Hydroxylysylpyridinoline Concentrations Research Articles

Organic Bone Matrix Component Type I and V Collagen Are Not Destructed in Bisphosphonate-Associated Osteonecrosis of the Jaws.

Background and objectives: The investigation of the pathophysiology behind medication-related osteonecrosis (MRONJ) of the jaw mostly focuses on alterations in osteoclast and osteoblast cell activity, but changes in the organic and inorganic bone matrix have rarely been studied. The aim of this study was to investigate whether collagen, the main organic component of extracellular bone matrix, is destructed in osteonecrosis of the jaw secondary to antiresorptive medication. Material and methods: Bone samples of patients with MRONJ (n = 15, control group n = 3) were demineralized, and collagen fragments were separated from intact collagen pellets by ultrafiltration. The quantification of mature collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) in pellets and ultrafiltrates was performed by high-performance liquid chromatography (HPLC). The detection of hydroxyproline (Hyp) was carried out using a spectrophotometric assay. In addition, collagen chains were analyzed by sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE). Results: The results revealed significantly higher concentrations of HP, LP and Hyp in pellet samples. In addition, there were no significant differences between samples from MRONJ patients and those of the control group. These results were paralleled by SDS- PAGE. Conclusion: These findings suggest that MRONJ does not involve the destruction of type I and V collagen molecules, in contrast to previously reported destruction by osteoradionecrosis.

Mechanical properties and collagen cross-linking of the patellar tendon in old and young men

Age-related loss in muscle mass and strength impairs daily life function in the elderly. However, it remains unknown whether tendon properties also deteriorate with age. Cross-linking of collagen molecules provides structural integrity to the tendon fibrils and has been shown to change with age in animals but has never been examined in humans in vivo. In this study, we examined the mechanical properties and pyridinoline and pentosidine cross-link and collagen concentrations of the patellar tendon in vivo in old (OM) and young men (YM). Seven OM (67 +/- 3 years, 86 +/- 10 kg) and 10 YM (27 +/- 2 years, 81 +/- 8 kg) with a similar physical activity level (OM 5 +/- 6 h/wk, YM 5 +/- 2 h/wk) were examined. MRI was used to assess whole tendon dimensions. Tendon mechanical properties were assessed with the use of simultaneous force and ultrasonographic measurements during ramped isometric contractions. Percutaneous tendon biopsies were taken and analyzed for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), pentosidine, and collagen concentrations. We found no significant differences in the dimensions or mechanical properties of the tendon between OM and YM. Collagen concentrations were lower in OM than in YM (0.49 +/- 0.27 vs. 0.73 +/- 0.14 mg/mg dry wt; P < 0.05). HP concentrations were higher in OM than in YM (898 +/- 172 vs. 645 +/- 183 mmol/mol; P < 0.05). LP concentrations were higher in OM than in YM (49 +/- 38 vs. 16 +/- 8 mmol/mol; P < 0.01), and pentosidine concentrations were higher in OM than in YM (73 +/- 13 vs. 11 +/- 2 mmol/mol; P < 0.01). These cross-sectional data raise the possibility that age may not appreciably influence the dimensions or mechanical properties of the human patellar tendon in vivo. Collagen concentration was reduced, whereas both enzymatic and nonenzymatic cross-linking of concentration was elevated in OM vs. in YM, which may be a mechanism to maintain the mechanical properties of tendon with aging.

