A multi-hierarchical approach reveals d-serine as a hidden substrate of sodium-coupled monocarboxylate transporters.

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

Development of a two-dimensional LC-MS/MS system for the determination of proline and 4-hydroxyproline enantiomers in biological and food samples.

A two-dimensional LC-MS/MS system has been developed for the enantioselective determination of proline (Pro), cis-4-hydroxyproline (cis-4-Hyp) and trans-4-hydroxyproline (trans-4-Hyp) in a variety of biological samples. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the NBD-derivatives were separated by a reversed-phase column (Singularity RP18) as their D plus L mixtures in the first dimension. The collected target fractions were then introduced into the second dimension where the enantiomers were separated by a Pirkle-type enantioselective column (Singularity CSP-001S) and determined by a tandem mass spectrometer (Triple Quad™ 5500). The method was validated by the standard amino acids and also by human plasma, and sufficient results were obtained for the calibration, precision and accuracy. The method was applied to human plasma and urine, bivalve tissues and fermented food/beverages. D-Pro was widely found in the human physiological fluids, bivalves and several fermented products. Although trans-4-D-Hyp was not found in all the tested samples, cis-4-D-Hyp was present in human urine and tissues of the ark shell, and further studies focusing on the origin and physiological significance of these D-enantiomers are expected.

Evaluation of Individual Variation of d-Amino Acids in Human Plasma by a Two-Dimensional LC-MS/MS System and Application to the Early Diagnosis of Chronic Kidney Disease.

For the discovery of sensitive biomarkers of kidney function focusing on chiral amino acids, a multiple heart-cutting two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS system has been designed/developed. As the target analytes, alanine (Ala), aspartic acid, glutamic acid (Glu), leucine (Leu), lysine, methionine, phenylalanine (Phe), proline (Pro), serine (Ser), and valine were selected considering the presence of their d-forms in mammals. The 2D LC-MS/MS system consisted of the nonenantioselective reversed-phase separation of the target amino acids, the separations of the d- and l-enantiomers, and detection using MS/MS. Using the method, the plasma chiral amino acids, precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, were isolated from other intrinsic substances, then determined without losing sensitivity by the fully automated whole-peak volume transfer operation from first to second dimension. In all of the tested plasma samples obtained from five healthy individuals and 15 patients with chronic kidney disease (CKD), the target chiral amino acids were determined without interference. In healthy individuals, the levels of all the tested d-amino acids were regulated in the low ranges. In contrast, the % d values of Glu, Leu, and Phe significantly increased with the progress of kidney dysfunction, besides the previously reported values of d-Ala, Pro, and Ser. Concerning Phe, the significant increase of the % d values (p < 0.05) was reported for the first time even in the mild CKD group compared to those of the healthy group; d-Phe might be a more sensitive marker than the previously reported d-forms. These results demonstrated the potential of these d-forms as the sensitive biomarkers of kidney function for the early diagnosis of CKD.

Development of a three-dimensional HPLC system for the determination of serine, threonine and allo-threonine enantiomers in the plasma of patients with chronic kidney disease.

A highly-selective three-dimensional high-performance liquid chromatographic (3D-HPLC) system was developed for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers in human plasma to screen the new biomarker of chronic kidney disease (CKD). d-Ser has been reported to be the candidate biomarker of CKD, however, multiple biomarkers are still required. Therefore, Ser analogs of hydroxy amino acids are the focus in the present study. For the sensitive analysis, the amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by their fluorescence. The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 × 250 mm), an anion-exchange column (Singularity AX, 1.0 × 150 mm) and a Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 × 250 mm). The developed method was validated and applied to the human plasma samples obtained from 15 healthy volunteers and 165 CKD patients. The concentrations of the d-forms were 1.13-2.26 (Ser), 0.01-0.03 (Thr) and 0.04-0.10 µM (aThr) for the healthy volunteers and 0.95-19.0 (Ser), 0-0.57 (Thr) and 0.04-1.02 µM (aThr) for the CKD patients. The concentrations and the %d values of all the target d-amino acids were increased along with the decreasing of renal function and further investigation for clinical applications are expected.

D-Amino acids differentially trigger an inflammatory environment in vitro.

