Normineralized and mineralized compartments of bone: The role of pyridinoline in nonmineralized collagen

Collagen tryptic peptides obtained from the nonmineralized and mineralized compartments of diaphyseal bone have different distributions of intermolecular crosslinks. Pyridinoline, a collagen crosslink thought to be associated with chronologically older bone, was detected in peptides from normineralized collagen but not from mineralized collagen. Mineralization may prevent collagen maturation; conversely, pyridinoline in nonmineralized collagen may decrease the intermolecular distances among collagen chains in fibrils and preclude mineralization.     

UPLC methodology for identification and quantitation of naturally fluorescent crosslinks in proteins: a study of bone collagen

Methods used to determine collagen crosslinks in different connective tissues require a relatively large amount of material and include a number of experimental steps. We addressed these issues by developing the first ultrahigh-pressure liquid chromatography (UPLC) methodology for detection and quantification of naturally fluorescent enzymatic (pyridinoline, deoxypyridinoline) and senescent (pentosidine) crosslinks using nanogram amounts of acid-hydrolyzed bone and purified bone collagen. Not only the developed set of UPLC methods relies on a single column analysis of all three fluorescent crosslinks in one separation step, but under different separation conditions, the same column is also used to determine hydroxyproline concentration necessary to calculate collagen contents in the samples making this a unique feature of our methodology. The determined detection limit was 10 fmol for the pyridinium crosslinks and 1.5 fmol for pentosidine. The smallest pieces of human cortical bones were 224-240 ng in weight and this is approx. 10(6)-fold less as compared to some high-pressure LC (HPLC) methods that need a minimum of approx. 0.50-1 mg of a bone sample. In general, our UPLC methodology can be applied to analysis of similar crosslinks in various collagenous tissues as well as purified/recombinant proteins of different origin. Thus, in addition to biomedical and bone research, this work is of general importance to other fields including biology, forensic, anthropology and archaeology, where samples could truly be rare, minute and precious.     

Simultaneous free and glycosylated pyridinium crosslink determination in urine: validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p<0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (相似文献   

Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.     

Cross-linking of collagen. Location of pyridinoline in bovine articular cartilage at two sites of the molecule.

The location of pyridinoline in 18-month-old bovine articular cartilage was investigated by fractionation of CNBr-derived peptides by ion-exchange chromatography and gel filtration. Two peptides, PCP1 and PCP2, were isolated and were shown to contain stoichiometric amounts of pyridinoline. From its amino acid composition and sequence studies, peptide PCP1 was shown to comprise two C-terminal non-helical chains (CB14) linked through pyridinoline to the alpha 1(II)-CB12 portion of the helix. The CB14 chains appeared to be labile at their C-terminal ends, resulting in lower-than-expected amounts of homoserine, and only the N-terminal portion of the peptide was sequenced. Similar studies of peptide PCP2 showed that it contained two N-terminal non-helical chains (CB4) linked to the alpha 1(II)-CB9,7 portion of the helix. The isolated peptides therefore confirmed the function of pyridinoline in stabilizing the 4D stagger of adjacent molecules. The possibility that the cross-link could act both as an intra- and an inter-microfibrillar cross-link was considered. A mechanism of formation of pyridinoline was postulated that, together with other evidence, appears to support the view that, in cartilage, pyridinoline acts primarily as an intramicrofibrillar cross-link and does not contribute to increased stability during maturation through lateral aggregation and bonding of filaments.     

Pyridinoline, a non-reducible crosslink of collagen. Quantitative determination, distribution, and isolation of a crosslinked peptide

Pyridinoline is a crosslink compound isolated from bovine Achilles tendon collagen. It is a 3-hydroxypyridinium derivative with three amino and three carboxyl groups (Fujimoto, D., Akiba, K., & Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129). The contents of pyridinoline in collagens from various sources were determined. The pyridinoline content of bovine Achilles tendon was 0.16 residue per 1,000 residues and that of rat Achilles tendon collagen was 0.017 residue per 1,000 residues. Besides Achilles tendon collagens, pyridinoline was found in collagens from costal cartilage, rib and femoral bone of rat. It was not found in collagens from the tail tendon and skin of rat. A crosslinked, triple-chained peptide containing pyridinoline was isolated from bovine Achilles tendon collagen after digestion with pronase. Its amino acid composition suggests that the peptide may be involved in an intermolecular crosslink among a carboxyterminal sequence, a sequence near the aminoterminus and a sequence in the helical region.     

