Evidence for natural existence of pyridinoline crosslink in collagen

Pyridinoline is a fluorescent crosslinking amino acid isolated from collagen. Recently it was claimed that this material is an artefact produced from contaminating proteins during acid hydrolysis. However, in our hands, bovine tendon collagen could not be depleted of pyridinoline by the suggested treatments. A peptide which had the same fluorescence properties as those of pyridinoline could be isolated from enzymic digests of collagen. After acid hydrolysis, presence of pyridinoline in the peptide could be demonstrated on amino acid analysis. The composition of the peptide suggests that it originates from the specific regions of collagen molecule. These results clearly indicate the existence of pyridinoline in collagen $\text{in}\text{vivo}$.     

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.     

Effect of vitamin D deficiency on bone formation in the chick.

1. The process of diaphyseal bone formation can be investigated by studying the rate of incorporation of radioactive precursors, administered in vivo into bone fractions of increasing density. 2. In the 4-week-old vitamin D-treated chick most of the osteoid becomes calcified within 12h and almost all within 2 days. The low-density calcified phase that is formed is converted into a higher density form and within 7 days the greater proportion of the calcified tissue is in the higher density form. 3. In the vitamin D-deficient chick of similar age the rate of calcification of osteoid is decreased, as is the rate of conversion into the higher density phase with the resultant accumulation of the lower density calcified form. 4. The higher density phase probably corresponds to hydroxyapatite and the lower density one to the ACP-pase described by Termine & Posner [(1967) Calcif. Tissue Res. 1, 8--23]. 5. The disorder in the process of calcification seems to be unrelated to the alteration in blood Ca2+ and phosphate concentrations, but related to the presence or absence of cholecalciferol.     

Influence of plasma calcium and vitamin D on bone collagen. Effects on lysine hydroxylation and crosslink formation.

In chick bone collagen the degree of lysine hydroxylation and the magnitude of the crosslink ratio dihydroxylysinonorleucine/hydroxylysinonorleucine were both found to be inversely related to the concentration of plasma calcium. Lysine hydroxylation was also affected by a second factor related to vitamin D status.     

The effect of vitamin D on the structural crosslinks and maturation of chick bone collagen.

The quantitative relationships were determined between the structural crosslinks, dihydroxylysinonorleucine (Lys(OH)2-Nle) and hydroxylysinonorleucine (Lys (OH) -Nle) in NaB 3H4-reduced diaphyseal bone collagen from 1-, 2-, 3- and 4-week-old chicks fed either a vitamin D-deficient diet, a normal-vitamin D diet or a high-, but non toxic, vitamin D diet from time of hatching. Chicks fed the normal diet showed a progressive decrease in the ratio of Lys(OH)2-Nle/Lys(OH)-Nle with age. This decrease was accelerated in chicks receiving the High-vitamin D diet. In the vitamin D-deficient group, the ratio was higher than controls at 1 and 2 weeks and increased further at 3 and 4 weeks. Similar changes in Lys(OH)2-Nle/Lys(OH)-Nle ratio did not occur in skin collagen. Compared to Control-vitamin D animals, the increased crosslink ratios in the vitamin D-deficient bone collagen occurred prior to changes in growth rate and could not be correlated with lysine hydroxylation or the hypocalcemia seen in this group. These results suggest that the type of crosslink analysis used in this study provides one of the earliest and most sensitive indications of a bone disturbance due to vitamin D deficiency and that vitamin D specifically acts to increase the rate of maturation of bone collagen.     

Ultraviolet light- and ozone-induced changes in pyridinoline, a trisubstituted 3-hydroxypyridinium crosslink of collagen

Pyridinoline photo-degraded with the formation of photoproducts absorbing diffusely around 260-290 nm (pH 7) and sharply at 232 nm (pH 1). Subsequent heating partially regenerated the original pyridinoline, also producing new products absorbing at 417/440 nm (pH 7) and 300/412 nm (pH 1). Pyridinoline (pH 7) and its new products (pH 7 and pH 1) also underwent ozone-induced degradation.     

Effects of dietary vitamin D and calcium on lysyl oxidase activity in chick bone metaphyses.

Activity of lysyl oxidase, an enzyme responsible for production of aldehydic precursors for lysine-derived collagen crosslinks, was measured in tibial metaphyses from chicks receiving different dietary levels of vitamin D and Ca for 2 weeks after hatching. Enzyme activities were increased twofold in D-deficient chicks compared to activities from chicks receiving control levels of vitamin D. Addition of Ca to the D-deficient diet had no effect on lysyl oxidase activity. It is suggested that vitamin D may play a role in the age-related decrease in lysyl oxidase activity that normally occurs in chick bone.     

Effect of cadmium on collagen content and solubility in rat bone

The toxic action of cadmium in the bone tissue is known, but its mechanisms are still unexplained. We examined whether Cd influences collagen content and its solubility in the femoral bone of three-week-old female rats exposed to 5 or 50 mg Cd/l in drinking water. Non-cross linked collagen was extracted with 0.5 M acetic acid, and two acid-insoluble collagen fractions were extracted with pepsin and 4.0 M guanidine hydrochloride, respectively. SDS/PAGE showed the presence of two collagen types, I and V, in all three extracted fractions. Exposure of rats to Cd for 6 months increased the amount of acid-soluble collagens type I and V and decreased the level of acid-insoluble collagens. The amount of total collagen extracted from the bones of rats exposed to 50 mg Cd/l was reduced by about 14% as compared to control and those intoxicated with 5 mg Cd/l. The solubility of type I bone collagen (determined as the percentage of acetic-soluble fraction of total collagen) was increased 2.9- and 3.0-fold in rats intoxicated with 5 and 50 mg Cd/l, respectively. Similarly, the solubility of type V collagen was increased 2.3- and 2.7-fold, respectively. Our results indicate that Cd treatment affects bone collagen by decreasing its content and increasing its solubility.     

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     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

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.     

Role of vitamin D in bone resorption.

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti-rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1 alpha,25(OH)2D3 in involved in bone resorption. 1 alpha,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte-macrophage lineage. 1 alpha,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1 alpha,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1 alpha,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1 alpha,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.     

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.