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Keywords:

  • type I collagen;
  • isomerization;
  • racemization;
  • fracture risk;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The Asp1211 residue of the1209AHDGGR1214 sequence of the C-terminal cross-linking telopeptide of type I collagen (CTX) can undergo spontaneous post-translational modifications, namely, racemization and isomerization, which result in the formation of four isomers: the native form (α-L) and three age-related forms, that is, an isomerized form (β-L), a racemized form (α-D), and an isomerized/racemized (β-D) form. Previous studies have suggested that changes in the pattern of type I collagen racemization/isomerization, which can be assessed in vivo by measuring the degradation products of the CTX isoforms, may be associated with alterations of bone structure. The aim of this study was to examine prospectively the value of the different urinary CTX isoforms and their related ratio in the prediction of osteoporotic fractures in 408 healthy untreated postmenopausal women aged 50-89 years (mean, 64 years) who were part of the OFELY cohort. During a median 6.8 years follow-up, 16 incident vertebral fractures and 55 peripheral fractures were recorded in 65 women. The baseline levels of the four CTX isoforms in women who subsequently had a fracture were compared with those of the 343 women who did not fracture. At baseline, women with fractures had increased levels of ratios of native α-L-CTX to age-related isoforms (β-L, α-D, and β-D) compared with controls (p < 0.01). In logistic regression analysis after adjustment for age, prevalent fractures, and physical activity, women with levels of α-L/β-L, α-L/α-D, and α-L/β-D-CTX ratios in the highest quartile had a 1.5- to 2-fold increased risk of fractures compared with women with levels in the three lowest quartiles with relative risk (RR) and 95% CI of 2.0 (1.2-3.5), 1.8 (1.02-2.7), and 1.5 (0.9-2.7), respectively. Adjustment of α-L/β-L and α-L/α-D-CTX ratios by the level of bone turnover assessed by serum bone alkaline phosphatase (ALP)- or femoral neck bone mineral density (BMD) decreased slightly the RR, which remained significant for the α-L/β-L-CTX ratio (RR [95%] CI, 1.8 [1.1-3.2] after adjustment for bone ALP, 1.8 [1.03-3.1] after adjustment for BMD, and 1.7 [0.95-2.9] after adjustment for both bone ALP and BMD). Women with both high α-L/β-L-CTX ratio and high bone ALP had a 50% higher risk of fracture than women with either one of these two risk factors. Similarly, women with both increased CTX ratio and low femoral neck BMD (T score < −2.5) had a higher risk of fracture with an RR (95% CI) of 4.5 (2.0-10.1). In conclusion, increased urinary ratio between native and age-related forms of CTX, reflecting decreased degree of type I collagen racemization/isomerization, is associated with increased fracture risk independently of BMD and partly of bone turnover rate. This suggests that alterations of type I collagen isomerization/racemization that can be detected by changes in urinary CTX ratios may be associated with increased skeletal fragility.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BONE STRENGTH depends on different components such as bone mineral density (BMD), but also other factors such as bone size, cancellous architecture, and bone quality, including the structure and orientation of type I collagen.(1) Currently, however, there is no validated noninvasive technique for assessing bone quality.

Modifications of type I collagen structure are likely to be involved in decreased bone strength associated with osteoporosis.(1) Indeed, several biochemical studies performed on bone specimens taken from patients with osteoporosis have shown abnormalities in post-translational modifications of type I collagen molecules. These, which result from enzymatic processes, include an overhydroxylation of lysine residues, an overglycosylation of hydroxylysine, and a reduction in the concentration of nonreducible cross-links that can be associated with decreased bone strength.(2–4) More recently, new nonenzymatic post-translational modifications of type I collagen, so-called racemization and isomerization, have been shown to occur in bone tissue and more specifically involve the aspartic acid residue in position 19 of the C-telopeptide of the α1-chain.(5,6) When type I collagen molecules are synthesized, the aspartic acid residue is in the native L-enantiomeric form and the peptide bound between aspartic acid and glycine residues involves the carboxyl group, which is in position α. During the aging process of type I collagen in the extracellular matrix, there are two types of spontaneous transformations. The racemization of the L-enantiomeric form to the biologically rare D-form and the isomerization, which is the transfer of the peptide backbone from the carboxyl group in position α to the side chain carboxyl group in position β (Fig. 1). Such transformations occur through an intermediate unstable succinimide ring. The hydrolysis of this intermediate generates four different isoforms: the native peptide isoform, so-called α-L C-terminal cross-linking telopeptide of type I collagen (CTX), which reflects newly synthesized collagen, and three age-related isoforms, the isomerized β-L-CTX, the racemized α-D-CTX, and the isomerized and racemized form β-D-CTX. These age-related spontaneous transformations can be detected in vivo by measuring the urinary excretion of the corresponding CTX using specific antibodies.(6)