Method for the isolation and purification of pyridinoline and deoxypyridinoline crosslinks from bone by liquid chromatographic techniques

Significant progress has been made in recent years in the development of new bone resorption markers, based principally on the urinary excretion of pyridinoline (Pyd) and deoxypyridinoline (Dpd) crosslinks. For their measurement, in spite of the recent development of immunoassays, HPLC remains the method of reference. However, the lack of an appropriate internal standard requires large amounts of pure crosslinks for external standardisation. Herein, we describe an efficient method for the isolation of both crosslinks from bone of adult turkey by isocratic semi-preparative HPLC. Demineralized bone is hydrolysed in hydrochloric acid, 9 M. A first liquid extraction step in butanol allowed to eliminate less polar compounds. The aqueous phase was concentrated and separated by gel filtration on Biogel P2 and eluted by acetic acid solution (10%). Fractions containing pyridinoline were pooled, concentrated, and purified on a CF1 cellulose column. Pyd and Dpd crosslinks were then separated isocratically by HPLC on a C18 reversed phase column (Vydac 218 TP 1010, 250x10 mm) and eluted with HFBA as the ion-pairing agent. Retention times of Pyd and DPD were 23.6 and 28.7 min, respectively. Both crosslinks prepared by HPLC were then transformed as hydrochloride to cellulose phosphate and desalted on Sephadex G-10 columns. These two further steps yielded highly purified compounds (the purity was greater than 98% evaluated by aminoacid analysis). In conclusion, the efficiency of this method allows to obtain rapidly Pyd and Dpd without interfering compounds as proven by spectral studies (NMR and mass spectroscopy).     

A comparative study of the distribution of the stable crosslink,pyridinoline, in bone collagens from normal,osteoblastoma, and vitamin D-deficient chicks

Tryptic peptides of bone collagens from 4-week-old normal, osteoblastoma and vitamin D-deficient chicks were studied using gel filtration chromatography. Absorbance at 230 nm and fluorescence (excitation at 330 nm, emission at 390 nm) of $\text{each}\text{fraction}$ were measured. The relative quantities of each peak from the absorbance and fluorescence patterns were semiquantified by planimetry. Osteoblastoma bone collagen had a prominent, fluorescent, crosslinked peptide that contained pyridinoline. Fluorescence of this pyridinoline-containing peak in AO collagen was much greater than in the vitamin D-deficient and normal bone collagen counterparts. A comparison of fluorescence patterns clearly showed that the distribution of pyridinoline in collagen from normal and diseased bone was totally dissimilar.The dissimilarities in distribution of pyridinoline in these bone collagens may be attributed to differences in the degree of lysine hydroxylation, to the degree of mineralization, or some other factor.     

Free radical involvement in hypertrophic scar formation

Hypertrophic scarring following thermal injury has become a major problem in Hong Kong. There is evidence that immunological and biochemical changes are associated with thermal injury, including pyridinoline crosslinks which are present in large quantities in hypertrophic scar, but the primary cause of hypertrophic scar formation still remains to be established. It has been reported that free radicals are assosciated with the formation of pyridinoline. In this study, attempts have been made to elucidate the involvement of free radicals in hypertrophic scar formation after thermal injury by determining the concentrations of Complement, free iron and pyridinoline crosslinks in collagen fibres. The results showed that the Complement activation product, C3d, was increased in the first week (i.e., day 7) postburn, indicating an acute inflammatory response. Free radicals, reported to be associated with the formation of pyridinoline crosslinks, and free iron content, were also found to have higher concentration in hypertrophic scar than in normal skin. The data suggest the involvement of free radical in hypertrophic scar formation. The observed increase in serum C3d concentration in about the first week indicates an acute inflammatory response to thermal injury. Both C3d and free iron concentrations (in vitro) are found higher in hypertrophic scar than in normal skin may suggest their roles in the generation of free radicals.     

