Miller L M, Vairavamurthy V, Chance M R, Mendelsohn R, Paschalis E P, Betts F, Boskey A L
Albert Einstein Center for Synchrotron Biosciences, Upton, NY 11973-5000, USA.
Biochim Biophys Acta. 2001 Jul 2;1527(1-2):11-9. doi: 10.1016/s0304-4165(01)00093-9.
Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.
原位测量骨矿物质含量和成分有助于深入了解骨矿物质沉积的化学过程。红外（IR）显微光谱法非常适合此目的。迄今为止，对骨磷灰石的红外显微（包括成像）分析主要集中在ν(1)、ν(3) PO₄³⁻ 谱带。ν(4) PO₄³⁻ 谱带（500 - 650 cm⁻¹），已被广泛用于监测匀浆骨样品中羟基磷灰石的结晶度，该谱带位于商用红外显微镜中碲镉汞探测器的截止频率以下，因此使得该振动无法用于成像研究。本研究报告了在ν(4) PO₄³⁻ 光谱区域中人类髂嵴横截面的首个红外显微光谱，该光谱是使用同步辐射源和与红外显微镜耦合的掺铜锗探测器获得的。绘制了人类骨单位的酸性磷酸盐（HPO₄²⁻）含量和矿物微晶完善程度（结晶度）图。为了建立光谱 - 结构相关性，从一系列不同物种和年龄的合成羟基磷灰石晶体以及天然骨粉中获得了X射线粉末衍射数据和常规傅里叶变换红外光谱。X射线粉末衍射数据表明，随着骨成熟，平均晶体尺寸增加，这与ν(4) PO₄³⁻ 轮廓内两个峰（603/563 cm⁻¹）的ν(4) PO₄³⁻ 傅里叶变换红外吸收峰比值增加相关。此外，红外结果表明，540 cm⁻¹ 附近的一个谱带可能归属于酸性磷酸盐。该谱带在新骨中浓度较高，并随着骨成熟而降低。ν(4) PO₄³⁻ 轮廓与ν(2) CO₃²⁻ 轮廓的相关性还表明，当酸性磷酸盐含量高时，A型碳酸盐含量（即占据羟基磷灰石晶格中OH⁻ 位点的碳酸盐）高。随着结晶度增加和酸性磷酸盐含量降低，碳酸盐取代向占据羟基磷灰石晶格中的PO₄³⁻ 位点转变。因此，对ν(4) PO₄³⁻ 轮廓的红外显微分析提供了相对矿物结晶度和酸性磷酸盐浓度的直接指标，可应用于骨样品的原位红外显微光谱分析，这对于理解正常和病理状态下骨沉积的化学机制具有重要意义。