Proof of Direct Radiogenic Destruction of Collagen in Vitro

Fibroses of vessels and soft tissue are side effects of radiotherapy. The authors assumed that there was an immediate direct radiogenic damage of collagen of bone, periosteum and skin. 15 porcine jaws samples (group 1) were exposed to a total dose of 60 Gy (cobalt-60, 2 Gy/day, five fractions/week). 15 jaws samples were stored accordingly (group 2, no irradiation, control). Collagen fragments of bone, periosteum and skin samples of groups 1 and 2 were isolated by ultrafiltration. Collagen types were characterized by SDS-PAGE measurement of the mature collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) by high-performance liquid chromatography (HPLC) and analysis of hydroxyproline (Hyp) was used to determine the ratio of the amount of collagen fragments from irradiated as opposed to nonirradiated samples. The concentrations of HP, LP and Hyp in ultrafiltrates of probes of irradiated bone, periosteum and skin were markedly increased (average factors for bone: 3.69, 1.84, and 3.40, respectively; average factors for periosteum: 1.55, 1.41, and 1.77, respectively; average factors for skin: 1.55, 1.60, and 2.23, respectively) as compared to nonirradiated probes. SDS-PAGE did show collagen types I and V in nonirradiated bone, I and III in nonirradiated skin, and I in nonirradiated periosteum samples. In irradiated samples, smeared bands illustrated fragmentation of the collagen molecule. The increased concentrations of HP, LP and Hyp in ultrafiltrates indicated increased concentrations of split collagen. Direct and instant radiogenic damage of (extracellular matrix of) bone, periosteum and skin tissue collagen could be demonstrated.

A new method of monitoring osteomyelitis

Chronic infections of bone such as osteomyelitis are frequent events, especially in immunocompromised or diabetic patients, and costly on a national level. Incorrect treatment or delayed diagnosis may lead to loss of the affected extremity or mandible. The aim of this study was to assess the possible value of urinary lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) concentrations in the monitoring of mandibular osteomyelitis. Patients were assigned to the following groups: group 1 ( n = 85), control; group 2a ( n = 38), patients with active disease; group 2b ( n = 25), patients of group 2a 6 months after successful treatment; group 2c ( n = 7), patients of group 2a with ongoing osteomyelitis 6 months after treatment. The range and upper limit of normal values (HP max and LP max) were determined in group 1. Levels of LP and HP were measured by high-performance liquid chromatography and fluorescence detection. There was a significant decrease (mean 45.43% for HP and 32.12% for LP) in samples of group 2b compared to 2a ( P < 0.001 for HP and LP). There was a significant increase in HP values in samples from group 2c compared to 2a ( P = 0.018). The urinary concentrations of HP and LP appear to act as a marker of disease activity, with a decrease reflecting treatment success and an increase or stable values indicating persistent disease. An inexpensive tool (US$5 per analysis) for the monitoring of osteomyelitis is described.

Increased excretion of collagen crosslinks in irradiated patients indicates destruction of collagen

Purpose: In a recent study we showed an instant radiogenic destruction of collagen in dental tissues. The hypothesis of this analysis was that there is a destruction of collagen directly in bone during irradiation. Our intention was to prove this assumption by the analysis of the intraindividual progression of urinary excretion of bone specific collagen crosslinks before, during and after radiotherapy (RT) of secondary malignant bone tumors.Material and methods: Forty-six patients were irradiated with a mean dose of 32 Gy (range 30 – 46 Gy). Four urine probes were collected from each patient before, during, at the end and 6 – 8 weeks after RT. Measurement of the mature collagen crosslinks hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) was performed by high performance liquid chromatography (HPLC).Results: In 43 patients we found increasing HP and LP concentrations by comparing the different chosen time-points. As regards to HP the urine excretion was significantly increased at the end (p = 0.02) and six weeks after RT (p = 0.01) and for LP six weeks after RT (p = 0.01). We observed significantly higher urinary HP levels in patients treated with doses of 35 – 46 Gy as compared to patients treated with 30 Gy (p = 0.05).Conclusions: The urinary excretion of HP and LP was increased directly after a course of RT. This finding may reflect either destruction of collagen or increased bone remodeling/resorption after radiotherapy. Either way these findings suggest a relation to the mechanical instability of bone directly after radiotherapy. Future investigations of irradiated patients without osseous metastases will further clarify this matter.

