Molecular age estimation based on posttranslational protein modifications in bone: why the type of bone matters.

Age-at-death estimation is of great relevance for the identification of unknown deceased individuals. In skeletonised corpses, teeth and bones are theoretically available for age estimation, but in many cases, only single bones or even only bone fragments are available for examination. In these cases, conventional morphological methods may not be applicable, and the application of molecular methods may be considered. Protein-based molecular methods based on the D-aspartic acid (D-Asp) or pentosidine (Pen) content have already been successfully applied to bone samples. However, the impact of the analysed type of bone has not yet been systematically investigated, and it is still unclear whether data from samples of one skeletal region (e.g. skull) can also be used for age estimation for samples of other regions (e.g. femur). To address this question, D-Asp and Pen were analysed in bone samples from three skeletal regions (skull, clavicle, and rib), each from the same individual. Differences between the bone types were tested by t-test, and correlation coefficients (ρ) were calculated according to Spearman. In all types of bone, an age-dependent accumulation of D-Asp and Pen was observed. However, both parameters (D-Asp and Pen) exhibited significant differences between bone samples from different anatomical regions. These differences can be explained by differences in structure and metabolism in the examined bone types and have to be addressed in age estimation based on D-Asp and Pen. In future studies, bone type-specific training and test data have to be collected, and bone type-specific models have to be established.

Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate.

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of early-branching Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable in our assays. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Chiral separations for d-amino acid analysis in biological samples.

It is widely accepted that some of the free d-amino acids play important biological role. d-Aspartate and d-serine formed in the central nervous system of higher vertebrates have neurotransmitter/neuromodulator function. Together with d-alanine they are distributed in various tissues and biological fluids. Studying their physiological and pathological significance requires their sensitive and accurate determination in biological samples. The various separation and detection methods used for their analysis are overviewed in the present paper. Our focus is mainly the quantitative performance and the analysis of real biospecimens.

A rapid and sensitive detection of D-Aspartic acid in Crystallin by chiral derivatized liquid chromatography mass spectrometry.

A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD<0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp76 and Asp84 in the αA-Crystallin and Asp96 in αB-Crystallin were racemized to D-Asp. These results show that this proposed chiral peptide LC-MS/MS method using chiral derivatization provides a rapid and sensitive analysis for post translational Asp racemization sites in aging proteins.

Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

Quantitative analysis of isomeric (l-α-, l-ß-, D-α-, D-ß-) aspartyl residues in proteins from elderly donors.

Homochirality is essential for life. For a long time, it was considered that d-amino acids were excluded from living systems. In the past 30 years, however, d-amino acids have been found in living organisms in the form of free amino acids, peptides and proteins, owing to advances in the analysis of optical isomers of amino acids. Free D-amino acids and D-amino-acid-containing peptides have been shown to have important physiological functions. The amount of D-aspartate (Asp) residues in protein spontaneously increases in metabolically inert tissues such as the eye and brain during aging, and may be related to cataract formation and the development of Alzheimer disease, suggesting that D-Asp might be a molecular marker of aging and age-related disorders. The presence of D-Asp in living organisms is thought to result from the isomerization of L-Asp residues in some proteins. Furthermore, the isomerization of Asp does not occur uniformly but only at specific sites. Therefore, it is necessary to determine the sites of isomeric Asp in these proteins in order to elucidate the mechanism of spontaneous Asp isomerization during aging. Herein, we summarize the localization and mechanism of D-amino acids in proteins of living tissues, and the effects of D-amino acid formation in proteins. Furthermore, we describe methods for the analysis of protein-bound D-amino acids including a conventional enantioseparation method based on HPLC and a new convenient method based on LC-MS that can identify the specific sites of D-Asp in proteins.

Evaluation of enantiomeric purity of magnesium-L-aspartate dihydrate.

Magnesium supplementation in form of organic magnesium salts is a very popular practice. We examined the enantiomeric purity of "Magnesium aspartate dihydrate" monographed in the European Pharmacopeia. A chiral capillary zone electrophoresis using (2-hydroxypropyl)-ß-cyclodextrin coupled to laser induced fluorescence detection and a HPLC-fluorescence method with chiral derivatization using o-phthaldialdehyde and N-acetyl-L-cysteine as an orthogonal method were developed and validated. Two batch samples of this substance and three drug products containing the salt were analyzed by means of both methods. The concentration of the D-enantiomer of aspartic acid ranged from 0.03 to 0.12%. Simulations of the synthesis revealed that the d-aspartic acid content is elevated if the dissolution of L-aspartic acid was performed at acidic pH values.

Gender differences in D-aspartic acid content in skull bone.

