The precision and torque production of common hip adductor squeeze tests used in elite football.

OBJECTIVES: Decreased hip adductor strength is a known risk factor for groin injury in footballers, with clinicians testing adductor strength in various positions and using different protocols. Understanding how reliable and how much torque different adductor squeeze tests produce will facilitate choosing the most appropriate method for future testing. In this study, the reliability and torque production of three common adductor squeeze tests were investigated. DESIGN: Test-retest reliability and cross-sectional comparison. METHODS: Twenty elite level footballers (16-33 years) without previous or current groin pain were recruited. Relative and absolute test-retest reliability, and torque production of three adductor squeeze tests (long-lever in abduction, short-lever in adduction and short-lever in abduction/external rotation) were investigated. Each participant performed a series of isometric strength tests measured by hand-held dynamometry in each position, on two test days separated by two weeks. RESULTS: No systematic variation was seen for any of the tests when using the mean of three measures (ICC=0.84-0.97, MDC%=6.6-19.5). The smallest variation was observed when taking the mean of three repetitions in the long-lever position (ICC=0.97, MDC%=6.6). The long-lever test also yielded the highest mean torque values, which were 69% and 11% higher than the short-lever in adduction test and short-lever in abduction/external rotation test respectively (p<0.001). CONCLUSIONS: All three tests described in this study are reliable methods of measuring adductor squeeze strength. However, the test performed in the long-lever position seems the most promising as it displays high test-retest precision and the highest adductor torque production.

Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal crosslinking.

Collagen was dehydrothermally treated (heat cured) by heating dry under vacuum at 60, 80, 100 and 120 degrees C. The change in stability was determined by subjecting to measurement of gross crosslinking, content of lysino-alanine and naturally occurring collagen crosslinks, shrinkage temperature (TM), susceptibility to digestion by lysosomal thiol proteases, and susceptibility to pepsin and trypsin. Morphological changes were examined by electron microscopy. The in vivo biodegradation of dehydrothermally treated collagen sponges was investigated using a rat lumbar muscle implantation model for up to 28 days. For all heat-cured collagens, the data strongly indicated that both crosslinking and denaturation/degradation was present in increasing quantities with increasing temperature of treatment, its level was too low (maximum 179 pmol mg-1) to account for the decreased solubility and increased molecular weight gross changes observed. Increasing resistance of treated collagen to both lysosomal cathepsins and pepsin correlated well with increased crosslinking and increasing temperature of the heat-curing process. However, increased denaturation/degradation of the collagen at higher temperatures was revealed by electrophoretic analysis, trypsin hydrolysis data and by electron microscopy. Differential scanning calorimetry (d.s.c.) correlated well with these results showing an increased level of denaturation in heated samples. The in vivo study showed little difference between control and heat-cured samples except for the material treated at 120 degrees C which was biodegraded in vivo at a significantly faster rate. The data shows, therefore, that crosslinking induced by the dehydrothermal treatment of collagen decreases its rate of proteolysis at acid pH in vitro. However, the simultaneous denaturation/degradation of the protein during the heat-cure process appears to be a more important factor in determining the fate of the material implanted into rat muscle.

The effect of modification on the susceptibility of collagen to proteolysis: I. Chemical modification of amino acid side chains.

A series of chemically modified collagens were subjected to proteolysis by lysozomal cathepsins, pepsin and trypsin. Modifications of the collagens included acetylation, succinylation, methylation and borohydride reduction. Changes in the integrity of the materials were also monitored by differential scanning calorimetry (DSC). All modified collagens were implanted intramuscularly to assess their relative biodegradation rates in vivo. Methylation of the collagen showed extensive denaturation as confirmed by DSC, pepsin solublization to small fragments and by increased susceptibility to trypsin. However, methylation and succinylation made little difference to hydrolysis by cathepsins. Acetylation and borohydride reduction gave increased resistance to cathepsins as well as to pepsin, this latter also being found with the succinylated substrate. In-vivo implantation data showed both succinylation and methylation increased the rate of biodegradation but that the other modifications did not affect the rate of breakdown when compared with control unmodified collagen. The results of this study showed that chemical modification of collagen can alter in vivo degradation rates and could aid in designing collagen-based prostheses.

The effects of conditioning on meat collagen: Part 3-Evidence for proteolytic damage to endomysial collagen after conditioning.

