An Ehrlich chromogen in collagen cross-links.

A well-characterized three-chain peptide [(Col1)2 X T9] from human type III collagen was a rich source of Ehrlich chromogen. The corresponding two-chain peptide [(Col1)2] was not, implying that the Ehrlich chromogen is a trifunctional cross-link. (Col1)2 X T9 also contained pyridinoline, which is not an Ehrlich chromogen. The 7S domain of type IV collagen also contained an Ehrlich chromogen.     

The changes in crosslink contents in tissues after formalin fixation

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.     

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.     

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.     

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.     

Thermomechanical analysis of collagen crosslinking in the developing ovine thoracic aorta

We have used hydrothermal isometric tension (HIT) techniques in a sheep model to assess collagen crosslink stability and its contribution to the mechanical properties of the ovine thoracic aorta during perinatal and postnatal development. Aortic tissue was studied from fetal sheep, lambs, and adult sheep. Strips of tissue were loaded under isometric tension and heated to a 90 degrees C isotherm which was sustained for 3 hours. The decrease in load at this temperature is associated with collagen peptide bond hydrolysis and chain slippage, and the rate of this decrease is an inverse indicator of collagen crosslinking. The half-time of load decay (t1/2) was computed before and after tissue was treated with NaBH4 which stabilizes immature, reducible crosslinks. We observed a two-fold increase in t1/2 of untreated tissue from the lamb to the adult, indicating that aortic collagen crosslinking increased during postnatal development. Furthermore, the t1/2 of NaBH4-stabilized lamb tissue was similar to that of the untreated adult tissue, suggesting that much of the immature crosslinking in the lamb is stabilized during postnatal development. These observations suggest (a) increased crosslinking occurs during postnatal development and (b) that this increase is largely due to a conversion of immature crosslinks into their mature heat stable form.     

The separation and amino acid analysis of collagen crosslinks on an extended basic ion-exchange column

The major reducible crosslinks found in collagen were separated and analyzed on an extended basic amino acid analyzer column. Reaction with ninhydrin allows the direct analysis of collagen crosslinks, including hydroxyaldol-histidine, a naturally occurring, nonreducible crosslink. In addition to known crosslinks, direct amino acid analysis of tissue hydrolysates reveals the presence of an unknown, ninhydrin-reactive component, in both NaB³H₄-reduced and unreduced collagenous tissues. Initial fractionation of hydrolysates on a Bio-Gel P-2 gel filtration column provides partial separaton of amino acids and crosslinks and enables more direct analysis of the crosslinks present in the samples, as well as detecting potential new crosslinks. The results also show that, prior to NaB³H₄ reduction, substantial amounts of known crosslinks are normally present in bovine skin and bone.     

Tensile properties, collagen content, and crosslinks in connective tissues of the immature knee joint

Background

The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.

Methodology/Principal Findings

The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.

Conclusions/Significance

To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues.     

p-Dimethylaminobenzaldehyde-reactive substances in tail tendon collagen of streptozotocin-diabetic rats: temporal relation to biomechanical properties and advanced glycation endproduct (AGE)-related fluorescence

In the present work, pepsin digests of tail tendons from streptozotocin-diabetic rats were found to contain material that reacted rapidly at room temperature with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to give an adduct with an absorbance spectrum characteristic of the Ehrlich chromogen of pyrrolic nature determined in ageing collagens. A significant correlation of the Ehrlich adduct with tendon mechanical strength and collagen fluorescence characteristic of advanced glycation endproducts was observed. Collagen content of the Ehrlich-positive material was found to be significantly elevated in tendons of diabetic rats compared with age-matched healthy controls. The results indicate that the p-dimethylaminobenzaldehyde-reactive pyrrole moieties may contribute to the increased cross-linking of diabetic matrix collagen. Profound inhibitory effect of aminoguanidine was observed, underlining the role of non-enzymatic mechanisms of advanced glycation in pyrrolisation and cross-linking of collagen exposed to hyperglycaemia. It is hypothesised that quantification of the p-dimethylaminobenzaldehyde-reactive material in matrix collagen may provide a tissue measure of integrated hyperglycaemia over prolonged periods of time. Further research is to assess the significance of p-dimethylaminobenzaldehyde-reactive substances in diabetic collagen tissues and to reveal their relationship to enzyme-mediated physiological pyrrolisation of ageing collagens.     

Thermal stability,mechanical properties and reducible cross-links of rat tail tendon in experimental diabetes

Thermal stability (measured as isometric contraction force), biomechanical properties and reducible cross-links were measured in tail tendons from streptozotocin diabetic rats, with and without insulin treatment. After 10 days of diabetes the maximum thermal contraction force was unchanged, but the relaxation following the maximal contraction was retarded. After 30 days the maximum contraction force was increased and the relaxation rate was decreased. The maximum strength and stiffness of the tendons were increased after 10 days of diabetes and even more after 30 days. There was no change in the density of reducible cross-links. However, diabetes increased the amount of glucose attached to the lysine and hydroxylysine residues of collagen. Insulin treatment prevented all changes in thermal stability and mechanical properties. The results indicate that stabilization of collagen fibres in diabetes does not follow the same pattern as that seen in normal ageing.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

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.     

Thermomechanical analysis of collagen crosslinking in the developing lamb pericardium

In a developing lamb model, we have used hydrothermal isometric tension (HIT) techniques to assess collagen crosslink stability and its contribution to the mechanical properties of the pericardium. Strip samples of tissue were either: (i) heated to a 90 degrees C isotherm or (ii) heated in 5 degrees C increments between 60-90 degrees C and then 93 and 97 degrees C isotherms. The half-life of stress relaxation associated with peptide bond hydrolysis (t1/2) was calculated at each isotherm. The activation energy, Eact, for the hydrolysis-associated relaxation process was also calculated using the data from the stepwise HIT tests--a technical improvement which significantly reduces the experimental time required to develop statistically valid measurements. Crosslinking in the pericardium increased during development and was demonstrated both by thermoelasticity and by resistance to enzymatic solubilization. We observed greater conversation to thermally stable crosslinks upon maturation, the ratio of the NaBH4-stabilized/unstabilized half-lives peaking at 21 days postnatal. Whereas tissue from lambs (119 day fetal, and 3 day and 21 day postpartum) showed an early maximum and rapid decay of force, NaBH4 stabilization significantly increased thermal stability and yielded profiles similar to those in adult tissue.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosenic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylyse, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonezymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot acount for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia.

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosemic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylysine, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonenzymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated with DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot account for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

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.     

Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15.     

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.     

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.     

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