Two-dimensional High-performance Liquid Chromatography Research Articles

Validation of An Efficient 2D-HPLC Method for The Determination of Pentazocine

Pentazocine is an opioid analgesic used to treat moderate to severe pains. The real analysis for the pharmaceutical products containing pentazocine is basically to ensure the correct dose and patient safety. This study developed and validated an improved high-performance liquid chromatography (HPLC) method in the selective and accurate quantification of pentazocine. Two-dimensional (2D) HPLC technique is employed to enhance the resolution and selectivity compared to conventional HPLC methods. The 2D-HPLC instrumentation consists of two C18 columns coupling with a switching valve to capture fractions from the first column, which is analyzed in the second column. The mobile phase was optimized to 45% acetonitrile and 55% water with 0.1% phosphoric acid. The method was validated in the International Conference on Harmonization guidelines and shows excellent linearity (R2=0.998), limit of detection of 1.58 μg/L, accuracy of 97.70–102.50%, and precision by relative standard deviation (RSD) of 1.02–4.20%. Selectivity was verified in resolving pentazocine from paracetamol, caffeine, ibuprofen, and oxycodone in laboratory mixtures. The utility of the 2D-HPLC method was demonstrated by accurate quantification of pentazocine in pharmaceutical injections and tablets unaffected in excipients. This research provides a rich validating technique to enhance the quality control testing of pharmaceuticals containing pentazocine.

Targeted Offline Two-Dimensional HPLC and UHPLC-Orbitrap-MS Combined with Molecular Networking Reveal the Effect of Processing on Chemical Constituents of Xuetong (the Stem of Kadsura heteroclita)

Xuetong, the dried stem of Kadsura heteroclita (Roxb.) Craib, is a traditional Tujia medicine extensively used to treat rheumatoid arthritis (RA). All traditional Chinese medicines (TCMs) necessitate a processing stage called “Paozhi” before clinical application. However, there is a dearth of research concerning the processing methods employed for Xuetong. To investigate the impact of vinegar and wine processing on the chemical constituents of Xuetong, this study devised a targeted offline two-dimensional (2D) high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-orbitrap-MS) method. By incorporating various MS data-processing techniques, such as molecular networking technology, fragment-ion similarity searching (FISh), online and offline database matching, and fragmentation pattern analysis, a total of 158 components were identified in Xuetong. Among them, 14 were verified by comparison with the reference standards. Notably, aside from triterpenoids and lignans, catechin derivatives were found to be the predominant constituents of Xuetong, and their levels exhibited a significant decrease following processing. This method significantly improved peak capacity and resolution, overcoming the limitations of 1D LC in simultaneously analyzing highly polar catechin derivatives and less polar triterpenoids and lignans. Moreover, the developed method shows promise for Xuetong’s quality control.

Impact of Preparative Isolation of C-Glycosylflavones Derived from Dianthus superbus on In Vitro Glucose Metabolism.

Dianthus superbus L. has been extensively studied for its potential medicinal properties in traditional Chinese medicine and is often consumed as a tea by traditional folk. It has the potential to be exploited in the treatment of inflammation, immunological disorders, and diabetic nephropathy. Based on previous studies, this study continued the separation of another subfraction of Dianthus superbus and established reversed-phase/reversed-phase and reversed-phase/hydrophilic (RPLC) two-dimensional (2D) high-performance liquid chromatography (HPLC) modes, quickly separating two C-glycosylflavones, among which 2″-O-rhamnosyllutonarin was a new compound and isomer with 6‴-O-rhamnosyllutonarin. This is the first study to investigate the effects of 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin on cellular glucose metabolism in vitro. First, molecular docking was used to examine the effects of 2″-O-rhamnosyllutonarin and 6″-O-rhamnosyllutonarin on AKT and AMPK; these two compounds exhibited relatively high activity. Following this, based on the HepG2 cell model of insulin resistance, it was proved that both of the 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin demonstrated substantial efficacy in ameliorating insulin resistance and were found to be non-toxic. Simultaneously, it is expected that the methods developed in this study will provide a basis for future studies concerning the separation and pharmacological effects of C-glycosyl flavonoids.

D -Alanine Affects the Circadian Clock to Regulate Glucose Metabolism in the Kidney.

