Nicholas C. Avery

Changes in collagen cross-linking in degenerative disc disease and scoliosis

Study Design. Biochemical study of the changes in the collagen cross‐link profile of human intervertebral discs collected at surgery from patients with either low back pain associated with disc degeneration or scoliosis. Objective. To determine whether changes occur in the collagen cross‐link profile in the disc of patients with either low back pain associated with disc degeneration or scoliosis, which may well influence matrix integrity. Such changes in the cross‐link profile of a tissue indicates increased matrix turnover and tissue remodeling and may have implications for the progression of these disorders. Summary of Background Data. The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. The stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc is dependent on the degree and type of cross‐links between the collagen molecules. This article reports results on the distribution of the different cross‐links in the disc and the changes that occur with age, degenerative disc disease, and scoliosis. Methods. Thirty‐three discs were obtained from patients with degenerative disc disease and 29 discs from patients with scoliosis. Samples were acid hydrolyzed and the collagen cross‐links analyzed by either fractionation on an amino acid analyzer configured for cross‐link analysis using ninhydrin postcolumn detection or fractionation by high‐pressure liquid chromatography with fluorescence detection. Results. The reducible cross‐links and the mature cross‐link all increased from the outer anulus fibrosus through into the nucleus pulposus. The highest levels of the mature cross‐link were found in the cartilage endplate. The nonenzymic derived cross‐link, pentosidine, in contrast, showed little difference across the disc, but did show the expected age‐related increase. In degenerative disc disease, no change in the levels of the reducible or mature cross‐links was found, but a decrease was observed in the levels of the age‐related cross‐link pentosidine in the more severe disease samples. In scoliosis, significantly higher levels of the reducible cross‐links were found on the convex than on the concave side of the scoliotic disc. Conclusions. These changes in the cross‐link profile of the intervertebral disc in degenerative disc disease and scoliosis are indicative of increased matrix turnover and tissue remodeling and likely to have implications for the progression of these disorders.

Quantitative Determination of Collagen Cross-links

The primary functional role of collagen is as a supporting tissue and it is now established that the aggregated forms of the collagen monomers are stabilised to provide mechanical strength by a series of intermolecular cross-links. In order to understand the mechanical properties of collagen, it is necessary to identify and quantitatively determine the concentration of the cross-links during their changes with maturation, ageing and disease. These cross-links are formed by oxidative deamination of the epsilon-amino group of the single lysine or hydroxylysine in the amino and carboxy telopeptides of collagen by lysyl oxidase, the aldehyde formed reacting with a specific lysine or hydroxylysine in the triple helix. The divalent Schiff base and keto-amine bonds so formed link the molecules head to tail and spontaneously convert during maturation to trivalent cross-links, a histidine derivative and cyclic pyridinolines and pyrroles, respectively. These latter bonds are believed to be transverse inter-fibrillar cross-links, and are tissue rather than species specific. We describe the determination of these cross-links in detail.Elastin is also stabilised by cross-linking based on oxidative deamination of most of its lysine residues to yield tetravalent cross-links, desmosine and iso-desmosine, the determination of which is also described.A second cross-linking pathway occurs during ageing (and to a greater extent in diabetes mellitus) involving reaction with tissue glucose. The initial product glucitol-lysine can be determined as furosine and pyridosine, and determination of advanced glycation end-products believed to be cross-links, such as pentosidine, are also described.

Identification of an Intermediate State in the Helix-Coil Degradation of Collagen by Ultraviolet Light*

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 °C following UV irradiation of collagen, compared with 39 °C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Collagen cross-linking in porcine m. longissimus lumborum: Absence of a relationship with variation in texture at pork weight.

The determination of all currently known intermolecular cross-links present in intramuscular collagen of porcine m. longissimus lumborum is described in relation to the texture of the meat as determined both objectively by instrumentation and subjectively by sensory panel. The variation in texture observed in the m. longissimus lumborum of pork weight pigs has been shown to be unrelated to the total collagen content or to the nature of the collagen intermolecular cross-links. We have also demonstrated a considerable error in the colorimetric method for quantitation of hydroxyproline when determining the very low values of collagen present in pig meat. During this study we have established a sound protocol for the determination of all the known cross-links in intramuscular collagen of meat from any meat animal species.

Identification of a New Cross-link and Unique Histidine Adduct from Bovine Serum Albumin Incubated with Malondialdehyde

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1ϵ-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nϵ-(2-carboxyl,2-aminoethane)-Nϵ-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.

Altered Collagen in Tartrate-Resistant Acid Phosphatase (TRAP)- Deficient Mice: A Role for TRAP in Bone Collagen Metabolism

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young’s modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.

An efficient method for the isolation of intramuscular collagen

Ultrasonic fragmentation of the myofibrils and subsequent solubilisation in buffer has been used to isolate intramuscular collagen (IMC) in high yield and purity. The method is superior to previously reported techniques in providing both a high yield of collagen and intact fibres. The material obtained is suitable for both physical and biochemical analysis in attempts to demonstrate its role in determining the tenderness of meat.

Thermal stabilization of collagen in skin and decalcified bone

The state of collagen molecules in the fibres of tail tendon, skin and demineralized bone has been investigated in situ using differential scanning calorimetry (DSC). Hydroxyproline analysis and tissue digestion with bacterial collagenase and trypsin were used to confirm that the common cause of all the DSC endotherms was collagen denaturation. This occurred within a narrow temperature range in tendons, but over a wide temperature range in demineralized bone and old skin and demonstrated that in tendon and demineralized bone at least the same type I collagen molecule exists in different thermal states. Hypothesizing that this might be caused by different degrees of confinement within the fibre lattice, experiments were performed to measure the effect of changing the lattice dimensions by extracting the collagen into dilute solution with pepsin, swelling the lattice in acetic acid, and contracting the lattice by dehydration. A theoretical analysis was undertaken to predict the effect of dehydration. Results were consistent with the hypothesis, demonstrating that collagen molecules within the natural fibres of bone and old skin are located at different intermolecular spacings, revealing differences between molecules in the magnitude of either the attractive or repulsive forces controlling their separation. One potential cause of such variation is known differences in covalent cross-linking.

Collagen in its fibrillar state is protected from glycation

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.