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.

Biochemistry and functional significance of collagen cross-linking.

The biophysical characteristics of vascular tissues are dependent largely on the properties of fibrillar collagens. Considering the predominant structural component, collagen type I, the present review describes the mechanisms of formation and maturation of lysyl oxidase-mediated cross-linking, leading to an understanding of how intracellular collagen-modifying enzymes affect the patterns of cross-links produced. An important distinction is made between the enzyme-mediated cross-linking, essential for optimum tissue function, and the non-enzymatic aging processes that generally lead to structural changes deleterious to function. Finally, the extracellular matrix of vascular tissue is a multicomponent system and the role of other major constituents, such as elastin and glycosaminoglycans, in modifying tissue properties should be considered. Some details of newer methods being developed to quantify these constituents will be presented.

Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts.

We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO2 was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by 3H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O2 were less organized and much less abundant than in 20% O2 cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia.

High-performance liquid chromatography method to analyze free and total urinary pyridinoline and deoxypyridinoline.

The pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of bone resorption measured in blood and urine and are used to investigate bone metabolism and manage bone diseases. Unfortunately, the currently observed interlaboratory variability caused by inconsistent assay calibration limits the optimal use of these markers. A high-performance liquid chromatography (HPLC)-based assay was developed using synthetic PYD and DPD as calibrators to analyze free and total PYD and DPD in urine. The spectroscopic characteristics of the synthetic calibrators were identical to those of calibrators isolated from bone. The mean intraassay variabilities of the HPLC method were 4.1 and 3.8%, respectively, for total DPD and PYD and 9.8 and 9.5%, respectively, for free DPD and PYD. The mean interassay variabilities were 9.1 and 8.2% for total DPD and PYD and 8.6 and 7.0% for free DPD and PYD, respectively. The mean recoveries were 98.1% for total DPD, 100.8% for total PYD, 98.6% for free DPD, and 94.9% for free PYD. The method exhibits a good correlation with a commercial immunoassay and with other HPLC assays currently used in hospital laboratories.

Sheep lumbar intervertebral discs as models for human discs.

OBJECTIVE: To determine the water content, collagen content and collagen orientation angle in different regions of sheep lumbar discs. DESIGN: A laboratory study of sheep discs obtained from an abattoir. METHODS: A total of 21 sheep lumbar discs were obtained from three lumbar spines. Water content was determined by oven drying (60 degrees C) to constant mass. Collagen content was determined by hydroxyproline analysis. Fibre orientation angles were determined by X-ray diffraction. RESULTS: Water content increased from 74% of total tissue mass in the outer annulus, to 82% in the inner annulus, to 86% in the nucleus. Collagen content decreased from 30% of total tissue mass in the outer region to 20% in the inner region of the anterior and lateral annulus; it was 16% in the posterior annulus. The orientation angle of the collagen fibres decreased from 59 degrees in the outer region to 56 degrees in the inner region of the anterior and lateral annulus; it was 51 degrees in the posterior annulus. CONCLUSIONS: Sheep lumbar intervertebral discs provide a reasonable model for human lumbar intervertebral discs. RELEVANCE: Sheep lumbar discs have been used to investigate the effects of removing and replacing the nucleus. These studies indicate that removal of nucleus may lead to further disc degeneration and indicate the material properties required for an implant material. The relevance of these previous studies is increased if human and sheep lumbar discs have a similar composition and structure.

Urinary excretion of pyridinium crosslinks in short children treated with growth hormone.

The aim of this study was to evaluate the effect of growth hormone (GH) treatment on bone resorption in children with GH deficiency and those with idiopathic short stature. The study population included seven children with subnormal spontaneous GH secretion and 13 children with idiopathic short stature, all of them pre-pubertal. Anthropometric measurements, free, protein-bound and total urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd), serum GH, and serum immunoreactive PTH were measured at baseline and months 1, 3, 6 and 12 of GH treatment. The urinary excretion of total Pyd and Dpd, standardized by the cube of height (m3) in overnight, 24-hour urine collections was not different from age-matched healthy controls at baseline in either group of patients. During treatment with human recombinant GH, both pyridinium crosslinks increased above normal values, reaching a peak after one month in children with GH deficiency and later (after 3-6 months) in children with short stature. Free and total crosslink forms were correlated, and GH treatment did not affect the proportion of free to bound crosslinks. Serum concentrations of iPTH showed a moderate but not statistically significant increase. This study provides no evidence of reduced bone resorption in untreated GH deficiency or in idiopathic short stature. GH treatment induced a marked, but temporary, increase of bone resorption in both groups of patients.