Detection of Mature Collagen in Human Dental Enamel

Mature dental enamel is the most mineralized of all mammalian tissues and considered to be free of collagen. Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are two nonreducible cross-links of mature collagen. Hydroxyproline (Hyp) is an amino acid that is believed to be indicative of the presence of collagen. We set out to assess the concentrations of Hyp, HP, and LP in dental enamel and dentin (control) to clarify whether there was minor collagen content in dental enamel. We studied 17.53 g of enamel and 22.12 g of dentin gained from 120 extracted human teeth. Enamel and dentin (control) were separated with a diamond dental drill under microscopic control by wasting a margin of enamel (Ca. 2 mm) at the dentin-enamel border. Collagen alpha-chains were analyzed by Sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) after decalcification and collagen extraction. Concentrations of HP and LP where measured by using high-performance liquid chromatography (HPLC). Hyp was analyzed by a spectrophotometric method. The pooled probe of enamel contained 0.23 mug/g of Hyp. This concentration was 49 times lower than that in dentin. Concentrations of HP and LP in enamel were 0.07 nmol/g and 0.02 nmol/g, respectively being 605.57 (HP) and 251.50 (LP) times lower in enamel as compared to dentin. Collagen type I was found in enamel; collagen types I and V were found in dentin samples. In reports of many studies and textbooks, collagen is considered to be completely absorbed in the course of the mineralization and maturation of dental enamel. We show that this is not the case. However, the concentration of collagen in enamel was considerably lower as compared to that in dentin.

Differential enhancement of collagen crosslink excretion in cases of osteosarcoma and chondrosarcoma

Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are markers of collagen absorption and LP is specific for collagen type I in bone. In the present study we evaluated the concentration of HP and LP in urine of patients with osteosarcoma ( n=20; age range 16-49 years) and chondrosarcoma ( n=15; age range 18-70 years). The values were compared with those obtained from 74 healthy controls (age range 16-83 years). The range and upper limit of normal values (HP(max) and LP(max)) were measured in our control group. High performance liquid chromatography (HPLC) was used to determine concentrations of HP and LP (nmol/mmol creatinine). The average urinary HP concentrations were significantly increased in patients with osteosarcoma ( p=0.001) and chondrosarcoma ( p<0.001), whereas HP remained within the normal range in approximately half of the patients. The average urinary LP concentrations were not increased in osteosarcoma and chondrosarcoma patients as compared with the control group. Further studies in a large group of patients are necessary to evaluate whether HP might be a valuable marker of prognosis, and if its urinary concentration can be correlated to tumour burden.

Elevated levels of collagen cross-link residues in gingival tissues and crevicular fluid of teeth with periodontal disease.

Lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) are collagen cross-link residues. Lysylpyridinoline is present in most tissues, whereas LP is present mainly in mineralized tissue. Both are elevated in tissue with increased collagen resorption. The purpose of this investigation was to assess if the concentrations of LP and HP are elevated in gingiva and gingival crevicular fluid (GCF) of teeth with advanced periodontitis (AP). We investigated human gingival biopsies of healthy teeth (n = 19) and teeth with AP (n = 43) in 49 individuals. Samples of GCF from 54 teeth with AP were collected in seven patients and compared with samples from 11 patients with experimentally induced gingivitis. Levels of LP and HP were measured by HPLC and fluorescence detection. Gingival concentrations of HP but not LP around teeth with advanced periodontitis were significantly elevated compared with teeth with healthy periodontium. While significant amounts of HP and LP were measurable in the GCF of teeth with AP, no HP and LP was identified 3 months following non-surgical periodontal therapy of the teeth or in fluid from teeth subjected to experimentally induced gingivitis. Elevated concentrations of HP and LP in GCF may serve as indicators of ongoing destruction of periodontal tissues and alveolar bone in advanced periodontitis.

Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross-linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44-88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross-links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross-links, that is, hydroxylysyl-pyridinoline (HP), lysyl-pyridinoline (LP), and pyrrole cross-link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross-link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.