In forensic medicine, the personal identification of cadavers is one of the most important tasks. One method of estimating age at death relies on the high correlation between racemization rates in teeth and actual age, and this method has been applied successfully in forensic odontology for several years. In this study, we attempt to facilitate the analysis of racemized amino acids and examine the determination of age at death on the basis of the extent of aspartic acid (Asp) racemization in skull bones. The specimens were obtained from 61 human skull bones (19 females and 42 males) that underwent judicial autopsy from October 2010 to May 2012. The amount of D-Asp and L-Asp, total protein, osteocalcin, and collagen I in the skull bones was measured. Logistic regression analysis was performed for age, sex, and each measured protein. The amount of D-Asp in the female skull bones was significantly different from that in the male skull bones (p = 0.021), whereas the amount of L-Asp was similar. Thus, our study indicates that the amount of D-Asp in skull bones is different between the sexes.

HPLC methods for determination of D-aspartate and N-methyl-D-aspartate.

D-Amino acids are stereoisomers or optical isomers of naturally occurring L-amino acids and thus possess the same chemical structure, but may differ in their biological/physiological properties. Until a half century ago, D-amino acids had been considered to be unnatural substances found only in microorganisms. However, improvements in analytical instruments and methods have revealed that D-amino acids are present in invertebrates and vertebrates, including humans, and that they possess important physiological functions. D-Aspartate (D-Asp) and its methylated form N-methyl-D-aspartate (NMDA) possess neuroendocrine properties in many species. Several methods have been developed for determination of D- and L-enantiomers of amino acids by high performance liquid chromatography (HPLC). We report here improved HPLC methods for the specific determination of D-Asp and NMDA in biological tissues.

Immunohistochemical localization of endogenous D-Aspartate in the marine brown Alga Sargassum fusiforme.

Immunohistochemical localization (cellular localization) of endogenous D-aspartate in the marine brown alga Sargassum fusiforme was investigated by the use of a specific polyclonal antibody raised against D-aspartate. D-Aspartate immunoreactivity was evident in the medullary layer in the blade of the alga, and weak staining was found in the cortical layer, whereas epidermal cells were found to lack D-aspartate. Within the cells of the layers, immunoreactivity was confirmed only in the cytosol and not in the cell wall, chloroplast, or vacuole. These results suggest that D-aspartate is present in S. fusiforme cells, and excludes the possibility that it is derived from attached or symbiotic organisms such as marine bacteria. This is the first report describing the localization of free D-aspartate in plant cells.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-ß-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-ß-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-ß-Asp and CML, immunohistochemical analysis using anti-D-ß-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-ß-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-ß-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.

D-Aspartate--an important bioactive substance in mammals: a review from an analytical and biological point of view.

It was long believed that D-amino acids were either unnatural isomers or laboratorial artifacts and that the important functions of amino acids were exerted only by l-amino acids. However, recent investigations have shown that a variety of D-amino acids are present in mammals and that they play important roles in physiological functions in the body. Among the free d-amino acids that have been identified in mammals, D-aspartate (D-Asp) has been shown to play a crucial role in the neuroendocrine and endocrine systems as well as in the central nervous system. Here, we present an overview of recent studies of free D-Asp, focusing on the analytical methods in real biological matrices, expression and localization in tissues and cells, biological and physiological activities, biosynthesis, degradation, cellular transport, and possible relevance to disease. In addition to frequently used techniques for the enantiomeric determination of amino acids, including high-performance liquid chromatography and enzymatic methods, the recent development of analytical methods is also described.

Simultaneous determination of D-aspartic acid and D-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure.

For a metabolomics study focusing on the analysis of aspartic and glutamic acid enantiomers, a fully automated two-dimensional HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column was developed. By using this system, a detailed distribution of D-Asp and D-Glu besides L-Asp and L-Glu in mammals was elucidated. For the total analysis concept, the amino acids were first pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to be sensitively and fluorometrically detected. For the non-stereoselective separation of the analytes in the first dimension a monolithic ODS column (750 mm × 0.53 mm i.d.) was adopted, and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm × 1.5 mm i.d.) was selected for the second dimension. In the rat plasma, RSD values for intra-day and inter-day precision were less than 6.8%, and the accuracy ranged between 96.1% and 105.8%. The values of LOQ of D-Asp and D-Glu were 5 fmol/injection (0.625 nmol/g tissue). The present method was successfully applied to the simultaneous determination of free aspartic acid and glutamic acid enantiomers in 7 brain areas, 11 peripheral tissues, plasma and urine of Wistar rats. Biologically significant D-Asp values were found in various tissue samples whereas for D-Glu the values were very low possibly indicating less significance.

Visible wavelength spectrophotometric assays of L-aspartate and D-aspartate using hyperthermophilic enzyme systems.

Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 µM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.

Thyroid hormones and D-aspartic acid, D-aspartate oxidase, D-aspartate racemase, H2O2, and ROS in rats and mice.

Total concentrations of thyroid hormones T(3) and T(4), and of their free forms, FT(3) and FT(4), D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H(2)O(2), and ROS (reactive oxygen species) were determined in rats and mice. T(3) and T(4) were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT(3) and FT(4) were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H(2)O(2) Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H(2)O(2) is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T(3) and T(4). Production of H(2)O(2) in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O(2)+H(2)O-->alpha-oxaloacetate+NH(3)+H(2)O(2)). D-Aspartate racemase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H(2)O(2) and consequently for the synthesis of thyroid hormones.