Biochemical markers of bone metabolism in growing thoroughbreds: a longitudinal study.

This study describes longitudinal changes in serum levels of biochemical markers of bone cell activity in a group of 24 thoroughbred foals from birth to 18 months of age. The markers of bone formation included the type I collagen carboxy-terminal propeptide (PICP), the bone-specific isoenzyme of alkaline phosphatase (BAP), and osteocalcin (OC). Levels of the cross-linked telopeptide of type I collagen (ICTP), a marker of bone resorption, and the N-terminal propeptide of type III collagen (PNIIIP), a marker of soft tissue turnover, were also measured. Levels of all markers fell significantly between birth and 18 months of age (70-80 per cent); this decrease being most marked between 0 and 6 months. However, a transient increase in levels of the markers then occurred between 6 and 14 months of age. The timing of this increase was specific for each parameter. ICTP and OC concentrations increased between October and December. PICP concentrations increased between December and April whereas the increase in PIIINP was coincident with the peak in weight gain between April and June. Changes in BAP concentration were less distinct at this time. Season was shown to have significant effects on the biochemical markers independent from the effect of age. Concentrations of all markers decreased with increasing body weight and at any given age heavier horses had lower marker levels. These results show that biochemical markers of bone cell activity and soft tissue turnover follow characteristic patterns of change in growing thoroughbreds influenced by age, season and bodyweight. The demonstration that the reference ranges for the biochemical markers change from month to month means that single samples from individuals are of little value for monitoring bone cell activity in growing thoroughbreds.

External surface display of proteins linked to DNA-binding domains.

A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein. In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle. The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively. Human kappa constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-kappa antibody. This display system may complement currently available bacterial selection techniques.

Changes in proteoglycan content of articular cartilage during avian degenerative joint disease.

OBJECTIVE: To determine the biochemical changes in articular cartilage composition associated with the development of avian degenerative joint disease (DJD) in ad libitum fed broiler fowl, in comparison to feed-restricted broilers and J-lin fowl (non-susceptible to DJD). METHODS: Articular cartilage from the distal tibiotarsus (DTT) was characterised up to age 180 days. Proteoglycan content was determined by uronic acid and sulphated glycosaminoglycan analysis, cellularity by assay for DNA content, and collagen content and crosslinking by hydroxyproline and pyridinoline analysis, respectively. RESULTS: Disease development was accompanied by increased hydration and proteoglycan content (particularly sulphated proteoglycans) and decreased cellularity, with no significant differences in either total collagen content or in mature collagen cross-linking. CONCLUSION: The biochemical features of avian DJD are similar to those observed in other animal models. This bipedal model is exceptional however since cartilage alterations occur spontaneously and in a load-dependent manner.

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.

Structural characterization of pyrrolic cross-links in collagen using a biotinylated Ehrlich's reagent.

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.

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.

[The biochemical markers of bone remodeling in cancer patients with skeletal involvement]. / Biokhimicheskie markery kostnogo remodelirovaniia u onkologicheskikh bol'nykh s porazheniem skeleta.

The report discusses a study of pyridinoline (Pyd) and deoxypyridinoline (Dpyd) as biochemical markers of bone resorption as well as total bone alkaline phosphatase level (APh) and that of its bone fraction as criteria of osteogenesis in skeletal lesions in breast, prostate and lung cancer and multiple myeloma. The investigation established a significantly enhanced Pyd and Dpyd excretion with urine and increased blood-serum APh levels in skeletal cancers (n = 271) as compared with healthy subjects (n = 173) and patients without bone metastases (n = 94). A case has been made for determination of total excretion of Pyd crosslinks of collagen to diagnose bone metastases. Most pronounced hyperenzymemia was found in prostate cancer which points to the leading role of APh as a bone metastasis marker. Pyd and Dpyd excretion and APh levels were significantly higher among patients multiple metastases with than in those with single bone metastases. The universality of pyridinoline crosslinks as skeletal damage markers has been confirmed by establishing a significant correlation between drug and therapeutic effect for Pyd and Dpyd only, in patients receiving ibandronate.

