desmosine and histidinohydroxylysinonorleucine

We have developed a novel chromatographic analytical method for the simultaneous quantitation of collagen crosslinks. Seven non-derivatised crosslinks could be separated on a Cogent Diamond Hydride HPLC column using either isocratic or gradient conditions then detected by mass spectrometry. The total run time was less than 10min which is significantly shorter than that previously reported. This is the first method in which histidinohydroxylysinonorleucine (HHL) and histidinohydroxymero-desmosine (HHMD) were separated and identified by mass spectrometry without the need for pre- or post-column derivatization. The CVs of the retention times of all seven crosslinks were less than 1% and the limit of detection (LOD) and the limits of quantitation (LOQ) were 0.07-0.13pmol/μL and 0.20-0.38pmol/μL, respectively. This novel method was used for the routine analysis and quantitation of crosslinks in different animal skins in which potential new collagen crosslinks were identified that are as yet undocumented.

Topics: Animals; Chemistry Techniques, Analytical; Chromatography, Liquid; Collagen; Desmosine; Dipeptides; Elastin; Histidine; Limit of Detection; Skin; Spectrometry, Mass, Electrospray Ionization

Keloid is a tissue with an excessive accumulation of collagen. In this study, we have partially characterized post-translational modifications of type I collagen in human keloid in order to pursue their potential involvement in this pathology. The levels of lysyl hydroxylation of the helical portions of alpha 1 and alpha 2 chains of type I collagen in keloid were significantly higher than those of normal, while the levels of prolyl hydroxylation were identical between these two groups. The contents of the major reducible cross-links in dermal collagen, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymero-desmosine, were both significantly higher in keloids (up to sixfold) than those of normal. In addition, significant amounts of hydroxylysine-aldehyde derived cross-links that are characteristic of skeletal tissue collagens, dehydro-dihydroxylysinonorleucine (about 0.3 mole/mole of collagen) and pyridinoline (about 0.1 mole/mole of collagen), were found in keloids. These results indicate that keloid-forming cells are phenotypically different from those in normal dermis and that the collagen produced is highly cross-linked. The increased cross-linking provides the fibrils with more stability that may result in an accumulation of collagen.

Topics: Adult; Amino Acids; Collagen; Cross-Linking Reagents; Desmosine; Dipeptides; Histidine; Humans; Hydroxylation; Hydroxylysine; Hydroxyproline; Keloid; Middle Aged; Protein Processing, Post-Translational; Protein Structure, Secondary; Skin

We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3 - 0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.

Topics: Aging; Animals; Cattle; Chromatography, High Pressure Liquid; Collagen; Cornea; Cross-Linking Reagents; Desmosine; Dipeptides; Histidine; Models, Structural; Skin

Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a "rejuvenation" phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.

Topics: Aged; Aging; Amyotrophic Lateral Sclerosis; Arm; Borohydrides; Collagen; Desmosine; Dipeptides; Female; Histidine; Humans; Male; Middle Aged; Muscular Diseases; Nervous System Diseases; Oxidation-Reduction; Skin