Bone Collagen Samples Research Articles

Carbon and nitrogen stable isotopic signatures of human dietary change in the Georgia Bight

Measurement of carbon and nitrogen stable isotope ratios (delta 13C and delta 15N) in samples of human bone collagen (n = 93) from a temporal series of four prehistoric (early preagricultural, late preagricultural, early agricultural, late agricultural) and two historic (early contact, late contact) periods from the Georgia Bight, a continental embayment on the southeastern U.S. Atlantic coast, reveals a general temporal trend for less negative delta 13C values and less positive delta 15N values. This trend reflects a concomitant decrease in emphasis on marine resources and increased reliance on C4-based resources, especially maize. This dietary reorientation is most apparent for the early agricultural sample (AD 1150-1300), coinciding with the Mississippian fluorescence in the eastern United States. There is, however, a shift toward the use of C3 (non-maize) foods during the last prehistoric period (AD 1300-1450), which is likely related to environmental stress and social disruption. A heavier use of maize and terrestrial resources in general after the establishment of mission centers on barrier islands is indicated. A reduced dietary breadth during the mission period may have contributed to the extinction of these populations in the eighteenth century.

The effect of complexing phosphoproteins to decalcified collagen on in vitro calcification.

Decalcified samples of chicken bone containing phosphoproteins of varying concentrations were used to assess the effect of phosphoproteins and of protein-bound Ser(P) and Thr(P) in the in vitro nucleation of a Ca-P solid phase from metastable solutions of Ca and P. Phosphoproteins of bone as well as the phosphoproteins from egg yolk (phosvitin) were used. Increasing concentrations of phosphoprotein [as measured by the amount of protein bound Ser(P) and Thr(P)] in the decalcified bone particles significantly reduced the time required for nucleation to occur after exposure to metastable solutions of Ca and P (decreased operational lag times). Treatment with wheat germ acid phosphatase markedly reduced the concentration of Ser(P) and Thr(P) in the decalcified bone samples and in the decalcified bone collagen samples complexed with phosphoproteins (almost to zero). The loss of the organic phosphate groups significantly increased the operational lag time, but did not abolish nucleation of apatite crystals by the bone collagen fibrils essentially devoid of Ser(P) and Thr(P). Bone phosphoproteins were not specific; substitution of phosvitin for bone phosphoproteins as complexes with bone collagen also proved to be effective facilitators of nucleation, which was interesting since both types of phosphoproteins have certain common chemical and structural characteristics. Noncollagenous components other than phosphoproteins were present in the decalcified bone samples. However, the marked dependence of the lag time on the Ser(P) and Thr(P) concentrations and the very marked diminution in the efficacy of the nucleation phenomenon as a result of treatment with wheat germ acid phosphatase, clearly suggests that the organic phosphate residues of the phosphoproteins play a direct and significant role in the process of in vitro nucleation of a solid phase of Ca and P (apatite) by bone collagen, and by implication, possibly in in vivo mineralization as well.

Extent and topography of the acetylation of calcified chicken-bone collagen.

Acetic anhydride in anhydrous acetone-triethylamine reacts with decalcified bone collagen in two ways. It completely acetylates the e-amino groups of lysine and hydroxylysine and causes a progressive change in the protein matrix, such that increasing amounts of degraded peptides become soluble in alkaline phosphate solution. The peptides produced have molecular weights ranging from 100,000 to less than 2,000 daltons. The number of peptides obtained indicates that there are about 10 bonds cleaved per chain. Ball-mill grinding, particle size of the decalcified bone fragments, and age of the chicken do not affect the fraction of collagen that can be solubilized. The appearance of peptides begins at the point where 90% of the e-amino groups are modified. Both the phosphate-soluble and phosphate-insoluble fractions were essentially completely N-acetylated. Calcified bone under the same conditions is rapidly acetylated to only 45% of the total e-amino groups. With further additions of reagent, the degree of modification of the phosphate-insoluble matrix stabilizes at 50 +/- 4%. At the same time, progressively larger amounts of peptides, soluble in alkaline phosphate solution, are produced. The peptides, which approach 100% N-acetylation, have a distribution of molecular weight from 2000 to 75,000 daltons, indicating 10-15 disrupted peptide bonds per chain. The peptides are initially derived from the more accessible regions of the matrix and subsequently are produced from increasingly inaccessible regions. Chromatography of samples of calcified bone collagen, which were acetylated nearly to completion, indicate that the lysyl and hydroxylysyl residues which are most difficult to modify in the calcified bone are located along continuous regions of the collagen chains. Chromatography of partially acetylated samples also show that all regions of the chains are not uniformly accessible to modification, and further, no single region of every alpha chain is exposed to an identical environment.