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d-Serine modulates excitatory neurotransmission by binding to N-methyl-d-aspartate glutamate receptors. d-Amino acid oxidase (DAO) degrades d-amino acids, such as d-serine, in the central nervous system, and is associated with neurological and psychiatric disorders. However, cell types that express brain DAO remain controversial, and whether brain DAO influences systemic d-amino acids in addition to brain d-serine remains unclear. Here, we created astrocyte-specific DAO-conditional knockout mice. Knockout in glial fibrillary acidic protein-positive cells eliminated DAO expression in the hindbrain and increased d-serine levels significantly in the cerebellum. Brain DAO did not influence levels of d-amino acids in the forebrain or periphery. These results show that astrocytic DAO regulates d-serine specifically in the hindbrain.

d-Alanine has protective effects on I/R-induced kidney injury. d-Ala inhibits ROS production and improves mitochondrial membrane potential, resulting in reduced TEC necrosis by hypoxic stimulation. The administration of d-Ala protects the tubules from I/R injury in mice. Moreover, the plasma level of d-Ala is conversely associated with eGFR in patients with AKI. Our data suggest that d-Ala is an appealing therapeutic target and a potential biomarker for AKI.

Mammals exhibit systemic homochirality of amino acids in L -configurations. While ribosomal protein synthesis requires rigorous chiral selection for L -amino acids, both endogenous and microbial enzymes convert diverse L -amino acids to D -configurations in mammals. However, it is not clear how mammals manage such diverse D -enantiomers. Here, we show that mammals sustain systemic stereo dominance of L -amino acids through both enzymatic degradation and excretion of D -amino acids. Multidimensional high performance liquidchromatography analyses revealed that in blood, humans and mice maintain D -amino acids at less than several percent of the corresponding L -enantiomers, while D -amino acids comprise ten to fifty percent of the L -enantiomers in urine and feces. Germ-free experiments showed that vast majority of D -amino acids, except for D -serine, detected in mice are of microbial origin. Experiments involving mice that lack enzymatic activity to catabolize D -amino acids showed that catabolism is central to the elimination of diverse microbial D -amino acids, whereas excretion into urine is of minor importance under physiological conditions. Such active regulation of amino acid homochirality depends on maternal catabolism during the prenatal period, which switches developmentally to juvenile catabolism along with the growth of symbiotic microbes after birth. Thus, microbial symbiosis largely disturbs homochirality of amino acids in mice, whereas active host catabolism of microbial D -amino acids maintains systemic predominance of L -amino acids. Our findings provide fundamental insight into how the chiral balance of amino acids is governed in mammals and further expand the understanding of interdomain molecular homeostasis in host-microbial symbiosis.

Plasma amino acids reflect the dynamics of amino acids in organs and their levels have clinical significance. Amino acids as clinical indicators have been evaluated as a mixture of D- and L-amino acids because D-enantiomers are believed to be physiologically nonexistent. However, it has become clear that some D-amino acids are synthesized by endogenous enzymes and symbiotic bacteria. Here, using a two-dimensional HPLC system, we measured enantiomers of all proteinogenic amino acids in plasma and urine and analyzed for correlation with other biochemical parameters in humans who underwent health checkups at our institutional hospital. Four D-amino acids (D-asparagine, D-alanine, D-serine, and D-proline) were detected in the plasma, amounting to less than 1% of the quantities of L-amino acids, but in the urine at several tens of percent, showing that D-amino acids have much higher fractional excretion than their L-counterparts. Detected plasma D-amino acids and D-/L-amino acid ratios were well correlated with renal parameters, such as blood urea nitrogen, creatinine, and cystatin C. On the other hand, a set of plasma L-amino acids were associated with body mass index and correlated with metabolic parameters such as liver enzymes, lipids, blood glucose, and uric acid. Thus, chiral resolution of plasma amino acids revealed totally different associations of the enantiomers with organ functions, and warrants further investigation for clinical and laboratory usefulness.