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Figure FIG. 1.. Pathway of racemization and isomerization of aspartic acid residue in type I collagen α1-C-telopeptide (CTX). The attack by the peptide backbone nitrogen of the side chain carbonyl group of an adjacent aspartic residue can result in the formation of a succinimide ring (A [RIGHTWARDS ARROW] B). The spontaneous hydrolysis of the succinimide produces either (A and D) peptides or (C and F) β-Asp peptides. The formation of D-Asp occurs primarily via the succinimide intermediates (B and E). Others pathways (A [RIGHTWARDS ARROW] D and C [RIGHTWARDS ARROW] F) also may contribute to racemization. The figures in parentheses represent the theoretical proportion of each of the CTX isoforms at equilibrium.

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We have shown previously that in patients with Paget's disease of bone there is a larger increase of urinary α-L-CTX, compared with β-L-CTX, resulting in a ratio α-L/β-L-CTX, which was 3-fold higher than in controls.(7) These data suggested that type I collagen within the abnormal woven pagetic bone, which is characterized by reduced strength, contains a lower degree of type I collagen isomerization than normal bone, as indicated by a higher urinary α-L/β-L-CTX ratio. These findings have been confirmed by an immunohistochemistry analysis of pagetic and normal bone tissue.(7) Recently, in a small retrospective cross-sectional study, Hoshino et al.(8) reported that in patients with hip fractures there was a larger increase over controls in urinary α-L-CTX compared with β-L-CTX, leading to an increased urinary α-L/β-L-CTX ratio, suggesting that the degree of type I collagen isomerization also could be altered in patients with osteoporosis. However, it is difficult from retrospective studies to determine if differences in the levels of these biochemical markers are related to the underlying mechanisms leading to fracture or to changes resulting from fracture healing and/or immobilization occurring after the fracture.

To test the hypothesis that the degree of isomerization/racemization of type I collagen is related to the risk of osteoporotic fractures, we compared baseline urinary concentrations of the different CTX isoforms in women who subsequently sustained a fracture with women who did not fracture during the follow-up. All women belonged to a population-based cohort of postmenopausal women with a wide age range of 50-89 years.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Subjects

Four hundred and eight healthy female volunteers who had been enrolled in a study of the determinants of bone loss (OFELY study) were included. The cohort of this study comprises 1039 women, 31-89 years of age, randomly selected from the regional section of a health insurance company (Mutuelle Générale de l'Education Nationale). Among the 672 postmenopausal women (menopause was defined as an absence of menses for at least 12 months) recruited at baseline, 264 women were excluded subsequently because of the presence of treatment or diseases that could influence bone metabolism. These included 189 women who had received hormone replacement therapy in the past 12 months, 7 patients on tamoxifen, 11 patients taking fluoride, 16 women receiving bisphosphonates, 4 women treated with calcitonin, 24 women receiving thyroid hormones, 5 women taking corticosteroid, and 8 women who had a disease known to affect bone metabolism (Paget's disease of bone, primary hyperparathyroidism, hyperthyroidism, or cirrhosis). The OFELY cohort has been described elsewhere.(9,10)

Identification of fractures

At baseline, women were submitted to a detailed questionnaire including history of fragility fractures, that is, fractures resulting from a low trauma since the age of 45 years; physical activity; calcium intake; and smoking habits. Past and present physical activity was registered by questionnaire. The monthly hours of physical exercise, the daily walking distance and number of stairs climbed, the weekly hours of home work, and the physical demands of professional work (setting, light, medium, and heavy) were recorded and used to calculate an individual physical activity score. Dietary calcium intake was assessed by a sequential self-questionnaire. Subsequently, each woman was seen every year for a maximum of 7 years (mean [SD], 5.9 (2) years). At each visit, the occurrence of fractures within the previous year was registered.