Key Points d-Alanine affects the circadian clock to regulate gluconeogenesis in the kidney. d-Alanine itself has a clear intrinsic circadian rhythm, which is regulated by urinary excretion, and acts on the circadian rhythm. d-Alanine is a signal activator for circadian rhythm and gluconeogenesis through circadian transcriptional network. Background The aberrant glucose circadian rhythm is associated with the pathogenesis of diabetes. Similar to glucose metabolism in the kidney and liver, d-alanine, a rare enantiomer of alanine, shows circadian alteration, although the effect of d-alanine on glucose metabolism has not been explored. Here, we show that d-alanine acts on the circadian clock and affects glucose metabolism in the kidney. Methods The blood and urinary levels of d-alanine in mice were measured using two-dimensional high-performance liquid chromatography system. Metabolic effects of d-alanine were analyzed in mice and in primary culture of kidney proximal tubular cells from mice. Behavioral and gene expression analyses of circadian rhythm were performed using mice bred under constant darkness. Results d-Alanine levels in blood exhibited a clear intrinsic circadian rhythm. Since this rhythm was regulated by the kidney through urinary excretion, we examined the effect of d-alanine on the kidney. In the kidney, d-alanine induced the expressions of genes involved in gluconeogenesis and circadian rhythm. Treatment of d-alanine mediated glucose production in mice. Ex vivo glucose production assay demonstrated that the treatment of d-alanine induced glucose production in primary culture of kidney proximal tubular cells, where d-amino acids are known to be reabsorbed, but not in that of liver cells. Gluconeogenetic effect of d-alanine has an intraday variation, and this effect was in part mediated through circadian transcriptional network. Under constant darkness, treatment of d-alanine normalized the circadian cycle of behavior and kidney gene expressions. Conclusions d-Alanine induces gluconeogenesis in the kidney and adjusts the period of the circadian clock. Normalization of circadian cycle by d-alanine may provide the therapeutic options for life style–related diseases and shift workers.

Two-Dimensional High-Performance Liquid Chromatography as a Powerful Tool for Bioanalysis: The Paradigm of Antibiotics.

The technique of two-dimensional high-performance liquid chromatography has managed to gain the recognition it deserves thanks to the advantages of satisfactory separations it can offer compared to simple one-dimensional. This review presents in detail key features of the technique, modes of operation, and concepts that ensure its optimal application and consequently the best possible separation of even the most complex samples. Publications focusing on the separation of antibiotics and their respective impurities are also presented, providing information concerning the analytical characteristics of the technique related to the arrangement of the instrument and the chromatographic conditions.

MP14-04 ESTABLISHMENT OF BLOOD-BASED DIAGNOSTIC MARKERS OF UROTHELIAL CARCINOMA USING CHIRAL AMINO ACIDS

You have accessJournal of UrologyCME1 Apr 2023MP14-04 ESTABLISHMENT OF BLOOD-BASED DIAGNOSTIC MARKERS OF UROTHELIAL CARCINOMA USING CHIRAL AMINO ACIDS Atsunari Kawashima, Akinaru Yamamoto, Sassi Nesrine, Toshihiro Uemura, Tomonori Kimura, Masashi Mita, Toshiki Oka, Yohei Okuda, Gaku Yamamichi, Eisuke Tomiyama, Yu Ishizuya, Yoshiyuki Yamamoto, Taigo Kato, Koji Hatano, Ryoichi Imamura, and Norio Nonomura Atsunari KawashimaAtsunari Kawashima More articles by this author , Akinaru YamamotoAkinaru Yamamoto More articles by this author , Sassi NesrineSassi Nesrine More articles by this author , Toshihiro UemuraToshihiro Uemura More articles by this author , Tomonori KimuraTomonori Kimura More articles by this author , Masashi MitaMasashi Mita More articles by this author , Toshiki OkaToshiki Oka More articles by this author , Yohei OkudaYohei Okuda More articles by this author , Gaku YamamichiGaku Yamamichi More articles by this author , Eisuke TomiyamaEisuke Tomiyama More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Taigo KatoTaigo Kato More articles by this author , Koji HatanoKoji Hatano More articles by this author , Ryoichi ImamuraRyoichi Imamura More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: There are no clinically established blood-based diagnostic biomarkers for urothelial carcinoma patients. Amino acids (AA) have two isoforms of L- and D-AA and D-AA have been believed to be physiologically nonexistent. However, it becomes clear that some D-AA has clinical significance in the human body. Here, we evaluated circulating D-AA levels in the blood and their clinical possibilities as diagnostic biomarkers in urothelial cancer patients. METHODS: We enrolled 345 patients just before surgery for urothelial carcinoma (UC) (n=123), renal cell carcinoma (RCC) (n=143), and healthy donor of kidney transplantation (n=79). We collected 345 serum samples just before surgery and 16 samples one week after surgery. D-AA levels in serum were measured using two-dimensional high-performance liquid chromatography. We evaluated its usefulness as a diagnostic biomarker of UC in the derivation cohort (n=22) and two independent validation cohorts in 254 (Cohort-1) and 69 (Cohort-2) participants, respectively. RESULTS: In the derivation cohort, only four D-AA (D-Asparagine (D-Asn), D-Serine (D-Ser), D-Alanine (D-Ala), and D-Proline (D-Pro)) could be measured in serum samples among 22 kinds of D-AA. D-Asn and D-Ala levels were significantly higher in UC patients compared to RCC patients and healthy donors (p<0.05 and p<0.01, respectively). The area under the curve (AUC) of each D-Asn to diagnose UC was 0.800. Sensitivity and specificity were 100% and 60% using a cut-off level of 0.233 IU/ml. In validation Cohort-1, D-Asn levels had a high accuracy of diagnosis for UC (AUC: 0.851). Sensitivity and specificity were 77.8% and 79.3% using the same cut-off level as the derivation cohort. In validation cohort-2, D-Asn levels had the high accuracy of diagnosis for UC (AUC: 0.836) and its sensitivity and specificity were 61.9% and 81.2% using the same cut-off level as the derivation cohort. Also, serum D-Asn levels of 16 UC patients just after surgery significantly decreased compared to those just before surgery (p=0.005). CONCLUSIONS: D-Asn levels in the blood could become the promising diagnostic markers of UC patients. Source of Funding: This work was supported by a KAKENHI grant (22H03213) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e183 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Atsunari Kawashima More articles by this author Akinaru Yamamoto More articles by this author Sassi Nesrine More articles by this author Toshihiro Uemura More articles by this author Tomonori Kimura More articles by this author Masashi Mita More articles by this author Toshiki Oka More articles by this author Yohei Okuda More articles by this author Gaku Yamamichi More articles by this author Eisuke Tomiyama More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Taigo Kato More articles by this author Koji Hatano More articles by this author Ryoichi Imamura More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

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.

A versatile strategy for high-resolution separation of reducing glycan mixtures as hydrazones by two-dimensional high-performance liquid chromatography

Glycomics analysis has been undermined by the lack of structurally defined individual glycans as model compounds. However, it is challenging to prepare individual glycans from natural resources, mainly due to separation difficulties caused by highly diverse structure, complicated mixture form and chromophore-free property of naturally-existing glycans. In this study, we report a simple, universal and low-cost glycan separation strategy, glycoselection, which allows preparation of individual reducing glycans from their mixtures through reversible chromogenic derivatization by hydrazide chemistry in combination with two-dimensional high-performance liquid chromatography (2D-HPLC). Investigations on reaction conditions using lactose and maltodextrin as model glycans showed the feasibility of reversible hydrazide labeling and one-pot hydrazone conversion under mild conditions, the good stability of hydrazone-form derivatives of glycans in solution and the difference among seven selected hydrazine-carrying chromogenic reagents in product yields during glycan labeling and post-column detagging. The 2D-HPLC separation conditions were established on maltodextrin, from which fourteen highly-purified individual reducing oligo-glucans were ultimately obtained. Using this strategy, we also successfully prepared and identified eleven individual neutral reducing N-glycans from chicken ovalbumin and thirteen individual neutral reducing oligosaccharides and eight individual sialylated ones from human milk, demonstrating its good applicability to different types of reducing glycans as well as biological samples. Given the compatibility of individual reducing glycans with almost all of glycan derivatization protocols and analytical techniques of glycans and the potential of the method for larger scale application, this work provides a universal approach to compound-specific analysis of natural glycans and has great significance for glycomics studies.

Two-Dimensional Chiral HPLC Analysis of Lactate, Hydroxybutyrates and Malate in Human Plasma

For the simultaneous determination of lactate (LA), 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB) and malate (MA) enantiomers, a new separation technique in a two-dimensional high-performance liquid chromatography (2D-HPLC) system following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been developed. The 2D-HPLC system was composed of the reversed-phase (1D) and enantioselective (2D) separations. In the first dimension, the target hydroxy acids were separated as their D plus L mixtures on a Singularity RP18 column (1.0 mm i.d. x 250 mm), and the enantiomers were separated in the second dimension using a polysaccharide-based chiral stationary phase, Chiralpak IG (2.0 mm i.d. x 250 mm). By using the Chiralpak IG column, the LA, 2HB, 3HB and MA enantiomers were separated with resolution values of 3.47, 3.63, 6.81 and 3.72, respectively. The developed 2D-HPLC system was applied to human plasma, and trace levels of D-LA and L-3HB in addition to their major antipodes were determined. The acquired %D of LA (the percentage of D-LA over total LA) and %L of 3HB (the percentage of L-3HB over total 3HB) were 1.0 and 2.7, respectively. For 2HB, only the L-form was detected, while the MA enantiomers were not found in the human plasma.

The Pharmacokinetics/Pharmacodynamics and Neurotoxicity of Tigecycline Intraventricular Injection for the Treatment of Extensively Drug-Resistant Acinetobacter baumannii Intracranial Infection.

ObjectiveThis study aimed to provide feasible suggestions for intraventricular injection of tigecycline to treat intractable Acinetobacter baumannii intracranial infections by studying its pharmacokinetics/pharmacodynamics and neurotoxicity.MethodsA simple and reliable two-dimensional high-performance liquid chromatography (2D-HPLC) method was used to determine tigecycline concentration. The pharmacokinetics (PK) of tigecycline in cerebrospinal fluid (CSF) was investigated by performing therapeutic drug monitoring (TDM). The pharmacodynamics (PD) of tigecycline was evaluated by its minimum inhibitory concentration (MIC) against XDR A. baumannii. CCK8 assay was used to evaluate the cytotoxicity of different concentrations of tigecycline effect on PC12 cells, and apoptosis assay was analyzed by flow cytometry.ResultsTigecycline retention time in 2D-HPLC was 7.636 min. The lower limit of quantitation (LLOQ) was 0.1mg/L, which met the requirements of concentration determination for TDM. The MIC50 and MIC90 values of tigecycline for A. baumannii were 2 and 4 mg/L, respectively. After a dose of 5mg tigecycline, Cmax in CSF was 37.894 mg/L which was high above the MIC values. The t1/2 of tigecycline was estimated to be 2.73 hours. Tigecycline significantly decreased cell viability as assessed and induced apoptosis of the PC12 cell. The IC50 value of PC12 cells treated with tigecycline was about 51.35 mg/L.ConclusionIntraventricular injection of tigecycline is a promising method for treating XDR A. baumannii intracranial infection. Since a high concentration of tigecycline in CSF may have potential neurotoxicity, and the t1/2 was short, giving small doses of less than 5 mg at least twice a day may be safer and more effective. Intraventricular injection of tigecycline must be selected cautiously and best carried out under TDM.

Identification of γ-Glutamyl-Selenomethionine as the Principal Selenium Metabolite in a Selenium-Enriched Probiotic, Bifidobacterium longum, by Two-Dimensional HPLC-ICP MS and HPLC-ESI Orbitrap MS.

Selenium (Se)-enriched probiotics are potential sources of organic Se in the human diet, but their application in food is debated because most selenized probiotics and their metabolites are not well-characterized. We analyzed a Se-enriched probiotic, Bifidobacterium longum DD98, to unveil its Se metabolite profiles by two-dimensional high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP MS) and HPLC-electrospray ionization Orbitrap MS. A major Se metabolite was identified as gamma-glutamyl-selenomethionine (γ-Glu-SeMet), which accounted for 42.5 ± 3.4% of water-soluble Se. Most of the remaining Se was present as SeMet (35.2 ± 0.6%) in a free or protein-bound form. In addition, 11 minor Se metabolites were identified, eight of which had not been reported before in probiotics. Six of the identified compounds contained γ-Glu-SeMet as the core structure, constituting a γ-Glu-SeMet family. This study demonstrates the presence of γ-Glu-SeMet in a probiotic, showing a different selenite metabolite pathway from that of Se-enriched yeast, and it offers an alternative and potentially attractive source of organic Se for food and feed supplementation.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

Measurement of glomerular filtration rate using endogenous d-serine clearance in living kidney transplant donors and recipients.