Dietary influences on bone mass and bone metabolism: further evidence of a positive link between fruit and vegetable consumption and bone health?

BACKGROUND: The role of nutritional influences on bone health remains largely undefined because most studies have focused attention on calcium intake. OBJECTIVE: We reported previously that intakes of nutrients found in abundance in fruit and vegetables are positively associated with bone health. We examined this finding further by considering axial and peripheral bone mass and markers of bone metabolism. DESIGN: This was a cross-sectional study of 62 healthy women aged 45-55 y. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at the lumbar spine and femoral neck and by peripheral quantitative computed tomography at the ultradistal radial total, trabecular, and cortical sites. Bone resorption was calculated by measuring urinary excretion of pyridinoline and deoxypyridinoline and bone formation by measuring serum osteocalcin. Nutrient intakes were assessed by using a validated food-frequency questionnaire; other lifestyle factors were assessed by additional questions. RESULTS: After present energy intake was controlled for, higher intakes of magnesium, potassium, and alcohol were associated with higher total bone mass by Pearson correlation (P < 0.05 to P < 0.005). Femoral neck BMD was higher in women who had consumed high amounts of fruit in their childhood than in women who had consumed medium or low amounts (P < 0.01). In a regression analysis with age, weight, height, menstrual status, and dietary intake entered into the model, magnesium intake accounted for 12.3% of the variation in pyridinoline excretion and 12% of the variation in deoxypyridinoline excretion. Alcohol and potassium intakes accounted for 18.1% of the variation in total forearm bone mass. CONCLUSION: The BMD results confirm our previous work (but at peripheral bone mass sites), and our findings associating bone resorption with dietary factors provide further evidence of a positive link between fruit and vegetable consumption and bone health.

[Role of biochemical indices as metastatic markers in patients with prostatic cancer invading skeleton]. / Znachenie biokhimicheskikh pokazatelei v kachestve markerov metastazirovaniia u bol'nykh rakom predstatel'noi zhelezy s porazheniem skeleta.

Biochemical markers of resorption and formation of bone tissue (piridinoline-PD, desoxypiridinoline-DPD and alkaline phosphatase-AP, respectively) were measured in blood serum and urine of 41 prostatic cancer (PC) patients with metastases to the bones and 24 PC patients free of such metastases as well as of 40 healthy males and 11 males with benign prostatic hyperplasia (BPH). It was found that PD, DPD levels, general AP activity and activity of its bone fraction were significantly higher in PC patients with bone metastases than in the others (p < 0.001). The former had a significantly higher percent of peptide-bound and lower percent of free forms of PD and DPD (p < 0.001) vs patients without metastases and controls. Higher biochemical serum and urine indices in PC patients with bone metastases reflect enhanced intensity of both formation and resorption of bone tissue in PC with bone metastatic lesions. Therefore, it is possible to use PD, DPD and AP values in diagnosis and monitoring of metastatic skeletal destruction in PC.

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.

Effects of variations in live weight gain on bone growth and composition and on markers of bone turnover in lambs.

Growing lambs were fed the same diet at intakes supporting mean live weight gains of 0.1, 0.2 and 0.3 kg day-1, representing slow, intermediate and fast growth groups, respectively. The effects on bone growth and composition, and on blood and urinary bone marker concentrations or excretion rates were monitored. Compared with the slow-growing lambs, the higher intake group grew twice as fast, had higher rates of bone growth (indicated by external metatarsal length), and larger and heavier bones at slaughter. Bones from fast-growing animals had higher collagen and deoxypyridinoline concentrations, and lower Ca:collagen, Ca :P and pyridinoline : deoxypyridinoline ratios, indicating a less mature bone compared with the slow-growing lambs. Bone growth rate had no effect on plasma osteocalcin, bone-specific alkaline phosphatase or growth hormone concentrations, nor on the urinary excretion of pyridinoline and deoxypyridinoline. The results for plasma markers may be explained by an increase in blood volume linked with increased body weight.