Maturation of Collagen Ketoimine Cross-links by an Alternative Mechanism to Pyridinoline Formation in Cartilage

The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH₄. On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.     

Collagen cross-links: location of pyridinoline in type I collagen

Collagen from bone, dentine and tendon (type I), all of which contain the pyridinoline cross-link at varying levels, were each digested with CNBr. The resulting peptide mixtures were resolved by gel filtration on A1.5m agarose and assayed for pyridinoline. The polymeric cross-linked peptide complex, poly alpha 1CB6 [(1980) Biochem. J. 189, 111] isolated from each of these tissues did not contain pyridinoline. Only one peptide fraction contained the pyridinoline cross-link; that identified as alpha 2CB3,5. However, this peptide showed only a small increase in Mr in its cross-linked form (approx. 2000-5000) demonstrating that pyridinoline is not involved in the formation of polymeric structures like poly alpha 1CB6. These data, considered in the light of the recent finding that pyridinoline is present in type I collagens from different sources in widely varying amounts, cast doubt on its role in collagen maturation.     

Collagen crosslinks and their relationship to the thermal properties of calf tendons

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.     

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.     

Age changes in the level of pyridinoline cross-linking and ratio of soluble and insoluble collagen in human rib collagen]

The changes in the content of mature crosslinks with pyridinoline structure and soluble/insoluble collagen ratio in the costal cartilage tissue of human beings aged from 1 month to 57 years were found to be age-dependent. The effect of the pyridinoline crosslink content on the soluble/insoluble collagen ratio in human costal cartilage tissue may constitute no less than 67% of the total influence of the sum of all factors. The pronounced nonlinearity of the studied dependencies points to a possible involvement of a factor(s) other than the pyridinoline crosslink content.     

An enzyme-linked immunoassay for the collagen cross-link pyridinoline.

An inhibition immunoassay method for the determination of pyridinoline was developed with the use of microtitre plates coated with a pyridinoline--gelatin conjugate and rabbit antisera directed against pyridinoline linked to bovine serum albumin. The sensitivity of the assay is about 2pmol of pyridinoline, and the presence of related pyridinium and lysine-derived compounds does not significantly interfere with the procedure. Its application to tissue and human urine samples is described.     

Analysis of age-associated changes in collagen crosslinking in the skin and lung in monkeys and rats

The present study was designed to address a specific question: can we define collagen aging in vivo in terms of alterations in collagen crosslinking? In order to assess the complete spectrum of change throughout life, tissues from rats, monkeys and (where available) humans were examined at ages ranging from fetal to old. Skin and lung were selected in order to include all of the crosslinks derived from lysyl oxidase-generated aldehydes that have been identified thus far, both reducible and nonreducible. Crosslinks analyzed included hydroxylysinonorleucine, dihydroxylysinorleucine, histidinohydroxymerodesmosine, hydroxypyridinium, lysyl pyridinium, and a deoxy analogue of hydroxypyridinium found in skin that differs structurally from lysyl pyridinium. Tissues from both a short-lived species (rats) and a long-lived species (monkeys) were analyzed to test further the hypothesis that changes in crosslinking are linked predominantly to biological age of the animal, rather than temporal aging. We found that biological aging seems to regulate certain predictable changes during the first part of the lifespan: the disappearance postnatally of dihydroxylysinonorleucine in skin, the rapid decrease in difunctional crosslink content in lung and skin during early growth and development, and the gradual rise in hydroxypyridinium and lysyl pyridinium in lung tissue. Changes in crosslinking were far less predictable during the second half of the lifespan. Although hydroxypridinium content continued to rise or reached a plateau in rat and monkey lungs, respectively, it showed a decrease in human lungs. The analogous trifunctional crosslink in skin, the so-called 'pyridinoline analogue', decreased dramatically in both rats and monkeys in later life. Our data suggest that caution must be taken in drawing inferences about human connective tissue aging from experiments performed in short-lived species such as rodents. Furthermore, the finding that there may be fewer total lysyl oxidase-derived crosslinks per collagen molecule in very old animals as compared with young animals suggests that we may need to expand our concepts of collagen crosslinking.     