d-Amino acids (d-AAs) have been associated with age-associated conditions in the general population but their relevance in people with HIV (PWH), who experience accentuated/accelerated aging has not been studied. We compared d-AA levels in HIV-infected and uninfected controls and explored their association with markers of immune activation, gut permeability and organ dysfunction.Case-control analysis.Plasma samples from 60 antiretroviral therapy-treated HIV-infected individuals and 59 uninfected controls were analysed. A three-dimensional HPLC system was used to measure d-and l-asparagine, serine, alanine and proline and presented as %d-AA. Additionally, cell-associated and soluble markers of immune activation and senescence were characterized. Kidney and liver functions were expressed as estimated glomerular filtration rate and fibrosis-4 scores, respectively. Mann-Whitney and Spearman rank correlation coefficients were used for statistical analysis.d-Asparagine, d-serine, d-alanine and d-proline were detectable in all plasma samples and correlated with age in HIV-infected and uninfected but not different between groups. Kynurenine/tryptophan ratio was positively correlated with all %d-AAs in PWH and with %d-serine and %d-proline in controls. %d-AAs were not consistently correlated with markers of gut permeability in both groups. All %d-AAs were also correlated with kidney function in both groups whereas age-associated accumulation of %d-asparagine, %d-serine and %d-proline were correlated with liver function and the VACS score in controls.Plasma d-AAs are associated with chronological age and correlated with markers of immune activation and organ decline, though variably, in PWH and controls. Their role in the biology of aging warrants further investigation.

Liquid chromatographic methods for the determination of amino acids and bioamines are outlined and discussed. In Section 4.2 , direct separation methods for amino acids with postcolumn derivatization or MS/MS detection are described. In Section 4.3 , typical indirect methods for amino acids are summarized. In this section, precolumn derivatization reagents for UV-VIS detection, fluorescence detection, and MS/MS detection are explained. Enantioselective analytical methods for amino acids are also described in Section 4.4 . Direct and indirect separation methods for bioamines are summarized in Sections 4.5 and 4.6 .

Activation of N-methyl

We performed in-situ analysis on a ~1 mm-sized Ryugu grain A0080 returned by the Hayabusa2 spacecraft to investigate the relationship of soluble organic matter (SOM) to minerals. The DESI-HRMS (desorption electrospray ionization-high resolution mass spectrometry) imaging using methanol spray identified more than 200 soluble organic compounds, which were assigned as CHN, CHO, CHO-Na (sodium adducted), and CHNO in molecular composition. Heterogeneous spatial distribution was observed for different compound classes of SOM as well as among the same alkylated homologues on the sample surface. The A0080 sample showed similar mineralogy to that of CI chondrite and contained two different lithologies, which are rich in magnetite, pyrrhotite, and dolomite (lithology 1) and poor in those minerals (lithology 2). CHN compounds were relatively concentrated in lithology 1 than in lithology 2, on the other hand, CHO, CHO-Na, and CHNO compounds were distributed in both lithologies. Such different spatial distribution of SOM is the result of interaction of the SOM with minerals, during precipitation of the SOM via fluid activity, or could be due to difference in transportation efficiencies of SOMs in aqueous fluid. However, organic-related ions measured by ToF-SIMS did not coincide with the spatial distribution revealed by DESI-HRMS imaging, indicating that the ToF-SIMS data would be mainly derived from methanol-insoluble organic matter in A0080. Alkylated homologues of CHN compounds with large C number appeared more abundant in lithology 2 than lithology 1. In contrast, fragments of the Murchison meteorite showed different features to of A0080, implying different formation or growth mechanisms for the alkylated CHN compounds by interaction with fluid and minerals on Murchison parent body and asteroid Ryugu. This difference might be mainly attributed to the carbonate grains, which would have played as a catalyst for CH2 growth of CHN compounds. Future in-situ analysis of CI chondrite will provide more reliable constraints for the history of soluble organic compounds in asteroid Ryugu.