The effects of conditioning on the endomysial fraction of bovine meat were investigated. Solubility studies on endomysial connective tissue and analysis of insoluble endomysial fractions remaining after conditioning were carried out. Yields of soluble endomysial fractions represented, on average, 94·5% of total extracted endomysial material for unconditioned muscles compared with 97·5% for conditioned muscles. Soluble endomysial fractions contained, on average, 0·13% collagen from unconditioned muscles and 0·22% collagen from conditioned muscles. The main peptide components observed on analysis of insoluble endomysial fractions after CN Br digestion were derived from types I and III collagen. Changes observed on the peptide maps, evident as the appearance of a number of new bands from conditioned samples, appeared to be muscle specific. The amount of type III collagen relative to type I decreased on conditioning, indicating that endomysial type III collagen was preferentially destroyed during conditioning. Two-dimensional polyacrylamide gel electrophoresis maps revealed new peptide material in the molecular weight range 40 000 to 50 000 on conditioning. These data provide direct evidence of the effect of proteolytic processing on endomysial collagen during conditioning.

The effects of conditioning on meat collagen: Part 4-The use of pre-rigor lactic acid injection to accelerate conditioning in bovine meat.

Injection of fresh bovine muscle with 0·1 m lactic acid (to a level of 10% of original muscle weight) resulted in a pH decline to a minimum pH of 5·33 at 15°C only 3 h after injection. Untreated muscle reached the same pH after 26 h when held at the same temperature. Fresh, unconditioned meat colour was unaffected by pre-rigor 0·1 m lactic acid injection as assessed by visual inspection. The percentage of perimysial collagen extracted as the soluble form was significantly higher (P < 0·05) from three muscles of varying quality when pre-injected with 0·1 m lactic acid and conditioned from 1 to 14 days, than from conditioned untreated muscles. SDS-polyacrylamide gel electrophoresis of CNBr peptides from insoluble perimysium obtained from three muscles of varying quality revealed no obvious differences due to pre-rigor lactic acid injection before conditioning. However, analysis of the high molecular weight perimysial collagen CNBr peptides from lactic acid treated muscles by two-dimensional SDS-polyacrylamide gel electrophoresis revealed an increased incidence of degradation in this region compared with untreated controls. These data strongly suggest that pre-rigor injection of beef muscle with lactic acid may accelerate conditioning. The implications of this finding are discussed.

Natural taxonomy of collagen based on amino acid composition.

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.

The effects of conditioning on meat collagen: Part 2-Direct biochemical evidence for proteolytic damage in insoluble perimysial collagen after conditioning.

The effect of proteolytic attack on unwashed and 6 M urea washed bovine perimysial collagen was examined using model systems. Pepsin and cathepsin solubilized collagen continuously over 24h (r = 0·95 andr = 0·88 for time course of pepsin and cathepsin solubilized collagen). Insoluble perimysium treated with pepsin over 24 h resulted in little damage to the insoluble collagenous residue remaining, as evidenced by one-dimensional SDS-polyacrylamide gel electrophoresis. Insoluble perimysium treated with cathepsin resulted in changes to the major peptide bands which were evident after 24 h treatment, visible as broadening and 'fuzziness' of bands, decreased staining intensity, loss of high molecular weight material and a significant reduction in quantity of all peptides when compared with untreated perimysium. The effects of proteolytic action on bovine perimysial collagen in vitro and in conditioned meat were investigated by means of two-dimensional polyacrylamide gel electrophoresis, which provided a more sensitive technique for elucidating changes than the one-dimensional method. The peptide maps obtained from conditioned insoluble perimysium and from insoluble perimysium treated with cathepsin for 24 h were altered relative to the unconditioned insoluble perimysium, with the loss of some peptides and generation of others. The in vitro case was extreme, but was comparable with samples of perimysium from conditioned muscles. The results provide direct biochemical evidence for the presence of proteolytic damage in the insoluble perimysial collagen matrix of meat after conditioning.

Type I and III collagen content and fibre distribution in normal human skin during ageing.

The content of type I and III collagen in normal human skin from subjects of different ages was studied by means of a new high performance liquid chromatography method and by SDS-polyacrylamide gel electrophoresis and scanning electron microscopy. The ratio of types I and III collagen in covered skin remained constant throughout childhood and young adult life and the proportion of type III was shown to be the same as previously reported. However, in the elderly, the proportion of type III collagen in the dermis increased to a variable degree. Scanning electron microscopic examination showed a decrease in the number of collagen fibre bundles with age. Average bundle width varied significantly with age. These results may reflect an impaired synthesis of type I collagen in aged skin.