Biochemical characterization of major bone-matrix proteins using nanoscale-size bone samples and proteomics methodology

There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows in-depth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation.     

Cross-linking of collagen. Isolation, structural characterization and glycosylation of pyridinoline.

A method for the isolation and purification of pyridinoline from bone collagen was developed, with the use of sulphonated polystyrene resins. The analytical techniques were used to quantify pyridinoline, for which hydroxyallysine is a known precursor, in a wide range of tissues. The structure of pyridinoline proposed by Fujimoto, Moriguchi, Ishida & Hayashi [(1978) Biochem. Biophys. Res. Commun. 84, 52-57] was confirmed by 13C-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry. At concentrations greater than about 0.1 mM, pyridinoline exhibited altered fluorescence properties that were consistent with excimer formation. From alkali hydrolysates of several different tissues, a fluorescent compound was purified by gel filtration and ion-exchange chromatography and was shown to be galactosylpyridinoline. This derivative was very labile to acid treatment compared with the bifunctional cross-link analogues, and was completely converted into free pyridinoline by heating at 108 degrees C for 8 h in 0.1 M-HCl. Galactosylpyridinoline was also partially converted into free pyridinoline by prolonged alkali hydrolysis. This lability, which could also apply to other multifunctional cross-link derivatives, may explain the fact that no disaccharide derivatives of pyridinoline were isolated.     

Identification of pyridinoline,a collagen crosslink,as a novel intrinsic ligand for the receptor for advanced glycation end-products (RAGE)

Advanced glycation end-products (AGEs) elicit inflammatory responses via the receptor for AGEs (RAGE) and participate in the pathogenesis of diabetic complications. An earlier study showed that 3-hydroxypyridinium (3-HP), a common moiety of toxic AGEs such as glyceraldehyde-derived pyridinium (GLAP) and GA-pyridine, is essential for the interaction with RAGE. However, the physiological significance of 3-HP recognition by RAGE remains unclear. We hypothesized that pyridinoline (Pyr), a collagen crosslink containing the 3-HP moiety, could have agonist activity with RAGE. To test this hypothesis, we purified Pyr from bovine achilles tendons and examined its cytotoxicity to rat neuronal PC12 cells. Pyr elicited toxicity to PC12 cells in a concentration-dependent manner, and this effect was attenuated in the presence of either the anti-RAGE antibody or the soluble form of RAGE. Moreover, surface plasmon resonance-based analysis showed specific binding of Pyr to RAGE. These data indicate that Pyr is an intrinsic ligand for RAGE.

Abbreviations: AGEs: advanced glycation end-products; RAGE: receptor for advanced glycation end-products; DAMPs: damage-associated molecular patterns; PRR: pattern recognition receptor; TLR: toll-like receptor; GLAP: glyceraldehyde-derived pyridinium; 3-HP: 3-hydroxypyridinium; Pyr: pyridinoline; HFBA: heptafluorobutyric acid; GST: glutathione S-transferase; SPR: surface plasmon resonance; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition     

An investigation of pyridinoline, and putative collagen cross-link.

A component, termed pyridinoline, has been reported to be derived from 'lysine aldehyde' (2,6-diaminohexanaldehyde) and designated as the stable cross-link of mature collagen. Commerically prepared collagen and freshly obtained mature bovine tendon collagen were both investigated with regard to their pyridinoline content. Both sources of material could be depleted of this component by mild washing procedures. Pepsin-solubilized collagen and peptides derived from CNBr cleavage of intact collagen did not contain the compound. Pure pyridinoline was isolated and shown to be hydrolysed by water, as previously reported, but neither hydroxylysine nor lysine could be ds not a cross-linking component of collagen.