Methyl groups were introduced on the acridine moiety in chemiluminescent acridinium esters that have electron-withdrawing groups (trifluoromethyl, cyano, nitro, ethoxycarbonyl) at the 4-position on the phenyl ester. The introduction of methyl groups at the 2-, 2,7-, and 2,3,6,7-positions on the acridine moiety shifted the optimal pH that gave relatively strong chemiluminescence intensity from neutral conditions to alkaline conditions. 4-(Ethoxycarbonyl)phenyl 2,3,6,7,10-pentamethyl-10λ4-acridine-9-carboxylate, trifluoromethanesulfonate salt showed long-lasting chemiluminescence under alkaline conditions. Acridinium esters to determine hydrogen peroxide concentration at pH 7-10 were newly developed.

In the chiral separation of amino acids, liquid chromatography has been mainly used because of the physicochemical properties of the analytes. To date, only few reports of the use of supercritical fluid chromatography (SFC) for the analysis of chiral amino acids exist, and there is much room for improvement in terms of the number of measurable amino acids, peak shape, and analysis time. In this study, we developed a novel method for the chiral analysis of native amino acids using a system combining SFC and tandem mass spectrometry. Specifically, the separation of amino acid enantiomers was investigated using a CROWNPAK CR-I(+) column with a chiral stationary phase of optically active crown ether. Methanol/water mobile phase with trifluoroacetic acid as a modifier based on supercritical carbon dioxide (CO

A highly-selective two-dimensional high-performance liquid chromatographic (2D-HPLC, off-line heart cutting mode) system was developed for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers in human physiological fluids. Ser, Thr and aThr have a hydroxy group in their side chains, and the development of a simultaneous analytical method with a practically sufficient enantio/chemo-selectivity has been required to clarify their amounts in human physiological fluids. The amino acids in the samples were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and were isolated by a reversed-phase column (Singularity RP18, 1.0 x 250 mm) in the first dimension. After the target amino acids were collected, the fractions were manually introduced into an enantioselective column in the second dimension and were detected by their fluorescence. For the second dimension, a Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 x 250 mm) was used. The resolution values of the enantiomers obtained by the Singularity CSP-013S column were 7.64 for Ser, 7.58 for Thr and 4.71 for aThr by using the mixture of methanol and acetonitrile containing formic acid as the mobile phases. The developed method was validated and applied to human plasma and urine. In the plasma, the obtained %d values (the percentage of d-form to total amino acid) were 1.7 for Ser, and trace levels of d-aThr and d-Thr were observed. In the urine, the %d values were 48.0 for Ser, 1.6 for Thr and 8.0 for aThr (calculated using d-aThr and l-Thr).

D-Tryptophan (D-Trp) is one of the minor D-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of free D-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinary D-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although only l-Trp was observed in a mixed urine sample of healthy volunteers, small amounts of D-Trp were detected in the C57BL/6J mice (n = 5, %D=6.18 ± 0.47). In B6DAO