Bone density and bone turnover in patients with osteoarthritis and osteoporosis.

OBJECTIVE: Osteoarthritis (OA) and osteoporosis (OP) are reported to be rare in the same patient. We examined bone mass, bone turnover, and radiological presence of OA in a group of patients with OA with previous hip fractures and age matched controls. METHODS: Bone mass was assessed by bone mineral density (BMD), using dual energy x-ray absorptiometry (DEXA) of the hip and total body, and quantitative ultrasound of the os calcis, measuring broadband ultrasound attenuation and velocity of sound. Bone turnover was assessed by measuring urinary pyridinium crosslinks and serum osteocalcin. RESULTS: There were differences in bone density, the patients with OP having lower bone density, while patients with OA had similar or increased bone density compared to controls, Serum osteocalcin showed no significant differences among the 3 groups of patients. Urinary pyridinium crosslinks excretion was significantly elevated in the OA group but not in the OP group compared with controls. CONCLUSION: Increased bone turnover was restricted to the OA group.

Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17.

Bruck syndrome is characterized by the presence of osteoporosis, joint contractures, fragile bones, and short stature. We report that lysine residues within the telopeptides of collagen type I in bone are underhydroxylated, leading to aberrant crosslinking, but that the lysine residues in the triple helix are normally modified. In contrast to bone, cartilage and ligament show unaltered telopeptide hydroxylation as evidenced by normal patterns of crosslinking. The results provide compelling evidence that collagen crosslinking is regulated primarily by tissue-specific enzymes that hydroxylate only telopeptide lysine residues and not those destined for the helical portion of the molecule. This new family of enzymes appears to provide the primary regulation for controlling the different pathways of collagen crosslinking and explains why crosslink patterns are tissue specific and not related to a genetic collagen type. A genome screen identified only a single region on chromosome 17p12 where all affected sibs shared a cluster of haplotypes identical by descent; this might be the BS (Bruck syndrome) locus and consequently the region where bone telopeptidyl lysyl hydroxylase is located. Further knowledge of this enzyme has important implications for conditions where aberrant expression of telopeptide lysyl hydroxylase occurs, such as fibrosis and scar formation.

Isoaspartyl bond formation within N-terminal sequences of collagen type I: implications for their use as markers of collagen degradation.

An ELISA was developed for the measurement of N-telopeptides of the alpha2(I) collagen chain containing an isomerized Asp-Gly bond (beta-peptide) using polyclonal antibodies raised against the synthetic peptide. The presence of this isomerized form in bone was confirmed by positive immunostaining of sections from human femoral head. The ELISA was used to measure isomerized peptide in both human bone digests and urine samples, showing that an isoaspartyl rearrangement occurs in the Asp-Gly sequence at the N-terminus of the alpha2(I) chain in an analogous fashion to that found in the C-terminal telopeptide of the alpha1(I) chain of collagen. Using this assay in conjunction with a monoclonal antibody ELISA to the non-isomerized alpha2(I) N-telopeptide (alpha-peptide), ratios of isomerized to normal peptides were estimated in the bone and urine samples. Urinary alpha2(I) N-telopeptides showed a higher degree of isomerization than the peptides derived from a human bone digest. This is possibly due to relative enrichment of the isoaspartyl-bonded peptide during metabolic processing due to the proximity of the isoaspartyl bond to a cross-link site. Urinary concentrations of isomerized and normal peptides were determined in normal adults, children, post-menopausal control subjects and subjects with osteoporosis. A lower ratio of beta-peptide to alpha-peptide was observed in children's urine, indicative of a higher rate of bone metabolism allowing less time for the isomerization to occur. No significant differences were found between the post-menopausal control and osteoporotic populations although the trends observed supported the hypothesis that a lower degree of isomerization may be associated with faster bone turnover.