Studies in vivo have demonstrated that the accumulation of D-amino acids (D-AAs) is associated with age-related diseases and increased immune activation. However, the underlying mechanism(s) of these observations are not well defined. The metabolism of D-AAs by D-amino oxidase (DAO) produces hydrogen peroxide (H2O2), a reactive oxygen species involved in several physiological processes including immune response, cell differentiation, and proliferation. Excessive levels of H2O2 contribute to oxidative stress and eventual cell death, a characteristic of age-related pathology. Here, we explored the molecular mechanisms of D-serine (D-Ser) and D-alanine (D-Ala) in human liver cancer cells, HepG2, with a focus on the production of H2O2 the downstream secretion of pro-inflammatory cytokine and chemokine, and subsequent cell death. In HepG2 cells, we demonstrated that D-Ser decreased H2O2 production and induced concentration-dependent depolarization of mitochondrial membrane potential (MMP). This was associated with the upregulation of activated NF-кB, pro-inflammatory cytokine, TNF-α, and chemokine, IL-8 secretion, and subsequent apoptosis. Conversely, D-Ala-treated cells induced H2O2 production, and were also accompanied by the upregulation of activated NF-кB, TNF-α, and IL-8, but did not cause significant apoptosis. The present study confirms the role of both D-Ser and D-Ala in inducing inflammatory responses, but each via unique activation pathways. This response was associated with apoptotic cell death only with D-Ser. Further research is required to gain a better understanding of the mechanisms underlying D-AA-induced inflammation and its downstream consequences, especially in the context of aging given the wide detection of these entities in systemic circulation.

Polycyclic aromatic hydrocarbons in samples of Ryugu formed in the interstellar medium.

Polycyclic aromatic hydrocarbons (PAHs) contain ≲20% of the carbon in the interstellar medium. They are potentially produced in circumstellar environments (at temperatures ≳1000 kelvin), by reactions within cold (~10 kelvin) interstellar clouds, or by processing of carbon-rich dust grains. We report isotopic properties of PAHs extracted from samples of the asteroid Ryugu and the meteorite Murchison. The doubly-13C substituted compositions (Δ2×13C values) of the PAHs naphthalene, fluoranthene, and pyrene are 9 to 51‰ higher than values expected for a stochastic distribution of isotopes. The Δ2×13C values are higher than expected if the PAHs formed in a circumstellar environment, but consistent with formation in the interstellar medium. By contrast, the PAHs phenanthrene and anthracene in Ryugu samples have Δ2×13C values consistent with formation by higher-temperature reactions.

Two-dimensional LC-MS/MS and three-dimensional LC analysis of chiral amino acids and related compounds in real-world matrices.

Amino acids normally have a chiral carbon and d/l-enantiomers are present. Due to the homochirality features on the present Earth, l-enantiomers are predominant in the living beings and the d-enantiomers are rare. Along with the progress and development of cutting edge analytical methods, several d-amino acids were found even in the higher animals including humans, and their biological functions and diagnostic values have also been reported. However, the amounts of these d-amino acids are much lower than the l-forms, and development/utilization of highly sensitive and selective methods are practically essential to avoid the disturbance from uncountable intrinsic substances. In the present review, multi-dimensional HPLC methods for the determination of chiral amino acids, especially two-dimensional LC-MS/MS and three-dimensional LC methods, and their applications to a variety of real-world matrices are summarized.

Mammals sustain amino acid homochirality against chiral conversion by symbiotic microbes.

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.

Soluble organic molecules in samples of the carbonaceous asteroid (162173) Ryugu.

The Hayabusa2 spacecraft collected samples from the surface of the carbonaceous near-Earth asteroid (162173) Ryugu and brought them to Earth. The samples were expected to contain organic molecules, which record processes that occurred in the early Solar System. We analyzed organic molecules extracted from the Ryugu surface samples. We identified a variety of molecules containing the atoms CHNOS, formed by methylation, hydration, hydroxylation, and sulfurization reactions. Amino acids, aliphatic amines, carboxylic acids, polycyclic aromatic hydrocarbons, and nitrogen-heterocyclic compounds were detected, which had properties consistent with an abiotic origin. These compounds likely arose from an aqueous reaction on Ryugu's parent body and are similar to the organics in Ivuna-type meteorites. These molecules can survive on the surfaces of asteroids and be transported throughout the Solar System.

Endogenous d-serine exists in the mammalian brain independent of synthesis by serine racemase.

Activation of N-methyl-d-aspartate receptors (NMDARs) requires binding of a co-agonist in addition to l-glutamate. d-serine binds to the co-agonist site on GluN1 subunits of NMDARs and modulates glutamatergic neurotransmission. While loss of GluN1 subunits in mice results in neonatal death due to respiratory failure, animals that lack a d-serine synthetic enzyme, serine racemase (SR), show grossly normal growth. However, SR-independent origins of d-serine in the brain remain unclarified. In the present study, we investigated the origin of brain d-serine in mice. Loss of SR significantly reduced d-serine in the cerebral cortex, but a portion of d-serine remained in both neonates and adults. Although d-serine was also produced by intestinal bacteria, germ-free experiments did not influence d-serine levels in the cerebral cortex. In addition, treatment of SR-knockout mice with antibiotics showed a significant reduction of intestinal d-serine, but no reduction in the brain. On the other hand, restriction of dietary intake reduced systemic circulation of d-serine and resulted in a slight decrease of d-serine in the cerebral cortex, but did not account for brain d-serine found in the SR-knockout mice. Therefore, our findings show that endogenous d-serine of non-SR origin exists in the brain. Such previously unrecognized, SR-independent, endogenous d-serine may contribute baseline activity of NMDARs, especially in developing brain, which has minimal SR expression.

