Fourier transform Infrared spectroscopic characterization of mineralizing type I collagen enzymatic trivalent cross-links.

The most abundant protein of bone's organic matrix is collagen. One of its most important properties is its cross-linking pattern, which is responsible for the fibrillar matrices' mechanical properties such as tensile strength and viscoelasticity. We have previously described a spectroscopic method based on the resolution of the Amide I and II Fourier transform Infrared (FTIR) bands to their underlying constituent peaks, which allows the determination of divalent and pyridinoline (PYD) collagen cross-links in mineralized thin bone tissue sections with a spatial resolution of ~6.3 µm. In the present study, we used FTIR analysis of a series of biochemically characterized collagen peptides, as well as skin, dentin, and predentin, to examine the potential reasons underlying discrepancies between two different analytical methodologies specifically related to spectral processing. The results identified a novel distinct FTIR underlying peak at ~1,680 cm(-1), correlated with deoxypyridinoline (DPD) content. Furthermore, the two different methods of spectral resolution result in widely different results, while only the method employing well-established spectroscopic routines for spectral resolution provided biologically relevant results, confirming our earlier studies relating the area of the underlying 1,660 cm(-1) with PYD content. The results of the present study describe a new peak that may be used to determine DPD content, confirm our earlier report relating spectroscopic parameters to PYD content, and highlight the importance of the selected spectral resolution methodology.

A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality.

Osteoporosis is a common disease with a strong genetic component. We previously described a polymorphic Sp1 binding site in the COL1A1 gene that has been associated with osteoporosis in several populations. Here we explore the molecular mechanisms underlying this association. A meta-analysis showed significant associations between COL1A1 "s" alleles and bone mineral density (BMD), body mass index (BMI), and osteoporotic fractures. The association with fracture was stronger than expected on the basis of the observed differences in BMD and BMI, suggesting an additional effect on bone strength. Gel shift assays showed increased binding affinity of the "s" allele for Sp1 protein, and primary RNA transcripts derived from the "s" allele were approximately three times more abundant than "S" allele--derived transcripts in "Ss" heterozygotes. Collagen produced from osteoblasts cultured from "Ss" heterozygotes had an increased ratio of alpha 1(I) protein relative to alpha 2(I), and this was accompanied by an increased ratio of COL1A1 mRNA relative to COL1A2. Finally, the yield strength of bone derived from "Ss" individuals was reduced when compared with bone derived from "SS" subjects. We conclude that the COL1A1 Sp1 polymorphism is a functional genetic variant that predisposes to osteoporosis by complex mechanisms involving changes in bone mass and bone quality.

Immunolocalization of extracellular matrix proteins during brain tumor invasion in BD IX rats.

The distribution of several native extracellular matrix proteins (type I, III, and IV collagens and fibronectin) using immunofluorescent localization is described for in two different malignant gliomas (BT4A and BT4An). In addition, antibodies against denatured forms of type I and III collagens were used to localize areas of active degradation within the tumors. We have shown that both tumors express the native connective tissue components studied, although the distribution of these components within and between the tumors was different. In addition, native type I and III collagens and fibronectin were overexpressed in the tumors compared to the normal brain. Morphometry on immunostained type IV collagen sections showed an increase in vascular elements in both tumors compared to normal brain tissue. The BT4A tumor, which by light microscopy showed a degradative mode of invasion, expressed denatured type I and III collagens at the tumor-brain border zone, suggesting that this tumor has collagenolytic activity. The present article suggests that the distribution and changes in extracellular matrix protein synthesis and degradation may play an important role in the progressive growth of brain tumors in vivo.

Pyridinium crosslinks of bone collagen and their location in peptides isolated from rat femur.

The relative proportions of pyridinoline and deoxypyridinoline in bone showed large species variations, although the total number of pyridinium crosslinks in rat, rabbit and bovine bone collagen was only 25-30% of that found in articular cartilage. Three pyridinium-containing peptides were isolated from cyanogen bromide digests of rat femoral bone and were characterized by their Mr values and amino-acid compositions. The results showed that pyridinoline and its deoxy analogue were equally distributed at two locations stabilizing the 4D stagger through interactions involving both the N- and C-terminal telopeptide regions. Less than stoichiometric amounts of pyridinium crosslinks were present in the peptides, suggesting that the isolated peptides contained additional (unidentified) maturation products of the bifunctional, reducible crosslinks.

Some observations on the ageing in vitro of reprecipitated collagen fibres.

The changes in solubility and amounts of reducible cross-links have been studied during "ageing" in vitro of reprecipitated rat skin collagen fibres by incubation at 37 degrees C. Fibres from pre-reduced collagen devoid of aldehyde precursors became insoluble at the same rate as that of normal fibres during "ageing". Insolubilization occurred at a much faster rate in the presence of oxygen than in air and was almost completely inhibited when oxygen was excluded. The rate of decline of the reducible cross-links was, however, unaffected by oxygen tension. The results indicate that, during "ageing" in vitro, conversion of the lysine-derived cross-links to a non-reducible form is not associated with solubility changes. The relationship of these in vitro changes to those ocurring in vivo is unknown.

Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate.

In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxypyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur.

Preparation and analysis of the products of non-enzymatic protein glycosylation and their relationship to cross-linking of proteins.

The presence of glycosylated protein-bound lysine residues has led to much speculation regarding changes in structure and function of the modified protein. The synthesis of hexose-lysine adducts and their separation using an amino acid analyser is described. These compounds are also produced during borohydride reduction and subsequent hydrolysis of modified proteins, and misidentification of these may occur depending upon precise chromatographic procedures. The possibility that glucose might participate in a cross-linking reaction between two protein-bound lysine residues was tested but no evidence for such a mechanism was found. The presence of 14C-labelled urinary hexosyllysine indicated that body protein breakdown in addition to ingested dietary hexosyllysine contributes to the excretion of this component.

Reducible components in the proteins of human erythrocyte membrane.

In contrast to a previous report, no collagen or elastin-type cross-linked derived from lysine-aldehydes were detected in human erythrocyte membranes. The major reducible components of erythrocyte membranes were shown to be hexosyllysines. From their structure it is clear that these components cannot act as cross-links between the protein subunits of the membrane. The components were also shown to be present in varying proportions in human serum albumin and haemoglobin. Whether the hexose attachments have any physiological significance or are artefacts of the analytical procedure has not yet been demonstrated. One other major reducible component was present but, although unidentified, this compound was shown to be unrelated to any of the known lysine-aldehyde-derived cross-links of collagen and elastin. A minor acidic component was identified as glucosylvaline derived from the N-terminus of the beta chain of haemoglobin A1c and not a lysine-aldehyde precursor of the collagen cross-links.

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.

Urinary pyridinium crosslinks of collagen: specific markers of bone resorption in metabolic bone disease.

The hydroxypyridinium compounds pyridinoline and deoxypyridinoline are specific constituents of mature skeletal collagens. They are released into the circulation and excreted in the urine. Their measurement in urine is a sensitive index of the extent of ongoing bone resorption. Currently, quantification of collagen crosslinks in urine is achieved by chromatographic techniques, but more convenient immunoassays will make these measurements more widely available in the near future. Clinical applications of hydroxypyridinium markers include numerous metabolic bone disorders such as osteoporosis, primary hyperparathyroidism, Paget's disease of bone, and metastatic bone disease. Urinary pyridinium crosslinks of collagen also show great promise as markers of therapeutic efficacy in bone disorders associated with accelerated bone resorption.

Biochemical investigation of possible lesions in human aorta that predispose to dissecting aneurysms: pyridinoline crosslinks.

Pyridinoline, a collagen specific covalent crosslink, was quantified in acid hydrolysates of human aorta using a non-equilibrium inhibition ELISA. The study was based on specimens from seven cases of aortic dissection and from seven control subjects whose death was unrelated to thoracic aortic dissection. There were no significant differences in the amounts or concentrations of pyridinoline in aortas with dissecting aneurysms compared with normal tissue, thus excluding the possibility of a causative relation between the degree of pyridinoline crosslinking of collagen molecules and dissection of the thoracic aorta. In all cases, however, the number of pyridinoline crosslinks per molecule of collagen in the ascending aorta and arch approached the theoretical maximum for lysyl derivatives and was as high as that present in cartilage. Thus in this region of the vessel pyridinoline represents the major stabilising crosslink of collagen. In contrast, the number of pyridinoline crosslinks per collagen molecule decreased maximally by a factor of 10 between the arch and the proximal regions of the descending thoracic aorta. This suggests a possible correlation between the rigidity of collagen fibres and the forces exerted on the aortic wall during diastole and systole.

Caspase-dependent cleavage of cadherins and catenins during osteoblast apoptosis.

As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of alpha-, beta-, and gamma-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and alpha-catenin, with either beta- or gamma-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and beta- and gamma- catenins but not alpha-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of beta-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of beta-catenin signaling.

Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption.

Several studies in recent years have shown that the pyridinium crosslinks of collagen provide good urinary markers of collagen degradation, primarily reflecting bone resorption. Most studies, however, were based on time-consuming HPLC assays of the crosslinks. We now describe the development of an immunoassay (ELISA) based on a monoclonal antibody for free deoxypyridinoline (Dpd) and its use in healthy individuals and patients with bone-related disorders to measure the urinary excretion of Dpd as an improved assessment of bone resorption rate. The Dpd antibody exhibited less than 1% cross-reaction with free pyridinoline and was shown to react only with free Dpd in urine, having no significant interaction with peptide forms of the crosslinks. The intra- and interassay variations were less than 10 and 15%, respectively. A total of 402 urine samples from patients and healthy volunteers were analyzed by both the immunoassay and HPLC. The ELISA results were highly correlated with those for total Dpd measured by HPLC over the full range of sample groups (r = 0.95). In normal adults, the excretion of Dpd (mean +/- SD) was 4.7 +/- 1.6 nmol/mmol creatinine, with about fivefold higher excretion rates in children. For 31 osteoporotic patients, the ELISA Dpd values (median 6.7; range 3.0-13.5 nmol/mmol Cr) were significantly higher (p < 0.0001) than the corresponding values for age- and sex-matched controls (median 4.0; range 1.8-7.4). The difference between the groups was similar for total Dpd by HPLC (osteoporotic: mean 12.8, range 4.8-30.7; controls: 6.6, range 3.0-18.1; p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary hydroxypyridinium crosslinks of collagen in population-based screening for overt vertebral osteoporosis: results of a pilot study.

The urinary pyridinium crosslinks pyridinoline (PYD) and deoxypyridinoline (DPD) have been shown to provide valid indices of bone resorption. At present, both crosslink components are determined by reversed-phase HPLC, a time-consuming method precluding the use of these markers for routine purposes. Therefore, efforts have been made to develop simple immunoassays for the rapid measurement of urinary crosslinks, and their application to large-scale osteoporosis screening has been proposed. To evaluate the applicability and diagnostic validity of pyridinium crosslink measurements for screening purposes, urinary concentrations of total and free PYD and DPD were determined by HPLC and immunoassay technique (ELISA) in a sample of 269 individuals (male to female ratio = 130:139; age 50-81 years) recruited at random within a population survey of vertebral osteoporosis. On a molar basis, ELISA measures of crosslink-related epitopes were highly correlated with both total and free PYD and DPD as determined by HPLC (r > 0.82, p < 0.001). Age-specific means for creatinine-corrected total and free pyridinium crosslinks were significantly higher in females than in males (p < 0.001). In both sexes, neither age nor anthropometric variables (weight, height, and body mass index) showed a linear effect on the urinary crosslink/creatinine ratio. On average, 50% of the total amount of urinary crosslinks were present in free form. For both PYD and DPD, this proportion was significantly higher in women than in men (p < 0.05), but no change was observed with age or anthropometric measures. The excretion of pyridinium crosslinks was higher in osteoporotic (n = 18) than in nonosteoporotic individuals (n = 208) from the same population.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary hydroxypyridinium cross-links of collagen in primary hyperparathyroidism.

