Curved mineral platelets in bone.

Bone is a composite material principally made up of a mineral phase (apatite) and collagen fibrils. The mineral component of bone occurs in the form of polycrystalline platelets 2-6 nm in thickness. These platelets are packed and probably glued together in stacks of two or more, ranging up to >30 platelets. Here we show that most of these stacks are curved flat sheets whose cylindrical axes are oriented parallel to the long axes of collagen fibrils. Consequently, the curvature of the platelets is not detectable in TEM sections cut parallel to the collagen fibril axes. The radius of curvature around these axes ranges from about 25 nm (the average radius of the collagen fibrils) to 100's of nm. The shapes of these curved forms contribute to the compressive strength of bone. STATEMENT OF SIGNIFICANCE: Bone, the material of which bones are made, is mainly composed of a protein, collagen, and the mineral apatite (calcium phosphate). The crystals have long been known to be flat plates about 5 nanometers (nm) thick. Here we show that the crystals are bound together in curved platelets with a radius of curvature between 25 and several hundred nm, which weave between fibrils of collagen. Some platelets wrap tightly around fibrils. The platelets form stacks of from two to up to 30. The crystals in the platelets are all oriented parallel to the cylindrical fibrils even though most crystals are not in contact with collagen. These curved structures provide greater strength to bone.

A search for apatite crystals in the gap zone of collagen fibrils in bone using dark-field illumination.

Bright-field transmission electron microscope (TEM) images of ion milled or focused ion beam (FIB) sections of cortical bone sectioned parallel to the long axis of collagen fibrils display an electron-dense phase in the gap zones of the fibrils, as well as elongated plates (termed mineral lamellae) comprised of apatite crystals, which surround and lie between the fibrils. Energy dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) studies by others have shown that the material in the gap zones is calcium phosphate. Dark-field (DF) images are capable of revealing the projected position of crystals of apatite in a section of bone. We obtained bright field (BF) images of ion milled sections of bovine femoral cortical bone cut parallel to fibril axes (longitudinal view), and compared them with DF images obtained using the (002) apatite reflection to test a widely held theory that most of the mineral in bone resides in the gap zones. Most apatite crystals which were illuminated in DF images and which projected onto gap zones were extensions of crystals that also project onto adjacent overlap zones. However, in BF images, overlap zones do not appear to contain significant amounts of mineral, implying that the crystals imaged in DF are actually in the interfibrillar matrix but projected onto images of fibrils. However a small number of "free" illuminated crystals did not extend into the overlap zones; these could be physically located inside the gap zones. We note that projections of gap zones cover 60% of the area of any longitudinal field of view; thus these "free" crystals have a high random probability of appearing to lie on a gap zone, wherever they physically lie in the section. The evidence of this study does not support the notion that most of the mineral of bone consists of crystals in the gap zone. This study leaves uncertain what is the Ca-P containing material present in gap zones; a possible candidate material is amorphous calcium phosphate.

Bone mineralization is elevated and less heterogeneous in adults with type 2 diabetes and osteoarthritis compared to controls with osteoarthritis alone.

PURPOSE: The purpose of this study was to determine whether trabecular bone mineralization differed in adults with type 2 diabetes compared to adults without type 2 diabetes. METHODS: Proximal femur specimens were obtained following a total hip replacement procedure from men and women ≥65 years of age with and without type 2 diabetes. A scanning electron microscope was used for quantitative backscattered electron imaging (qBEI) analysis of trabecular bone samples from the femoral neck. Gray scale images (pixel size=5.6 µm(2)) were uploaded to ImageJ software and gray level (GL) values were converted to calcium concentrations (weight [wt] % calcium [Ca]) using data obtained with energy dispersive X-ray spectrometry. The following bone mineralization density distribution (BMDD) outcomes were collected: the weighted mean bone calcium concentration (CaMEAN), the most frequently occurring bone calcium concentration (CaPEAK) and mineralization heterogeneity (CaWIDTH). Differences between groups were assessed using the Student's t-test for normally distributed data and Mann-Whitney U-test for non-normally distributed data. An alpha value of <0.05 was considered significant. RESULTS: Thirty-five Caucasian participants were recruited (mean [standard deviation, SD] age, 75.5 [6.5]years): 14 adults with type 2 diabetes (years since type 2 diabetes diagnosis, 13.5 [7.4]years) and 21 adults without type 2 diabetes. In the adults with type 2 diabetes, bone CaMEAN was 4.9% greater (20.36 [0.98]wt.% Ca versus 19.40 [1.07]wt.% Ca, p=0.015) and CaWIDTH was 9.4% lower (median [interquartile range] 3.55 [2.99-4.12]wt.% Ca versus 3.95 [0.71]wt.% Ca, p<0.001) compared to controls. There was no between-group difference in CaPEAK (21.12 [0.97]wt.% Ca for type 2 diabetes versus 20.44 [1.30]wt.% Ca for controls, p=0.121). CONCLUSION: The combination of elevated mean calcium concentration in bone and lower mineralization heterogeneity in adults with type 2 diabetes may have deleterious effects on the biomechanical properties of bone. These microscopic alterations in bone mineralization, which may be mediated by suppressed bone remodeling, further elucidate higher fracture risk in adults with type 2 diabetes.

ESR of E' centres in unstrained geological quartz grains.

E1' centres are detectable by ESR spectroscopy in unstrained quartz extracted from weathered rock of Cretaceous age. These centres completely disappear when the samples are irradiated with an artificial gamma-dose of about 200 Gy. The concentration of E1' centres thermally (re)generated at 310 degrees C can be reduced drastically under the influence of gamma-quanta, too. The gamma-radiation induces thermally unstable components in the quartz matrices that start to decay at around 100 degrees C.

Infant feeding and weaning practices in Roman Egypt.

Current knowledge of infant feeding and weaning practices during the Roman period in Egypt is limited to scanty documentary and iconographic evidence. Stable nitrogen and carbon isotope analysis provides another avenue to explore this question. A sample of 49 infant and juvenile human skeletal remains from the Kellis 2 cemetery in the Dakhleh Oasis, Egypt, was used to determine patterns of infant feeding and weaning. delta(15)N values indicate that supplementary foods were introduced at around 6 months of age, and that weaning was complete by 3 years of age. By 6 months of age, delta(13)C values become increasingly enriched over adult values, and reach peak enrichment at approximately 1.5 years of age. Beyond this age, delta(13)C gradually declines to approach adult values. This enrichment in infant delta(13)C values is indicative of consumption of (13)C-enriched supplementary foods. Based on isotopic study of faunal and botanical remains from the ancient village of Kellis, we conclude that at approximately 6 months of age, infants were fed milk of goat and/or cow.

Glucose ingestion and substrate utilization during exercise in boys with IDDM.

This study was intended to compare exogenous [(13)C]glucose (Glu(exo)) oxidation in boys with insulin-dependent diabetes mellitus (IDDM) and healthy boys of similar age, weight, and maximal O(2) uptake. In a control trial with water intake (CT) and in a (13)C-enriched glucose trial (GT), subjects cycled for 60 min (58.8 +/- 0.9% maximal O(2) uptake) while the utilization of total glucose, total fat, and Glu(exo) was assessed. In CT, total glucose was 84.7 +/- 9.2 vs. 91.3 +/- 6.6 g/60 min (not significantly different) and total fat was 13.3 +/- 2.2 vs. 11.1 +/- 1.7 g/60 min (not significantly different) in IDDM vs. healthy boys, respectively. In GT, Glu(exo) was 10.4 +/- 1.7 vs. 14.8 +/- 1.1 g/60 min, corresponding to 9.0 +/- 1.0 vs. 12.4 +/- 0.5% of the total energy supply in IDDM and healthy boys, respectively (P < 0.05). Endogenous glucose was spared in both groups by 12.6 +/- 3.5% (P < 0.05). Blood glucose and plasma insulin concentrations were two- to threefold higher in IDDM vs. healthy boys in both trials. In conclusion, Glu(exo) is impaired in exercising boys with IDDM, even when plasma insulin levels are elevated.

ESR dating of the Die Kelders Cave 1 site, South Africa.

ESR measurements were made on ten enamel subsamples from six teeth recovered in layers 4-5, 6, 10, and 12 in the site of Die Kelders Cave 1, South Africa. The teeth (enamel and dentine) contained significant concentrations of uranium and therefore the U uptake model has a large influence on the computed ages. Variations in moisture content in the sediment had a smaller effect on the dose rate and calculated ages. For any given model of U uptake and moisture content, all the teeth gave very similar ages, implying that the entire deposit was formed over a short interval (<10,000 y). Comparison with OSL ages for the sediments suggests that the teeth experienced early U uptake, in which case the average age of the deposit is 70+/-4 ka (assuming a moisture content of 10%). Agreement between replicate subsamples was excellent.

Substrate utilization in boys during exercise with [13C]-glucose ingestion.

The influence of glucose ingestion on substrate utilization during prolonged exercise in children and adolescents is currently unknown. In the present study we determined the effect of intermittent exogenous glucose (GLUexo) ingestion on substrate utilization during prolonged exercise, in adolescent boys ages 13 17 years. Healthy untrained volunteers performed four 30-min exercise bouts on a cycle ergometer, separated by 5-min rest periods (approximately equal to 60% maximum O2 consumption), on two occasions spaced 1-4 weeks apart. Two trials were performed, a control trial (CT), in which subjects ingested water intermittently during the exercise, and a glucose trial (GT), in which subjects ingested a 13C-enriched GLUexo drink (approximately egual to glucose kg body mass(-1)), also intermittently during the exercise. Total free fatty acids (FATtotal), glucose (GLUtotal) and carbohydrate (CHOtotal) oxidation was determined from indirect calorimetry, while GLUexo oxidation was calculated from the 13C/12C ratio in expired air after 5-10 min and 25-30 min of exercise in each bout. Heart rate and rating of perceived exertion (RPE) were determined at the same time intervals. The oxidation of CHOtotal was 169.1 (12.9) g x 120 min(-1) and 203.1 (15.9) g x 120 min(-1) (P < 0.01) and that of FATtotal was 31.0 (4.2) g x 120 min(-1) and 17.1 (2.5) g x 120 min(-1) (P < 0.01) in CT and GT, respectively. GLUexo oxidation in GT was 57.8 (4.3) g x 120 min(-1), or 34.2 (2.2)% of that ingested. Endogenous glucose oxidation was 169.1 (12.9) g x 120 min(-1) and 145.3 (11.9) g x 120 min(-1) (P < 0.01) in CT and GT, respectively. Insulin and glucose concentrations were higher in GT than in CT by 226% and 37%, respectively (both P < 0.05). Free fatty acids and glycerol concentrations were lower in GT than in CT, by 27% and 79%, respectively (both P < 0.05). Heart rate was similar between trials, but RPE was lower in GT vs CT at both 115 and 135 min. Thus, under these experimental conditions, GLUexo intake spares endogenous carbohydrate and fat by 16% and 45%, respectively, contributes to approximately 25% of the total energy demand of exercise, and lowers the RPE.

Stable carbon and oxygen isotopes in human tooth enamel: identifying breastfeeding and weaning in prehistory.

This paper investigates the utility of stable carbon and oxygen isotopes in human dental enamel to reveal patterns of breastfeeding and weaning in prehistory. Enamel preserves a record of childhood diet that can be studied in adult skeletons. Comparing different teeth, we used delta13C to document the introduction of solid foods to infant diets and delta18O to monitor the decline of breastfeeding. We report enamel carbonate delta13C and delta18O of 33 first molars, 35 premolars, and 25 third molars from 35 burials from Kaminaljuyú, an early state in the valley of Guatemala. The skeletons span from Middle Preclassic through Late Postclassic occupations, ca. 700 B.C. to 1500 A.D. Sections of enamel were removed from each tooth spanning from the cusp to the cemento-enamel junction. Stable isotope ratios were measured on CO2 liberated by reaction of enamel with H3PO4 in an automated carbonate system attached to a VG Optima mass spectrometer. Within a skeleton, teeth developing at older ages are more enriched in 13C and more depleted in 18O than teeth developing at younger ages. Premolars average 0.5/1000 [corrected] higher in delta13C than first molars from the same skeleton (P = 0.0001), but third molars are not significantly enriched over premolars. The shift from first molars to premolars may be due to the shift to solid foods from lipid-rich milk. After 2 years, when premolars begin to mineralize, the delta13C in childhood diets did not change systematically. First molars and premolars are similar in delta18O, but third molars average 0.7/1000 [corrected] lower than first molars (P = 0.0001) and 0.5/1000 [corrected] lower than premolars (P = 0.0003). First molar and premolar delta18O is heavier, because breast milk is more enriched in 18O than is drinking water. Hence, many children continued to nurse during the period of premolar formation. Together, these results indicate that Kaminaljuyú children had begun to eat solid maize foods before the age of 2 years but continued to drink breast milk until much later.

Neanderthal skeleton from Tabun: U-series data by gamma-ray spectrometry.

The Neanderthal hominid Tabun C1, found in Israel by Garrod & Bate, was attributed to either layer B or C of their stratigraphic sequence. We have used gamma-ray spectrometry to determine the 230Th/234U and 231Pa/235U ratios of two bones from this skeleton, the mandible and a femur. The ages calculated from these ratios depend on the uranium uptake history of the bones. Assuming a model of early U (EU) uptake the age of the Tabun C1 mandible is 34+/-5 ka. The EU age of the femur is 19+/-2 ka. The femur may have experienced continuous (linear) U uptake which would give an age of 33+/-4 ka, in agreement with the mandible's EU age, but implies marked inhomogeneity in U uptake history at the site. These new age estimates for the skeleton suggest that it was younger than deposits of layer C. This apparent age is less than those of other Neanderthals found in Israel, and distinctly younger than the ages of the Skhul and Qafzeh burials. This suggests that Neanderthals did not necessarily coexist with the earliest modern humans in the region. All of the more complete Neanderthal fossils from Israel are now dated to the cool period of the last glacial cycle, suggesting that Neanderthals may have arrived in this region as a result of the southward expansion of their habitable range. The young age determined for the Tabun skeleton would suggest that Neanderthals survived as late in the Levant as they did in Europe.

Latest Homo erectus of Java: potential contemporaneity with Homo sapiens in southeast Asia.

Hominid fossils from Ngandong and Sambungmacan, Central Java, are considered the most morphologically advanced representatives of Homo erectus. Electron spin resonance (ESR) and mass spectrometric U-series dating of fossil bovid teeth collected from the hominid-bearing levels at these sites gave mean ages of 27 +/- 2 to 53.3 +/- 4 thousand years ago; the range in ages reflects uncertainties in uranium migration histories. These ages are 20,000 to 400,000 years younger than previous age estimates for these hominids and indicate that H. erectus may have survived on Java at least 250,000 years longer than on the Asian mainland, and perhaps 1 million years longer than in Africa. The new ages raise the possibility that H. erectus overlapped in time with anatomically modern humans (H. sapiens) in Southeast Asia.

Temporal trends in stable isotopes for Nubian mummy tissues.

From Meroitic to Christian times (350 B.C.-A.D. 1400), Sudanese Nubia experienced political, economic, cultural, and environmental upheaval. Change in any one of these aspects of ancient lifeways can affect subsistence. Dietary patterns from this period are reconstructed by measuring stable carbon and nitrogen isotope ratios in tissue samples from 146 mummies excavated from five sites in the Wadi Halfa area. On average, delta 13C values of bone collagen, muscle, and skin indicate high consumption levels of C3 plants (presumably wheat or barley staples, mixed vegetables, and fruits) throughout the sequence. However, during the X-Group period (A.D. 350-550), there is a statistically significant increase in consumption of C4 plants (millet or sorghum), which are predominant in both the archeological record and in modern crop production for most of the Northern Sudan. The X-Group period was also associated with a low Nile and political and economic restructuring. Increased use of C4 plants on a seasonal basis is also indicated by shifting delta 13C values along hair shafts for both X-Group and Christian periods. delta 15N values suggest that the major source of protein for all time periods came from herbivorous animals. A small, but significant increase in 15N over the 1,000-year sequence could be the result of fertilization.

ESR dating tooth enamel from the Paleolithic site at Longola, Zambia.

A single radiation-sensitive ESR signal at g = 2.0018 occurs in fossil tooth enamel, but not in modern teeth. In dating fossil teeth, the equivalent radiation dose (AD) needed to produce the observed ESR signal is the integral with respect to time of the natural, environmental dose rate (ED) experienced by the tooth during burial. Since the age depends on the U uptake history assumed, three dates are normally calculated assuming early U uptake (EU), continuous (linear) U uptake (LU), or recent U uptake (RU). Generally the LU ages agree best with known ages determined by other methods, but the EU and RU ages are respectively the minimum and maximum ages. Longola Spring Mound, in Central Zambia, contains a Late Stone Age collection occurs on the mound surface. Embedded in layer near the base is a much older layer containing Middle Stone Age artifacts and bone material. Four ungulate teeth collected from the lower layer were ESR dated. EU, LU, and RU ages for each tooth agree very closely, but ages range from 14 to 96 ka. Although the layer may be a two component deposit with teeth averaging 18 +/- 2 ka and 91 +/- 3 ka, high sedimentary Th concentrations and ESR isochrons suggest that gamma ext dose estimates are in error. LU dates estimated from isochron plots average 204 +/- 86 ka, while LU ages calculated with the average isochron-derived gamma ext = 10.79 +/- 1.89 mrad/a average 220 +/- 62 ka. More excavation and dating are necessary to determine if the isochron data is reasonable.(ABSTRACT TRUNCATED AT 250 WORDS)

ESR isochron dating for teeth: a brief demonstration in solving the external dose calculation problem.

The radiation sensitive ESR signal at g = 2.0018 in fossil enamel can be used to date teeth absolutely, providing a suitable U uptake model can be selected, and the external dose rate, Dext, can be accurately calculated. Apart from the uptake model, the most obvious uncertainty lies in Dext. With the isochron method, Dext can be derived easily during the ESR analysis. By plotting the AD vs. the total internal dose rate for teeth where multiple subsamples have been analyzed, a regression line, the isochron, can be determined for each model age calculation. Each isochron slope represents the sample age, while its y-intercept gives the total external dose, Aext.

Evaluation of protein requirements for trained strength athletes.

Leucine kinetic and nitrogen balance (NBAL) methods were used to determine the dietary protein requirements of strength athletes (SA) compared with sedentary subjects (S). Individual subjects were randomly assigned to one of three protein intakes: low protein (LP) = 0.86 g protein.kg-1.day-1, moderate protein (MP) = 1.40 g protein.kg-1.day-1, or high protein (HP) = 2.40 g protein.kg-1.day-1 for 13 days for each dietary treatment. NBAL was measured and whole body protein synthesis (WBPS) and leucine oxidation were determined from L-[1-13C]leucine turnover. NBAL data were used to determine that the protein intake for zero NBAL for S was 0.69 g.kg-1.day-1 and for SA was 1.41 g.kg-1.day-1. A suggested recommended intake for S was 0.89 g.kg-1.day-1 and for SA was 1.76 g.kg-1.day-1. For SA, the LP diet did not provide adequate protein and resulted in an accommodated state (decreased WBPS vs. MP and HP), and the MP diet resulted in a state of adaptation [increase in WBPS (vs. LP) and no change in leucine oxidation (vs. LP)]. The HP diet did not result in increased WBPS compared with the MP diet, but leucine oxidation did increase significantly, indicating a nutrient overload. For S the LP diet provided adequate protein, and increasing protein intake did not increase WBPS. On the HP diet leucine oxidation increased for S. These results indicated that the MP and HP diets were nutrient overloads for S. There were no effects of varying protein intake on indexes of lean body mass (creatinine excretion, body density) for either group. In summary, protein requirements for athletes performing strength training are greater than for sedentary individuals and are above current Canadian and US recommended daily protein intake requirements for young healthy males.

