The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process

Serum periostin is associated with cancer mortality but not cancer risk in older home-dwelling men: A 8-year prospective analysis of the STRAMBO study

International audienceBackground: Periostin (POSTN) regulates multiple biological behaviors of tumor cells. We studied the association of serum POSTN with mortality in home-dwelling men.Methods: POSTN was measured at baseline using immunoassay (USCN life science, China) in 815 home-dwelling men aged 60-87 followed-up for 8 years.Results: In the entire cohort, higher serum POSTN was associated with higher all-cause mortality [Hazard Ratio (HR) = 1.30 per Standard Deviation (SD), 95% Confidence Interval (CI): 1.13-1.50, p < .001] after adjustment for potential confounders. In a similar model, cancer mortality (n = 69) increased with POSTN levels (HR = 1.44 per SD, 95%CI: 1.16-1.78, p < .001). Cardiovascular mortality (n = 55) and non-cardiovascular-non-cancer mortality (n = 44) was not associated with POSTN levels (p = .28 and p = .94 respectively). In 107 men with prevalent cancer, all-cause mortality (HR = 1.93 per SD, 95%CI: 1.30-2.87, p < .005) and cancer mortality (HR = 2.07 per SD, 95%CI: 1.23-3.47, p < .01) increased with the increasing POSTN concentrations. In 613 men with incident cancer, higher POSTN concentrations were associated with higher cancer mortality (HR = 1.40 per SD, 95%CI: 1.12-1.76, p < .005) but not with the risk of cancer (HR = 1.16 per SD, 95%CI: 0.89-1.46, p = .21).Conclusions: Higher serum POSTN is associated with higher cancer mortality, but not with the cancer risk in older home-dwelling men

Effect of 72 hrs UV photolysis on enzymatic cross-links on cortical bovine bone measured by HPLC, and on the 1660/1690 cm⁻¹ ratio by FTIRM.

<p>Despite a high decrease of mature PYD cross-links after UV photolysis (and not immature DHLNL cross-links), no difference in the 1660/1690 cm⁻¹ area ratio was observed by FTIRM analysis.</p

Study design.

<p><b>A.</b> Animal study design. <b>B.</b> Body weight of control (full line) and lathyritic (dotted line) rats: the body weight in lathyritic rats started to decrease from the 14th day of ßAPN injection.</p

Biochemical dosages of pyridinoline (PYD), and PYD+DPD (D-pyridinoline) in radii and tibiae.

<p>*Mann Whitney, p≤0.0006.</p><p>PYD and PYD+DPD contents decreased in lathyritic bones compared to the control bones.</p

Image representations in false color of 1660/1690 cm⁻¹ and 1030/1110 cm⁻¹ of a control (left) and a lathyritic radius (right).

<p>The background is set to 0 (blue color). No discrimination between the 2 groups (control and lathyritic bones) was observed in the 1660/1690 cm⁻¹ ratio. Inside a given bone region, the variations in false color from the boundary to the inside emphasize the differences in the collagen maturity and mineral maturity according to the location.</p

Comparison of histological images showing a major exostosis on lathyritic radius.

<p>FTIRM analysis of 1660/1690 cm⁻¹ area ratio revealed no significant difference between control and lathyritic bone.</p

FTIRM analysis.

<p><b>A.</b> Sections of bone (radii) from control (left) and lathyritic (right) rat, stained with Trichrome Goldner. □: zones of measurements (35×35 µm) performed by infrared microspectrocopy either in inner bone (near bone marrow) in white, or in central bone in yellow (10 measurements in each cortical, 4 shown). The dash line indicates the exostosis in lathyritic bone, in lateral part of bone. <b>B.</b> Infrared spectra of center and inner bone in control and lathyritic bones, with curve fitting of amide I and ν₁ν₃PO₄ vibrations. For a given mineral maturity (1030/1110 cm⁻¹ area ratio), there was no difference in the collagen maturity (1660/1690 cm⁻¹) between control and lathyritic bones. However, collagen maturity and mineral maturity area ratios were higher in the center bone compared to the inner bone in both control and lathyritic bones.</p

The C-terminal intact forms of periostin (iPTN) are surrogate markers for osteolytic lesions in experimental breast cancer bone metastasis

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (− 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer