Aspartic Acid Racemization Research Articles

Aspartic acid racemization reveals a high turnover state in knee compared with hip osteoarthritic cartilage

We investigated tissue turnover in healthy and osteoarthritic cartilage. We challenge long held views that osteoarthritis (OA) is dominated by a similar turnover process in all joints and present evidence that hip and knee cartilage respond very differently to OA. d- and l-Aspartate (Asp) were quantified for whole cartilage, collagen and non-collagenous components of cartilage obtained at the time of joint replacement. We computed the Asp racemization ratio (Asp-RR = d/d + l Asp), reflecting the proportion of old to total protein, for each component. Compared with hip OA, knee OA collagen fibrils (P < 0.0001), collagen (P = 0.007), and non-collagenous proteins (P = 0.0003) had significantly lower age-adjusted mean Asp-RRs consistent with elevated protein synthesis in knee OA. Knee OA collagen had a mean hydroxyproline/proline (H/P) ratio of 1.2 consistent with the presence of type III collagen whereas hip OA collagen had a mean H/P ratio of 0.99 consistent with type II collagen. Based on Asp-RR, the relative age was significantly different in knee and hip OA (P < 0.0005); on average OA knees were estimated to be 30 yrs 'younger', and OA hips 10 yrs 'older' than non-OA. The metabolic response to OA was strikingly different by joint site. Knee OA cartilage evinced an anabolic response that appeared to be absent in hip OA cartilage. These results challenge the long held view that OA cartilage is capable of only minimal repair and that collagen loss is irreversible.

Aminostratigraphy and taphonomy of ostrich eggshell in the sedimentary infill of Apollo 11 Rockshelter, Namibia

The extent of racemization (total hydrolysable amino acids) of aspartic acid, glutamic acid and valine provide a geochronological framework to assign relative and numeric ages, and examine the taphonomy of the sedimentary infill of Apollo 11 Rockshelter, Namibia. Based on the analysis of 37 ostrich (Struthio camelus) eggshell samples from the rockshelter sedimentary succession, three aminogroups are identified and may be correlated with oxygen isotope records from marine and ice cores. Aminogroup A spans the interval from 80,000±12,000years ago [late Marine Isotope Stage (MIS) 5a] through to the late Holocene (2000±300years). A significant diastem is identified below −65cm separating the lower portion of the rockshelter sediments (that were deposited predominantly by natural depositional processes), from the overlying succession. Although a human presence is generally evident throughout the whole succession, the post-diastem succession records an increasing influence of humans on sediment accumulation. Two aminogroups were identified in the sediments beneath the diastem based on contrasting extents of racemization. Pooled mean ages (representing minimum ages) for aminogroups B and C are 180,000±10,000years and 236,000±18,000years respectively, and are significantly different at the 2-sigma level. The amino acid derived numeric ages indicate an early Middle Stone Age (MSA) for the lower portion of the sedimentary succession within the rockshelter. Collectively, these ages attest to a long, but punctuated record of sediment accumulation within Apollo 11 Rockshelter. At several levels within the anthropogenically-dominated succession (layers A to T), fragments of ostrich eggshell show a disproportionately high extent of racemization in relation to their depth of burial and signify the combined effects of heating and their reworking. The reworked eggshell fragments represent a small component of the assemblage, and the overall results for aminogroup A indicate that the sedimentary succession shows a high degree of stratigraphical integrity. Reworking of sedimentary constituents is not evident in aminogroups B and C. The age for the basal layers Y and Z from Apollo 11 is one of the oldest dates for an Early MSA inventory without Acheulean elements in Southern Africa, a period that is otherwise poorly known.

Age estimation based on aspartic acid racemization in human sclera.

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

Life history parameters of narwhals (Monodon monoceros) from Greenland

Life history parameters for narwhals (Monodon monoceros) were estimated based on age estimates from aspartic acid racemization of eye lens nuclei. Eyes, reproductive organs, and measures of body lengths were collected from 282 narwhals in East and West Greenland in the years 1993, 2004, and 2007–2010. Age estimates were based on the racemization of L-aspartic acid to D-aspartic acid in the nucleus of the eye lens. The ratio of D- and L-enantiomers was measured using high-performance liquid chromatography. The age equation used, 420.32X — 24.02·year where X is the D/L ratio, was determined from data from Garde et al. (2012). Asymptotic body length was estimated to be 405 ± 5.8cm for females and 462 ± 16.2cm for males from East Greenland, and 399 ± 5.9 cm for females and 456 ± 6.9 cm for males from West Greenland. Due to several indeterminant age class estimates, age at sexual maturity was subjectively assessed based on data from reproductive organs and was estimated to be 8–9 years for females and 12–20 ye...

PLEISTOCENE HUMAN OCCUPATION AT BEGINNERS LUCK CAVE, FLORENTINE VALLEY, TASMANIA

EVIDENCE of human occupation dated at approximately 20,000 years BP is recorded from a cave site in the Florentine Valley in south central Tasmania. The artifact assemblage and faunal content are described and the factors giving rise to bone accumulation are examined. Occupation has been dated by 14C analysis of associated charcoal and aspartic acid racemization dating of bone with close agreement between the two methods. The interior river valleys of southern Tasmania were being utilized by hunter-gatherers when the Last Glacial was reaching its climax and Tasmania was a peninsula of the Australian mainland. Suggestions by recent authors that late Pleistocene occupation was tightly coastal and that the interior was uninhabited are refuted. Under the open vegetation conditions prevailing during the late Pleistocene the broad river valleys of southern Tasmania provided easy routes for population movement and a suitable environment for game. In contrast dense wet forest was the dominant vegetation throughout the Holocene making these areas less attractive for both human occupation and the larger elements of the fauna.

STOWING AWAY ON SHIPS THAT PASS IN THE NIGHT: SCLEROBIONT ASSEMBLAGES ON INDIVIDUALLY DATED BIVALVE AND BRACHIOPOD SHELLS FROM A SUBTROPICAL SHELF

ABSTRACT This study evaluates encrustation and bioerosion of brachiopods (Bouchardia rosea) and bivalves (Semele casali) occurring on the inner shelf of the Southeast Brazilian Bight, accounting for differences in water depth, sediment type, host size, and time averaging. Frequencies of colonization covary across sites, but brachiopods are more frequently encrusted than bivalves at all sites, although this difference may disappear after standardization for shell size, depending on the chosen metric. Size selectivity during sclerobiont colonization appears to change as a function of their population density, rather than substrate differences between hosts. Sediment grain size and composition do not appear to exert environmental controls on encrustation or bioerosion, nor does either vary as a function of water depth alone. Radiocarbon-calibrated aspartic acid racemization dating of individual host valves shows similar age ranges and age structures for both hosts. Both epifaunal brachiopods and infaunal biv...

The free D-Aspartic acid and D-Glumatic acid content of sheep milk and sheep milk products

The role of D-amino acids of foods in the human health is a strongly discussed topic and usually, data came from the investigation of cow milk. We have studied the free D-aspartic acid and free D-glutamic acid content of sheep milk, heat-treated sheep milk at various temperatures and various products of sheep milk. Raw sheep milk didn’t contain free D-aspartic and D-glutamic acid in remarkable amount and ratio 5.92% free D-aspartic acid; 2.62% free D-glutamic acid). Our heat-treatments didn’t cause major change in the free D-aspartic and D-glutamic acid content (max.: 7.8% free D-aspartic acid; 5.3% free D-glutamic acid in total free aspartic and glutamic acid). Contrary, all of the investigated products contained high level of free D-amino acids. The free D-aspartic acid and free D-glutamic acid content of the products were 16,9-39,5%,and 13,3-27% in the percent of total free amino acids. The racemization of aspartic acid was higher, than that of glutamic acid in every product. The D-amino acid content of fermented milk products was higher than in different cheeses.

Racemization of aspartic acid in root dentin as a tool for age estimation in a Kuwaiti population

Estimation of age is one of the most significant tasks in forensic practice. Amino acid racemization is considered one of the most reliable and accurate methods of age estimation and aspartic acid shows a high racemization reaction rate. The present study has investigated the application of aspartic acid racemization in age estimation in a Kuwaiti population using root dentin from a total of 89 upper first premolar teeth. The D/L ratio of aspartic acid was obtained by HPLC technique in a test group of 50 subjects and a linear regression line was established between aspartic acid racemization and age. The correlation coefficient (r) was 0.97, and the standard error of estimation was ±1.26 years. The racemization age "t" of each subject was calculated by applying the following formula: ln [(1 + D/L)/(1 - D/L)] = 0.003181 t + (-0.01591). When the proposed formula "estimated age t = ln [(1 + D/L)/(1 - D/L)] + 0.01591/0.003181" was applied to a validation group of 39 subjects, the range of error was less than one year in 82.1% of the cases and the standard error of estimation was ±1.12. The current work has established a reasonably significant correlation of the D-/L-aspartic acid ratio with age, and proposed an apparently reliable formula for calculating the age in Kuwaiti populations through aspartic acid racemization. Further research is required to find out whether similar findings are applicable to other ethnic populations.

Aspartic acid racemization as a dating tool for dentine: A reality

Given the interest in dating sediments from numerous caves, lakes and fluvial terraces containing fossils and lithic components in Europe, here we provide a complete revision of the amino acid racemization (AAR) (aspartic acid in dentine) dating method in vertebrates. To examine the reliability of this method, which is based on a straightforward sample preparation (previous 3.5 kDa dialysis), we used a range of dental material. We examined human dentine collagen from living donors and remains from historical (16th and 19th centuries) and prehistorical (Neolithic) periods. On the assumption that genus does not affect collagen racemization rates, we also studied Neanderthal material and material from carnivores (cave bear) and several other mammals. To validate our age calculation algorithm, we used a wide series of radiometric datings (ESR and 14C), along with thermoluminescence and AAR dating on invertebrate (ostracode) samples. Our results demonstrate that AAR shows satisfactory correlation between age and the extent of aspartic acid racemization for material from modern humans and for ancient (Pleistocene) mammal remains (cave bears, horses and Neanderthals) and highlight a strong correlation between ages derived from dentine collagen aspartic acid and other dating methods. However, in samples from burial sites (19th and 16th century and Neolithic samples from Syria), it was impossible to establish age at death. We assume that taphonomic processes (time and geochemistry) greatly contribute to the denaturation of the collagen triple helix and higher order structures, thereby allowing the racemization of peptide-bound aspartic acid.

Isomerization and Autolysis at Specific Amino Acid Residues of the Tau Protein and its Relations to Alzheimer's Disease

Alzheimer's Disease is the most common form of dementia that more than 5 million Americans are suffering from. With no known cure and no known cause, it is an urgent matter that researcher's are desperately attempting to tackle. Alzheimer's disease is clinically characterized by memory loss and histopathologically characterized by the high prevalence of amyloid plaques and neurofibrillary tangles (tau tangles). In a healthy brain, the tau protein performs the role of stabilizing microtubules in healthy neurons. However, due to their long turn-over rates, they are susceptible to many non-enzymatic, post-translational protein modifications such as racemization and isomerization. While in many cases aged or non-enzymatically damaged proteins can be repaired or replaced, some changes can inhibit protein turnover resulting in an accumulation of damaged protein over time. One of the modifications that very-long lived proteins have been shown to undergo is racemization of aspartic acid and asparagine residues to the non-natural D-form from the common L-form via succinimide intermediate. In this study we have employed quantitative label-free mass spectrometry to study isomerization/racemization reactions site specifically in the repeat-1 domain of Tau protein. We found that Asp-265 isomerizes into four different isoforms. We also found that the Tau peptide undergoes an auto-catalytic cleaveage without the use of a protease. A possible mechanism for the formation of the four isomers is proposed. This data may provide useful information on the in vivo rates of tau racemization, providing insights into the mechanisms and potentially opening a door to finding biomarkers for Alzheimer's disease.

Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89years for 1km depth and 27°C and 1-2years for 3km depth and 54°C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85°C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis

Age estimation based on aspartic acid racemization (AAR) has been applied successfully to various tissues. For routine uses, AAR is analyzed in dentine. For cases in which teeth are unavailable, analyzing AAR in purified elastin has been shown to be an alternative method. The suitability of elastic cartilage from the epiglottis as an elastin source for age estimation based on AAR was tested. A total of 65 tissue samples (cartilage) of epiglottis and 45 samples of elastin purified from the elastic cartilage of epiglottis samples were analyzed. While the D-aspartic acid content of total tissue samples increased with age only slowly, its increase with age in purified elastin samples was similar to that in purified elastin from other tissues. The relationship between the D-aspartic acid content and age was shown to be close enough for age estimation based on AAR in purified elastin from the elastic cartilage of the epiglottis, provided a sufficient quality of elastin purification. Age estimation based on AAR in purified elastin from the epiglottis might serve as a valuable alternative in cases in which other tissues (e.g., teeth) are unavailable.

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10−5–11×10−5 yr−1. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.

Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc

The intervertebral disc (IVD) is a complex cartilaginous structure which functions to resist biomechanical loads during spinal movement. It consists of the highly viscous cartilaginous nucleus pulposus, which is surrounded laterally by a thick outer ring of fibrous cartilage-the annulus fibrosus-and sandwiched inferiorly and superiorly by the cartilage end-plates. The main extracellular matrix molecules of the disc are collagens, proteoglycans, glycoproteins and elastin. The disc also contains appreciable amounts of water, matrix-degrading protease enzymes and their inhibitors, soluble signalling molecules and various metabolic breakdown products. This review provides a comprehensive description of the biochemical composition of the extracellular matrix of the IVD and, specifically, the proteases involved in its molecular turnover. Quantitation of the turnover rates using racemization of aspartic acid as a molecular clock is also discussed. Molecular turnover rates of the major constituent matrix macromolecules of the IVD are found to be particularly slow, especially in the case of collagen. Over a normal human life span, this slow turnover may compromise the structural integrity of the IVD extracellular matrix essential for normal physiological functioning.

Estimation of age using aspartic acid racemisation in human dentin in Indian population

AimThe quest to identify an accurate method of age estimation, had lead to the evaluation of aspartic acid racemisation in hard tissues of the human remains using high performance liquid chromatography (HPLC). Our study is aimed at the applicability of the racemisation technique to use dentin as the sample to estimate the age in South Indian sub-population. Materials and methodsThirty-six non-carious teeth from living individuals distributed among 6 age groups (6 each), sexes (18 each) and jaws (18 each) were analysed for dextro (d) and levo (l) forms of aspartic acid using high performance liquid chromatography (HPLC) technique and their racemisation ratio were calculated for each tooth sample. ResultsHigh correlation was obtained between the aspartic acid racemisation rates in dentin and age of the individual with an error limited to ±3 years. Racemisation rates in teeth did not significantly differ between the sexes or jaws. ConclusionThe d-aspartic acid accumulation in dentin is synchronous with the aging of an individual and can be used as an accurate method of age estimation in our population.

Two-water-assisted racemization of the succinimide intermediate formed in proteins. A computational model study

Racemization of aspartic acid (Asp) residues in proteins plays an important role in the molecular biology of aging. In the widely accepted mechanism of the Asp racemization, a succinimide (SI) intermediate is the species which actually undergo the direct racemization. In the present study, a two-water-assisted mechanism of the SI racemization was computationally investigated using a model compound in which an aminosuccinyl (Asu) residue is capped with acetyl and NMe groups on the N-and C-termini, respectively. The two water molecules catalyze the enolization of the Hα-Cα-C=O portion in the Asu residue by mediating proton relay from the α-carbon atom to the carboxyl oxygen atom. After the enolization, migration of the water molecules and conformational change lead to the mirror image of the initially formed enol two-water complex, and the racemization is completed by the following ketonization. The overall activation barrier (28.2 kcal·mol-1) corresponds to the enolization and ketonization steps, and falls within the available experimental activation energies (21.4-29.0 kcal·mol-1). Therefore, the two-water-assisted mechanism investigated here is plausible for the in vivo and in vitro racemization reactions of the SI intermediates formed in peptides and proteins.