BackgroundEndogenous molecules that provide an unbiased and a precise evaluation of kidney function are still necessary. We explored the potential of clearance of d-serine, a rare enantiomer of serine and a biomarker of kidney function, as a measure of glomerular filtration rate (GFR).MethodsThis was a cross-sectional observational study of 200 living kidney transplant donors and recipients enrolled between July 2019 and December 2020 in a single Japanese center, for whom GFR was measured by clearance of inulin (C-in). Clearance of d-serine (C-dSer) was calculated based on blood and urine levels of d-serine, as measured by two-dimensional high-performance liquid chromatography. Analytical performance was assessed by calculating biases. Utilizing data from 129 participants, we developed equations for C-in based on C-dSer and C-cre using a linear regression model, and the performance was validated in 68 participants.FindingsThe means of C-in and C-dSer were 66.7 and 55.7 mL/min/1.73 m2 of body surface area, respectively, in the entire cohort. C-dSer underestimated C-in with a proportional bias of 22.0% (95% confidence interval, 14.2–29.8%) and a constant bias of -1.24 (-5.78–3.31), whereas the proportional bias was minor to that of C-cre (34.6% [31.1–38.2%] and 2.47 (-1.18–6.13) for proportional and constant bias, respectively). Combination of C-dSer and C-cre measured C-in with an equation of 0.391 × C-dSer + 0.418 × C-cre + 3.852, which reduced the proportional bias (6.5% [-0.2–13.1%] and -4.30 [-8.87–0.28] for proportional and constant bias, respectively). In the validation dataset, this equation performed well with median absolute residual of 3.5 [2.3–4.8], and high ratio of agreement (ratios of 30% and 15% different from C-in [P30 and P15] of 98.5 [91.4–100] and 89.7 [80.0–95.2], respectively).InterpretationThe smaller proportional bias compared to that of C-cre is an advantage of C-dSer as a measure of C-in. Combinational measurement of d-serine and creatinine, two endogenous molecules, has the potential to serve as a measure of GFR with precision and minor biases and can support important clinical decisions.FundingJapan Society for the Promotion of Science (JSPS, grant number 17H04188), Japan Agency of Medical Research and Development (AMED, JP20gm5010001), Osaka Kidney Bank (OKF19-0010), Shiseido Co., Ltd and KAGAMI Inc.

Characterization of a New Impurity in Metaraminol Bitartrate for Injection by 2D HPLC-Q/TOF-MS, Chemical Synthesis and NMR

Background: Impurities in pharmaceutical compounds can influence their clinical effects and represent a potential health risk. To ensure the safety and effectiveness of a drug, it is necessary to investigate potential impurities. Methods: In this paper, a new impurity was separated and characterized by two-dimensional high performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (2D HPLC-Q/TOF-MS) in negative electrospray ionization mode. The peak containing the new impurity, eluted from the first dimension chromatographic system, was selectively trapped by a switching valve based on its retention time and transferred to the second dimension chromatographic system, which was connected to the mass spectrometer. We obtained MET-TA by chemical synthesis, and its structure was characterized by MS/MS and further confirmed by nuclear magnetic resonance (NMR). Results: The impurity was found to be (2S, 3S)-2,3.-dihydroxy-4-((1R,2S)-1-hydroxy-1-(3-hydroxyphenyl) propan-2-yl)amino)-4-oxobutanoic acid, labelled as MET-TA. In this study, we investigated the mechanism of formation of MET-TA, and found that it was the amidation product of metaraminol and tartaric acid. Conclusions: The identification, structural elucidation, synthesis and most probable mechanism of formation of MET-TA are discussed in detail in this paper.

