Bone
´ TH,1 B. FORNET,2 and G. POO ´ R1 J. DONA 1 National Institute of Rheumatology and Physiotherapy, Hungary and 2 St. Imre Hospital, Hungary Introduction Our purpose was to assess the use of quantitative bone scintigraphy (QBS) in the monitoring of treated patients suffering from Paget’s disease of bone and to evaluate the relationship between biochemical marker of bone turnover and bone scan indices of disease activity.
Methods Ten patients were treated with 100 mg/day intramuscular salmon calcitonin for 1 year, 17 patients with pamidronate infusion for 30 mg/day for 6 days, and 3 patients with oral tiludronate 400 mg/day for 3 months. Serum samples were obtained from 30 patients with Paget’s disease to determine the levels of total alkaline phosphatase (total AP). QBS was performed in all patients and the results expressed as a ratio, obtained by comparing isotope uptake at an affected and an unaffected control site. Reduction in bone pain was assessed using a pain scale. Efficacy and side effects were monitored for a follow up period of up to 1 year.
Results Total AP levels fell to minimum 14.7 6 7.4% in calcitonin-treated patients and to 41.2 6 8.2% in bisphosphonate-treated patients. There was a difference in biochemical response parameters between the two therapy (Mann–Whitney U test, p 5 0.018). Total AP levels correlated significantly with the bone scan score (coefficient of correlation 5 0.515, p 5 0.005). The pain scale score decreased significantly in both treatment of calcitonin (p 5 0.03) and bisphosphonate (p 5 0.0009). Side
Clinical aspects: Abstracts
Bone Vol. 24, No. 5, Supplement May 1999:51S–53S
A Marker for Type I Collagen Degradation Identified in Urine From Patients With Paget’s Disease of Bone
A New Radioimmunoassay for C-Terminal Telopeptide of Type I Collagen in the Diagnosis and Follow-Up of Paget’s Disease
S. M. KRANE Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
S. GONNELLI,1 R. PALMIERI,1 C. CEPOLLARO,1 R. MONACO,1 S. PACINI,1 A. MONTAGNANI,1 and C. GENNARI1 1 Institute of Internal Medicine, University of Siena, Siena, Italy
In patients with Paget’s disease, resorption of components of the extracellular matrix may occur at high rates and urinary excretion of hydroxyproline (Hyp)containing peptides may exceed 1 g (7.6 mmol) per day. Analysis of Hypcontaining peptides from Paget’s disease could therefore provide clues as to mechanisms of collagen turnover in bone disease. We reasoned that defined collagen peptides of more than three or four amino acids could be formed and eventually excreted in the urine. We therefore isolated Hyp-containing peptides from urine of patients with severe Paget’s disease (alkaline phosphatase .;1000 IU) by sequential dialysis, Sephadex G-75 chromatography in 1 mol/L CaCl2, and chromatofocusing. Fractions eluting with pI ; 6.2 were resolved by HPLC and peaks collected and amino acid sequencing performed. Several peaks could not be sequenced because of blocked N-termini. One fraction, a major peak isolated by HPLC, was sequenced and comprised a peptide identical to residues 620 – 633 of the collagen a1(I) chain (Ala-Hyp-Gly-Asp-ArgGly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Ala) and was therefore called “peptide/ a1(I)620 – 633.” As described by Ju et al. (J Bone Miner Res 12:S178, 1997), at Metra Biosystems, a KLH-SMCC-Cys N-terminal conjugate of a synthetic peptide with the “peptide/a1(I)620 – 633” sequence was used as immunogen to generate monoclonal antibodies for an ELISA with an alkaline phosphatase-SPDP-Cys conjugate. Intra-assay coefficient of variation was 4%–7%, interassay coefficient of variation was 5%–7%, and mean linearity was 104 6 8% over dilutions of 1:2–1:16. The mean urinary level of peptide/a1(I)620 – 633 in healthy young men and premenopausal women was 30.0 6 17.9 ng/mmol creatinine. The mean level in 30 patients with Paget’s disease was 241.7 with 27 of 30 .60.0 ng/mmol creatinine (vs. normal, p , 0.0001). Measurement by ELISA of the urinary excretion of peptide/ a1(I)620 – 633, first identified in Paget’s disease, is a promising marker for skeletal turnover. Address for correspondence and reprints: Dr. S. M. Krane, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114. PII S8756-3282(99)00054-X
Urinary Crosslinked N-Telopeptide of Type I Collagen and Crosslaps as Markers of Bone Resorption in Patients With Paget’s Disease of Bone S. J. IQBAL, P. WHITTAKER, and T. DAVIES Department of Chemical Pathology, Leicester Royal Infirmary, Leicester, UK Paget’s disease of bone is characterized by focal areas of increased bone remodeling. Total serum alkaline phosphatase (TALP) and urinary hydroxyproline/ creatine (OHP/Cr) have long been used as markers of bone formation and resorption, but these measures lack sensitivity and specificity. We measured urinary cross-linked N-telopeptide of type I collagen (NTx) and the C-terminal peptide of collagen (Crosslaps; CLAPs) as markers of bone resorption. Twenty-one patients, age range 46 – 87 years (13 males, 8 females), with X-ray and biochemical evidence of Paget’s