Aspartic acid racemization in dentine: new insights

Aspartic acid racemization in dentine: new insights

Age estimates based on aspartic acid racemization for bowhead whales (Balaena mysticetus) harvested in 1998–2000 and the relationship between racemization rate and body temperature

AbstractFifty‐two eyes were collected and analyzed to estimate ages of 42 bowhead whales using the aspartic acid racemization aging technique. Between‐eye and within‐eye variance components for the ratio of the D and L optical isomers (D/L ratio) were estimated via analysis of variance using multiple measurements from nine whales with both eyes sampled and analyzed. For whales with more than one (D/L)act value, an inverse variance weighted average of the values was used as (D/L)act for the whale. Racemization rate (kAsp) and D/L ratio at birth (D/L)0 were estimated using (D/L)act from 27 bowhead whales with age estimates based on baleen or ovarian corpora data and two term fetuses. The estimates were kAsp = 0.977 × 10−3/yr and (D/L)0 = 0.0250. The nonlinear least squares analysis that produced these estimates also estimated female age at sexual maturity as ASM = 25.86 yr. SE(age) was estimated via a bootstrap that took into account the SE of (D/L)act and the variances and covariance of kAsp and (D/L)0. One male exceeded 100 yr of age; the oldest female was 88. A strong linear relationship between kAsp and body temperature was estimated by combining bowhead data with independent data from studies of humans and fin whales. Using this relationship, we estimated kAsp and ASM for North Atlantic minke whales.

Comparative analysis of aspartic acid racemization methods using whole-tooth and dentin samples

One way to estimate biological age is to use the aspartic acid (Asp) racemization method. Although this method has been performed mostly using enamel and dentin, we investigated whether an entire tooth can be used for age estimation. This study used 12 pairs of canines extracted from both sides of the mandible of 12 individuals of known age. From each pair, one tooth was used as a dentin sample and the other as a whole-tooth sample. Amino acids were extracted from each sample, and the integrated peak areas of d-Asp and l-Asp were determined using a gas chromatograph/mass spectrometer. Statistical analysis was performed using the d/l-Asp ratio. Furthermore, teeth from two unidentified bodies, later identified as Japanese and Brazilian, were examined in the same manner. Results showed that the d/l ratios of whole-tooth samples were higher overall than those of dentin samples. The correlation coefficient between the d/l ratios of dentin samples and their age was r=0.98, and that of the whole-tooth samples was r=0.93. The difference between estimated age and actual chronological age was −0.116 and −6.86years in the Japanese and Brazilian cases, respectively. The use of whole teeth makes the racemization technique easier and can standardize the sampling site. Additionally, using only a few tooth samples per analysis made it possible to reanalyze known-age samples. Although the difficulty in obtaining a proper control sample has prevented racemization from being widely used, the method described here not only ensures the availability of a control tooth, but also enables the teeth to be used for other purposes such as DNA analysis. The use of a whole tooth will increase the application of the racemization technique for age determination.

Application of a novel method for age estimation of a baleen whale and a porpoise

AbstractEyeballs from 121 fin whales (Balaenoptera physalus) and 83 harbor porpoises (Phocoena phocoena) were used for age estimation using the aspartic acid racemization (AAR) technique. The racemization rate (kAsp) for fin whales was established from 15 fetuses (age 0) and 15 adult whales where age was estimated by reading growth layer groups (GLGs) in the earplugs. The (kAsp) for harbor porpoises was derived from 15 porpoises (two calves and 13 &gt; 1 yr old) age‐estimated by counting GLGs in the teeth and two calves classified to age based on length. The (kAsp) values were estimated by regression of GLGs against D/L ratios. For the fin whales an (kAsp) of 1.15 × 10−3/yr (SE ± 0.00005) and a D/L ratio at birth [(D/L)0] of 0.028 (SE ± 0.0012) were estimated, which is in agreement with rates for other mysticeti. For the harbor porpoises a (kAsp) of 3.10 × 10−3/yr (SE ± 0.0004) and a (D/L)0 value of 0.023 (SE ± 0.0018) were estimated, which is considerably higher than found for other cetaceans. Correlation between chosen age estimates from AAR and GLG counts indicated that AAR might be an alternative method for estimating age in marine mammals.