Chemistry of the collagen cross-links. Origin and partial characterization of a putative mature cross-link of collagen.

The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen.

The effects of conditioning on meat collagen: Part 1-Evidence for gross in situ proteolysis.

The relative effectiveness of various reagents was investigated to determine the best method for extracting damaged collagen or collagen fragments from conditioned meat. SDS- and urea-washing were showd to yield clean connective tissue preparations and to extract the largest quantities of soluble collagen from perimysial preparations. Neutral 6m urea was used thereafter to extract soluble material from perimysial and endomysial preparations made from unconditioned and conditioned beef muscles. Eight bovine muscles of varying quality were examined. Conditioned muscles yielded significantly greater quantities of solubilized perimysial material than unconditioned muscles (P = 0·096) although the total collagen solubilized was very low (3·2-5·6% of total perimysial collagen). The amount of collagen or collagen fragments extracted from the eight perimysial preparations from conditioned muscles was significantly higher (P = 0·015) than from unconditioned muscles. Considerable variability in the percentage solubility of collagen from conditioned muscle perimysium was seen. This appeared to be site-specific. It was not possible to demonstrate similar changes in endomysial preparations due to the difficulty in quantitating the relatively low amounts of collagen in these fractions.

Collagen and elastin changes in D-penicillamine-induced pseudoxanthoma elasticum-like skin.

A patient with cystinuria who was treated with large doses of D-penicillamine for 19 years developed skin abnormalities resembling those seen in pseudoxanthoma elasticum. Biochemical and histological examination of the dermis showed that the collagen content, the ratio of the major genetic forms of collagen and the distribution of collagen types was normal. Light microscopy demonstrated the presence of vastly increased amounts of elastin in the dermis, and the individual elastin fibres were shown by electron microscopy to be abnormal; chemical analysis showed the elastin to be poorly cross-linked. Some of the collagen also appeared structurally abnormal, and biochemically resembled that seen in the dermis of a young child with respect to cross-linking and hexosyl-lysine content. The therapy led to an increased deposition of collagen and elastin fibres which appeared abnormal, and resulted in an increase in total skin surface area. These data indicate that D-penicillamine was not fully effective in inhibiting collagen and elastin cross-linking, and appeared to prevent or inhibit the natural maturation process of the collagen.

Use of galactose oxidase in labelling hydroxylysine glycosides of collagen.

Commercial preparations of galactose oxidase were shown to contain a proteolytic component active against collagen and collagen CNBr peptides which could not be inhibited by reagents such as EDTA, PMSF, PCMB and TLCK nor activated by Ca++ or Mn++. 30% of the total galactose oxidase activity could be recovered free of proteolytic activity in a simple one-step combined gel filtration/affinity chromatography procedure. The biological activity of such preparations was tested with reference to collagen hydroxylysine glycosides. It was shown that over 70% of the covalently linked tritium label introduced by KB3H4 reduction after galactose oxidase treatment was not associated with galactose but was present in the same non-specifically labelled components in control experiments. The enzyme could be used in a controlled manner to label hydroxylysine glycosides in collagen by immobilising it on agarose beads. In this form it was easy to store, re-usable and retained 95% of its original activity even after prolonged (18 h) incubation with substrates.

Mitral valve prolapse, aortic compliance, and skin collagen in joint hypermobility syndrome.

Mitral valve prolapse was sought clinically and with phonocardiography and M mode and sector echocardiography in 15 women aged 22-57 years with joint hypermobility syndrome. The type III:III + I collagen ratio was measured in skin biopsy specimens and was found to be raised in seven of 10 patients sampled. Thirteen patients had increased aortic wall compliance measured by the continuous wave Doppler ultrasound technique. Ten (67%) patients had mitral valve prolapse shown by auscultatory signs or echocardiography or both--a prevalence at least three times greater than that in the general adult population. It is concluded that if the abnormality of collagen biosynthesis found in skin biopsy samples in these patients is also present in their mitral valve tissue this may predispose them to prolapse of the valve.

The borohydride-reducible compounds of human aortic elastin. Demonstration of a new cyclic amino acid in alkali hydrolysate, and changes with age and in patients with annulo-aortic ectasia including one with Marfan syndrome.