Dynamic Landscape of Extracellular Vesicle-Associated Proteins Is Related to Treatment Response of Patients with Metastatic Breast Cancer.

Breast cancer is the leading cause of cancer death in women. The majority of these deaths are due to disease metastasis, in which cancer cells disseminate to multiple organs and disrupt vital physiological functions. It is widely accepted that breast cancer cells secrete extracellular vesicles (EVs), which contain dynamic molecular cargo that act as versatile mediators of intercellular communication. Therefore, Evs. secreted by breast cancer cells could be involved in the development of metastatic disease and resistance to treatment. Moreover, changes in EV cargo could reflect the effects of therapy on their parent tumor cells. The aim of this feasibility study was to quantitatively profile the proteomes of Evs. isolated from blood samples taken from treatment sensitive and resistant metastatic breast cancer patients to identify proteins associated with responses. Three serial blood samples were collected from three patients with metastatic breast cancer receiving systemic therapy including a responder, a non-responder, and a mixed-responder. Evs. were isolated from plasma using size exclusion chromatography and their protein cargo was prepared for tandem mass tag (TMT)-labelling and quantitative analyses using two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. After filtering, we quantitatively identified 286 proteins with high confidence using a q value of 0.05. Of these, 149 were classified as EV associated candidate proteins and 137 as classical, high abundant plasma proteins. After comparing EV protein abundance between the responder and non-responder, we identified 35 proteins with unique de-regulated abundance patterns that was conserved at multiple time points. We propose that this proof-of-concept approach can be used to identify proteins which have potential as predictors of metastatic breast cancer response to treatment.

Identification of Diabetic Nephropathy in Patients Undergoing Kidney Biopsy through Blood and Urinary Profiles of d-Serine.

The diagnosis of diabetic nephropathy (DN), the major cause of ESKD, requires kidney biopsy. d-Serine, present only in trace amounts in humans, is a biomarker for kidney diseases and shows potential to distinguish the origin of kidney diseases, whose diagnoses usually require kidney biopsy. We extended this concept and examined the potential of d-serine in the diagnosis of DN. We enrolled patients with biopsy sample-proven DN and primary GN (minimal change disease and IgA nephropathy) and participants without kidney disease. A total of 388 participants were included in this study, and d-serine levels in blood and urine were measured using two-dimensional high-performance liquid chromatography, and urinary fractional excretion (FE) of d-serine was calculated. Using data from 259 participants, we developed prediction models for detecting DN by logistic regression analyses, and the models were validated in 129 participants. A d-serine blood level of >2.34 μM demonstrated a high specificity of 83% (95% CI, 70% to 93%) for excluding participants without kidney diseases. In participants with a d-serine blood level >2.34 μM, the threshold of 47% in FE of d-serine provided an optimal threshold for the detection of DN (AUC, 0.85 [95% CI, 0.76 to 0.95]; sensitivity, 79% [95% CI, 61% to 91%]; specificity, 83% [95% CI, 67% to 94%]). This plasma-high and FE-high profile of d-serine in combination with clinical factors (age, sex, eGFR, and albuminuria) correctly predicted DN with a sensitivity of 91% (95% CI, 72% to 99%) and a specificity of 79% (95% CI, 63% to 80%), and outperformed the model based on clinical factors alone in the validation dataset (P<0.02). Analysis of d-serine in blood and urinary excretion is useful in identifying DN in patients undergoing kidney biopsy. Profiling of d-serine in patients with kidney diseases supports the suitable treatment through the auxial diagnosis of the origins of kidney diseases.

D-Serine Mediates Cellular Proliferation for Kidney Remodeling.

d-serine, a long-term undetected enantiomer of serine, is a biomarker that reflects kidney function and disease activity. The physiologic functions of d-serine are unclear. The dynamics of d-serine were assessed by measuring d-serine in human samples of living kidney donors using two-dimensional high-performance liquid chromatography, and by autoradiographic studies in mice. The effects of d-serine on the kidney were examined by gene expression profiling and metabolic studies using unilateral nephrectomy mice, and genetically modified cells. Unilateral nephrectomy in human living kidney donors decreases urinary excretion and thus increases the blood level of d-serine. d-serine is quickly and dominantly distributed to the kidney on injection in mice, suggesting the kidney is a main target organ. Treatment of d-serine at a low dose promotes the enlargement of remnant kidney in mouse model. Mechanistically, d-serine activates the cell cycle for tissue remodeling through an mTOR-related pathway. d-serine is a physiologic molecule that promotes kidney remodeling. Besides its function as a biomarker, d-serine has a physiologic activity that influences kidney function.