Aims Coronary arteritis is a life-threatening complication that may arise in the acute stage of Kawasaki disease (KD), the leading cause of systemic vasculitis in childhood. Various microorganisms and molecular pathogens have been reported to cause KD. However, little is known about the key molecules that contribute to the development of coronary arteritis in KD. Methods and results To identify causative molecules for coronary arteritis in KD, we prospectively recruited 105 patients with KD and 65 disease controls in four different parts of Japan from 2015 to 2018. During this period, we conducted lipidomics analyses of their sera using liquid chromatography–mass spectrometry (LC-MS). The comprehensive LC-MS system detected a total of 27 776 molecules harbouring the unique retention time and m/z values. In the first cohort of 57 KD patients, we found that a fraction of these molecules showed enrichment patterns that varied with the sampling region and season. Among them, 28 molecules were recurrently identified in KD patients but not in controls. The second and third cohorts of 48 more patients with KD revealed that these molecules were correlated with inflammatory markers (leucocyte counts and C-reactive proteins) in the acute stage. Notably, two of these molecules (m/z values: 822.55 and 834.59) were significantly associated with the development of coronary arteritis in the acute stage of KD. Their fragmentation patterns in the tandem MS/MS analysis were consistent with those of oxidized phosphatidylcholines (PCs). Further LC-MS/MS analysis supported the concept that reactive oxygen species caused the non-selective oxidization of PCs in KD patients. In addition, the concentrations of LOX-1 ligand containing apolipoprotein B in the plasma of KD patients were significantly higher than in controls. Conclusion These data suggest that inflammatory signals activated by oxidized phospholipids are involved in the pathogenesis of coronary arteritis in KD. Because the present study recruited only Japanese patients, further examinations are required to determine whether oxidized PCs might be useful biomarkers for the development of coronary arteritis in broad populations of KD.

We experienced a case of a 36-year-old female with rapidly progressive glomerulonephritis (RPGN) due to anti-neutrophil cytoplasmic antibody (ANCA)-associated nephritis and systemic lupus erythematosus (SLE) nephritis. Chiral amino acid metabolomics revealed a prominent profile of d-serine in this patient. At the fulminant period of RPGN, the level of plasma d-serine, a potential biomarker in CKD that reflects actual glomerular filtration ratio (GFR), was extremely high. On the other hand, urinary fractional excretion (FE) of d-serine, which was usually much higher than that of l-isoform, was 0% in this patient. These abnormal d-serine profiles normalized in response to the intensive treatment. Normalizations of blood d-serine levels were in parallel with those of blood creatinine levels and potentially reflect the recovery of GFR. FE of d-serine increased transiently before the normalization of d-serine profile, suggesting that kidney promotes urinary excretion of d-serine for the normalization of plasma d-serine level. These unexplored clinical features of d-serine well reflected the clinical course of this patient. Blood d-serine level can also serve as a biomarker in acute kidney injury (AKI) or RPGN, and, in combination with FE of d-serine, may render the clinical practitioners to judge the efficacy of intensive treatments. Electronic supplementary material The online version of this article (10.1007/s13730-019-00411-6) contains supplementary material, which is available to authorized users.

D-Amino acids, long-term undetected enantiomers of L-amino acids, are now emerging as potential biomarkers, especially for kidney diseases. Management of chronic kidney disease (CKD), a global problem with its high prevalence and poor prognosis, is currently unsatisfactory due to the difficulty in estimating kidney function and in early detection of diseases. We now show that intra-body dynamics of D-serine reflect kidney function and diseases. The blood level of D-serine correlated well with the actual glomerular filtration ratio, a key kidney function. This correlation was compatible with those of conventional kidney markers, and blood level of D-serine was relatively unaffected by such clinical factors as body size. The balance between excretion and reabsorption of amino acids by the kidney was controlled with chiral selectivity, and the reabsorption of D-serine was sensitive to the presence of CKD. The combination of blood level and urinary dynamics of D-serine effectively distinguished CKD from non-CKD. These lines of evidence provide new insights into the enantioselective amino acid dynamics in the human body that reflect disease pathophysiology. D-Serine may serve as a vital biomarker that suppress CKD onset through the precise assessment of kidney function and the diagnosis of CKD.