Lateral X-ray films of the thoracic and lumbar spine were obtained at baseline in all women and at follow-up for 79% of them after an average of 3.8 years (2.8-5.3 years). All vertebral prevalent and incident fractures were identified by semiquantitative morphometry by two individuals who were unaware of the baseline assay results. A vertebra was classified as having a prevalent fracture on the baseline radiograph if any vertical height (anterior, middle, and/or posterior) was reduced by >20%. A new fracture was defined by a decrease of 20% or more and of at least 4 mm in any vertebral height of one or more thoracic or lumbar vertebrae between follow-up and baseline X-ray films.(11,12)

Nonvertebral and symptomatic vertebral fractures were recorded throughout the study in all women including after the follow-up X-ray and by annual mail in women who did not come back to the follow-up visit. All fractures were confirmed by radiographs.

Measurements of urinary CTX isoforms

For each woman, total 24-h urinary excretion was collected at baseline without any preservative for measuring urinary CTX isoforms. Urine samples were stored frozen at −70°C until assayed.

Assay for urinary α-L-CTX

Urinary native nonisomerized and nonracemized-CTX (α-L-CTX) was measured with a two-site ELISA using a monoclonal antibody (MabF44) raised against the linear α-L-EKAHDGGR peptide, a sequence specific for a part of the C-telopeptide of α1-chain of human type I collagen. The intra- and interassay CV are <4.5% and 11%, respectively.(6)

Assays for urinary β-L-CTX

Urinary isomerized nonracemized β-L-isomerized CTX (β-L-CTX) was measured by an ELISA (CrossLaps ELISA; Osteometer Biotech A/S AS, Herlev, Denmark).(13) This assay uses a polyclonal antiserum raised against the β-L-EKAH DGGR sequence. The intra- and interassay CVs are <5% and 8%, respectively.

Assay for urinary α-D-CTX

Urinary racemized α-D-CTX was measured by a competitive ELISA based on the use of a polyclonal antibody raised against the α-D-EKAH DGGR sequence as previously described.(6) The intra- and interassay CVs are <9% and 10%, respectively.(6)

Assay for urinary β-D-CTX

Urinary isomerized and racemized β-CTX (β-D-CTX) was measured by a competitive ELISA based on the use of a polyclonal antibody raised against the β-D-EKAH DGGR sequence as previously described.(6) The intra- and interassay CVs are <8% and 11%, respectively.(6)

Using a mixture of the four AHDGGR isoforms peptides (α-L, β-L, α-D, and β-D) it has been shown that the cross-reactivity of each immunoassays toward the respective “nonreactive” isoforms was below 1.2% for all assays.(6)

Urinary CTX data were corrected by the urinary creatinine (Cr) concentration measured by a standard colorimetric method.

Assay for serum bone alkaline phosphatase

Bone alkaline phosphatase (ALP) was measured with a human specific immunoradiometric assay using two monoclonal antibodies raised against the human bone ALP isoenzyme and bone ALP purified from SAOS-2 osteosarcoma cells as a standard (Ostase; Beckman-Coulter, San Diego, CA, USA).(14) The intra- and interassay CVs are lower than 10%.(14)

BMD

BMD of the femoral neck was measured by dual-energy X-ray absorptiometry (DXA) on a QDR 2000 device (Hologic, Inc., Waltham, MA, USA) with a short-term CV of 0.6%.(10)