Proteomic Analysis of Circulating Extracellular Vesicles Identifies Potential Biomarkers for Lymph Node Metastasis in Oral Tongue Squamous Cell Carcinoma.

Lymph node metastasis is the most reliable indicator of a poor prognosis for patients with oral tongue cancers. Currently, there are no biomarkers to predict whether a cancer will spread in the future if it has not already spread at the time of diagnosis. The aim of this study was to quantitatively profile the proteomes of extracellular vesicles (EVs) isolated from blood samples taken from patients with oral tongue squamous cell carcinoma with and without lymph node involvement and non-cancer controls. EVs were enriched using size exclusion chromatography (SEC) from pooled plasma samples of patients with non-nodal and nodal oral tongue squamous cell carcinoma (OTSCC) and non-cancer controls. Protein cargo was quantitatively profiled using isobaric labelling (iTRAQ) and two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. We identified 208 EV associated proteins and, after filtering, generated a short list of 136 proteins. Over 85% of the EV-associated proteins were associated with the GO cellular compartment term “extracellular exosome”. Comparisons between non-cancer controls and oral tongue squamous cell carcinoma with and without lymph node involvement revealed 43 unique candidate EV-associated proteins with deregulated expression patterns. The shortlisted EV associated proteins described here may be useful discriminatory biomarkers for differentiating OTSCC with and without nodal disease or non-cancer controls.

Intra-body dynamics of d-serine reflects the origin of kidney diseases

Introductiond-Serine, present only in trace amounts in humans, is now recognized as a biomarker of chronic kidney disease (CKD). CKD is heterogeneous in its original kidney diseases, whose diagnoses require kidney biopsy. In this study, we examined whether the intra-body dynamics of d-serine, indexed by its blood and urinary levels, reflects the origin of kidney diseases.MethodsPatients with six kinds of kidney disease undergoing kidney biopsy were enrolled in a single center. Levels of d- and l-serine were measured using two-dimensional high-performance liquid chromatography. The associations between the origin of kidney diseases and the intra-body dynamics of d-serine were examined using multivariate cluster analyses.ResultsUnlike the non-CKD profile, patients with CKD showed broadly-distributed profiles of intra-body dynamics of d-serine. The plasma level of d-serine plays a key role in the detection of kidney diseases, whereas a combination of plasma and urinary levels of d-serine distinguished the origin of CKD, especially lupus nephritis.ConclusionIntra-body dynamics of d-serine have the potential to predict the origin of kidney diseases. Monitoring of d-serine may guide specific treatments for the origin of kidney diseases.

Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application

Objective To establish a two-dimensional high-performance liquid chromatography method for the determination of tigecycline in human cerebrospinal fluid, which can be used for the drug monitoring in patients with intracranial infection. Methods The quantification was carried out by an external standard method. The first-dimension column was a Aston SNX5 phenyl chromatographic column (50 mm×4.6 mm, 5 μm) with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.5)-methanol (45∶55, V/V) as the mobile phase and the flow rate was 1.2 ml/min. The second-dimension chromatographic column was Aston SC5 C18 (275 mm×4.6 mm, 5 μm), with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.4)-ammonium phosphate (pH was adjusted with ammonium hydroxide to 3.0)- acetonitrile (30∶50∶20, V/V/V) as the mobile phase and the flow rate was 1.0 ml/min. The detection wavelength was 340 nm. The temperature was 40 ℃ and the injection volume was 200 μl. Results The calibration curve of tigecycline showed good linearity from 64.5 to 1 290.0 ng/ml in human cerebrospinal fluid (r=0.999 8). The RSD of intra and inter-day precision were less than 5.0% with the detection accuracy of 98.80%−106.51%. Conclusion This method is simple, quick, accurate, specific and sensitive. It meets the requirements of tigecycline determination in clinical human cerebrospinal fluid, which offers the individualized therapeutic assurance for patients with intracranial infection.