Human aortic elastin reduced with [3H]borohydride was analysed by ion-exchange chromatography after alkali or acid hydrolysis. Alkali hydrolysates of elastins contained a radioactive peak that was eluted between proline and leucine. This peak was not present in foetal elastin, but its proportion increased steadily during aging. Aortic samples from patients with annulo-aortic ectasia (aneurysm of the ascending aorta), including one with classical Marfan syndrome, contained less elastin (CNBr-insoluble material) than did the age-matched controls. The proportion of radioactivity in the new peak of all these aortas was low when compared with age-matched controls. Gas-chromatographic/mass-spectrometric analysis suggested that it contained a cyclic derivative of a hydrated aldol-condensation product. The concentration of the cross-link precursors, lysine aldehyde and aldol-condensation product (estimated from the acid-hydrolysis product 6-chloronorleucine and the acid-degradation product of reduced aldol-condensation product) was high in very young aortas but remained quite stable after childhood. No differences were observed in cross-link profiles of acid hydrolysates between pathological and control aortas. A low proportion of radioactivity in the new peak may indicate the presence of young or immature elastin in the pathological aortas.

Lipoid proteinosis: an inherited disorder of collagen metabolism?

The dermal collagen of a patient with lipoid proteinosis was investigated by immunohistochemistry and biochemical analysis. The affected skin was found to contain significantly less collagen per unit dry weight than normal dermis but showed elevated levels of type 3 collagen with respect to type I. Purification of collagen types from affected skin after pepsin digestion showed no novel forms, but a doubling in the yield of type 5 collagen. These results correlated well with those of immunohistochemistry which showed a patchy, diffuse, widely distributed type 3 collagen and an increase in types 4 and 5 collagens associated with 'onion skin' endothelial basement membrane thickening. Estimation of collagen cross-links showed an abnormal pattern with a preponderance of the keto-imine form not normally associated with skin. These results strongly suggest that lipoid proteinosis involves a primary perturbation of collagen metabolism.

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.

Evidence for glucose-mediated covalent cross-linking of collagen after glycosylation in vitro.

Rabbit forelimb tendons incubated for 15 or 21 days at 35 degrees C in the presence of 8 or 24 mg of glucose/ml were shown to change their chemical, biochemical and mechanical characteristics. The tendons treated with glucose contained up to three times as much hexosyl-lysine and hexosylhydroxylysine as did control tendons as judged by assay of NaB3H4-reduced samples. Measurement of the force generated on thermal contraction showed significant increases in glycosylated tendons compared with controls, indicating the formation of new covalent stabilizing bonds. This conclusion was supported by the decreased solubility of intact tendons and re-formed fibres glycosylated in vitro, and by the evidence from peptide maps of CNBr-digested glucose-incubated tendons. The latter, when compared with peptide maps of control tendons, revealed the presence of additional high-Mr peptide material. These peptides appear to be cross-linked by a new type of covalent bond stable to mild thermal and chemical treatment. This system in vitro provides a readily controlled model for the study of the chemistry of changes brought about in collagen by non-enzymic glycosylation in diabetes.

Intermolecular cross-linking in fibrotic collagen.

The extent, nature and location of the cross-links involved in the stabilization of collagen in fibrotic lesions are crucial to its subsequent removal, naturally or induced by treatment. Stabilization is achieved initially by divalent aldimine and keto-imine intermolecular cross-links located at the end-overlap region in the quarter-stagger alignment of the molecules in the fibre. Elucidation of the location of the cross-links also provides chemical evidence for the organization of the collagen molecule in the fibre. All the fibrous collagens are stabilized by these cross-links, the more stable keto-imine cross-link predominating in the types I and II collagens present in the initial stages of fibrosis. Further stabilization of the lesion usually follows, increasing the resistance to degradative enzymes, thus rendering the fibrosis irreversible. This maturation process, which also occurs in normal ageing, involves the formation of multivalent cross-links derived from the initial aldimine and keto-imine cross-links to form a three-dimensional network through a polymeric peptide (poly-alpha 1CB6 in type I collagen). The nature of these cross-links has not yet been elucidated. The so-called mature cross-link, 3-hydroxypyridinoline, could not be identified in this polymeric network. A secondary process involving non-enzymic glycosylation of lysine residues and subsequent intermolecular cross-linking has also been demonstrated, although the nature and extent of this type of cross-link remain to be determined.