Statistical analyses

Comparisons of baseline characteristics between fracture group and control group were assessed by unpaired student t-tests after logarithmic transformation of urinary CTX data. The relationships between baseline levels of CTX isoforms, related CTX ratios, and fracture incidence were analyzed by logistic regression analysis after adjustment for potential confounding variables such as age, prevalent fractures, and physical activity. Because the risk is relative to those without the risk factor rather than the risk relative to the general population, we also calculated adjusted relative risk (RR) as suggested by Kanis et al.(15) The adjusted RR is calculated as [RR/(pRR +[1 − p])], where RR is the unadjusted RR and p is the prevalence of the risk factors in the studied population. Correlation between urinary CTX ratios, bone ALP, and BMD was assessed by Pearson correlation. All statistical analyses were carried out using SAS (SAS Institute, Inc., Cary, NC, USA).(16)

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

During follow-up, 16 vertebral fractures and 55 peripheral fragility fractures (16 wrist, 9 hip, 7 rib, 9 ankle, 3 patella, 3 humerus, 3 metatarsi, 1 pelvis, 1 sacrum, 1 elbow, 1 clavicle, and 1 knee) were recorded in 65 women. Women who had fractures during the study were older, had a slightly lower physical activity, and had a lower femoral neck BMD (Table 1). As expected, the proportion of women with prevalent fragility fractures also was higher among women who fractured during the study (Table 1).

Table Table 1.. Baseline Characteristics of Women With Incident Osteoporotic Fractures and Controls
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Baseline levels of urinary CTX isoforms and the corresponding ratios are shown in Table 2. Women who had osteoporotic fractures during follow-up had significantly higher urinary α-L-CTX levels than the controls. In contrast, no significant differences were observed for the age-related CTX isoforms (Table 2). The ratios between the native α-L-CTX and the age-related isoforms (β-L, α-D, and β-D) were significantly increased in women who had fracture during follow-up compared with controls.

Table Table 2.. Baseline Levels of Urinary CTX Values and Ratios of Women With Incident Osteoporotic Fractures and Controls
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After adjustment for age, prevalent fractures, and physical activity, women with levels of urinary CTX isoforms in the highest quartile had a greater risk of osteoporotic fracture than women who had lower values, with an increased risk of 1.2-2.0 according to the CTX isoform (Table 3). Women with baseline values of α-L/β-L-CTX or α-L/α-D-CTX ratio in the highest quartile had a significant RR of fracture of 2.0 and 1.8, respectively, compared with the rest of the population. Increased urinary α-L/β-D-CTX ratio was associated also with an increased risk of fracture, although the RR did not reach significance.

Table Table 3.. Adjusted Association Between Increased Urinary CTX Levels and Ratios and the Risk of Osteoporotic Fracture in Postmenopausal Womena
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At baseline, the different urinary ratios between native and age-related CTX modestly correlated with bone turnover rate as assessed with bone ALP (Table 4). Thus, a higher bone turnover rate was associated with increased CTX ratios, that is, with a lower rate of post-translational modification of CTX. Similarly, increased ratios—except α-L/β-L-CTX—were slightly associated with decreased femoral neck BMD, with r values ranging from −0.06 to −0.25 (Table 4). To investigate whether the association between increased CTX ratios and the risk of osteoporotic fractures was mediated by the correlation between CTX ratios and bone turnover and/or BMD, adjustment for serum bone ALP and femoral neck BMD was performed. After adjustment for serum bone ALP or femoral neck BMD, RRs decreased only marginally and remained significant for α-L/β-L-CTX ratio (Table 5). After adjustment for both bone turnover and BMD, increased α-L/β-L-CTX ratio was still associated with increased fracture risk, which was, however, not significant (RR (95% CI), 1.7 [0.95-2.9]). Women with both high bone ALP and high α-L/β-L-CTX ratio had a higher risk than women with either bone ALP or high α-L/β-L-CTX ratio with a 6-year probability of fracture, which increased by 18% compared with one risk factor alone. Women with high α-L/β-L-CTX ratio and low femoral neck BMD (T score < −2.5) had increased risk of fracture with prevalence-adjusted RR of 3.9 (Table 6) and a 6-year probability of fracture of 56% (Table 6). Combining serum bone ALP levels, α-L/β-L ratio and BMD did not improve the RR compared with that obtained when combining two risk factors, although the very low number of women (2.5%) in that category rendered the estimation very unstable.