Growth, slaughter and intra-muscular collagen characteristics in Garganica kids

Growth performance, ossification of metacarpal growth plate, and intra-muscular collagen characteristics in three muscles of 10 intact male Garganica kids were studied. Each week, starting from the 47th day of age to slaughter (75th day of age), kids were weighed and blood samples taken for plasma non-protein hydroxyproline analysis. At slaughter, metacarpal, metatarsal bones and metacarpal growth plate thickness were measured. The semitendinosus (ST), semimembranosus (SM), and rectus medialis (RM) muscle samples were analysed for collagen concentration and hydroxylysyl pyridinoline (HP) cross-links. ST muscle had a collagen amount higher than SM and RM ( P<0.01), and RM muscle had the highest ( P<0.01) HP concentration. Tenderness index was the highest ( P<0.01) in RM muscle. Animal growth rates slowed, starting from 61st day of age ( P<0.01); this trend coincided with a marked reduction in concentration of plasma non-protein hydroxyproline ( P<0.01). In addition, plasma non-protein hydroxyproline concentration was positively correlated ( r=0.61, P=0.001) with growth rate of animals, suggesting that collagen turnover decreases during a slow growth phase. Based on the metacarpal growth plate width, the rate of bone ossification appears to be slower in goats than sheep.

Regional differences in LV collagen accumulation and mature cross-linking after myocardial infarction in rats.

To determine the extent of and any regional differences in remodeling response of the extracellular matrix (ECM) to myocardial infarction (MI), moderate-to-large transmural infarcts were surgically produced in left ventricular (LV) free wall of rats. Animals were killed 13 wk after surgery. In comparison to age-matched controls, infarction was associated with an overall increase in heart weight, which included hypertrophy of both the right ventricle and LV. Although the remaining viable myocardium in LV free wall was significantly reduced, the interventricular septum was hypertrophied some 30% compared with control tissues (247 +/- 9 vs. 189 +/- 8 mg). Collagen concentration more than doubled in remaining viable free wall (8.92 +/- 0.59 vs. 3.95 +/- 0.25 mg/100 mg, P < 0.0001), and a smaller but still highly significant 27% increase occurred (P < 0.01) in the more remote septum. Degree of covalent cross-linking of collagen fibrils as assessed by hydroxylysylpyridinoline (HP) concentration also revealed regional differences in response of the ECM to infarction. Although HP concentration was increased 60% in viable free wall (P < 0.05) post-MI, it was unchanged in the septum. With respect to collagen characteristics of the transmural infarct per se, the scar exhibited still further increases in both collagen and HP concentrations compared with the already elevated values for these two parameters in viable free wall. The results indicate that any evaluation of the remodeling response of viable myocardium post-MI must include not only the myocyte but also the ECM, the principal component of which is collagen.

Non-Reducible Collagen Cross-Linking in Cartilage from Broiler Chickens with Tibial Dyschondroplasia

Recent work has shown that hydroxylysylpyridinoline (HP), a non-reducible cross-link that stabilizes the collagen fibril network, is significantly greater (over 10-fold) in dyschondroplastic cartilage than in normal growth-plate cartilage in the tibiotarsi of chickens with homocysteine-induced tibial dyschondroplasia (TD). In the present study, broiler chicks with a genetic disposition to TD, as well as normal broiler chicks on a copper-deficient diet alone or supplemented with copper and thiram, were raised for 3 to 4 weeks (Expts. 1 and 2). Their dyschondroplastic cartilage from the proximal tibiotarsus was collected and analyzed for HP as well as lysylpyridinoline (LP) cross-links. Normal growth plate cartilage was obtained from chicks on the copper-deficient diet supplemented with copper. In a third experiment, another set of broiler chicks was raised on a corn/soybean meal-based diet with or without homocysteine, and their articular and sternal cartilage were isolated for cross-link analysis. In the first two experiments, dyschondroplastic cartilage from all birds with induced TD had higher HP and LP concentrations than growth-plate cartilage from birds without TD, although the ratios of HP to LP varied. In the third experiment, the sternal and articular cartilage from birds with homocysteine-induced TD appeared normal, having similar HP concentrations in the same types of cartilage in birds without TD.