disease of bone were studied. On fasting urine samples, CLAPs (micrograms per milliliter) were measured by a competitive enzyme immunoassay using a microtiter plate (using a CIS kit) and NTx (units as bone collagen equivalents, nanometers BCE) measured by a competitive inhibition enzyme-linked immunosorbent assay (ELISA) using the Osteomark kit. The results for TALP, OHP/Cr, NTx, and CLAPs urine measures were measured per millimole of creatinine (median values and range): TALP, 271, IU/L (56 –1114); OHP/Cr, 32 nmol (7–145); NTx, 423 BCE (22– 4199); and CLAPs, 630 mg (15–2319). Correlation coefficients (r) on log-transformed data were: TALP: vs. NTx, 0.86; vs. CLAPs, 0.69, vs. OHP, 0.84. OHP: vs. NTx, 0.80; vs. CLAPs, 0.72; NTx vs. CLAPs, 0.88. p , 0.001 for all comparisons. NTx and CLAPs appear to reflect bone turnover and require further clinical evaluation in patients with Paget’s disease. Address for correspondence and reprints: Dr. S. J. Iqbal, Department of Chemical Pathology, Leicester Royal Infirmary, Leicester LE1 5WW, UK. PII S8756-3282(99)00055-1
Paget’s disease of bone is a focal disorder characterized by high bone turnover. The activity of Paget’s disease is usually followed by total alkaline phosphatase (TALP). Measurement of new more specific markers of bone turnover would be expected to reflect disease activity more accurately, but it is unclear whether this would affect clinical management. The aim of this study was to evaluate the clinical usefulness of a new radioimmunoassay (RIA) method for the determination of C-terminal telopeptide of type 1 collagen (a-Crosslaps). We studied 34 pagetic patients (25 males and 9 females, age 70.1 6 9.4 years) without treatment and 34 age- and gender-matched controls. A subgroup of 20 patients were treated for 3 months with oral clodronate (800 mg/day). Fasting serum samples, 24 h urine specimens, and second voided 2 h urine specimens were collected for each patient. In all, we measured TALP, bone alkaline phosphatase (BALP; Tandem-R Ostase, Hybritech), osteocalcin (OC; Incstar Corp), hydroxyproline (HOP; Organon), and a-Crosslaps with a new RIA method using a monoclonal antibody (Osteometer BioTech). In our laboratory, the intra- and interassay coefficient of variations for a-Crosslaps were 2.9% and 6.9%, respectively. Receiver operator characteristic (ROC) curve analysis showed that, in Paget’s disease, both serum TALP and BALP had the highest accuracy to a similar extent among markers of bone formation and urinary a-Crosslaps had the most discriminatory power of all markers of bone resorption. No significant difference was found between 24 h and 2 h urine specimens for HOP and a-Crosslaps. Baseline values of a-Crosslaps were markedly higher in pagetic patients than in controls (1894.3 6 909.1 mg/mmol and 281.7 6 119.0 mg/mmol, respectively). All markers, except OC, were significantly correlated (p , 0.01) with the extent of the disease as assessed by bone scan. In patients treated for 3 months with clodronate the decrease of a-Crosslaps was more marked than that of HOP (median 259.5% vs. 238.5%; p , 0.05). Our results indicate that a-Crosslaps measured by this new RIA method may be considered a highly suitable marker of bone resorption to ascertain the extent and activity of Paget’s disease. Address for correspondence and reprints: Dr. S. Gonnelli, Institute of Internal Medicine, University of Siena, 53100 Siena, Italy. PII S8756-3282(99)00056-3
Quantitative Bone Scintigraphy in the Management of Paget’s Disease of Bone
´ TH,1 B. FORNET,2 and G. POO ´ R1 J. DONA 1 National Institute of Rheumatology and Physiotherapy, Hungary and 2 St. Imre Hospital, Hungary Introduction Our purpose was to assess the use of quantitative bone scintigraphy (QBS) in the monitoring of treated patients suffering from Paget’s disease of bone and to evaluate the relationship between biochemical marker of bone turnover and bone scan indices of disease activity.
Methods Ten patients were treated with 100 mg/day intramuscular salmon calcitonin for 1 year, 17 patients with pamidronate infusion for 30 mg/day for 6 days, and 3 patients with oral tiludronate 400 mg/day for 3 months. Serum samples were obtained from 30 patients with Paget’s disease to determine the levels of total alkaline phosphatase (total AP). QBS was performed in all patients and the results expressed as a ratio, obtained by comparing isotope uptake at an affected and an unaffected control site. Reduction in bone pain was assessed using a pain scale. Efficacy and side effects were monitored for a follow up period of up to 1 year.
Results Total AP levels fell to minimum 14.7 6 7.4% in calcitonin-treated patients and to 41.2 6 8.2% in bisphosphonate-treated patients. There was a difference in biochemical response parameters between the two therapy (Mann–Whitney U test, p 5 0.018). Total AP levels correlated significantly with the bone scan score (coefficient of correlation 5 0.515, p 5 0.005). The pain scale score decreased significantly in both treatment of calcitonin (p 5 0.03) and bisphosphonate (p 5 0.0009). Side