Increased levels of oral Streptococcus-derived D-alanine in patients with chronic kidney disease and diabetes mellitus.

The number of patients on hemodialysis is increasing globally; diabetes mellitus (DM) complications is the major cause of hemodialysis in patients with chronic kidney disease (CKD). The D-amino acid (AA) profile is altered in patients with CKD; however, it has not been studied in patients with CKD and DM. Furthermore, bacteria responsible for altering the D-AA profile are not well understood. Therefore, we examined the D-AA profiles and associated bacteria in patients with CKD, with and without DM. We enrolled 12 healthy controls and 54 patients with CKD, with and without DM, and determined their salivary, stool, plasma, and urine chiral AA levels using two-dimensional high-performance liquid chromatography. We performed 16S rRNA gene sequencing analysis of the oral and gut microbiota to determine the association between the abundance of bacterial species and D-AA levels. Plasma D-alanine and D-serine levels were higher in patients with CKD than in healthy adults (p < 0.01), and plasma D-alanine levels were higher in patients with CKD and DM than in those without DM. The abundance of salivary Streptococcus, which produced D-alanine, increased in patients with CKD and DM and was positively correlated with plasma D-alanine levels. Patients with CKD and DM had unique oral microbiota and D-alanine profiles. Plasma D-alanine is a potential biomarker for patients with CKD and DM.

Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice.

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- mice lacking the activity of d-amino acid oxidase (DAO), relatively high levels of D-Trp were observed (n = 6, %d=27.43 ± 3.26). The obtained %d values of Trp in the urine of the C57BL/6J mice and B6DAO- mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinary D-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsic D-Trp, are expected.

Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry.

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 (CO2) was used. At a low modifier concentration of 30% for the separation of hydrophilic compounds, 18 proteinogenic amino acid enantiomers except glycine and proline were successfully separated with resolution (Rs) = 1.96-33.62 within 6.5 min. In attempt to shorten the analysis time, the flow rate was increased; using a CO2/modifier ratio of 60/40 at a flow rate of 3 mL/min, ultrafast chromatography of 17 amino acid enantiomers, except histidine, was achieved with retention time ≤ 1 min and resolution ≥ 1.5. The developed ultrafast chiral separation method was verified by analyzing a commercially available black vinegar, which detected eight kinds of d-amino acids. The present method has thus confirmed to be successful and practical in terms of both analyte coverage and throughput.

Astrocytic d-amino acid oxidase degrades d-serine in the hindbrain.

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.

Development of an off-line heart cutting two-dimensional HPLC system for enantioselective analysis of serine, threonine and allo-threonine in human physiological fluids.

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).

Protective effect of d-alanine against acute kidney injury.

Recent studies have revealed the connection between amino acid chirality and diseases. We have previously reported that the gut microbiota produces various d-amino acids in a murine acute kidney injury (AKI) model. Here, we further explored the pathophysiological role of d-alanine (d-Ala) in AKI. Levels of d-Ala were evaluated in a murine AKI model. We analyzed transcripts of the N-methyl-d-aspartate (NMDA) receptor, a receptor for d-Ala, in tubular epithelial cells (TECs). The therapeutic effect of d-Ala was then assessed in vivo and in vitro. Finally, the plasma level of d-Ala was evaluated in patients with AKI. The Grin genes encoding NMDA receptor subtypes were expressed in TECs. Hypoxic conditions change the gene expression of Grin1, Grin2A, and Grin2B. d-Ala protected TECs from hypoxia-related cell injury and induced proliferation after hypoxia. These protective effects are associated with the chirality of d-Ala. d-Ala inhibits reactive oxygen species (ROS) production and improves mitochondrial membrane potential, through NMDA receptor signaling. The ratio of d-Ala to l-Ala was increased in feces, plasma, and urine after the induction of ischemia-reperfusion (I/R). Moreover, Enterobacteriaceae, such as Escherichia coli and Klebsiella oxytoca, produce d-Ala. Oral administration of d-Ala ameliorated kidney injury after the induction of I/R in mice. Deficiency of NMDA subunit NR1 in tubular cells worsened kidney damage in AKI. In addition, the plasma level of d-Ala was increased and reflected the level of renal function in patients with AKI. In conclusion, d-Ala has protective effects on I/R-induced kidney injury. Moreover, the plasma level of d-Ala reflects the estimated glomerular filtration rate in patients with AKI. d-Ala could be a promising therapeutic target and potential biomarker for AKI.NEW & NOTEWORTHY 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.

Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV.

BACKGROUND: 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. DESIGN: Case-control analysis. METHOD: 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. RESULTS: 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. CONCLUSION: 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.