Uranium-series dating and the origin of modern man.

Uranium-series dating is based on measurement of the radioactivity of short-lived daughter isotopes of uranium formed in samples which initially contained only the parent uranium. Materials suitable for U-series dating are found in many prehistoric archaeological sites, and include stalagmitic layers (flowstones), and spring-deposited travertines. Some marls and calcretes are also datable using isochron methods, whereas dates on molluscan shells, bones and teeth are less reliable. Ages obtained using alpha counting to determine isotope ratios have errors greater than 5%, and can range from 1 to 350 ka. Mass spectrometric methods slightly increase the range (0.1-500 ka) but greatly decrease the error to less than 1%, making this the optimal method for high-precision dating of the origin of modern man.

Electron spin resonance (ESR) dating of the origin of modern man.

Many materials found in archaeological sites are able to trap electronic charges as a result of bombardment by radioactive radiation from the surrounding sediment. The presence of these trapped charges can be detected by electron spin resonance (ESR) spectroscopy: the intensity of the ESR signal is a measure of the accumulated dose and thus of the age. Tooth enamel is ubiquitous at archaeological sites and is well suited for ESR dating, with a precision of about 10-20%. This method has now been used to date many sites critical to the biological and cultural evolution of modern man. Dates for sites in Israel and Africa have demonstrated the existence of anatomically modern humans more than 100 ka ago.

ESR dates for the hominid burial site of Es Skhul in Israel.

The Middle East has been critical to our understanding of recent human evolution ever since the recovery of Neanderthal and early anatomically modern fossils from the caves of Tabun and Skhul (Mount Carmel) over 50 years ago. It was generally believed, on archaeological and morphological grounds, that middle eastern Neanderthals (such as those from Tabun, Amud and Kebara) probably dated from more than 50,000 years ago, whereas the earliest anatomically modern specimens (from Skhul and Qafzeh) probably dated from about 40,000 years. Recent thermoluminescence and electron spin resonance (ESR) determinations, however, have supported biostratigraphy in dating the Qafzeh deposits to an earlier part of the late Pleistocene, probably more than 90,000 years ago. These dates have been questioned on unspecified technical grounds, and it has also been argued that they create explanatory problems by separating the morphologically similar Qafzeh and Skhul samples by some 50,000 years, thus implying a long-term coexistence of early modern humans and Neanderthals in the area. Here we report the first radiometric dating analysis for Skhul, using ESR on bovine teeth from the hominid burial levels. Early uptake and linear uptake ages average 81 +/- 15 and 101 +/- 12 kyr respectively. These analyses suggest that the Skhul and Qafzeh samples are of a similar age and therefore it is possible that the presence of early modern humans in the area was episodic, rather than long-term during the early late Pleistocene.

The measurement of whole body water by H2(18)O dilution in newborn pigs.

Whole body water was measured in newborn piglets by isotope dilution with H2(18)O. The results were compared with those obtained by direct analysis of whole body water made by freeze-drying. When the tracer dose of H2(18)O was given to 30 piglets by intragastric gavage, post-equilibration values measured in urine were highly variable (average equilibration error equalled 9%) and the random error of the technique in predicting body water was 13%. When the dose was given to 49 animals intravenously, the post-equilibration values in urine were less variable (equilibration error equalled 4%), the random error of the technique was 7%, and body water was overestimated by 2%. The random error was reduced to 6% if data from animals in which equilibration errors exceeded 10% were excluded. When 18O enrichment in plasma instead of urine was measured, the technique was less precise (random error 11%). The estimation of body water from H2(18)O overestimates body water by 2%. Isotope dilution with 18O is a safe, accurate and repeatable method for the estimation of whole body water, and is suitable for use in adult and newborn humans.