Table Table 4.. Correlation Between Urinary CTX Ratios, Bone Turnover Markers, and Bone Mineral Density in 408 Postmenopausal Women
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Table Table 5.. Adjusted Association Between Increased Urinary CTX Ratios and the Risk of Osteoporotic Fractures in Multivariate Modelsa
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Table Table 6.. Combination of Risk Factors to Predict the Risk of Vertebral and Nonvertebral Fractures in Postmenopausal Women
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When we restricted the analysis to women who sustained only nonvertebral fractures (n = 55), we found that increased α-L/β-L- and α-L/α-D-CTX ratios were significantly associated with an increased risk of fracture with RR of 2.5 and 1.9, respectively (Table 5). Adjustment for bone ALP levels, femoral neck BMD, or both only marginally decreased the RRs, which remained significant for α-L/β-L-CTX ratio (Table 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This is the first study analyzing the relationship between the urinary excretion of different age-related isoforms of type I collagen degradation products and fracture risk. Using these new biochemical parameters, we confirmed on a longer follow-up period that increased bone resorption rate assessed by the total urinary excretion of CTX was associated with increased fracture risk of all types of osteoporotic fractures independently of the level of BMD in a large cohort of healthy untreated postmenopausal women from 50 to 89 years of age. More importantly, we found that an increased urinary ratio between native (α-L) and age-related modified forms of CTX was associated with increased fracture risk independently of BMD and partly of bone turnover rate, suggesting that this new parameter could represent an additional risk factor for the risk of fracture.

We found that the urinary excretion of native α-L-CTX was more predictive of fracture risk than the age-related isoforms (β-L, α-D, and β-D) of type I collagen. The accumulation of the age-related CTX isoforms in bone matrix depends on both the inversion rate of the different isoforms (Fig. 1) and the rate of bone turnover.(6) This indicates that for a given inversion rate, a higher bone turnover rate will be associated with lower levels of age-related CTX isoforms and higher levels of native α-L-CTX, a concept that is in agreement with the positive correlation we found between the urinary ratios of native α-L-CTX on age-related isoforms and serum bone ALP. Thus, the measurement of native α-L-CTX may be more sensitive to detect small increases of bone turnover, which have been shown to be associated with increased risk of fracture.(17–20)

We found that not only an overall increase of bone resorption, which can be assessed by the total urinary excretion of the different CTX isoforms, but also changes in the degree of racemization and isomerization of type I collagen could be associated with increased fracture risk. Indeed, women with baseline values of α-L/β-L (or α-D-CTX) in the highest quartile of the population had about a 2-fold higher risk of fracture than the other women and a 6-year probability of fracture, which was increased over the population risk by 38%. This risk is of the same order of magnitude as those reported for levels of bone resorption in the highest quartile and for each SD decrease in BMD, suggesting that this new parameter represents a potential important risk factor for osteoporotic fractures. One important issue is whether the association between increased urinary CTX ratios and fracture risk is mediated by the correlation of CTX ratio with bone turnover as discussed previously. Although the correlation was significant, bone turnover as assessed by serum bone ALP explained <7% of the interindividual variability of the α-L/β-L-CTX ratio, suggesting that this ratio reflects at least in part biological processes different from those captured by conventional biochemical indices of overall bone turnover rate. This hypothesis was supported by the fact that when CTX ratios were adjusted for the overall level of bone turnover as assessed by serum bone ALP, the RR of fracture decreased only slightly (from 2.0 to 1.8) and remained significant for the α-L/β-L-CTX ratio. In addition, women with increased values of both α-L/β-L-CTX ratio and bone ALP had a 50% higher risk of fracture (RR adjusted for prevalence increasing from 1.6 to 2.1) than women with either high CTX ratio or high bone turnover rate. However, this increase in the risk is lower than that expected if the two risk factors were completely independent (+80%), suggesting that part of the relationships between increased CTX ratio and fracture risk is mediated through changes in bone turnover. After adjustment of CTX ratios by femoral neck BMD, the RR decreased only slightly and women with both α-L/β-L-CTX ratio in the highest quartile and BMD of 2.5 SD below the mean of young adults had a 170% higher risk than women with either increased CTX ratio or low BMD, in agreement with a complete independence of these two risk factors. Thus, altogether these data suggest that urinary ratios between native and age-related CTX isoforms may represent a new risk factor for osteoporotic fracture, which is independent of BMD and partly independent of bone turnover rate.