Urinary concentrations of the collagen cross-links, pyridinoline (PYD) and deoxypyridinoline (DPD), were determined in 87 patients with untreated or surgically treated primary hyperparathyroidism (PHPT). Eighty-four healthy individuals, matched for age and sex, constituted the control group for the excretion of pyridinium cross-links. In addition, a subgroup of 25 patients with PHPT was followed longitudinally for up to 2 yr after successful parathyroidectomy. Mean urinary excretion of PYD (46.8 +/- 2.7 nmol/mmol creatinine) and DPD (17.6 +/- 1.3 nmol/mmol creatinine) was significantly higher in patients with untreated PHPT than in normal subjects (P less than 0.001). In the group undergoing successful parathyroidectomy, mean urinary concentrations of PYD (34 +/- 2.5) and DPD (9.4 +/- 0.8) were similar to those in normal controls and significantly lower than those in the untreated patient population (P less than 0.001). The urinary concentration of both cross-links was significantly correlated with serum levels of both alkaline phosphatase and PTH. Mean urinary concentrations of both cross-link compounds decreased significantly within 6 months in patients followed longitudinally and as early as 2 weeks after surgery in individual patients compared to presurgical baseline values. These changes preceded the reduction in serum alkaline phosphatase and hydroxyproline by approximately 6 months. The results demonstrate that urinary hydroxypyridinium cross-links of collagen are useful indices in the clinical assessment of bone involvement in PHPT.

Role of calcium intake in modulating age-related increases in parathyroid function and bone resorption.

Serum parathyroid hormone (PTH) and bone resorption increase in elderly women and contribute to age-related bone loss. Whether these abnormalities are caused by calcium deficiency resulting from age-related decreases in absorption and renal conservation is unclear. We studied 28 normal elderly women (mean +/- SD, age 69.3 +/- 2.7 yr) who were maintained for 3 yr on usual calcium intake levels (20.4 +/- 7.2 mmol/day [815 +/- 289 mg/day]; n = 15) (known as the usual calcium group) or high calcium intake levels (60.4 +/- 6.5 mmol/day [2414+/260 mg/day]; n = 13) (known as the high calcium group) and a reference group of 12 normal young adult women (age 30.1 +/- 4.4 yr), whose calcium intake was 23.0 +/- 4.8 mmol/day (918 +/- 193 mg/day) (known as the young group). Serum PTH was measured every 2 h, and urinary excretion of deoxypyridinoline (Dpd), a new marker for bone resorption, was measured in 4 h collections. Parathyroid gland secretory capacity was assessed during induced hypocalcemia. The mean 24 h serum PTH was 40% lower (P < 0.001), and the mean 24 h urinary Dpd was 35% lower (P < 0.005) in the high than in the usual calcium group. Mean parathyroid gland secretory capacity also was 47% lower (P < 0.005) in the high calcium group than in the usual calcium group. However, the usual calcium group had a mean 24 h serum PTH level that was 70% higher (P < 0.001) and a mean 24 h urinary Dpd level that was 30% higher (P < 0.005) than the young group, whereas the high calcium group was indistinguishable from the young group. Thus, failure of elderly women to increase their calcium intake to offset age-related increases in calcium requirement contributes substantially to their development of increased parathyroid activity and increased bone resorption, whereas a high calcium intake can reverse both abnormalities.

Measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers.

UNLABELLED: Urinary excretion of the bone collagen derived pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) was measured in 19 patients (4 M:15 F) with untreated thyrotoxicosis, and 20 pre-, and 20 postmenopausal women taking T4 100-200 micrograms daily for autoimmune hypothyroidism. Both PYD and DPD excretion (nanomoles per mmol creatinine) was elevated in the thyrotoxic patients compared to 287 controls; median 131 vs. 26 and 37.5 vs. 7.2, respectively, P less than 0.0001. In premenopausal women mean urinary pyridinium cross-link excretion and serum osteocalcin levels were similar in both T4-treated and matched control groups, despite suppression of serum TSH concentrations to below 0.1 mU/L in 14 of the 20 taking T4. In postmenopausal women mean (+/- 1 SE) urinary PYD excretion (nanomoles per mmol creatinine) was raised in those taking T4, relative to euthyroid controls; 40.0 +/- 2.7 vs. 32.1 +/- 2.3, P less than 0.05. DPD excretion and serum osteocalcin levels were also higher, but not significantly. When only the T4-treated women with a subnormal serum TSH were considered the difference in PYD excretion was more marked, and mean DPD excretion was also significantly elevated; 13.7 +/- 1.3 vs. 10.1 +/- 0.8, P less than 0.05. CONCLUSION: bone collagen breakdown is increased in thyrotoxicosis, and in postmenopausal women taking sufficient T4 to suppress serum TSH. Similarly treated premenopausal women appear to be at lower risk.