The Long-Evans Agouti (LEA/Tohm) rat has recently been established as a new rat model of type 2 diabetes. The onset of diabetes mellitus was observed only in male LEA/Tohm rats; however, urinary glucose appeared before the onset of diabetes. To clarify the genetic basis of urinary glucose, we performed genetic linkage analysis using (BN × LEA) F2 intercross progeny. A recessively acting locus responsible for urinary glucose excretion (ugl) was mapped to a 7.9 Mb region of chromosome 10, which contains the cystinosin (Ctns) gene. The Ctns gene encodes the cystine transporter, which transports cystine out of the lysosome and is responsible for nephropathic cystinosis in humans. Sequence analysis identified a 13-bp deletion in the Ctns gene, leading to a truncated and loss-of-function protein, which cause cystine accumulation in various tissues. We also developed a novel congenic rat strain harboring the Ctnsugl mutation on the F344 genetic background. Phenotypic analysis of F344-Ctnsugl rats indicated that the incidence of urinary glucose was 100% in both males and females at around 40 weeks of age, and marked cystine accumulation was observed in the tissues, as well as remarkable renal lesions and cystine crystals in the lysosomes of the renal cortex. Furthermore, treatment with cysteamine depleted the cystine contents in F344-Ctnsugl rat embryonic fibroblasts. These results indicated that the F344-Ctnsugl rat provides a novel rat model of cystinosis, which allows not only a better understanding of the pathogenesis and pathophysiology of cystinosis but will also contribute to the development of new therapies. Electronic supplementary material The online version of this article (10.1007/s00335-018-9790-3) contains supplementary material, which is available to authorized users.

Molecular chirality of amino acids is linked to bacterial recognition by mammals to modulate IgA for bacterial symbiosis., Organisms use l-amino acids (l-aa) for most physiological processes. Unlike other organisms, bacteria chiral-convert l-aa to d-configurations as essential components of their cell walls and as signaling molecules in their ecosystems. Mammals recognize microbe-associated molecules to initiate immune responses, but roles of bacterial d-amino acids (d-aa) in mammalian immune systems remain largely unknown. Here, we report that amino acid chirality balanced by bacteria-mammal cross-talk modulates intestinal B cell fate and immunoglobulin A (IgA) production. Bacterial d-aa stimulate M1 macrophages and promote survival of intestinal naïve B cells. Mammalian intestinal d-aa catabolism limits the number of B cells and restricts growth of symbiotic bacteria that activate T cell–dependent IgA class switching of the B cells. Loss of d-aa catabolism results in excessive IgA production and dysbiosis with altered IgA coating on bacteria. Thus, chiral conversion of amino acids is linked to bacterial recognition by mammals to control symbiosis with bacteria.

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine &beta, synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

Nephrotoxic serum (NTS) nephritis occurs in three stages: inflammation, early kidney damage, and severe kidney damage. We quantified the temporal changes in the enantiomers of lactate (LA) and 3-hydroxybutyrate (3HB) in serum and urine during the progression of autoimmune kidney damage in mice with NTS nephritis. Two-dimensional and three-dimensional HPLC were used to quantify the enantiomers. The serum and urinary levels of LA and 3HB enantiomers significantly changed during the progression of NTS nephritis. Specifically, d-LA was significantly higher in the serum (131.8 ± 30.6, 123.7 ± 27.2, 109.3 ± 15.6 vs. 51.2 ± 7.5 μM; p 0.05) and urine (222.2 ± 34.8, 197.4 ± 53.9, 214.8 ± 68.9 vs. 100.8 ± 37.7 μmol/g creatinine; p 0.05) of the week 0 (W0), week 1 (W1), and week 2 (W2) groups than the normal group. The l-3HB/d-3HB ratio was significantly lower in the W0, W1, and W2 groups than the normal group in serum (0.0362 ± 0.0082, 0.0346 ± 0.0065, 0.0323 ± 0.0033 vs. 0.0602 ± 0.0214; p 0.05) and urine (0.0591 ± 0.0304, 0.1524 ± 0.0365, 0.1232 ± 0.1066 vs. 0.3273 ± 0.1394; p 0.05). The changes in serum and urinary d-LA and l-3HB/d-3HB ratios were stable before severe kidney damage. In conclusion, we successfully determined the levels of LA and 3HB enantiomers in NTS nephritis by HPLC. Serum and urinary d-LA contents and l-3HB/d-3HB ratios may have potential as biomarkers of early autoimmune kidney injury.