These findings raised the question of the potential mechanisms underlying the association between changes in CTX ratios and fracture risk. It has been shown a close relationships between CTX ratios assessed directly in bone specimens and in matched urine samples(5–7) indicating that the measurement of urinary CTX reflects adequately the degree of racemization and isomerization of type I collagen in bone tissue. Bone strength is determined by the relative amounts and properties of the mineral (apatite) and organic matrix (mostly type I collagen), as well as the organization of the bone at both the microscopic and the macroscopic level (microarchitecture and anatomy, respectively). The involvement of BMD in bone strength is well established, although the correlation between these two parameters is not perfect.(21) Conversely, the existence of an association between type I collagen structure and bone strength has been well documented in osteogenesis imperfecta (OI) and other clinical syndromes attributable to mutations of type I collagen genes encoding for both α1- and α2-chains.(22–24) Although most OI patients have subnormal BMD, some have decreased bone strength but normal BMD,(25) providing evidence that type I collagen structure may be related to changes in mechanical strength independently of BMD.

There is some evidence that racemization and isomerization may induce changes in the structure, properties, and function of affected proteins. Isomerization and racemization introduces a kink in the peptide backbone and racemization reorients the angles of the peptide bonds and the α-carbon substituent. Such structural alterations will affect the electrostatic interactions responsible for protein conformation, that is, hydrogen bonding and the dipole moments of the peptide chain,(26) which may alter the conformation of the whole protein. Isomerization and racemization processes also have been suggested to be involved in the pathogenesis of degenerative diseases. For example, racemization and isomerization of aspartic acid residues of the β-amyloid protein have been shown to affect the conformation of this protein leading to a decreased sensitivity to proteolytic degradation and, consequently, accumulation of this protein in plaque cores of the brain of patients with Alzheimer disease.(27,28) However, because racemization affects the Asp-Gly sequence of the C-telopeptide and collagen fibrils are formed shortly after synthesis probably before racemization has occurred, it is unlikely that this post-translational modification by itself will induce important changes of structure and/or alignment of type I collagen molecules. Thus, the precise biological impact of this post-translational modification remains unclear and further experimental studies should explore the impact of these collagen modifications on skeletal strength.

Our study has some limitations. We may not have identified all vertebral fractures, because spine X-ray evaluation was performed only once after 3.8 years in 79% of the subjects. However, all symptomatic vertebral and nonvertebral fractures were recorded and the presence of some nonsymptomatic vertebral fractures in the control group would have resulted in an underestimation of the association between increased CTX ratios and fracture risk. The follow-up was different for vertebral and nonvertebral fractures, although when the analyses were restricted to nonvertebral fractures, similar results were obtained. Finally, we presented an association between increased urinary CTX ratio and increased risk of fragility fractures, but did not provide data on the mechanisms underlying this relationship.

In summary, we found that increased urinary ratios between native and age-related forms of type I collagen CTX are associated with increased fracture risk independently of BMD and partly in dependent of bone turnover rate. This suggests that a decreased degree of bone type I collagen isomerization/racemization, which can be assessed noninvasively by urinary biochemical markers may be associated with alterations of bone strength properties. This hypothesis requires confirmation by studies correlating the degree of type I collagen racemization/isomerization with mechanical properties of bone specimens.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was supported in part by a contract INSERM-MSD Chibret (the OFELY Study).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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