Urinary pyridinium collagen cross-links predict growth performance in children with idiopathic short stature and with growth hormone (GH) deficiency treated with GH. Skeletal metabolism during GH treatment.

GH is able to promote longitudinal growth in children with GH-deficiency (GHD) and in some children with idiopathic short stature (ISS). The objectives of this study were to evaluate the predictive value of bone and collagen markers on the growth response to GH therapy in children with ISS and with GHD, and to characterize the effects of GH treatment on bone and collagen turnover in children with ISS and with GHD. Twenty prepubertal short, slowly growing, children treated with GH, 15 IU/m2 per week, were studied; of them 13 (10 males) had ISS and 7 (5 males) had GHD. An overnight 12-h urinary collection and a fasting morning blood sample were obtained at baseline, 1, 3, 6, and 12 months of treatment. Urinary levels of collagen cross-links, pyridinoline (Pyd) and deoxypyridinoline (Dpd), and circulating levels of osteocalcin, intact PTH, calcitonin, procollagen type III aminoterminal propeptide (PIIINP), insulin-like growth factor-I, and alkaline phosphatase were determined. Urinary collection was also obtained from 127 healthy children (51 males) aged 6-13 yr. In children with ISS, the changes in Dpd over 1 month of GH therapy were related to the changes in height velocity (HV) over 1 yr of therapy (r = 0.67; P < 0.05); the changes in Pyd after 1 month of GH treatment were related to the changes in HV at 6 months of GH treatment (r = 0.57; P < 0.05). All the other markers evaluated were not related to the HV changes in children with ISS. In children with GHD, the changes in Pyd and in Dpd after 1 month of GH treatment were positively related to the changes in HV after 12 months of therapy (r = 0.82; P < 0.05, and r = 0.82; P < 0.05, respectively). The changes in Pyd after 1 month were also related to the HV changes after 6 months of GH (r = 0.77; P < 0.05). Positive relationships between the HV after 6 months of GH and the increases of PIIINP (r = 0.80; P < 0.05) and osteocalcin (r = 0.77; P < 0.05) after 3 months of GH therapy were observed. All patients showed urinary Dpd and Pyd excretions in the normal range. In patients with ISS, Pyd (P < 0.05), Dpd (P < 0.05), osteocalcin (P < 0.01), PIIINP (P < 0.01), and alkaline phosphatase (P < 0.01) increased longitudinally during the GH treatment and the increments reached a maximum after 3-6 months of therapy. Patients with GHD showed an increase of the same markers but the increases occurred earlier, after 1 month of GH therapy. The collagen cross-links, Pyd and Dpd, could be helpful early markers in predicting the responsiveness to GH therapy in children with ISS and with GHD. GH treatment stimulates bone and collagen metabolism.

Identification of lysine-derived crosslinks in porcine collagen type X from growth plate and newly mineralized bone.

Intact collagen type X cannot readily be extracted from the growth plate. Both the use of pepsin to release this molecule from tissue and the relative solubility of collagen type X following treatment of chick embryos with beta-aminopropionitrile (Chen et al., 1992) suggest that the insolubility may by brought about by the formation of lysine-derived crosslinks. By immunocytochemical labelling using antibodies specific for collagen type X, we have shown that this collagen type persists in the cartilaginous spicules present in metaphyseal bone and appears to be colocalized with collagen type II. The combined concentration of the reducible bifunctional crosslinks, dihydroxylysinonorleucine and monohydroxylysinonorleucine, in collagen type X isolated from the premineralized and newly mineralized growth plate was about 0.6 residues/ molecule, a level which might explain the relative intractability of collagen type X. Pyridinoline and deoxypyridinoline were present in very small amounts in collagen type X; this suggests that, unlike the situation in other types of collagen, few of the bifunctional crosslinks undergo maturation to pyridinium compounds. Although it is clear that collagen type X contains lysinederived crosslinks, work is in progress to establish which molecule also participates in the formation of these crosslinks.

Primary structure of the helical domain of porcine collagen X.

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.